key: cord-331421-rioeke67 authors: valentowitsch, johann title: flattening the covid-19 curve: the impact of contact restrictions on the infection curve in germany date: 2020-07-22 journal: gesundheitswesen doi: 10.1055/a-1194-4967 sha: doc_id: 331421 cord_uid: rioeke67 although germany is coping well with the coronavirus crisis, many voices are currently being raised that fundamentally question the success of the contact restriction strategy to contain the virus. i show in this study that there is no justification for such criticism. in fact, contact restrictions have flattened the infection curve and were possibly decisive for the good german performance in the crisis. in many parts of the world, germany is seen as a model and exemplary country in the fight against the new cornavirus. compared to the rest of the world, germany has one of the lowest case fatality rates and handles the coronavirus crisis very well [2] . germany also seems to have got the infection rate under control relatively quickly. according to estimates by the robert koch institute, the effective basic reproductive number dropped below the critical mark just a few weeks after the outbreak of the disease [6] . in addition to the good german health care system, the massive political crackdown of social life is held responsible for the rapid success in disease control. in order to stop the spread of the coronavirus, german politics has largely shut down large parts of public and cultural life. schools and daycare centers were closed, companies were asked to send their employees home, and extensive contact restrictions were put in place -in short: public life was torn out of normality and, wher ever possible, put into an artificial deep sleep. however, from an economic point of view, the consequences of the social lockdown are gigantic and cannot be compared to any other crisis in the post-war period. economists recently predicted a drop in german gross domestic product up to 10 % this year [3] . in the end, the social lockdown will most likely rob many people of their economic existence and drive many companies into bankruptcy. against this background, voices were rasied early, just like in other countries that pursue a strategy similar to germany, which called for a rapid end to the drastic measures or even questioned the usefulness of the contact restriction strategy as a whole. thus, given the current situation, it is important to know how effectively the lockdown actually helps to contain the epidemic. based on this question, i will show that the contact restriction strategy has fundamentally not missed its target. even if many people question the success of the strategy, social distancing has actually led to a massive reduction in the infection rate and thus made it possible to flatten the infection curve. according to the results of my estimate, the infection rate in germany would be many times higher without contact restrictions. the temporary lockdown of social life was therefore an important and correct step to contain the disease. in this study, i look at the daily infection rate in germany and, based on a set of plausible basic reproduction numbers, estimate how the infection rate would have developed had the social lockdown not occurred. i therefore estimate a counterfactual scenario based on the actual infection dynamics that could be observed in the first weeks before the first political interventions. for the calibration of the estimate i use the number of cases documented by the robert koch institute, an independent german fe-deral authority for infectious diseases and non-communicable diseases. in my study, the calibration sample began at the end of february with the appearance of the first local outbreaks, which, unlike in january, could no longer be completely controlled by the authorities through quarantine measures. the end of the calibration sample marks march 30 th . the first political decisions that worked towards a comprehensive restriction of social contacts in germany can be dated back to march 16 [3] . for my investigation, i assumed that the effects of the corona regulations on the case number statistics will manifest themselves about 2 weeks later. i am guided by the official assessment of the robert koch institute, according to which the case statistics in germany document the situation around 2 weeks later [6] . the robert koch institute gives 2 main reasons for the delay in statistics. on the one hand, in most cases it takes about 5-7 days between being infected and going to the doctor, and on the other hand, another 7 days pass from the laboratory detection of the infection to the notification of the illness to the robert koch institute. in germany, the federal organization of health authorities can be held responsible for the comparatively long transmission of infection numbers. the german health authorities first aggregate the data at the level of individual regional districts and only then report the case numbers to the robert koch institute in berlin [6] . for the counterfactual simulation of the disease development from march 30 th , i used a branching process model based on the renewal equation [1] . the methodological approach used in this study builds on nouvellet et al. [4] . the model simulation is based on the estimation of the serial interval, which represents the duration between symptom onset of a secondary case and that of its primary case, and the estimation of the basic reproduction number, which describes the expected number of cases directly generated by one case in a population where all individuals are susceptible to infection. the serial interval was assumed to be gamma distributed with parameters taken from the literature. initial studies indicate a serial interval between 4 and 8 days for the novel coronavirus [5] . the basic reproduction number for the simulation estimates was also borrowed from the literature. the world health organization estimates that the basic reproductive count for the new type of coronavirus would be between 2 and 2.5 without state intervention [7] . based on this, each infected person would have to infect about 2 more people with the virus on average, which would lead to an exponential increase in the number of cases. simulation results are summarized in ▶ fig. 1 . as can be seen from the illustration, the social lockdown broke the infection line and greatly weakened the exponential increase in the number of infections. without political intervention, the number of infections should have been 20 to 50 times higher than at the current level. these days, not only in germany, there is much controversy about the usefulness of government interventions in social life. the debate is often emotional and is much more based on guesswork than fact. i have shown that there is currently no reason to doubt the effectiveness of the social lockdown measures. based on the results of my analysis, it is obvious that contact restrictions are contributing to the slowing down of the virus epidemic. nevertheless, the question must be asked how long such measures can be maintained without the economy collapsing or acceptance of the population disappearing. moreover, the social lockdown effect may not prove to be sustainable because people do not want to stick to the strict rules for long and neglect the risk of infection over time. however, since the disease is not yet completely contained, such negligence can overturn the positive development of infection dynamics at any time and quickly bring the development of case numbers back to the point of exponential increase. germany is currently well positioned in the coronavirus crisis, which can be attributed not least to the effect of the official contact restriction measures. other countries that decided social distancing too late or simply did not consider it as a real option for action are now much worse off and have significantly increased mortality rates. while the utilization of intensive care beds did not yet reach its capacity limit in german hospitals even at the height of the crisis, triage decisions had to be made elsewhere and patients had to be ventilated selectively [2] . the german special route, of which the rest of the world often speaks with admiration, is actually not very puzzling in itself. rather, the current situation in germany testifies to the effectiveness of the measures taken at an early stage, the success of which, however, is fragile and prone to setbacks. estimating individual and household reproduction numbers in an emerging epidemic predicting the prevalence of covid-19 pandemic in germany an economic policy exit strategy from the corona lockdown a simple approach to measure transmissibility and forecast incidence a systematic review of covid-19 epidemiology based on current evidence lagebericht des rki zur coronavirus-krankheit-2019 (covid-19) report of the who-china joint mission on coronavirus disease 2019 (covid-19) the authors declare that they have no conflict of interest. key: cord-027027-2vxnmiyj authors: schartau, patricia; kirby, mike title: male mortality and the german response: lessons from covid‐19 date: 2020-06-04 journal: nan doi: 10.1002/tre.752 sha: doc_id: 27027 cord_uid: 2vxnmiyj the current covid‐19 outbreak has raised many questions, amongst them the higher mortality rates in men and the low overall mortality rates in germany compared to other european countries. here the authors explore some of the reasons behind both these phenomena and outline what we can learn from them for the future. o n average, men die younger and are at higher risk of life-threatening ailments, including heart disease and many forms of cancer. the sars-cov-2 coronavirus appears to be following suit. in all six of the countries that, up to 20th march, had sex-specific records of deaths from covid-19, the proportion of men was higher than women. over time this was confirmed by data collected by global health 50/50 (may 6th) in countries that had a high covid-19 caseload; with death rates of 62% men and 38% women in italy, 58% and 42% (respectively) in spain, and 80% and 20% (respectively) in greece. 1 in a very recent (29th april) preliminary study published in frontiers in public health, 2 beijing researchers explored the role of gender in morbidity and mortality of a small sample of 43 patients with a covid-19 diagnosis. they concluded that while men and women have the same prevalence of infection, men with covid-19 are at higher risk for worse outcomes and death, independent of their age. the authors further investigated the data of 37 patients who had died of covid-19 from the chinese public health science data centre and found that the number of men who died was 2.4 times that of women. given the small sample sizes, no generalisation of the results is possible at this stage; however, the widely noted tendency that men are more severely affected by covid-19 and have a higher mortality rate than women holds. 2 uk data collected and analysed from 1st february-25th april 2020 confirmed (amongst other factors) a male hazard ratio of 1.99 in covid-19 related deaths when compared to females. 3 there are possible biological and behavioural explanations for this trend, as discussed in this issue's 'journal watch', which include: hormones, the immune system, genetics and the fact that older men have more (severe) comorbidities than their female counterparts. male lifestyle factors, such as a higher ratio of smoking and alcohol intake, lower compliance with handwashing advice, and delays in presentation, may play a role in explaining the above findings. evidently, poor hand hygiene in men could expose them more frequently to the virus, and to a higher viral load, which in turn can affect severity of the infection. a uk survey of >2000 adults from march 2020 found that both men and women had taken steps to protect themselves from the virus by improving their personal hygiene such as hand washing; however, fewer men (67%) than women (74%) had up-scaled their hand washing routine. 4 more research is required in order to fully explore why men are more vulnerable to covid-19. a greater in-depth understanding of the underlying biological and behavioural processes will help to inform targeted measures -a form of precision medicine where the goal is to better understand sex and gender differences in disease and drug response in order to tailor preventive measures and treatment. however, it must be remembered that covid-19 affects all of us significantly, regardless of sex and gender. in the next section, we will move to discuss how governmental and public the current covid-19 outbreak has raised many questions, amongst them the higher mortality rates in men and the low overall mortality rates in germany compared to other european countries. here the authors explore some of the reasons behind both these phenomena and outline what we can learn from them for the future. no there has been much debate about why germany has one of the lowest fatality rates in europe. as per the 11th may 2020, 171 879 cases of covid-19 have been identified and 7569 deaths registered, giving a fatality rate of 4.4%. as shown in figure 1 , this compares with, for example, fatality rates of the uk (14.53%) and france (15.04%). 5 as it happens, one of the authors (ps) of this article was in the small town of landsberg for a meeting on the day when germany's first covid-19 case was recorded there: the patient was a male who worked for a company that has two car plants in wuhan in china. the author spent the following days in munich and was particularly struck by the speed at which the local health department and federal authorities acted. within a couple of days of the first case being recorded, contacts had been identified and quarantined, and the company closed its bavarian plant in addition to the ones based in china in order to contain the outbreak. the public was informed locally and nationally about the case and measures were taken without any delay. this early action is an example of the coordinated and rapid response that defines how the german authorities have dealt with the current covid-19 situation. the german healthcare system has been persistently modernised over the last 20 years, which certainly put germany in a good position to deal with the covid-19 pandemic. the result of this was more hospital beds, more ventilators, more intensive care unit (icu) beds and more hospital doctors per capita than any other comparable country in europe. 6, 7, 8 in addition, community specialist practices and a dense network of primary care physicians provided a strong backbone to support hospital care during the outbreak. germany was one of the first countries to initiate so-called social distancing measures. this allowed the early shielding of the elderly population while a meticulous tracing of the chain of infections was undertaken in order to suppress spread. the government made optimal use of the time available after the first cases emerged in southern germany, upscaling bed capacity, joining the ppe eu procurement scheme, and mobilising the diagnostics industry that was already well established. furthermore, the government listened to scientific advisors and drew them in early to the decision-making process. as mentioned, in germany the diagnostics infrastructure was readily in place and scaled up upon the emergence of covid-19 cases. this allowed germany to become one of the first countries to develop a reliable covid-19 test as early as january, and to initiate widespread testing. from early on, for example, germany tested >20 people per thousand (as compared to the uk, which tested 5.54 per 1000). 9 in the bmj, 10 christian drosten, virologist at the berlin university hospital charité, highlighted that in germany testing is done across an array of quality-controlled labs rather than relying on a central lab for all processing and testing. as a result, early widespread testing resulted in up-to-date analysis of current infection trends and timely countermeasures, increased identification of mild cases and, therefore, a lower overall case fatality rate. in germany, regular national updates were held where the current situation was summarised, scientific evidence discussed, and the rationale for decisions laid open to the public. 11, 12 broader society, possibly as a result of the transparent communication strategy, mostly followed the measures implemented or recommended by the government. however, there is admittedly no reliable source of data to suggest that this was adhered to more (or less) in germany as compared to other european countries. an additional reason for the robust management of the covid-19 crisis by the german healthcare system up to this point is the digitalisation that has taken place, particularly over the past two years and more rapidly over the past weeks and months. this has started to move germany to a partially digitalised system. at the end of last year, the german government passed the digital care act (dca). 13 this enables, amongst others, an expansion of telemedicine to all medical specialties and affiliated healthcare professionals -offered free of charge in the covid-19 related context. the german government developed strategies to encourage production and widespread usage of ce-certified covid-19 chatbots and triage apps by established authorities (for example, the robert-koch-institut), all of which happened within days and weeks. digital prescribing is following suit. in light of the dca and covid-19, a vibrant german digital health ecosystem has emerged rapidly, covering anything from screening, prevention and diagnosis to treatment and rehabilitation. and many of these companies have switched to a free service in covid-19 times. 13 the german national health innovation hub that was formed a couple of years ago by the ministry of health has incorporated an up-to-date covid-19 section on their website that summarises, signposts and evaluates digital applications relating specifically to covid-19 and distributes this in a daily newsletter. 14 furthermore, in search for covid-19 solutions, the german government organised a covid-19related hackathon (an event where people collaborate to try and initiate solutions to technical problems within a certain time frame, in this case within 48 hours) in march. this led to the selection of 20 out of 1500 projects aiming, for example, to optimise national ppe distribution, the delivery of food and medicines, communication relating to infection control, and local business support, to name but a few. 15 over the last months, germany has been catching up fast with other countries, such as the uk, where digital healthcare solutions such as telemedicine and digital prescribing were already well embedded prior to covid-19. in addition to developing digital health interventions and system optimisations, researchers and developers in the uk and across europe have been working to determine whether existing technologies can be given new applications to assist with the pandemic. examples of such innovation include: mobile phone location data to predict disease spread and the impact of interventions such as social distancing; robots designed to clean hospitals; drones to deliver food to patients; health tracker apps to monitor for potential covid-19 symptoms; new equipment designs, such as the ventilator challenge uk consortium to provide more than 10 000 ventilators; the involvement of private industry experts from formula one, dyson, and 3d printer companies to develop new devices in record time; and the use of ai algorithms for speedy drug development. 16, 17, 18 the successful management of covid-19 is clearly multifactorial and will change as more deaths across europe are unfortunately inevitable. nevertheless, we can conclude that up to this point, germany's covid-19 management has been exemplary and there is a lot to learn from the country's healthcare, communication and policy strategies. across the world, once the pandemic starts to subside, further comparative and collaborative analysis of strategies and policies is required in order to better prepare us globally for future pandemics. what is evident from the current situation is that an effective future response requires the sharing of information and collaboration across nations. mike kirby has received funding from the pharmaceutical industry for research, travel and educational initiatives; however, no funding was directly related to the writing of this article. gender differences in patients with covid-19: focus on severity and mortality gender differences in patients with covid-19: focus on severity and mortality opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients most americans are worried about covid-19-but not republicans covid-19) death rate in countries with confirmed deaths and over 1,000 reported cases as of the countries with the most critical care beds per capita leads europe in hospital bed capacity. statista germany has a low coronavirus mortality rate: here's why what can data on testing tell us about the pandemic covid-19: why germany's case fatality rate seems so low the leader of the free world gives a speech, and she nails it making the fight against the coronavirus pandemic sustainable here to stay: digital health in times of covid-19 -a german deep dive corona -sars cov 2 -covid-19 the #wirvsvirus hackathon. wirvsvirus china's tech fights back ventilator challenge uk to start production in covid-19 fight ai-designed drug to enter human clinical trial for first time key: cord-337037-xpj17vn4 authors: weigel, ralf; krüger, carsten title: global child health in germany time for action date: 2020-10-09 journal: global health action doi: 10.1080/16549716.2020.1829401 sha: doc_id: 337037 cord_uid: xpj17vn4 child health is central to the sdg agenda. universities in the uk and other european countries provide leadership in research and education for global child health to inform related policy and practice, but the german contribution is inadequate. german paediatricians and other child health professionals could make more substantial contributions to the debate at home and internationally, but lack opportunities for scholarship and research. we argue, that there is a momentum to advance global child health in academia and call on german universities to realise this potential. 'viruses don't need visas, pathogens don't need passports' -the world health organization (who) director-general's urgent message to the participants of the world health summit in berlin in 2017 is more relevant today than ever [1] . the impact of the sars-cov-2 pandemic on children is a powerful reminder in this regard [2] and other threats are looming [3, 4] . germany, like other highincome countries, is a beneficiary of globalisation. however, benefits come with responsibilities: as a signatory of the sustainable development goals (sdg) 2016-2030, germany committed to advance health globally [5] . child health and well-being are central to the sdg agenda illustrating our responsibility for future generations [6, 7] . unfortunately, global child health in germany is somewhat neglected in research and education. we need a major effort to improve the situation. in germany, global child health institutions and the scientific debate are still in their infancy compared to other european countries. in the uk (uk), the centres at the university college london, the london school of hygiene and tropical medicine, the liverpool school of tropical medicine, and other universities have active research groups in global child health as an integral part of maternal, newborn, child and adolescent health. the royal college of paediatrics and child health annual meetings regularly devote entire days to global child health research and training. global child health topics regularly feature in the college's scientific journal. universities in italy, the netherlands, norway and sweden have institutes dedicated to international maternal and child health. at the universities in utrecht, london and liverpool, under-and postgraduates can attend various courses on global child health. in sweden, the institute for global health transformation initiated a multidisciplinary forum hosted by the royal swedish academy of sciences, which resulted in a roadmap on global child health with five priority areas in the context of the sdgs [8] . although germany has successful research groups in maternal and child public health that collaborate internationally, for example at the universities in hamburg, heidelberg and munich, there is no such overarching forum to share ideas, to develop strategies and to provide direction. it is the private witten/ herdecke university that has the only professorship for global child health, funded by the friede springer foundation [9] . the german society for tropical paediatrics and international child health (gtp) is a professional society established almost 40 years ago with about 400 current members which brings together paediatricians with different backgrounds at its annual meetings and offers a range of trainings, but its mandate for research is limited [10] . the academic global child health landscape in germany is fragmented, without a dedicated chair at a statefunded university and with little collaboration between different actors. however, there are also deeper and more systemic reasons why german global health research and education as a whole are underdeveloped [11, 12] . for contact ralf weigel ralf.weigel@uni-wh.de friede springer endowed professorship for global child health, witten/herdecke university, witten 58448, germany example, the abuse of public health by the nazi regime for their racial hygiene policies and atrocities descredited the field and left a stain that still affects perceptions today [13, 14] . currently, public health research is concentrated at several federal institutions, such as the robert koch institute (rki) and the federal centre for health education (bzga). but, compared with universities, their scholarly role is limited. at the local level, public health interventions are implemented by public health offices that have no formal academic role [15] . furthermore, global health policy programmes in germany are distributed over six ministries and international health programmes are funded to a large degree not by the ministry of health but the federal ministry for economic cooperation and development. its main implementers, the society for international cooperation (giz) and the kreditanstalt für wiederaufbau (kfw), a promotional bank owned by the state, have little focus on academic research and education. thus, the historical heritage, and the policy and funding structure appear to be barriers that may have contributed to the slow development of an academic base in global health in general [16] and global child health specifically. within this historical and structural context and with weakly organised public or global health institutions, it is not surprising that german paediatricians are hard to find in scientific landmark publications, guidelines and reports of global relevance. in the 108page global strategy 2016-2030 for the health of women, children and adolescents [17] , a groundbreaking document for the global health of mothers and children, no german name is found in the recognition and author lists, similar to the 16 review articles in the bmj special issue 2015, which provides the scientific background for the strategy [18] . of the 471 organisations that contributed to the development process of the strategy, only five came from germany [19] . the same applies to the who publications 'standards to improve the quality of care for mothers and newborn babies in health care institutions' from 2016 [20] and 'standards to improve the quality of care for children and adolescents in health care institutions' from 2018 [21] . among the authors from more than 100 institutions, only three and seven, respectively, are from germany, and only in one case from a paediatric professional society. similarly, of the institutions involved in 'the 2019 report of the lancet countdown on health and climate change: ensuring that the health of a child born today is not defined by a changing climate', 10 come from the us, 11 from the uk, five from other european countries, including one from germany, and five from other, non-european countries [4] . although this lack of representation is not necessarily a sign of a lack of participation in the international scientific debate, the few opportunities german researchers have to engage in global child health research and education at universities suggest that this is, in fact, the case. without academic leadership, a lively exchange of ideas, a research agenda and funding, it is hard to participate and to be heard. without global child health institutes, students and young researchers have few opportunities and academic career prospects, preventing them from engaging in research and applying for funding. our research and educational institutions need to provide a better environment for child health professionals that they can move the global scientific and policy debate forward and contribute more substantially to the global research agenda. many opportunities exist for paediatricians and other health workers caring for children to engage with the realities of global child health in research and education. for example, in 2015/2016, some 350,000 children and their families came to germany to seek refuge, many of them vulnerable with multiple risks and in urgent need of health care [22, 23] . their physical and mental health needs and strategies to meet them are important to share [24, 25] . what are the enablers and barriers to their integration in the health care and education system, viewed from a child rights perspective [26] ? germany's development cooperation focus on health systems strenghtening offers further opportunities. the initiative 'hospital partnerships -partners strengthen health' financed by the federal ministry for economic cooperation and development and the else kröner-fresenius foundation, supports 181 projects with institutions from 51 low-and middle-income countries, several of them focusing on mother and child health [27] . the german academic exchange service (daad) has helped to establish 28 cooperations between universities in germany and low-and middle-income countries with its 'partnership for health care in developing countries' programme [28] , some addressing maternal and child health. rigorous evaluation of the short and long term effects of interventions implemented within these partnerships, for example on human resources or on child health outcomes, would also make a substantial contribution to the field. it is time for german universities to use this potential to strengthen research and education in global child health -there is momentum to realise this. the sars-cov-2 pandemic has fuelled a debate of how social determinants, such as access to education, affect health, well-being and development of children in germany and elsewhere [29, 30] . children are leading in advocacy for their own for their right to health in the context of climate change, holding world leaders accountable in the fridays-for-future movement. the experiences of families while educating their children at home during lock-downs due to the pandemic as well as the voices of children concerned about climate change are making headlines in the media [31, 32] . this may represent an opportunity to leverage global child health concepts, such as social and environmental determinants of health and child rights, higher on the policy and research agenda. as germany is updating its global health strategy, receiving valuable advice from various professional organisations [33, 34] , global child health has to become a core element of this strategy, building on and developing further existing initiatives. a recent discussion paper, published by the commission for global child health of the german academy for child and adolescent medicine (dakj), listed several recommendations for improving the landscape of global child health research and education [35] . in addition, the german society of tropical paediatrics and international child health and the named dakj commission will continue to lobby for the inclusion of global child health into the planned german centre for child health, funded by the federal ministry of education and research [36] . and the recently founded global health hub germany [37] and the german alliance for global health research [38] are also prime opportunities for building institutional capacity. to date, the global child health agenda has had limited visibility in germany. we call on the academic leadership of paediatric professional societies in germany to provide a forum for the scientific and political aspects of global child health, to provide leadership and to lobby for funding from the government. paediatric researchers should respond more actively to calls from multilateral agencies like who [39, 40] and make public their positions on issues such as child rights [41] . medical faculties need to strenghten their academic base by offering under-and postgraduate education in global child health through institutes and chairs so that students and young researchers see a path for their careers. we must now seize the opportunities unfolding for urgently needed engagement in this important field in research and education. german universities can and should play a much more active part in advancing the health and well-being of children throughout the world. viruses don't need visas, pathogens don't need passports early estimates of the indirect effects of the covid-19 pandemic on maternal and child mortality in low-income and middle-income countries: a modelling study climate change and global child health: what can paediatricians do? the 2019 report of the lancet countdown on health and climate change: ensuring that the health of a child born today is not defined by a changing climate united nations department of economic and social affairs. the sustainable development goals report placing children and adolescents at the centre of the sustainable development goals will deliver for current and future generations a future for the world's children? a who-unicef-lancet commission swedish institute fo global health transformation. a new roadmap on global child health germany's expanding role in global health german society for tropical paediatrics and international child health global health education in germany: an analysis of current capacity, needs and barriers global health research and education at medical faculties in germany results presented of the research project confronting the past: contemporary german paediatric response to medical practice in the third reich statutory health insurance in germany: a health system shaped by 135 years of solidarity, self-governance, and competition germany's expanding role in global health every women every child. the global strategy for women's, children's and adolescents health towards a new global strategy for women's, children's and adolescents' health participating organizations: fhi360 standards for improving quality of maternal and newborn care in health facilities geneva standards for improving the quality of care for children and young adolescents in health facilities accumulated environmental risk in young refugees-a prospective evaluation recommendations for the diagnosis and prevention of infectious diseases in pediatric and adolescent refugees in germany: statement of the german society of pediatric infectious diseases, the society of tropical pediatrics and international child health mental health needs of refugee children in specialized early education and care programs in germany immunization coverage among refugee children in berlin unaccompanied refugee minors in germany: attitudes of the general population towards a vulnerable group else kröner fresenius-stiftung. initiative hospital partnerships pagel -partnerships for the health sector in developing countries covid-19 and its impact on child and adolescent psychiatry -a german and personal perspective children and adolescents in the covid-19 pandemic: schools and daycare centers are to be opened again without restrictions. the protection of teachers, educators, carers and parents and the general hygiene rules do not conflict with this after two years of school strikes, the world is still in a state of climate crisis denial we swallowed our misgivings'; 2020 statement of the international advisory board on global health: global health centre, the graduate institute of international and development studies deutsche gesellschaft für public health deutschland und sein engagement für die gesundheit der kinder weltweit federal ministry of education and research. startschuss für zwei neue deutsche zentren der gesundheitsforschung deutsche gesellschaft für internationale zusammenarbeit (giz) gmbh. global health hub germany berlin charité global health. german alliance for global health research berlin who hospital care for children guidelines: what do users need? new who standards for improving the quality of healthcare for children and adolescents the budapest declaration for children and youth on the move-comment in the lancet child and adolescent health we thank william christopher buck for proofreading the manuscript. rw wrote the draft manuscript, which ck reviewed. both authors read and approved the final version. rw holds the friede springer endowed professorship for global child health at the witten/herdecke university. ck is currently the chairperson of the german society of tropical paediatrics and international child health and the spokesperson of the committee of global child health of the german academy of child and adolescent medicine. not applicable. the authors have no funding to report. this call to action addresses child health professionals and stakeholders to engage in research and education for global child health at germany's higher education institutions. universities should realise the momentum and recognise the importance of global child health to enable substantial contributions to the scientific and policy debate at the national and global levels. http://orcid.org/0000-0001-9034-2634 carsten krüger http://orcid.org/0000-0001-7936-7689 key: cord-028201-x57bhyhr authors: platz, thomas; bender, andreas; dohle, christian; gorsler, anna; knecht, stefan; liepert, joachim; mokrusch, thomas; sailer, michael title: german hospital capacities for prolonged mechanical ventilator weaning in neurorehabilitation – results of a representative survey date: 2020-07-01 journal: neurol res pract doi: 10.1186/s42466-020-00065-1 sha: doc_id: 28201 cord_uid: x57bhyhr a brief survey among members of the german neurorehabilitation society aimed to document the hospital capacities (“beds”) for prolonged weaning from a mechanical ventilator for patients with neuro-disabilities that require simultaneous multi-professional neurorehabilitation treatment. sixty-eight institutions declared to have capacities with a broad distribution across germany and its federal states. overall, 1094 “beds” for prolonged weaning (and neurorehabilitation) were reported, 871 together with further information regarding their identification and hence regional location. these units had on average 16.1 beds for prolonged weaning (95% confidence interval 12.6 to 19.6) with a range from 2 to 68 beds per organization. the data indicate substantial capacities for the combined prolonged weaning and neurorehabilitation treatment in germany. for most “beds” included in this analysis a basic validation was possible. while a reasonable coverage of these specialized service capacities by the survey is likely, the number reported could still be biased by underreporting by non-response. both the broad variation of number of “beds” for prolonged weaning per unit and their unequal geographical distribution across federal states (per capita rate) warrant a more refined follow-up survey that will provide insights into reasons for the observed pattern of variation for these specialized hospital capacities. weaning is the medical process of withdrawing ventilator support. prolonged weaning describes a situation of initial weaning failure, i.e. when more than three spontaneous breathing trials (sbt) or 7 days from the first sbt are required, and hence prolonged weaning care [1] . in specialized pulmonologic weaning centers, about 50% of all patients with initial weaning failure can be liberated from mechanical ventilation [2] . however, a substantial subset of patients in need for prolonged weaning treatment is also affected by neurodisabilities and requires the combination of prolonged weaning treatment and multi-professional neurorehabilitation to address their various needs for improving both their health, body functions, and autonomy with activities of daily living [5] . in a german cohort, 26% of 754 patients admitted for "early neurorehabilitation" were on mechanical ventilation commencing their neurological rehabilitation; their weaning rate from mechanical ventilation was 65% during their stay [4] . while there is a considerable need for such a specialized combined service with proven effectiveness, there is a lack of knowledge about such hospital capacities that are currently available in germany. the german neurorehabilitation society (deutsche gesellschaft für neurorehabilitation, dgnr e.v.) conducted a survey among its members to document hospital capacities ("beds") for prolonged weaning for patients with neuro-disabilities that require simultaneous multiprofessional neurorehabilitation treatment ("neuro-weaning beds"). by email invitation end of december 2019 and a repeated invitation at the beginning of january 2020, 381 members of the dgnr were invited to participate in a short online survey. they were asked to answer two questions hosted on the platform invote.de (provided by netzmanufaktur gmbh, theaterstraße 4, 01067 dresden): 1. number of "neuro-weaning beds" in their hospital 2. combined question to indicate number of "neuroweaning beds", organizational background (acute care hospital versus rehabilitation facility), name of the hospital, and name of head of department. by repeating the question for number of beds and by asking for more detailed (identifying) information, the validation of entries was sought to be promoted. in addition, society members were encouraged to make sure within their hospital by contact with their head of department that data entry was provided only once per hospital to prevent reporting in duplicate. all entries were screened for validity. based on hospital name and head of department name the location of each unit within one of 16 german federal states was coded. the number of "neuro-weaning beds" per federal state was divided by population statistics for that state as published by the german federal agency for statistics [7] to obtain the rate of "neuro-weaning beds" per 1.000.000 inhabitants. descriptive statistics were generated using the software package sas. sixty-nine of a total of 123 survey respondents indicated that their hospital provides capacities for combined prolonged weaning and neurorehabilitation ("neuro-weaning beds"). one entry was regarded as "invalid entry": the entry stated "135" (beds) without further information and was not used for the descriptive statistics (as stated below). the 68 remaining units had a total of 1094 "neuroweaning beds", on average 16.1 beds (95% confidence interval 12.6 to 19.6) with a range from 2 to 68 beds per organization. given a total population of 82.792 thousand inhabitants in germany [7] this statistic would imply a capacity of 13.2 "neuro-weaning beds" per 10 6 inhabitants. as a sensitivity analysis we further analyzed the subset of data from units that gave more detailed (identifying) information (n = 57). collectively these units reported 871 "neuro-weaning beds" with on average 15.3 beds for prolonged weaning (95% confidence interval 11.7 to 18.8) and a range from 2 to 68 beds per organization. three of these units were specialized for health care in children and adolescents (22 beds), two units for people with spinal cord injury (12 beds). the (subset of) units that reported "neuro-weaning beds" together with their identification served as basis to describe their distribution across federal states in germany (see table 1 ). this representative survey indicated substantial hospital capacities for combined prolonged weaning and neurorehabilitation with a total of 1094 "neuro-weaning beds" in germany. for 871 of these "neuro-weaning beds" identifying information was available supporting the survey's validity. as with any voluntary survey, there is a relevant risk of underreporting. thus, the true number of "neuroweaning beds" in germany is likely to be higher than the one reported here. the number of "neuro-weaning beds" within individual reporting hospitals varies considerably (from 2 to 68 beds) with an average of 16 beds and a 95% confidence interval ranging from 12 to 19. this indicates both a central tendency for organizational size and substantial differences in organizational settings. the analysis of the geographical distribution across federal states of germany was based on the 80% of "neuro-weaning beds" reported with identifying information. hence, this analysis suffers from an "incomplete data" bias and absolute numbers should be interpreted with great caution. the data nevertheless points to a huge variability of population-based density of "neuroweaning beds" (per 10 6 inhabitants for german federal states) in germany. the survey generated a crude estimate of hospital capacities ("beds") for prolonged weaning from a mechanical ventilator for patients with neuro-disabilities that require simultaneous multi-professional neurorehabilitation treatment. the substantial variability in size of units and their geographical distribution warrants a more refined follow-up survey to learn about the setting and organizational structures of such units before further conclusions can be drawn. however, this survey already confirms the high relevance of these "neuro-weaning" capacities for the recovery from breathing failure and hence the avoidance of long-term intensive home care. the number of people in need for invasive long-term ventilation in germany dramatically increased over the past 15 years to an estimate of currently 20.000 patients, implying additional health care costs of around 4 billion euros per year [3] . it is estimated that approximately 10.000 patients with neuro-disabilities in need for weaning from mechanical ventilation can be taken care of each year with the capacity of 1094 "neuro-weaning beds" of this survey. given a success rate of 65 to 75% [4, 6] , they collectively might prevent an estimated 7000 new cases of long-term ventilation per year, let alone the other neurorehabilitation achievements in terms of disability reduction and re-gaining autonomy with activities of daily living. weaning from mechanical ventilation s2k guideline "prolongiertes weaning tremendous increase of home care in ventilated and tracheostomized patients -reasons, consequences, solutions rehabilitationsverlauf von patienten in der neurologisch-neurochirurgischen frührehabilitation: ergebnisse einer multizentrischen erfassung im jahr 2014 in deutschland. nervenarzt prolonged weaning during early neurological and neurosurgical rehabilitation: s2k guideline published by the weaning committee of the german neurorehabilitation society (dgnr) factors influencing weaning from mechani-cal ventilation in neurological and neurosurgical early rehabilitation patients kapitel 2 bevölkerung, familien springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the help of katherina rupp, medical documentation assistance is gratefully acknowledged. authors' contributions tp collected, analyzed, and interpreted the data regarding "neuro-weaning beds" and wrote a first draft of the manuscript. all authors read and edited the manuscript, and approved the final manuscript.authors' information thomas platz acts as president of the dgnr, christian dohle as president elect, thomas mokrusch as past president. this work was supported by the bdh bundesverband rehabilitation e.v. (charity for neuro-disabilities) by a non-restricted personal grant to tp. the sponsors had no role in the decision to publish or any content of the publication. the datasets generated during this survey are not publicly available, since no consent to share the hospital-based information has been obtained. confidential inspection of the data is possible at the site of the corresponding author on reasonable request.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord-252343-a85wz2hs authors: skoda, eva-maria; teufel, martin; stang, andreas; jöckel, karl-heinz; junne, florian; weismüller, benjamin; hetkamp, madeleine; musche, venja; kohler, hannah; dörrie, nora; schweda, adam; bäuerle, alexander title: psychological burden of healthcare professionals in germany during the acute phase of the covid-19 pandemic: differences and similarities in the international context date: 2020-08-07 journal: j public health (oxf) doi: 10.1093/pubmed/fdaa124 sha: doc_id: 252343 cord_uid: a85wz2hs background: healthcare professionals (hps) are the key figures to keep up the healthcare system during the covid-19 pandemic and thus are one of the most vulnerable groups in this. to this point, the extent of this psychological burden, especially in europe and germany, remains unclear. this is the first study investigating german hps after the covid-19 outbreak. methods: we performed an online-based cross-sectional study after the covid-19 outbreak in germany (10–31 march 2020). in total, 2224 hps (physicians n = 492, nursing staff n = 1511, paramedics n = 221) and 10 639 non-healthcare professionals (nhps) were assessed including generalized anxiety (generalized anxiety disorder-7), depression (patient health questionnaire-2), current health status (eq-5d-3l), covid-19-related fear, subjective level of information regarding covid-19. results: hps showed less generalized anxiety, depression and covid-19-related fear and higher health status and subjective level of information regarding covid-19 than the nhps. within the hp groups, nursing staff were the most psychologically burdened. subjective levels of information regarding covid-19 correlated negatively with generalized anxiety levels across all groups. among hps, nursing staff showed the highest and paramedics the lowest generalized anxiety levels. conclusions: in the context of covid-19, german hps seem to be less psychological burdened than nhps, and also less burdened compared with existing international data. the covid 19 pandemic reached germany in late february 2020. it brought not only objective medical challenges for healthcare professionals (hps), but also reports and findings from other more affected countries. due to exponentially increasing case numbers and large numbers of patients requiring intensive care, those more affected countries are facing unexpected challenges. countries such as china, italy, spain, brasil and the usa were and are currently reaching the limits of their healthcare systems in the context of this pandemic: something that was previously unimaginable in industrialized countries. 1 such a development seems to have been avoided in germany but is not completely ruled out for the future. in the face of an ever-renewing european and a further worldwide escalation, there is no shortage of uncertainty and concern among hps. it is already known from countries other than germany that hps are under elevated psychological stress during the covid-19 pandemic and show increased levels of various psychometric values, including anxiety and depression. 2-5 existing evidence, e.g. from china, already shows the extent of the psychological burden on hps. front-line healthcare workers were identified as bearing a particularly heavy psychological burden. 2,6 however, these studies were conducted during the extreme stress phase of the covid-19 epidemic in china. only few data in the context of other studies suggest that, e.g. in the uk, a heightened psychological burden for the hps may exist. 7 there is, as yet no comparable data, especially from a time when the health system is still mainly coping normally, alongside already population-wide uncertainty, particularly in europe. the german situation to this point is 2-fold: continuing and past restrictions in public life, contact restrictions, empty supermarket shelves and daily updated increasing case numbers are still coupled with a hospital system that is and was largely still able to cope normally. this is combined with mortality rates, which are, for the moment, low when compared internationally. 8, 9 though, the german population shows itself already burdened in terms of generalized anxiety, depression and distress, which is in line with evidence from other countries, 10,11 customized low-threshold interventions, offline as well as online, are needed and already implemented. [12] [13] [14] the aim of this study was to close the research gap and provide initial findings on psychological burden of german hps after the covid-19 outbreak. it is hypothesized that the group of hp in germany will mirror the existing, population-wide elevated psychological burden 15 to an even greater extend by being in the 'front line', as already could be observed in previous studies in other countries. 2,3 a nationwide, online-supported cross-sectional survey was conducted. participants were recruited via online channels and official channels e.g. websites of clinics. the survey period was from the 10-31 march 2020. it was during this period that the first increased numbers of covid-19 cases in germany, increasingly restrictive government regulations, the closure of european borders and the restriction of individual freedoms occurred. in total, 12 863 people completed the questionnaire, of which we identified 2224 people in the medical sector as hps and 10 639 as non-healthcare professionals (nhps). hps were from three different groups: physicians, nursing staff and paramedics. the sample description can be seen in table 1 . all participants gave their written consent to participate in the survey and the evaluation of the collected data. the study was conducted in accordance with the ethical guidelines from the declaration of helsinki and was approved by the local ethics committee of the faculty of medicine. details of general socio-demographic variables were asked. validated psychometric instruments were used to assess psychological burden. the generalized anxiety disorder-7 (gad-7) to measure generalized anxiety symptoms over the course of the last 2 weeks (gad-7, 7 items, 4-point likert scale meaning 0 = never to 3 = nearly every day), 16 the patient health questionnaire-2 (phq-2) to screen for depression symptoms over the course of the last 4 weeks (phq-2, 2 items, 4-point likert scale meaning 0 = never to 3 = nearly every day) 17 and the visual analogous scale of the euroqol eq-5d-3l scale to assess current health status (ranging from 0 [worst imaginable health status] to 100 [best imaginable health status]). 18 additionally, based on scientific and media reports, multiple items and item scales were formed in expert consensus with regard to 'covid-19-related fear' (one item, 7-point likert scale meaning 1 = very low to 7 = extremely high), 'the subjective level of information regarding covid-19' (3 items: i feel informed about covid-19; i feel informed about measures to avoid an infection with covid-19; i understand the health authorities' advice regarding covid-19. seven-point likert scale, meaning 1 = complete disagreement to 7 = complete agreement). scale reliability for was tested using cronbach's α for internal consistency. 'the subjective level of information regarding covid-19' showed high internal consistency (cronbach's α = 0.801). the descriptive and inferential statistics were performed with r3.6.1 (r core team, 2019). sum scores for the gad-7 and phq-2 and mean scores for all other scales were calculated. to assess the hypotheses, the 95% confidence of the association measures are reported; for each difference between the groups after having assessed the global mean difference in the respective scale. hence, the assumptions were assessed based on their precision. [19] [20] [21] generally, test statistics and p values are not reported given that at this sample size even the slightest deviation from equivalence results in extremely low p values. when the confidence interval (ci) of the effect size covers 0, we assume there is no effect. as soon as this is the case, we use the guidelines by sawilowsky 22 to evaluate the importance of the effect; a cohen's d ∼0.2 is considered a small, a d ∼0.5 is considered medium-sized and d ∼0.8 is regarded as large effects. due to the large sample size and the intuitive and common interpretation of the effect sizes, parametric methods were also used for violation of the normality assumption. 23 for mean comparisons welch's t-test with the cohen's d association measure was used, for multiple mean comparisons and between-subject analysis of variance with the association measure η 2 with subsequent t-tests for post hoc comparisons with tukey error correction. a complete summary of all post hoc group comparisons after calculation of the variance analyses and post hoc tests can be assessed in the supplementary materials. to clear the association of subjective level of information regarding covid-19 and other variables, spearman correlations between variables were performed. to subsequently test the interdependence of variables a robust linear mestimator regression was performed (rlm from the r package mass, 2002). all spearman correlations including confidence between the measures are provided in the supplemental material. following the results of the correlation analyses, prevalence ratios for the amount of participants with moderate generalized anxiety in relation to the subjective level of information regarding covid-19 were explored. levels of generalized anxiety were divided by using the gad-7 sum score of ≥10 24 as a split into low levels of generalized anxiety (<10) and moderate to high levels of generalized anxiety (≥10). this was compared with a pre-covid-19 standard population, where 5.9% of the population scored above ≥10. 25 the subjective level of information regarding covid-19 was split by the median into high (≥median) and low (<median) levels. prevalence ratios were calculated using unconditional maximum likelihood. cis were calculated using normal approximation (via the r-packages epitools, 2020). concerning generalized anxiety measured by the gad-7, differences between the different hp groups and between the hps and the nhps could be found (η 2 = 0.009, ci = subjective level of information regarding covid-19 correlates with generalized anxiety. the association of high or low subjective levels of information regarding covid-19 and low or moderate to high generalized anxiety (cutoff of ≥10) is shown in table 2 . the proportion of moderately anxious participants regardless of level of information is 15.99% in the nhps, 4.25% in physicians, 11.41% in nursing staff and 4.55% in paramedics. the probability of reaching the pathological gad-7 cutoff score of ≥10 thus diminishes across almost all hp groups and the nhps with the degree of subjective level of information regarding covid-19 (fig. 3) . for physicians, nursing staff and others, the prevalence of having generalized anxiety when sufficiently informed is critically reduced (with cis of the prevalence ratio deviating significantly from 1). however, reduced moderate generalized anxiety in participants with high subjective levels of information can also observed when the sample is analyzed as a whole. assessing the psychological burden and covid-19-related concerns of people working in the healthcare system is of high relevance in the light of the covid-19 pandemic events. to this point, this study is the first to assess those points in the german healthcare system. in this study, nhps reported overall higher levels of psychological burden than the hps, which is particularly pronounced in generalized anxiety and depression scores. within the hp groups there was, in relative terms, less psychological burden on physicians and paramedics than on nursing staff. health status was better in the hp groups and within those, physicians reported the best health status, followed by paramedics. hps also showed less covid-19-related fear than nhps, paramedics showing the least fear. subjective levels information regarding covid-19 was high overall hps and nhps. high levels of subjective levels of information regarding covid-19 correlated negatively with high levels of generalized anxiety symptoms globally. in the group of individuals with low subjective levels of information regarding covid-19, 17.88% of the nhps, 8.60% of the physicians, 14.69% of the nursing staff and 5.00% of the paramedics scored above the cutoff of ≥10, indicating the presence of gad symptoms. even without the division in subjective levels of information regarding covid-19, still 15.99% of the nhps, 5.89% of the physicians, 11.41% of the nursing staff and 4.55% of the paramedics scored above this cutoff. especially concerning the nhps and the nursing staff, this is a massive elevation of prevalence in comparison to a pre-covid-19 sample, where only 5.9% of the population scored above this cutoff. 25 the psychological burden of hps during the covid-19 pandemic has already been investigated in the early hotspots of the pandemic, especially in china and southeast asia. high psychological burden of front-line workers could be found in china 4-6,26 and other countries (e.g. tan et al . 3 ). in detail, at the height of the crisis in china, 50.7% of healthcare workers showed symptoms of depression, 44.7% those of generalized anxiety and 73.4% showed increased stress symptoms. 27 the psychological response of hps during epidemics has already been investigated during other virus outbreaks, e.g. sars and h1n1, drawing similar pictures. 28,29 a clear picture concerning the hps in europe, especially in the beginning of the virus outbreak still does not exist. in the present study, it was possible to show a rare picture of a healthcare system in the early outbreak stages of a pandemic in the western world. during this phase of the outbreak in germany, in which uncertainty and anxiety prevailed but covid-19 cases and death rates were still moderate and low, hps were less psychologically burdened than the nhps and less psychologically burdened than comparable hp groups in other countries. [3] [4] [5] [6] 26 this poses the question why the german hps coped and cope so well in the light of the current pandemic and in comparison to the nhps. the finding that physicians in the current situation are among the least burdened by generalized anxiety and depression can maybe be understood by the fact that generalized anxiety was most correlated with the subjective level of information regarding covid-19. this may explain why physicians and hps show lower overall levels of generalized anxiety and covid-19related fear, which also is in line with findings from tan et al . 3 where nonmedical healthcare workers had higher prevalence of anxiety than medical healthcare workers. this, in a second step, points to the inevitable need for the population to be informed transparently and evidence based in order to experience the covid-19 pandemic with a low level of fear as possible. 15, 30 the present findings of this study also urge especially western countries to conduct still more research concerning the psychological burden of their hps. existing literature is majorly consisting of studies from southeast asia and during the early, dramatic scenes of the pandemic. to meet the needs of the hps, especially as soon as the situation worsens again or a much feared 'second wave' hits europe, the mental health of hps will have to be given high priority in supporting measures. 31 limitations need to be considered. data were obtained via an anonymous online, self-reported questionnaire in a crosssectional study design. a potential selection bias may exist. during the short survey period, a series of various governmental restrictions were put into place, which may represent different manifestations of psychological states at different points in time. data were acquired during a rather early period of the outbreak, which poses the risk of an under-or even overestimation of psychological burden. on the other hand, this exceptional early assessment period might be a major strength of posed study-a thus large sample as the present on the beginning of the current outbreak is scarce and adds a piece to the covid-19 puzzle in order to gain a clearer picture of the unprecedented covid-19 pandemic. in conclusion, hps in germany show less psychological burden compared with hps in other countries and compared with the general population in germany after the outbreak of covid-19. in the investigated sample, nursing staff seems to be the most vulnerable group for mental health burden during the covid-19 pandemic, whereas a high subjective level of information seems to be associated with less psychological burden. supplementary data mentioned in the text are available to subscribers in pubmed online. this study was supported by the essen university medicine foundation. the funder had no role in the design and conduct of the study; management, collection, analysis and interpretation of the data; preparation, review or approval of the manuscript and decision to submit the manuscript for publication. the authors declare that they have no competing interests. covid-19 in europe: the italian lesson psychological support in times of covid-19: the essen community-based cope concept an e-mental health intervention to support burdened people in times of the covid-19 pandemic: cope it techniques, methods, and dissemination of community based psychological support strategies in times of the covid-19-pandemic not all world leaders use twitter in response to the covid-19 pandemic: impact of the way of angela merkel on psychological distress, behaviour and risk perception validation and standardization of the generalized anxiety disorder screener (gad-7) in the general population detecting and monitoring depression with a two-item questionnaire (phq-2) euroqol-a new facility for the measurement of healthrelated quality of life heuristic thinking and inference from observational epidemiology sifting the evidence-what's wrong with significance tests? moving to a world beyond new effect size rules of thumb the importance of the normality assumption in large public health data sets a brief measure for assessing generalized anxiety disorder: the gad-7 psychometric evaluation of the generalized anxiety disorder screener gad-7, based on a large german general population sample the mental health of medical workers in wuhan, china dealing with the 2019 novel coronavirus online mental health services in china during the covid-19 outbreak psychological impact of severe acute respiratory syndrome on health workers in a tertiary hospital long-term psychological and occupational effects of providing hospital healthcare during sars outbreak history in a crisis -lessons for covid-19 timely mental health care for the 2019 novel coronavirus outbreak is urgently needed key: cord-318900-dovu6kha authors: pitschel, t. title: sars-cov-2 proliferation: an analytical aggregate-level model date: 2020-08-22 journal: nan doi: 10.1101/2020.08.20.20178301 sha: doc_id: 318900 cord_uid: dovu6kha an intuitive mathematical model describing the virus proliferation is presented and its parameters estimated from time series of observed reported covid-19 cases in germany. the model replicates the main essential characteristics of the proliferation in a stylized form, and thus can support the systematic reasoning about interventional measures (or their lifting) that were discussed during summer and which currently become relevant again in some countries. the model differs in form from elementary sir models, but is contained in the general kermack-mckendrick (1927) model. it is maintained that (compared to elementary sir models) the model is more faithfully representing real proliferation at the instantaneous level, leading to overall more plausible association of model parameters to physical transmission and recovery parameters. the main policyoriented results are that (1) mitigation measures imposed in march 2020 in germany were absolutely necessary to avoid health care resource exhaustion, (2) fast response is key to containment in case of renewed outbreaks. a model generalization aiming to better represent the true infectiousness profile is stated. construction of the model has been motivated in course of the analysis of intensive-care capacity expenditure to be expected from sector-specific lifting of restrictions. this former analysis used a budget-oriented argument to arrive at an indicative estimate of the resource expenditure, but did not analyze dynamics. (concretely it assumed a constant rate of new infections.) a reasonable question to be posed is: if a sector was allowed to reopen, what would the trajectory of infections actually look like, when surely it is not a linear increase? further, can parameters of the local transmission behaviour be derived from the aggregate observed numbers? in the present text, a model capturing the dynamics of the number of infections is developed towards answering these questions. it deliberately contains only few parameters and is in fact not designed to a specific stage of the virus proliferation. though models for tracing the trajectory of infectious diseases exist, for example the intuitive sir model [ken56] which is formulated as a system of scalar differential equations, we believe that physically more realistic descriptions are possible, which, moreover, lead to increased accuracy of estimated parameters. we assume a homogeneous set of individuals which act as unwitting agents in the proliferation. we assume that infection spreads probabilistically from the infected (and still contagious) individuals to any other individual of the set, wherein we assume that each individual is connected randomly to others, but such that all individuals approximately have an equal number of neighbours (=: a1). (in graph-theoretic terminology, the graph of contacts between individuals is a random undirected graph where each node has about the same edge degree d.) no other assumptions are imposed on the global topology of interconnections. individuals who were once infected cannot be infected again (=: a2). finally, an assumption here made is that contagiousness lasts only for a duration t c , i.e. an infected individual is contagious for the period [0, t c ] after its infection and then not at all afterwards (a3). this simplified characteristic is motivated by results on infectiousness found in epidemiological and clinical investigations: in [hlwea20] , infection incidence data of the wuhan area is examined and combined with clinical data to derive an infectiousness profile which has most of its weight located at about 7 consecutive days around the symptom onset 1 . [wcg + 20] recorded viral rna load data in sputum, throat swab and stool and report of nine patients viral peak loads of 2.35 · 10 9 copies per ml sputum, declining rapidly starting from the first day of presentation in almost all patients, decreasing to 10 5 copies per ml within about 10 to 16 days after symptom onset. (a level of below 10 5 copies per ml sputum, combined with no symptoms and past day 10, has been regarded as warranting discharge of the patient from clinical care with ensuing home isolation.) [ttl + 20] (fig 2) report viral load in posterior oropharyngeal saliva samples decreasing monotonously to below 10 4 copies per ml in day 21 after symptom onset, for the majority of 20 non-intubated patients (out of n = 23). changes in population size due to non-disease effects will be ignored, instead n will be considered constant; similarly the changes in proliferation characteristic due to disease-related reduction of the population will be deemed negligible. assumptions a1 to a3 will be "baseline" assumptions throughout the text and substantial deviations from them will be discussed in the appendix only. we aim for a numerical formulation of the aggregate evolution in which the randomness is averaged out. for this, let n be the number of agents, and let at t = 0 the number of infected agents x(t) be given as x 0 < n . before t = 0, the number of infected agents shall be zero. to develop the model incrementally, lets momentarily assume that all infected agents are contagious infinitely long. in a unit time interval, all infected agents are deemed to infect each of respectively d other neighbours -stochastically independently -with probability p. the expected total number of virus receivers, per unit time interval, then is x(t) · d · p. but not all receivers get infected because some are already infected. the share of non-infected receivers among all agents is (1 − x(t)/n ); therefore the expected number of new infections in unit time is approximating the model evolution as continuous process even at small time intervals 1 caution in the usage of numbers from pure incidence analysis is required: as consequence of the way the raw data is obtained in [hlwea20] , only infectiousness around the moment of symptom onset is in fact fully observed. this is because earlier transmission are likely usually not properly associated to the real primary case because the primary case does not show symptoms yet. later transmissions are simply inhibited because the primary case is put into quarantine. an epidemiological analysis of incidence data alone therefore necessarily is insufficient to determine "pure" infectiousness. to emphasize the distinction between "pure"/"medical" infectiousness and infectiousness after taking into account the population's sociocharacteristics (household structures, current mitigation policies), it is worthwhile to call the density of the latter an "infection incidence profile". (reasonable given the size of the numbers involved), one concludes, under assumption of infinitely enduring contagiousness, that x(t) followṡ for t ≥ 0, with x((−∞, 0)) = 0 and x(0) = x 0 . obviously the function x(t) is non-decreasing. for incorporating the finite duration contagiousness, one determines the number of contagious individuals as the difference of the accumulated number of infected at time t minus the accumulated number of infected prevailing at the earlier time t − t c , because that share of agents had the infection already for at least duration t c , and will cease to be infectious at t. consequently, the expected total number of virus receivers is refined towards the model with the finite duration contagiousness thus reads, in expectation,ẋ with initial conditions as before. both differential equations respectively have a unique solution. 2 the here presented model is not representable by the elementary sir models that involve only instantaneous evaluations of the state variables on the right-hand side of the differential equation, as e.g. equation (2) in [ken56] (see [zml + 20] for a current example of its usage). it is therefore also necessarily different for example from [mb20] . the reason for this is a restriction imposed by such formulations, namely that the individual's transition from infection to recovery is modelled using a rate of transition proportional to the number of infected individuals, which corresponds to a stochastic recovery occurrence and yields an exponential decay characteristic on average. it is known however that, in reality, the sars-cov-2 shows a rather deterministic disease progression with regards to infectiousness in time, leading to end of the infectiousness after about two to three weeks after begin of infection, based on cell culture (see earlier citations). this clinically supported characteristic is properly represented in equation (2), but not in elementary sir models. on the other hand the here presented model is conceptually contained in the original (i.e. general) compartmental model of kermack and mckendrick [km27] (which involves a formulation using integrals; see comments in [bra17] also), for example by setting there ψ = 0. 3 this holds also for the refinement given in section b. the advantage of the here given formulation is that it allows for a mathematically relatively simple description while still fully allowing accomodation of the infectiousness characteristic in generalized form. this simplicity gives some room to incorporate other, hithertho 2 instead of considering only a temporally finite and uniform infectiousness, more detail can be incorporated into the differential equation using a convolution term, as shown in appendix b. unconsidered, effects into the model and still retain a model complexity which is amenable to simulation for parameter identification. 3 analysis of the model and exploratory simulation for later simulation, it is helpful to make use of the scale invariances inherent in the above differential equations. if one denotes the equation (2) parametrized with d · p and t c and n and initial value x 0 as "ode(dp, t c , n, x 0 )", then we have the following fact: if t → x(t) is a solution to ode(dp, t c , n, x 0 ), then t → x(at) is a solution to ode(a · dp, t c /a, n, x 0 ) for any a > 0. this means we can restrict analysis for example to t c = 1 and vary only d · p and x 0 . the other scale invariance is described by "x(·) solution of ode(dp, t c , n, instead of a further analytical proceeding, the above equation's evolution was examined via computer simulation, for various parameter choices d · p and initial values. the purpose is first to explore the general (i.e. not real-data matched) behaviour of equation (2) (next subsection), then to fit the parameters to observed real data (section 4). throughout it was used n = 1.0, t c = 1 and a (forward euler) discretization step size of 0.01 (corresponding to resolution=100 in code). the below discusses general features of the model and its behaviour under parameter variations. this is for demonstration only, and arguments on the proliferation phenomena should be taken as schematic. (whether the phenomena occur in the real parametrization is to be discussed in section 4.) fig 1a shows the evolution behaviour for some arbitrary but temporally constant parameter set. the most striking feature at this graph is that the number of infections asymptotically does not reach the total number n of agents. rather, the limit is a value x(∞) < n which depends on the d · p and the initial value. for comparison, the evolution of the number of infections as would arise when observing eqn. (1) [with same d · p parameter] is depicted as grey dashed line; in it, the x(t) converges to n independent of the choice of d · p. (in subsequent text, this will be referred to as "bounded exponential growth".) the reason for including this curve here and in following graphs is that it can give a hint on trajectories of future viruses that may have a much more extended infectiousness interval. in fact, this curve would result if infected individuals remained infinitely long infectious and were not quarantined. in simulations, the dependence of the limit x(∞) on d · p appeared to be generally overproportional (see fig 1b) . this is well-known behaviour also in the instantaneous-state models. on the other hand, the dependence of the limit on x 0 was linear or sub-linear. in instantaneous-state models, the limit does not depend on the size of the initiating jump of noteworthy is (in both cases) that even though the same number of exogenously infected was used as initially, the contagion effect is much smaller. the reason for this is that already about one fifth of the population had been infected (thus was immune in this model). 6 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.20.20178301 doi: medrxiv preprint we use here the number of reported covid-19 cases (as aggregated by the robert-koch-institut [1]) as a proxy for the number of infections in germany. 4 we fit parameters for the interval until beginning of may 2020, assuming that the evolution proceeded within two different parameter regimes: first a d · p corresponding to no restrictions, then a d · p corresponding to the restrictions posed by contact disencouragement and store closure. (the observational interval used for parameter estimation does cover only a few days of the time of obligatory indoor face mask wearing.) we can derive parameters and based on them predict the trajectory of infections way forward. because of the simplicity of the examined model, there is the risk of a high model error existing. therefore, at the present state of this text, such estimation can only serve to determine reasonable bounds on the parameters of the model, rather than to give a reliable forecast of expect number of eventual infections. fitting of parameters is here conducted manually, focussing on moments in the time series that are indicative of parameter changes. at the beginning of april 2020, the number of weekly new covid-19 cases stood at about 40000 in germany. if we regard the modelling time unit to correspond to a real duration of 2 weeks (implying that each individual newly infected is non-contagious two weeks after and onwards), then we have a new-infections rate of 80000 individuals per such time unit which corresponds to an increment of approximately ∆x = 0.001 per unit time after normalizing to n = 1.0. identifying the moment which was one week after the initial wider lock-down in germany (i.e. around 29th march) as moment t = 3 in the modelling, parameters consequently need to be fitted such thatẋ(3.0+) = 0.001 (green line). (the t = 3.0 also implies that the model assumes around 6 weeks of initial evolution under a low-restrictions scenario, which matches the timeline of the outbreak in germany approximately.) fig 3a shows the trajectory of the system evolution using initially d·p = 1.42 and switching to d·p = 0.7 afterwards. fig 3b shows the evolution if no parameter switch (i.e. no intervention) had happened at t = 3.0. note: the matching is overly simplified for the interval t ∈ [0, 3.0], leading to an overestimated x(t), since for example x(3.0) = 0.0025 -corresponding to 200000 individuals-, while the actually reported number was around 52550. in reality, the d · p must have been larger than 1.42 at the beginning of the interval, but on the other hand closer to (but above) 1.0 in the second half of [0, 3.0]. assuming an infected individual occupies an intensive-care bed with ventilator (icu) for one to two weeks, the icu capacity in germany currently is about 12500 to 25000 icu cases per week. this allows for a maximum of 87500 to 175000 reported infections per week (assuming share of cases needing intensive care around 14.28%), i.e. 175000 to 350000 reported infections per two weeks. this in turn corresponds to a normalized increment of 0.0021875 to 0.0043750 per time unit (a horizontal line somewhere in the upper half of the graphs in fig 3) . it is necessary to remark that the conclusion drawn in connection with fig 2e and 2f i.e. that a second outbreak of similar magnitude as initially would not effect a substantial increase in the accumulated number of infected individuals -cannot be affirmed for the current scenario (in germany and elsewhere), since that number is rather about 0.25% to 0.5% of total population currently, rather than the 1/5 prevailing in the demo scenario in fig 2e and 2f at the onset of the second outbreak. the challenge with lockdown measures for the current corona virus is the following: when imposing them, they will show effect only if the basic reproduction number is pushed below 1 sufficiently enough. then, after the number of infected individuals has eventually dwindled, a lift of the lockdown is tempting -however even a slight increase of r 0 above one opens the way to renewed catastrophic infections increase. one therefore has a binary evolution characteristic; to control r 0 by policy such that a steady stream of just managable new infections is maintained is daunting, and likely impossible (in practice) if a policy requiring a constant set of restrictions is targetted. the natural answer, at least from a theoretical point of view, is to consider phases of lifted restrictions interleft with repeated adaptively switched phases of more stringent restrictions or more stringent enforcement of existing restrictions. the need for such strategy is not in principle altered by the local aspect of transmission, except that switched lockdowns only need to be local and thus do not affect the whole population. another point that needs to be mentioned is that the graphs suggest that a future virus having infectiousness lasting much longer than the about two to three weeks for sars-cov-2 and also being as highly infectious would pose serious challenges for containment, because of resource exhaustion in the mid-stages of the pandemic. 8 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in in this study a novel model for virus proliferation dynamics was developed and with it the sars-cov-2 outbreak in germany retraced on an aggregate level, using covid-19 case count data by the robert-koch institute in berlin. elementary properties of the model were identified. predictions by the model for different levels of mitigation measures were hinted at or stated in approximate manner, and put into context of available health care resources in germany. future policy oriented work would need to address better understanding of fine-grained and adaptively activated mitigation measures, for which a spacial model should be favoured over purely aggregate models as the present one. further, for purpose of improving parameter and state estimates, the issue of underreporting (i.e. #actual > #reported cases) must be taken into account appropriately. ideally, one can develop an estimate for the factor of underreporting from more exact spacial analyses. on the mathematical side, a more rigorous formulation of the instantaneous proliferation dynamics is desirable, which allows to link parameters of the aggregate model to well-defined elementary parameters and results in more systematic parameter estimation. the ultimate goal is to be able to estimate more local structure from the observed time series. 10 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.20.20178301 doi: medrxiv preprint a additional graphs a.1 data series on daily newly reported covid-19 cases figure 5 : a "smoothed" derivate of numbers of daily newly reported covid-19 cases in germany published by [rob] . the blue squares and green triangles series show (for comparison) the sum of daily new cases over a moving 7-day window. orange diamonds and triangles show daily new cases after scaled with a weekday-specific weight factor to remove the weekly pattern seen in the original data. the weight factors were estimated from data corresponding to the squares and diamonds series, i.e. from the interval from 1st april until 6th may 2020. germany imposed face-mask wearing in stores starting from 27th april and allowed certain (moderate) shop reopening starting from 4th may 2020. the "bend" at around 14th april is remarkable because no changes in measures were effected at that time or within the preceding one week. b refinement of the infectiousness mechanism, including a model generalization so far, a crude specification of the infectiousness has been used, putting focus on the main infectiousness interval of a few days. an additional aspect in the virus transmission which should be accounted for in a refinement is the transmission from longer lived remnants of the virus in otherwise cured individuals. for this, we imagine that individuals infected at time t 0 remain contagious until t 0 + t c2 with reduced probability, additionally to the previously used interval [0, t c ]. concretely, let p 2 be the probability that an individual which has been infected for a duration exceeding t c but not exceeding t c2 , will transmit the virus in a unit time step. withp 2 := p 2 /p the adjusted model equation then readṡ x(t) = d · p · (x(t) − x(t − t c )) +p 2 (x(t − t c ) − x(t − t c2 )) · (1 − x(t)/n ), 11 all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . mathematical epidemiology: past, present, and future temporal dynamics in viral shedding and transmissibility of covid-19 deterministic and stochastic epidemics in closed populations a contribution to the mathematical theory of epidemics effective containment explains subexponential growth in recent confirmed covid-19 cases in china covid-19: fallzahlen in deutschland und weltweit temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study katrin zwirglmaier, christian drosten, and clemens wendtner. clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster early prediction of the 2019 novel coronavirus outbreak in the mainland china based on simple mathematical model since those individuals must be added to the instantaneous reservoir from which infections are generated. the equation is better written aṡif we denote by i(t) the infectiousness profile, which shall describe the relative infectiousness of an infected individual 5 at time increment +t after the infection moment (relative to infectiousness at t = 0), then the above used specification for sars-cov-2 is expressed asits derivative is (with dirac notation) i = δ 0 − (1 −p 2 ) · δ tc −p 2 · δ t c2 . we therefore find that the model equation (6) in fact is generally best written aṡwhere " * " denotes the function convolution. a dirac notation-free representation deriveshere the last integral signifies the well-known stieltjes integral.note: the infection from contaminated surfaces of objects can be represented in the same framework. this is because initially and during the evolution of the spread, viruses are on surfaces mostly there where infected individuals previously had been. key: cord-299988-jaekryq5 authors: karte, claudia; platje, nadine; bullermann, johannes; beer, martin; höper, dirk; blome, sandra title: re-emergence of porcine epidemic diarrhea virus in a piglet-producing farm in northwestern germany in 2019 date: 2020-09-10 journal: bmc vet res doi: 10.1186/s12917-020-02548-4 sha: doc_id: 299988 cord_uid: jaekryq5 background: porcine epidemic diarrhea (ped) is a viral enteric disease of pigs. it affects all age classes of animals but lethality is mainly seen in suckling piglets. after its first appearance in england in 1971, porcine epidemic diarrhea virus (pedv) has spread worldwide. while sporadic outbreaks prevailed in europe, the disease had high impact in asia. following particularly severe outbreaks in 2011, high impact cases were also reported in the united states and neighboring countries in 2013. subsequently, outbreaks were also reported in several european countries including germany. these outbreaks were less severe. this case report describes a recent case of ped re-emergence in germany and the sequence analyses of the causative pedv. case presentation: in spring 2019 5 years after re-introduction of ped into central europe, a piglet-producer in northwestern germany experienced an outbreak that affected sows, their suckling piglets, and weaners. after initial confirmation of pedv by real-time rt-pcr, fecal material and small intestine samples from affected pigs were subjected to metagenomic analyses employing next-generation sequencing. phylogenetic analyses showed high identities among the pedv sequences obtained from samples of different animals and a close relation to recent strains from hungary and france. compared to the pedv strains analyzed in 2014, genetic drift could be confirmed. changes were mainly observed in the spike protein encoding s gene segment. in addition, metagenomic analyses showed multiple picobirnavirus reads in all investigated samples. conclusion: this case report shows that pedv is still circulating in europe. the causative strains are moderately virulent and are still closely related to the so-called indel strains reported previously in europe, including germany. however, a genetic drift has taken place that can be seen in a novel cluster comprising strains from germany, hungary and france in 2019. relevance and impact of the detected picobirna sequences need further investigations. porcine epidemic diarrhea (ped) is an acute and highly contagious enteric disease of swine that results in severe enteritis, diarrhea, vomiting, and dehydration. especially in suckling pigs, lethality can be very high [1] [2] [3] . the causative agent, porcine epidemic diarrhea virus (pedv), is an enveloped positive single-stranded rna virus that belongs to the family coronaviridae, genus alphacoronavirus [4] . the complex coronavirus particles are pleomorphic and possess club-shaped surface projectors [5] . the length of the genome ranges from 27 to 31 kilobases [6] . coronaviruses have a low tenacity [7] but are shed in high amounts and are thus easily transmitted by the fecal-oral route [8] . after its first recognition in the 1970s in europe [7, 9] , the disease caused considerable economic losses especially in asia, where the disease remains endemic [10, 11] . in europe, the disease disappeared quickly, and from most countries, only very sporadic cases were reported over the last three decades. after reports from asia, that a new pedv variant caused considerable losses [12, 13] , that highly virulent pedv variant emerged also in the united states (us) in 2013, with swine farms experiencing explosive epidemics affecting all age classes of animals, with up to 95% mortality in suckling pigs [2, 14] . in 2014, several cases of ped were also reported from southern and western germany. in most cases, fattening pigs were affected showing high morbidity with almost non-existent mortality [15] . however, some breeding herds reported high mortality rates with up to 85% losses in suckling piglets [1] . similar outbreaks were observed in several other central european countries including france, the netherlands, italy, slovenia, belgium, romania, portugal, spain, and austria [16] [17] [18] [19] [20] [21] [22] [23] . the characterization of the involved virus strains revealed that so-called s-indel variants of the virus were involved in central europe, which, in contrast to the highly virulent non-indel strains from asia and the usa, are characterized by deletions and insertions in the spike protein encoding s gene [23] . in the majority of cases, the s-indel variants are associated with milder ped courses. in the absence of reporting obligations, and following the confirmation that the pedv strains in the eu did not belong to the highly virulent non-inde l type, notification and broader follow-up of cases decreased. however, sporadic outbreaks, sometimes with severe problems to get rid of the disease, were still reported from all production systems from different regions of germany and other countries (personal communications and unpublished data). from this time, pedv sequence information is largely missing. when a new wave of ped struck a piglet producer in northwestern germany in 2019, questions were raised to what extent the virus might have changed and if a new emerging variant was causing the clinical case in sows, piglets and weaners. here, we report on the clinical presentation and the whole-genome sequencing of the causative 2019 pedv strain. the affected piglet producer is located in northwestern germany. the farm keeps approximately 350 sows in seven groups (50 sows each), and has a total of 2200 piglet rearing places (1000 on site and 1200 in a leased farm). on the premise, sows and piglets are kept in the same building complex. the farm also includes a fattening unit with 1500 fattening slots. this unit is in close proximity to the above-mentioned units but has its own building with a hygiene lock. three other pig farms are in the radius of 500 m around the holding. prior to the disease event, the farm recorded 33 weaned piglets per sow and year with suckling losses below 10%. loss in piglet rearing and fattening was < 2%. the animals were routinely screened for enteric pathogens and only rotavirus types a and c were detected every now and again (rotavirus type c only very sporadically). the farm was unsuspicious for dysentery and was tested negative for tgev and pdcov prior, during and after the disease event. the routinely applied immunization scheme included maternal vaccinations against colibacillosis, oedema disease, and necrotic enteritis. depending on the infection pressure, rotavirus a vaccines were used in gilts. piglets received vaccinations against porcine circovirus type 2 (pcv-2), mycoplasma, and shiga toxin producing e. coli. sows in integration and reproduction received additional vaccination e.g. against porcine respiratory and reproductive syndrome virus, influenza virus, and parvovirus. in spring 2019, massive diarrhea occurred in sows and suckling pigs. at first, nursing sows (60% of the sows in the unit) in the farrowing unit showed inappetence and shortly afterwards mushy diarrhoea. fever or increased temperature were not detected. the sows recovered completely after three to 4 days. the suckling piglets, which were about 14 days old, showed the first signs of diarrhea two to 3 days after the mothers. about 70% of the litters of this first affected farrowing group showed diarrhea and losses rose to 10% (see table 1 ). immediate investigations confirmed pedv (rt-qpcr from fecal samples and organs). sick piglets were treated with commercial electrolyte solution and additional water supply was given to sows. to increase maternal immunity, infection was enforced in the waiting area. weaned piglets with secondary infections received antibiotic treatment. in total, three farrowing groups (sows and suckling pigs) showed clinical signs of ped and increased loss rates in suckling pigs (10 to 30%). morbidity reached 60 to 90% in sows and 70 to 100% in piglets (see table 1 ). some of the weaned piglets also showed diarrhea, wasting, and growth retardation. the overall losses in piglet rearing rose to 5 to 10% (details see table 1 ). in the fourth farrowing group (approx. eight weeks after the first clinical signs) no clinical signs indicative for ped were recorded and up to now no further ped suspicions arose. in autumn, five suckling piglets were randomly selected and subjected to necropsy and ped screening. all samples were negative for pedv. in the connected fattening unit, no ped signs were recorded at any time. serological checks in the sow rearing unit (separate building) gave negative results. during the disease event, intensive cleaning and disinfection was carried out in the farrowing unit, on driveways, in the waiting areas, and all related stables. disinfectants were chosen in accordance with the list recommended by the germany veterinary society (dvg) for enveloped viruses. purchase of gilts was stopped and replaced by self-remounting. follow-up investigations showed that neighboring farms were also affected by ped shortly before the onset in the described farm. upon initial confirmation of ped by a private laboratory, fecal samples from five sows and feces and intestines from two affected piglets were sent to the friedrich-loeffler-institut (fli) for further analyses and nextgeneration sequencing. ribonucleic acids were extracted from fecal samples or supernatants from homogenized intestines using trizol reagent (lifetechnologies, darmstadt, germany) in combination with the rneasy mini kit (qiagen, hilden, germany) and dnase digestion on the spin column. all rnas were confirmed to be pedv positive by rt-qpcr [24, 25] . subsequently, all samples were subjected to whole genome sequencing and metagenomic analyses using the illumina miseq platform as previously described [26] . in brief, nucleic acids were processed into shotgun dna libraries and then deep-sequenced. the resulting raw data was taxonomically classified using the software pipeline riems [27] . the obtained sequence reads were assembled to determine the genomes in full length. all sequences are available from the insdc databases under study accession prjeb38314. with the help of the geneious prime software suite (v. 2019.2.3; biomatters ltd., auckland, new zealand), phylogenetic analyses were performed (for details see legend fig. 1 ). the genomes originating from the reported german case form a new and distinct cluster within the s-indel strains (see fig. 1 ). close relatives are three virus strains reported in 2019, two from hungary [16] (accessions mh593900 and kx289955) and one from france (accession mn056942). identity among the new german pedv strains was almost 100% (> 99.9%) whereas identities of > 98.8% were found with regard to the hungarian and french sequences. comparing the german strains from 2014 with the german strains detected in 2019, identities are higher than 99.5%. comparisons between german prototype strains from 2014 (the first reported strain, bh76/14-01_l00719_ farm a) and 2019 (894_3_l03204_ger) show high similarities in the nucleotide sequence (see supplementary figure 1 ). in total, 135 nucleotides exchanges are the rna-shotgun sequencing approach allowed metagenomic analyses using riems. in this analysis, several reads were classified taxonomically as picobirnaviridae sequences. multiple reads of the rna-dependent rnapolymerase gene as well as the gene segment encoding the capsid were found in the fecal but not the intestine samples. porcine epidemic diarrhea can have a tremendous impact on the pig industry as was seen in the us following the introduction of pedv in 2013 [2, 29] . critical losses occurred especially in piglet rearing companies and the losses impacted the whole pork industry [15, 29] . following the devastating outbreaks on the american continent, re-emergence of pedv was also reported from europe after intensified surveillance [23] . however, here, strains of lower virulence were circulating and the reporting and follow-up of cases abated quickly despite ongoing cases. one reason for the subsiding of official follow-up is that ped is neither notifiable nor reportable but still has impact on trade and reputation. against this background, most farmers had no interest to make their cases public. thus, official and published information on the german ped situation in general and viral evolution in particular is missing roughly from 2016 onwards. when pedv was introduced in a piglet-producing farm in northwestern germany in 2019, clinical disease and losses were rather disturbing and the farmer and responsible veterinarian initiated a closer follow-up. one hypothesis for the observed impact was a change in virulence and thus, next-generation sequencing was employed to test this hypothesis. our data show that the causative virus strains are still s-indel variants with close relationship to those found in 2014 and the following years. however, viral evolution has taken place and the drift gave rise to a new cluster that comprises recent fig. 1 phylogenetic tree of current pedv strains. phylogenetic tree of 2019 pedv strains from germany, hungary, and france as well as 2014 pedv strains from germany, usa, and china. the complete genome sequences were aligned using mafft and a phylogenetic analysis was performed using phyml, with a gtr substitution model and tree reconstruction supported by 1000 bootstrapping replicas [28, 29] . green branches show the 2019 pedv isolates from germany, blue branches highlight the isolates from hungary and france and red branches are the highly virulent non-indel strains from the usa and china strains from germany, hungary, and france. given the accordant drift, one can speculate that pedv is still circulating in europe. there is no indication that these variants have a higher virulence per se. the previously observed variation seems still present. the affected farm described in this report was finally able to control the outbreak by forced infection in the waiting unit of sows, biosecurity and strict cleaning and disinfection. yet, the history of ped in neighboring fattening farms also shows that the virus was able to enter the farm and room for improvement was given in veterinary hygiene and biosafety. the exact route of introduction remained unclear. supplementary studies into the metagenomic data set showed picobirnaviral sequences in the fecal material. picobirnaviruses are non-enveloped double-stranded rna viruses. they are bisegmented with segment one consisting of 2.3 to 2.6 kilobases and segment two of 1.5 to 1.9 kilobases [30] . picobirnaviruses are often associated with cases of gastroenteritis or infections in the respiratory tract [31] . the role in diarrhea diseases in piglets is unclear, since picobirnaviruses were found in piglets with and without diarrhea [32] . the transmission is fecal-oral [33] and these viruses have so far been detected mainly in feces in various species [30, 33] . impact and relevance of these findings remains to be clarified by future studies. in conclusion, ped re-emerged in northwestern germany in 2019 leading to high morbidity and substantial impact in a piglet-producing farm. the causative virus strains are still s-indel variants but a genetic drift occurred since 2014. this drift is accordant with the evolution in other european countries. the relevance of picobirnavirus detections in fecal samples from pedvpositive animals remains unclear. supplementary information accompanies this paper at https://doi.org/10. 1186/s12917-020-02548-4. emergence of porcine epidemic diarrhea virus in southern germany emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences genetic properties of endemic chinese porcine epidemic diarrhea virus strains isolated since 2010 the springer index of viruses a new coronavirus-like partiele assoeiated with diarrhea in swine diagnostic notes: update on porcine epidemic diarrhea verlaufsuntersuchung über die ausscheidung von porcine epidemic diarrhea virus (pedv) und die serokonversion nach feldinfektion bei saugferkeln und mastschweinen porcine epidemic diarrhoea (ped) -neuausbrüche in deutschen mastschweinebeständen chinese-like strain of porcine epidemic diarrhea virus pig farming. letter to the editor new variant of porcine epidemic diarrhea virus the prevalence of intestinal trichomonads in chinese pigs isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states comparison of porcine epidemic diarrhea viruses from germany and the united states isolation and characterisation of porcine epidemic diarrhoea virus in hungary -short communication complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium porcine epidemic diarrhea virus (pedv) introduction into a naive dutch pig population in 2014 outbreak of porcine epidemic diarrhea virus in portugal first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in austria porcine epidemic diarrhoea virus in italy: disease spread and the role of transportation porcine epidemic diarrhea in europe: in-detail analyses of disease dynamics and molecular epidemiology genomnachweis des porzinen epidemischen diarrhoe virus (pedv) mittels real-time rt-pcr. avid-methodensammlung: avid-methode vir03 evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral rna at long distances from infected herds a versatile sample processing workflow for metagenomic pathogen detection riems: a software pipeline for sensitive and comprehensive taxonomic classification of reads from metagenomics datasets mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform factors associated with time to elimination of porcine epidemic diarrhea virus in individual ontario swine herds based on surveillance data molecular detection and characterization of picobirnaviruses in piglets with diarrhea in thailand detection and molecular characterization of porcine picobirnavirus in feces of domestic pigs from kolkata, india publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank patrick zitzow, robin brandt and ulrike kleinert for technical assistance. authors' contributions ck performed next-generation sequencing and was a major contributor in writing the manuscript, np and jb collected and analyzed clinical data, mb interpreted the overall dataset and critically revised the manuscript, sb conceived the study, assisted in interpreting the data and contributed in writing the manuscript, dh analyzed and interpreted whole-genome data and contributed to the manuscript. all authors read and approved the final manuscript. this study was funded by federal excellence initiative of mecklenburg western pomerania and european social fund (esf) grant koinfekt (esf_14-bm-a55-00xx_16). the excellence initiative covered consumables for next-generation sequencing and the personnel costs of the major contributor (ck). open access funding provided by projekt deal.availability of data and materials sequence information was deposited at the european nucleotide archive (ena) under study id prjeb38314 (accessions lr812928; lr812932; lr812927; lr812929, lr812930, lr812926, and lr812931). additional metadata are available from the authors upon reasonable request. the sample material was submitted to the friedrich-loeffler-institut for enhanced diagnostics and was taken by the responsible farm veterinarian in the context of the health monitoring program of the respective farm (in accordance with the regulation on hygiene requirements for the keeping of pigs; online available at http://www.gesetze-im-internet.de/schhalthygv/). in germany, every keeper of pigs must have his herd supervised by a veterinarian as part of the on-farm inspections, this includes clinical and laboratory checks to maintain and improve the health status of the herd. no permissions were necessary to collect the mainly non-invasive specimens. the farmer approved in-detail analyses of the ped case and shipment of samples (verbal agreement with the responsible veterinarian). all procedures were carried out in accordance with the relevant regulations. the owner of the case farm approved submission of the report. all authors have approved submission of the manuscript. the authors declare that they have no competing interests. key: cord-298469-0sny9dit authors: schlickeiser, reinhard; schlickeiser, frank title: a gaussian model for the time development of the sars-cov-2 corona pandemic disease. predictions for germany made on march 30, 2020 date: 2020-04-02 journal: nan doi: 10.1101/2020.03.31.20048942 sha: doc_id: 298469 cord_uid: 0sny9dit for germany it is predicted that the first wave of the corona pandemic disease reaches its maximum of new infections on april 11th, 2020 +5.4-3.4 days with 90 percent confidence. with a delay of about 7 days the maximum demand on breathing machines in hospitals occurs on april 18th, 2020 +5.4-3.4 days. the first pandemic wave ends in germany end of may 2020. the predictions are based on the assumption of a gaussian time evolution well justified by the central limit theorem of statistics. the width and the maximum time and thus the duration of this gaussian distribution are determined from a statistical ξ2-fit to the observed doubling times before march 28, 2020. in these days there is a very high interest in the societal, economical and political world to understand the time evolution of the first wave of infections of the population by the current sars-cov-2 (corona) virus. the most important issues are the total duration and the peak time of the infection evolution as well as the maximum number of daily infections. it would be most helpful for many people to have a reproducable, crude, but reliable estimate when this pandemic wave is over. it is the purpose of this manuscript to provide such an estimate based on a simplified gaussian model for the time development of the pandemic outburst. the best justification for the gaussian or normal distribution for the virus time evolution is given by the central limit theorem of statistics 1 . the central limit theorem states that in situations, when many n 1 independent random variables are added, their properly normalized sum tends toward a normal or gaussian distribution function of the form (1) even if the original variables themselves are not normally distributed. the spread of the virus infection of populations with high number of persons certainly is such a random process to which the central limit theorem ia applicable. each person in a given population has a probability distribution (normalized to unity) as a function of time of being infected: it is a very noncontinuous distribution being 1 at the day of infection and 0 on all other days. if one adds up these discrete distributions of persons living in villages and districts of towns of typical size of about 1000 persons one obtains quasi-continous probability distributions for be* rsch@tp4.rub.de, schlickeiser@gmail.com ing infected which certainly will be different in hotspots of the disease and isolated rural areas. if we then add up a large number of these village probability distributions for all of germany we obtain the daily infection rate distribution which according to the central limit is close to a gaussian distribution. the analysis of gaussian distribution functions plays a central role in many problems of statistical physics and plasma physics. e.g. in plasma kinetic theory they are referred to as drift-maxwellian 2 or counterstreaming bi-maxwellian 3,4 velocity distribution functions. both authors of this manuscript are not virologists but theoretical physicists in plasma physics and astrophysics (rs) and solid state physics (fs) with past experience in analyzing normal or gaussian distribution functions. apart from consulting several reviews 5-7 , as nonvirologists we are not familiar with the recent relevant virology literature. nevertheless, it is our hope that in these hard times an estimate by unbiased non-experts might be welcomed by specialists as well as the broad population, especially if some positive information and outlook is provided. we base our parameter estimates on publicly available information, especially by the excellent podcast by prof. c. drosten 8 and the recent sophisticated modelling study for germany 7 . numerical simulations and the empirical data of earlier epidemies 8 indicate that the time evolution of epidemic waves is characterized by an early exponential rise until a pronounced maximum is reached followed by a rapid decrease. as argued above we adopt a simple gaussian model for the time evolution of infections and explore its consequences. if i(t) denotes the number of infections . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.31.20048942 doi: medrxiv preprint per day, we assume that its time evolution is given by the gaussian function shown in fig. 1 , where i 0 denotes the maximum value at time e and ∆ denotes the width of the gaussian. by monitoring the new daily infections one easily derives the relative change in the infections per day where we used the distribution (1). the monitored data are often given in terms of the doubling time d of the corresponding exponential function at any time using the distribution (3) in equation (2) provides for the relative change in daily infection rate equating the two results (2) ans (4) leads to the timedependent gaussian doubling time figure 2 shows the monitored doubling times for germany 9 starting on march 15, 2020 until march 28,2020. we assume that every value has an error of 15 percent. it starts at d(t = 0) = 2.6 days and increases to d(t = 12) = 4.8 ± 0.24 on march 28, 2020. the gaussian doubling time modeling then provides d(t = 0) = a/e = 2.6 days, corresponding to moreover, eq. (5) reduces to we determine the value of the only free parameter e in equation (7) by performing a χ 2 -fit to the data shown in fig. 2 . if m(t i ) denote the observed doubling times at days t i , δm(t i ) = 0.15m(t i ) its error and d(e, t i ) the theoretical doubling time for given values of e, we calculate the best fit with the minimum value of χ 2 min = 9.51 is provided for e = 27.5 days. the χ 2 min,p.d.f = 9.51/13 = . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint correponding to april 11, 2020 +5.4 −3.4 days. consequently, the best fit gaussian doubling time for germany is given by as an aside we note that the variation (10) becomes infinitely large as t → 27.5. moreover, for times t > e the doubling times becomes a decay half-life approaching 0 for very large times t e. moreover, after inserting the values (9) equation (6) yields with 90 percent confidence in fig. 3 we show the prediction of the doubling times in germany until day 25 corresponding to april 8th, 2020. fig. 3. the same as in fig. 2 but now the predicted doubling times until day 25 corresponding to april 8, 2020. it is known that during the whole duration of the first wave of the virus evolution 70 percent of the total population are infected 8 , if nothing is done to reduce the number of infections. scaling the total population in units of 10 5 n 5 , we estimate that 0.7q10 5 n 5 are infected during the whole duration of the first virus wave, where the quarantaning factor q accounts for the currently taken political actions such as quarantining of elder and infected people, social distancing actions in the society as well as the closure of schools and daycare facilities. integrating the gaussian (1) over all times we then obtain where we used the integral ∞ −∞ dx e −x 2 = √ π. equation (12) yields for the maximum value with the 90 percent confidence value for ∆ from equation (11) we obtain with the same confidence level for the maximum value (13) we assume as typical medical parameters those suggested by the recent modeling by the robert-koch-institute 7 (see their fig. 1 ): only about 20 percent of the infected people are seriously infected, 5 percent have to be hospitalized and 1α percent need access to breathing apparati for typically 1w week, corresponding to 7w days. we refer to the latter as nssps standing for new seriously sick persons per day. as these numbers are uncertain we keep their scalings with the typically adopted numbers. in germany there are at most 56000 breathing apparati in total available, corresponding for a population of 80 million people to 70b 70 breathing apparati available per 10 5 n 5 people. as a consequence, every day the hospitals can handle i n nssps with the infections are handable by hospitals for all times if at the maximum of the virus evolution 0.01αi 0 , denoting the maximum number of seriously sick persons per day needing access to breathing apparati, is less or equal to i n , i.e. . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.31.20048942 doi: medrxiv preprint inserting the value (14) for the maximum value i 0 , the quantity n 5 cancels out and we obtain with 90 percent confidence the condition which is equivalent to in order to handle all serious infections in german hospitals the condition (18) has to be fulfilled. it seems that german hospitals can only ensure the best treatment of all nssps at the maximum of first wave if either (1) the number of available breathing apparati can be increased by a factor of 3, corresponding to b = 210 per day. or (2) the quarantaining factor q can be reduced to q = 0.36 +0.03 −0.02 1/3. the first option is unrealistic on short time scales. to achieve the second option of the reducing the quarantaining factor q to about 0.3 by the currently taken social distancing and quarantaining activities seems to be realistically achievable in germany. we therefore will adopt this optimistic value q = 0.3q 0.3 in our further predictions. however we note that with such a small quarantaining factor only 21000n 5 will be infected during the whole first wave of the virus, so that additionally waves are likely to occur in the future. it is important to notice that the outbreak of serious sickness syndroms of nssps is delayed to the infection time by about τ = 7 days 8 . this delay time has to be added to the above derived maximum time scale e, so that e + τ = 34.5 +5.4 −3.4 days, corresponding to april 18, 2020 +5.4 −3.4 is the predicted day in germany when the maximum number of nssps has to be treated. the number of infections are signifantly reduced by a factor 10 3 compared to the maximum i 0 at the time with 90 percent confidence. consequently, the first pandemic wave will be over in germany not before 65 days or about 2 months with the indicated uncertainty. this corresponds to 38 ± 4 days after the time of maximum. according to our predictions, the first pandemic wave in germany will reach its maximum by april 18, 2020 when about about (8.3 ± 0.78)q 0.3 n 5 nssps have to be treated in the hospitals. the wave has a broad distribution from april 6 to april 30, 2020 with more more than (4.2 ± 0.39)q 0.3 n 5 but less than (8.3 ± 0.78)q 0.3 n 5 nssps per day. the number of nssps needed to be treated at hospitals will sharply drop to less than (0.0083 ± 0.00078)q 0.30 n 5 nssps by may 26th, 2020. as germany has a population of about 80 million persons we have n 5 = 800. therefore in absolute numbers german hospitals will have to cope with (6640 ± 624)q 0.3 nssps at the maximum of the outburst on april 18th, 2020, more than (3360±312)q 0.3 but less than 6640±624 nssps per day between april 6 and april 30, 2020, before the total number per day drops below (6.64 ± 0.63)q 0.3 nssps after the end of may 2020. all errors have 90 percent confidence. our analysis can be applied to other countries too if reliable information on the early doubling times are available. we plan to test our modeling with the data from the past first corona wave in china. we end with a sentence of caution: although the central limit theorem provides us with a very good justification of the adopted gaussian time distribution function it is not guaranteed that the actual virus time evolution follows this behavior. we will only know for sure after the first pandemic wave is over. so it is possible that our estimates and we are wrong. we take this risk because we are convinced that many persons will welcome our optimistic estimate that the first wave is over by end of may 2020. there is light at the end of a long tunnel. our estimate might also help decision makers when to lift the current societal and economical lockdown. die erste welle wird gegen ende mai 2020 in deutschland enden mit einem tausendstel kleineren neuinfektionsraten. diese vorhersagen basieren auf der annahme einer gauss-förmigen zeitverteilung der infizierungsrate, die gut durch den zentralen grenzwertsatz der statistik begründet ist. die breite und die zeit des maximums der gauss-verteilung und damit deren gesamtdauer werden durch einen statistischen χ 2 -fit an die in deutschland beobachteten verdopplungszeiten vor dem 28.märz 2020 bestimmt. die behandlung aller schwer erkrankten patienten mit beatmungsgeräten ist gewährleistet, wenn es durch die andauernden quarantäne-und soziale distanzierungs-massnahmen gelingt, die anzahl der infizierten personen in der bevölkerung durch die erste welle unter 30 prozent zu halten. . cc-by 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.31.20048942 doi: medrxiv preprint an introduction to probability theory and its ppplications infectious diseases of humans: dynamics and control modellierung von beispielszenarien an der sars-cov-2 epidemie 2020 in deutschland coronavirusupdate (ndr.de/coronaupdate, 2020) süddeutsche zeitung online key: cord-331336-4kf2jn8c authors: aravindakshan, a.; boehnke, j.; gholami, e.; nayak, a. title: restarting after covid-19: a data-driven evaluation of opening scenarios date: 2020-05-30 journal: nan doi: 10.1101/2020.05.28.20115980 sha: doc_id: 331336 cord_uid: 4kf2jn8c to contain the covid-19 pandemic, several governments introduced strict non-pharmaceutical interventions (npi) that restricted movement, public gatherings, national and international travel, and shut down large parts of the economy. yet, the impact of the enforcement and subsequent loosening of these policies on the spread of covid-19 is not well understood. accordingly, we measure the impact of npi on mitigating disease spread by exploiting the spatio-temporal variations in policy measures across the 16 states of germany. this quasi-experiment identifies each policy's effect on reducing disease spread. we adapt the seir (susceptible-exposed-infected-recovered) model for disease propagation to include data on daily confirmed cases, intraand inter-state movement, and social distancing. by combining the model with measures of policy contributions on mobility reduction, we forecast scenarios for relaxing various types of npis. our model finds that, in germany, policies that mandated contact restrictions (e.g., movement in public space limited to two persons or people co-living), initial business closures (e.g., restaurant closures), stay-at-home orders (e.g., prohibition of non-essential trips), non-essential services (e.g., florists, museums) and retail outlet closures led to the sharpest drops in movement within and across states. contact restrictions were the most effective at lowering infection rates, while border closures had only minimal effects at mitigating the spread of the disease, even though cross-border travel might have played a role in seeding the disease in the population. we believe that a deeper understanding of the policy effects on mitigating the spread of covid-19 allows a more accurate forecast of the disease spread when npis are (partially) loosened, and thus also better informs policymakers towards making appropriate decisions. in response to the covid-19 pandemic, governments around the world implemented varying degrees of non-pharmaceutical interventions (npis) to control the spread of the disease (1,2,3,4). these policies severely restricted movement, public gatherings, national and international travel, and shut down large parts of the economy including schools and non-essential businesses. while the shutdowns helped delay the spread and reduce the severity of the epidemic, they also created tremendous hardships for individuals and businesses (5, 6, 7) . as the spread of covid-19 decelerated across countries, governments have started relaxing npis to help balance the need for economic security and the risk of growing infection numbers (8). nevertheless, there is limited understanding of the effect that loosening policies might have on the spread of the disease. to determine this effect, we quantify each npi's contribution to disease mitigation, permitting the forecasting of disease spread under different policy scenarios. the proposed model, then, will allow policymakers to forecast the impacts of the removal of different types of restrictions. initial analysis of the impact of policy restrictions in china suggests that npis that significantly affected human mobility (e.g., household quarantine) reduced the spread of the disease (7, 10) , even more than restrictions that limited national and international travel (11) . additionally, simulations of npis in wuhan (6) show that maintaining restrictions helped delay the epidemic peak. the results also imply that an early end to such interventions leads to an earlier secondary peak, which can be flattened by relaxing the social mixing at varying rates (6) . nevertheless, to the best of our knowledge, no study quantifies the effects of the types and timings of the implementation and relaxation of government policy interventions in reducing mobility and in turn decreasing the spread of covid-19. our estimates allow for projections of the impact of easing individual interventions on disease spread. these predictions act as decision aids for policy makers to judge how lifting certain policies changes social mobility rates and in turn the number of new covid-19 cases. using data from the 16 states of germany, we explore the effectiveness of different npis ( figure 3 ) in reducing social mobility, and in turn affecting the spread of the disease. because german states enforced (and relaxed) policies to varying degrees and at different points in time, the variations in implementation allow us to capture the incremental effectiveness of these policies at reducing social mobility amongst the general population. to determine how policy enforcement impacted mobility and disease spread, we associate the type and timing of the policy intervention to actual social mobility as recorded in the data released by google (14) . next, using our predictions of social mobility based on the policy interventions, we predict the spread of covid-19 by modifying the seir model presented in (9) to include social distancing and other forms of mobility data (e.g., travel by air, bus, rail, and road). finally, we project the impact of relaxing a policy on the number of new cases across germany and compare how differences in start times for policy relaxations alter the cumulative number of expected cases over a six-week time span. we find that not implementing social distancing in germany, would have resulted in a 37.8-fold (iqr: 27 to 52-fold) increase in cumulative infected case counts as of may 7, 2020. in other words social distancing reduced case counts by about 97.3% (iqr: 96.3-98.07%). we also find that policies are not equal in their effectiveness at reducing new cases. contact restrictions were the most effective at lowering infection rates in germany (51%, iqr: 50.7-51.2), while border closures had only minimal effects at mitigating the spread of the disease (2%, iqr: 0 -4%), even though they might have played a role in seeding the disease in the population. interconnected air, land, and sea transportation networks led to the spreading of covid-19 from wuhan, china to the rest of china and eventually to most countries around the world (12, 13). to accurately model the spatial spread of the disease into germany, we collected three types of daily mobility data: (i) daily air transportation data to capture the movement within and between germany and 142 other countries; (ii) daily ground transportation data between the nine countries that share borders with germany; and (iii) daily inter-state ground transportation. the daily covid-19 case data were obtained from the johns hopkins coronavirus resource center (15) and robert koch institute (16) for all countries in our dataset as well as the 16 german states (see figure 1 ). figure 2 shows the cumulative case numbers for all states in germany and globally. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. to encourage and enforce physical distancing, governments across all 16 states introduced a variety of npis at different points in time ( figure 3 ). data for these policies were collected from (17, 18). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 30, 2020. . https://doi.org/10.1101/2020.05.28.20115980 doi: medrxiv preprint figure 3 . timeline for implementation of npis across states. these policies include border closures (closing international borders), contact restriction (movement in public space is limited to two persons or people co-living), educational institutes closure (e.g. schools and . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. . https://doi.org/10.1101/2020.05.28.20115980 doi: medrxiv preprint universities), initial business closure (e.g. restaurants), non-essential business closure (e.g. trade shows), stay at home order and retail store closure. border closure applies to 10 states sharing international borders. we use data from march 1, 2020 to april 30, 2020 in our study. every policy was not implemented by every state as of april 26, 2020. also, none of the implemented policies were relaxed until april 20. state governments start relaxing these policies from april 20, 2020. google's covid-19 community mobility reports (14) detail how movement trends change over time as public awareness increases and npis are introduced ( figure 4 ). the report tracks movement trends over time by geography, across different categories of places such as retail and recreation, groceries and pharmacies, parks, transit stations, workplaces, and residential. we consider community mobility trends in retail and recreation as a measure of social distancing. we define social distancing as sdi = -ci/100 where ci is the community mobility trend in state i. while the community mobility data provides information on changes in local movement, it does not provide information on inter-state movement and international travel. ground transportation accounts for the vast majority of the movement, with cars accounting for 85% of total ground transportation in germany (19). we collected detailed traffic data from jan 1, 2013 to dec 31, 2018 from the german bundesanstalt für straßenwesen (federal institute for roadways). the dataset contains the hourly count of the number of vehicles crossing different checkpoints along highways. the institute used sensors to identify the type of vehicle, which we include in our analysis to estimate the number of individuals. we construct a linear regression model to predict . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. . https://doi.org/10.1101/2020.05.28.20115980 doi: medrxiv preprint hourly traffic for jan 1, 2020 to april 30, 2020 (details in supplementary material). the model includes year, public holidays, day of the week, and state population as control variables. to control for changes in car movement during the period of the study, we adjust the predicted daily traffic using google's community mobility data for workplaces ( figure 5 ). we used deutsche bahn's timetables (www.bahn.com) to estimate the number of daily rail travelers moving across states in germany and arriving from neighboring countries. to account for the changes in movement due to covid-19 and cancelations of several trains, we adjust the number of passengers moving across states by using the community mobility data for transit stations ( figure 6 ). is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. . https://doi.org/10.1101/2020.05.28.20115980 doi: medrxiv preprint we obtained the search history of a large european bus and train comparison platform to estimate the number of passengers moving across cities (states) in germany and passengers traveling to germany from neighboring countries. we set bus transport to zero after march 16, 2020 as all bus movement in germany stopped on that day. last, we obtained flight transportation information from the opensky network (20) . this database utilizes automatic dependent surveillance broadcast (ads-b) flight trajectories to identify the departure and arrival airport of a flight (figure 7) . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. finally, we use additional controls to isolate the effect of individual policies. we use cumulative google trends data for the search term "covid-19 in deutschland" to control for increased awareness over time (figure 7(c) ). severe movement restrictions due to increased enforcement of these policy restrictions led to a greater sense of unease and dissatisfaction amongst some sections of the population (e.g. (21)). while such protests are small, prolonged enforcement of restrictions could increase dissatisfaction. we use weather data (max temperature in degree celsius) from wetterkontor.de as a control to account for the propensity of the population to leave their home as summer peaks. we also include an index that measures the propensity to violate (ptv) npis using the arctan function. as the policy enforcement prolongs, the ptv index increases, in turn potentially increasing social mobility (figure 7(d) ). to determine the impact of different state policies, we use a penalized linear regression (lasso regression) model to predict changes in community mobility, sdi due to a policy p that is active in state i on a given day. we explain the regression model in detail in the supplementary material. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. figure 8 (b) shows the predictions of mobility in a sample state (bavaria) from our model. based on these results, we note the policies that significantly affect changes in mobility to project the number of new cases when the policy is relaxed. from the graph, we see that educational facilities closures have a very large negative effect on mobility (29.26 percentage point drop in mobility, iqr: 29.25 to 29.95). even though it is plausible that the closure of educational facilities precipitates this drop (for example, children staying home without caregivers forces parents to work from home), we do not include this variable when projecting the lifting of policy restrictions scenarios. this is due to the potential confounding between initial awareness of the disease, population preparedness for a shutdown, and the closure of the educational facilities. the estimates for the other policy restrictions capture the average incremental effect of these policies across the 16 the model also finds that the longer the policies remain in place and restrict movement, the likelier it is for ptv to grow, which can lead to individuals breaking the policy restrictions on their own. we use the predicted mobility from the linear regression to determine the impact of social distancing on new case counts. we investigate the contribution of each policy to the mitigation of disease spread by determining the role of social distancing in the estimation of the number of susceptible and exposed individuals in a given population. we modify the seir model (equations 1 -5 in the supplementary material) used in (9) to include different transportation networks and predicted mobility for each state. using the estimation procedure in (9), we find the model parameters that we use to predict disease spread for all 16 states and for germany. we note that the model accounts for documented as well as undocumented infected cases. as shown in supplementary material figure s14 , the proportion of documented infected (i d ) as a function of total cases increases over time. this finding comports with expectations because of the rapid increase in testing across germany (22). figure 9 (a) shows the actual disease progression in germany, the disease spread as predicted by our model in the presence of predicted social distancing, as well as disease spread as predicted by our model when mobility remained unchanged with no social distancing measures. similar predictions for the states are provided in figure 9 (c). we use the time period of feb 18, 2020 -apr 20, 2020 to infer model parameters. this period includes early stages of the covid-19 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. . epidemic in germany and the time that state policies are enacted. we use these parameters to estimate the number of daily documented cases during the time interval of feb 18, 2020 -may 7, 2020, which corresponds to 17 days out of sample forecasts. the model finds 179,487 (iqr: 144,590 -211,771) cumulative documented cases in germany as of may 7, 2020 (actual reported cases: 165,991) with the estimated average error rate of 8%. figure 9 (b) shows the amount of expected increase in the number of cases across the states of germany and the nation, if no social distancing was observed. across germany one would expect a 37.8-fold (iqr: 27 to 52) increase in the number of cases without any social distancing (i.e., # = 0), the effect varying significantly by states from a low of 13.7-fold (iqr: 7 to 21) in berlin to a high of 45.2fold (iqr: 31 to 63) in bavaria. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. we simulate what-if scenarios to determine the impact of lifting restriction on new cases in each state. under the scenario that a restriction has been relaxed while others remained operational, we forecast mobility using equation (11) in the supplementary material. we subsequently project the new case count using the predicted mobility. this step is repeated across all restrictions, each relaxed individually. due to the confounding noted earlier, we do not report the case of educational facilities being reopened. in scenario 1, we project all changes from april 21, 2020 to june 2, 2020, assuming that the restriction was relaxed on april 21, 2020. figures 10 (a) and (c) shows the projections of case counts over a six-week period if a restriction was relaxed exclusively. figures 10 (b) and (d) show scenario 2, which projects all changes from april 21, 2020 to june 2, 2020, assuming that the restriction was relaxed on april 28, 2020. because the two scenarios are exactly one week apart, it allows us to determine the impact of delaying the lifting of a restriction by one week. from the analysis, the lifting of contact restrictions, i.e., the rule limiting movement in public . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. . https://doi.org/10.1101/2020.05.28.20115980 doi: medrxiv preprint spaces, had the biggest impact on new case counts. compared to keeping the restrictions in place, lifting contact restrictions will result in a 51.5% (iqr: 51.0-52.1%) increase in daily case numbers in scenario 1 and a 27.4% (iqr: 26.8-27.8%) increase in scenario 2. however, lifting restrictions on initial business closures leads to a 29.2% (iqr: 28.6-29.7%) increase in daily case numbers in scenario 1 and a 15.8% (iqr: 15.4-16.3%) increase in scenario 2. easing nonessential service closures increases daily case numbers by 6.6% (iqr: 6.2-7.0%) in scenario 1 and 3.7% (iqr: 3.2-4.0%) in scenario 2, and the opening of retail outlets increases daily case numbers by 5.6% (iqr: 5.1-6.0%) in scenario 1 and 3.4% (iqr: 3.0-3.8%) in scenario 2. these results show that npis have differential impacts on lowering disease spread, and suggest a measured approach to lifting restrictions. for example, the opening of retail outlets could be balanced by maintaining the restrictions around limiting the number of individuals in a given place or store (e.g., controlling entry) -thereby allowing for the resumption of economic activity while limiting the risk of contagion. figure 10 (e) shows the increases in the expected number of cases if a restriction was lifted on april 21 versus on april 28. delaying the lifting of certain restrictions by one week could also have a significant impact on the total case counts. this occurs not only due to the delay, but also because the number of infected individuals that a person could come in contact with decreases over the week. for example, delaying the lifting of contact restrictions by one week reduces the number of new cases over the six-week forecast by on average 19% (iqr: 16% to 21%). we also observe that lifting restrictions on the opening of retail outlets and non-essential services leads to an average 2% (iqr: 0% to 6%) and 3% (iqr: 0% to 4%) increase in total case numbers over the six-week forecast period. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 30, 2020. this study explores the role of npis in reducing the spread of covid-19. we extend the spatiotemporal seir model in (9) by incorporating daily social distancing numbers from transportation data and mobility patterns. our model finds that without npis in place, covid-19 cases would likely have shown a 37.8-fold increase across germany. we also investigate the marginal impact of each of the various npis implemented by state governments in germany by determining the differential impacts of the policies on reducing social mobility. we relate these reductions to disease spread, reconstructing patterns of spread across the 16 states. finally, we forecast new cases when policies are relaxed, one-at-a-time. we find that certain policies have a larger impact on disease spread than others. our model forecasts find that early relaxation of some npis could lead to an increase in the number of cases, potentially leading to a second wave. this observation is confirmed by an estimated increase in the effective reproduction number ' (supplementary material). we also compare case counts if the policies were relaxed with a oneweek delay. keeping some npis in place for an extra week could reduce total covid-19 cases by up to 19% (as of june 2, 2020). the results confirm that policy restrictions are not all equal in their ability to affect disease spread. the policy of restricting mass gatherings (contact restriction) is estimated to be the most effective npi to contain covid-19, followed by closures of various businesses and stay-at-home orders. due to this variation in effect, it is advisable to lift restrictions with minimal effects first, gradually easing restrictions that potentially lead to higher case numbers. this study presents a comprehensive quantitative analysis that includes individual effects of npis on the transmission of covid-19. to the best of . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 30, 2020. . https://doi.org/10.1101/2020.05.28.20115980 doi: medrxiv preprint our knowledge, this is the first study that uses variations in policy interventions by governments to discover their differential impacts at reducing mobility that in turn reduces disease spread. prolonged lockdowns and restrictive policies can have devastating social and economic consequences. however, opening too soon could result in rapid disease spread. therefore, governments need to develop cautious approaches to lifting restrictions in a bid to return to normalcy (23). the approach presented in this paper allows for a deeper understanding of the policy effects on mitigating the spread of covid-19. the forecasts of disease spread when npis are (partially) loosened guide policymakers towards the appropriate strategy when reversing the interventions. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus from patients with pneumonia in china how will country-based mitigation measures influence the course of the covid-19 epidemic? the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study effect of non-pharmaceutical interventions to contain covid-19 in china substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) the effect of human mobility and control measures on the covid-19 epidemic in china the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak google covid-19 community mobility reports current situation report of the rki to covid-19 bringing up opensky: a large-scale ads-b sensor network for research key: cord-318766-vx0dnnxh authors: wendt, ralph; nagel, stephan; nickel, olaf; wolf, johannes; kalbitz, sven; kaiser, thorsten; borte, stephan; lübbert, christoph title: comprehensive investigation of an in-hospital transmission cluster of a symptomatic sars-cov-2–positive physician among patients and healthcare workers in germany date: 2020-06-03 journal: infection control and hospital epidemiology doi: 10.1017/ice.2020.268 sha: doc_id: 318766 cord_uid: vx0dnnxh we investigated potential transmissions of a symptomatic sars-cov-2–positive physician in a tertiary-care hospital who worked for 15 cumulative hours without wearing a face mask. no in-hospital transmissions occurred, despite 254 contacts among patients and healthcare workers. in conclusion, exposed hospital staff continued work, accompanied by close clinical and virologic monitoring. on january 27, 2020, the first infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was diagnosed in germany. 1 by may 20, 2020, the number of cases had increased to 176,000. 2 to address the large number of patients at a given time, hospital capacity, especially the availability of intensive care facilities and the number of healthcare workers (hcws), particularly doctors and nurses, are cornerstones and essential pillars in the struggle against the covid-19 pandemic. disease transmission among infected hcws is a major threat that could adversely affect the capacity of hospitals to care for patients and might even endanger patients. 3 we report on a symptomatic sars-cov-2-infected physician who worked in a large 1,030-bed municipal hospital in leipzig, germany. at the time of the report, coronavirus disease 2019 (covid-19) cases in germany were rapidly increasing. the index case physician had traveled to the part of germany with the highest covid-19 rates at that time, thereby visiting pubs and restaurants in the city of stuttgart (federal state of baden-wuerttemberg) on march 12-13, 2020. after returning home, she felt unwell for 2 days and had a sore throat, cough, and fever. despite these symptoms, she went to work at the hospital without wearing a face mask or other protective devices. she remained symptomatic, particularly with subfebrile temperature and frequent coughing. on march 16, 2020, she was working an 8-hour shift in addition to a 4-hour on-call shift. she was making rounds at the hospital, caring for patients, doing admissions, discussing treatments with colleagues, having frequent contact with nurses and other healthcare staff, having lunch and coffee breaks in a small lounge area, and even sitting in a crowded lecture room along with other hcws (supplemental fig. 1 online), as well as listening to employee information on the management of covid-19 patients. during the on-call shift, she saw patients all over the hospital. the next day, she stayed at home, but she returned the following day for another 3 hours of hospital work, still coughing heavily and apparently ill. when noticed, she was immediately sent home after undergoing coronavirus testing (combined nose and throat swab), which was positive for sars-cov-2. to assess sars-cov-2 infection, either copan liquid amies swabs (copan, brescia, italy) or pharyngeal lavage (10 ml saline solution) was used for sampling the nasopharyngeal material of the index physician and all contacts. rna extraction and real-time to further investigate potentially missed transmissions, we attempted to detect iga and igg antibodies against sars-cov-2 in sera, withdrawn on days 15 or16 and 22 or 23 after exposure, by an in vitro diagnostic labeled anti-sars-cov-2 enzyme-linked immunosorbent assay (elisa, euroimmun, lübeck, germany), following the manufacturer's instructions. only descriptive statistics were applied. numerical variables were summarized as means, and categorical variables were given as frequencies or proportions. ethical approval was not required for this study because only anonymous aggregated data were used, and no medical interventions were made on human subjects. sampling of hcws or patients was part of hospital policy. we identified 187 contacts with hcws and 67 contacts with patients. of these, 23 were identified as high-risk contacts, as defined by the world health organization guidance document on covid-19 global surveillance. 4 table 1 summarizes the characteristics of each high-risk contact. all high-risk contacts were subject to active symptommonitoring and committed to wearing a face mask during work. we tested all 254 potential contacts of the symptomatic sars-cov-2-positive index physician, including 67 patients, and 187 nurses and doctors, technical and medical assistants, and other healthcare staff, on day 5 after the exposure by specific rt-pcr from nose and throat swabs or pharyngeal lavage, irrespective of reported symptoms. of 187 tested hcws, 30 (16%) reported minor unspecific symptoms of upper airway infection (sore throat, coughing, sniffing). all tested persons turned out to be sars-cov-2 negative. the 23 high-risk contacts were investigated again 10 days after exposure by specific rt-pcr from nose and throat swabs. test results were negative, again. additionally, all high-risk contacts and the index physician were examined serologically on days 15 or 16 and days 22 or 23 after exposure. despite some iga positive-to-inconclusive ratios, none showed positivity for sars-cov-2 igg antibodies at follow-up except the index physician featuring seroconversion (table 2 ). we tested a large number of possible contact persons of a symptomatic sars-cov-2-infected physician among hcws and patients on day 5 after exposure; all were negative. after a comprehensive investigation of all contact clusters, we identified 23 highrisk contacts (22 hcws and 1 patient) and tested them again on day 10 after exposure. all rt-pcr tests remained negative for sars-cov-2, confirming that there was no transmission of the virus. extensive investigation and testing were performed because viral shedding of sars-cov-2 has been shown in completely asymptomatic individuals, prompting the hypothesis that clinical status is not reliable for triage and further testing. 5 sars-cov-2 has frequently been detected in asymptomatic carriers, for instance, during a cruise ship outbreak in which most of the passengers and staff were tested irrespective of symptoms: 51% of the laboratory-confirmed cases were asymptomatic at the time of confirmation. 6 for further analysis and confirmation of our results, we investigated the serum of all high-risk contacts (n = 23) on days 15 or 16 and 22 or 23 for sars-cov-2-specific antibodies. we found positive iga antibodies at both times but no igg antibodies, confirming the rt-pcr results of zero transmission. the specificities for iga and igg against sars-cov-2 were 91.3% and 100%, respectively. although the calculated performance values were obtained in a small study cohort (n = 24), the specificities were similar to those reported in a previous study and in accordance with the manufacturer's specifications. 7 these results are unexpected. considering an active sars-cov-2 transmission source with a presumably high viral burden and many high-risk contacts inside a hospital, massive spread was anticipated, particularly since a protective face mask was not in use. sars-cov-2 has been shown to persist (at least under experimental circumstances) for up to 72 hours depending on the surface type. 8 in hospitals, surfaces are frequently cleaned and disinfected, and all hcws reported regular handwashing, disinfection, and strict adherence to hygiene rules. recently, the importance of presymptomatic transmission (r p ) has been stressed (r p = 0.9 of an r 0 of 2), and the proportion of symptomatic transmission (r s ) to the basic reproduction number r 0 was calculated to be only 0.8 of an r 0 of 2. 9 a low percentage of transmission to high-risk contacts (5%) has been reported in nonhousehold members. 10 another study in the united states investigated the high-risk contacts of a patient among healthcare personnel (n = 32) and did not find any transmission, confirming our results. however, testing was only done in symptomatic persons after clinical monitoring, and asymptomatic transmission could have been missed. 11 importantly, not every infected person with sars-cov-2 is a super spreader, and not every infected individual in a closed room triggers a superspreading event, although this situation has the potential to do so and therefore must be dealt with as such. 12 in this context, our data support the recommendation to keep high-risk contacts among the hospital staff at work (especially in these difficult times with personnel shortages) when strictly using a protective mask, accompanied by close clinical and virologic monitoring. transmission of 2019-ncov infection from an asymptomatic contact in germany covid-19). world health organization website strengthening icu health security for a coronavirus pandemic global surveillance for human infection with coronavirus disease (covid-2019)-interim guidance. world health organization website evidence of sars-cov-2 infection in returning travelers from wuhan, china japan national institute of infectious diseases website severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing investigation of a covid-19 outbreak in germany resulting from a single travel-associated primary case: a case series first known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the usa the role of superspreaders in infectious disease acknowledgments. we kindly acknowledge the enormous personal commitment of ulrike schmidt (study department), and ines geßner as well as gerit görisch, md (both from hospital hygiene department).financial support. no financial support was provided relevant to this article. all authors report no conflicts of interest relevant to this article. key: cord-326223-q6e60nf8 authors: gembardt, florian; sterner-kock, anja; imboden, hans; spalteholz, matthias; reibitz, franziska; schultheiss, heinz-peter; siems, wolf-eberhard; walther, thomas title: organ-specific distribution of ace2 mrna and correlating peptidase activity in rodents date: 2005-02-16 journal: peptides doi: 10.1016/j.peptides.2005.01.009 sha: doc_id: 326223 cord_uid: q6e60nf8 biochemical analysis revealed that angiotensin-converting enzyme related carboxy-peptidase (ace2) cleaves angiotensin (ang) ii to ang-(1–7), a heptapeptide identified as an endogenous ligand for the g protein-coupled receptor mas. no data are currently available that systematically describe ace2 distribution and activity in rodents. therefore, we analyzed the ace2 expression in different tissues of mice and rats on mrna (rnase protection assay) and protein levels (immunohistochemistry, ace2 activity, western blot). although ace2 mrna in both investigated species showed the highest expression in the ileum, the mouse organ exceeded rat ace2, as also demonstrated in the kidney and colon. corresponding to mrna, ace2 activity was highest in the ileum and mouse kidney but weak in the rat kidney, which was also confirmed by immunohistochemistry. contrary to mrna, we found weak activity in the lung of both species. our data demonstrate a tissueand species-specific pattern for ace2 under physiological conditions. in the regulation of heart function and blood pressure, different peptide systems are involved, e.g. the renin-angiotensin system (ras), the kallikrein-kinin system, and the natriuretic peptide system. in these systems, proteases like angiotensin-converting enzyme (ace) or neutral endopeptidase (nep) have the distinction of generating or catabolizing biologically active peptides [10, 39, 42] . the newly discovered angiotensin-converting enzyme-related carboxypeptidase (ace2) has considerable sequence homology to ace (40% identity and 61% similarity), contains a hexxh zinc-binding domain, and conserves other critical residues typical of the ace family [12, 37] . the first step in generating angiotensin peptides is the cleavage of angiotensinogen to angiotensin (ang) i by renin. ang i is hydrolyzed by either ace or chymase to ang ii, which mediates its biological actions via the at1 and at2 receptors [15, 21] . ang i is also metabolized by nep to ang-(1-7) [15] , which mediates distinct effects through its receptor mas [35] . importantly, ang-(1-7) can also be directly metabolized from ang ii by ace2, whereas aminopeptidase a converts ang ii to ang iii [18] . ace2 also hydrolyzes ang i to ang-(1-9), although there is no hydrolysis of ang-(1-9), ang-(1-7), and ang(1) (2) (3) (4) (5) . moreover, ace2 hydrolysis is also specific for des-arg [9] bradykinin and its shorter fragments, although it cleaves neither bradykinin nor bradykinin-(1-7) [40] . ace2 mrna is expressed in many tissues but shows a less ubiquitous profile than ace. first studies in mice detected the highest expression in the ileum by quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) [23] . ace2 is an important part of the ras, which counteracts the function of ace. it was also shown that ace2 expression can be upregulated by blockade of at 1 -receptors [27] . the importance of ace2 in cardiovascular regulation was confirmed by targeted disruption of ace2 in mice. the absence of ace2 in mice leads to a severe cardiac contractility defect, increased ang ii levels, and upregulation of hypoxia-induced genes in the heart [11] . in addition to its peptidolytic function, recent investigations have discovered that ace2 is a functional receptor for the coronavirus, which causes the severe acute respiratory syndrome (sars) [30] . in this investigation, we (i) measured the mrna distribution of ace2 through different tissues in both species. moreover, we (ii) quantified ace2 protein by western blot using a commercial polyclonal antibody to ace2. we (iii) measured ace2 activity in different tissues of mice and rats. we (iv) established a monoclonal antibody against ace2 to complete the investigation of tissue distribution by immunohistochemistry. finally, we compared (v) the distribution of ace2 in both species on the mrna and protein level. all experiments were done according to the guidelines of the federal law on the use of experimental animals in germany and were approved by the local authorities. for this investigation we used c57bl/6 mice and sprague-dawley (sd) rats in an age of 3-5 months. animals were killed by cervical dislocation. for rnase protection assay (rpa), ace2 activity assay and western blot, the tissues were snap frozen in liquid nitrogen. the samples were stored at −80 • c until further processing (all organs in total, heart divided into atria and ventricles). the tissues for immunohistochemistry were put in 4% formalin. after 24 h they were embedded and processed to paraffin sections. the polymerase chain reaction (pcr) amplified a 358 bp fragment (probe: mmace2) from mouse kidney cdna using the 5 -primer ctc agt gga tgg gat ctt gg (mmace25) and the 3 -primer tgt agc cat ctg ctc cct ct (mmace23), respectively a 342 bp fragment (probe: rnace2) from rat lung cdna using the 5 -primer cgg gga aag atg tca agc tcc tgc (rnace25) and the 3 -primer ctt gtc tgg tga cag cgc (rnace23), which were subcloned in a t-vector (promega gmbh, mannheim, germany). a sp6 polymerase transcribed a radioactive probe complementary to mmace2 (resp. rnace2) mrna, and a rna complementary to 127 nucleotides of the rl32 mrna was used as positive control [2] . ace2-specific mrna for mouse and rat were identified by rnase protection assay (rpa) using the ambion rpa ii kit (ambion (europe) ltd., huntingdon, uk). total rna was isolated from tissues using the trizol reagent (invitrogen gmbh, karlsruhe, germany) with subsequent chloroform-isopropanol extraction according to the manufacturer's instructions. a 15 g total rna fraction of each sample was hybridized with approximately 50 000 cpm for ace2 and 50 000 cpm for rl32 of the radiolabeled antisense probes in the same assay. equal loading has been insured by mrna measurements and mrna gel electrophoresis using 1 g of each sample (not shown). the hybridized fragments protected from rnase a + t1 digestion were separated by electrophoresis on a denaturing gel (5%, w/v polyacrylamide, 8 m urea) and analyzed using a fujix bas 2000 phospho-imager system (raytest gmbh, straubenhardt, germany) to perform quantitative analysis by measuring the intensity of the ace2 bands. the blots of each species were calculated to ace2 mrna expression in kidney, which was present on both blots of each species. the expression level in the lung was set to 100%. ace2 activity was measured similar to the method by vickers et al. [40] . tissue was homogenized in assay buffer (50 mm 2-morpholinoethanesulfonic acid, 300 mm nacl, 10 m zncl 2 , 0.01% brij-35, ph 6.5). protein concentration was determined using roti-quant (carl roth gmbh and co. kg, karlsruhe, germany) by the manufacturers instruction. we used mca-apk(dnp) (biosynthan gmbh, berlin, germany) dissolved in dmso (50 m, final concentration) as the ace2 substrate. the assay was performed in assay buffer and was started by adding 10 l of tissue homogenate. after 2 h incubation at ambient temperature (24 • c), the reaction was suppressed by adding 100 m o-phenanthrolin (final concentration). parallel control tests were performed in the presence of 1 m dx 600 (data not shown) [25] . after centrifugation (10 min, 10 000 × g) the fluorescence was measured at 320 nm (excitation) and 405 nm (emission) with the perkin-elmer fluorescence reader lambda 5 (perkin-elmer las gmbh, rodgau, germany). the molecular standardization was performed with mca-ap (biosynthan gmbh, berlin, germany) and calculated per mg protein. the functionality of the assay was proven by a standardized solution with defined, recombinant ace2 activity (r&d systems gmbh, wiesbaden, germany). tissue was homogenized in phosphate-buffered solution (pbs) containing protease inhibitor mixture (complete, roche diagnostics gmbh, mannheim, germany). protein concentration was determined with bca protein assay kit (perbio science gmbh, bonn, germany). sample proteins (10 g/lane) and a prestained protein-weight marker (amersham biosciences gmbh, freiburg, germany) were size fractionated by sds-polyacrylamide gels (10%) and transferred to pvdf membranes with a pegasus semidry-blotter (phase gmbh, lübeck, germany). equal loading has been insured by staining control gels with simply-blue safe stain (invitrogen gmbh, karlsruhe, germany) using 10 g of each sample (not shown). the membranes were blocked at room temperature in 5% dry milk powder (blotting grade, non-fat dry milk, bio-rad laboratories gmbh, munich, germany) prepared with tris-buffered saline containing 0.1% tween 20 (ttbs) for 1 h, incubated with goat polyclonal antibody against ace2 (santa cruz biotechnology inc., heidelberg, germany, 1:250 diluted in 5% dry milk powder ttbs, 1 h), and then washed three times with ttbs (15 min each). subsequently, the membranes were incubated with horseradish peroxidase-conjugated antigoat igg (dakocytomation a/s, glostrup, denmark, 1:1000, 1 h) and washed three times. specific immunoreactive proteins were detected by enhanced chemiluminescence (amersham biosciences gmbh, freiburg, germany). the bands on the x-ray film were quantified by densitometry scanning and expressed as percentage of the kidney protein signal. monoclonal antibodies against the synthetic peptide avgeimslsaat (aa 403-414 of murine ace2) have been raised. for immunization of the mice peptide was cross-linked with glutaraldehyde to albumin fraction v from bovine serum. balb/cj female mice were injected with the conjugate. following four booster injections the spleen lymphocytes were fused with fo myeloma cells by using polyethylene glycol 1500 (roche diagnostics gmbh, mannheim, germany) following the manufacturers instructions. the different hybridoma supernatants were screened for specific antibodies by using the synthetic peptide in the nctest [26] . for production of monoclonal antibodies, positive hybridoma cells were grown in celline incubators (integra biosciences gmbh, fernwald, germany). the mouse monoclonal antibodies were affinity purified on a mabtrap g ii column (amersham-pharmacia gmbh, otelfingen, switzerland) from cell culture supernatants. immunoglobulin class and subclasses were determined with the immuno type kit (sigma-aldrich chemie gmbh, taufkirchen, germany). paraffin sections of mouse tissues were prepared and stained using standard histology procedures. for immunostainings, deparaffinized and rehydrated tissue slides were first treated for 30 min with 30% h 2 o 2 to block the endogenous peroxidase. after rinsing in ddh 2 o and soaking in pbs for 5-10 min, slides were treated with 10% (w/v) bsa in pbs to eliminate non-specific protein binding sites. the slides were then exposed (overnight, 4 • c) to the monoclonal ace 2 antibodies (clone 7e7, 1d3) at concentrations of 1 and 4 g/ml, respectively. after removing excess antibody, slides were treated with biotin-labeled anti-mouse (dianova gmbh, hamburg, germany) antibody for 30 min at 37 • c and finally with horse-radish peroxidase (hrp) labeled streptavidine (zymed laboratories inc., san francisco, usa) for 20 min at 37 • c. after washing, slides were incubated in aminoethylcarbazol (sigma-aldrich co., st. louis, usa) for 10 min at room temperature. slides were counterstained with hematoxylin, and cover slipped according to conventional procedures. slides were examined under a conventional microscope after removing the excess substrate in ddh 2 o. negative controls were performed without the primary antibody, just applying dilution buffer of the primary antibody. data were analyzed by t-test using spss11 software (spss benelux bv, gorinchem, the netherlands). each value was expressed as the mean ± s.e.m., and statistical significance was accepted for p < 0.05. ace2 mrna could be detected in all investigated organs, but with profound distinction between different organs. in both species, only a low amount was found in ventricle, liver, testis, forebrain, and spleen ( figs. 1 and 2) , whereas in the lungs a moderate and comparable expression of ace2 mrna was found and set to 100%. the highest levels were found in the ileum of both species (fig. 3) . between the species several differences in tissue specific expression of ace2 mrna were found. the expression in mouse was most pronounced higher than in rat in kidney (∼31.9-fold), colon (∼18.6-fold), and ileum (∼12.0-fold) (fig. 3) , whereas in bladder (∼2.5-fold) and ventricle (∼2.1-fold) ace2 expression in rat exceeded the mouse. in accordance with the rna expression data, highest activity for ace2 was found in the ileum of mouse and rat (table 1) , whereas the activity in the mouse was 3.2fold higher. lowest ace2 activity was found for both species in spleen. low activity was also found for liver of mice and thymus of rats. corresponding to the differences on mrna levels in the kidney the ace2 activity fig. 3 . quantification of the rpas of mice (white columns) and rats (black columns). the mrna amount of the lungs is set to 100% (n ≤ 4) . the values are shown as mean + s.e.m. 1. ventricle, 2. kidney, 3. lung, 4. liver, 5. testis, 6. bladder, 7. forebrain, 8. spleen, 9. thymus, 10. stomach, 11. ileum, 12. colon, 13. brainstem, 14. atrium, 15. adipose tissue. * p < 0.05, ** p < 0.01, *** p < 0.0001 compared mouse vs. rat. was much higher in mice than in rats (∼13.9-fold). the activity of ace2 in the lung was different to mrna and 2.6-fold higher in rats than in mice. in contrast to rpa data the activity in colon was comparable between both species. using a commercial polyclonal antibody in western blot for the quantification of protein levels in mouse and rat tissues (fig. 4) a pattern completely different from rna expression and ace2 activity was found. a moderate and comparable expression could be detected in the kidney of both species and was set to 100%. thus, the highest amount of protein could be detected in atrium of both species (mouse: 124.5%; rat: 131.5%) and ventricle (mouse: 131.7%; rat: 143.3%). for the mouse less ace2 protein was found in lung (19.7%) and testis (28.7%), whereas no protein was detectable in these two tissues in rat. in thymus (mouse: 44.4%; rat: 50.6%) and forebrain (mouse: 87.9%; rat: 80.7%) of both species a moderate expression was detectable, whereas no ace2 protein was found in spleen of mouse and rat. to further clarify the discrepancy between rpa and activity on one side and western blot on the other, immunohistochemistry was performed in lung, kidney (fig. 5) , and testis (data not shown) of mice and rat with new monoclonal ace2 antibodies (clones 7e7 and 1d3), we generated. the antibodies were determined to belong to the igg1 subclass. in the lungs of both species alveolar macrophages and type 2 cells (fig. 5 , upper row) were stained with both monoclonal ace2 antibodies (data for clone 1d3 not shown). the epithelium of the renal tubuli was strongly stained (fig. 5 , lower row, left) in the kidney of mice. in rats only a weak signal, but the same pattern as in mouse, was detected, what aligned with mrna and ace2 activity (fig. 5, lower row, right) . in recent investigations it was shown that peptidases like ace and nep are important regulators of cardiovascular and endothelial function as well as myocardial remodelling [1, 7, 36, 41] . consequently, after its discovery in 2000, ace2 became an enzyme of interest for scientific investigation of its impact in cardiovascular physiology and pathophysiology [11, 12, 37] . to elucidate some of its physiological functions we investigated the tissue distribution of mrna and protein in a variety of tissues of c57bl/6 mice and sprague-dawley rats. while we could see correlating patterns of mrna and ace2 activity in most of the examined tissues, we also found significant divergences between the investigated species. the huge difference between mrna and protein levels in the lung may be due to shedding as demonstrated for ace [4, 12, 32] . this shedding leads to an increased secretion of ace2 and lowered its protein content in the lung by even high mrna expression. the significant differences that we found between the species on ace2 protein and mrna levels in kidney could be explained by the varying interspecies regulation and expression of peptidases, as shown in the literature for nep activity in rat and rabbit kidneys [14] . comparing our mrna and activity data with the western blot pattern, we have to conclude that the commercial polyclonal antibody is not detecting ace2 protein in organ homogenates and is not suitable for ace2 staining. in contrast, using immunohistochemistry our new monoclonal ace2 antibodies produce staining patterns comparable to our mrna and activity data. we have shown that ace2 expression in rodents is highest in ileum among the examined organs. it was shown for other peptidases of ras like ace and nep that they are also present at high levels in the intestine [29] . however, the distinct function of these peptidases in the ileum is not yet known. further investigations have to clarify the physiological and pathophysiological functions of the peptidases in the gastrointestinal tract. beside its physiological function as a peptidase, ace2 is used by coronavirus as a co-receptor in severe acute respiratory syndrome (sars) [30] . it was shown that the sars coronavirus only can enter cells which express ace2 [24] . ace2 distribution in the small intestine, lung and vascular endothelium may offer a point of entry for the sars coronavirus, but does not reflect its basic function [22, 30, 38] . interestingly, the distribution patterns we found for mrna and ace2 activity contradict investigations using a commercial northern blot for detecting mrna [12, 37] but have been confirmed by recent papers using rt-pcr [23] . this discrepancy may be a species-specific alteration of tissue distribution, since they used human tissue for northern blot, or it may be due to technique differences (commercial northern versus rpa and activity assay). the first possibility is at least supported by our finding that significant differences in ace2 expression patterns exist between the close relatives mouse and rat. recent investigations revealed biological activity for angiotensin peptides other than angii, like ang-(1-7) [16, 33, 34] . ace2 can generate ang-(1-7) by cleaving the cterminal amino acid from angii [40] . ace2 is also involved in another pathway leading to the generation of ang-(1-7). it cleaves angi to ang-(1-9) [12] . ang-(1-9) is then hydrolyzed by ace to ang-(1-7) . we demonstrated that ang-(1-7) is an endogenous ligand for the g protein-coupled receptor (gpcr) mas [35] . mrna of the gpcr mas was found at high levels in testis and certain brain regions and at fig. 5 . immunohistochemical visualization of ace2 positive cells. sections of lungs (upper row) and kidneys (lower row) from mouse (left panel) and rat (right panel). in the lungs of both species alveolar macrophages and type 2 cells were stained positive. the tubulus epithelium in mouse kidney was stained positive, whereas in the rat kidney only weak staining was seen. moderate levels in kidney and heart [2, 3, 31] . it was shown that high concentrations of ang-(1-7) were present in heart, kidney, and brain [5, 6, 8, 28] . in recent investigations, it was demonstrated that ace2, mas, and its endogenous ligand ang-(1-7) are present in the same cells of the kidney [9] . as we recently postulated, this indicates a relevant impact of the ace2/ang-(1-7)/mas axis on blood pressure regulation and cardioprotection. actual investigations indicate an upregulation of ace2 in heart failure, pointing to the relevance of ace2 in cardiac function [20, 32, 43] . however, there was a high incidence of sudden death in animals overexpressing ace2. electrophysiology revealed severe, progressive conduction and rhythm disturbances with sustained ventricular tachycardia that progressed to fibrillation and death [13] . while anti-arrhythmic actions were demonstrated for ang-(1-7) in low concentra-tions (0.22 nm) by stimulating its own receptor, 100-fold higher concentrations of ang-(1-7) lead to arrhythmias by stimulating the at1 receptor [17, 19] . therefore, the overexpression of ace2 may lead to a high increase in the production of ang-(1-7), turning its cardioprotective actions into effects causing arrhythmias by unspecific at1 stimulation. in future studies, the actions of ang-(1-7) and its concentrationdependency on ace2 expression on heart rhythm have to be proven in in vivo experiments with at1-and mas-deficient animals. our data on tissue and species-specific ace2 expression point to the fact that the ras becomes increasingly complex. since we identified an expression pattern markedly different from ace, we conclude that the expression levels of the involved peptidases like ace, ace2, and nep that generate and/or degrade the bioactive peptides of the ras are predic-tive of either the occurrence of vasoconstriction or dilatation or the dominance of pathophysiological stimuli over beneficial conditions. the acute infarction ramipril efficacy (aire) study investigators, effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure imprinting of the murine mas protooncogene is restricted to its antisense rna cell type-specific expression of the mas proto-oncogene in testis a point mutation in the juxtamembrane stalk of human angiotensin i-converting enzyme invokes the action of a distinct secretase cardiac angiotensin-(1-7) in ischemic cardiomyopathy immunocytochemical localization of angiotensin-(1-7) in the rat forebrain vasopeptidase inhibitors: an emerging class of cardiovascular drugs enhanced renal immunocytochemical expression of ang-(1-7) and ace2 during pregnancy novel aspects of the renal renin-angiotensin system: angiotensin-(1-7), ace2 and blood pressure regulation vasopeptidase inhibitors: a new therapeutic concept in cardiovascular disease? angiotensin-converting enzyme 2 is an essential regulator of heart function a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 heart block, ventricular tachycardia, and sudden death in ace2 transgenic mice with downregulated connexins distribution of neutral endopeptidase activity along the rat and rabbit nephron novel angiotensin peptides counterregulatory actions of angiotensin-(1-7) angiotensin-(1-7): cardioprotective effect in myocardial ischemia/reperfusion brain renin-angiotensin system blockade by systemically active aminopeptidase a inhibitors: a potential treatment of salt-dependent hypertension effects of angiotensin ii and angiotensin-(1-7) on the release of [3h]norepinephrine from rat atria ace2 gene expression is up-regulated in the human failing heart insights into angiotensin ii receptor function through at2 receptor knockout mice tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor novel peptide inhibitors of angiotensin-converting enzyme 2 immunocytochemistry in brain tissue upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors angiotensin-(1-7) immunoreactivity in the hypothalamus of the (mren-2d)27 transgenic rat burrell lm. differential tissue and enzyme inhibitory effects of the vasopeptidase inhibitor omapatrilat in the rat angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus expression of the mouse and rat mas proto-oncogene in the brain and peripheral tissues the role of ace2 in cardiovascular physiology vasodilator action of angiotensin-(1-7) on isolated rabbit afferent arterioles angiotensin-(1-7): an update angiotensin-(1-7) is an endogenous ligand for the g protein-coupled receptor mas remodeling of myocardium and arteries by chronic angiotensin converting enzyme inhibition in hypertensive patients a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase exploring the pathogenesis of severe acute respiratory syndrome (sars): the tissue distribution of the coronavirus (sars-cov) and its putative receptor, angiotensin-converting enzyme 2 (ace2) the angiotensin-converting enzyme gene family: genomics and pharmacology hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase at1 receptor blockade increases cardiac bradykinin via neutral endopeptidase after induction of myocardial infarction in rats vasopeptidase inhibitors: will they have a role in clinical practice? increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace2 florian gembardt is paid by a grant from the "deutsche forschungsgemeinschaft" (german research foundation)[grk865]. this study was also supported by the "stiftung zur förderung der wissenschaftlichen forschung an der universität bern". we thank helmut würdemann and susanne gygax for their technical assistance. key: cord-304930-gf3cptnt authors: hinz, sebastian; ellmann, daniel; wegner, christian; bömicke, wolfgang; bensel, tobias title: the digital abutment check: an improvement of the fully digital workflow date: 2020-10-24 journal: case rep dent doi: 10.1155/2020/8831862 sha: doc_id: 304930 cord_uid: gf3cptnt by using modern digitalization techniques, an existing denture can be digitized and aid the provision of a new implant-supported denture according to a fully digital workflow. this includes fully navigated implant surgery and results in an immediately provided prosthetic restoration. however, even with the current digital workflow, it is challenging to achieve a definitive prosthetic restoration in a single treatment session. in order to achieve a definitive denture in as few treatment sessions as possible, we have implemented the digital abutment test. this test modified the existing data set and determined the final restoration. in the present case, the preexisting maxillary removable complete denture was converted into a fixed immediate restoration using the fully digital workflow. the workflow is divided into two treatment phases, each with three treatment sessions, where part of the second phase involves an innovative digital abutment check. the illustrated case shows an effective use of current digital possibilities. special attention was also paid to a minimally invasive course of therapy. once the hard and soft tissues of the oral cavity have been digitally recorded, e.g., by intraoral scanners and modern 3d radiograph technology, these data can serve as the basis for further digital process steps [1] . before implant surgery, the final position of the implants can be determined virtually using planning software and 3d radiograph images. the position of the inserted implants is of crucial importance for the final design of the anchored prosthesis and the longterm survival of the dentures [2] [3] [4] . in this context, template-guided implant surgery allows for the accurate placement of dental implants [2, 3] . a precise surgical template can be fabricated by merging the intraoral-scan data with the 3d radiographic data [5] [6] [7] . for the surgeon, the use of a surgical template results in a predictable and safe treatment procedure. patients additionally benefit from a shorter treatment time, fewer postoperative restrictions, and an overall increase in comfort [8] . as a consequence, template-guided implant placement is becoming increasingly popular. [8] [9] [10] [11] . the implants were placed according to the comfour® system (camlog vertriebs gmbh, wimsheim, germany). four implants are used to immediately rehabilitate the edentulous jaw with a fixed interim restoration. the advantages of the system lie in the maximum expansion of the support polygon through a precisely planned implant position, but especially implant angulation; the distal implants are usually inserted at an angle between 15 and 30 degrees [3, 4, [12] [13] [14] . this allows the prosthetic support field to be expanded posteriorly without compromising relevant anatomical structures and without having to perform extensive bone augmentation measures. the template used in the present report provided a precise implementation of the digitally determined implant position [5, 9-11, 15, 16] . the navigated procedure offered further advantages by eliminating the need for intraoperative flap formation, thus reducing surgical trauma and reducing the postoperative need for analgesics [8, 17] . however, the main challenge for the prosthetic treatment team (dentist, dental technician) in the implementation of a fully digital all-on-x workflow is to transfer the final implant position to the definitive restoration. the deviations between the virtually planned and the real implant position must be balanced out even with guided implantation [18] [19] [20] [21] [22] [23] [24] [25] . digital impressions represent a reliable impression method for the fabrication of implant-supported full-arch frameworks [26] . usually, scan-bodies are screwed onto the implants for digital impression making. these can negatively affect the precision of the digital impression [27] [28] [29] . however, transmission errors due to the use of scan-bodies must be taken into account. a transfer without the use of scan-bodies could increase the precision of the prosthetic restoration. in this present case report, we successfully demonstrate the realization of the final prosthodontic restoration while avoiding inaccuracies caused by screwed-in scan-bodies. this was accomplished by the implementation of the digital abutment check without the use of scan-bodies. the 55-year-old patient first came to the department of prosthodontics (faculty of medicine, martin luther university halle-wittenberg) during an outpatient consultation. the patient reported smoking about 10 cigarettes a day but otherwise had an uneventful medical history. the maxilla was edentulous, and the remaining teeth in the mandible were stable from a periodontological and endotontological point of view. the existing prosthetic and conservative restorations were found to be sufficient and restored a continuous dental arch, which extended from the second left to the second right premolar. according to the patient, the last maxillary teeth had been extracted about one year prior to the initial consultation. since then, he has been wearing a conventionally made removable complete denture. the patient stated that the stability of the prosthesis was not sufficient to eat properly. he also complained about the extensive coverage of the palate. in the course of weighing up the different therapy options, the patient decided on an implant-supported, fixed full-arch prosthesis without palatal coverage. the mandible shortened dental arch was to be maintained in agreement with the patient. case reports in dentistry the aim of the decided upon therapy was to achieve stable, palate-free care, involving little surgical effort and with as few treatment sessions as possible. we decided to use a gentle, minimally invasive procedure without any additional augmentations. the final planning included the insertion of four implants in the upper jaw and a fixed, provisional immediate restoration, which should be transferred to a definitive fixed partial denture (fpd) after the healing period of six months. the treatment was divided into two independent phases. the first treatment phase involves diagnostics and therapy planning, as well as surgical intervention and the immediate provision of an interim restoration. the second treatment phase consists of the transfer of the interim fpd to the definitive fpd after the successful healing period. we decided to use the comfour® system (camlog, wimsheim, germany) to maximise patient comfort during the treatment. the advantages of the comfour® system are the possibility of immediate loading with appropriate primary stability of the implants (>35 ncm) and the targeted avoidance of augmentation by angulation of the posterior implants. 4.1. pretreatment. as the first treatment step, the mandible and the maxillomandibular relationship were digitally recorded intraorally (trios 3 intraoral scanner, 3shape a/s, copenhagen, denmark). the correct fit of the maxillary removable complete denture was checked in advance using low viscosity polyvinyl siloxane (gc fit checker® advanced, gc europe n.v., leuven, belgium). the correct maxillomandibular relationship was checked clinically based on the resting position of the mandible. if the measured parameters are inadequate, dentures would have to be adjusted in advance. alternatively, if the patient does not wear any removable dentures or the maxillomandibular relationship has to be changed, the maxillomandibular relationship can be adjusted with digital measurement systems (jmanalyser +, zebris medical gmbh, isny, germany), due to the fact that these systems offer a digital interface for computer-aided design/computer-aided manufacturing (cad/cam). in the present case, the existing maxillary removable complete denture was scanned in the dental laboratory (rüberling & klar dental laboratory, halle (saale), germany) with a laboratory scanner (e4 lab scanner, 3shape a/s, copenhagen, denmark) ( figure 2) . it was then used as a template for the radiographic and the surgical template and the provisional fpd. the standard triangulation/-tesselation language (stl) data records of the mandible and the maxillomandibular relationship record were matched with the data of the existing complete denture (exocad den-talcad, r+k cad/cam technologie gmbh & co.kg, berlin, germany). the soft tissue situation of the edentulous maxilla was picked up using the base area of the complete denture. this procedure resulted in the manufacturing of the radiographic template. the base of the radiographic template was milled from clear polymethylmethacrylate using a silicone occlusion key (shera-duett-soft, shera werkstoff-technologie gmbh & co.kg, lemförde, germany), the surgical template was placed in the patient and fixed in its definite position with anchor pins (guided anchor pin, nobel biocare ag). for a flapless surgery, the mucous membrane was punched through the drill sleeves and removed. afterwards, the implant bearings were reprocessed using 6-13 mm drill bits. for the correct transmission of the planned threedimensional implant position, the implants (guide cam-log®sl promote plus, camlog vertriebs gmbh) were inserted using the torque wrench up to the marking of the rotation indicator on the drill sleeves. the bone quality corresponded to d2 and the implants performed primary stability (>35 ncm). immediate loading was therefore possible ( figure 6 ). abutments compensating for the implant angulation were connected to the implants (bar abutments, camlog vertriebs gmbh). a flexible handle (comfour®, cam-log vertriebs gmbh) was used to screw in the posterior implants (figure 7 ). 4.6. immediate restoration. titanium adhesive bases (titanium adhesive base for bar abutment, passive fit, camlog, wimsheim, germany) were screwed onto the bar abutments. this resulted in an intraoral and tension-free bonding of the provisional fpd. the static and dynamic occlusion was checked and adjusted. the provisional fpd was then drained and cleaned using alcohol. afterwards, the provisional fpd case reports in dentistry was bonded to the titanium adhesive bases (titanium adhesive base for bar abutment, passive fit, camlog) using autopolymerizing prosthesis repair resin (qu resin, bredent gmbh & co.kg, senden, germany). the basal surface of the interim fpd was elaborated and polished, and then, the interim fpd was tightened to the implants at 15 ncm and the occlusion finally checked. the screw channels were closed with foam pellets and a gypsum-based sealing material (cavit™, 3m deutschland gmbh, seefeld, germany), and a postoperative orthopantomogram was then performed (figure 8 ). (figure 9) 5.1. abutment scan. after a six-month implant healing period, the interim fpd had to be replaced by a definitive screw-retained fpd. for this purpose, the occlusion of the existing interim situation and the maxillomandibular relationship were reevaluated. a digital maxillomandibular relation record was made with the interim restoration in place using an intraoral scan-ner (trios 3 intraoral scanner, 3shape a/s). then, the interim fpd was unscrewed in order to scan the bar abutments screwed onto the implants and the adjacent soft tissues. after the scan was completed, the provisional fpd was screwed back on. check. the stl scan data were sent to the dental laboratory for further processing. in the dental laboratory, the existing planning data record is matched with the new maxillomandibular relation record scan and the abutment and soft tissue scan. the incisive papilla, palatine raphe, and palatine rugae served as points of reference for matching the scans. as a result, changes in the jaw relation and soft tissue, as well as minimal positional deviations of the abutments, can be transferred to the definitive fpd ( figure 10) . the cobalt-chromium alloy (cocrmo) fpd framework (organic cocr, organical dental implant, r+k cad/cam technologie gmbh & co.kg, berlin, germany) was designed in accordance with the generated data set (exocad dentalcad, r+k cad/cam technologie gmbh & co.kg) and subsequently milled (organical® 5x dental milling try-in. the interim fpd was unscrewed in order to try it in the definitive fpd framework and to check the passivity of fit using the sheffield test [30] . in the present case, the fpd scaffold fitted without the need for any adjustment ( figure 11 ). the provisional fpd was then screwed back on, and the definitive fpd scaffold was sent to the dental laboratory for final veneering. inclusion of the fpd. the cocrmo fpd framework was veneered individually in the laboratory using composite resin material (sr chromasit, ivoclar vivadent, schaan, liechtenstein). in the final treatment session, the provisional fpd was removed and the definitive and veneered fpd was screwed on with 15 ncm. the fit of the fpd was optimal; the occlusion was checked and optimized with minimal grinding measures. finally, the screw channels were covered with foam pellets and composite resin (crb-bonding, shofu inc., kyoto, japan; tetric evoflow, ivoclar vivadent, schaan, liechtenstein) ( figure 12 ). the final orthopantomogram was performed (figure 13 ). the present case report demonstrates the effective use of the available modern digital manufacturing processes in dentistry. the treatment procedure integrated consequent digital backward planning, fully navigated implantation, and completely digital dental prosthesis production. the procedure described here is in contrast to most of the other all-on-x concepts, which do not involve purely digital processes [6, 7, [12] [13] [14] . as conventional impressions could be avoided completely in this workflow, the number of individual treatment sessions (session for maxillomandibular relationship record and try-in) and individual session time could be significantly shortened. for example, the entire surgical procedure up to the installation of the provisional fpd could be carried out by an experienced practitioner in about 75 minutes. the relatively short duration of treatment in combination with a minimally invasive procedure lowers the risk of postoperative complaints such as swelling and pain [8, 17] . the key innovation of this case report is the digital abutment check, which is carried out directly using an intraoral scanner without screwed-in scan-bodies. this is possible because the exact geometry of the bar abutments is stored in the cad software databases (organical® dental implant, r+k cad/cam technologie gmbh & co.kg). the direct scan of the bar abutments without the use of scan-bodies again offers advantages in terms of digital impression precision. possible errors due to incorrect positioning of the scan-body on the implants can thus be excluded [27] [28] [29] . ultimately, this in turn influences the exact fit of the final fpd. in the present case report, an indication-oriented application of both 3d printing methods and cam milling methods is also demonstrated. nowadays, surgical templates can be printed with a clinically acceptable fit [31, 32] . if anchor pins have to be used, the 3d printing process is ideal when compared to milling because even with the most 11 case reports in dentistry modern 5-axis milling machines, the tool angle is limited. with 3d printing, the drilling channels for the anchor pins, which are often at an angle of 90°to the actual machining axis, can be more easily realized. the printing material, like the milling material, offers the possibility of sterilization before insertion in the patient during the surgery. the ability to sterilize any used dental laboratory materials in order to avoid the chain of infection during the surgical treatment is essential and not just since the beginning of the covid-19 pandemic situation [33] . the construction of the interim fpd is an additional advantage of the performed digital workflow. the interim fpd was deliberately manufactured using pmma. in contrast to more stable materials like zirconium dioxide, pmma offers potential material-specific benefits. these benefits result in the ability to check and adapt the case reports in dentistry maxillomandibular relationship during the provisional restoration phase. this means that the occlusion of the provisional pmma-fpd could be either easily reduced by grinding or increased by adding self-curing resin material. the higher abrasiveness of the material offers some protection against overloading the implants during the healing process. finally, the definitive fpd may be veneered with ceramic instead of composite resin. in contrast, it is also possible to produce a fully anatomical milled fpd, which can be inserted as the definitive dental restoration. this procedure could avoid the previous scaffold try-in. in this case report, one of the objectives of the treatment procedure was that the denture should be easily repairable. therefore, the veneering material chosen for the definitive fpd was composite resin. however, compared to ceramic veneering material, composite resin enables easier occlusal adaptation to the opposing jaw. in contrast to a conventional treatment process, not only are sessions for the maxillomandibular relationship and possibly the scaffold try-in avoided, but the process described here also creates an accurate adjustment of the definitive restoration. the treatment concept shown in this present case report combines a safe and time-saving digital workflow with demanding, predictable therapy results. in addition, the surgical intervention was not a major burden for the patient. the enormous gain in quality of life exceeds the manageable treatment effort for the patient and is clearly in focus. the patient's wish to switch from a removable complete denture to a fixed, palate-free prosthetic restoration could be fulfilled after three sessions following the end of the first treatment phase. in addition, both the original tooth position and aesthetics could be transferred to the provisional and definitive fpd, producing a harmonious appearance that was familiar to the patient. the data used to support the findings of this study may be released upon application to the department of prosthodontics, martin-luther-university halle-wittenberg, which can be contacted at dr. christian wegner, department of prosthodontics, university hospital halle, magdeburger straße 16, 06112 halle (saale), germany. relationship between the ct value and cortical bone thickness at implant recipient sites and primary implant stability with comparison of different implant types the accuracy of single-tooth implants placed using fully digitalguided surgery and freehand 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implant surgery with the codiagnostixtmsoftware precision and accuracy of a digital impression scanner in full-arch implant rehabilitation comparison of postoperative intraoral scan versus cone beam computerised tomography to measure accuracy of guided implant placement-a prospective clinical study methods used to assess the 3d accuracy of dental implant positions in computer-guided implant placement: a review evaluation of impression accuracy for a four-implant mandibular model-a digital approach improving the fit of implant-supported superstructures using the spark erosion technique comparison of the accuracy of implants placed with cad-cam surgical templates manufactured with various 3d printers: an in vitro study the impact of the fabrication method on the three-dimensional accuracy of an implant surgery template preventing sars-cov-2 transmission in rehabilitation pools and therapeutic water environments the authors declare that they have no conflict of interest. the study was performed as part of the employment of the authors of the department of prosthodontics, faculty of medicine, martin-luther-university halle-wittenberg, halle (saale), germany. key: cord-029402-5gun91ep authors: celi, giuseppe; guarascio, dario; simonazzi, annamaria title: a fragile and divided european union meets covid-19: further disintegration or ‘hamiltonian moment’? date: 2020-07-17 journal: j doi: 10.1007/s40812-020-00165-8 sha: doc_id: 29402 cord_uid: 5gun91ep despite being symmetric in its very nature, the covid-19 shock is affecting european economies in a very asymmetric way, threatening to deepen the divide between core and peripheral countries even more. it is not covid-19 itself, however, but the contradictions within the eu’s growth model and institutional architecture that would be to blame for such an outcome. the dramatic impact of the economic crisis brought on by the pandemic and the threat that it poses to eurozone survival seem to have forced a reluctant germany into action: a minor step, but an important signal. this note analyses the crossroads currently facing europe—the risk of disintegration vis-a-vis the opportunity for a ‘hamiltonian moment’—discussing possible future scenarios in the light of past developments. like viruses, crises too can rapidly change their dna: the financial crisis of 2008 changed from international to regional, from financial to real, eventually turning into an existential threat to the whole european integration project. in the institutional context of the eurozone (ez), the financial crisis soon developed into a sovereign debt crisis, dragging the banks along with it. in the austerity environment that followed, the southern periphery (sp) never completely recovered the losses in output, employment, and fiscal sustainability. thus, the "symmetric" coronavirus shock hit countries that were in highly asymmetric conditions. in fact, not all the countries of the union have the resources needed to intervene in support of their economy, prompting concern that countries with the deepest pockets might be getting an unfair advantage in the eu's single market. far from triggering mutual protection, the covid-19 crisis seems to be paving the way for the same mistakes that followed the 2008 financial crisis. the centrifugal forces threatening disintegration of the european monetary union (emu) seem to have been defused, albeit only in part and only in extremis, at least for the time being. however, the survival of the union depends not only on responding to the severe financial problems caused by the epidemic, but also means addressing the long-term, structural problems that led to the increasing divergences among her members. as chancellor merkel herself acknowledged, "it is in nobody's interest for germany alone to be strong after the crisis". 1 convergence is essential to put the union on a more solid basis so as to guarantee its long-term sustainability. what policies and what reforms should be implemented to pursue this objective? and are they economically and politically feasible? trying to answer these questions, we shall briefly review the institutional and structural causes of the increasing divergence between core and sp, shedding light on three momentous events: the creation of the monetary union, the 2008 financial crisis and the covid-19 shock. the first decade following the introduction of the emu saw continuity in the process of europeanisation embarked upon as from the formation of the common market, based on financial liberalization and market globalization. as argued in celi et al. (2018 celi et al. ( ,2019 , europeanisation meant eu-wide application of a policy of deregulation of goods, labour and capital markets that affected the timing, shape and direction of the european integration process, halting the process of convergence between the core and the sp of the eu. the more developed core (centred on germany) increased its productive and technological capacity; the sp, caught between product competition within the eu and cost competition from emerging economies in the international markets, saw a decline in its manufacturing capacity. 2 with the fall of the soviet union and the entry of the former socialist countries of central and eastern europe in the eu, the eastern periphery (ep) became a key gear of germany's manufacturing matrix (stehrer and stollinger 2015) . a huge flow of direct investments, primarily in the automotive sector, transformed the economies 1 merkel: germany must help other eu states get back on their feet, euractiv.com with reuters 13 mag 2020 https ://www.eurac tiv.com/secti on/econo my-jobs/news/merke l-germa ny-must-help-other -eustate s-get-back-on-their -feet/. 2 these diverging trends are likely to increase as a result of the slow, small and asymmetric response that europe is giving to the ongoing pandemic-driven economic crisis, as confirmed by the macroeconomic evidence provided in this forum by heimberger et al. of the visegrad pact (poland, hungry, slovakia, and check republic) into an essential source of intermediate goods (medium and medium-high quality) for the german industry. a well-qualified, extremely cheap workforce, generous subsidies and tax breaks, as well as geographical proximity and historical links, are among the determining factors of the increasingly tight links between the core and its ep. the impressive growth in manufacturing capacity in the east led to a restructuring in the hierarchical organization of the supply chains across europe: the weaker suppliers in the south were displaced by their cheaper competitors in the east, while the highly specialised suppliers of components in the industrial regions of the south maintained, and even increased, their close links with the german producers. 3 the crowding-out of the less dynamic firms in the sp did not take the form of efficiencyenhancing market selection but rather a generalized reduction of production capacity, contributing to fuel a well-documented (see, among the others, guarascio and simonazzi 2016; dosi et al. 2019 ) process of 'poor tertiarisation' of the sp. on the other hand, the ep's industrial miracle was created by foreign, mostly german, direct investment, with the automotive sector taking the lion's share. so far, we have seen no comparable development of other productive sectors, nor has the automotive sector created spill-over effects in the rest of the economy (krzywdzinski 2019). on the contrary, the surge in the production of components for the automotive sector has partly displaced other productions, leading to an increasing 'mono-specialization' of these economies. despite a growing shortage of skilled labour, wages have remained modest. threats of production shifting further east, to romania, turkey, or to north africa, (pavlinek et al. 2017) 4 are reflected in the adoption of a wage containment policy at home, driving young people with high educational qualifications to emigrate, and weakening the countries' skills base. with domestic demand subdued, the high growth rates recorded by these countries are entirely led by the growth in exports of local production by foreign multinationals (i.e., the so-called "integrated peripheral markets"). while their intensive specialisation in the automotive industry makes them totally dependent on the health of the german automotive industry, the foreign control of production decisions, innovation processes and markets makes it extremely difficult to undertake an independent, less unbalanced development path (celi et al. 2018) . to conclude, the two peripheries-the southern one, made up of the mediterranean economies, and the eastern one, with the prominent role of the visegrad countries-suffer from different fragilities, which descend from their common, albeit diverse, economic and financial dependence on the core. however, the core itself is dependent for its growth on the pattern of specialisation within the eu: the southern markets providing an outlet for its increasing surplus of manufactures, the eastern countries supplying cheap inputs for its industries. this combination of structural divergence and economic interdependence lies behind the fragility of the union as well as of the improbability of its disintegration given the high costs it would entail for core and peripheries alike. in the first period of the emu (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) , the core-sp structural divergence was partly hidden by massive financial flows to the periphery. the 2008 financial crisis, and the ensuing international liquidity crunch, prompted a "sudden stop" of capital flows and a collapse in demand and imports. at that point, the structural and institutional flaws of the emu became evident: the reaction to the crisis aggravated the divergence. with the blame for the crisis put squarely on borrowers, austerity policies were advocated (or imposed) to ensure debtor countries' public and private solvency. with austerity killing demand, growth and imports in the sp, germany, which had built most of its huge trade surplus between 2003 and 2008 by exporting to the periphery, had to find new outlets for its goods. special international conditionsnamely, china's huge growth, which gobbled up german capital goods and highquality durable consumer products (particularly cars), and the vigorous american recovery-supported germany's ability to redirect its trade flows, expand its market shares outside the emu, and make a speedy return to its pre-crisis production levels. the united kingdom, the united states, but above all china, became the most important markets for german exports. the rapid recovery of the german economy pulled the ep along with it: the visegrad countries recorded unparalleled growth in europe. with the abrupt change in the international scenario in 2016, germany's (and the entire emu's) mercantilist strategy was up against the ropes. the brexit referendum, trump's election, and the u-turn in chinese economic policy inaugurated a phase of retreat in international trade. trade with the uk began to suffer due to the increasing uncertainty in future trade relations. when the united states took action to reduce the external deficit, china and germany, the countries with the largest trade surpluses vis-à-vis the united states, were caught in the crosshairs. trade tensions between the us and china put further pressure on international trade. the export-led growth model that had so far supported germany's leadership began to creak. the change in world trade took its toll on german (and eu) growth rates. from the second quarter of 2017, the slowdown in german exports hit industrial production and the gdp, widening the growth gap with china and the usa and dragging the whole emu along with it ( fig. 1) . as the escalation of trade disputes affected relations between the united states and germany 5 (and by extension the eu), the negative effects on europe's (exportled) growth intensified. in the last quarter of 2019, just a few months before the outbreak of covid-19 in the eu, germany's growth rate zeroed. income growth estimates for the rest of europe were consequently reduced. the pandemic arrived in europe from the south: italy was the first country to suffer the contagion. its abrupt, dramatic effects exposed the fragility of the periphery and the crippling effects of austerity policies. since 2010, across the board cuts in social spending had hit the entire range, from health to education, from social assistance to social investment. 6 figures 2, 3 and 4 show the evolution of the share of public expenditure on education and health (divided between general expenditure and hospitals) relative to gdp in the emu, germany and the sp between 2008 and 2018. many hospitals had been closed, the number of beds reduced, medical and nursing staff cut back (for a detailed analysis of the impact that austerity policies had on the italian health care system, see prante et al. 2020 ). it is not surprising that the death toll was higher where intensive care facilities were scarcer. on the eve of the covid crisis, public health accounted for 6.5 percent of the social product in italy and spain, and almost 10% in germany, where per capita healthcare spending did not suffer cuts due to austerity (though it was not completely spared self-imposed restrictions). the covid-19 exposed another aspect of the 'divisive' union (celi et al. 2020) : different capacities to respond to the pandemic crisis. economic ideology shares with austerity the responsibility for the scant endowment of medical equipment and health staff. efficiency, understood as cost reduction, has been taken as the guiding principle. the obsession with competitiveness and reliance solely on the export-led growth model accounts for the almost exclusive emphasis on "tradable" sectors, to the detriment of "non-tradable" sectors (housing, health, education, welfare services in general), considered of lesser importance for international competition. this means that, in the era of austerity, these items have been the first to be sacrificed, in debtor and creditor countries alike. chazan (2020) reports that for years, politicians and health economists in germany have complained that the country has too many hospitals, with the bertelsmann foundation recommending halving the number of hospital, from 1400 to fewer than 600 (chazan 2020 ). only such a radical consolidation-the bertelsmann study arguedwould "improve patient care and mitigate the shortage of doctors and nursing staff". the pandemic succeeded in transforming this "oversupply" into an asset. the same logic of pursuing the lowest cost guided the international location of production, which displaced domestic production and weakened production capacity in the sp. from a regional (european) point of view, this process resulted in a reorganisation of production and trade relations between core, ep and sp. on a global scale, core and peripheries entered into very long and complex gvcs that proved extremely vulnerable in the face of the interruptions prompted by the pandemic. personal protective equipment, respirators, medicines: the emergency has made it clear what it means to lose the capacity to produce domestically, both in quantity and quality, what is urgently needed, bringing the problem of self-sufficiency back to the attention of economists and policymakers. there is no such thing as a symmetric shock. in addition to the grim toll of victims and the incredible pressure on the health systems of all countries, the lockdown of activities to reduce contagion meant a tremendous plunge in production and incomes and enormous pressure on public finances all over the world. however, the lockdown is expected to affect economies differently. the central and eastern european countries have been less affected by covid than the western european countries: not trusting the resilience of their fragile health systems, they have had to rely on rigid social distancing (walker and smith 2020) . even within this group of countries there are differences: thanks to their more robust health systems, the czech republic and slovenia were less constrained by rigid social distancing and able to start economic recovery earlier. moreover, due to their strong productive links with austria-a country relatively less affected by the pandemic which came out of the lockdown earlier-and their favourable positioning in the development of digital economy (wiiw 2020) , their economic outlook is rather better. conversely, it will be tougher for the economies, like those of the sp, which are more dependent on services-tourism and hospitality in particular (fig. 5) -and for cee countries and southern regions that rely to a greater extent on production of intermediate products for final producers, since the latter can better defend themselves from fall in demand by cutting down orders to their suppliers (the so-called "whip effect"). policies have also differed widely across countries and regions. while all the central banks of the developed world promptly intervened to provide almost unlimited . although the stability pact has been temporarily suspended, 7 there are obvious differences in how much member states can spend, depending on their fiscal space. member states are making use of the new flexibility granted by the ec on state aid rules, strictly enforced beforehand to ensure fair competition within the internal market (rios 2020) . germany, which accounts for about a quarter of the eu's gdp, accounts for more than half (52%) of the emergency coronavirus state aid approved by the ec, prompting concerns that countries with the deepest pockets might be getting an unfair advantage by such a sudden (and temporary) abandonment of one of the common market's key pillar (france and italy each account for 17% of the total). an eu official, speaking on condition of anonymity, observed that "if you look at the scale of what germany in particular, but also some others, are doing-any notion of level playing field or single market integrity has gone out of the window." 8 these concerns underpin the ailing south's demand for a joint eu financial plan. in the absence of a prompt and massive common effort, the sp will pay the highest price to the health crisis. indeed, the different firepower will entail a still greater asymmetry in the economic and power relations between the various member states. the ecb, alone among the eurozone institutions, is doing as much as it can to avoid breakdown of the emu. to address the covid-19 crisis, it launched a new asset purchasing programme: the eurosystem's balance sheet shot up from 4692 billion euros on 28 february to 5395 billion by 1st may 2020. despite this massive monetary injection (700 billion in two months) the spread on italian bonds, which had fallen in mid-march following the ecb's announcements, again rose very rapidly, fluctuating in response to political developments. indeed, as tooze and schularick (2020) point out, if, in the 2008 crisis, the liquidity injected into the system by the ecb was enough to prevent deflagration of the banking system, 9 the current crisis would require a coordinated fiscal policy of enormous proportions. despite some recent moves (inaugurated by a merkel-macron agreement), this still does not seem to be looming on the horizon. the newly released 'next generation' (ng) plan, based on the 2021-2027 budget, celebrated by some as a "hamiltonian moment", has yet to qualify as forerunner of an eu-wide up-to-the-challenge fiscal capacity. 10 first of all, it is meant to be temporary and, moreover, it is too little, too late. the plan should mobilize 750 billion euros, 500 in the form of grants and 250 in loans. 8 quote reported by the website euractiv.com. 9 the eurosystem balance sheet (the network of european central banks, guided by the ecb) rose from 1150 billion euros at the beginning of 2007 to 4675 billion euros by the end of 2018; that is, from barely 10% to almost 40% of the euro zone gdp (12 000 billion euros). 10 the ng plan money will be spent over the 2021-2024 period. with an even subdivision over the period, the package amounts to an annual 0.56% of the eu's 2019 gdp, over four years. 7 several parties, including most recently the president of the ecb, christine lagarde, are urging the ec to review the pact before its temporary suspension expires on december 31, 2020. apart from the fact that these are gross figures-once the member states' contributions to the eu budget are subtracted, the net amount received by the neediest countries is much smaller-their disbursement will not start before 2021, will be distributed over a 4-year period, with amounts that grow over time, and, as stated in the ec's "proposal for a regulation" the financial contribution will "be paid in instalments once the member state has satisfactorily implemented the relevant milestones and targets identified in relation to the implementation of the recovery and resilience plan" (ec 2020, art. 17.4.a). as darvas (2020) emphasizes, the incorporation of the ng plan into the eu's next multiannual budget would take advantage of a well-established framework, 'already subject to various checks and balances'. on the other hand, ng resources risk to be trapped in a 'slow-moving machine'. in order to be financed, ng-related projects need to be designed, approved and implemented as part of a process that can take several years. as a result, the timing of disbursements is just the opposite of what would be required to respond to the urgency imposed by the current situation and, even more so, by the expected collapse of incomes that the european economies are going to face. 11 however, the commission expects that barely 24.9% of the total new firepower for grants would be spent in 2020-2022, when the recovery needs will be greatest (darvas 2020) . far from being a tool to counter the immediate effects of the crisis, the ng plan is more similar to the juncker plan, and shares all its weaknesses. 12 it is highly unlikely that countries like italy, severely hit by the pandemic and in persistent financial distress, will be able to afford to refrain from asking for other funds (namely, esm, sure and others for a total amount of about 59 billion euros) which could be paid out immediately, subject to the usual conditionality. the merkel-macron agreement has been hailed as the first step towards a more supportive union. behind the good intentions, there are the concrete interests of both france and germany for the survival of the emu: they look with growing concern at the rise of euroscepticism in the sp. the french economy has been hit hard by the pandemic, and was already in difficulty before. gdp forecasts for 2020 vary widely, but all agree in estimating a fall in the french gdp of much the same proportions as in the case of italy. on the other hand, germany was, together with the netherlands, the main beneficiary of the creation of the euro, and italy and france were the main losers (gasparotti and kullas 2019) . 13 as chancellor merkel told the german lawmakers, "it is essential for germany, as an export nation, that its eu partners also do well". 14 indeed, the history of the eu has taught that excessive german surpluses are deleterious for the south of the eurozone. greater government action, retreat from hyper-globalism, and lower growth rates predate the pandemic. the covid-19 crisis has given yet more voice to calls for protectionist and "beggar thy neighbours" types of policies. it has led countries to prioritize resilience and autonomy in production over cost savings and efficiency through global outsourcing. the same powerful german production platform, so disproportionately export-oriented and dependent on imports of intermediate goods, finds itself vulnerable to a type of shock (the covid-19 pandemic) that disrupts gvcs and threatens to change the existing economic order through permanent disruption of the patterns of demand and production. although transition from an industrial platform designed for export to one for the internal market (a sort of transition from a war to a peace economy) is a formidable challenge, this transformation would benefit germany itself, considering the winds of trade war and the growing uncertainty about the future developments of the global value chains. the european countries are at a crossroad between either letting the union dissolve or radically reforming it. today's darkened geopolitical environment requires europe to act as a whole. however, the emu will remain fragile as long as it chooses to continue to delegate control over its policies to market surveillance. a true "hamiltonian moment", which involves adopting a common fiscal policy in support of the common monetary policy is a matter of urgency. we still have a long way to go. divisions between member countries marked by opposition between debtors and "frugal" creditors, as well intra-country political struggles and conflicting interests, have-even in the face of this dramatic crisisled to the paralysis of the european institutions, with the one exception of the ecb. faced with what she sees as a serious threat to the eu's survival, the german chancellor (and the commission's president ursula von der leyen) have been driven to action. however, as we argued in sect. 3, little can be expected from the ng plan for immediate support. the ability of the sp to emerge from the crisis will increasingly depend on its ability to take advantage of the greater flexibility of eu rules for an efficient use of industrial policy, helping companies and the whole economy to respond to the challenge posed by social and technological innovation, the restructuring of production and the reorganization and shortening of gvcs. the pandemic will have significant repercussions on the international organization of production and gvcs (on this point, see also the contributions to this forum by strange and coveri et al.) . indeed, the countries initially most affected by covid (china, korea, italy) are among the most important suppliers of intermediate goods at the international level. studies on the propagation of economic shocks triggered by natural disasters (such as the earthquake that hit japan in 2011) along the value chains (boehm et al. 2019 ; inoue and todo 2019) found significant supplier substitution effects. anecdotal evidence signals numerous cases of supplier substitution in some countries as a result of the coronavirus (baldwin and tomiura 2020). the extent of these effects depends on the degree of complexity of the production chains, which affects the degree of input substitutability. propagation effects also depend on the presence of "hub" companies interconnected with a large number of supplier and customer firms (inoue and todo 2019) . future developments are uncertain, depending on the relative strength of two opposite effects. on the one hand, greater coordination afforded by digitalisation of production networks could favour substitution effects (especially in cases where value chains are less regionalised and the search for new suppliers is more difficult) (zhenwei quiang et al. 2020 ). on the other hand, processes of reshoring and shortening of value chains could occur, especially where production chains are less complex or automation is more advanced. the second possibility could represent an opportunity to reverse the processes of deindustrialization that have impoverished, above all, the productive fabric of the peripheral countries. a third perspective, probably utopian, could contemplate coordination of coalitions of producers across eu member states. in a situation of strong productive complementarities between countries, the fortunes of the producers (workers and firms) in one country are bound to those in the other. this would call for a coordinated industrial policy at the european level aiming at ensuring a balanced development of the economies of its members through their integration in the european production networks. in emergency situations where production activities are reduced or temporarily suspended (as in the case of coronavirus shock), bilateral agreements (mediated by governments) between producers in different countries should aim at stabilizing employment levels and pre-existing supply contracts between firms through "mutualisation" of the required financial effort. after all, having surprisingly spoken out in favor of the eurobonds, the ceo of volkswagen herbert diess could-at one remove-be also supportive of such a project! material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. thinking ahead about the trade impact of covid-19 input linkages and the transmission of shocks: firm-level evidence from the 2011 tōhoku earthquake crisis in the european monetary union. a core-periphery perspective unravelling the roots of the emu crisis. structural divides, uneven recoveries and possible ways out un'unione divisiva. una prospettiva centro-periferia della crisi europea germany's oversupply of hospital beds aids pandemic fight, the financial times next generation eu: 75% of grants will have to 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investment and the development of the automotive industry in eastern and southern europe decades of tight fiscal policy have left the health care system in italy ill-prepared to fight the covid-19 outbreak eu countries use looser state aid rules to uphold troubled firms the central european manufacturin core: what is driving regional production sharing? (no. 2014/15-02) the shock of coronavirus could split europe-unless nations share the burden. the guardian why has eastern europe suffered less from coronavirus than the west? the vienna institute for international economic studies foreign direct investment and global value chains in the wake of covid-19: lead firms of gcv acknowledgements open access funding provided by università di foggia within the crui-care agreement. key: cord-168710-a5pst4gf authors: jalilian, abdollah; mateu, jorge title: a hierarchical spatio-temporal model to analyze relative risk variations of covid-19: a focus on spain, italy and germany date: 2020-09-28 journal: nan doi: nan sha: doc_id: 168710 cord_uid: a5pst4gf the novel coronavirus disease (covid-19) has spread rapidly across the world in a short period of time and with a heterogeneous pattern. understanding the underlying temporal and spatial dynamics in the spread of covid-19 can result in informed and timely public health policies. in this paper, we use a spatio-temporal stochastic model to explain the temporal and spatial variations in the daily number of new confirmed cases in spain, italy and germany from late february to mid september 2020. using a hierarchical bayesian framework, we found that the temporal trend of the epidemic in the three countries rapidly reached their peaks and slowly started to decline at the beginning of april and then increased and reached their second maximum in august. however decline and increase of the temporal trend seems to be sharper in spain and smoother in germany. the spatial heterogeneity of the relative risk of covid-19 in spain is also more pronounced than italy and germany. started from wuhan, the capital of hubei province, china in december 2019, the outbreak of 2019 novel coronavirus disease has spread rapidly across more than 200 countries, areas or territories in a short period of time with so far over 4.4 million confirmed cases and 296 thousand confirmed deaths (world health organization, 2020) . the spread of covid-19 across and within countries has not followed a homogeneous pattern (giuliani et al., 2020) . the causes of this heterogeneity are not yet clearly identified, but different countries have different levels of national capacity based on their abilities in prevention, detection, response strategies, enabling function, and operational readiness (kandel et al., 2020) . besides, different countries have implemented different levels of rigorous quarantine and control measures to prevent and contain the epidemic, which affect the population movement and hence the spread pattern of covid-19. given the highly contagious nature of covid-19, the spatial pattern of the spread of the disease changes rapidly over time. thus, understanding the spatio-temporal dynamics of the spread of covid-19 in different countries is undoubtedly critical. the spatial or geographical distribution of relative location of incidence (new cases) of covid-19 in a country is important in the analyses of the disease risk across the country. in disease mapping studies, the spatial domain of interest is partitioned into a number of contiguous smaller areas, usually defined by administrative divisions such as provinces, counties, municipalities, towns or census tracts, and the aim of the study is to estimate the relative risk of each area at different times (lee, 2011; lawson, 2018) . spatio-temporal models are then required to explain and predict the evolution of incidence and risk of the disease in both space and time simultaneously (anderson and ryan, 2017) . estimation of area-specific risks over time provides information on the disease burden in specific areas and identifies areas with elevated risk levels (hot spots). in addition, identifying the changes in the spatial patterns of the disease risk over time may result in detecting either regional or global trends, and contributes to make informed and timely public health resource allocation (wakefield, 2007) . to account for the underlying temporal and spatial autocorrelation structure in the spread of covid-19, available data on the daily number of new cases and deaths in different countries/regions have already been analyzed in a considerable number of studies. for example, kang et al. (2020) used moran's i spatial statistic with various definitions of neighbors and observed a significant spatial association of covid-19 in daily number of new cases in provinces of mainland china. gayawan et al. (2020) used a zero-inflated poisson model for the daily number of new covid-19 cases in the african continent and found that the pandemic varies geographically across africa with notable high incidence in neighboring countries. briz-redón and serrano-aroca (2020) conducted a spatio-temporal analysis for exploring the effect of daily temperature on the accumulated number of covid-19 cases in the provinces of spain. they found no evidence suggesting a relationship between the temperature and the prevalence of covid-19 in spain. gross et al. (2020) studied the spatio-temporal spread of covid-19 in china and compare it to other global regions and concluded that human mobility/migration from hubei and the spread of covid-19 are highly related. danon et al. (2020) combined 2011 census data to capture population sizes and population movement in england and wales with parameter estimates from the outbreak in china and found that the covid-19 outbreak is going to peak around 4 months after the start of person-to-person transmission. using linear regression, multilayer perceptron and vector autoregression, sujath et al. (2020) modeled and forecasted the spread of covid-19 cases in india. as pointed out in alamo et al. (2020) , there are many national and international organizations that provide open data on the number of confirmed cases and deaths. however, these data often suffer from incompleteness and inaccuracy, which are considerable limitations for any analyses and modeling conducted on the available data on covid-19 (langousis and carsteanu, 2020) . we highlight that we are yet in the center of the pandemic crisis and due to the public health problem, and also to the severe economical situation, we do not have access to all sources of data. thus reseachers know only a portion of all the elements related to covid-19. in addition, data on many relevant variables such as population movement and interaction and the impact of quarantine and social distancing policies are not either available or accurately measured. combined with the unknown nature of the new covid-19 virus, any analysis such as the present study only provides an approximate and imprecise description of the underlying spatio-temporal dynamic of the pandemic. nevertheless, having a vague idea is better than having no idea, and the results should be interpreted with caution. currently, a wealth of studies have appeared in the very recent literature. many of them follow the compartmental models in epidemiology, partition the population into subpopulations (compartments) of susceptible (s), exposed (e), infectious (i) and recovered (r), and fit several variations of the classical deterministic sir and seir epidemiological models (peng et al., 2020; roda et al., 2020; bastos and cajueiro, 2020) . we believe that considering stochastic components is important, if not essential, to explain the complexity and heterogeneity of the spread of covid-19 over time and space. for this reason, in the present work we propose a spatio-temporal stochastic modeling approach that is able to account for the spatial, temporal and interactions effects, together with possible deterministic covariates. we acknowledge that the proposed model in its current form requires development and refinements as more information becomes available, but at the stage of the pandemic we are now, it can provide a reasonable modeling framework for the spatio-temporal spread of covid-19. this is illustrated by modeling the daily number of new confirmed cases in spain, italy and germany from late february to mid august 2020. the r code for implementing the proposed model can be made available upon request. we also provide a shiny web application (chang et al., 2020 ) based on the model discussed in this paper at https://ajalilian.shinyapps.io/shinyapp/. the structure of the paper is the following. the open data resources used in this study are introduced in section 2. a model for the daily number of regional cases is considered in section 3. as described in section 4, this model explains the spatio-temporal variations in the relative risk of each country in terms of a number of temporal, spatial and spatio-temporal random effects. the results of fitting the considered model to the number of daily confirmed cases in spain, italy and germany are given in section 5. the paper concludes in section 6 with a few last remarks. governmental and non-governmental organizations across the world are collecting and reporting regional, national and global data on the daily number of confirmed cases, deaths and recovered patients and provide open data resources. incompleteness, inconsistency, inaccuracy and ambiguity of these open data are among limitations of any analysis, modeling and forecasting based on the data (alamo et al., 2020) . particularly, the number of cases mainly consist of cases confirmed by a laboratory test and do not include infected asymptomatic cases and infected symptomatic cases without a positive laboratory test. in this study, we focus on the daily number of confirmed cases in spain, italy and germany and used the following open data resources. italy: data on the daily accumulated number of confirmed cases in the 20 regions of italy are reported by the civil protection department (dipartimento della protezione civile), a national organization in italy that deals with the prediction, prevention and management of emergency events. these data are available at the github repository https://github.com/ pcm-dpc/covid-19 and are being constantly updated every day at 18:00. germany: the robert koch institute, a federal government agency and research institute responsible for disease control and prevention, collects data and publishes official daily situation reports on covid-19 in germany. data on the daily accumulated number of confirmed cases in the 16 federal states of germany extracted from the situation reports of the robert koch institute are available at the github repository https: //github.com/jgehrcke/covid-19-germany-gae and are being updated on a daily basis. table 1 summarizes the number of regions, study period and country-wide daily incidence rate of the data for each country. data on distribution population of the considered countries are extracted from the gridded population of the world, version 4 (gpwv4), which provides estimates of the number of persons per pixel (1 degree resolution) for the year 2020 (center international earth science information network (ciesin) columbia university, 2018). these data are consistent with national censuses and population registers. 3 modeling daily regional counts suppose that a country, the spatial domain of interest, is partitioned into regions a 1 , . . . , a m , defined by administrative divisions such as states, provinces, counties, etc (see table 1 ). let y it denote the number of new covid-19 cases in region the expected number of new cases is given by e it = p i it , where p i is the population of region a i and it is the incidence rate of covid-19 in region a i at time t. under the null model of spatial and temporal homogeneity of the incidence rate it = 0 and provides an estimate for e it , where is an estimate of the country-wide homogeneous daily incidence rate (waller et al., 1997) . the estimated daily incidence rate per million population (10 6 0 ) so far is around 68, 46 and 29 for spain, italy and germany, respectively (see table 1 ). 3.2 distribution of daily regional counts consul and jain (1973) introduced a generalization of the poisson distribution, which is a suitable model to most unimodal or reverse j-shaped counting distributions. given nonnegative random rates λ it , i = 1, . . . , m, t = 1, . . . , t , we assume that y it 's are independent random variables following the generalized poisson distribution with and the parameterization (zamani and ismail, 2012 ) thus ϕ is the dispersion parameter and the case ϕ = 0 represents the ordinary poisson distribution (no dispersion) with here, parameter α controls the shape (power) of the relation between the conditional variance of y it |λ it and its conditional mean. for example, the relation between (zamani and ismail, 2012) . the underlying random rates λ it , i = 1, . . . , m, t = 1, . . . , t , account for the extra variability (overdispersion), which may represent unmeasured confounders and model misspecification (wakefield, 2007) . variations of the random rate λ it relative to the expected number of cases e it provide useful information about the spatio-temporal risk of covid-19 in the whole spatial domain of interest during the study period. in disease mapping literature, the nonnegative random quantities are called the area-specific relative risks at time t (lawson, 2018, section 5.1.4). obviously eθ it = 1 and which means that the temporal and spatial correlation structure of the underlying random rates λ it determine the spatio-temporal correlations between θ it 's. temporal correlation ar(2) ζ i spatial correlation due to distance between regions gmrf ξ i spatial correlation due to neighborhood relation between regions bym by ignoring these correlations, the standardized incidence ratio θ it = y it / e it provides a naive estimate for the relative risks (lee, 2011) . however, in a model-based approach the variations of the relative risks are often related to regional and/or temporal observed covariates and the correlation between θ it 's are explained in terms of regional and/or temporal random effects using, for example, a log linear model (wakefield, 2007; lee, 2011; lawson, 2018) . in the present study, we consider the log linear model where µ is the intercept and d i is the population density of region a i , i.e. the population of a i , p i , divided by the area of a i . the population density is standardized to have mean 0 and variance 1 and β is its regression coefficient. moreover, η it is a zero mean random effect which represents spatio-temporal variations in relative risks due to temporal and spatial trend and correlation. among many different possibilities, we assume that η it takes the additive form where δ i represents the temporal trend, ε t accounts for temporal correlation and ζ i and ξ i explain spatial correlation due to spatial distance and neighborhood relations among regions a 1 , . . . , a m , respectively (see table 2 ). the latent (stochastic) temporal trend δ t is expected to be a smooth function of t. since the second order random walk (rw2) model is appropriate for representing smooth curves (fahrmeir and kneib, 2008) , δ = (δ 1 , . . . , δ t ) is assumed to follow a rw2 model, i.e., where 2 , . . . , t −1 are independent and identically distributed (i.i.d.) zero mean gaussian random variables with variance 1/τ δ . here the precision parameter τ δ > 0 acts as a smoothing parameter enforcing small or allowing for large variations in δ t (fahrmeir and kneib, 2008) . to account for temporal correlation, we assume that ε t follows a stationary autoregressive model of order 2, ar(2); i.e., ε t = ψ 1 (1 − ψ 2 )ε t−1 + ψ 2 ε t−2 + t , t = 2, . . . , t, where −1 < ψ 1 < 1 and −1 < ψ 2 < 1 are the first and second partial autocorrelations of ε t and 2 , . . . , t are i.i.d. zero mean gaussian random variables with variance 1/τ ε . on the other hand, to account for spatial correlation, we assume that ζ = (ζ 1 , . . . , ζ m ) follows a gaussian markov random field (gmrf). more specifically, we assume that ζ is a zero mean gaussian random vector with the structured covariance matrix where i m is the m × m identity matrix, 0 ≤ ω < 1 and e max is the largest eigenvalue of the m × m symmetric positive definite matrix c = [c ii ]. the entry c ii of matrix c represents to what extend the regions a i and a i are interconnected. for example, c ii can be related to a data on commuting or population movement between regions a i and a i . in absence of most recent and reliable movement data between the regions of spain, italy and germany, we set c ii to be the euclidean distance between the centroids of a i and a i . in addition to interconnectivity and correlations due to spatial distance, the neighbourhood structure of regions a 1 , . . . , a m may induce spatial correlation among relative risks of regions because neighbouring regions often tend to have similar relative risks. to include spatial correlation due to neighborhood structure of regions in the model, we assume that ξ = (ξ 1 , . . . , ξ m ) follows a scaled version of the besag-york-mollié (bym) model (besag et al., 1991) ; i.e., ξ is a zero mean gaussian random vector with (riebler et al., 2016) here q − denotes the generalized inverse of the m × m spatial precision matrix q = [q ii ] with entries where n i is the number of neighbors of region a i and i ∼ i means that regions a i and a i share a common border. the parameter τ ξ > 0 represents the marginal precision and 0 ≤ φ ≤ 1 indicates the proportion of the marginal variance explained by the neighborhood structure of regions (riebler et al., 2016) . in a bayesian framework, it is necessary to specify prior distributions for all unknown parameters of the considered model. the gaussian prior with mean zero and variance 10 6 is considered as a non-informative prior for the dispersion parameter of generalized poisson distribution, log ϕ, and for the parameters of the log linear model for the relative risks µ, β, log τ δ , log τ ε , log τ ζ , log τ ξ , log ω 1−ω , 1−ψ1 and log 1+ψ2 1−ψ2 . the prior distribution for the α parameter of the generalized poisson distribution is considered to be a gaussian distribution with mean 1.5 and variance 10 6 . table 3 summarizes the model parameters and their necessary transformation for imposing the non-informative gaussian priors. since all random effects of the model are gaussian, the integrated nested laplace approximation (inla) method (rue et al., 2009) can be used for deterministic fast approximation of posterior probability distributions of the model parameters and latent random effects (martins et al., 2013; lindgren et al., 2015) . the r-inla package, an r interface to the inla program and available at www.r-inla.org, is used for the implementation of the bayesian computations in the present work. the r code can be made available upon request. the initial values for all parameters in the inla numerical computations are set to be the mean of their corresponding prior distribution. the initial value of α is chosen to be one (see table 3 ). for count data y it and in a bayesian framework, a probabilistic forecast is a posterior predictive distribution on z + . it is expected to generate values that are consistent with the observations (calibration) and concentrated around their means (sharpness) as much as possible (czado et al., 2009) . following a leave-one-out cross-validation approach, let be the event of observing all count values except the one for region a i at time t. dawid (1984) proposed the cross-validated probability integral transform (pit) for calibration checks. thus, pit it is simply the value that the predictive distribution function of y it attains at the observation point y it . the conditional predictive ordinate (cpo) is another bayesian model diagnostic. small values of cpo it (y it ) indicate possible outliers, high-leverage and influential observations (pettit, 1990) . for count data, czado et al. (2009) suggested a nonrandomized yet uniform version of the pit with which is equivalent to the mean pit can then be comparing with the standard uniform distribution for calibration. for example, a histogram with heights table 4 presents the bayesian estimates (posterior means) for every parameter of the considered model fitted to the daily number of new covid-19 cases in spain, italy and germany. the corresponding 95% credible intervals of the model parameters are also reported in parentheses. comparing the estimated parameters among different countries, it can be seen that the dispersion parameter ϕ of the generalized poisson distribution for italy is higher than spain and germany, but its shape parameter α is around 1.5 for the three countries, which implies that the variance of the daily counts in each region is approximately a quadratic function of their mean. the coefficient of the population density is not significantly different from zero for spain and italy, but is positive for germany which indicates that regions with higher population density have larger relative risks. the precision parameters of the temporal random effects imply that the temporal trend δ t has at least 35 times larger contribution (smaller precision) than ε t which represents temporal correlation. the opposite signs of ψ 1 and ψ 2 indicate rough oscillations in ε t . the spatial random effect ζ i has larger contribution (smaller precision) than ξ i in the total variations of the relative risks only in spain, while for italy and germany it is the opposite. this could be a result of large euclidean distance between spain continental european territory from two archipelagos territories, which is affecting the considered covariance structure of ζ i . in summary, the higher contribution (lower precision) in the total variations of the relative risks for spain, italy and germany is due to the temporal trend, spatial correlation and finally temporal correlation, respectively. this may hint that spatial correlations have a greater impact on the relative risks of covid-19 than temporal correlations. the bayesian estimates and 95% credible intervals for the temporal trend δ t , t = 1, . . . , t , are shown in figure 1 . these plots can be interpreted as a smoothed temporal trend of the relative risk in the whole country. in fact, figure 1 suggests that the covid-19 epidemic in all three countries rapidly reached their peaks and slowly started to decline at the beginning of april and then increased and reached its maximum in august. in addition, the second wave of the epidemic seems to be stronger in spain and germany shows a more smoother trend during the study period. figure 2 shows the the posterior means of the spatial random effects ζ i and ξ i , i = 1, . . . , m, on the corresponding map of each country. the plot illustrates spatial heterogeneity of the relative risk of covid-19 across regions in each country, particularly in spain. regions with positive (negative) ζ i + ξ i values are expected to have elevated (lower) relative risks than the the baseline country-wide risk during the study period. in order to see how the estimated relative risks under the fitted model are in agreement with the observed data, figure 3 shows the spatially accumulated daily number of cases m i=1 y it , t = 1, . . . , t , and their expected values under the fitted model, namely the posterior mean and 95% credible interval of m i=1 e it θ it , t = 1, . . . , t . except some discrepancies for spain and italy, the observed values are inside the 95% credible intervals and close to the expected values under the fitted model. figure 3 in addition shows 4-days ahead forecasts of the total daily number of new cases at the end of study period of each country. finally, histograms of the normalized pit values described in section 4.4 are obtained using j = 20 from the fitted models and plotted in figure 4 . the normalized pit values for the fitted models to data do not show a clear visible pattern and the histograms seems to be close to the standard uniform distribution. the above results and more details on observed and predicated values from the fitted model are also provided in an interactive shiny web application at https://ajalilian.shinyapps.io/shinyapp/. there are some limitations in the analyses and modeling of data on the number of new cases of covid-19, including data incompleteness and inaccuracy, unavailability or inaccuracy of relevant variables such as population movement and interaction, as well as the unknown nature of the new covid-19 virus. nevertheless, understanding the underlying spatial and temporal dynamics of the spread of covid-19 can result in detecting regional or global trends and to further make informed and timely public health policies such as resource allocation. in this study, we used a spatio-temporal model to explain the spatial and temporal variations of the relative risk of the disease in spain, italy and germany. despite data limitations and the complexity and uncertainty in the spread of covid-19, the model was able to grasp the temporal and spatial trends in the data. however, the posterior predictive checks using the normalized probability integral transform (pit) showed that there is room for the model improvements. obliviously, there are many relevant information and covariates that can be considered in our modeling framework and improve the model's predictive capabilities. one good possibility would be considering most recent and accurate human mobility amongst regions. we would expect our model would benefit from this information, which right now can not be accessed. moreover, the considered spatio-temporal model in this paper is one instance among many possibilities. for example, one possibility is to include a random effect term in the model that represents variations due to joint spatio-temporal correlations; e.g., a separable sptaio-temporal covariance structure. however, the considered model was adequate and no joint spatio-temporal random effect term was considered to avoid increasing the model's complexity. we focused here on a stochastic spatio-temporal model as a good alternative to existing deterministic compartmental models in epidemiology to explain the spatio-temporal dynamics in the spread of covid-19. however, it should be emphasized that one step forward would be considering a combination of a deterministic compartmental model in terms of differential equations for the number of susceptible, exposed, infectious and recovered cases with our sort of stochastic modeling approach. this is a clear novelty and a direction for future research. open data resources for fighting covid-19 a comparison of spatio-temporal disease mapping approaches including an application to ischaemic heart disease in new south wales, australia modeling and forecasting the covid-19 pandemic in brazil bayesian image restoration, with two applications in spatial statistics a spatio-temporal 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authors declare that they have no conflict of interest. key: cord-294815-mhqe3xjz authors: kuì�chenhoff, h.; guenther, f.; hoì�hle, m.; bender, a. title: analysis of the early covid-19 epidemic curve in germany by regression models with change points date: 2020-10-30 journal: nan doi: 10.1101/2020.10.29.20222265 sha: doc_id: 294815 cord_uid: mhqe3xjz we analyze the covid-19 epidemic curve from march to end of april 2020 in germany. we use statistical models to estimate the number of cases with disease onset on a given day and use back-projection techniques to obtain the number of new infections per day. the respective time series are analyzed by a poisson trend regression model with change points. the change points are estimated directly from the data without further assumptions. we carry out the analysis for the whole of germany and the federal state of bavaria, where we have more detailed data. both analyses show a major change between march 9th and 13th for the time series of infections: from a strong increase to a stagnation or a slight decrease. another change was found between march 24th and march 31st, where the decline intensified. these two major changes can be related to different governmental measures. on march, 11th, chancellor merkel appealed for social distancing in a press conference with the robert koch institute (rki) and a ban on major events with more than 1000 visitors (march 10th) was issued. the other change point at the end of march could be related to the shutdown in germany. our results differ from those by other authors as we take into account the reporting delay, which turned out to be time dependent and therefore changes the structure of the epidemic curve compared to the curve of newly reported cases the first phase of the covid-19 pandemic in germany was managed relatively successful in comparison to other countries in europe. therefore, it is worth taking a closer look at the course of the pandemic in germany, which has already led to controversial discussions. this particularly concerns the important question about the effectiveness of various control measures. there are several publications using data from different countries on the effects of control measures, see, e.g., [1] , [2] and [3] . as [4] point out, many of such studies are undermined by unreliable data on incidence. many papers use data provided from the johns hopkins university (jhu) [5] . these data are based on cumulative registered cases in different countries, which induces several problems, particularly the fact that not all cases are reported and that there is delay between the day of infection and the reporting day. furthermore, the systems of reporting vary between countries, which makes comparisons between countries difficult. therefore we focus on the analysis of the epidemic curve in bavaria and germany. the availability of case based data for bavaria and detailed data for disease onset for germany is essential for our analysis. in a recent paper on germany by [6] , the authors use a complex bayesian modeling approach based on the daily registrations in the jhu data. an important claim by [6] is that the lock-down-like measures on march 23rd were necessary to stop exponential growth, however, this result contradicts for example results by the german rki [7] . furthermore, these approaches were critically questioned by [8] and [9] , where the latter emphasized the importance of taking into account the delay by reporting and incubation time, when analyzing the possible effect of non pharmaceutical interventions. we follow this line of argument and use a statistical model to estimate daily numbers of infected and of persons with disease onset on a certain day. for bavaria, we use detailed case-based data, while a modeling approach is utilized for german data. we analyze the respective epidemic curves using a segmented regression model with change points. the paper is organized as follows. in section 2, we present the data and the the strategy of estimating the relevant daily counts. then the segmented regression model, which is the basis for further analyses, is presented. in section 3, we present the results followed by a discussion in section 4. the latter is not always known: partly because it could not be determined and partly because the case did not (yet) have any symptoms at the time of entry into the data base. a procedure for imputation of missing values regarding the disease onset has been developed by [10] , using a flexible generalized additive model for location, scale and shape (gamlss; [11] ), assuming a weibulldistribution for time t d > 0 between disease onset and reporting date. we estimate the delay time distribution from data with disease onset and impute missing disease onsets based on this model. for the german data, no individual case data were available, so instead we used estimated disease onset data provided by the robert koch institute (rki), see [12] and [7] . the method used by the rki is similar to our approach applied to bavarian data [10] . to interpret the course of the epidemic and possible effects of interventions, case based data on time of infection is essential. however, as such data is generally not available, one simple approach is to shift the curve to the past by the average incubation period. the average incubation period for covid-19 is about five days [13] . a more sophisticated approach is to use the incubation period distribution as part of an inverse convolution, also known as backpropagation, in order to estimate the number of infections per day from the time series of disease onsets [14, 15] . we assume a log-normal distribution for the incubation time with a median of 5.1. days and a 95% percentile at 11.5 days [13] . these are the same values as used by [6] . for our calculation, we use the back-projection procedure implemented in the r package surveillance [16] . to analyze the temporal course of the infection we use the following poisson regression model with over-dispersion and change points (see [17] , [18] ): where e(y t ) is the expected number of new reported cases at time t, k is the number of change points, and x + = max(x, 0) is the positive part of x. the change points are used to partition the epidemic curve y t into k + 1 phases. these are characterized by different growth parameters. in the phase before the first change point cp 1 the growth is characterized by the parameter β 1 , in the 2nd phase between cp 1 and cp 2 by β 2 = β 1 + γ 1 . the next change is then at time cp 2 . in the 3rd phase between cp 2 and cp 3 the growth parameter is given by β 3 = β 1 + γ 1 + γ 2 . this applies accordingly until the last phase after cp k . the quantities exp(β j ), j = 1, . . . , k + 1 can be interpreted as daily growth factors. since model (1) is a generalized linear model given the change points, the parameters of the model (including the change points) can be estimated by minimizing the quasi likelihood function for the poisson model. due to the estimation of the change points the numerical optimization problem is not straight forward. for the estimation of the model we use the r-package segmented, see [19] . the starting values are estimated by discrete optimization using all possible integer suitable combinations of change points. the number of change points k is varied step-wise up to a maximum of k = 4. it is examined whether the increase of the number of break points leads to a relevant improvement of the model fitting (over-dispersion parameter). models with more than 4 change points since they are hardly interpretable and the danger of overfitting is high. we apply the segmented regression model to time series of the estimated daily numbers of infections for bavaria and germany. since the back propagation algorithm yields an estimate for the expected values of the number of daily infections and does so by inducing a smoothing effect, as a sensitivity analysis for the location of the breakpoints, we also apply the model to the time series of the daily number of disease onsets. when comparing the results, it should be taken into account that the onset of the disease is on average 5 days after infection. in figure 1 , the three different time series of daily cases (reported, disease onset and estimated infection date) are presented. the delay between the three time series for bavaria and germany is evident. furthermore, the curves do not just differ by a constant delay, but there is some change in structure of the curves. the curve relating to the date of infection is clearly smoothed due to the back projection procedure (cf. section 2.2) and has a clear maximum both for bavaria and germany. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 30, 2020. ; for the bavarian data on disease onset, the model with k = 4 change points gives the best result with an estimate of the over-dispersion parameter of 3.8, i.e., the variance of y t is 3.8 times higher than the value of var(y t ) = e(y t ) otherwise expected under the assumption of the poisson regression model. the over-dispersion for a model with k = 3 change points is substantially higher (4.5), which suggests that the model with four change points should be preferred. table 1 , left panel. the model delivers five phases starting with a steep increase, which is slowed down in the second phase. in the third phase starting at 15th-17th of april, the increase is stopped and there is decrease in number of disease onsets in the fourth phase, which is accelerated in the fifth phase. the poisson model for the infection date gives a substantially better fit than the disease onset model. the model with four change points has an underdispersion by the the factor 0.39 compared to 0.79 for the model with three change points. therefore, we use the model with three change points to avoid overfitting. the result can be seen in figure 2 (right panel) and in table 1 (right panel). the phases are similar to those for the disease onset, but the change from increasing curve (phase 1 and 2) to a decreasing phase (phase 3) is direct without a plateau in between. taking the mean incubation period of five days into account, we combine the results for the two models for bavaria, which implies four phases of the epidemic: 1st phase there is a substantial increase in new infections in both models. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 30, 2020. ; https://doi.org/10.1101/2020.10.29.20222265 doi: medrxiv preprint the results for the german data are presented in figure 3 and in table 2 . for the disease onset model, the overdispersion is substantially lower for the model with four change points than with three change points. however, the overdipersion of the model with four change points is rather high (20.1) and the confidence intervals for the change points are rather big, especially for the first part of the time series where two change points are estimated. while the distinction in the first three phases is unclear, there is a clear turning point between march 14th and march 18th. the fifth phase with a further slowdown starts at the end of april. as can be seen from figure 3 there are further estimated change points in both models, which have wide confidence intervals and do not fit well together in the models. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 30, 2020. ; https://doi.org/10.1101/2020. 10.29.20222265 doi: medrxiv preprint the present analysis is a retrospective, exploratory analysis of the german and bavarian covid-19 reporting data during mar-apr 2020. the analysis does not include cases that have not been recorded. if the proportion of undetected cases changes over time, this can distort the curve and thus the determination of the change points. therefore, additional data on daily deaths and hospital admissions and the number of tests performed should be considered. furthermore, it is possible to estimate the proportion of undetected cases with the help of representative studies such as the one currently conducted in munich, see [20] . our analysis is based to a considerable extent on imputed data, see [10] , which is a results of missing data w.r.t. the disease onset. since changes in behavior do not occur abruptly, the assumption of change points is also problematic in itself. therefore, the interpretation of change points should always be done in conjunction with a direct observation of the epidemic curve. our analysis is based on the onset of the disease (more precisely: the onset of symptoms) and a back projection to the date of infections, and therefore, despite its limitations, is better suited to describe the course of the epidemic than the more common analysis of daily or cumulative reported case numbers. in the analysis of the bavarian and the german data in different settings, the main result is the change point, where the exponential growth was stopped and is clearly visible between march 9th and 13th. the timing of this change point coincides to the implementation of the first control measures: the partial ban of mass events with more than 1000 people. furthermore, in a press conference on march 11th chancellor merkel and the president of the rki appealed to self-enforced social distancing (https://www.bundesgesund heitsministerium.de/en/coronavirus/chronologie-coronavirus.html. furthermore, the extended media coverage from bergamo, italy, as well as the voluntary transition to home-office work could be related to this essential change in the course of the pandemic. in bavaria and in germany, the change point at the end of march of infection date is apparent. this change point is associated with different measures taken in march (closing of schools and stores on march 16th and the shutdown including contact ban on march 21st). since there were many measures administered simultaneously, it is not possible to attribute individual measures to the development of the epidemic curve. the claim by [6] , that the shutdown on march 21st was necessary to stop the growth of the epidemic is not supported by our analysis. there is a change 10 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 30, 2020. point in the epidemic curve after that date, but the major change from an exponential growth to a decrease was before the shutdown. the difference in results can be explained by the different data bases used for the respective analyses. while [6] use data based on daily registered cases, in our analysis, data on disease onset are included. as can be seen from figure 1 and from the results of our data analysis, the delay distribution of the time between disease onset and reporting day changed over time. using this information is a crucial difference between our analysis and that of [6] . in a recent technical addendum [21] the authors re-fit their model on more appropriate data. these analysis -in our opinion -clearly show that the effective reproduction number decreased earlier than in their initial analysis, however, they attribute the decrease to a sir model peculiarity, where a linear decrease in the contact rate can lead to the incidence curve dropping despite r(t) > 1. the above discussions illustrate how complex the interpretation of even simple sir models is and the question is, if such sir modeling is not too simple to really allow for questions to be answered model based (no age structure, no time varying reporting delay, no incubation delay). in contrast, our approach is more data driven with a minimum of modeling assumptions and without the need to include strong prior information about the change points. directly using a segmented curve with exponential growth (decline) is in line with common models of infectious diseases in its early stages, where the limitation of the spread by immune persons plays no role. the problem of using complex models with many parameters for the evaluation of governmental measures has also been highlighted by [22] . our approach is similar to that of [9] . however, using change point analysis for variables derived from daily new infections appears problematic, since assumptions modeling the reproduction number r(t) or the cumulative numbers are questionable. more specifically, the use of the time-varying reproduction number r(t), a standard measure to describe the course of an epidemic is challenging, as different definitions have been proposed in the literature that also imply different interpretations (see [23, 24] ). however, the analysis of r(t) as a relative measure can be useful, when one wants to analyze data from different countries with non comparable reporting systems, see [3] . we prefer the direct use of a poisson regression model with a more plausible assumptions about the error terms instead of using ols for logarithmic case numbers. furthermore, we apply a direct maximum likelihood estimation of the change points of the segmented regression model. for the interpretation of the model based on disease onsets, we also use a simple difference of five days to take the incubation time into account. our results are similar to that of [9] . however, the claim that there is no evidence for the effect of governmental measures is not supported by our analysis. our result is in line with that of [25] , where a stop of exponential growth in great britain has been before the shutdown. furthermore, the effect of governmental measures as whole is clearly documented in the literature, see, e.g., [1] and [2] .our result on a possible effect of the ban of mass events is also in line with the results of [3] . . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 30, 2020. ; the temporal connection between the change points in our analysis and various control measures should be interpreted as an association, rather than a direct causal relationship. in the end many other explanations exists and from a simple time series analysis it is not possible to say to what extent the population already had changed their behavior voluntarily, as for example observed in mobility data [26] , and in what way the measures contributed to this. more speculative alternative explanations would include the possibility of a seasonal effect on coronavirus activity (e.g. related to temperature) or changes in test capacity or the case detection ratio. however, given the re-emergence of the epidemic in the fall of 2020 at high test capacity and at relatively high temperatures shows that contact behavior is the major explanatory factor for virus activity. nevertheless, any analysis of observational time series data including only a limited amount of explanatory factors has to be interpreted with care and with respect to the many uncertainties which remain regarding covid-19 [27] . despite the limitations of the approach, we argue that it is advantageous and important to directly interpret the epidemic curve and the absolute number of cases, rather than indirect measures like the r(t). furthermore, the reproduction rate does not contain information about how many people are currently affected, or whether the infected persons belong to risk groups. the course of the time-varying reproduction number calculated by us for bavaria fits well with the change point analysis [10] . a value of r(t) >1 corresponds to a rate of increase >1, noting that the time delays in the interpretation of r(t) must be kept in mind. it should be noted, that the presented analysis is retrospective. control measures have to be decided based on a completely different level of information than what the retrospectively established epidemic curve suggests. the simple observation of the course of the reported case numbers by reporting date is also problematic because this course is strongly influenced by the reporting behavior and the methods and capacities of the test laboratories. typically, substantially fewer cases are reported at weekends than during the week. therefore, the estimation [10] is an important step to estimate the better interpretable curve of new cases, but is limited by assumptions and limitations itself, that need to be considered when interpreting the results. since the impact of the measures also depends on how they are implemented by the population (compliance), the results cannot be directly transferred to the future. nevertheless, it remains a remarkable result that the clear turning point of the early covid-19 infection data in germany is associated with non drastic measures (no shutdown) and strong appeals by politicians. all data used for the analyses and all code to reproduce the models, figures and tables in the manuscript are openly and freely available from https://gi thub.com/adibender/covid19-changepoint-analysis-germany-bavaria. all 12 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 30, 2020. ; analyses were performed using the r programming language [28] . figures were created using r package ggplot2 [29] . estimating the effects of non-pharmaceutical interventions on covid-19 in europe physical distancing interventions and incidence of coronavirus disease 2019: natural experiment in 149 countries the temporal association of introducing and lifting non-pharmaceutical interventions with the time-varying reproduction number (r) of sars-cov-2: a modelling study across 131 countries. the lancet infectious diseases lockdown-type measures look effective against covid-19 but evidence is undermined by unreliable data on incidence an interactive web-based dashboard to track covid-19 in real time. the lancet infectious diseases inferring change points in the spread of covid-19 reveals the effectiveness of interventions the limits of estimating covid-19 intervention effects using bayesian models a phenomenological approach to assessing the effectiveness of covid-19 related nonpharmaceutical interventions in germany nowcasting the covid-19 pandemic in bavaria. medrxiv flexible regression and smoothing: using gamlss in r. the r series schätzung der aktuellen entwicklung der sars-cov-2-epidemie in deutschland -nowcasting the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application a method of non-parametric backprojection and its application to aids data associations of age and sex on clinical outcome and incubation period of shiga toxin-producing escherichia coli o104:h4 infections monitoring count time series in r: aberration detection in public health surveillance estimating regression models with unknown breakpoints modelling covid-19 outbreak: segmented regression to assess lockdown effectiveness segmented: an r package to fit regression models with broken-line relationships. r news protocol of a population-based prospective covid-19 cohort study munich model-based and model-free characterization of epidemic outbreaks -technical notes on dehning the limits of estimating covid-19 intervention effects using bayesian models a new framework and software to estimate time-varying reproduction numbers during epidemics association of public health interventions with the epidemiology of the covid-19 outbreak in wuhan, china did covid-19 infections decline before uk lockdown ? estimating the impact of mobility patterns on covid-19 infection rates in 11 european countries covid-19's known unknowns r: a language and environment for statistical computing elegant graphics for data analysis we would like to thank katharina katz and manfred wildner from the bavarian state office for health and food safety (lgl) for providing the data and for useful discussions. we also thank nadja sauter for help with visualizations. key: cord-033219-uwzgbpeo authors: naumann, elias; möhring, katja; reifenscheid, maximiliane; wenz, alexander; rettig, tobias; lehrer, roni; krieger, ulrich; juhl, sebastian; friedel, sabine; fikel, marina; cornesse, carina; blom, annelies g. title: covid‐19 policies in germany and their social, political, and psychological consequences date: 2020-09-28 journal: nan doi: 10.1002/epa2.1091 sha: doc_id: 33219 cord_uid: uwzgbpeo many policy analyses on covid‐19 have been focusing on what kind of policies are implemented to contain the spread of covid‐19. what seems equally important to explore are the social and political consequences of the confinement policies. does the public support strict confinement policies? what are the social, political, and psychological consequences of the confinement policies? the question of how legitimate a policy is among the public is at the core of democratic theory. its relevance also stems from the expected consequences of public support on behavior: the more someone supports a policy, the more someone is likely to follow the policy even if the policy is not strictly enforced. in this paper, we will focus on germany, briefly summarize the main policies during the first 6 weeks of confinement and then explore political attitudes, risk perceptions, and the social consequences of the lockdown. many policy analyses of the covid-19 pandemic have focused on what kind of policies are implemented to contain the spread of the disease and on how effective these policies are in reducing the number of new infections and deaths. what seems equally important to explore are the political, social, and psychological consequences of the containment policies. in this paper, we focus on germany and provide an overview of the main policies that were implemented in an attempt to halt the spread of covid-19 during the first 8 weeks of confinement. we complement this with results of a daily survey, the german internet panel (gip), which allows us to capture the immediate political, social, and psychological consequences of the confinement. first, does the public support strict confinement policies, and how does this support change over time? the question of how legitimate a policy is among the public and how public opinion and policies are linked is a prominent and widely researched topic in political and social sciences. theoretical and empirical research support the claim that it is, in fact, an interrelationship: public opinion affects policy-making (brooks & manza, 2006; burstein, 2003; page & shapiro, 1983) and policies affect public opinion (ebbinghaus & naumann, 2017; mettler & soss, 2004; pierson, 1993) . these policy feedback effects then, in turn, alter attitudes (kumlin & stadelmann-steffen, 2014) . as lockdown policies have been so quickly introduced and as the public most likely did not have any attitude regarding these policies before the onset of the covid-19 pandemic, this seems to be one of the few examples where policies were clearly enacted before the emergence of public opinion. hence, we focus rather on how the lockdown policies might have affected public attitudes. skocpol (1992:58) refers to policy feedback as the ways "policies, once enacted, restructure subsequent political processes," and pierson (1993) distinguishes two main types of feedback effects. first, resource and incentive effects link policies to the self-interest of people since they determine how resources are distributed, provide incentives, and thus shape the costs and benefits of actors. hence, we would expect that the more threatened people feel by the pandemic, the higher they perceive the risk of being infected to be, and the lower their support is. moreover, the more apparent the economic costs of the lockdown become over time, the lower the support should be. second, interpretative effects provide a mechanism to link policies and attitudes via values since they serve as sources of information and meaning. for example, policies "frame the meaning and origins of societal problems by identifying target groups for government action and defining solutions" (mettler & soss, 2004:62) . let us take the labor market and possible reasons for unemployment as an example. when the government promotes job-training programs, the focus is on a lack of skills, and when wage subsidies are introduced, the focus is on the structural limitations of the labor market and potential consequences of globalization, whereas strict regulations of the access to unemployment benefits shifts the focus to individual behavior and self-discipline (gusmano, schlesinger, & thomas, 2002) . so, policies can alter frames and the way people think about an issue (chong & druckman, 2007) and we expect that policies themselves shape the framing and perception of the pandemic and should have an effect on reform preferences but also on risk perceptions. second, we explore the social consequences of the lockdown and particularly focus on the labor market. the closure of non-essential businesses but also macro-economic consequences of the global covid-19 crisis had an immediate effect on the labor market and led to lay-offs and short-time work (see for example fuchs-schündeln, kuhn, & tertilt, 2020) . moreover, many employers extended possibilities for remote work and flexible work arrangements so that employees had the opportunity to work and to stay at home. at the same time, employees in essential areas of the economy who are not able to work remotely face an increased risk of being infected. do these negative consequences affect everyone equally? we would rather expect the opposite. germany's labor market is characterized by a pronounced "insider-outsider" divide and has a considerable amount of low wage and fixed-term employment, while the core workforce in standard employment is well protected by labor regulations (emmenegger, häusermann, palier, seeleib-kaiser, 2012) . hence, immediate lay-offs, for example, are not possible for those in regular employment. furthermore, labor market measures to buffer the negative crisis consequences for employees and companies are mostly targeted at the core workforce. as for the possibilities to work remotely, most jobs in which this is a viable option are in well-paid occupations that require a high level of education (dingel & neiman, 2020) . in summary, we expect that existing social inequalities will rather be increased by the confinement policies. third, we examine the psychological consequences of the lockdown. we explore the extent to which people feel threatened by covid-19. feelings of threat play an important role in shaping attitudes and behavior. if threat perceptions are too low, people might not support confinement policies or might not follow them if they are not strictly enforced. too strong threat perceptions might impair people in their daily lives if, for example, they do not leave their home any more. moreover, there is a growing political economy literature that argues that subjective (mis-)perceptions of societal trends are very important in explaining political attitudes and behavior. for example, alesina, miano, and stantcheva (2018) show that natives' usually overestimate the share of migrants and underestimate their labor market integration. these misperceptions are then one important factor in understanding anti-migrant attitudes. similarly, we would expect that people rather overestimate their own infection risk (overestimation of small probabilities) but over time they might adapt their perceptions to reality (see breznau, 2020) . in what follows, we will first provide a brief overview of how the covid-19 pandemic unfolded in germany and how the government reacted to it. this provides the background against which we will then present our results and will show how public attitudes and perceptions changed during this period. in figure 1 , we provide a summary of the first half of 2020 and show daily case numbers and the cumulative numbers of deaths in germany. moreover, the government response index shows the reaction of the german government to the pandemic aggregating containment and closure policies, economic policies, and health system policies (hale, webster, phillips, & kira, 2020) . the initial period of the covid-19 pandemic in germany resembles very much the experience of many other european countries. the government knew about covid-19 in china and its potential threat also for europe already in late december 2019. yet, the first confirmed case in germany was found on january 27, 2020. the government response to this was the introduction of contact tracing systems, the mandated isolation of the few infected people, and a first information campaign mainly recommending good hand hygiene and reassuring the population that the isolation of the infected worked well in keeping the virus at bay. the situation did not change until the end of february when the number of confirmed infections started to increase, in particular in some local hotspots. moreover, the italian experience with the pandemic led to doubts that it would be possible to contain the virus in germany by the initial measures that had been taken. "the situation in italy also changes our assessment of the situation: corona has arrived in europe as a pandemic" german health minister jens spahn remarked on february 24th. yet, the government was still reticent to introduce strict lockdown measures. it was recommended to cancel big public events of more than 1,000 participants, and it was also recommended to close schools and childcare if confirmed infections were established. at the same time, large public events like the carnival in cologne took place as planned with several thousand attendees. this changed very quickly in mid-march, when the number of daily cases increased within 2 weeks from less than 100 to 4,000 (see figure 1 ) and as also the first covid-19-related deaths were recorded. on march 12, schools and childcare facilities were closed and the government issued recommendations regarding social distancing. on march 17th, the borders were closed, and on march 18th, chancellor angela merkel announced the general lockdown with stay-at-home orders. "this is serious," she remarked in her speech to the nation "please, take it seriously, too." these measures went into effect on march 21st and were then further extended twice until the beginning of may. at the same time, the government reassured the population that they would do everything necessary to buffer the negative consequences of the lockdown. the debt brake was suspended, and the government announced an extra federal budget of 150 billion euros. short-time work ("kurzarbeit") was introduced which allows firms to temporarily reduce hours worked while providing employees with income support from the state for the hours not worked. on april 22nd, the subsidy was increased to up to 80% of the regular salary. in mid-april, some states started to make the wearing of face coverings obligatory in public transportation and shops. by april 27th, face masks were obligatory in public transportation and shops in all german states. germany reached its peak of the first wave in terms of daily infections in early april when the 5-day moving average of new infections per day reached almost 6,000. yet, the lockdown and stayat-home orders which had gone into effect on march 21st seem to have been very effective since case numbers decreased quickly in the second half of april, and also, the increase in the number of deaths slowed down in the beginning of may when it has reached about 8,000 deaths. the government (and the public) started to discuss re-opening plans at the end of march, and the first restrictions were lifted in may. as we are mainly interested in the public reactions to the lockdown, we restrict the following analysis to the time between march 20 (i.e. from when survey data is available) and may 14 when the first re-openings occurred (marked in gray in figure 1 ). in summary, having a high performing medical system but little past experience with pandemics, germany tended to show a later, slower, and weaker policy response compared to some other countries (capano, howlett, jarvis, ramesh, & goyal, 2020) . to capture public reactions of the german population, we use data from the german internet panel (gip). the gip is based on a random probability sample of the general population in germany aged 16 to 75. the study started in 2012 and was supplemented with additional participants in 2014 and 2018. the panel participants were recruited offline using strict statistical procedures (blom, gathmann, & krieger, 2015) . every other month, panel participants are invited to take part in a voluntary online survey. for the mannheim corona study (mcs), the gip launched a special survey on march 20th. the sample was divided into eight random subsamples. the subsamples 1-7 were assigned to a specific day of the week, while the eighth subsample serves as control group and is not surveyed. within one week, the questionnaire remains exactly the same for all participants. between 411 and 643 (on average 489) respondents take part in the study every day, allowing for the analysis of daily but also weekly changes in attitudes and behavior. in our analysis, we use a response propensity weight, which projects the characteristics of the msc participants to the general gip study using data on employment status and occupational sector. moreover, a raking weight was used to extrapolate the characteristics of msc participants to those of the general population of germany based on age, gender, marital status, level of education, household size, and federal state. we use two survey questions as indicators for the "public support for" and "evaluation of" the lockdown policies. respondents are asked whether they think that the societal benefits of the current policies outweigh their economic costs. answer categories ranged from 1, the societal benefits are greater than the economic costs to 7, and the economic costs are greater than the societal benefits. we also provided respondents with a list of lockdown policies, and respondents should choose all the policy measures they think are appropriate to deal with the covid-19 pandemic (see appendix for the exact wording of questions). to examine the economic consequences of the lockdown, we explore how the employment situation changes over time. we distinguish four different employment status (employed, short-time work, furloughed with and without pay, and unemployed) and also whether the employed work on-site or remotely. finally, we explore the psychological reactions of the public focusing on how threatened people feel by the covid-19 pandemic, how they rate their individual infection risk and their ability to control an infection, and the perceived likelihood of severe illness if infected (see appendix for the exact wording of questions and response scale). daily data on policies are available from january 1, 2020, and the survey data are available on a daily basis starting on march 20, 2020. as we are primarily interested in the lockdown policies and their effect on the public, we restrict our analysis to the period between march 20, 2020, to may 14, 2020. in mid-may, germany began to gradually lift some of the lockdown measures and started to re-open the country. how did the public react to the lockdown measures? we first look at political reactions and distinguish between performance evaluations and support for specific policies. figure 2 shows how the public evaluates the consequences of the lockdown policies. we reversed the scale in the questionnaire so that higher values on the 7-point scale mean that the population thinks that the societal benefits are greater than the economic costs. we see that at the beginning of our period of observation, at the peak of the first wave and briefly after strict lockdown measures had been introduced, that the public evaluates the policies quite positively and in general supports the evaluation that the societal benefits of the lockdown outweigh its economic costs. this evaluation remains stable at around 4.6 during the first two weeks but then begins to steadily decline already in early april and continues to drop weekon-week until the beginning of may. at the end of our period of observation, the evaluation dropped below 4 on the 7-point scale as around 50% of the population in germany felt that the lockdown had more negative than positive consequences. in figure 3 , we move from the general performance evaluation to support for specific lockdown measures. the graph shows which share of the population supported each of six different policies. we see that most of the policies receive tremendous support in the first weeks of the lockdown. more than 90% of the population supported closing public facilities (like schools and universities), closing borders, and prohibiting public gatherings with more than 100 participants. a lockdown and strict stay-at-home order also received majority support. the public was more critical toward closing public transportation and also toward tracking mobile phones of infected people without their consent, although notably about a third did support such a policy. over time, with declining case numbers and higher awareness for the economic costs of the lockdown measures, support steadily declined for all policies. we observe the steepest decline in support for closing public facilities which fell from 95% support in week 1 to around 40% support at the beginning of may. the population in germany became equally critical toward a stay-at-home order and slightly less than 10% of the population supported a stay-at-home order in may. other measures lost support, including banning international travel (−25 percentage points) and prohibiting gatherings of more than 100 participants (−5 percentage points). briefly before the first re-opening started, a majority still supported these two measures. also, only very few people (around 5%) did not support any of the lockdown measures. overall, during the lockdown, the public in germany became most critical about those policies which had the strongest | 7 negative impact on their daily lives, such as the measures regarding the closures of public facilities and stay-at-home orders, whereas banning international travel or prohibiting larger public events were still supported by a majority. although our empirical data only provide evidence on one very strict version of a tracking and tracing policy, the results suggest that the public sees a benefit in these kinds of measures as support did not decline very much over time. in addition to the political consequences, the lockdown also had negative effects on the economy and the labor market in particular. here, we focus on the workforce and explore who is affected most by the negative consequences of the lockdown. does the covid-19 crisis increase existing social inequalities? our results suggest that it does. figure 5 shows the employment situation depending on the level of school education, and we distinguish between low (without or with basic school-leaving qualifications-hauptschulabschluss), middle (intermediate school-leaving qualification-mittlere reife), and high (with higher education entrance qualification (fach-hochschulreife) education) levels. we restrict our analysis to those respondents who were employed before the lockdown (i.e., in january 2020). hence, our results show which groups were able to keep their jobs, who switched to remote work, but who lost their job, were furloughed or were sent on short-time work. overall, our results show that the lower the level of education, the higher the proportion of people who changed to short-time work, to furlough without pay, or who were laid off (figure 4 ). job loss for someone with a low or middle education is twice as likely as for someone with a high level of education. among those who kept their jobs and were able to work about the same hours as in january 2020, employees with a high level of school education are more often working remotely (more than 40 percent in march and april) than employees with a lower level of education. the majority of those with a low or medium school education work on-site (between 62 -64 percent in march). in summary, high education reduces the risk of job loss and at the same time provides the privilege to work remotely. the negative consequences of the covid-19 lockdown hence increase existing social inequalities along two dimensions. the lower educated lost their jobs, were suspended without pay, or experienced a partial income loss by short-time work to a much higher degree than higher educated workers. what's more, if they were able to keep their jobs, they tended have to work on-site, facing higher risks of infection. these social inequalities are linked to political attitudes and policy evaluations. additional analyses (not shown here) confirm that the lower educated are much more critical of the lockdown policies and rather tend to say that the economic costs are higher than the societal benefits. to better understand public reactions to the lockdown in germany, we argue that it is crucial to look more specifically at public perceptions. in figure 5 , we show how threatened the public felt by the covid-19 pandemic and how the population rated its own infection risk (both on the left y-scale). moreover, we explore whether people thought that they were at risk of a severe illness if infected and whether they felt in control of their own infection risk. in the first two weeks, shortly after the lockdown had been introduced but before the peak of the first wave of infections, threat perceptions were highest and the population rated the degree they felt personally threatened above 5 on the 11-point scale ranging from 0 (indicating no perceived threat at all) to 10 (indicating an extreme perceived threat to me). also, the population thinks that almost 15 out of 100 people will get infected in the next 7 days. this is a very major overestimation of the actual infection risk. both the threat perception and the individual infection risk then steadily decreased over time. in contrast, the subjective risk assessment of having a severe illness in case of infection and also the feeling of having control over an infection remains more stable over time. in summary, the steady decrease of feelings of threat and perceived risk might be one explanation for why the lockdown slowly lost public support over time. additional analyses show that such a correlation also exists at the individual level, as the more someone felt threatened, the higher degree of support they indicated for lockdown policies and the more positive was their overall evaluation of the benefits of the lockdown. in this paper, we have summarized the political response to the covid-19 pandemic in germany and focused on the period between the occurrence of the first case in january to the end of the lockdown in mid-may 2020. our analysis shows that germany followed a containment strategy for the first 2 months until mid-march, focusing on isolation of the infected and on contact tracing but refraining from stricter policy measures which would affect the daily life of those not infected. this changed very quickly during the first two weeks of march when the daily new cases increased from below 100 to around 1,000. in response, germany very quickly moved from a containment strategy to a strategy focused on delaying the spread of the virus and within 3 weeks moved from prohibiting large events with more than 1,000 participants to a lockdown with a national stay-at-home order. our analysis of survey data starting at the peak of the first wave briefly after the lockdown was introduced shows very high approval rates of these policy measures which might explain their success in effectively delaying the spread and reducing new infections to below 1,000 per day by mid-may. also, germany managed to keep the number of deaths at a very low level throughout the crisis (see contributions by malandrino (2020) on italy and by colfer (2020) , covering the uk, in this issue). yet, our analysis also shows that the widespread support for the containment and delay policy measures steadily decreased over time as did feelings of threat and subjective risk perceptions. moreover, the negative economic consequences of becoming unemployed or of moving to short-time work did not affect everyone equally but did track existing social inequalities. while economic and social policies provided support to buffer these negative consequences in the short run, it is unclear whether the policies will also be able to adequately address medium-and long-term consequences of the lockdown. hence, the conclusions and policy implications drawn from our analysis are clearly limited to these short-term effects and are clearly hampered by the fact that we are trying to explore an ongoing phenomenon. the external validity of our findings for other countries and institutional settings is of course also hampered by the specificities of the german case. germany has a strong economy and labor market, a comparably well-functioning welfare state, and a stable government that has been in office for several years. this might explain both the success but also the high approval of the lockdown policies in the country (breznau, 2020) . in summary, our analysis clearly shows that the lockdown policies feed back into the political process by altering political attitudes and public risk perceptions (pierson, 1993; skocpol, 1992) . this raises some doubts that the acceptance of policies and the willingness of citizens to comply with lockdown measures during a potential second wave would be as widespread and as strong as they were during the first wave of the pandemic. information campaigns, a societal discussion reflecting on the experiences of the lockdown, but also social policies that address some of the social inequalities the pandemic has exposed (see for example lynch, 2020) might help to prepare the population for subsequent waves and for subsequent pandemics. how to cite this article: naumann e, möhring k, reifenscheid m, et al. covid-19 policies in germany and their social, political, and psychological consequences. eur policy anal. 2020;00:1-12. https://doi.org/10.1002/epa2.1091 question text (english translation) what do you think about the consequences of the current confinement policies in germany: are the economic costs greater than the societal benefits, or are the societal benefits greater than the economic costs? 1-the societal benefits are greater than the economic costs. … 7-the economic costs are greater than the societal benefits. policy support which of the following policy measures do you think are appropriate for dealing with the current situation? please tick all that apply. -closing public facilities (e.g., universities, schools, and nursery schools) -closing boarders -prohibit events and public gatherings with more than 100 participants -general stay-at-home order -closing of public transportation -tracking of mobile phones of infected persons for contact tracing (without consent) -i do not support any of these measures. to what degree do you feel personally threatened by the coronavirus pandemic? no threat at all to me … 10 extreme threat to me we would like to know how likely you think it is that you or someone like you will be infected with covid in the next 7 days. please think of 100 persons who are very similar to you, that is, they have a similar age, a similar health, live in your neighborhood, have a similar occupation and a similar lifestyle. what do you think, how many of these 100 persons will be infected with covid in the next 7 days? immigration and redistribution (no. w24733) setting up an online panel representative of the general population: the german internet panel the welfare state and risk perceptions: the novel coronavirus pandemic and public concern in 70 countries social policy responsiveness in developed democracies the impact of public opinion on public policy: a review and an agenda mobilizing policy (in)capacity to fight covid-19: understanding variations in state responses framing theory herd-immunity across intangible borders: public policy responses to covid-19 in ireland and the uk. european policy analysis. forthcoming how many jobs can be done at home? welfare state reforms seen from below: comparing public attitudes and organized interests in britain and germany the age of dualization: the changing face of inequality in deindustrializing societies the short-run macro implications of school and child-care closures policy feedback and public opinion: the role of employer responsibility in social policy oxford covid-19 government response tracker health equity, social policy, and promoting recovery from covid-19 conflict in decision-making and variation in public administration outcomes in italy during the covid-19 crisis the consequences of public policy for democratic citizenship: bridging policy studies and mass politics effects of public opinion on policy when effect becomes cause protecting soldiers and mothers https://orcid.org/0000-0003-1415-0678 roni lehrer https://orcid.org/0000-0002-9202-9278 key: cord-253720-s6hwui6n authors: andraz, jorge m.; rodrigues, paulo m.m. title: monitoring tourism flows and destination management: empirical evidence for portugal date: 2016-03-26 journal: tour manag doi: 10.1016/j.tourman.2016.03.019 sha: doc_id: 253720 cord_uid: s6hwui6n we propose the use of a tool recently introduced by gayer (2010), known as the “economic climate tracer”, to analyze and monitor the cyclical evolution of tourism source markets to portugal. considering the period 1987–2015, we evaluate how tourism to portugal has been affected by economic cycles. this tool is useful as it clearly illustrates the evolutionary patterns of different markets, and allows us to identify close relationships with economic fluctuations. we found that german tourism plays a leading role, since its movements are followed with delays by tourism flows from other countries, and exhibits higher resilience to shocks. also, domestic and spanish tourism have both displayed less irregular behaviors than tourism from other source markets. on the contrary, tourism from the netherlands and the uk, have displayed irregular patterns, which demonstrates the urgency to diversify tourism source markets to reduce the country's vulnerability to external shocks and economic cycles. business cycles are recurrent phenomena in all economies and are transversal to all economic sectors. tourism is not an exception, as this sector is particularly vulnerable to economic fluctuations. this vulnerability reinforces the relevance of developing tools that allow for a clear understanding of the sector's cyclical stance in order to inform the authorities and to manage the adoption of measures to diversify and, simultaneously, to reduce the country's dependency on a reduced number of tourism source markets. this issue is of particular importance for portugal as tourism plays a central role on the country's economic performance. according to the world travel and tourism council [wttc] (2013), the total contribution of tourism to gdp in 2012 was usd 26.4 billion, corresponding to 15.9% of gdp, and it is expected to grow by 1.6% per annum to usd 31.0 billion, corresponding to 6.3% of gdp, by 2023. the total contribution to employment was 860,500 jobs in 2012, or 18.5% of total employment and it is expected to grow 1.0% per annum to 954,000 jobs, or 20.7% of total employment, by 2023. about a quarter of foreign investment is motivated by tourism trade. these figures provide an overall picture of the importance of tourism for portugal. both the increasing number of tourists and the sector's strategic importance have led portuguese economic and political agents to pay special attention to this sector by taking active measures towards its sustainability. it is therefore not surprising that private and public organizations are increasingly interested in obtaining a deeper understanding of the tourism cycles of the main origin markets and monitoring their evolution. over the last decades business cycle regimes have been implicitly taken into account in the analysis of tourism with different objectives (see, inter alia, andraz, gouveia and rodrigues, 2009; collins & tisdell, 2004; crouch, 1996; witt & witt, 1995; lim & mcaleer, 2002; lim, 1997; and ramos & rodrigues, 2013) . most of these studies have focused on the tourism demand side and little attention has been devoted to the effects of different regimes on the tourism supply side. research on tourism cycles was, to the best of our knowledge, first considered by gouveia and rodrigues (2005) . this article provides a dating approach of the tourism demand cycle based on the method described in harding and pagan (2003) . using concordance and recursive concordance indices, gouveia and rodrigues (2005) establish a strong degree of cycle synchronization between tourism and economic cycles and identify delay effects between them. in a recent study guizzardi and mazzocchi (2010) , using a structural time series approach, also conclude that tourism demand is driven by delay effects of the overall business cycle. therefore, a better understanding of the cyclical stance of tourism to portugal and how each market has reacted to the major turning points, accounting for their heterogeneous behavior to regime changes, is of great relevance to tourism agents when looking for source markets. this paper aims to provide such valuable information regarding domestic tourism and tourism coming from the main international source markets to portugalegermany, the netherlands, spain and the united kingdom (hereafter uk). methodologically speaking, in this paper we consider the "economic climate tracer" proposed by gayer (2010) and apply it to the series of the monthly number of overnight stays of tourists in portugal. the approach consists on the graphical representation of the standardized level of a smoothed indicator, in this case based on the hodrick and prescott (1997) filter in order to eliminate shortterm fluctuations, on its month-on-month changes. the resulting diagrams can be divided into four quadrants, allowing for the association of the temporal evolution of the smoothed variables to the different phases of the tourism cycle. the rest of the paper is organized as follows. in section 2 we present the data and a rigorous description of the methodology used in the analysis. in section 3 we discuss the main results in terms of each market position with respect to the tourism cycle and briefly describe the major events that have likely been at the origin of the major turning points. in section 4 we report the main conclusions and lessons for tourism destination management as well as the limitations of our approach. the data used in this paper correspond to monthly overnight stays in hotels, apartment hotels, tourist apartments, tourist villages, motels, bed and breakfasts, inns, guest houses and camping parks of domestic tourists and international tourists coming from the uk, the netherlands, germany and spain. these series are used as a proxy for tourism activity (hereafter tourism). the option for using the number of overnight stays as a proxy for tourism was dictated by the lack of consistent information on other variables, such as, e.g. tourists' expenditures. however, this variable has been widely used in literature focused on the evaluation of tourism (see, for example, aguayo, 2011; dritsakis, 2004; and archer & fletcher, 1996) . this proxy for tourism has also been used in recent works (see e.g. paci & marrocu, 2014; and cort es-jim enez, 2008) , as it reflects the length of stay and therefore provides information about the occupation rate of tourism facilities. in this way, it can be more informative than other variables, such as the number of arrivals, which do not provide information on such dimensions, or tourism expenditures, for which decisions on the adoption of price deflators can be an issue. this study uses data covering a long period, from january 1987 to september 2015, during which several economic downturns have occurred. this period provides a rigorous picture about the reactions of tourism from different source markets to portugal and, thereby, allows us to identify general trends. the data were collected from the annual issues of tourism statistics (ine, 2008 (ine, , 2009 (ine, , 2010 (ine, , 2011 , published by statistics portugal (the national institute of statistics in portugal). summary statistics of these variables are provided in table 1 and they reflect the relevance of the source markets considered. overall, the five source markets considered represent around 75.0% of the total number of overnight stays in the country. the first position belongs to domestic tourism with an average of 30.9% over the period under analysis, and is followed by the uk with an average of 19.7% and germany with an average of 12.5%. the last positions are shared by the spanish and the dutch markets, which together represent an average close to 12.0% of the total number of overnight stays in the country. by considering three sub-periods, we identify interesting trends, which are quite informative on the reactions of the different tourism segments. we notice steadily decreasing patterns in tourism coming from germany, the uk and the netherlands, and increasing trends in the number of overnight stays of domestic and spanish tourists. the methodology used to analyze the cyclical evolution of tourism in portugal is inspired in the economic climate tracer developed by gayer (2010) . the approach consists of the graphical representation of the standardized level of a smoothed indicator computed using the hodrick and prescott (1997) filter (in order to eliminate short-term fluctuations) on its month-on-month changes. the standardized levels of tourism on the y-axis are plotted against their month-on-month changes on the x-axis. this approach provides an attractive visual tool for the inspection of tourism series through circular movements across the four quadrants of the graphs, corresponding to the four growth cycle phases. these phases can be characterized in a counter-clockwise rotation as follows: above average and increasing (upper-right quadrant, corresponding to "expansion"), above average but decreasing after having reached the peak (upper-left quadrant, corresponding to "downswing"), below average and decreasing (lower-left quadrant, corresponding to "recession") and below average but increasing after having passed the trough (lower-right quadrant, corresponding to "upswing"). the peaks occur in the upper center of the graph, in the transition from "expansion" to "downswing", while the troughs are located in the lower center, in the transition from "recession" to "upswing". therefore, the resulting diagrams can be divided into four quadrants allowing, in this way, for the association of the temporal evolution of the smoothed variables to the different phases of the tourism cycle: the first quadrant corresponds to the expansion phase and is observed when the standardized series are above their means and increasing; the second quadrant indicates that the cycle entered in downswing, i.e., when the standardized series are above their means but decreasing; the third quadrant indicates recession since it corresponds to the case where the standardized series are below their means and decreasing; and finally, the fourth quadrant indicates that the cycle entered into an upswing as the standardized series are below their mean but increasing. this classification follows the conventional notion of the business cycle and offers a simple and clear method to characterize the development of economic indicators throughout the cycle and may also be used as a monitoring tool for destination management when applied to the number of overnight stays. the tourism industry is not immune to shocks caused by economic fluctuations or financial instability (see neumayer, 2004) . on the contrary, it is very sensitive to these shocks as they impact negatively on tourists' confidence and income. the uncertainty about the economic evolution in each country is regarded with caution and imposes serious limitations to tourism flows. however, the reaction of economies to shocks can be quite diverse given the structural and political differences they exhibit and consequently the impact on destinations can differ depending on the composition of the tourism source markets "portfolio". to understand the evolution of tourism it is important to review the world events that occurred over the last decades and which clearly marked the tourism world cycle. fig. 1 displays the cycle of visitor exports 1 . the early 1990's were characterized by successive fluctuations of tourism worldwide. this instability translated into a deep recession in the early 2000's. after a recovery period until 2007, tourism suffered again a downturn, but from 2010 onwards there was an oscillating recovery. the evolution of the tourism cycle ( fig. 1 ) mirrors the chronology of the economic crisis of the national bureau of economic research (www.nber.org/cycles.html). the recessive periods in tourism observed in fig. 1 were motivated, or at least were partially explained by a series of unusually deep and sequential negative events that hit the world economy in the 1990s and the 2000s. among them there is the european monetary system (ems) currency crisis in 1992 and 1993, which were precipitated by the german reunification in 1990, followed by the subsequent raise of german interest rates, which seriously affected large european economies like the uk and france. the 1990s were also the stage for crises that started in specific regions of the world but soon spread across the globe, such as the asian crisis in 1997, the russian crisis in 1998 and the brazilian crisis in 1999 which, in turn, spread out to argentina. in general, these crises were caused by short-term commercial bank debt and/or securities market investment and were followed by huge capital outflows and severe currency speculation. in the case of the asian crisis, banks, non-banks and corporations over-borrowed and foreign banks and private investors over-lent. all these episodes originated significant down and upswings of the global economy impacting countries differently, and this evidence is not surprising given, on the one hand, the economic and financial globalization and the dimension of the us economy and, on the other hand, the specific structural and cyclical conditions that prevailed in each country. in what concerns the tourism cycles, we can report three major crisis periods from fig. 1 , 2004) and also the severe acute respiratory syndrome (sars) outbreak in november, 2002. the recent financial crisis that broke in the us in 2007 has spread to all economies generating serious effects on employment and domestic demand. therefore, the impacts on the tourism industry were unavoidable. all these events have lead tourists to reduce their travels or change destinations and, therefore, they deeply marked tourism demand. the impacts of crisis on destinations are more or less pronounced depending on the impact of events on the source markets responsible for the main tourism flows. it is unquestionable that all countries were impacted by the worldwide events responsible for the up-and downswings in world tourism. that is, in all countries there are downswings in tourism flows over the periods 1990e1995, 2000e2005 and recently since 2007. these movements are possibly more pronounced in some countries than in others, which clearly suggests that countries have reacted differently to external shocks. if this is the case, this information is obviously of valuable importance to tourism agents in portugal since it can be a guide to identify which source markets are less sensitive to external shocks and to endeavor efforts to maintain and/or increase tourism flows from those countries, and which markets are more prone to those shocks in order to look for market diversification. the tracer that is used in this paper can therefore be a useful tool for the analysis of the degree of resilience of the source markets to crises. fig. 2 illustrates the evolution of the domestic tourism cycle in portugal. we observe that domestic tourism remained in the expansion quadrant over the 2000s, reaching the peak by the beginning of 2010. since then, and after a short decline as a result of the economic and financial crisis that culminated with political and economic austerity measures, we notice a recovery reflected in the steady location of the demand in the first quadrant, exhibiting increasing growth movements. this evolutionary behavior of domestic tourism to portugal reflects, although with different magnitudes, the negative effects of the different crises that impacted the world tourism cycle. however, the negative effects of the recent financial crisis on domestic tourism were not very pronounced when compared to foreign tourism. domestic tourism in portugal is obviously not independent of the structural economic context the country has experienced. since 2002 portugal has been going through a problem of economic stagnation with an annual economic growth of less than 2.0% (lower than the eu average). it recorded a zero growth in 2008, followed by a 2.9% contraction in 2009 (european commission, 2014). due to the economic downturn and the fiscal stimulus packages in response to the crisis, the fiscal balance deteriorated to a record deficit of 9.9% and 10.2% of gdp in 2009 and 2010, respectively (oecd, 2014). since 2009 portugal has faced an economic recession together with a continuous growth of public debt, austerity policies, nationalization of banks, and difficulties in deficit control. the low growth rates and the successive deficits implied an increase of the public debt from 50.7% in 2000 to 108.2% in 2011 (european commission, 2014). as a result, the moody's investors service and other rating agencies cut portugal's sovereign bond rating in the summer of 2010, which led to an increased pressure on the portuguese government bonds. this brief description highlights the devastating effects of the global crisis on the portuguese public finances and economy. from the late 2009, fears about the ability of the country to fulfill its sovereign debt liabilities dictated the raise of risk premiums to a point where the access to capital markets was no longer an option and a debt default soon became imminent. this context dictated the urgency in negotiating a bailout in the form of a memorandum of understanding with the consortium composed by the european commission, the european central bank and the international monetary fund (known as "troika") and severe austerity measures that have led to a substantial increase of unemployment and significant wage reductions. this crisis has been by far the most devastating to the portuguese economy, and it has originated inevitable effects on domestic tourism, leading to a significant reduction of the demand for foreign destinations by portuguese tourists and consequently to an increase in domestic tourism, as reflected by the positive evolution in recent years; see fig. 2 . fig. 3 illustrates the evolution of the international demand cycle dynamics of the four main source markets to portugal from january 1987 to september 2015. as before, all series were smoothed through the hodrick-prescott filter to eliminate short-term fluctuations. the smoothed series are then plotted against their month-on-month variation. the graphs in fig. 3 report the dynamics of tourism flows from the international source markets considered, which drive tourism from the lower quadrants to the upper quadrants. two main distinctive aspects emerge from the graphs, which cannot be dissociated from the economic and political context experienced by markets over the sample period. the first is related to the distinctive movements reported by tourism flows in their trajectory across quadrants. starting from the upswing quadrant (fourth quadrant), in the early 1987 tourism flows to portugal enacted positive growth trends with level increases over the 1990s. these movements toward the expansionary quadrant (first quadrant) were shared by tourism flows from all sources, although with episodes of short cycles that constitute the main distinctive feature among source markets. these short cycles were observed in tourism from all sources, with the exception of tourism coming from germany, which exhibited positive and increasing growth rates (fig. 3a) . these short cycles translated into rather irregular paths in the case of tourism from the netherlands and spain in their movement towards the expansion phase ( fig. 3b and c, respectively) . in fact, their paths were characterized by the occurrence of successive and persistent mini-cycles with recessive movements towards the recession (third quadrant) and upswing (fourth quadrant) quadrants, reporting reductions in growth and levels, until they finally reached the expansionary quadrant by mid of 2000. tourism from the netherlands continued to report large volatility over the 2000s. over the first half of 2000 it moved to the downswing quadrant and then to the expansion quadrant, reaching the peak by the end of 2013. we also notice a strengthening of this ascending movement up to 2015. tourism from spain moved to the expansion quadrant in 2005, reached its peak in 2010, and after a short passage through the downswing quadrant, it registered an inversion towards the expansion quadrant. the behavior of tourism from germany and the uk (fig. 3a and d, respectively) also followed similar, but smoother, patterns. in fact, tourism from germany entered the expansion phase by the middle of the 1990s and reached the peak by the end of 1999. tourism from the uk entered the expansion phase in the second half of the 1990s and after a short cycle, reached a peak in 2006. these countries followed a similar path over the 2000s moving together to the recession quadrant by the end of 2007. tourism from these countries to portugal enacted a steadily recovery trajectory by 2010 until 2015. the second aspect that emerges from the data is the leading role of tourism flows from germany, motivated by a smooth evolutionary path and continuous high growth rates over the 1990s. this growth engine explains the fact that german tourism flow path is one-step ahead of the other source markets. tourism from germany moved to the expansion quadrant and reached the peak in the 90s, long before tourism flows from other markets. we also notice that the recession period tourism flows experienced over the first half of 2000s was felt by german tourism earlier and with shorter duration. therefore, it seems that tourism from germany is more resilient to crises and that its trajectories are followed by tourism from other source markets. to better understand the cyclical dynamics just described, as well as the apparent leading role of the german tourism flows to portugal, it is useful to briefly characterize the economic context of each source market. the ascending path of tourism from germany over the 90s (fig. 3a) coincides chronologically with the huge economic growth observed in the aftermath of the country's reunification (gr€ omling, 2008). between 1990 and 1992, the german average real gdp growth was 4.5%, which exceeded its long-term average. however, in the following period, germany enacted an adjustment process towards its long-term potential growth with lower growth rates. over the second half of the 1990s, the average gdp growth rate was almost 1 and 2 percentage points lower than that of the emu/eu countries and the united states, respectively, while the annual average increase in employment in germany fell short of that of the us. this context coincides with an inversion path of tourism flows to portugal, which dictated the end of the expansionary path and the occurrence of the peak by the end of the decade. at the beginning of the 2000s, the german economy practically stagnated. the lowest gdp growth figures were observed in 2002 (þ1.4%), in 2003 (þ1.0%) and in 2005 (þ1.4%), together with high unemployment rates. these problems, together with the country's aging population led to the implementation of a set of reforms, which have become known as agenda 2010. the contraction of the german nominal gdp in the second and third quarters of 2008 drew a scenario of technical recession following the path observed at the global and european scales. the german government's response to this downturn path translated into the approval of a package of economic stimulus measures to prevent the unemployment rates to rise. over this period, we observe a downturn of tourism flows to portugal and the first signs of economic recovery started to emerge in 2010 and are reflected in fig. 3a through persistent movements towards the boom quadrant. we notice that the german tourism cycle is similar to those of the uk and the netherlands. they all exhibit a contraction at the beginning of the 2000s which came to an end ten years later, around 2010. the british economy registered a period of more than 10 years of continuous economic growth, from the mid-1990s to 2005. the downturn that followed imposed huge oscillations in british tourism to portugal, which attained the peak just before the recent financial crisis (fig. 3d) . tourism from the netherlands and spain were also particularly affected between 1990 and 1995 (see fig. 3b and c, respectively). in both cases, there were significant reductions in levels and growth rates and both end in the upswing quadrant in the second half of the 1990s. the dutch tourism moved to the boom quadrant and reached the peak around 2000. this movement is coincident with the strong performance of the dutch economy in the second half of the 1990s, which is referred to as the "dutch miracle". real gdp growth reached 3.7% on average (clearly above the european union average of 2.7%). the unemployment rate dropped from 6.6% in 1996 to 2.5% in 2001. this positive conjuncture is reflected in a positive evolution of tourism to portugal. this economic boom appears to have been mainly motivated by an overheating of the economy, but the slowdown that followed seems to have been the result of the global downturn and the turmoil in financial markets, and consequently tourism suffered a reduction in levels and growth. in fact, we observe that tourism flows moved to the downswing quadrant around 2001 recording continuous reductions in levels up to 2005. however, as a result of the efforts on public finances' consolidation, the country reached a government surplus of 0.6% by the end of 2006. this economic performance also tourism from spain exhibits a similar pattern. after having stepped backwards in the periods 1990e1995 and 2005e2010 it entered the boom quadrant in the second half of the 2000s. however, the recent financial crisis has imposed a slowdown by 2007. we observe a reduction in levels and growth rates and the peak was reached in 2010 after which tourism moved to the downswing quadrant. however, the spanish tourism flows rapidly returned to the expansion quadrant, having registered increasing growth rates in the last years. our results show that tourism source markets to portugal react differently to economic adverse shocks depending on internal economic and political particularities and that tourism from germany seems to have a leading reaction to economic fluctuations. in this paper we illustrate the usefulness of the approach introduced by gayer (2010), known as the "economic climate tracer" to analyze and monitor the cyclical evolution of tourism demand, through an application to portugal. we analyze the tourism flows of domestic tourists and tourists coming from the main international source markets -germany, the netherlands, spain and the uk -which together represent on average 75.0% of the total number of overnight stays in the country. it is shown, through the analysis of the economic evolution in the tourism source economies from 1987 to 2015 that the climate tracer is a useful tool to illustrate the evolutionary patterns of the different markets and therefore an important source of information to support economic and policy agents in decision making. this analysis highlights three relevant issues. first, we concluded that tourism flows from germany, the netherlands and the uk exhibit decreasing trends over the period under analysis, whereas domestic tourism and spanish tourism report increasing trends. second, tourism from all source markets, including the domestic market, reported levels below the average with increasing growth rates by the end of the 1980s, and all are currently reporting growth rates and levels above their averages. third, in-between, tourism flows from different source markets to portugal depicted different behavior paths which are not independent of the economic and financial environments in each country. domestic tourism and tourism from spain exhibited the less irregular patterns in their evolutionary path between 1987 and 2015. despite the two recessive episodes in 1995 and 2007, which are certainly a result of economic recessions, they followed rather smooth and similar trajectories. possibly, the crises episodes over the 1900s and 2000s have impacted on the decisions of traveling, leading tourists to choose closer destinations. this could possibly explain the less irregular patterns of the portuguese and spanish tourism. tourism from the netherlands, the uk and germany reported larger oscillations, revealing thereby higher sensitivity to economic cycles. we also noticed that tourism from germany has played a leading role since its movements seem to have been followed by other source markets. in general, this paper provides evidence that there is a narrow relation between economic context and tourism flows to portugal. recession periods dictate tourism contractions, while economic expansions are reflected in persistent increases of tourism flows. it also provides evidence that markets react differently to economic and political, or even terrorist events. the domestic and spanish tourism are segments that may mitigate external adversities, and therefore, the investment in advertising campaigns should be directed to these markets, while promotional actions should be reinforced in order to guarantee the sustainability of other international segments, for which portugal has been a traditional holiday destination. notwithstanding, these results should be interpreted with caution. the source markets here considered are in different maturity stages. the evolution of tourism from different sources is perhaps not independent from their destination life cycle stages. portugal has been a traditional destination for british tourists since the 1960s, while tourism from the netherlands, or even germany or spain is more recent. therefore, linking the evolutionary paths with the corresponding destination life cycle stages of each market may provide additional insights on the cyclical behavior of tourism and is left for future research. research. international journal of forecasting, 11, 447e475. world travel and tourism council. (2013) . travel and tourism economic impact 2013-portugal. jorge m. andraz ph.d in economics and professor of economics and econometrics at the university of algarve, he is a member of the centre for advanced studies in management and economics of the university of evora (cefage-ue). his main research interests are focused on tourism, applied macro econometrics, time series econometrics, economic growth, financial economics and tourism economics. he has published several books about the economic effects of public investment in portugal. he is also the author of several publications in influential international journals. he belongs to the referee board of several international scientific journals. paulo m. m. rodrigues senior economist at the economics and research department of the bank of portugal and professor of econometrics at nova school of business and economics of universidade nova de lisboa (lisbon, portugal). he was a jean monnet fellow at the european university institute in florence, italy and visiting scholar at the institute for advanced studies in vienna, austria, the university of british columbia, vancouver, canada, the university of navarra, spain and the university of the balearic islands, spain. research interests include timeseries econometrics, financial econometrics, empirical finance and macroeconomics, and tourism economics. he has published a number of peer-reviewed articles in several internationally renowned scientific journals. impact of tourism on employment: an econometric model of 50 ceeb regions. regional and sectoral economic studies modelling and forecasting the uk tourism growth cycle in algarve the economic impact of tourism in the seychelles which type of tourism matters to the regional economic growth? the cases of spain and italy cointegration analysis of german and british tourism demand for greece report: the economic climate tracer: a tool to visualize the cyclical stance of the economy using survey data wirtschaftswissenschaftliche beitr€ age from julius-maximilians-universit€ at würzburg, lehrstuhl für volkswirtschaftslehre, insbes tourism demand for italy and the business cycle a comparison of two business cycle dating methods post-war us business cycles. an empirical investigation tourism statistics tourism statistics tourism statistics 2010 tourism statistics review of international tourism demand models a cointegration analysis of annual tourism demand by malasia for australia the impact of political violence on tourism: dynamic crossnational estimation tourism and regional growth in europe the importance of online tourism demand forecasting tourism demand: a review of empirical we are grateful to two anonymous referees and editor professor steve page for useful comments and suggestions. the first author is pleased to acknowledge financial support from fundação para a ciência e a tecnologia (grant uid/eco/04007/2013) and feder/ compete (poci-01-0145-feder-007659). key: cord-303489-ve1fgnyg authors: klabunde, thomas; giegerich, clemens title: how high and long will the covid-19 wave be? a data-driven approach to model and predict the covid-19 epidemic and the required capacity for the german health system date: 2020-04-17 journal: nan doi: 10.1101/2020.04.14.20064790 sha: doc_id: 303489 cord_uid: ve1fgnyg background an objective: in march 2020 the sars-cov-2 outbreak has been declared as global pandemic. most countries have implemented numerous social distancing measures in order to limit its transmission and control the outbreak. this study aims to describe the impact of these control measures on the spread of the disease for italy and germany, forecast the epidemic trend of covid-19 in both countries and estimate the medical capacity requirements in terms of hospital beds and intensive care units (icus) for optimal clinical treatment of severe and critical covid-19 patients, for the germany health system. methods: we used an exponential decline function to model the trajectory of the daily growth rate of infections in italy and germany. a linear regression of the logarithmic growth rate functions of different stages allowed to describe the impact of the social distancing measures leading to a faster decline of the growth rate in both countries. we used the linear model to predict the number of diagnosed and fatal covid-19 cases from april 10th until may 31st. for germany we estimated the required daily number of hospital beds and intensive care units (icu) using clinical observations on the average lengths of a hospital stay for the severe and critical covid-19 patients. results: analyzing the data from germany and italy allowed us to identify changes in the trajectory of the growth rate of infection most likely resulted from the various social distancing measures implemented. in italy a stronger decline in the growth rate was observed around the week of march 17th, whereas for germany the stronger decline occurred approximately a week later (the week of march 23rd). under the assumption that the impact of the measures will last, the total size of the outbreak can be estimated to 155,000 cases in germany (range 140,000-180,000) and to 185,000 cases in italy (range 175,000-200,000). for germany the total number of deaths until may 31st is calculated to 3,850 (range 3,500-4,450). based on the projected number of new covid-19 cases we expect that the hospital capacity requirements for severe and critical cases in germany will decline from the 2nd week of april onwards from 13,500 to ~2500 hospital beds (range 1500-4300) and from 2500 to ~500 icu beds in early may (range 300-800). conclusion: the modeling effort presented here provides a valuable framework to capture the impact of the social distancing measures on the covid-19 epidemic in european countries and to forecast the future trend of daily covid-19 cases. it provides a tool for medical authorities in germany and other countries to help inform the required hospital capacity of the health care system. germany appears to be in the middle of the (first) covid-19 outbreak wave and the german health system is well prepared to handle it with the available capacities. on march 11 th 2020 the world health organization (who) declared the covid-19 outbreak as pandemic. the steep increase of covid-19 cases in china, iran, italy, france, germany and other european countries resulted in to tens of thousands of severe cases requiring hospitalization as well as in thousands of critical cases due to progression to life-threatening respiratory distress that require intensive care unit (icu) admission (fig. 1) . the dramatic rate of cases that progresses has created a significant strain in the national health systems of these countries and has led to a significant shortage of icus equipped with extra-corporeal membrane oxygenation (ecmo) to save the lives of very critical covid-19 cases. since early march several european governments have implemented numerous control measures to reduce the transmission of the disease and decrease the number of new daily cases of covid-19 so that fewer patients need to seek treatment at any given time and avoid overwhelming hospital capacity, commonly referred as "flattening the curve". the strategy to achieve of the flattening of the curve of covid-19 cases in europe has consisted of (1) early detection of new cases and primary contacts followed by self-quarantine or hospitalization, (2) promoting the adherence to hygiene standards like washing hands and (3) . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint parts of the population by "social distancing". in germany these "social distancing" measures have evolved over time from banning gatherings of more than 1000 participants (issued on march 9 th ), university and school closures (issued on march 16 th ), to prohibiting gatherings of more than two people (issued on march 23 rd ). these restrictions have put a significant burden on german society and have reduced the quality of life for most. there has been a mounting interest on how long these measures and restrictions will last and when can they be lifted safely. on the other hand, medical professionals are expecting a surge of covid-19 patients reaching the hospitals soon and wonder when and how hard it will hit them. germany has significantly expanded its hospital capacity by increasing the number of icu beds and ecmo units in order to prepare for the expected wave of covid-19 patients and to prevent overwhelming the health system. a registry has been formed to track all occupied and readily available intensive care beds in germany to ensure that possible regional peaks of critical covid-19 cases can be treated by transferring the influx of patients to nearby health systems with availability [1] . as of april 12 th this registry covers approximately 19,700 icu beds with 11,376 occupied beds and 8325 available beds. this indicates that there is available capacity for any upcoming influx of new critical and severe cases. over the past weeks mathematical modeling of the covid-19 pandemicespecially sir (susceptible, infectious, recovered) modelshas provided important insights to better understand the spread of virus sars-cov-2 and to evaluate how various potential scenarios of control measures could impact the dynamics of the covid-19 out-break [2, 3] . these simulation studies have informed decision making for national governments on how to react to this coming covid-19 epidemic. simulated estimates of the size of the epidemic in germany if no control measures were implemented [4] and have provided a strong rationale for the closures of school and universities in the uk [5] . other simulations have shown that a contact-tracing app that builds a memory of proximity contacts and immediately notifies contacts of positive casesand potentially asks for self-isolationmight significantly reduce the transfection rate especially by pre-symptomatic covid-19 cases thus reducing the spread of the virus [6] . due to the underlying mechanistic model structure of sir-based models that include representations of mobility information of society it is possible to explore multiple control measure scenarios and thus estimate the impact of certain measures at a very early stage of the epidemic. usually, an epidemic follows an exponential growth at an early stage, peaks and then at the inflection point of the total case curve the growth rate declines again. this decline of the growth rate is either achieved by reaching herd immunity or by implementation of measures to hinder the transmission of the virus. as soon as the impact of these measures become apparent in the decline of the daily growth rate, empirical or so-called top-down methods can be applied to model the data (e.g. logistic growth model, gompertz model, exponential growth rate decline model). these top-down modeling methods can then provide . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint insights regarding the stage of the epidemic, allow the prediction of future trends of the outbreak and estimate its final size [7] [8] [9] . since early april 2020 the numbers of daily new covid-19 cases in germany and italy appear to be either constant with similar numbers of new infections each day or declining (see figure 1 ), indicating that the epidemic has moved from the exponential growth phase into a linear growth phase in these countries. with this new data becoming available, we have used an exponential decline of the growth rate model to analyze the trajectory of the daily growth rate [10] . thus, we have captured and quantified the dynamics of the epidemic in these countries and described the impact on the growth rate measures have had taken. using this approach, we have captured a specific date for both countriestwo to three weeks after the start of these measurementswhen the spread of the epidemic significantly slowed down. in a second step we have used the model to forecast the dynamics of the epidemic in italy and germany and to estimate the number of daily and cumulative total covid-19 cases. this allowed us to address the question of how long the epidemic will last in both countries. in a third step we have used these forecasts of diagnosed covid-19 cases to estimate the number of severe and critical cases for each day of april and may to provide an estimate for the daily required capacity of hospital beds, in particular icu beds with ecmo equipment to treat the predicted severe and critical covid-19 cases in germany. we have used an exponential decline of growth rate model to analyze the trajectory of the daily growth rate in germany and italy. we also evaluated two well established growth models, the logistic function and the gompertz function, but decided in favor of the exponential decline model as the model structure allows to easily capture differences in the growth curve that may result from the social distancing measures. the exponential growth rate decline model defines the growth rate as ratio of new cases today versus total cases yesterday as shown in equation (1) and assumes the growth rate is following an exponential decline function given in equation (2) . the parameter  characterizes how fast the growth rate is decreasing. high values of  indicate a slow decline of the growth rate, whereas low values indicate that the growth rate is declining fast and that the ratio of new cases versus total cases will reach zero more rapidly. with the given model a logarithmic plot of the daily growth rate trajectory thus provided a straight line and allowed us to derive  from the slope by linear regression. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint assuming the measures have an impact on the decline of the growth rate, we would expect a discontinuity in the logarithmic plot of the data. for each country we obtained two fits with different values for , capturing the dynamics before and after the impact of the implemented control measures became visible. for germany data was obtained from the website of the robert koch institute (rki) [11] . for italy the data was taken from the coronavirus covid-19 global cases published by the center for systems science engineering (csse) at johns hopkins university (jhu) [12] . for germany and italy, we used the reported data up to april 9 th for modeling and parameter estimation. as a second step we used the exponential decline of growth rate model to predict the cumulative diagnosed covid-19 and the daily new cases for germany and italy beyond april 9 th . as described before a linear regression of the logarithmic plot of the growth rate data between march 23 rd and april 9 th for germany and between march 17 th and april 9 th for italy, respectively, allowed us to model the growth rate using equation (2) . here we could identify the values for  and the intercept as well as their standard deviation and confidence intervals. this allowed us to estimate the expected growth rate for each day beyond april 10 th , as well as an upper and lower limit of the growth rate based on the 95% confidence interval of the linear model. for the prediction of the number of total cases for april 10 th onwards by equation (3) we calculated the expected number of total covid-19 cases using the calculated growth rate value and its upper and lower limit. this allowed us to project the uncertainty in the parameter identification from the logistic regression model into a confidence interval for the predicted covid-19 cases. (3) we have used the predicted number of covid-19 cases (including the confidence intervals of these predictions) in the remaining of april and may to estimate the daily required capacity of hospital beds, in particular icu beds with ecmo equipment to treat the predicted severe and critical covid-19 cases in germany. we used assumptions adapted from thomas-rüddel et al. [13] on the fraction of diagnosed covid-19 cases requiring hospitalization and the subset of cases that progress to intensive care along with the average length of each disease state of an covid-19 patient (summarized in figure . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint 2). after first symptoms appear on day 0 for an average covid-19 patient, we assume ~7 days to reported diagnosis (labeled with d in figure 2 ) [14] . for severe cases we assume ~7 days from first symptoms to hospitalization [15] , for critical cases ~8 days from first symptoms to icu and for fatal cases ~14 days from first symptoms to death (labeled with m in figure 2 ) [16] . thus we expect a time period of ~7 days between reported diagnosis and death. we assume an average duration of hospital stay for severe and critical cases of 12 days [17] , an average duration of a stay in intensive care unit of 6 days for fatal critical cases and of 12 days for critical cases that recover [17] . we assume that ~20 % of covid-19 cases are severe and require hospitalization, ~25% of the severe cases developed into critical cases requiring intensive care and ~50% of the icu patients have a fatal outcome [17] . we have used an exponential decline of the growth rate modelwith the growth rate being defined by daily new cases today versus daily total cases yesterdayto analyze the trajectory of the daily growth rate in germany and italy. figure 3 shows the plot of the daily growth rateusing a logarithmic function for linearizationfrom march 1 st to april 9 th in italy and germany. by fitting the data using linear regression, we obtained two fits of the data that are shown in blue and orange. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint with the value for  being identified we predicted the trend for new daily cases and accumulated total cases for germany and italy assuming that the same control measures remain in place until the last day of may. we projected beyond april 10 th using a value for  of 10.3 and 12.4 days for germany and italy, respectively, that has been determined from the regression analysis using data from the day the impact of the measures have become visible until april 9 th . figure 4 shows the observed data in blue for the total and daily new covid-19 cases and the projected cases in the same graph in red. for the projections of the total covid-19 cases an estimate of the total case range (upper and lower bound) is given that results from the uncertainty in the parameter identification from the linear regression. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint based on these projections the total number of cases in germany by end of may can be predicted to be 140,000 to 180,000 with a peak in the cases around april 1 st . for italy the estimated total size of the epidemic is predicted to be 175,000 to 200,000 cases. evidently these projections assume that the decline of the daily number of new cases continues to follow the exponential trend due to successful control measures taken by both countries. how high will be the covid-19 patient wave for the german health system? when will it hit? how many people will die? we have used the prediction of the newly diagnosed covid-19 cases for germany to estimate the daily capacity requirements for hospital beds and icus as well as daily death rates for april and may in germany. mrz. apr. apr. apr. mai. mai. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint estimated calculated to 3850 (range 3508 to 4450 using the growth rate confidence interval of 95% for the expected diagnosed patients beyond april 10 th ). next, figure 6 provides an estimate for every day in april or may for the required capacity of hospital beds, in particular icu beds with ecmo equipment, to adequately treat the predicted severe and critical covid-19 cases in germany. we made assumptions on the fraction of mild, severe, critical or fatal covid-19 cases and on the average length of the required hospitalization and/or intensive care for each disease state (see methods and figure 2) . we used the observed number of diagnosed covid-19 cases as input before april 10 th and the number of projected covid-19 cases after april 10 th . for the projections beyond april 10 th a confidence interval is given that results from the uncertainty in the parameter identification from a linear regression. according to the model the number of severe cases requiring hospitalization is expected to peak in the first week of april at ~13,000. the maximum of ~2500 critical cases requiring intensive care with ecmo capability is expected at in the first week of april. this is in good agreement with the reported number of covid-19 cases on icus in germany of 2.405 on april 12 th [1, 11] . based on the expected number of new covid-19 cases we would expect that the capacity needs for severe and critical cases will decline from the 2 nd week of april onwards from ~13,500 to ~2500 (range 1500 to 4300) for hospital beds and from ~2500 to ~500 (range 300 to 800) for icu units, respectively. predicted severe and cricial cases in hospital at given day . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint in this study we have captured the trajectory of the daily growth rate of covid-19 cases in germany and italy with an exponential decline model. analyzing the data from germany and italy allowed us to identify changes in the trajectory of the growth rate that most likely are the result from the various "social distancing" measures that have been implemented to reduce the transmission of covid-19. for italy a stronger decline in the growth rate has been observed starting around the week of march beyond the analysis of the given data the data-driven modeling approach presented allowed to describe the observed data with a mathematical model and to provide predictions of new and cumulative covid-19 cases for both countries. the predictions suggest that the peak of the epidemic in terms of newly diagnosed cases has passed not only in italy (in late march), but that also in germany (early april). under the assumptions that the impact of the control measures on the transmission of the virus will last for the duration of the simulated time period, the total size of the epidemic can be estimated to be 155,000 cases for germany (range 140,000 to 180,000) and to be 185,000 cases for italy (range 175,000 to 200,000) for italy. evidently these projections assume that the decline of the daily number of new cases will continue to follow the exponential trend due to successful measures taken in both countries. we have used the predicted daily new diagnosed covid-19 cases in germany to estimate the number of daily new severe, critical and fatal covid-19 cases allowing us to estimate the actual requirements for hospital beds and icus at any given day for april and may. based on the projected number of new covid-19 cases we would expect that the capacity needs for severe and critical cases will decline from the 2 nd week of april onwards from ~13,500 to ~2500 hospital beds in early may (range 1500 to 4300) and from ~2500 to ~500 icu beds in early may (range 300 to 800), respectively. the german hospital register tracks all occupied and available intensive care beds in germany [1] . as of april 12 this register covers approximately 19,700 icu beds with 11,376 occupied beds and 8,325 available beds. compared to the expected now decreasing demand for icu beds this indicates that there is sufficient capacity for the new critical and severe covid-19 cases. in other words, we are just in with respect to the requirements for the german hospitals middle of the wave -that has been expected to hit and possibly overwhelm the german health system -has been reached and it can be well covered by the . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint available capacities. our analysis covers germany as a single entity and does not offer the granularity that may be needed to identify regional short comings of hospital capacities in areas that are more severely effected by the covid-19 epidemic. evidently projections in the future come with certain limitations. first the projection for new covid-19 cases assume a continuation of (similar) control measures of social distancing having the same impact on the growth rate of new cases. they can only provide a trend, can't be applied to project further out into the future and obviously are not able to predict the size of a second wave (or whether there would be one at all). in addition, for the calculations used above to estimate the risk for the overwhelming of the german health care system, several assumptions are based on clinical data from the epidemic in china. evidently due to differences in the age distribution of patients and in the health system between both countries these can only be estimates for the situation in germany. nevertheless, it appears that several of these assumptions translate into projections of death rate and capacity needs for icu beds that are in-line with the observed clinical findings in germany. we estimate the total rate of fatalities per diagnosed covid-19 case to be 2.5% for germany, resulting from the assumption that 20% of the diagnosed cases are severe, 25% of the severe cases get critical and 50% of the critical cases are fatal. on april 12 th for 2.2% of diagnosed covid-19 cases in germany a fatal outcome has been reported [11] . while the epidemic is still ongoing, this formula to calculate the final death rate can be misleading and may lead to a too low number as of today the outcome is unknown for a non-negligible proportion of these diagnosed patients [18] . in other words, current deaths belong to a total case figure of the past, not to the current case figure in which the outcome (recovery or death) of a proportion (the most recent cases) hasn't yet been determined. applying the recommended equation to calculate the case fatality ratio (cfr) by death at day d / cases at day d-x and assuming a time of 7 days between reported diagnosis and death an average death rate for germany of ~2.9 % can be obtained from the data up to april 12 th (total death by april 12 th / total cases by april 5 th ) [10] . assuming 2.5% of diagnosed cases will have a fatal outcome 7 days later we calculated the expected daily deaths in germany (see figure 5 ). the maximum of new deaths per day of ~150 is expected for april 10 th . as shown in figure 5 most of the daily death calculations match well the data. some differences between the predictions and observed data are evident for the daily new fatalities, which have unexpected high values from april 8 to april 10. this difference may be attributed to covid-19 outbreaks in nursing homes leading to unexpectedly high case numbers in this period. it is also noteworthy that with the number of diagnosed covid-19 cases as input, the assumptions on the length of hospital stays for critical patients and the assumed fraction of 5% critical cases of all diagnosed covid-19, we expected the maximum of ~2500 critical cases requiring intensive care with ecmo capability at the first week of april. this is in good agreement with the reported number of covid-19 cases currently in icus in germany of 2405 on april 12 th [1, 11] . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.04. 14.20064790 doi: medrxiv preprint in conclusion, we are convinced that regardless of numerous included assumptions derived from clinical observations and regardless of the given uncertainty of the model projections beyond april 10 th , the model predictions can help to capture the impact of social distancing measures on the epidemic in european countries, to understand the dynamics of the epidemic with respect to diagnosed and fatal cases and to estimate the required daily capacity for german hospitals. thus, it appears to be a valuable tool for germany or other countries to provide guidance and support decision making in the health care system with respect to how much hospital capacity is required to be well prepared for the (next) wave. forecasting the worldwide spread of covid-19 based on logistic model and seir model quantifying undetected covid-19 cases and effects of containment measures in italy modellierung von beispielszenarien der sars-cov-2-epidemie 2020 in deutschland on behalf of the imperial college covid-19 response team. impact of nonpharmaceutical interventions (npis) to reduce covid19 mortality and healthcare demand quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing rational evaluation of various epidemic models modeling and forecasting trend of covid-19 estimation of the final size of the second phase of the coronavirus epidemic by the logistic model generalized logistic growth modeling of the covid-19 outbreak in 29 provinces in china and in the rest of the world covid-19) daily situation report of the robert koch institute an interactive web-based dashboard to track covid-19 in real time coronavirus disease 2019" (covid-19): update für anästhesisten und intensivmediziner märz 2020 schätzung der aktuellen entwicklung der sars-cov-2-epidemie in deutschland -nowcasting clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumoniain wuhan, china updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan for the china medical treatment expert group for covid-19 /funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint we are thankful to munkhjargal schöpfel (klinikum koblenz) for sharing her own professional experience with covid-19 patients, for being a sounding board while this work has been performed and for the supportive daily discussions. we gratefully acknowledge susana zaph (sanofi us) for her thorough and mindful revision of the manuscript. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/10.1101/2020.04.14.20064790 doi: medrxiv preprint key: cord-278508-h145cxlp authors: streng, andrea; prifert, christiane; weissbrich, benedikt; liese, johannes g. title: continued high incidence of children with severe influenza a(h1n1)pdm09 admitted to paediatric intensive care units in germany during the first three post-pandemic influenza seasons, 2010/11–2012/13 date: 2015-12-18 journal: bmc infect dis doi: 10.1186/s12879-015-1293-1 sha: doc_id: 278508 cord_uid: h145cxlp background: previous influenza surveillance at paediatric intensive care units (picus) in germany indicated increased incidence of picu admissions for the pandemic influenza subtype a(h1n1)pdm09. we investigated incidence and clinical characteristics of influenza in children admitted to picus during the first three post-pandemic influenza seasons, using active screening. methods: we conducted a prospective surveillance study in 24 picus in bavaria (germany) from october 2010 to september 2013. influenza cases among children between 1 month and 16 years of age admitted to these picus with acute respiratory infection were confirmed by pcr analysis of respiratory secretions. results: a total of 24/7/20 influenza-associated picu admissions were recorded in the post-pandemic seasons 1/2/3; incidence estimates per 100,000 children were 1.72/0.76/1.80, respectively. of all 51 patients, 80 % had influenza a, including 65 % with a(h1n1)pdm09. influenza a(h1n1)pdm09 was almost absent in season 2 (incidence 0.11), but dominated picu admissions in seasons 1 (incidence 1.35) and 3 (incidence 1.17). clinical data was available for 47 influenza patients; median age was 4.8 years (iqr 1.6–11.0). the most frequent diagnoses were influenza-associated pneumonia (62 %), bronchitis/bronchiolitis (32 %), secondary bacterial pneumonia (26 %), and ards (21 %). thirty-six patients (77 %) had underlying medical conditions. median duration of picu stay was 3 days (iqr 1–11). forty-seven per cent of patients received mechanical ventilation, and one patient (2 %) extracorporeal membrane oxygenation; 19 % were treated with oseltamivir. five children (11 %) had pulmonary sequelae. five children (11 %) died; all had underlying chronic conditions and were infected with a(h1n1)pdm09. in season 3, patients with a(h1n1)pdm09 were younger than in season 1 (p = 0.020), were diagnosed more often with bronchitis/bronchiolitis (p = 0.004), and were admitted to a picu later after the onset of influenza symptoms (p = 0.041). conclusions: active screening showed a continued high incidence of a(h1n1)pdm09-associated picu admissions in the post-pandemic seasons 1 and 3, and indicated possible underestimation of incidence in previous german studies. the age shift of severe a(h1n1)pdm09 towards younger children may be explained by increasing immunity in the older paediatric population. the high proportion of patients with underlying chronic conditions indicates the importance of consistent implementation of the current influenza vaccination recommendations for risk groups in germany. influenza is one of the most common vaccine-preventable viral diseases, with the highest morbidity reported for children and elderly patients [1, 2] . influenza infections during childhood usually present as mild respiratory upper airway disease, but severe complications and fatalities also occur, especially in children less than 2 years of age and in children with underlying chronic conditions [2] [3] [4] [5] [6] [7] . however, 40-50 % of influenza-associated fatalities occur in previously healthy children [4, 8] . before the influenza a(h1n1)pdm09 pandemic in 2009/ 2010, comparisons of clinical characteristics between patients infected with different influenza types (a vs. b) and between patients infected with different influenza a subtypes showed only small differences when controlling for age [9, 10] . during the pandemic, however, some studies observed increased morbidity and mortality among children compared to previous seasonal influenza [6, [11] [12] [13] , while other studies described the clinical features of a(h1n1)pdm09 as being similar or even milder [14, 15] . acute respiratory distress syndrome (ards) and fatal viral pneumonia was observed more frequently during the pandemic [16] . post-pandemic surveillance was recommended, as circulation of a(h1n1)pdm09 was expected to continue for several years, gradually assuming a seasonal influenza pattern [16] . in germany, influenza sentinel surveillance on outpatients of all ages [17] confirmed that the first postpandemic season 2010/11 was dominated by influenza a(h1n1)pdm09 (62 %), co-circulating with influenza b (37 %) whereas a(h3n2) was rare (<1 %). during the second season 2011/12, a(h1n1)pdm09 was rare (1 %), whereas a(h3n2) was diagnosed in 75 % of cases and co-circulated with influenza b (24 %). in the third season 2012/13, all three types/subtypes co-circulated in similar proportions (34 % a(h1n1)pdm2009, 31 % a(h3n2), and 35 % b). information on the incidence and clinical characteristics of severe paediatric influenza resulting in intensive care treatment and/or fatal outcome is still limited in germany, and post-pandemic data is thus far available only for the season 2010/11 [18] [19] [20] . based on cases recorded by a nation-wide paediatric intensive care unit (picu) reporting system, the pre-pandemic (2005/06-2007/08), pandemic (2009/10) and post-pandemic (2010/ 11) annual incidence of severe influenza cases per 100,000 children below 15 or 17 years of age was estimated as 0.05, 0.8-1.0, and 0.4, respectively [18] [19] [20] . the data so far available indicated a shift towards younger children in a(h1n1)pdm09 cases from the pandemic to the first post-pandemic season [20] . in these studies, it remained unclear whether the higher pandemic and post-pandemic incidence in children was caused by higher influenza activity, heightened physician awareness, more frequent or more sensitive influenza testing, or a more severe course of disease of a(h1n1)pdm09 [18] . furthermore, all these previous studies may have been affected by underreporting, as influenza cases were reported at the discretion of the picu physician without systematic screening for influenza in patients with severe acute respiratory infection. in the study presented here, we used active screening to estimate the incidence of laboratory-confirmed influenzaassociated picu admissions in one of germany's largest federal states during the first three post-pandemic seasons. furthermore, we described the clinical characteristics of influenza picu patients and compared patients with severe a(h1n1)pdm09 disease between the post-pandemic seasons. prospective, active surveillance was conducted in picus of paediatric hospitals in bavaria, germany. on december 31 st , 2010 roughly 2,001,700 children <17 years of age were registered in bavaria [21] , representing 16 % of the german population in this age group [22] . the annual study population was defined as the sub-group of all children in bavaria at least 1 month and <17 years of age. all 30 paediatric hospitals in bavaria equipped for paediatric intensive care treatment of children older than 1 month of age were invited to participate. these picus reported a total of 432 intensive care beds (median 14, iqr [11] [12] [13] [14] [15] [16] , including 207 beds (median 9, iqr 6-12) equipped with ventilation facilities. , all patients who fulfilled the following inclusion criteria were enrolled: i) admission to a participating picu with suspected acute respiratory infection (ari) of the upper or lower respiratory tract, with arirelated symptoms (for example, coryza, cough, or sore throat); ii) age at picu admission due to ari at least 1 month and below 17 years of age; iii) parental written informed consent. enrolled children with pcr-confirmed influenza were classed as influenza-associated ari. the picu physician documented demographic characteristics, underlying chronic medical conditions, influenza vaccination status, diagnostic findings, ari-associated diagnoses and complications, treatment, duration of hospital and picu stay, and outcome in a structured questionnaire. a respiratory sample, usually a flocked nasopharyngeal or pharyngeal swab, was collected on the day of picu admission for pcr-confirmation of influenza. microbiological testing for bacteria or fungi was at the discretion of the picu physician; pathogens detected at usually sterile sites or in tracheal aspirates were classified as bacterial or fungal co-infection. pcr confirmation of influenza was performed either at the local laboratories of the participating picus using influenza-specific pcr, or (in the majority of cases) at the central laboratory at the institute of virology and immunobiology of the university of würzburg using multiplex pcr for respiratory viruses. for the latter, respiratory samples were placed in a viral transport medium (mast diagnostica gmbh, reinfeld, germany). at the central laboratory, they were tested using the commercial multiplex pcr 'ftd® respiratory pathogens 21' (fast track diagnostics, luxembourg) to screen for respiratory viruses (sensitivity and specificity of 99-100 % compared to singleplex pcr assays for all included viruses in clinical samples). pathogens detected by the test kit included influenza a and b viruses, respiratory syncytial virus (rsv), parainfluenza virus (piv) 1-4, coronavirus (cov) nl63, oc43, hku1, and 229e, human metapneumovirus (hmpv), human bocavirus (hbov), adenovirus (adv), rhinovirus (rhv), enterovirus (ev), parechovirus (pv), and additionally mykoplasma pneumoniae. samples positive for influenza a and b virus rna in the multiplex pcr were further analysed to determine the subtype and lineage, respectively. primers and probes specific for influenza a(h1n1)pdm09 were included in the 'ftd respiratory pathogens 21' kit. all samples positive for influenza a virus rna but negative for influenza a virus (h1n1)pdm09 rna were tested by a pcr specific for influenza a virus h3. all data was entered into a microsoft access database and transferred to ibm spss 21.0 for statistical analysis. data was analysed descriptively (percentages, or median with inter-quartile range, iqr). comparisons between groups were assessed for significance (p < 0.05, twosided) using pearson's chi 2 -test or fisher's exact test for categorical data, and the mann-whitney u-test for continuous data. the minimum incidence of influenza-associated picu admissions per 100,000 children <17 years of age was calculated for each season based on the observed number of influenza picu patients with a residential address in bavaria. to correct for non-participating picus, the estimates of the total number of picu influenza cases treated in all eligible picus in bavaria per season were derived taking into account the annual percentage of participating picus. the annual study population was used as denominator. a similar questionnaire and case definition had been used in previous studies on influenza-related picu admission [18, 19] . key variables were extracted from these publications for comparison purposes. data from streng et al. [18] and the present study were pooled for statistical comparison of pre-and post-pandemic seasons. the study was approved by the ethical committee of the medical faculty, university of würzburg, germany. based on the observed cases, the minimum incidence for pcr-confirmed influenza-associated picu admission per 100,000 children <17 years of age in bavaria was calculated as 1.15/0.36/1.03 for seasons 1/2/3, respectively. taking into account that the observed cases were based on data from 67 %/47 %/57 % of all eligible picus, the total number of influenza-associated picu admissions in bavaria was estimated and corrected incidences were calculated as 1.72/0.76/1.80 per 100,000 children <17 years. subtype-specific corrected incidences were 1. 35 (table 2) . after onset of ari symptoms, children were admitted to hospital after a median interval of 3.0 days; 83 % were transferred to the picu on the day of hospital admission or the following day (table 2) . two long-term hospitalized children (4.2 %) required picu treatment due to ari and were diagnosed with suspected nosocomial influenza a(h1n1)pdm09 infection. median length of picu stay was 3.0 days and median length of total hospital stay was 7.5 days (table 2) . underlying chronic medical conditions were reported for a total of 36 influenza picu patients (76.6 %) ( table 3) . chronic neurological diseases were most frequent (34.0 %), followed by chronic lung disease (25.5 %), preterm birth (21.3 %), cardiac malformations (17.0 %), obesity (10.6 %), genetic disorders (8.5 %), and immunocompromising conditions (8.5 %). of 36 influenza picu patients with underlying chronic conditions, four (11.1 %) were too young (<6 months of age) to have been immunized against influenza, and for two patients (5.6 %), data on their influenza vaccination status was unavailable. twenty-nine (80.6 %) patients from this risk group had not been vaccinated against influenza although they would have been eligible. one immunocompromised child (2.8 %) had been vaccinated in october 2012, but was diagnosed with a(h3n2) in january 2013. one or more specific influenza-associated diagnoses/complications were reported for 42 (89.4 %) of the 47 children ( table 4 ). the most frequent diagnosis was influenzaassociated pneumonia (61.7 %), followed by bronchitis/ bronchiolitis (31.9 %), and secondary bacterial pneumonia (25.5 %). ards was reported for 10 (21.3 %) and sepsis for six children (12.8 %); other complications were rare. thirty-nine of the 47 patients (83.0 %) underwent a chest radiograph. in addition to influenza, laboratory-confirmed co-infections were reported for 16 children (34.0 % out of 47 (1)). the majority of the 47 picu patients were treated intravenously with antibiotics (72.3 %), and with antipyretics (70.2 %) ( five children (10.6 %), infected with a(h1n1)pdm09, died at an age of 4 to 11 years; four were male patients ( table 6 ). four of these children suffered both from severe neurological conditions (two children with previous peripartal asphyxia and spastic tetraparesis; one child with cerebral paresis and tetraspasticity; one child with congenital cerebral disorder), and from chronic pulmonary conditions; two of these four children were also born pre-term. influenza-associated pneumonia was diagnosed in all four of these children; three additionally had secondary bacterial pneumonia, and one child also developed sepsis. for the fifth child, obesity was reported as the only risk factor; and sepsis and suspected encephalitis as complications. bacterial co-pathogens were detected in three of these five children and suspected in one child; two viral and two fungal co-infections were also reported. all five children received intratracheal ventilation, antibiotics and catecholamines; two were additionally treated with antiviral medication. death occurred 1, 2, 4, 19, and 26 days after picu admission, with ards reported as cause of death in three children. sequelae were reported for five patients (10.6 %): state after surgery due to pleural effusion/empyema in two children; increased oxygen requirements in two children who had previously already received oxygen therapy at home; damage of the lung after high-pressure ventilation in one child. table 2 ). figure 1 shows the difference in age distribution between both seasons, and the high proportion of children below 2 years of age as opposed to low proportions in all other age groups in season 3. after onset of symptoms, children were admitted to a picu after a significantly shorter period, with a median of 3 days (iqr 1-4) in season 1 compared to 6 days (iqr 2.0-7.5) in season 3 ( table 2 ). in season 1, significantly fewer children were diagnosed with bronchitis/bronchiolitis (table 4 ), and they tended to require cpap treatment less frequently than in season 3 (11.1 % vs. 41.7 %, p = 0.084, table 5 ). in the pre-pandemic period, median duration of picu stay was longer (19 days) , and children were more often diagnosed with encephalitis/encephalopathy (25 %) and co-infections (65 %) than in later periods ( table 7) . the proportion of children with influenza-associated pneumonia was highest (74 %) during the pandemic, whereas secondary bacterial pneumonia (17 %), bronchitis/ bronchiolitis (12 %) and sepsis (6 %) were reported less frequently during the pandemic than in the pre-and post-pandemic seasons. oseltamivir treatment decreased significantly in the post-pandemic period (table 7) . during the first three post-pandemic seasons 2010/11, 2011/12 and 2012/13, active screening of children with acute respiratory infection admitted to 24 paediatric intensive care units in bavaria identified a total of 51 pcr-confirmed influenza cases, resulting in annual incidence estimates of 1.7, 0.7, and 1.8 influenza-associated picu admissions per 100,000 children, respectively. these figures would, by extrapolation, correspond to a total number of 559 children with influenza-associated picu admission in germany within the 3-year post-pandemic period, with an annual average of 186 children. this is almost 28 times as high as the annual average of six to seven influenza-associated picu admissions detected by nation-wide picu surveillance in germany during three pre-pandemic years without active screening [18] . furthermore, the incidence estimates for the subtype a(h1n1)pdm09 derived from our active screening study were higher in the first and third post-pandemic seasons (1.35 and 1.17, respectively) than previous incidence estimates for picu patients in the pandemic (0.8-1.0) and the first post-pandemic (approximately 0.4) season in germany [19, 20] . thus, our results indicate possible underreporting in previous studies, and show a continued high level of a(h1n1)pdm09-associated picu admissions even 3 years after the pandemic. in our study, the proportions of children with bacteriaassociated complications (secondary bacterial pneumonia, sepsis) were similar to the proportions observed during the pre-pandemic period, but appeared higher than those observed during the pandemic 2009/10 [19] . the lower proportions observed during the pandemic might be explained by the time shift of the peak of influenza cases, which was observed as early as november 2009 in germany [19] . thus, the pandemic influenza peak did not coincide with the seasonal peak of streptococcus pneumoniae, the bacterial pathogen most frequently associated with community-acquired influenza [23] . antiviral treatment patterns changed considerably during the post-pandemic period, with a decrease in the proportion of paediatric influenza cases receiving oseltamivir from previously 50 % [18] and 61 % [19] to 19 %. oseltamivir is considered to be most advantageous when administered within the first 48 h of influenza disease. the reduced use in the post-pandemic period may therefore be partly due to the fact that median time between onset of influenza symptoms and picu admission was longer than during the pandemic (3 vs. 2 days [19] ). increasing uncertainty regarding the effectiveness of oseltamivir in the treatment of paediatric influenza may also have played a role [24, 25] . post-pandemic oseltamivir treatment was associated with co-infections and longer picu stay, suggesting that it were mainly children with severe complications or with serious underlying conditions who received this medication. in our study, about two-thirds of influenza cases and all fatalities were a(h1n1)pdm09-associated. during the postpandemic seasons 1/2/3, the proportion of a(h1n1)pdm09 cases among the picu patients was 79 %/14 %/65 % and, thus, considerably higher than the proportion of this subtype reported among outpatients by national influenza surveillance (65 %/1 %/34 %) [17] . this observation suggests that a(h1n1)pdm09 may be associated more often with a severe course of influenza requiring picu treatment than other influenza types/ subtypes. similar observations on the proportion of a(h1n1)pdm09-associated picu admissions have been reported in the united states [9] . comparison of picu patients with a(h1n1)pdm09 between the post-pandemic seasons showed that median age was 1.7 years in the third season and, thus, significantly lower than in the first season. a significant age shift towards younger children, from a median age of 5 to 3 years, had already been observed in a comparison of the pandemic and the first post-pandemic season in germany [20] . the continued shift towards younger patients in the third season is likely to be due to increasing immunity in the older paediatric population, after previous contact with a(h1n1)pdm09. seroprevalence data from germany had already shown evidence for a(h1n1)pdm09 infection in as many as 25 % of children aged 1-4 years and 48 % of 5-17 year-old children for the pandemic season 2009/10 [26] . a similar shift towards younger hospitalized children [27, 28] and towards younger children with severe paediatric a(h1n1)pdm09-associated influenza from the pandemic season to the first post-pandemic season had also been detected in other european countries [28] [29] [30] [31] . in germany, paediatric influenza vaccination for pandemic influenza a(h1n1)pdm09 was recommended and funded for all children as monovalent vaccination from october 2009 to july 2010 [32] . for seasonal influenza, however, paediatric influenza vaccination was and is currently recommended only for specific risk groups with underlying chronic conditions [33] . vaccination uptake was low, even in this target group. pre-pandemic vaccination rates were 5 % for all children and about 15 % for children with chronic underlying conditions in 2007/2008 [34] . for the pandemic and post-pandemic seasons, no data on vaccination rates is available for children, but vaccination rates as low as 14 % (2009/10) and 11 % (2010/11) were reported for adults, with a vaccination rate of only 17 % even for risk group adults [35] . in our study, more than 75 % of influenzaassociated picu patients were children with underlying chronic conditions. analysis of their reported influenza [20] ). data are given in %, by age group and season. season 1: oct10-sep11 (n = 18), season 3: oct12-sep13 (n = 13); season 2: oct11-sep12 (n = 1) is not shown vaccination status showed that among these were a high proportion of vaccine-eligible but unvaccinated children. patients with chronic conditions too young to be vaccinated and other paediatric risk groups, such as otherwise healthy children below 2 years of age, are not covered by the current german recommendation. all these groups could profit considerably from universal influenza vaccination for children, either directly or by herd protection. in contrast to the situation in germany, in the united states universal influenza vaccination for all children older than 6 months of age has been established, and vaccination coverage reached a level of approximately 41 % in 2013 [36] . compared to 348 influenza-associated paediatric deaths observed in the united states during the pandemic 2009/10, only 79 were observed in the strong a(h1n1)pdm09 season 2013/14 [37] . this might partly be explained by increasing immunity in children after previous a(h1n1)pdm09 infection, but may in part also be a result of the influenza vaccination program [37] . in england, a universal childhood vaccination programme with a new live attenuated influenza vaccine (laiv) with intra-nasal application was started in the 2013/14 influenza season [38] . first results showed an overall uptake of 53 % in primary school aged children, indicating a good acceptance of laiv, and suggesting direct and indirect impacts on disease incidence, including reduction of paediatric influenza-associated hospitalisations. to our knowledge, our study is the first in europe to investigate paediatric influenza in picu patients during the first three post-pandemic seasons after the 2009/10 pandemic. the strengths of our study include the multicentre design covering the majority of picus in bavaria, the active screening for influenza in patients admitted to picus, and pcr-confirmation of all influenza cases. a limitation is that the corrected incidence estimates were based on the assumption that participating and nonparticipating picus treated a similar number of severe paediatric influenza patients. although picus of both groups were of similar size, some of the non-participating picus, where paediatricians indicated lack of time as reason for non-participation, may have treated a higher number of patients, or patients with higher acuity. further limitations include potential over-and underreporting in participating picus. on the one hand, due to different hospitalization rules some children may have been admitted to picus mainly for the purpose of monitoring their course of influenza disease more closely, thus resulting in an overestimate of severe cases. on the other hand, some parents of children with severe influenza may have refused study participation, or children with a fulminant course of influenza disease may have died before they were admitted to a picu [4] . thus, the high incidence estimates derived in this study may still underestimate the true burden of severe influenza. [19] b key data from this study were pooled with data from streng et al. 2011 [18] and compared using fisher's exact test or mann-whitney u-test, respectively the incidence estimates of influenza a(h1n1)pdm09associated picu admissions, derived from active screening of picu patients with acute respiratory infections, reached similarly high levels in the first and third postpandemic seasons. both incidence estimates were higher than those previously reported by nation-wide picu surveillance for the pandemic and the first post-pandemic season, suggesting possible underreporting in previous studies without active screening. comparison of the first and third post-pandemic seasons indicated an age shift of severe a(h1n1)pdm09 towards younger children, which might be explained by increasing immunity in the older paediatric population. the large proportion of children with underlying chronic conditions indicates the need for a more consistent implementation of the current recommendations for influenza vaccination of specific risk groups in germany. these children could also profit from herd protection, if universal influenza vaccination was successfully introduced in germany. authors' contributions as designed the study, coordinated data collection, performed the analysis, interpreted the data, and drafted the manuscript. bw and cp performed multiplex pcr and subtyping on laboratory specimens, interpreted virological data, and revised the manuscript. jgl supervised the study, supported data interpretation, and revised the manuscript. the clinical data were collected by the bavarian picu study group on influenza and other viral ari, from oct 2010 to september 2013. all authors read and approved the final manuscript. group on influenza and other viral ari participants and their affiliations while participating during the study period städtisches klinikum münchen gmbh, klinikum harlaching städtisches klinikum münchen gmbh, klinikum schwabing christoph schmidtlein (kinderklinik dritter orden tobias trips (kliniken südostbayern ag missionsärztliche klinik ggmbh, kinderklinik; würzburg) references 1. heikkinen t. influenza in children the underrecognized burden of influenza in young children risk factors associated with severe influenza infections in childhood: implication for vaccine strategy influenzaassociated pediatric deaths in the united states identification of children at risk of influenza-related complications in primary and ambulatory care: a systematic review and meta-analysis the burden of seasonal and pandemic influenza in infants and children the burden of influenza illness in children with asthma and other chronic medical conditions implications for immunization recommendations patients hospitalized with laboratory-confirmed influenza during the 2010-2011 influenza season: exploring disease severity by virus type and subtype the burden of influenza b: a structured literature review hospitalized children with 2009 pandemic influenza a (h1n1): comparison to seasonal influenza and risk factors for admission to the icu influenza a (ph1n1) infection in children admitted to a pediatric intensive care unit: differences with other respiratory viruses clinical characteristics of pediatric hospitalizations associated with influenza a (h1n1) in northern bavaria the role of infections and coinfections with newly identified and emerging respiratory viruses in children comparative analysis of clinical characteristics of pandemic influenza a/h1n1 and seasonal influenza a infections in hospitalized children forward look risk assessment -likely scenarios and uncertainties in the 2010/2011 influenza season in europe and beyond bericht zur epidemiologie der influenza in deutschland saison 2012/13 severe influenza cases in paediatric intensive care units in germany during the pre-pandemic seasons severe cases of pandemic (h1n1)pdm2009 in children unchanged severity of influenza a(h1n1)pdm09 infection in children during first postpandemic season bavarian state office for statistics and data processing bacterial and viral infections associated with influenza. influenza other respir viruses neuraminidase inhibitors for preventing and treating influenza in healthy adults and children effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h1n1pdm09 virus infection: a meta-analysis of individual participant data post-pandemic seroprevalence of pandemic influenza a (h1n1)pdm2009 infection (swine flu) among children <18 years in germany clinical features of influenza disease in admitted children during the first postpandemic season and risk factors for hospitalization: a multicentre spanish experience influenza in hospitalized children in ireland in the pandemic period and the 2010/2011 season: risk factors for paediatric intensive-care-unit admission critical care surveillance: insights into the impact of the 2010/11 influenza season relative to the 2009/10 pandemic season in england burden and characteristics of influenza a and b in danish intensive care units during the 2009/10 and 2010/11 influenza seasons first influenza season after the 2009 pandemic influenza: characteristics of intensive care unit admissions in adults and children in vall d'hebron hospital änderung der empfehlung zur impfung gegen influenza analyse regionaler unterschiede der influenza-impfraten in der impfsaison influenza a(h1n1)pdm09 antibodies after pandemic and trivalent seasonal influenza vaccination as well as natural infection in update: influenza activity -united states influenza activity -united states, 2013-14 season and composition of the 2014-15 influenza vaccines uptake and impact of a new live attenuated influenza vaccine programme in england: early results of a pilot in primary school-age children, 2013/14 influenza season the authors thank all participating hospitals, and picu and university staff involved in data collection and virological testing. karin seeger we thank for helpful comments on the manuscript. the study was supported by an unrestricted grant from glaxosmithkline gmbh & co. kg, munich, germany. apart from financial support, the company was not involved in any part of the study. the publication was funded by the german research foundation (dfg) and the university of wuerzburg in the funding programme open access publishing. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-333413-8buawes0 authors: liebing, j.; völker, i.; curland, n.; wohlsein, p.; baumgärtner, w.; braune, s.; runge, m.; moss, a.; rautenschlein, s.; jung, a.; ryll, m.; raue, k.; strube, c.; schulz, j.; heffels-redmann, u.; fischer, l.; gethöffer, f.; voigt, u.; lierz, m.; siebert, u. title: health status of free-ranging ring-necked pheasant chicks (phasianus colchicus) in north-western germany date: 2020-06-16 journal: plos one doi: 10.1371/journal.pone.0234044 sha: doc_id: 333413 cord_uid: 8buawes0 being a typical ground-breeding bird of the agricultural landscape in germany, the pheasant has experienced a strong and persistent population decline with a hitherto unexplained cause. contributing factors to the ongoing negative trend, such as the effects of pesticides, diseases, predation, increase in traffic and reduced fallow periods, are currently being controversially discussed. in the present study, 62 free-ranging pheasant chicks were caught within a two-year period in three federal states of germany; lower saxony, north rhine-westphalia and schleswig-holstein. the pheasant chicks were divided into three age groups to detect differences in their development and physical constitution. in addition, pathomorphological, parasitological, virological, bacteriological and toxicological investigations were performed. the younger chicks were emaciated, while the older chicks were of moderate to good nutritional status. however, the latter age group was limited to a maximum of three chicks per hen, while the youngest age class comprised up to ten chicks. the majority of chicks suffered from dermatitis of the periocular and caudal region of the head (57–94%) of unknown origin. in addition, intestinal enteritis (100%), pneumonia (26%), hepatitis (24%), perineuritis (6%), tracheitis (24%), muscle degeneration (1%) and myositis (1%) were found. in 78% of the cases, various mycoplasma spp. were isolated. mycoplasma gallisepticum (mg) was not detected using an mg-specific pcr. parasitic infections included philopteridae (55%), coccidia (48%), heterakis/ascaridia spp. (8%) and syngamus trachea (13%). a total of 8% of the chicks were avian metapneumovirus (ampv) positive using rt-pcr, 16% positive for infectious bronchitis virus (ibv) using rt-pcr, and 2% positive for haemorrhagic enteritis virus (hev) using pcr. all samples tested for avian encephalomyelitis virus (aev), infectious bursal disease virus (ibdv) or infectious laryngotracheitis virus (iltv) were negative. the pool samples of the ten chicks were negative for all acid, alkaline-free and derivative substances, while two out of three samples tested were positive for the herbicide glyphosate. pheasant chick deaths may often have been triggered by poor nutritional status, probably in association with inflammatory changes in various tissues and organs as well as bacterial and parasitic pathogens. theses impacts may have played a major role in the decline in pheasant populations. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the original distribution area of the ring-necked pheasant (phasianus colchicus) ranged from the black sea over the dry areas of central asia to the east of asia to south korea and siberia [1] . the romans introduced the pheasant to europe around 500 ad, from where it spread through regular release throughout central and western europe [2] . according to current published data, the pheasant mainly prefers structurally semi-open land, using trees and hedges as cover, and also occupies adjacent sparse forests and reedy areas [3] . most pheasants seek shelter under trees to be protected from natural predators. however, some subspecies spend the night on the ground or among dense reeds. their resting places during the day are usually well-hidden hedges, where sand-baths are taken in carved hollows [1] . adult pheasants mainly feed on plants, consuming different parts of the plant such as seeds, berries, tubers, root shoots and leaves, as well as green sprouts. however, on occasions, their diet is supplemented by animal protein, preferably in the form of insects [1] . for chicks, smaller ground-level insects are especially important during the first weeks of life. they feed on a variety of species of insects such as spur cicadas (delphacidae), bugs (heteroptera), sawfly wasps (tenthredinidae) and butterfly caterpillars (lepidoptera larvae) [1, 4, 5] . this diversity is particularly important for a healthy growth [6, 7] . for example, a diet based only on aphids can lead to delayed plumage development due to inadequate amino acid supply [8] . in germany, the pheasant is a typical soil-breeding bird of the agricultural landscape. the main part of the german population is found in southwest lower saxony, north rhine-westphalia and schleswig-holstein. the population level reached its plateau between 1960 and 1970 in lower saxony. during this period, the hunting bag statistics (state registered numbers of hunting animals, in this case pheasants), i.e. the absolute number of pheasants killed, amounted to approximately 300,000 pheasants in germany [9] . in the severe winter of 1970 and the following wet spring of 1971, the population of pheasants and many other wild living animals declined [9, 10] . the hunting bag was reduced to an average of about 80,000 pheasants and declined further. not only was the pheasant population subjected to this decline, but also that of many other farmland birds [11, 12, 13] . around 2007/2008, the population showed another severe decline of unknown cause. in germany, the renewable energy sources act (erneuerbare-energie-gesetz: renewable energy sources act describes the implementation of ecological energy generation in germany) amendment of 2004 with an advancement in biogas, triggered the doubling of corn cultivation. consequently, huge areas of fallow land disappeared in lower saxony [14] . the contributory factors to the ongoing decline in the pheasant population, such as the effects of pesticides, infectious agents, predation, increasing traffic and human populations as well as reduced fallow periods, are currently the subject of controversial discussion among different stakeholders [15, 16, 17, 18] . some authors see a correlation between the changes in agriculture and the decline in the populations of many farmland birds [19, 20] . in the third week of life, 70% of the chicks' diet consists of insects. gradually, the insect percentage is reduced. from the sixth week of life, the diet is similar to that of adult birds. previous studies [21, 22] associated the decline in the number of many farmland birds with the use of insecticides. if chicks are unable to find a sufficient number of insects during the first weeks of life, they have to search a larger range of their habitat, which can lead to malnutrition and weakening. thus, harmless ubiquitous pathogens may have negative effects on chicks [23, 24, 25, 26, 27, 28] . investigations carried out led to the assumption that there is no specific epidemic infectious agent currently circulating in the adult pheasant population [29] . many hunters report that especially the number of chicks has declined, with more older birds making up the hunting bag. however, the authors found serological evidence of certain viruses (infectious bronchitis virus (ibv), avian encephalomyelitis virus (aev) and infectious bursal disease virus (ibdv)) which typically cause chick mortality. these pathogens infected adult and young pheasants, but the pathogenicity in chick and subadult populations is considerably more serious than in adult birds [29, 30] . in addition, other factors may weaken the population and pathogens become more important. based on these findings, our study focused on pheasant chicks up to eleven weeks of age. previous studies on pheasants indicated that the most sensitive age class for infectious diseases was pheasant chicks, possibly due to a higher susceptibility [30, 31] . the aim of our research was to assess the health state of free-living pheasant chicks in order to check the animals for lesions indicative of infections or toxic substances. the findings should contribute to understanding the causes for the decline in the pheasant population in north-western germany. in 2014 and 2015, the institute for terrestrial and aquatic wildlife research (itaw), university of veterinary medicine hannover, foundation, hannover and the wildlife research institute, state office for nature, environment and consumer protection of north rhine-westphalia caught free-living ring-necked pheasant chicks from lower saxony (cuxhaven, grafschaft bentheim, emsland, osnabrück, vechta), north rhine-westphalia (coesfeld, warendorf) and schleswig-holstein (dithmarschen) to assess the health state by means of pathological, microbiological, virological, parasitological and toxicological investigations. the caught chicks were grouped into three age classes (ac) based on the feather markings of the hand-wings. age class one (ac1) included chicks up to three weeks of age, ac2, chicks from four to six weeks of age and ac3, chicks older than six weeks and up to 11 weeks. an animal experiment permit was obtained from the responsible veterinary office of the lower saxony state office for consumer protection and food safety (laves) (permit number: 33.14-42502-04-14/1486). the study areas comprised 11 hunting regions with 15 traps in lower saxony (hemmoor, meppen, neuenkirchen, osten, strücklingen, vechta, wilsum,)11 regions with 10 traps in north rhine-westphalia (ahlen, dülmen, lippstadt, welte) and 4 districts with 4 traps in schleswig-holstein (warwerort) (fig 1) . the catching period lasted from may until august. in 2014, the investigated chicks were three to 11 weeks old. in 2015, the age of the chicks varied from one-day-old to eleven-week-old chicks. at the age of 11 weeks, the young pheasants were considered as sexually mature. after the catch, the mother hen was released and at maximum, half of the chicks in the trap were taken for analysis (mostly onethree chicks at random). in 2014, the traps had a size of 2.3 m 2 and were covered with iron bars with a mesh-size of 1 cm 2 . in 2015, the traps were slightly adapted based on experience from 2014, using a cover made of loose polyethylene netting with a mesh-size of 1 cm 2 . a piece of string was used as a trigger so that both trap doors closed when the chicks moved forward. corn was used to attract the hen and her chicks. afterwards, the chicks were transported alive to the university of veterinary medicine hannover for examination. the time span from catch to examination took on average about five hours. in 2014, the chicks were stunned by a head blow and killed by exsanguination. in 2015, the chicks were euthanised with an intravenous injection of pentobarbital-sodium (boehringer-ingelheim, ag & co. kg, ingelheim, germany). the nutritional condition score was evaluated macroscopically by the thickness of the pectoral muscles and the body fat percentages as good, moderate, poor or cachectic. as described by curland et al. [29] , animals in a good body condition revealed a vast amount of fatty tissue within the thoracic and abdominal regions, whereas animals with a moderate body condition demonstrated reduced amounts of body fat tissue. animals in a poor body condition possessed only low amounts of fat reserves, these frequently associated with pectoral muscle atrophy. in contrast, cachectic animals lacked fat reserves and exhibited serous atrophy of the coronal myocardial fatty tissue. the necropsy was carried out in accordance with the standard protocol [31] . representative samples of the following tissues and organs were collected, fixed in 10% neutral-buffered formalin and routinely embedded in paraffin wax: the skin of the head and abdomen, skeletal muscle (musculus pectoralis, musculus quadriceps), ischiadic nerve, brachial plexus, nose with infraorbital sinus, eye with lacrimal gland, bone with bone marrow, trachea, thymus, thyroidal gland, lung, heart, liver, pancreas, spleen, kidney, crop, proventriculus, gizzard, intestine, adrenal gland, gonads, bursa of fabricius and brain. paraffin sections of 3-5 μm were stained with haematoxylin and eosin (he) for histological examination. in selected cases, periodic acid schiff (pas) reaction, ziehl-neelsen stain, brown-brenn stain and turnbull's blue stain were performed [32] . for parasitological examinations, samples of the small intestine were collected from all 62 chicks at necropsy. at the same time, skin and plumage of the chicks were macroscopically examined for ectoparasites. for coproscopical examination, the combined sedimentation-flotation method was performed: the faecal sample was filled into a tea strainer (mesh size 1 mm) and rinsed in a beaker with a jet of water. the filtrate containing helminth eggs and protozoan oocysts was allowed to sediment for 30 min. afterwards, the supernatant was decanted and the sediment transferred to a 15-ml centrifuge tube filled with saturated zinc sulphate solution (znso 4 , specific gravity 1.30) and centrifuged at 450 x g for 5 min. the liquid surface was transferred onto a slide with a wire eyelet and examined microscopically. if at least one egg or oocyst was detected, the sample was classified as positive. a semiquantitative classification was applied using the following key: one-two eggs or oocysts were categorised as mild, six-ten eggs or oocysts as moderate, 11-20 eggs or oocysts as severe; if more than 20 eggs or oocysts were detected, the shedding intensity was classified as by mass [29] . the fresh samples, consisting of brain, trachea and caecal tonsils, as well as the bursa of fabricius were placed in rnalater1 (sigma-aldrich chemie gmbh, münchen, germany). samples were analysed by rt-pcr for avian metapneumovirus (ampv), infectious bronchitis virus (ibv), avian encephalomyelitis virus (aev) and by pcr for infectious bursal disease (ibdv) and infectious laryngotracheitis as described in [33, 34] . serum was taken from all birds to check for antibodies against avian influenza virus (aiv) subtypes h5, h7 and h9. eight additional liver samples from chicks with hepatitis were analysed for the presence of haemorrhagic enteritis virus (hev) by pcr [29] . for microbiological investigations for mycoplasma, 13 tracheal swabs, six tracheal tissue samples and three periorbital skin tissue samples were taken [29] . the samples were directly transferred to mycoplasma cultivation medium (sp4). detection of mycoplasma by pcr. for dna extraction, swabs were soaked and rubbed in 350 μl phosphate buffered saline (pbs). using the dneasy 1 blood & tissue kit (qiagen gmbh, hilden, germany) in accordance with the manufacturer's instructions, 100 μl of the liquid was taken for dna extraction. for dna extraction of tissue samples and the single colony subcultures, the fluid medium from culturing (2 ml) was centrifuged at 4000 x g for 45 minutes. the remaining pellet was incubated with 180 μl lysis buffer (atl buffer, qiagen, gmbh) and 20 μl proteinase k (qiagen gmbh) for two hours at 56˚c. all samples and single colony subcultures were screened via mycoplasma-genus-specific pcr (target: 16s rrna gene sequence) for dna of mycoplasma spp. as described by [35] and modified [36] . from all single colony subcultures, an additional pcr (target: 16s-23s rrna sequence (intergenetic transcribed spacer region)) was performed [37] . furthermore, all samples were examined via mycoplasma gallisepticum-specific pcr [38] . the pcr products were sequenced by a commercial dna sequencing service (lgc genomics gmbh, berlin, germany). the sequences of the pcr products were aligned with the 16s rrna gene and 16s-23s rrna isr sequences of mycoplasma spp. in the ncbi database using blast (ncbi, bethesda, md, usa) algorithm [39] . mycoplasma culture. the samples were cultured using sp4 liquid and agar media produced in house as described previously [34] . each sample was immersed in the sp4 broth and afterwards removed and stored for further investigations. the broth was diluted (ten-fold dilution up to 10 −2 ) and an aliquot of 50 μl each was transferred onto agar media. both, liquid and solid media were incubated at 37˚c with 5% co 2 in a humidified environment for up to ten days. broth was examined for colour change and agar plates for colony growth daily. in case of colour change, or after five days, an additional "subculture" on agar media was performed. in case of mycoplasma growth, several single colony subcultures were performed at least twice in order to ensure pure species cultures. each third single colony subculture was stored at -80˚c until further investigation by molecular biological methods [36, 40] . liver samples of nine pheasants were screened for herbicide glyphosate and other pollutants (for details see s1 table) . of these nine samples, one sample was taken from a ten-chick ratchet in ac1, while the remaining eight samples were single-samples from ac3 with liver or kidney inflammation. the samples were stored directly after autopsy at -80˚c. toxicological samples (n = 10), 7 g liver pool samples, were used to detect substances by means of the gas chromatography-mass spectrometry ( during the two-year-period, a total of 62 chicks were caught: 29 birds in lower saxony, 27 in north rhine-westfalia and six in schleswig-holstein. fourteen chicks were allocated to ac1, 16 to ac2 and 32 to ac3. of the investigated animals, 34 were female, 17 male and for 11 chicks, macroscopic gender estimation was unknown; histological samples were not taken. the nutritional status in ac1 was predominantly poor (n = 12; 85.7%) or cachectic (n = 2, 14.3%). in ac2, approximately nine (56.3%) of the chicks were well fed and seven (43.8%) were moderately fed. the majority of birds in ac3 were well fed (n = 25, 78.1%), some were moderately fed (n = 6, 18.8%) and one chick (3.1%) was in a poor body condition (table 1) . to an excessive amount, mild to severe cutaneous abrasions with feather loss, lacerations and/ or subcutaneous haemorrhages of the head were noticed in one out of two chicks (50%) in ac1, nine out of 16 chicks (56%) in ac2 and 11 out of 20 chicks (55%) in ac3 trapped with the tt1. one out of 12 animals (8%) in ac1 and nine out of 12 individuals (75%) in ac3 that had been trapped with tt2 were more mildly affected by those lesions. histological examination of the skin from the head revealed various types of inflammatory alterations which occurred solely or concurrently in one individual (table 2 ). in all age classes and independent of trap type, mainly perivascular predominantly lympho-histiocytic dermatitis admixed with occasional heterophils and plasma cells of varying degrees was present (fig 2) . this type of inflammation was in some cases accompanied by follicular aggregations of lymphocytes sometimes with secondary follicle formation (ac3, tt2). in addition, ulcerative (fig 3) , occasionally necrotising, suppurative and pustular inflammatory changes were found more often in chicks trapped with tt1 than with tt2, these in many cases being associated with dermal and/or subcutaneous haemorrhages of varying degrees. only a few animals showed no cutaneous alteration in this localisation. the abdominal skin of the chicks was rarely affected by perivascular dermatitis; single individuals were mainly affected by lymphohistiocytic or pustular dermatitis. crops, glandular stomachs and gizzards were variably filled (table 3 ). however, it should be noted that the chicks had spent up to five hours in the traps. the mentioned parts of the digestive tract contained grains, green food, and, inside the gizzard, grit stones. in one chick (7%) in ac1, in seven chicks (44%) in ac2 and in ten chicks (31%) in ac3, the quality of the intestinal content was associated with hyperaemic intestinal mucosa and perianal attachment of faeces suggestive of catarrhal enteritis. histologically, one animal (3%) in ac3 showed focally severe ulcerative stomatitis at the gums and one chick in ac3 focally moderate lympho-histiocytic ingluvitis. in single chicks in ac3, nematodes without reactive inflammatory changes were found in the crop. furthermore, single individuals showed erosive and heterophilic inflammation of the gizzard, occasionally associated with intralesional nematodes which were not differentiated here. the intestinal mucosa in all animals showed a mild to moderate infiltration with eosinophils, lymphocytes and a few plasma cells. in eight out of 16 chicks (50%) in ac2 and in four out of 32 chicks (13%) in ac3, reproduction stages of protozoal organisms, most likely coccidia sp., were found histologically within the epithelium (fig 4) . predominantly mild focal lymphohistiocytic hepatitis was observed in one out of 14 chicks (7%) in ac1, in four out of 16 chicks (25%) in ac2, and in ten out of 32 chicks (31%) in ac3. single individuals in ac3 showed multifocal granulomatous hepatitis with severe acute coagulation necrosis (fig 5) . in eight out of 14 chicks (57%) in ac1, a mild to severe diffuse fatty change in hepatocytes was present. nematodes with the morphology consistent with syngamus trachea were found in the trachea of none of the 14 chicks in ac 1, in five out of 16 chicks (31%) in ac2 and in ten out of 32 (31%) chicks in ac3. histologically, tracheal parasitism in most animals was associated with multifocal lympho-histiocytic, occasionally granulomatous or ulcerative tracheitis of variable extent. in single animals, subepithelial lymphoid follicles were found. in the lung, focal or multifocal interstitial, mild to moderate lymphohistiocytic pneumonia was observed in one out of 14 chicks (7%) in ac1, in three out of 16 chicks (19%) in ac2, and in six out of 32 chicks (19%) in ac3. focal or multifocal, mild to moderate granulomatous, occasionally necrotising pneumonia was present in three individuals (19%) in ac2 and in two individuals (6%) in ac3. one animal in ac3 suffered from severe suppurative to necrotising pneumonia. there was no evidence of viral, bacterial, fungal or parasitic agents in these lungs, even in the histological special stains. hyperplasia of bronchus-associated lymphoid tissue was noticed in two chicks (13%) in ac2 and in three chicks (9%) in ac3. numerous lungs showed acute haemorrhages. the kidneys displayed focally mild interstitial infiltrations consisting mainly of lymphocytes and macrophages in two chicks (13%) in ac2 and five chicks (16%) in ac3. independent of the age class, a mostly moderate diffuse infiltration with plasma cells was observed in almost all examined lacrimal glands. miscellaneous findings included focally mild lymphocytic myocarditis in one chick (3%) in ac3 (fig 6) , focally moderate lymphohistiocytic perineuritis (n. ischiadicus) in one chick in both ac2 (19%) and ac3 (3%), severe subacute hyaline degeneration of skeletal muscles with histiocytic infiltration in one chick (3%) in ac3, focal chronic suppurative myositis in another chick (3%) in ac3, and single protozoal cysts, most likely sarcosporidia sp., in the skeletal musculature of two individuals (6%) in ac3 without inflammatory changes. in many brains, perivascular and parenchymatous haemorrhages were observed without reactive changes. of all 62 chicks tested, 8% of the chicks were positive for avian metapneumovirus (ampv) using rt-pcr, 16% positive for infectious bronchitis virus (ibv) using rt-pcr, and 2% for haemorrhagic enteritis virus (hev) using pcr. none of the 37 chicks tested for hev were positive using pcr. tracheae of 33 chicks and caecal tonsils of ten birds were tested by the coronavirus-rt-pcr and were negative for the respective virus. all samples tested for avian encephalomyelitis virus (aev), infectious bursal disease virus (ibdv), or infectious laryngotracheitis virus (iltv) were negative. the pool samples of the ten chicks were completely negative for all acid, alkaline-free and derivative substances as listed in s1 table which summarises the substances tested and the detection limits. two out of three samples tested for the herbicide glyphosate were positive (0.044 mg/kg, 0.095 mg/kg). since the 1970s, a population decrease in ring-necked pheasants has been observed. especially in 2007/2008, the population decline intensified [41] . the present investigation revealed that the randomly trapped pheasant chicks displayed inflammatory lesions in different organs. in association with environmental stressors and a depleted nutritional status, these health changes may increase pheasant chick mortality, thus contributing to the population decrease. the dermatitis detected was often of a non-purulent character, mostly perivascularly accentuated with different cellular compositions of gradual variable infiltrations by lymphocytes, plasma cells and macrophages. especially on the head, this alteration additionally displayed pustular and lymphocytic inflammation. it was possibly itch-or parasite-induced. avian pox was excluded due to lack of pathognomonic and histological changes [42] . these alterations occurred in chicks as young as one or two days of age. six out of 12 chicks (50%) in ac1 already showed these alterations, with different types and degrees of inflammation. m. gallisepticum (mg) is an important etiological differential diagnosis, especially inducing periocular dermal swelling with lymphocytic inflammation [43] . however, this pathogen was ruled out by the investigations. nevertheless, various mycoplasma spp. were isolated in 15 out of 21 (71.4%) of the investigated chicks. however, the role of these mycoplasma spp. as a potential cause of periorbital skin alterations in pheasants is still unclear, but should be considered in following investigations in pheasants. as some birds were rt-pcr positive for ibv or ampv, it has to be elucidated further whether these viruses may have contributed to these lesions, as they are known to be respiratory disease associated. the inflammations might be itch induced following insect or tick bites. furthermore, the head injuries with lacerations and haemorrhages resulted from catching caused by the chicks jumping against the iron bars of the traps. these injuries did not appear anymore after exchanging these bars for loose nylon mesh. a total of 65% of the 26 pneumonia cases were of an eosinophilic character and were most likely caused by syngamus trachea in ten cases. all cases of granulomatous inflammation were free of acid-fast bacteria as shown by the ziehl-neelsen stain. therefore, the cause of this granulomatous inflammation remains unknown. other possible agents able to induce pneumonia, bronchopneumonia, tracheitis and bronchitis were not detected. a prevalent eosinophilia tracheitis (94%) occurred in almost all cases in connection with detected parasites at different stages including coccidia, heterakis/ascaridia spp. and syngamus trachea. the degrees of inflammation were mainly mild up to moderate so that the clinical relevance is rather subordinate. the proventriculitis can have many origins. a histologically similar disease, that of gizzard erosion in broilers is often caused by an interaction between vitamin deficiency, fungal infections and stress situations after consuming mycotoxins. with periodic acid schiff reaction (pas) and brown-brenn stain, fungi, gram-positive and gram-negative bacteria were excluded [44] . as ampv, ibdv, coronavirus and siadenovirus were excluded by pcr, a viral cause is relatively unlikely. marek's disease is doubtful as well due to the lack of other typical organ changes [45] . it is possible that the birds may have been exposed to mycotoxins or pesticides that caused proventriculitis. based on localisation, size and shape of the eggs found in the proventriculus, a nematode-infection with dyspharynx nasuta probably resulted [46] . the inflammation of the livers showed lymphocytic and lymphohistiocytic characters. the causes for these inflammatory changes are manifold and may include infectious as well as noninfectious agents. in three cases, the inflammation was granulomatous and necrotising. using ziehl-neelsen stain, acid-resistant bacteria were excluded. differential diagnoses for granulomatous and necrotising hepatitis include toxic, ischemic or infectious causes [47, 48] . in the presented investigations, only a limited number of samples could be investigated for pesticides. therefore, it is difficult to directly link pathological findings to any of the investigated chemicals. further investigations are needed to elucidate the role played by pesticides in the declining pheasant populations as their habitat is regularly exposed to different chemicals used in agriculture. the main findings in the study were the poor nutritional status in the younger age groups and the increasing occurrence of various inflammation when the birds were ageing. as no direct cause for the inflammation was found and the inflammation affected various organs, it might be more a sign of various pathogens affecting the chicks. this seems to be more a sign of a weakened immune system, unable to defeat facultative pathogenic organisms. this is in line with the poor nutrition status, which triggers the development of diseases. no suspected virus infection was detected though. virus infections cannot be ruled out completely as a cause as viruses obviously circulate in the adult pheasant population and infected chicks die quickly. therefore, such cases were not among the sampled animals as the study focused on live chicks which still followed the hen. due to predation, decomposition and vegetation in the field, diseased pheasants are difficult to retrieve for health examinations and therefore were not included here. concerning parasites, low coccidian infections can be regarded as desirable to build up a protective immunity against reinfections. however, intestinal changes of the chicks show that coccidia sometimes considerably damage the intestinal mucosa due to severe infections, which may lead to a reduction in nutrient uptake. furthermore, a severe syngamus sp. infection can occlude the tracheal lumen, resulting in suffocation of the chicks, or their general condition deteriorates to such an extent that they become easy prey for predators. all these findings point to an effective complexity that either chicks die of starvation or their immune system becomes weakened. it seems that when the effects of maternal antibodies slowly diminish and the chicks have to mobilise their own immune system, the chicks become weakened as their immune system is not sufficiently developed. also, it is known from poultry that the development of the immune system is influenced by nourishment and that malnutrition can negatively influence the immune system [31, 49, 50] . this hypothesis has not been confirmed yet in pheasants. not only pheasants, but also many other farmland birds have to cope with the intensive agricultural landscape. this change in habitat and the use of pesticides make it increasingly difficult to find insects that are vital for the chicks during their first weeks of life. a whole concatenation of circumstances could be explained by this connection; namely, the rather poor nutritional status, a possibly weakened immune system and the increased susceptibility to diseases. additionally, it is possible that the chicks are easier prey for predators due to various inflammations or poor physical condition, too. also, the weather can have a greater influence. supporting information s1 table. toxicological investigation of substances and limits of detection. highlighted in bold are the substances found in the pheasant samples. (docx) s1 data. (pdf) data curation: j. liebing. formal analysis handbuch der vö gel mitteleuropas edition parasitoses of pheasants (phasianus colchicus) in confined system the pheasant: ecology, management and conservation phasianus colchicus) seine naturgeschichte, aufzucht und hege effect of protein levels in the diet on the growth of pheasants early nutrition causes persistent effects on pheasant morphology importance of insect prey quality for grey partridge chicks perdix perdix: a self-selection experiment analyse der rückgangsursachen der fasanenbesätze in niedersachsen. (unpublished) jägerstiftung natur+mensch. institute for terrestrial and aquatic wildlife research retrospektive zum rückgang des fasans agricultural intensification and the collapse of europe's farmland bird populations the second silent spring? farming and birds in europe: the common agricultural policy and its implications for bird conservation eeg stellt kulturlandschaft auf den kopf effects of cropping practices on declining farmland birds during the breeding season responses of plants and invertebrate trophic groups to contrasting herbicide regimes in the farm scale evaluations of genetically modified herbicide-tolerant crops weeds in fields with contrasting conventional and genetically modified herbicide-tolerant crops. i. effects on abundance and diversity influence of autumn applied herbicides on summer and autumn food available to birds in winter wheat fields in southern england the swiss agri-environment scheme promotes farmland birds: but only moderately birds as useful indicators of high nature value farmlands: using species distribution models as a tool for monitoring the health of agro-ecosystems declines in insectivorous birds are associated with high neonicotinoid concentrations host mortality, predation and the evolution of parasite virulence parasites and the dynamics of wild mammal populations differential body condition and vulnerability to predation in snowshoe hares prevalence, intensity and aggregation of intestinal parasites in mountain hares and their potential impact on population dynamics. international host manipulation by parasites in the world of dead-end predators: adaptation to enhance transmission? interactions between sources of mortality and the evolution of parasite virulence investigation into diseases in free-ranging ring-necked pheasants (phasianus colchicus) in northwestern germany during population decline with special reference to infectious pathogens effects of early feeding and dietary interventions on development of lymphoid organs and immune competence in neonatal chickens: a review romeis mikroskopische technik kompendium der geflü gelkrankheiten modified live infectious bursal disease virus (ibdv) vaccine delays infection of neonatal broiler chickens with variant ibdv compared to turkey herpesvirus (hvt)-ibdv vectored vaccine experimental haematobiochemical alterations in broiler chickens fed with t-2 toxin and co-infected with ibv prevalence of mycoplasmas in eggs from birds of prey using culture and a genus-specific mycoplasma polymerase chain reaction genus-and species-specific identification of mycoplasmas by 16s rrna amplification high inter-species and low intra-species variation in 16s-23s rdna spacer sequences of pathogenic avian mycoplasmas offers potential use as a diagnostic tool das vorkommen von mykoplasmen bei storchnestlingen in brandenburg und sachsen-anhalt basic local alignment search tool recovery of mycoplasmas from birds wild und jagd-landesjagdbericht 2010/2011. ni ministerium für ernä hrung, landwirtschaft, verbraucherschutz und landesentwicklung a retrospective studie of skin lesions in wild turkeys (meleagris gallopavo) in the eastern usa, 1975-2013 mycoplasma gallisepticum in pheasants and the efficacy of tylvalosin to treat the disease hard af segerstad c. mycotic proventriculitis in gray partridges (perdix perdix) on two game bird farms causes of gizzard erosion and proventriculitis in broilers first report of five nematode species in phasianus colchicus linnaeus (aves, galliformes, phasianidae) in brazil / primeiro registro de cinco espécies de nematóides em phasianus colchicus linnaeus (aves, galliformes, phasianidae) no brasil necrotizing hepatitis in a domestic pigeon (columba livia) hepato nephropathology associated with inclusion body hepatitis complicated with citrinin mycotoxicosis in a broiler farm early nutrition programming (in ovo and posthatch feeding) as a strategy to modulate gut health of poultry nutrition-mechansims of immunosuppression we wish to thank the hunting associations of lower saxony, north rhine-westphalia and schleswig-holstein for supporting the study. furthermore, special thanks go to the laboratory personnel for their excellent technical assistance in the laboratory investigations. key: cord-279557-hk77e3pp authors: drosten, christian; seilmaier, michael; corman, victor m; hartmann, wulf; scheible, gregor; sack, stefan; guggemos, wolfgang; kallies, rene; muth, doreen; junglen, sandra; müller, marcel a; haas, walter; guberina, hana; röhnisch, tim; schmid-wendtner, monika; aldabbagh, souhaib; dittmer, ulf; gold, hermann; graf, petra; bonin, frank; rambaut, andrew; wendtner, clemens-martin title: clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection date: 2013-06-17 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70154-3 sha: doc_id: 279557 cord_uid: hk77e3pp background: the middle east respiratory syndrome coronavirus (mers-cov) is an emerging virus involved in cases and case clusters of severe acute respiratory infection in the arabian peninsula, tunisia, morocco, france, italy, germany, and the uk. we provide a full description of a fatal case of mers-cov infection and associated phylogenetic analyses. methods: we report data for a patient who was admitted to the klinikum schwabing (munich, germany) for severe acute respiratory infection. we did diagnostic rt-pcr and indirect immunofluorescence. from time of diagnosis, respiratory, faecal, and urine samples were obtained for virus quantification. we constructed a maximum likelihood tree of the five available complete mers-cov genomes. findings: a 73-year-old man from abu dhabi, united arab emirates, was transferred to klinikum schwabing on march 19, 2013, on day 11 of illness. he had been diagnosed with multiple myeloma in 2008, and had received several lines of treatment. the patient died on day 18, due to septic shock. mers-cov was detected in two samples of bronchoalveolar fluid. viral loads were highest in samples from the lower respiratory tract (up to 1·2 × 10(6) copies per ml). maximum virus concentration in urine samples was 2691 rna copies per ml on day 13; the virus was not present in the urine after renal failure on day 14. stool samples obtained on days 12 and 16 contained the virus, with up to 1031 rna copies per g (close to the lowest detection limit of the assay). one of two oronasal swabs obtained on day 16 were positive, but yielded little viral rna (5370 copies per ml). no virus was detected in blood. the full virus genome was combined with four other available full genome sequences in a maximum likelihood phylogeny, correlating branch lengths with dates of isolation. the time of the common ancestor was halfway through 2011. addition of novel genome data from an unlinked case treated 6 months previously in essen, germany, showed a clustering of viruses derived from qatar and the united arab emirates. interpretation: we have provided the first complete viral load profile in a case of mers-cov infection. mers-cov might have shedding patterns that are different from those of severe acute respiratory syndrome and so might need alternative diagnostic approaches. funding: european union; german centre for infection research; german research council; and german ministry for education and research. in june, 2012, a coronavirus belonging to a group of viruses that had previously only been detected in bats was cultured from respiratory secretions of a patient who had died from severe acute respiratory infection. 1 the same agent was retrospectively detected in clinical samples from a hospital outbreak of severe acute respiratory infection that occurred in jordan in april, 2012, marking the fi rst known occurrence of the virus in people. 2 the agent has been named middle east respiratory syndrome coronavirus (mers-cov). 3 as of june 10, 2013, 55 laboratory-confi rmed cases had been reported in jordan, saudi arabia, the uk, france, italy, germany, and tunisia. 3 31 individuals with laboratory-confi rmed infection had died. few virological data have become available for mers-cov cases, and there is no information about the viral genome sequence, which could identify important epidemiological characteristics. [4] [5] [6] here, we provide a full description of a fatal case of mers-cov infection imported to munich, germany, from abu dhabi, including a chronological profi le of virus concentrations in diverse body compartments. we fully sequenced the mers-cov genome, and therefore could do a chronologically calibrated phylogenetic analysis with all available mers-cov genome sequences. these data were complemented by novel sequence data from an unlinked case treated in germany in 2012. 7, 8 we report data for a patient who was admitted to the klinikum schwabing (munich, germany) in march, 2013. investigation was done as part of a public health intervention according to the german infection protection act. written consent for scientifi c assessment was obtained from the patient's spouse as part of the patient treatment contract. we did diagnostic rt-pcr and indirect immunofl uorescence, following who recom mendations. 7, 9 for serum neutralisation tests, we grew vero b4 cells to subconfl uence in 24-well plates. pre-incubation reactions contained 25 plaque-forming units of mers-cov (emc strain) in 100 μl of medium, mixed one-to-one with serum samples from the patient prediluted in medium. the starting dilution was a tenth. after 1 h incubation at 37°c, each well was infected for 1 h at 37°c with the total 200 μl pre-incubation reaction. supernatants were removed and overlaid with avicell resin as described by herzog and colleagues. 10 assays were terminated and stained after 3 days. we defi ned neutralisation titres as the serum dilution reducing the number of plaques in four parallel wells in summary by greater than 90%. antibodies were tested by immunofl uorescence assay. 7 all clinical materials stored in the ward and laboratories were gathered and submitted for virological diagnostic tests. from the time of laboratory diagnosis, respiratory, faecal, and urine samples were obtained. we designed two diff erent sets of primers generating overlapping amplicons (available on request). the fi rst set consisted of 70 amplicons, 386-800 bp in length, with all primers containing two strong watson-crick bps at their 3 ends, so as to bind the template with high affi nity. the second set consisted of 68 amplicons, 415-761 bp in length, with primers that had no more than two strong bps in their fi ve 3 terminal nucleotides and no strong pairings in the two 3 positions. this method of primer design can decrease sensitivity, but it prevents mispriming within the product, which can improve the success of amplifi cation. after rt-pcr, we sequenced all fragments on a roche 454 junior instrument (roche, penzberg, germany) and assembled in geneious (version 6.1.2). virus quantifi cation was done with standard calibration curves that were based on quantifi ed in-vitro transcribed rna for the upe target gene. 9 we constructed a maximum likelihood tree of the fi ve available complete mers-cov genomes with phyml 11 and the gtr+gamma model of molecular evolution; we assessed phylogenetic support with 1000 bootstrap replicates. we inferred a timescale by linear regression of genetic divergence from the root against time of collection of the samples. the root was placed such that the correlation coeffi cient was maximised. a phylogenetic tree based on all available mers-cov sequences was calculated with phyml 11 on a concatenated 4012 bp dataset with the hky substitution model. reduction of the dataset was determined by the small number of sequence fragments that could be retrieved from a stored clinical sample containing a small amount of the virus, derived from a patient treated in essen, germany. the sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and fi nal responsibility to submit for publication. on march 8 (day 0), the patient-a 73-year-old man from abu dhabi, united arab emirates-abruptly developed fl u-like symptoms, with fever and non-productive cough. he was admitted to mafraq hospital (abu dhabi) on day 2 (fi gure 1), and was diagnosed with pneumonia. he was intubated on day 9 because of progressive hypoxia and acute respiratory distress syndrome (fraction of inspired oxygen 60%; positive end-expiratory pressure 10 cm h 2 o). the patient had received intensive antimicrobial treatment with meropenem, levofl oxacin, vancomycin, caspofungin, aciclovir, and oseltamivir during his stay in an intensive care unit in abu dhabi, without major improvement in his pulmonary function. the patient was transferred to klinikum schwabing (munich, germany) on march 19, 2013 (fi gure 1). the patient had been diagnosed with multiple myeloma in 2008, and had received several lines of treatment in the previous few years, such as high-dose chemotherapy with autologous stem-cell transplantation in 2009. at relapse of his multiple myeloma in november, 2012, he was given lenalidomide plus dexamethasone. relatives reported that the patient owned camels, and had taken care of a diseased animal shortly before onset of symptoms. no animal samples, or further details about potential sources or exposures could be retrieved. during his stay in munich, we recorded thrombocytopenia (table 1) . thrombocytopenia was also reported in the fi rst described case of mers-cov infection, 1 in two of four patients from a family cluster in saudi arabia, 12 and in the two cases reported from france. 5 the patient developed renal insuffi ciency on day 14, and required dialysis. despite continuous invasive ventilation and antibiotic treatment, the patient's health status deteriorated. death occurred on day 18 and was due to septic shock, with signs of haemolysis and acute coagulation disorder (fi gure 1, table 1). after hospital admission in munich, infection with mers-cov was suspected on the basis of treatment-refractory acute respiratory distress syndrome, combined with the geographical origin of the patient. bronchoalveolar fl uid was obtained on march 20 and 22 (days 12 and 14). mers-cov was detected in both samples by rt-pcr. we also detected herpes simplex virus type 1 dna 13 (6·4 × 10⁴ to 1·9 × 10⁷ copies per ml) and rhinovirus rna 14 (3·7 × 10⁵ to 2·1 × 10⁹ copies per ml) by (rt-)pcr in both samples. mers-cov rna concentrations in respiratory samples ranged from 933 to 1·2 × 10⁶ genome copies per ml. virus concentrations seemed to be higher in samples taken earlier in the course than in those obtained later (fi gure 2). concentrations were more variable in tracheobronchial samples than in bronchoalveolar lavage samples (fi gure 2), which was ascribed to variation in volumes of saline solution applied during removal of tracheobronchial samples. notably, suction catheters without opening at point of care and stored for as long as 5 days at 8°c in a refrigerator in the intensive care unit tested consistently positive but yielded up to roughly 3·5log 10 lower rna concentrations than did those in fresh tracheobronchial aspirates taken on the same days (fi gure 2). immunofl uorescence assays yielded endpoint titres on day 16 of infection (table 2 ). an igm-specifi c immunofl uorescence assay confi rmed recent infection in the same serum sample (table 2) . plaque-reduction neutralisation test confi rmed mers-cov specifi city of detected antibody titres (table 2) . these titres were somewhat lower than those recorded for serum samples from an unlinked non-fatal case of mers-cov treated in germany in 2012. 8 serum samples from this patient had been taken later than they were for our patient (table 2) . we tested two urine samples on day 12, one on day 13, and one on day 16. one of the two samples on day 12, and the sample from day 13 were positive, meaning that the virus was not present in urine after renal failure (day 14), with a maximum virus concentration of 2691 rna copies per ml on day 13. both stool samples obtained on day 12 and the fi ve on day 16 were positive, with up to 1031 rna copies per g, which is a concentration close to the lowest detection limit of the assay. we recorded a low virus concentration in one of two oronasal aspirate samples taken from the intubated patient on day 16 (5370 copies per ml). one dialysate sample and two serum samples on day 16 , and one serum sample on day 18 were negative. although we obtained several isolates for the herpes simplex virus type 1, repeated attempts to isolate mers-cov were unsuccessful. herpes simplex virus is a frequent bystander infection in intubated patients, and is known to not aff ect the cardiorespiratory prognosis and outcome. 15 we sequenced the full mers-cov genome directly from respiratory samples (genbank accession number kf192507). we subjected all available mers-cov genome sequences to phylogenetic analysis, including a correlation and regression analysis of known dates of virus isolation versus tree branch lengths (fi gure 3). we estimated the rate of evolution as 1·6 × 10 -³ substitutions per site per year. the time of the common ancestor of all fi ve viruses for which genomes are available was halfway through 2011 (fi gure 3). the virus in our patient clustered with a sequence from a virus imported into the uk from qatar. 16 to compare this sequence with that of another virus from the same region, we reanalysed a stored clinical sample from another case of mers-cov infection imported into germany in october, 2012. this sample contained low concentrations of rna, so the genome of the virus had not been successfully sequenced previously. 8 after many attempts to recover rt-pcr fragments from the available bronchoalveolar lavage sample, we could sequence 12 fragments, covering 4012 nucleotides of the mers-cov genome (genbank accession number kc875821). a concatenated alignment of homologous sequence portions of all available mers-cov sequences was subjected to phylogenetic analyses, confi rming a clustering of sequences from qatar and the united arab emirates (fi gure 3). a sequence from a patient with a history of travel to pakistan and saudi arabia branched next to this cluster. during and up to 10 days after the course of treatment, 14 health-care workers who had direct contact with our patient or patient-derived materials reported mild respiratory symptoms. samples were taken from the upper respiratory tract and tested by two diff erent rt-pcr assays for mers-cov. none yielded positive results. by contrast, one patient who had had direct contact with the patient with mers-cov was infected with hcov-nl63, a common human coronavirus, and four patients were infected with rhinoviruses. these rhinoviruses were not all mutually related, and none was related to the rhinovirus detected in the patient with mers-cov (appendix). follow-up of all contact patients, including investigation for subclinical infections, is in progress. we have outlined the chronological follow-up of a patient with mers-cov, in which we used quantitative virological diagnostic tests (panel). viral loads were highest in the lower-respiratory tract. the viral sequence from this patient clustered with sequences from nearby qatar. laboratory data are crucial for diagnostic recommendations, to make projections about prognosis, and to estimate infection risks. without quantitative laboratory data from well documented cases of mers-cov infection, most considerations had been made on the basis of an assumed analogy to severe acute respiratory syndrome (sars). 12, [18] [19] [20] however, elementary traits of the virus, such as its receptor usage and sensitivity to type i and type iii interferon, diff er substantially from that of the sars coronavirus, suggesting that diff erences in disease patterns (eg, in organ tropism or in virus shedding) might exist. [21] [22] [23] [24] [25] we focused on these aspects with quantitative virus testing in all relevant body compartments, including viral loads in non-respiratory samples. 5, 12 however, our patient-like most other cases anecdotally reported so far-had an underlying disease that could aff ect virus shedding patterns. only analysis of a large number of patients can yield general fi gures about qualitative virus data. faecal shedding was of particular interest, because patients with sars regularly showed high virus concentrations and prolonged virus excretion in stools that led to the use of stool samples, even for routine sars diagnostic tests. 18, 20, 26 diarrhoea was reported in two descriptions of mers-cov clusters, and it was speculated that faecal virus shedding might have occurred. 5, 12 however, no laboratory data for virus in stool samples were provided. our patient had low faecal virus concentrations that were close to the lowest detection limit on days 12 and 16 of illness. in the only other description so far, one stool sample from a patient with mers-cov had a negative result. 16 stool samples from many patients, including those with early stages of disease, should be tested to assess whether faecal sources could have a role in transmission, or whether mers-cov diff ers from sars in this aspect. another important fi nding was that we recorded low concentrations of virus in urine samples. this fi nding is surprising, because early kidney failure during the course of mers-cov infection has been reported, and kidney cells in laboratory models are highly permissive for mers-cov replication. 5, 12, 23 the fact that the virus was present in urine but not in the blood suggests autonomous virus replication in the kidneys, potentially without active secretion of virus into the urine. however, renal failure due to specifi c viral infection or immunopathogenesis is not necessarily indicated, because the patient had received several doses of potentially nephrotoxic antimicrobial agents in a setting of underlying multiple myeloma. post-mortem examinations are urgently needed to clarify whether kidney failure in mers-cov infection is a primary and preventable result of viral infection, or a secondary complication of severe systemic disease. 27, 28 quantitative virus data are needed to orient diagnostics and hospital infection control measures. the recorded viral load profi le, with highest rna concentrations in bronchoalveolar lavage and tracheobronchial aspirates, confi rms suggestions made in another report about the preferential use of lower-respiratory-tract samples for virus diagnostic tests. 5 notably, the reported overall stability of detectable virus rna in closed suction catheters indicates a straightforward and non-contagious way to collect diagnostic samples even from non-intubated patients. oronasal swabs should not be preferentially submitted for testing, especially in patients presenting late in their disease course with substantial lower respiratory involvement. 5 our data for stool, urine, and blood samples suggest a fairly low infection risk during non-respiratory care procedures. the absence of detectable virus in blood matches reports made in an earlier case of mers-cov infection. 16 however, guery and colleagues 5 reported low semi-quantitative virus measurements in the blood of one of their patients. moreover, initial experimental studies suggested that mers-cov infected vascular endothelial cells. 25 however, quantitative data suggest a low risk from general laboratory procedures involving blood. by contrast, the low virus concentrations and failure to isolate infectious virus from respiratory secretions should not be taken as a general indication of airway-associated infection risks. virological monitoring of the patient started only late in the disease course, at a time when the infectiousness of the virus could already have been reduced (as suggested by the occurrence of neutralising antibodies). the fact that we could not isolate mers-cov could have been due to the concomitant presence of herpes simplex virus type 1, which overgrew some of the diagnostic cell cultures. we searched pubmed for reports published in english at any time before june 7, 2013. we used the search term "mers-cov or hcov-emc". we identifi ed 29 reports linked to middle east respiratory syndrome coronavirus (mers-cov), starting with zaki and colleagues' initial report, 1 in which a previously unknown coronavirus isolated from the sputum of a 60-year-old man is described. when we used the search term "mers-cov", we identifi ed four reports, 2,5,12,17 none of which provided quantitative viral load profi les of infected patients. our report provides the fi rst complete viral load profi le in a case of mers-cov infection. the distribution of viral loads in the respiratory tract suggests lower-respiratory-tract samples should be taken preferentially. low concentrations of the virus in stool, urine, and blood samples suggests little virus excretion-at least in our patient-from body compartments other than the respiratory tract. furthermore, coronaviruses that infect people are generally diffi cult to isolate, particularly in late phases of disease. only two successful isolations of mers-cov have been reported worldwide so far. 1, 5 these isolations were done on day 4 of disease, 5 and day 7 of disease. 1 our samples were taken on days 12 and 14, and the sample from day 12 was stored for 3 days before cell culture. isolation success cannot provide any information about infectiousness of the patient. even a highly concentrated rhinovirus does not seem to have been transmitted from the patient, suggesting that eff ective protective measures were in place during treatment of the intubated patient in the intensive care unit. the sequence data from this and another patient treated in germany enable an extended analysis of phylogeny, hinting at a geographical structure of the mers-cov tree. specifi cally, viral sequences from the eastern part of the arabian peninsula cluster together and stem from one common ancestor whose date of existence is projected to be after that of viruses from jeddah and jordan. date estimates will probably be refi ned when more sequences become available. moreover, whether the reported geographical structure represents repeated transfer from a geographically structured viral reservoir population and limiting chains of person-to-person transmission or multiple sustained lineages of human infections is unclear. with only fi ve complete genome sequences available as yet, genetic data are urgently needed to establish the spatial and temporal distribution of cases, estimate the number of independent human chains of transmission, and thus better assess the threat that mers-cov poses to world health. four sequences from a continuing hospital outbreak in al-hasa, saudi arabia, have now been deposited in genbank. preliminary phylogenetic analyses confi rm the clustering of viruses from the eastern part of the arabian peninsula (qatar: strains england2 and essen; abu dhabi: strain munich; al-hasa: genbank accession numbers kf186564-kf186567). cd designed the virological studies, phylogenetic analysis, and virological data analysis. ms, whar, gs, ss, wg, hgu, tr, ms-w, fb, and c-mw contributed to clinical data generation and analysis. ms, vmc, rk, dm, sj, and ar contributed to fi gures. vmc, rk, dm, sj, mam, sa, and ud generated and analysed virological data. ar did phylogenetic analysis. cd, whaa, hgo, and c-mw interpreted data. cd, ms, vmc, fb, ar, and c-mw wrote the report. whaa advised the public health intervention. hgo and pg coordinated the public health intervention. we declare that we have no confl icts of interest. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov); announcement of the coronavirus study group middle east respiratory syndrome coronavirus (mers-cov)-update severe respiratory illness caused by a novel coronavirus clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections contact investigation of a case of human novel coronavirus infection treated in a german hospital detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction plaque assay for human coronavirus nl63 using human colon carcinoma cells a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood family cluster of middle east respiratory syndrome coronavirus infections detection of herpes simplex virus dna by real-time pcr real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses respiratory herpes simplex virus type 1 infection/colonisation in the critically ill: marker or mediator? severe respiratory illness caused by a novel coronavirus genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus the severe acute respiratory syndrome the aetiology, origins, and diagnosis of severe acute respiratory syndrome identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome effi cient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures severe acute respiratory syndrome multiple organ infection and the pathogenesis of sars acute renal impairment in coronavirus-associated severe acute respiratory syndrome this work was supported by a european research project on emerging diseases detection and response (emperie; contract no 223498). cd has received infrastructural support from the german centre for infection research, which included full funding of the position of vc. virological analyses were partly support by the german ministry for key: cord-255465-sc1yzzsn authors: krasteva, gabriela; pfeil, uwe; drab, marek; kummer, wolfgang; könig, peter title: caveolin-1 and -2 in airway epithelium: expression and in situ association as detected by fret-clsm date: 2006-08-11 journal: respir res doi: 10.1186/1465-9921-7-108 sha: doc_id: 255465 cord_uid: sc1yzzsn background: caveolae are involved in diverse cellular functions such as signal transduction, cholesterol homeostasis, endoand transcytosis, and also may serve as entry sites for microorganisms. hence, their occurrence in epithelium of the airways might be expected but, nonetheless, has not yet been examined. methods: western blotting, real-time quantitative pcr analysis of abraded tracheal epithelium and laser-assisted microdissection combined with subsequent mrna analysis were used to examine the expression of cav-1 and cav-2, two major caveolar coat proteins, in rat tracheal epithelium. fluorescence immunohistochemistry was performed to locate caveolae and cav-1 and -2 in the airway epithelium of rats, mice and humans. electron-microscopic analysis was used for the identification of caveolae. clsm-fret analysis determined the interaction of cav-1α and cav-2 in situ. results: western blotting and laser-assisted microdissection identified protein and transcripts, respectively, of cav-1 and cav-2 in airway epithelium. real-time quantitative rt-pcr analysis of abraded tracheal epithelium revealed a higher expression of cav-2 than of cav-1. immunoreactivities for cav-1 and for cav-2 were co-localized in the cell membrane of the basal cells and basolaterally in the ciliated epithelial cells of large airways of rat and human. however, no labeling for cav-1 or cav-2 was observed in the epithelial cells of small bronchi. using conventional double-labeling indirect immunofluorescence combined with clsm-fret analysis, we detected an association of cav-1α and -2 in epithelial cells. the presence of caveolae was confirmed by electron microscopy. in contrast to human and rat, cav-1-immunoreactivity and caveolae were confined to basal cells in mice. epithelial caveolae were absent in cav-1-deficient mice, implicating a requirement of this caveolar protein in epithelial caveolae formation. conclusion: these results show that caveolae and caveolins are integral membrane components in basal and ciliated epithelial cells, indicating a crucial role in these cell types. in addition to their physiological role, they may be involved in airway infection. caveolae are omega-shaped invaginations of the plasma membrane measuring 50 to 100 nm in diameter. they are found in numerous cell types such as type i pneumocytes, endothelial cells, adipocytes, fibroblasts, smooth muscle cells, cardiac and striated muscle cells [1] . caveolar formation is dependent on the expression of caveolins. three caveolins (cav) are known. cav-1 and cav-2 are widely expressed, whereas cav-3 is thought to be restricted to muscle cells [2] . cav-1 is expressed in two isoforms, cav-1α and cav-1β, exhibiting a cell type-specific distribution (endothelial vs. alveolar type-1 cells) in the alveolar region [3] . caveolae are involved in diverse cellular functions such as organizing signal transduction mechanisms, endocytosis and intracellular transport [2] . several pathogenic microorganisms selectively use caveolae to enter cells [4] . after accumulation in the caveolae, they are delivered to the endoplasmatic reticulum bypassing the classical endosome-lysosome trafficking and thereby preventing inactivation [5, 6] . it has been shown that the infectivity of ctype human adenovirus can be greatly reduced by the expression of a dominant negative cav-1 mutant in plasmocytic cells [7] , indicating that caveolae are involved in this process. in addition, it was recently shown for chlamydia pneumoniae that it co-localizes intracellularly with cav-1 and cav-2 after infection, and a role of these proteins for the developmental cycle of chlamydiae is discussed [8] . also, the human coronavirus 229e that is known to induce respiratory tract infections enters cells via a caveolae dependent mechanism [9, 10] . although the airway epithelium serves as entry site for microbes, fulfils functions that are associated with caveolae such as endo-and transcytosis, and harbors receptors that are associated with caveolae [1] , the expression of caveolins, their interaction, and the presence of caveolae in tracheal and bronchial epithelial cells have not yet been determined. interestingly, the presence of "vesicles that sometimes are connected with the membrane" has earlier been described at the electron-microscopic level in mouse basal cells [11] . moreover cav-1 and cav-2 were detected in cell lines derived from bronchial epithelium [12] , pointing to the presence of caveolae in the airway epithelium. both cav-1 and cav-2 exhibit a similar expression, but seem to have different functions. cav-1 is sufficient to drive caveolar formation [13] . in general, it is thought that cav-2 alone is not sufficient for caveolae formation, and the absence of caveolae in cav-1-deficient mice is associated with marked reduction in cav-2 levels [14] . in contrast, although caveolae are still present in cav-2 deficient mice, these mice show the marked pathological alveolar phenotype of cav-1 deficient mice [15] . this indicates that cav-2, although not able to form caveolae on its own, has profound influences on caveolar function. since a selective association of cav-2 but not cav-1 was described with chlamydia species other than chlamydia pneumoniae it is likely that both proteins can have divergent functions during infectious processes making it necessary to examine the presence and localization of both proteins. in view of these facts, it is pivotal to gain insight in the cellular expression of caveolae and caveolins in bronchial and tracheal epithelium. we therefore examined the expression of cav-1 and cav-2 on the mrna and protein level, determined the distribution of cav-1α, cav-1β, and cav-2 by immunohistochemistry and examined the presence of caveolae by electron microscopy. to address the molecular composition of caveolae, we determined the molecular association of cav-1α and cav-2 in tracheal epithelial cells in tissue sections by double-labeling indirect immunofluorescence combined with confocal laser scanning microscopy (clsm) and fluorescence resonance energy transfer (fret) analysis. this study was performed on 1) wistar rats (150-250 g) of either sex, kept either under standard laboratory conditions or under specified pathogen-free (spf) conditions, and 2) cav-1-deficient mice [14] and the corresponding c57/bl6 wild-type mice that were kept under spf conditions. the animals were held according to the german guidelines for the care and use of laboratory animals. they were killed by inhalation of an overdose of isoflurane (abbott, wiesbaden, germany). total rna from abraded tracheal epithelial cells of adult wistar rats (n = 6) was isolated by using the rneasy method according to the manufacturer'sprotocol (qiagen, hilden, germany). the epithelial cells from the trachea were abraded using cotton swabs that were carefully rolled over the epithelial layer. contaminating dna was degraded using 1 u dnase-i (invitrogen, karlsruhe, germany) per µg of total rna, and reverse transcription was done for 50 min at 42°c using 200 u superscript ii reverse transcriptase (invitrogen) per µg of rna. rt-pcr was performed by adding 1 µl cdna, 0.5 µl of each genespecific intron-spanning primer pair for cav-1 or cav-2 (20 pm; mwg biotech, ebersberg, germany, total rna was isolated from abraded tracheal epithelial cells of rats (n = 6, spf; n = 3, standard conditions) and reverse-transcribed as described above. real-time pcr was performed in an i-cycler (bio-rad, munich, germany) using a quantitec sybr green pcr kit (qiagen). primer sets for cav-1 and -2 amplifying the sequences corresponding to nucleotides 25-147 and 392-497, respectively, were used ( table 1 ). the pcr conditions included initial denaturation in one cycle of 10 min at 95°c followed by 40 cycles of 20 s at 95°c, 20 s at 59°c, and 20 s at 72°c. all analyses were done in triplicate. as a basis for the relative mrna quantification, the mean cycle thresholds (ct) for cav-1 and cav-2 were calculated. the corresponding threshold cycles of the target gene were subtracted from mean β-mg-ct according to: the relative expression (re) of cav-2 compared to that of cav-1 was calculated as follows: the pcr products were analyzed by electrophoresis on a 2% tris-acetate-edta agarose gel. laser-assisted microdissection (using a microbeam system, p.a.l.m. microlaser technologies, bernried, germany) was used to isolate epithelial cells from cryosections of tracheae of rats (spf, n = 6; normal conditions, n = 2). serial cryosections (6 µm) were collected on membrane slides (p.a.l.m. microlaser technologies), previously radiated with uv-light (254 nm) for 30 min. for each cup, the amount equal to 50% of the epithelium of a transverse section of a trachea was collected within 2 h after preparing the sections. rna isolation and purification were performed using rneasy micro kit (qiagen) according to the manufacturer's protocol, but omitting the dna digestion step. ten µl rna were incubated at 70°c for 10 min. rt-mix was added (2 µl 10 × pcr buffer ii, 100 mm tris-hcl, 500 mm kcl, ph 8.3; 4 µl mgcl 2 , 25 mm; 1 µl dntps, 10 mm; 1 µl random hexamers, 50 mm; 0.5 µl rnase inhibitor, 20 u/µl; 1 µl mulv reverse transcriptase, 50 u/µl; 0.5 µl h 2 o; all reagents from applied biosystems). rna was reverse-transcribed for 75 min at 43°c, followed by inactivation of the reverse transcriptase by heating the rna samples for 5 min at 99°c. for subsequent pcr, 4 µl cdna, 2.5 µl 10 × pcr buffer ii, 2 µl mgcl 2 (15 mm), 0.5 µl dntps (10 mm), 0.5 µl of each primer (20 pm; primer sets spanning the region 25-147 for cav-1 and 392-497 for cav-2), 0.2 µl amplitag gold polymerase (5 u/µl, all reagents from applied biosystems) and 14.8 µl h 2 o were applied. cycling conditions were 4 min at 95°c, 50 cycles with 20 s at 95°c, 20 s at 59°c, 20 s at 73°c, and a final extension at 73°c for 7 min. to control for smearing of rna during the cutting procedure, areas of dried o.c.t. compound (sakura, zoeterwoude, the netherlands) similar in number and size to the samples of picked epithelial cells and directly adjacent to the luminal side of the tracheal epithelium were applied. control reactions for each primer pair included the absence of template. the pcr products were separated by electrophoresis on a 2% tris-acetate-edta agarose gel. sequencing of the pcr products was done by mwg biotech. antibodies and their sources were as follows: anti-caveolin-1α (anti-cav-1α; immunohistochemistry (ihc) 1:400, western blotting (wb) 1:500), polyclonal from rabbit (sc-894; santa cruz biotechnology, heidelberg, germany); anti-caveolin-1αβ (anti-cav-1αβ; ihc 1:200; wb 1:500), monoclonal from mouse (clone 2297; transduction laboratories, heidelberg, germany); anti-caveolin-2 (anticav-2; ihc 1:200), monoclonal from mouse (clone 65; transduction laboratories); anti-endothelial nitric oxide synthase (anti-enos; ihc 1:100), monoclonal from mouse (clone 3; transduction laboratories); anti-surfactant protein d (anti-sp-d; ihc 1:100), monoclonal from mouse (clone vi f11, dianova, hamburg, germany), anti-villin, polyclonal from rabbit (ihc 1:2,000, [16] ). secondary antibodies used in this study for immunohistochemistry were: fitc-conjugated donkey antimouse-lg, f(ab') 2 fragments (1:200; dianova), cy3-conjugated donkey anti-rabbit-ig (1:2,000; chemicon, temecula, ca, usa), cy5-conjugated donkey anti-rabbit-ig, f(ab') 2 fragments (1:50; dianova), and cy3-conjugated donkey anti-mouse-ig (1:1,000; dianova). secondary antibodies used in this study for western blotting were: horseradish peroxidase-conjugated goat anti-rabbit-igg or horseradish peroxidase-conjugated goat anti-mouse-igg (both 1:10,000; pierce, rockford, usa). for conventional electron microscopy, tracheae of wistar rats (normal conditions, n = 2), cav-1 deficient mice (spf, n = 2), and c57/bl6 mice (spf, n = 3) were prepared as follows: the vascular system was flushed via the ascending aorta with a rinsing solution containing heparin (2 ml/l; 10,000 u; ratiopharm, ulm, germany), polyvinylpyrrolidone (25 g/l, mw 40.000; roth, karlsruhe, germany) and procaine hydrochloride ( unfixed tissue of wistar rats, cav-1-deficient mice (spf, n = 2), wild-type mice (spf, n = 2), and 10% neutral formalin-fixed and paraffin-embedded human bronchi (n = 4) were used for immunohistochemical analysis. lungs from rats and mice were inflated via the trachea with o.c.t. compound diluted with an equal amount of 0.1 m phosphate buffer (ph 7.4), orientated on a piece of filter paper, and shock-frozen in melting isopentane. cryosections (10 µm) were cut, fixed (either with acetone at -20°c and air dried for 10 min or with 4% pfa for 20 min and washed) and incubated for1 h in 5% normal goat serum containing 5% bsa in 0.005 m phosphate-buffered saline (pbs). primary antibodies were diluted in 0.005 m phosphate buffer containing 0.01 % nan 3 and 4.48 g/l nacl and applied overnight at room temperature. these antibodies were appliedeither singly or in combination for doublelabeling immunofluorescence. primary antibody combinations were as follows: mouse anti-cav-1αβ/rabbit anticav-1α; mouse anti-cav-2/rabbit anti-cav-1α; mouse anti-enos/rabbit anti-cav-1α; mouse anti-sp-d/rabbit anticav-1α; mouse anti-cav-2/rabbit anti-villin. after a washing step, cy3-conjugated donkey anti-rabbit-ig was applied for 1 h and after a second washing step the slides were incubated with fitc-conjugated f(ab') 2 donkey antimouse-ig. sections wererinsed, postfixed for 10 min in 4% pfa, rinsed again and coverslipped with carbonatebuffered glycerol (ph 8.6). sections from human bronchi (6 µm) were deparaffinated and incubated with anti-cav-1α and anti-cav-2 antibody as described above. slides were evaluated with an epifluorescence microscope (zeiss, jena, germany) using appropriate filter sets and with a confocal laser scanning microscope (leica-tcs sp2 aobs;leica, mannheim, germany). specificity of the anti-cav-1α antibody was validated by incubation of cryosections from cav-1 deficient mice. the specificity of the anti-cav-1αβ and anti-cav-2 antibodies was previously shown by other groups in experiments with cav-2 deficient mice [17] . here, we characterized these antibodies by western blotting. additional controls included omission of the primary antibodies. for western blot analysis, abraded tracheal epithelial cells of wistar rats (n = 5), hearts and lungs from wistar rats, lungs from cav-1-deficient mice and from wild-type mice (each n = 2) were homogenized by a mixer mill (mm 300, qiagen) with lysis buffer containing 10 mm tris (ph 7.5), 50 mm nacl, 1% triton x-100, 60 mm octylglucoside (sigma-aldrich chemie gmbh, munich, germany), and one complete mini protease inhibitor cocktail tablet (roche diagnostics gmbh, mannheim, germany) per 10 ml buffer. after incubation of the protein solutions at 4°c for 1 h, they were centrifugedfor 5 min at 14,000 rpm. equal amounts of proteins for each tissue were applied as judged by staining of gels with simply bluetm safe stain (invitrogen, carlsbad, usa) stained gels. ten µl appropriately diluted protein solution and 2 µl of 5 × sample buffer (320 mm tris-hcl, ph 6.8, 5% sds, 50% glycerol, 0.25 mg/ml bromphenol blue and 1% β-2-mercaptoethanol) were boiled for 5 min at 95°c. the samples were subjected to 15% sds-page under reducing conditions and subsequently transferred to a nitrocellulose membrane (bio-rad) by semidry blotting. the nitrocellulose membranes were stained with ponceau s to confirm the protein transfer from the gels to the membrane. after subsequent washing in 25 mm tris-buffered saline with 0.05% tween-20 (ttbs), the unspecific binding sites were saturated by incubation with 10% non-fat dry milk in ttbs for1 h at room temperature. the membrane was incubated overnight at 4°c with anti-cav-1α, -1αβ and -2, primary antibodies, diluted in 5% non-fat dry milk in ttbs. the secondary antibodies were diluted in 2.5% non-fat dry milk in ttbs and incubated for 1 h at room temperature. super signal west pico chemiluminescence substrate (pierce) was used for visualization. controls were done by omitting the primary antibody, and the specificity of the anti-cav-1α and anti-cav-2 antibodies was verified in lung homogenates from cav-1-deficient mice. fret is a nonradiative energy transfer between two fluorophores (a donor and an acceptor) that can be detected only if the two fluorophores are less than 10 nm apart. we used fret combined with clsm and double-labeling indirect immunofluorescence as a technique for measuring close spatial association of proteins in tissue sections [18] . cav-1 and cav-2 were labeled using conventional indirect double-labeling immunofluorescence technique (anti-cav-1α from rabbit labeled with cy5-conjugated secondary reagent, anti-cav-2 antibody labeled with cy3conjugated secondary reagent). both primary antibodies were applied simultaneously. after a washing step, cy3conjugated donkey anti-mouse-ig was applied for 1 h and after a second washing step the slides were incubated with cy5-conjugated f(ab') 2 donkey anti-rabbit-ig. for control of the species-specificity of the secondary reagents, only the anti-cav-1 antibody and both secondary antibodies were applied. fret was quantified by the acceptor photobleaching method using a clsm (tcs-sp2 aobs, leica). in this method, fret is detected by measuring the intensity of fluorescence of the donor before and after bleaching of the acceptor. the clsm settings were as follows: detection of cy3: 52% laser power at 543 nm, detection at 555-620 nm; cy5: 20% laser power at 633 nm, detection at 639-738 nm. a region of interest was photobleached 10 times at 100% activity using the 633 nm laser and maximal zoom to destroy the acceptor fluorophore (cy5) and the change in cy3 signal (∆if) was determined in the photobleached area where ∆if = d da -d db , d da is the fluorescence intensity of the donor after photobleaching of the acceptor, and d db is the fluorescence intensity of the donor before photobleaching of the acceptor. differences among experimental group and control group in the fret-experiments were analysed with the kruskal-wallis test followed by mann-whitney test using spss software, version 11.5.1 (spss gmbh software, munich, germany), with p ≤ 0.05 being considered as significant and p ≤ 0.01 as highly significant. rt-pcr analysis of total mrna isolated from rat lungs and abraded tracheal epithelial cells revealed expression of cav-1 and cav-2. pcr products were of the expected size and the sequence was verified by sequencing. no bands were observed in control reactions that included absence of dna template or the reverse transcriptase ( figure 1a) . we quantified the relative expression of cav-1 and cav-2 in tracheal epithelial cells in rats held under standard conditions or spf conditions. in both groups, cav-2 expression was higher than that of cav-1. cav-2 expression levels were 22.2 times higher than that of cav-1 under standard conditions and 25.8 times higher in animals housed under spf conditions ( figure 1b) . in accordance to the results obtained in abraded epithelial cells, mrna for cav-1 was detected in 17/18 samples of microdissected tracheal epithelial cells collected from 5 rats (spf). cav-2 mrna was present in all microdissected samples (n = 15, 4 spf rats). the identity of the pcr products was validated by sequencing. no mrna for cav-1 and cav-2 was detected in areas next to positive tissue that contained o.c.t. compound only, but no cells. this result indicates that the mrna signal detected in the epithelium for cav-1 and cav-2 was not caused by contamination originating from caveolin-containing adjacent cells ( figure 1a ). using the anti-cav-1α antibody, we detected a single band of approximately 22 kd in rat abraded tracheal epithelial cells (figure 2a) . a band of 22 kd and a band of 18 kd, corresponding to cav-1β, were detected in abraded tracheal epithelial cells using an anti-cav-1αβ antibody (figure 2b) . bands of the same molecular weight were detected in lung and heart homogenates. using an antibody to cav-2, we detected a band of 15 kd (γ-isoform) that was close to the detection limit, an 18 kd band (αisoform), and a 21 kd band (β-isoform) ( figure 2c ). in lung homogenates of wild-type mice, cav-1α-immunoreactive bands of 22 kd, 37 kd, and 48 kd were detected, which were absent in lung homogenates from cav-1 deficient mice ( figure 2d ). using an antibody to cav-2 for western blot analysis of lung homogenates from wild-type mice we detected 18 kd and 21 kd bands. no cav-2 bands were detected in lung homogenates from cav-1deficient mice ( figure 2e ). in rat, cav-1-immunoreactivity was detected in epithelial cells of the large airways ( figure 3a ). in contrast to large cartilaginous airways, no cav-1-immunolabeling was detected in the epithelium of the small non-cartilaginous airways of the rat ( figure 3d ). in addition, cav-1-immunoreactivity was observed in tracheal and bronchial smooth muscle cells, in vascular endothelial cells and in unidentified cells in the lamina propria, probably fibroblasts. in the alveolar region, we observed cav-1-immunoreactivity in endothelial cells and in type i alveolar epithelial cells. differences in the labeling patterns obtained with the anti-cav-1α and the anti-cav-1αβ antibody were noted. the labeling with the anti-cav-1α antibody was stronger in the endothelium, whereas labeling of the anti-cav-1αβ antibody was stronger in the epithelium (cf. figures 3a, b) . also, the number of the epithelial cells that were immunoreactive for cav-1αβ was higher than that of the cells immunoreactive for cav-1α ( figures 4a-c) . cav-2-immunoreactivity was found in the same cell types as cav-1-immunoreactivity. interestingly, not all of the cells that showed cav-2-immunofluorescence were cav-1α-immunoreactive ( figures 4d-f) . the cav-2-immunolabeling of the epithelium was stronger than that of smooth muscle cells ( figure 3c ). as for cav-1, no labeling for cav-2 was observed in the epithelial cells of the small bronchi ( figure 3f ). throughout the rat trachea and large bronchi, we found cav-1α-and cav-1αβ-immunoreactivity 1) in the ciliated cells, identified by their typical morphology and by immunolabeling with a monoclonal antibodyto enos [19] (figure 5a ), and 2) in the basal cells, identified by their typical morphology. unlike ciliated cells, brush cells immunoreactive for villin were not labeled for cav-2 (figure 5b ). most of the secretory cells that were labeled with anti-sp-d [20] were not cav-1-immunoreactive ( figure 5c ). in human bronchi, the ciliated and the basal cells were immunoreactive for cav-1α ( figures 5d-f) . the anti-cav-2 antibody gave no signal in paraffin-embedded human tissue. punctate cav-1α labeling was localized at the basolateral membrane and in the apical area of the ciliated cells underneath the luminal plasma membrane. in the mouse respiratory epithelium, cav-1α-immunoreactivity was confined to basal cells ( figure 6d ). the specificity of the cav-1-immunostaining was confirmed by the absence of cav-1α-immunolabeling in cav-1-deficient mice ( figure 6g ). in agreement with the immunohistochemical results, we observed caveolae at the basolateral cell membrane of the ciliated cells and at the cell membrane of the basal cells in rat trachea (figures 6a-c) . caveolae were more frequent in basal cells. in agreement with the immunohistochemical findings in the mouse trachea, caveolae were only found in the basal cells ( figures 6d-f) . basal cells were devoid of caveolae in tracheae of cav-1-deficient mice ( figures 6g-i) . conventional indirect double-labeling immunofluorescence with subsequent fret-clsm analysis was conducted to determine whether cav-1 and cav-2 are in close apposition in airway epithelial cells in situ, thereby indicating an association of both proteins and formation of hetero-oligomers. distinct increase of fluorescence (∆if) was observed in the bleached area with a median value of 2.72 (n = 16 regions of interest of tracheae obtained from 4 rats; figure 7) . a false-positive fret signal that can be caused by cross-reactivity of secondary antibodies was excluded by applying both secondary antibodies to sections incubated with anti-cav-1 α antibody only (median ∆if = 0.287). since the caveolins are membrane proteins, we measured ∆if in the region of the basolateral plasma membrane. ∆if measured in this region was higher (∆if = 4.795) than that in the whole bleached area. the ∆if measured in the controls in the same region was low (∆if = 0.55). the difference between ∆if observed in the experimental group as compared to the corresponding controls was highly significant (p < 0.001, mann-whitney test; figure 7f ). no increase in the donor fluorescence (∆if) could be detected in the airway epithelial cells that were immunoreactive for cav-2, but not for cav-1α (data not shown). the present study demonstrates for the first time the expression of cav-1 and -2 in epithelial cells of the trachea and large bronchi. electron microscopy demonstrated that, indeed, ciliated and basal cells possess caveolae. it has been shown that muscarinic receptors as well as βadrenergic receptors may translocate to caveolae upon agonist binding [21, 22] . these receptors are involved in the regulation of ciliary function [23, 24] . in addition, several proteins involved in ca 2+ -dependent signaling processes, including the ca 2+ -pump and ca 2+ -atpase, are localized to caveolae as shown in renal and intestinal epithelial cells [25] . therefore, caveolae are likely to be involved in the fine-tuned regulation of epithelial cytosolic ca 2+ -concentration and in regulation of ciliary function. several pathogenic microorganisms selectively use caveolae to enter cells [4] . since the caveolae are localized at the basolateral surface of the ciliated cells, it is conceivable that they may be involved in the process of endocytosis of infectious agents after epithelial damage. indeed, adenoviruses require damage of the integrity of the epithelia or of the tight junctions to get access to the basolateral membrane of the ciliated cells to be infectious [26] . basal cells are much more susceptible to infection with adenoviruses [7, 27] . accordingly, we observed more caveolae in basal cells than in ciliated epithelial cells. it has been shown for the adenovirus2 that it can enter the cell via its receptor car that is also localized basolaterally in ciliated cells and in basal cells. since adenovirus2-car is endocytosed via clathrin-coated pits, caveolae, at first sight, seem not to be western blotting important in this process. nevertheless, another receptor for adenovirus2 is major histocompatibility complex (mhc) class 1. this also is localized in the basolateral membrane of ciliated cells and in basal cells. mhc class 1 is also the receptor for simian virus 40 that is endocytosed via caveolae. this indicates a role for caveolae in the infection of airway epithelium by these adenoviruses. furthermore, the infectivity of c-type human adenovirus can be greatly reduced by the expression of a dominant negative cav-1 mutant in plasmocytic cells indicating that caveolae can serve as an alternative entry site for these viruses [28] . in addition, it was recently shown for chlamydia pneumoniae that it co-localizes with cav-1 and cav-2 in the cytosol in hela cells after cellular entrance [8] , indicating a role for caveolins in infectious processes after microbial entry. since the loss of cav-1 was accompanied with loss of caveolae in tracheal epithelial cells in cav-1-deficient mice, cav-1 is required for the formation of caveolae in these cells. in addition, the stabilization and the transport of cav-2 to the plasma membrane are dependent on the expression of cav-1 [29, 30] . our results from fret experiments also prove that cav-1α and cav-2 are closely associated in ciliated and in basal cells, indicating that both proteins are involved in the formation of caveolae. interestingly, we found considerably higher expression of cav-2 mrna than cav-1 mrna, indicating that cav-2 could immunohistochemistry, rat have roles independent from cav-1 once it has reached the plasma membrane. indeed, it has been shown for cav-2 that it associates with chlamydial inclusions independently of cav-1 [8] . we observed differences in the labeling intensities for cav-1α and cav-1αβ among cell types. cav-1α-immunoreactivity was stronger in endothelial cells compared to epithelial cells. in contrast, cav-1αβ-labeling was stronger in the epithelium than in the endothelium of the large airways. in line with this observation, western blots showed a strong protein expression of cav-1β in abraded tracheal epithelium but not in lung and heart homogenates. cav-1β is less efficient in the formation of caveolae [31] , which could be an explanation for the lower number of caveolae found in epithelial cells compared to endothelium where the α-isoform is predominantly expressed [3] . the β-isoform of cav-1 is derived from an alternative translational starting site that creates a protein truncated by 32 amino acids [32] and lacks the phosphorylation site tyr 14. this site has been shown to be phosphorylated upon stimulation of cultured cells by epithelial growth factor (egf), leading to neoformation of caveolae [33] . since egf is considered as a key factor for repair of the bronchial epithelium [34] , caveolae containing the α-isoform might be involved in this process. it is tempting to speculate that certain caveolae may exist that contain predominantly the β-isoform. such caveolae might be involved in other signaling cascades. if both isoforms are present in caveolae, cav-1β could be a negative regulator for signaling cascades relying on the phosphorylation of cav-1α. we found caveolin-1 and -2 only in the large airways of mice and rats, limiting caveolar function to larger airways in these species. it has to be kept in mind, however, that many human intrapulmonary bronchi are considerably double-labeling immunofluorescence, clsm, large airways, rat figure 4 double-labeling immunofluorescence, clsm, large airways, rat. (a-c) cav-1α-(a) and cav-1αβ-immunoreactivity (b) were colocalized in the basal cells (doubled arrowheads) and basolaterally in a subset of columnar epithelial cells (arrows). some cells exhibited cav-1αβ-but not cav-1α-immunoreactivity (arrowheads). (d-f) cav-1α (d) and cav-2 (e) were localized at the basolateral membrane. co-localisation of cav-1α and cav-2 (f, arrowheads). we observed cells that were only cav-2-immunoreactive (f, arrows). epithelium = e larger than the rat trachea. since we have found cav-1 also in human intrapulmonary bronchi, caveolins are likely to be present throughout a substantial part of the human bronchial tree. in summary, we conclude that ciliated and basal cells of the trachea and large bronchi possess caveolae resulting from an association between cav-1α and cav-2. since caveolae are implied in a variety of different functions in other cell types, they are likely to be important also for these functions in the airway epithelium. cav-immunoreactive epithelial cell types the author(s) declare that they have no competing interests. pk and gk conceived and designed the study. gk performed the immunohistochemical analysis, the western blot experiments and the laser-assisted microdissection with subsequent rt-pcr analysis. gk and pk performed detection of close association of cav-1 and cav-2 in epithelial cells by double-labeling indirect immunofluorescence and fret in tissue sections of the rat trachea and large bronchi the fret analysis. up and gk carried out the quantitative rt-pcr. wk, gk, and pk analyzed the electron microscopic specimens. md generated and genotyped the cav-1 deficient mice. pk and gk performed the statistical analysis. the manuscript was drafted by gk, wk, and pk. caveolae: from cell biology to animal physiology role of caveolae and caveolins in health and disease cell type-specific occurrence of caveolin-1alpha and -1beta in the lung caused by expression of distinct mrnas caveolae in the uptake and targeting of infectious agents and secreted toxins caveolar endocytosis of simian virus 40 is followed by brefeldin a-sensitive transport to the endoplasmic reticulum, where the virus disassembles caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the er lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1 development of a transgenic mouse model susceptible to human coronavirus 229e human coronavirus 229e binds to cd13 in rafts and enters the cell through caveolae untersuchungen am trachealepithel verschiedener säuger towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line cfbe41o de novo formation of caveolae in lymphocytes by expression of vip21-caveolin loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities evidence for the association of villin with core filaments and rootlets of intestinal epithelial microvilli caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae fret-clsm and double-labeling indirect immunofluorescence to detect close association of proteins in tissue sections distribution of the novel enos-interacting protein nosip in the liver, pancreas, and gastrointestinal tract of the rat increased surfactant protein d in rat airway goblet and clara cells during ovalbumin-induced allergic airway inflammation redistribution of muscarinic acetylcholine receptors on human fibroblasts induced by regulatory ligands internalization of beta-adrenergic receptor in a431 cells involves non-coated vesicles atp regulation of ciliary beat frequency in rat tracheal and distal airway epithelium effects of beta-agonists on airway epithelial cells calcium pump of the plasma membrane is localized in caveolae basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia efficient adenovirusmediated gene transfer to basal but not columnar cells of cartilaginous airway epithelia efficient species c hadv infectivity in plasmocytic cell lines using a clathrin-independent lipid raft/caveola endocytic route expression of caveolin-1 is required for the transport of caveolin-2 to the plasma membrane. retention of caveolin-2 at the level of the golgi complex caveolin-2 localizes to the golgi complex but redistributes to plasma membrane, caveolae, and rafts when co-expressed with caveolin-1 isoforms of caveolin-1 and caveolar structure caveolin isoforms differ in their n-terminal protein sequence and subcellular distribution. identification and epitope mapping of an isoform-specific monoclonal antibody probe epithelial growth factor-induced phosphorylation of caveolin 1 at tyrosine 14 stimulates caveolae formation in epithelial cells epithelial damage and response we thank k. michael for expert technical help with the figures and g. kripp from the electron microscopy unit of the institut für anatomie und zellbiologie for the embedding and cutting of the electron microscopic specimens. the laser-assisted microdissection for subsequent rt-pcr analysis was done in cooperation with the z-project of the sfb 534. this study was supported by the deutsche forschungsgemeinschaft (sfb 547, c1; gk 534, both to wk) and by a young scientist grant from the faculty of medicine of the justus-liebig-universität giessen to pk. we also thank prof. dr. robert leroy snipes for linguistic correction of the manuscript. key: cord-226245-p0cyzjwf authors: schneble, marc; nicola, giacomo de; kauermann, goran; berger, ursula title: nowcasting fatal covid-19 infections on a regional level in germany date: 2020-05-15 journal: nan doi: nan sha: doc_id: 226245 cord_uid: p0cyzjwf we analyse the temporal and regional structure in mortality rates related to covid-19 infections. we relate the fatality date of each deceased patient to the corresponding day of registration of the infection, leading to a nowcasting model which allows us to estimate the number of present-day infections that will, at a later date, prove to be fatal. the numbers are broken down to the district level in germany. given that death counts generally provide more reliable information on the spread of the disease compared to infection counts, which inevitably depend on testing strategy and capacity, the proposed model and the presented results allow to obtain reliable insight into the current state of the pandemic in germany. in march 2020, covid-19 became a global pandemic. from wuhan, china, the virus spread across the whole world, and with its diffusion, more and more data became available to scientists for analytical purposes. in daily reports, the who provides the number of registered infections as well as the daily death toll globally (https://www.who.int/). it is inevitable for the number of registered infections to depend on the testing strategy in each country (see e.g. cohen and kupferschmidt, 2020) . this has a direct influence on the number of undetected infections (see e.g. li et al., 2020) , and first empirical analyses aim to quantify how detected and undetected infections are related (see e.g. niehus et al., 2020) . though similar issues with respect to data quality hold for the reported number of fatalities (see e.g. baud et al., 2020) , the number of deaths can overall be considered a more reliable source of information than the number of registered infections. the results of the "heinsberg study" in germany point in the same direction (streeck et al., 2020) . a thorough analysis of death counts can in turn generate insights on changes in infections as proposed in flaxman et al. (2020) (see also ferguson et al., 2020) . in this paper we pursue the idea of directly modelling registered death counts instead of registered infections. we analyse data from germany and break down the analyses to a regional level. such regional view is apparently immensely important, considering the local nature of some of the outbreaks for example in italy (see e.g. , france (see e.g. massonnaud et al., 2020) or spain. the analysis of fatalities has, however, an inevitable time delay, and requires to take the course of the disease into account. a first approach on modelling and analysing the time from illness and onset of symptoms to reporting and further to death is given in jung et al. (2020) (see also linton et al., 2020) . understanding the delay between onset and registration of an infection and, for severe cases, the time between registered infection and death can be of vital importance. knowledge on those time spans allows us to obtain estimates for the number of infections that are expected to be fatal based on the number of infections registered on the present day. the statistical technique to obtain such estimates is called nowcasting (see e.g. höhle and an der heiden, 2014) and traces back to lawless (1994) . nowcasting in covid-19 data analyses is not novel and is for instance used in günther et al. (2020) for nowcasting daily infection counts, that is to adjust daily reported new infections to include infections which occurred the same day but were not yet reported. we extend this approach to model the delay between the registration date of an infection and its fatal outcome. we therefore analyse the number of fatal cases of covid-19 infections in germany using district-level data. the data are provided by the robert-koch-institute (www.rki.de) and give the cumulative number of deaths in different gender and age groups for each of the 412 administrative districts in germany together with the date of registration of the infection. the data are available in dynamic form through daily downloads of the updated cumulated numbers of deaths. we employ flexible statistical models with smooth components (see e.g. wood, 2017) assuming a district specific poisson process. the spatial structure in the death rate is incorporated in two ways. first, we assume a spatial correlation of the number of deaths by including a long-range smooth spatial death intensity. this allows to show that regions of germany are affected to different extents. on top of this long-range effect we include two types of unstructured region specific effects. an overall region specific effect reflects the situation of a district as a whole, while a short-term effect mirrors region specific variation of fatalities over time and captures local outbreaks as happened in e.g. heinsberg (north-rhine-westphalia) or tirschenreuth (bavaria). in addition we include dynamic effects to capture the global changes in the number of fatal infections for germany over calendar time. this enables us to investigate the impact of certain interventions, such as social distancing, school closure, complete lockdowns and lockdown releases, on the dynamic of the infection and hence on the number of deaths. modelling infectious diseases is a well developed field in statistics and we refer to held et al. (2017) for a general overview of the different models. we also refer to the powerful r package surveillance . since our focus is on analysing the district specific dynamics of fatal infections we here make use of poisson-based models implemented in the mgcv package in r, which allows to decompose the spatial component in more depth. the paper is organized as follows. in section 2 we describe the data. section 3 highlights the results of our analysis. the remaining sections provide the technical material, starting with section 4 where we motivate the statistical model, which is extended by our nowcasting model in section 5. extended results as well as model validation are given in section 6, while section 7 concludes the paper. we make use of the covid-19 dataset provided by the robert-koch-institute for the 412 districts in germany (which also include the twelve districts of berlin separately). the data are updated on a daily basis and can be downloaded from the robert-koch-institute's website. we have daily downloads of the data for the time interval from march 27, 2020 until today. the subsequent analysis was conducted on may 14, 2020, and was performed considering only deadly infections with registration dates from march 26, 2020 until may 13, 2020 (the day before the day of analysis). the data contain the newly notified laboratory-confirmed covid-19 infections and the cumulated number of deaths related to covid-19 for each district of germany, classified by gender and age group. each data entry has a time stamp which corresponds to the registration date of a confirmed covid-19 infection. this means that the time stamp for a fatal outcome always refers to the registration date and not to the death date. due to daily downloads of the data we can derive the time point of death (or to be more specific, the time point when the death of a case is included in the database). we obtain the latter by observing a status change from infected to deceased when comparing the data from two consecutive days. the robert-koch-institute collects the data from the district-based health authorities (gesundheitsämter). due to different population sizes in the districts and certainly also because of different local situations, some health authorities report the daily numbers to the robert-koch-institute with a delay. this happens in particular over the weekend, a fact that we need to take into account in our model. we refrain from providing general descriptive statistics of the data here, since these numbers can easily be found on the rki webpage, which also gives a link to a dashboard to visualize the data (see also https://corona.stat.uni-muenchen.de/maps/) before we discuss our modelling approach in detail, we want to describe our major findings. first, table 1 shows that age and gender both play a major role when estimating the daily death toll. as is generally known, elderly people exhibit a much higher death rate which is for the age group 80+ around 100 times higher than for people in the age group 35-59. a remarkable difference is also observed between genders, where the expected death rate of females is around 40% (≈ 1 − exp(−0.503)) lower than the death rate for males. furthermore, we see that significantly less deaths are attributed to infections registered on sundays compared to weekdays, due to the existing reporting delay during weekends. our model includes a global smooth time trend representing changes in the death rate since march 26th. this is visualized in figure 1 . the plotted death rate is scaled to give the expected number of deaths per 100.000 people in an average district for the reference group, i.e. males in the age group 35 -59. overall, we see a peak in the death rate on april 3rd and a downwards slope till end of april. however, our nowcast reveals that the rate remains constant since beginning of may. note that this recent development cannot be seen by simply displaying the raw death counts of these days. the nowcasting step inevitably carries statistical uncertainty, which is taken into account in figure 1 by including best and worst case scenarios. the latter are based on bootstrapped confidence intervals, where details are provided in section 6.3 later in the paper. our aim is to investigate spatial variation and regional dynamics. to do so, we combine a global geographic trend for germany with unstructured region-specific effects, where the latter uncover local behaviour. in figure 2 we combine these different components and map the fitted nowcasted death counts related to covid-19 for the different districts of germany, cumulating over the last seven days before the day of analysis (here may 14, 2020). while in most districts of germany the death rate is relatively low, some hotspots can be identified. among those, traunstein and rosenheim (in the south-east part of bavaria) are the most evident, but greiz and sonneberg (east and south part of thuringia) stand out as well, to mention a few. a deeper investigation of the spatial structure is provided in section 6, where we show the global geographic trend and provide maps that allow to detect new hotspot areas, after correcting for the overall spatial distribution of the infection. on the day of analysis, we do not observe the total counts of deaths for recently registered infections, since not all patients with an ongoing fatal infections have died yet. we therefore nowcast those numbers, i.e. we predict the prospective deaths which can be attributed to all registration dates up to today. this is done on a national level, and the resulting nowcast of fatal infections for germany is shown in figure 3 . for example, on may 14, 2020 there are 25 deaths reported where the infection was registered on may 5th (red line on may 5th). we expect this number to increase to about 50 when all deaths due to covid-19 for this registration date will have been reported (blue line on may 5th). naturally, the closer a date is to the present, the larger the uncertainty in the nowcast. this is shown by the shaded bands. details on how the statistical uncertainty has been quantified are provided in section 5 below. the fit of this model has been incorporated into the district model discussed before, but the nowcast results are interesting in their own right. the curve confirms that the number of fatal infections is decreasing since the beginning of april. note that the curve also mirrors the "weekend effect" in registration, as less infections are reported on sundays. further analyses and a detailed description of the model are given in the following sections. let y t,r,g denote the number of daily deaths due to covid-19 in district/region r and age and gender group g with time point (date of registration) t = 0, . . . , t . here t = t corresponds to the day of analysis, which is may 14, 2020 and t = 0 corresponds to march 26, 2020. note that time point t refers to the time point of registration, i.e. the date at which the infection was confirmed. even though the time point of infection obviously precedes that of death, registration can also occur after death, e.g. when a post mortem test is conducted, or when test results arrive after the patient has passed away. we set the day of death to be equal to the day of registered infections in this case. the majority of fatalities with registered infection at time point t have not yet been observed at time t, as these deaths will occur later. we therefore need a model for nowcasting, which is discussed in the next section. for now we assume all y t,r,g to be known. we model y t,r,g as (quasi-)poisson distributed according to where we specify λ t,r,g through λ t,r,g = exp{(β 0 + age g β age + gender g β gender + weekday t β weekday the linear predictor is composed as follows: • β 0 is the intercept. • β age and β gender are the age and gender related regression coefficients. • β weekday are the weekday-related regression coefficients. • m 1 (t) is an overall smooth time trend, with no prior structure imposed on it. • m 2 (s r ) is a smooth spatial effect, where s r is the geographical centroid of district/region r. • u r0 and u r1 are district/region-specific random effects which are i.i.d. and follow a normal prior probability model. while u r0 specifies an overall level of in the death rate for district r over the entire observation time, u r1 reveals region specific dynamics by allowing the regional effects to differ for the last 14 days. • pop r,g is the gender and age group-specific population size in district/region r and serves as an offset in our model. we here emphasize that we fit two spatial effects of different types: we model a smooth spatial effect, i.e. m 2 (s r ), which takes the correlation between the death rates of neighbouring districts/regions into account and gives a global overview of the spatial distribution of fatal infections. in addition to that we also have unstructured district/region-specific effects u r = (u r0 , u r1 ) , which capture local behaviour related to single districts only. the district specific effects u r are considered as random with a prior structure for r = 1, . . . , 412. the prior variance matrix σ u is estimated from the data. the predicted values u r (i.e. the posterior mode) exhibit districts that show unexpectedly high or low death tolls when adjusted for the global spatial structure and for age-and gender-specific population size. model (1) belongs to the model class of generalized additive mixed model, see e.g. wood (2017) . the smooth functions are estimated by penalized splines, where the quadratic penalty can be comprehended as a normal prior (see e.g. wand, 2003) . the same type of prior structure holds for the region-specific random effects u r . in other words, smooth estimation and random effect estimation can be accommodated in one fitting routine, which is implemented in the r package mgcv. this package has been used to fit the model, so that no extra software implementation was necessary. this demonstrates the practicability of the method. the above model cannot be fitted directly to the available data, since we need to take the course of the disease into account. for a given registration date t, the number of deaths of patients registered as positive on that day, y t,r,g , may not yet be known, since not all patients with a fatal outcome of the disease have died yet. this requires the implementation of nowcasting. we do this on a national level, and cumulate the numbers over district/region r and gender and age groups g. this allows to drop the corresponding subscripts in the following and we simply notate the cumulated number of deaths with registered infections at day t with y t . let n t,d denote the number of deaths reported on day t + d for infections registered on day t. assuming that the true date of death is at t+d, or at least close to it, we ignore any time delays between time of death and its notification to the health authorities. we call d the duration between the registration date as a covid-19 patient and the reported day of death, where d = 1, . . . , d max . here, d max is a fixed reasonable maximum duration, which we set to 30 days (see e.g. wilson et al., 2020) . the minimum delay is one day. in nowcasting we are interested in the cumulated number of deaths for infections registered on day t, which we define as the total number of deaths with a registered infection at t is apparently unknown at time point t and becomes available only after d max days. in other words, only after d max days we know exactly how many deaths occurred due to an infection which was registered on day t. we define the partial cumulated sum of deaths as on day t = t , when the nowcasting is performed, we are faced with the following data constellation, where na stands for not (yet) available: we may consider the time span between registered infection and (reported) death as a discrete duration time taking values d = 1, . . . , d max . let d be the random duration time, which by construction is a multinomial random variable. in principle, for each death we can consider the pairs (d i , t i ) as i.i.d. and we aim to find a suitable regression model for d i given t i , including potential additional covariates x t,d . we make use of the sequential multinomial model (see agresti, 2010) and define π(d; t, x t,d ) = p (d = d|d ≤ d; t, x t,d ) let f t (d) denote the corresponding cumulated distribution function of d which relates to probabilities π() through f t (d) = p t (d ≤ d) = p(d ≤ d|d ≤ d + 1) · p (d ≤ d + 1) = (1 − π(d + 1; ·)) · (1 − π(d + 2; ·)) · . . . · (1 − π(d max ; ·)) for d = 1, . . . , d max − 1 and f t (d max ) = 1. the available data on cumulated death counts allow us to estimate the conditional probabilities π(d; ) for d = 2, . . . , d max . in fact, the sequential multinomial model allows to look at binary data such that where • s 1 (t) is an overall smooth time trend over calendar days, • s 2 (d) is a smooth duration effects, capturing the course of the disease, • x t,d are covariates which may be time and duration specific. assuming that d, the duration between a registered fatal infection and its reported death, is independent of the number of fatal covid-19 infections, we obtain the relationship note further that if we model y t with a quasi-poisson model as presented in the previous chapter, we have no available observation y t for time points t > t − d max . instead, we have observed c t,t −t , which relates to the mean of y t through (7). including therefore log f t (t − t) as additional offset in model (2), allows to fit the model as before, but with nowcasted deaths included. that means, instead of λ t,r,g as in (2), the expected death rates are now parametrized by λ t,r,g = λ t,r,g exp(log f t (t −t)), where the latter multiplicative term is included as additional offset in the model. we fit the nowcasting model (5) with parametrization (6). we include a weekday effect for the registration date of the infection with reference category "monday". the estimates of the fixed linear effects are shown in table 2 . the fitted smooth effects are shown in figure 4 , where the top panel shows the effect over calendar time, which is very weak and confirms that the course of the disease hardly varies over time. this shows that the german health care system remained stable over the considered period, and hence survival did not depend on the date on which the infection was notified. the bottom panel of figure 4 shows the course of the disease as a smooth effect over the time between registration of the infection and death. we see that the probabilities π(d; ·) decrease in d, where this effect is the strongest in the first days after registration. thus, most of the covid-19 patients with fatal infections are expected to die not long after their registration date. the effect of d becomes easier to interpret by visualizing the resulting distribution function f t (d). this is shown in figure 5 for two dates t, i.e.. april 14th and may 13th. the plot also shows how the course of the disease hardly varies over calendar time: in fact, the small differences between the two distribution functions is dominated by the weekday effect, since the red curve is related to a tuesday while the blue one is from a wednesday. in figure 3 above we have shown the nowcasting results along with uncertainty intervals shaded in grey. these were constructed using a bootstrap approach as follows. given the fitted model, we simulate n = 10 000 times from the asymptotic joint normal distribution (7), where c t −t is the observed partial cumulated sum of deaths at time point t − t. the pointwise lower and upper bounds of the 95% prediction intervals for the nowcast for y t are then given by the 2.5 and the 97.5 quantiles of the set { y (i) t , i = 1, . . . , n}, respectively. in section 3 we presented the fitted death rate, which is the convolution of a smooth spatial effect as well as region specific effects. it is of general interest to disentangle these two spatial components. this is provided by the model. we visualize the fitted global geographic trend figure 7: long term region specific level (left hand side) and short term dynamics (right hand side) of the covid-19 infections m 2 (·) for germany in figure 6 . the plot confirms that up to may 2020 the northern parts of the country are less affected by the disease in comparison to the southern states. the two plots in figure 7 map the region specific effects, i.e. the predicted long term level of a district u r0 (left hand side) and the predicted short term dynamics u r1 (right hand side). both plots uncover quite some region-specific variability. in particular, the short term dynamics captured in the right hand side plot (u r1 ) pinpoint districts with unexpectedly high nowcasted death rates in the last two weeks, after correcting for the global geographic trend and the long term effect of the district. some of the noticeable districts have already been highlighted in section 3 above, but we can detect further districts, which are less pronounced in figure 1 . for instance, steinfurt (in the north-west of north rhine-westphalia), olpe (southern north rhine-westphalia) or gotha (center of thringen) presently show a high rate of fatal infections. a large number of the registered deaths related to covid-19 stem from people in the age group 80+. locally increased numbers are often caused by an outbreak in a retirement home. such outbreaks apparently have a different effect on the spread of the disease, and the risk of an epidemic infection caused by outbreaks in this age group is limited. thus, the death rate of people in the age group 80+ could vary differently across districts when compared to regional peaks in the death rate of the rest of the population. in order to respect this, we decompose the district-specific effects u r in (2) into u 80− r = (u 80− r0 , u 80− r1 ) for the age group 80-and u 80+ r = (u 80+ r0 , u 80+ r1 ) for the age group 80+, where the age group 80-consists of the aggregated age groups 15-34, 35-59 and 60-79. we put the same prior assumption on the random effects as we did in (3), but now the variance matrix that needs to be estimated from the data has dimension 4 by 4. the fitted age group-specific random effects are shown in figure 8 , where the u 80− r are shown in the top panel and the u 80+ r in the bottom panel. most evidently, the variation of the random effects is much higher in the age group 80+ when compared to the younger age groups, as more districts occur which are coloured dark blue or dark red, respectively. when comparing the district-specific short term dynamics of the last 14 days (u r1 ) in figure 8 to those in figure 7 , we recognize that in most of the districts which recently experienced very high death intensities (with respect to the whole period of analysis), these stem from the age group 80+. as mentioned before, this can often be explained by outbreaks in retirement homes. when fitting the mortality model (1) we included the fitted nowcast model as offset parameter. this apparently neglects the estimation variability in the nowcasting model, which we explored via bootstrap as explained in section 5.3 and visualized in figure 3 . in order to also incorporate this uncertainty in the fit of the mortality model, we refitted the model using (a) the upper end and (b) the lower end of the prediction intervals shown in figure 3 . it appears that there is little (and hardly any visible) effect on the spatial components, which is therefore not shown here. but the time trend shown in figure 1 does change, which is visualized by including the two fitted functions corresponding to the 2.5% and 97.5% quantile of the offset function. we can see that the estimated uncertainty of the nowcast model mostly affects the last ten days, with a strong potential increase in the death rate mirroring a possible worst case scenario. in figure 9 we show a normal qq-plot of the pearson residuals in the nowcasting model. apart from some observations in the lower tail, the pearson residuals are distributed very closely to a standard normal distribution when considering the estimate φ = 1.766 of the dispersion parameter in the quasi-poisson model (7). overall, the model seems to fit to the available data quite well. the paper presents a model to monitor the dynamic behaviour of covid-19 infections based on death counts. it is important to highlight that the proposed model makes no use of new infection numbers, but only of observed deaths related to covid-19. this in turn means that the results are less dependent on testing strategies. the nowcasting approach enables us to estimate the number of deaths following a registered infection today, even if the fatal outcome has not occurred yet. moreover, the district level modelling uncovers hotspots, which are salient exclusively through increased death rates. a differential analysis of the number of current fatal infections on a regional level allows to draw conclusions on the current dynamics of the disease assuming a constant case fatality rate, i.e. a stable proportion of death compared to the true number of infections when adjusting for age and gender. a natural next step would now be to consider the nowcasted deaths in relation to the number of newly registered infections, which is, in contrast, highly dependent on both testing strategy and capacity. we consider this as future research, and the proposed model allows us to explore data in this direction. this might ultimately help us in shedding light on the relationship between registered and undetected infections as well as on the effectiveness of different testing strategies. there are several limitations to this study which we want to address as well. first and utmost, even though death counts are, with respect to cases counts, less dependent on testing strategies, they are not completely independent from them. this applies in particular to the handling of post-mortem tests. we therefore do not claim that our analysis of death counts is completely unaffected by testing strategies. secondly, a fundamental assumption in the model is the independence between the course of the disease and the number of infections. overall, if the local health systems have sufficient capacity and triage can be avoided, this assumption seems plausible, but it is difficult or even impossible to prove the assumption formally. finally, the nowcasting itself is not carried out on a regional level, though the model focuses on regional aspects of the pandemic. while it would be desirable to fit the nowcast model regionally, the limited amount of data simply prevents us from extending the model in this direction. analysis of ordinal categorical data real estimates of mortality following covid-19 infection countries test tactics in 'war' 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of publicly available case data covid-19: forecasting short term hospital needs in france. medrxiv spatio-temporal analysis of epidemic phenomena using the r package surveillance quantifying bias of covid-19 prevalence and severity estimates in wuhan, china that depend on reported cases in international travelers infection fatality rate of sars-cov-2 infection in a german community with a super-spreading event smoothing and mixed models case-fatality risk estimates for covid-19 calculated by using a lag time for fatality generalized additive models: an introduction with r we want to thank maximilian weigert and andreas bender for introducing us to the art of producing geographic maps with r. moreover, we would like to thank all members of the corona data analysis group (codag) at lmu munich for fruitful discussions. key: cord-256635-zz58w3ro authors: beermann, sandra; allerberger, franz; wirtz, angela; burger, reinhard; hamouda, osamah title: public health microbiology in germany: 20 years of national reference centers and consultant laboratories date: 2015-08-21 journal: int j med microbiol doi: 10.1016/j.ijmm.2015.08.007 sha: doc_id: 256635 cord_uid: zz58w3ro in 1995, in agreement with the german federal ministry of health, the robert koch institute established a public health microbiology system consisting of national reference centers (nrcs) and consultant laboratories (cls). the goal was to improve the efficiency of infection protection by advising the authorities on possible measures and to supplement infectious disease surveillance by monitoring selected pathogens that have high public health relevance. currently, there are 19 nrcs and 40 cls, each appointed for three years. in 2009, an additional system of national networks of nrcs and cls was set up in order to enhance effectiveness and cooperation within the national reference laboratory system. the aim of these networks was to advance exchange in diagnostic methods and prevention concepts among reference laboratories and to develop geographic coverage of services. in the last two decades, the german public health laboratory reference system coped with all major infectious disease challenges. the european union and the european centre for disease prevention and control (ecdc) are considering implementing a european public health microbiology reference laboratory system. the german reference laboratory system should be well prepared to participate actively in this upcoming endeavor. public health microbiology laboratories play a central role in detecting infectious disease, monitoring outbreak response and providing scientific evidence to prevent and control disease. they have important roles and responsibilities associated with accurate diagnosis, resistance testing and prevention of the spread of infectious disease. for example, outbreak investigations often depend on confirming cases by methods that are not commonly available in a routine laboratory setting. the scientific community, policy makers and pharmaceutical companies rely on advice and information from reference laboratories in order to adjust vaccine and antibiotic production (witze et al., 2014) . according to the european centre for disease prevention and control (ecdc), the five key activities of public health microbiology reference laboratories are reference diagnostics; reference material resources; scientific advice; collaboration and research; and monitoring, alerting and responding (european centre for disease prevention and control, 2010) . in the various european countries, microbiology reference laboratories are defined, organized, maintained and operated differently. we present an overview of germany's public health reference laboratory system. germany is a highly industrialized country with 82 million inhabitants, and is made up of 16 federal states ("länder"). the principal responsibility for public health lies with the 16 states, or with their ministries of health, and with the almost 400 local public health departments. since the 1980s, the federal government ("bundesregierung"), federal assembly ("bundestag") and federal council ("bundesrat") have increasingly taken responsibility for healthcare reform and legislation. specific health issues, such as infectious diseases that threaten public safety and life cycle management of pharmaceuticals, are within the jurisdiction of the federal government. for example, the german protection against http://dx.doi.org/10.1016/j.ijmm.2015.08.007 1438-4221/© 2015 elsevier gmbh. all rights reserved. infection act ("infektionsschutzgesetz," ifsg) as a federal law regulates the prevention and management of infectious diseases in humans. federated states are responsible for all primary aspects of public health, but there are also responsible for the implementation of federal laws, including federal social and labour laws. the robert koch institute (rki) is a federal institute within the portfolio of the federal ministry of health (bundesministerium für gesundheit, bmg). as such the rki is the central federal reference institution in the public health sector responsible for disease monitoring, control and prevention and conducting applied and response-oriented research in the field of disease control and prevention at the federal level. the research activities of the rki are partly directly related to the activity fields of a ministry. although robert koch and his contemporaries built a strong tradition for infectious disease epidemiology in germany in the late 19th and early 20th centuries, this tradition had all but disappeared in the 1930s and 1940s (allerberger, 2013) . in former west germany, the work of the rki as part of the then federal health office (bundesgesundheitsamt, bga) mainly focused on basic science research. the aids epidemic demanded a national public health response which resulted in the creation of the national aids centre in 1988. in 1994, when the bga was dissolved and the rki was assigned additional spheres of competence a combined aids center and infectious disease epidemiology division was created at the rki. in 1995, representatives of the rki, the federal ministry of health and the federal ministry for education and research developed the concept of a network of collaborators whose goal was to intensify epidemiological research and improve infectious disease surveillance (fock et al., 1995) . as part of this concept, the rki implemented a weekly epidemiological bulletin, formed the committee for infectious disease epidemiology, trained epidemiologists for surveillance and outbreak investigation and set up a system of national reference laboratories: national reference centers (nrcs) and consultant laboratories (cls) (petersen et al., 2000) . they were responsible for laboratory surveillance of important pathogens and syndromes. these laboratories are considered national centers of excellence in the field of laboratory science for a particular pathogen or group of pathogens. nrcs establish and use reference methods, and can validate and verify test results from other laboratories (confirmatory testing). nrcs also produce and distribute reference materials for external quality control and assurance. owing to the high level of expertise, resources and infrastructure, nrcs and cls are involved in training and in providing expert advice to national health authorities and other laboratories. moreover, these laboratory scientists work closely together with their epidemiologist counterparts at the rki as well as those at the federal, state and local levels. the nrcs focus on outbreak detection and response and advice the rki in the preparation of case definitions according to the protection against infection act (ifsg). furthermore, the reference laboratories conduct or are involved in laboratory surveillance systems which provide additional information complementing statutory notifications. nrcs and cls are also involved in developing rki guidelines for physicians ("ratgeber für ärzte") as well as investigating outbreaks and conducting epidemiological studies. the following are the basic tasks of nrcs and cls, which include detailed requirements referring to specific pathogens or syndromes as listed in the respective calls for tenders: general catalogue of nrc tasks (1) developing or improving diagnostic procedures; coordinating standardization and distribution of generally accepted test procedures; initiating investigations for quality assurance. (2) diagnosing and subtyping pathogens beyond routine measures, including molecular biological studies to elucidate the epidemiological context. (3) maintaining a strain collection and distributing reference strains or diagnoses of specific reference strains, with the exception of commercially available isolates, such as from the american type culture collection (atcc) and the german collection of microorganisms and cell cultures (dsmz). (4) organizing and coordinating the upkeep of a network of diagnostic facilities. (5) providing a consulting service for public health services laboratories, practicing physicians, hospitals and research institutes; implementing continuing education and handling public relations. (6) collaborating with reference laboratories of other countries as well as collaborating centers of the who, including participating in international ring trials. (7) evaluating and interpreting data in coordination with the rki with the aim of best describing the epidemiological situation relevant for germany; initiating and participating in surveillance projects. (8) monitoring incoming data with the goal of timely detection of outbreaks or outbreak hazards as well as immediate communication with the rki; support of public health services and the rki with complementary studies during outbreak investigations. (9) epidemiological analysis and evaluating the development of resistance and virulence. (10) reporting routinely to and consulting with the rki on relevant issues; participating in developing rki recommendations for diagnostics, therapies and prevention as well as for applied epidemiology of infectious diseases in general. general catalogue of cl tasks 1. consulting (especially with the public health services as well as laboratories, practicing physicians, hospitals and research institutes). 2. working within the framework of quality assurance (participating in studies and inter-laboratory tests, e.g., in cooperation with instand (german eqas), who, eu, and professional associations and participating in further education). 3. improving or developing diagnostic procedures. 4. participating in epidemiological evaluations of the current situation by the rki. 5. carrying out studies within the network of diagnostic facilities. 6. consulting with the rki in developing scientific materials concerned with pathogens or symptoms (e.g., case definitions, rki guidelines for physicians). the number of nrcs increased from 12 in 1995 to 15 in 2009. presently, 19 nrcs have been appointed (table 1) . five laboratories are situated at the rki; the others are located at various universities and research facilities in germany. since 1996, 46 cls have decreased to 40 designated cls, mainly devoted to providing scientific advice (table 2) . currently a total of 59 nrcs and cls located at universities, federal or state institutes and private laboratories are supported for this function by the rki. the high relevance of nrc and cl work for the surveillance of infectious diseases is evident by the wide range of national and international publications. for example, the nrc for mycobacteria and the rki performed analyses of routine laboratory diagnosis data of pediatric tuberculosis in the european union/european economic area (sanchini et al., 2014) . the nrc for helicobacter pylori and the rki examined h. pylori resistance to antibiotics in europe and its relationship to antibiotic consumption (megraud et al., 2013) . another example is the work of the streptococci nrc, which studied the epidemiology of streptococcus pneumoniae serogroup 6 isolates from invasive pneumococcal disease in children and adults in germany (van der linden et al., 2013) . nrcs and cls are also involved in outbreak investigations and epidemiological studies. for instance, the cl for coronaviruses performed contact investigation for an imported case of middle east respiratory syndrome (reuss et al., 2014) , and the influenza nrc was involved in detecting local influenza outbreaks (schweiger and buda, 2013) . the rki and the nrc for surveillance of nosocomial infections examined the question, "how many outbreaks of nosocomial infections occur in german neonatal intensive care units annually?" (schwab et al., 2014) . additionally, the cl for legionella was involved in examining a legionnaires' disease outbreak associated with a cruise liner in august 2003 (beyrer et al., 2007) . dengue virus infections in a traveler returning from croatia to germany were analyzed by the nrc for tropical infection agents (schmidt-chanasit et al., 2010) . the nrc and the rki for meningococcal diseases and h. influenzae examined a cluster of invasive meningococcal disease in young men who have sex with men in berlin (marcus et al., 2013) . nrcs and cls are also involved in evaluating implemented vaccination recommendations and analyzing the effectiveness of the vaccines (kalies et al., 2009; ruckinger et al., 2009 ). for which pathogen a reference laboratory is to be established is decided based on the public health relevance of the pathogen as appraised by the rki and on the needs expressed by the national public health services ("öffentlicher gesundheitsdienst," ögd) . in addition, medical professional societies, the federal ministry of health and other third parties can approach the rki with perceived needs for additional reference laboratories. in the next step, the advisory board for public health microbiology (formerly called the committee for infectious disease epidemiology) assesses the proposal and provides the rki with a recommendation on whether to set up a new laboratory. in addition to the epidemiological relevance, and a declared need from national public health services, the availability of financial resources is another essential criterion. the decision to establish or continue an nrc or a cl is made by the rki, which considers recommendations given by the advisory board for public health microbiology, and must be confirmed by the federal ministry of health. appointments are restricted to three-year periods. the advisory board consists of up to 14 experts, appointed by the rki for periods of three years. the members of this advisory forum are renowned experts in the fields of microbiology, virology, hygiene, epidemiology and public health. occasionally, other national and international professional societies and experts are consulted to achieve a solid appraisal of the candidate laboratories. from 2009 to 2012, numerous important modifications were made to improve the transparency of the tendering and selection processes for the nrcs and the cls. a strict prioritization process, based upon necessity and not upon offer, was implemented. the evaluation process became more rigorous. essential evaluation criteria are public health needs and public health relevance, successful network activities, attestable quality assurance, publications as well as a positive appraisal of the advancement of diagnostic procedures. at the end of each appointment period, an evaluation of the laboratories is performed by the rki in cooperation with the advisory board for public health microbiology, which again consults national and international professional societies and experts. based on the evaluation results, the president of the rki, in cooperation with the federal ministry of health, appoints and reappoints the nrcs and cls. the 2013 evaluation of the cls resulted in the reappointment of 40 cls and the shutdown of nine cls. reasons for closing were, for example, the retirement of the laboratory head (appointments are based on the combination of personal and institutional expertise), decreased public health relevance of the pathogen or an overlapping of the functional areas of responsibility with other cls or nrcs. in the 2013 evaluation of the nrcs, all 19 nrcs were reappointed. in 2007, the 16 nrcs were supported with d1540,000 in total. in 2008, the available funding increased to d2173,000. the nrcs received between d57,000 and d241,000 per year. the decision on the level of funding of individual nrcs is made by the rki, based on criteria such as high consultation effort, high sample appearance and extraordinary public health relevance of the pathogen. in contrast to the nrcs, which have always been financially supported, the cls initially performed their work (mainly consultation) without any financial support. from april to december 2009, the cls received basic funding of d5000 per year (total amount in 2009: d2173,000). in 2010, the available funding increased to d2612,000. the increase in funding was used to upgrade the cls' basic funding to around d10,000 per year. beyond that, the funds had to cover new national networks. in 2014, the 19 nrcs received between d60,000 and d253,000 per year. thirty-three of the cls get d10,200, and seven cls with a high number of samples and extraordinary public health relevance of the pathogen received d16,000 per year. the network projects were funded with approximately d380,000 in 2014. the nrcs have a more comprehensive work package than the cls and therefore the nrc receive a higher funding. public funding does not and cannot cover the total costs of the reference laboratories. since 2010, the sphere of action and the workload of the laboratories have increased due to advancement of methods. at the same time costs in general have increased, but the grant total has remained unchanged. currently, funding increases for individual laboratories can occur only through money shifting from one laboratory to another or through giving up funding for existing nrcs or cls. in order to maintain the current quality and scope an increase in funding for nrcs and cls is urgently needed. since 2005, the rki, in close cooperation with the advisory board for public health microbiology, has worked to foster collaboration between and among the nrcs and the cls. this concept was amended in a workshop with representatives of public health microbiology laboratories of other eu member states in 2008. ten nrc networks were launched at a work conference of the standing working group of nrcs and cls ("ständige arbeitsgruppe nrz/kl") in stuttgart in 2009. these networks covered the following topics: respiratory tract infections; enteral infections; infections in patients with immune deficiency or pregnancy; invasive bacterial infections; zoonoses; mycoses; sexual and blood transmitted infections; infections of the nervous system; antimicrobial resistance; and parasitoses, tropical and vector-borne infections. the aim of these networks was to facilitate the exchange of diagnostic methods among the nrcs and cls, to improve collaboration in planning and performing studies and to enlarge the geographic coverage of these services. furthermore, these networks should provide opportunities to work on issues beyond single pathogens. scientific coordination and administration are supervised by the rki. the advisory board for public health microbiology and external experts play a pivotal role in selecting the proposed network projects. essential selection criteria are public health relevance and the scientific quality of the proposal, the prospect of success and the cost efficiency of the planned network project. moreover, it is important that these projects contribute to expanding the network's characteristics. an exclusion criterion is if the project addresses established nrc or cl tasks. in 2010, the network projects were funded with d400,000 per year, allocated to ten projects (duration 1.5 years). as of 2011, network projects ran for three years. in the funding period 2011-2013, the rki supported eight projects. within the scope of the projects, common database infrastructures were set up, such as tissue material and serums. other projects performed cross-sectional studies to ascertain data on the prevalence and incidence of different pathogens. in 2014, the rki evaluated the present composition and structure of the networks. the evaluations revealed that difficulties with ethical approval and with compliance with data protection and juridical aspects were the most commonly experienced hurdles during the study planning process. recruiting participating laboratories was also a challenge for some projects. it became clear that early involvement of epidemiological and statistical experts is necessary to further optimize study design and case number planning for the specific research questions and to raise the prospects of success for the projects. in addition to this evaluation, the rki organized a network meeting in 2014, in which all nrcs and cls were represented. members of the advisory board for public health microbiology and representatives of the federal ministry of health participated. at this meeting, the networks shared their experiences. potential for improvement from the perspective of the members of the nrcs and cls as well as from the rki and the advisory board for public health microbiology was identified and discussed. as a consequence of this meeting, the rki initiated the following changes: (1) regular network meetings with all nrcs and cls to address the stated need for regular face-to-face meetings, the rki will organize network meetings every three years. the meetings will take place one year before the start of the upcoming funding period for network projects, so that the nrcs, the cls and the rki can elaborate on the content and structure of project submissions. (2) basic funding for the networks the rki will provide annual basic funding to allow for separate meetings of the respective networks, to facilitate exchange among network participants regardless of successful project applications. these meetings can be used for more intensive preparation of new project proposals and should strengthen network cohesion. (3) stronger presence of the networks on the internet to satisfy network demand for the presentation of network projects to a larger professional audience, accepted projects will be presented on the rki's internet site. (4) decrease in the number of funded projects in the past, the rki funded up to eight projects; in 2014, the institute decided to decrease the number of projects funded per period in the future. in the current funding period four projects were selected for funding. in the following period only two projects will be financed. that implies higher financial support for single projects, which could be used to employ a study coordinator. (5) two-stage application procedure for network projects the rki installed a two-stage application procedure for network projects. as the first step, the networks formulate short pre-applications. the rki screens these short concepts for network projects with the help of the advisory board for public health microbiology and external experts. in the case of positive assessment, the networks are asked to submit a detailed project proposal for final evaluation. during the last 20 years, the field of public health microbiology has seen many changes. the everyday work of local public health agencies depends on the professional expertise of national reference centers (nrcs) and consultant laboratories (cls). meanwhile, the public often sees the relevance of public health microbiology only within the context of serious health events. during periods of restricted financial resources, the need for public health infrastructures is consistently questioned. the large ehec o104 outbreak in hamburg during 2011 provides an example of the importance of public health laboratory infrastructures (frank et al., 2011) . during the hamburg outbreak caused by fenugreek sprouts, the german public health system successfully investigated and controlled the outbreak, which would not have been possible without support from the nrc for enteral infections and the cl for hemolytic uremic syndrome (hus). this support would not have been possible without these highly specialized laboratory structures. the work of all other nrcs and cls is also highly relevant, since their work and expertise help in the efforts to contain and prevent higher levels of infectious disease. nevertheless, there is also room for improvement in germany. for example, the anticipating of new outbreak situations that might require cooperation with the responsible veterinarian and food authorities or with other national authorities should be the focus of optimization plans. the creation of a prospective network of eu-wide public health microbiology reference laboratories is currently being discussed within the european union, which will have consequences for the public health laboratory systems of each member state. from this perspective, considerable future challenges to the german public health laboratory system can already be foreseen. thus, the structures established during the past 20 years should be adaptable so that the responding public health infrastructures can react adequately to the upcoming challenges. sandra beermann, franz allerberger, angela wirtz, osamah hamouda and reinhard burger have no financial disclosures to declare. structural requirements and conditions for effective microbiological diagnostics in disease outbreaks legionnaires' disease outbreak associated with a cruise liner core functions of microbiology reference laboratories for communicable diseases epidemiology of infection in germany large and ongoing outbreak of haemolytic uraemic syndrome prioritisation of infectious diseases in public health: feedback on the prioritisation methodology invasive haemophilus influenzae infections in germany: impact of non-type b serotypes in the post-vaccine era a cluster of invasive meningococcal disease in young men who have sex with men in berlin helicobacter pylori resistance to antibiotics in europe and its relationship to antibiotic consumption developing national epidemiologic capacity to meet the challenges of emerging infections in germany contact investigation for imported case of middle east respiratory syndrome reduction in the incidence of invasive pneumococcal disease after general vaccination with 7-valent pneumococcal conjugate vaccine in germany laboratory diagnosis of paediatric tuberculosis in the european union/european economic area: analysis of routine laboratory data dengue virus infection in a traveller returning from croatia to germany how many outbreaks of nosocomial infections occur in german neonatal intensive care units annually? detection of local influenza outbreaks and role of virological diagnostics epidemiology of streptococcus pneumoniae serogroup 6 isolates from ipd in children and adults in germany scientific advice: crisis counsellors we thank all nrcs and cls for their excellent work during the last 20 years. key: cord-285162-srkd3wh0 authors: jung, f.; krieger, v.; hufert, f.t.; küpper, j.-h. title: how we should respond to the coronavirus sars-cov-2 outbreak: a german perspective date: 2020-06-05 journal: clinical hemorheology and microcirculation doi: 10.3233/ch-209004 sha: doc_id: 285162 cord_uid: srkd3wh0 background: in the early phase of the covid-19 pandemic germany missed to set up efficient containment measures. consequently, the number of cases increased exponentially until a lockdown was implemented to suppress the spread of sars-cov-2. fortunately, germany has a high capability for coronavirus lab testing and more than 30,000 icu beds. these capabilities and the lockdown turned out to be an advantage to combat the pandemic and to prevent a health-system overload. aim: the aim was to predict the plateau day of sars-cov-2 infections or deaths. results: the effect on the viral spread of the german measures taken and the impact on the peak of new infection cases is shown. by normalizing daily case numbers, the plateau day of the current outbreak in germany could be calculated to be reached at april 12, 2020 (day 103 of 2020). conclusion: normalized case number curves are helpful to predict the time point at which no further new infections will occur if the epidemic situation remains stable. upon reaching the plateau day during a lockdown phase, a residual time-period of about 2-3 weeks can be utilized to prepare a safe unlocking period. as can be learned from asian countries such as south korea and taiwan there must be strict rules to keep the risk of infection low. those include social distancing, face mask wearing in combination with digital contact tracing and serosurveillance studies. following those rules, a safe dance around the infection curve allows to keep the population at a reduced infection rate. in december 2019, a novel coronavirus emerged in the metropolis of wuhan, china, causing a severe lung disease. on december 31, china informed the who of a total of 27 patients with pneumonia, and already on january 7, 2020, chinese scientists succeeded in identifying the infectious agent. the new coronavirus sars-cov-2 is highly related to the well-known bat-borne sars-cov which emerged in february 2003 [1, 2] and to the middle east respiratory syndrome coronavirus (mers-cov) detected in 2015 [3] . the 2003 global sars outbreak spread to more than two dozen countries in north america, south america, europe, and asia before it was contained. more than 8,000 cases with a mortality of 10-50% depending on age occurred globally [4, 5] . on january 11, 2020, china reported the first death from the new disease covid-19. china reacted with severe counter measures including quarantine and complete highly controlled lockdown of the affected areas. in the following week first cases outside of china were reported from thailand and japan which were imported from wuhan and first evidence of human to human transmission was reported. on january 21, the first imported case appeared in the usa and on january 24, sars-cov-2 emerged globally in many other countries including europe where first cases were reported from france [6] [7] [8] . on january 26, china reported 2000 confirmed cases and 56 covid-19 deaths and measures to contain the spread were strengthened. already on january 23, the chinese government ordered the complete lockdown of social and economic life in wuhan city, later followed by nationwide closure of schools and universities. on january 27, the infection was detected in germany for the first time. an employee of the bavarian company webasto was infected by a chinese visitor to the company who later tested positive for sars-cov-2 after her return home to china and was apparently almost symptom-free in germany. on january 30, the who declared the status of health emergency because of covid-19. however, the federal authority for infectious diseases in germany, robert koch institute (rki), still defined the risk for germany as being low and did not recommend to close borders and stop incoming flights to germany. the experts believed that all emerging sars-cov-2 cases were under control and contact persons quarantined. however, from that time point on the outbreak within germany increased rapidly because dozens of sars-cov-2 infected people returned from ski vacation in tyrol and from italy. failure to impose an early ban on entry into the country from the risk areas in austria, italy and china was a serious mistake, particularly when the strategy to combat the outbreak is based on eradication. besides that, in germany the federal structures of the public health service hampered a straight-forward approach to fight the pandemic. despite the fact that there was strong evidence of rapid person-to-person transmission [9] even before classical clinical symptoms of a respiratory disease were present [10] carnival meetings were held in different regions such as in the district of heinsberg and other cities in the west and southwest of germany pouring oil into the fire of the outbreak. as a result, on march 10, over 300 people in the heinsberg district tested positive for sars-cov-2. on march 17, the rki classified the risk situation for germany as moderate to high. until this point, there were already more than 9,000 confirmed sars-cov-2 cases and 26 covid-19-related deaths in germany. the german public learned about the strategy of herd immunity meaning that at least 60% of the population will be infected to create a protective barrier. at this stage, there was no reliable information on covid-19 mortality. the who calculated the case fatality rate to be 3-4 %, with the true infection fatality rate to be much lower (who situation report 46 as of march 6, 2020). assuming an infection fatality rate of 0.5 % for sars-cov-2, herd immunity of the german population would generate about 250,000 deaths -by covid-19 only. in addition, there would have been further deaths due to massive overload of the german health system. on march 18, german chancellor angela merkel for the first time addressed the population directly in a speech on the coronavirus outbreak. she described the situation as follows: "it is serious. take it seriously, too!" since world war ii, there has been no challenge to the country where national solidarity was so important as right now, she said. on march 22, following a consultation with the federal state's prime ministers, the german chancellor tightened up the measures and announced a total of nine rules of conduct for germany to be valid from midnight on monday, march 23. the central point was "to reduce public life as far as it is justifiable". this included limiting contacts to persons other than those living in the same household to the bare minimum, keeping a minimum distance of at least 1.5 m in public, only two persons not living in the same household are allowed to meet, people are still allowed to go to work, to the doctor, to shop, to do outdoor sports alone, but parties in groups or meetings in parks were not allowed any longer. service and catering establishments as well as restaurants were closed. these guidelines were initially valid for two weeks. universities, schools, and kindergartens were already closed on march 16. until the first day of lockdown in germany on march 23 (day 83; day zero: 01/01/2020), about 29,000 people were already tested positive for the virus. until april 12 (day 103), 127,459 cases and 2996 deaths due to covid-19 were identified in germany. figure 1 shows that until march 20 (day 80), the daily cases of new confirmed infections increased with doubling times between 1-5 days, showing a strong exponential rise of positive tests for sars-cov-2 infections in germany. however, it is unlikely that the obvious decline of the curve after day 80 already reflects official counter measures of the german government. there is a delay of at least 10 days between an infection event and the registration of a positive test due to the virus incubation time of at least 5 days, the test time and the time until the positive result is reported to the authorities. cumulative cases reported until march 20 reflect infection events until march 10, i.e. at a time point when the german public was not officially warned about the covid-19 risks. however, it is possible that the number of positive tests at day 80 was still limited by the overall capacity of pcr-based sars-cov-2 detection. one week after the initial lockdown, on march 30 (day 90), the highest number of new cases per day was reported (fig. 1) . thereafter, the number of new daily cases started to decline continuously. doubling times show a flat course over the first 90 days. then they started to increase strongly by about day 100 (april 9, 2020). at this time point, the test capacity was almost doubled in germany. thus, the declining number of new cases of persons with covid-19-like symptoms should not have been affected any longer by the pcr testing capacity. this result should thus reflect the counter measures of the german government, especially the lockdown since march 23, and the substantial discussions of experts and politicians in public media of germany. doubling times were then steadily increasing, reaching 30 days or more since day 106. korea and japan to document the different strategies followed during the covid-19 crisis. it is obvious that in the east-asian countries measurements were taken right at the beginning of the sars-cov-2 pandemic to contain the virus spread. taiwan and south korea used their knowledge from the first sars pandemic in 2003 and the 2015 outbreak of mers-cov. in south korea, where a religious community initiated a fatal infection cluster in the city of daegu, schools were closed soon, infected persons were efficiently tracked with smartphone apps and rigorous testing for sars-cov-2 infections were performed [3, 11] . taiwan used a combination of big data analytics, community protection and rigorous testing to combat the crisis. as being closely located to the mainland of china, taiwan was at high risk for outbreak of covid-19, but the country was able to implement fast and efficient counter measures [12, 13] . by the end of february 2020, the government of japan recommended closing of schools, entry ban of people from coronavirus risk regions and a stop of sports and cultural events. these early reactions and the fact that the japanese are used to wearing face masks during seasonal influenza [14] seemed to help combat the sars-cov-2 outbreak until end of march 2020. after a period of stagnation, cases in japan were reported to increase again as people were reducing their social distancing in public. however, the total number of confirmed cases is still much lower than reported for european countries. common elements of these asian states were the immediate action of governments to implement certain social distancing strategies and the wearing of face masks in public to reduce the number of new cases, which has proven to be effective to prevent transmission from infected individuals [15] . by contrast, the three european states had some delay in their national responses to the sars-cov-2 pandemic. at the starting points of the outbreak during the end of january 2020, there were neither discussions on travel entry bans nor recommendations on social distancing, and wearing of face masks in the public was also not recommended. this led to a longer phase of exponential growth of sars-cov-2 infections and deaths in germany, france and italy and caused cumulative case numbers to grow significantly higher in comparison to the east-asian countries (fig. 2) . the data were obtained from the following sources: taiwan, south-korea: and japan: www.ecdc. europa.eu/en/publications-data/download-todays-data-geographic-distribution-covid-19-cases-world wide; germany: https://www.rki.de/de/content/infaz/n/neuartiges coronavirus/ fallzahlen.html; france: who.sprinklr.com/region/euro/country/fr, italy: github.com/pcm-dpc/covid19) . the data obtained from the above listed sources is put in to a context described herein with. our policy regarding the information format is prioritizing open source and free software. we therefore make all data retrieved and analyzed hereby available at corona.milliways.online. due to the imperative of social distancing and the lockdown decreed in european countries, the increase in case numbers flattened out considerably. figure 2 shows that for germany the lockdown could allow to keep the cumulative number of cases below 150-200 thousand. this clearly would prevent the collapse of the health system in germany. this is best seen in logarithmic representation. the scope of this work is primarily to provide a forecast for the time when theoretically there will be no more growth of confirmed cases. at that time point the growth of values (e.g. corona cases confirmed) is zero -resulting also in zero slope of the curves in fig. 2 . however, it is not possible to read from this cumulative diagram the exact point in time when no more cases should occur, as the slope at the peak is getting flatter. to overcome this problem, one can plot normalised growth rates (corona cases at day n -corona cases at day n-1) / corona cases at day n) against a linear timeline. this normalization keeps each rate of change in the range between 0% and 100%. by plotting these normalized change rates against the standardized day counts, an approximate linear behaviour can be observed. the approximation lines meet the x-axis at the day when no further infections or deaths will occur -provided that no systematic changes in the underlying social epidemic behaviour occur in the following days. we call this day the "plateau day". this type of analysis enables health-policy makers to adjust in time to the point at which both new cases and deaths will end. figure 3 shows that germany, france and italy reached their calculated plateau days, i.e. the days when no further confirmed sars-cov-2 cases should be found, at day 103, 107 and 101, respectively. the respective plateau day of deaths is 7-14 days later for germany and france, but only 3 days for italy. it can be seen from the curves for germany and italy, that there were still new cases detected at the time point of the plateau day, when the approximation line meets the x-axis. of course, the infection events that led to those newly confirmed cases occurred at least 10 days before and could reflect variations in western-oriented societies tending to behave individually rather than collectively. in contrast, south korea has achieved the fastest descent with only very few further cases detected at the expected plateau date. the same course is to be expected from taiwan. this points out that efficient measures along with a high compliance of a population can lead very quickly to success. in the case of japan, it is different. this country always showed low numbers (see day rate), but there was also a moderate rate of testing (less than 10,000 tests per day). using the actions described above, japan fought their way down to zero on day 82, but then popular events such as the cherry blossom festival occurred, and people started to behave more careless. subsequently, more action such as regional or general lockdown, social distancing etc. is required for japan to keep sars-cov-2 infections low. the coefficient of determination (r 2 ) assesses the quality of fit of the chosen linear model and thus its ability to predict an outcome. since the zero line is reached for taiwan and also south korea and hardly any new cases occur, a prediction of the linear correlation is no longer possible. regarding japan, the fluctuations are too large for successful model fitting (only 8% of the fluctuations are due to time). thus, there are strong other factors that must explain the 92% fluctuation in the "normalized rate of change per day". however, the data show that outcome prediction by a simple linear model is possible for italy, france and germany. a forecast can thus be made when no more cases will occur if social behaviour does not change. table 1 shows times of plateau of corona infections (f(0) in table 1 ) and of deaths calculated according to fig. 3 . in addition, the time delay between plateau of infections and deaths is shown. for those countries, table 1 provides the relevant data in relation to the cumulated cases, population sizes and median age. since march 23 (day 83) a strict lockdown was started in germany. public life was shut down almost completely, schools, kindergartens and universities were closed. many service providers such as hairdressers and all restaurants were closed in germany. because of the lockdown, as many people as possible worked from home. in contrast, not retarding the exponential virus spread in germany characterized by short doubling times in the first weeks of march would have resulted in more than 600,000 sars-cov-2 cases by the end of the month (fig. 4) . that clearly would have knocked out the german health system due to the limited capacity of 30,000 icu beds, because about 5% of infected persons need intensive medical care according to rki information. thus mentioning the dramatic covid-19 risks on march 18 by the german chancellor angela merkel was one of the last chances to address the attention of the german population in order to slow down the sars-cov-2 spread preventing the breakdown of the german health care system. at the beginning of the sars-cov-2 outbreak, the strategy of herd immunity was pursued in germany, the uk and in sweden. the aim was simply to order measures that would flatten the curve in order to limit the number of people infected simultaneously to a level acceptable to the health care system. this strategy is also called mitigation. however, as mentioned above, this mitigation strategy would have caused at least 250 thousand deaths in germany assuming 60% of the population to become infected based on a fatality rate of only 0.5%, this is not comparable to the death toll to be paid yearly for seasonal influenza, but rather to an armed conflict. a comparison with seasonal influenza outbreak is not possible, since the population is immune naïve to sars-cov-2 and the mortality is at least 5 to 10 times higher compared to seasonal influenza. and even the influenza viruses have a high potential to cause severe outbreaks of public concern as documented in the 1918, seasonal influenza outbreaks after 1918 have never brought the german health care system to a collapse. the analysis of available clinical data on covid-19 clearly revealed that symptoms and diagnostic tests could not be explained by impaired pulmonary ventilation alone. what is special about this disease is that it is a kind of microcirculatory disorder, which is obviously associated with generalized endothelial dysfunction [16] . this highly thrombotic syndrome leads to thrombosis and embolism. in many organs such as lung, liver, kidney, brain and myocardium, vascular occlusions occur in branch arteries as well as in branch veins, which can have a hereditary effect on local microcirculation and thus on organ function [16] [17] [18] [19] . in contrast to influenza -which is often erroneously used for comparison -there is a considerable difference in clinical significance here, particularly in irreversible vascular damage and residual organic impairments. the alternative strategy to mitigation is called suppression. germany as well as many other countries initiated this suppression phase with the decision to lock down. this is a decision that has probably saved hundreds of thousands of lives in germany and other states. in the long run, however, the lockdown would entail serious economic and social costs. the lockdown can therefore only be temporary. in order to have a vision of a situation afterwards, it is helpful to compare the development of sars-cov-2 infections in germany with that in asian countries. immediately the main difference of the development can be seen in march. the asian countries south korea, japan and taiwan had moderate increases in case numbers, far below the critical values for their respective health care systems. while in europe the epidemic was contained much too late, taiwan shows how successful early measures can be. following the sars experience of 2003, a national health command centre (nhcc) was established with the central epidemic command centre (cecc) as the central coordinating body. the cecc has rapidly produced and implemented a list of at least 124 action items including border control from the air and sea, case identification (using new data and technology), quarantine of suspicious cases, proactive case finding, resource allocation (assessing and managing capacity), reassurance and education of the public while fighting misinformation, negotiation with other countries and regions, formulation of policies toward schools and childcare, and relief to businesses [12] . these measures were so effective that only 6 patients died from a total of 397 confirmed infections in a population of more than 23 million people. in the case of south korea there was almost no increase any longer at this time. in contrast, germany, italy and france recorded very steep increases from march 5 to 21, with increases being exponential over a period of several weeks. as described above, the curves flattened out with calculated plateau days until mid of april 2020 ( fig. 3 and table 1 ). another comparison is interesting: germany and france on the one hand and japan on the other hand had roughly the same numbers of confirmed cases at the beginning of march. until the end of march (day 91), japan, however, has managed to stabilize these at under 5,000 confirmed cases, while germany had almost 71,000 and france almost 52,000 confirmed sars-cov-2 infections. the charts show that the asian countries have so far coped well with the crisis. however, in the case of japan, it is noticeable that the trend curve has been rising more strongly again since the end of march. the situation in countries like italy, france and spain (not shown) was more than worrying by the end of march 2020. germany, with its very efficient health care system and a high number of icu beds, has managed to achieve the lockdown just in time and prevented an overload of the health care system. what was the reason for these different developments in europe and asia: 1) until the turnaround, europe mainly pursued the strategy of mitigation, with the aim of gradually achieving herd immunity. this led to an exponential increase in case numbers over weeks, thousands of deaths, and a supercritical strain on health care systems in several countries. 2) the asian strategy was different to that: there was a very rapid lockdown to contain the infection and then the countries implemented follow-up measures with the aim of suppressing the virus spread. examples are the complete lockdown in china, and a moderate lockdown in japan (e.g. schools closed, restaurants open). in china, the number of cases was stabilized at under 100,000 confirmed cases (not shown) -at 1.4 billion people, and in japan at under 5,000 infected people -at 126 million. consequently, the number of sars-cov-2 infected persons compared to the total population was low. however, the asian strategy is also based on the aim to avoid any exponential increase of sars-cov-2 cases at any time. the combination of strong suppression with controlled release was elegantly described as "hammer and dance" strategy [20] . virus replication is stopped when the basic reproduction number (r-value) of the virus drops below 1. in the exponential course of infection, the average of r is 2-3, i.e. each infected person infects at least 2-3 people. from the epidemiological side, r must be below 1 to stop the outbreak. however, this contrasts with the civil liberties of citizens. thus it is a "dance" around the curve, since a sensible and democratically legitimate balance must be constantly struck between the medically and epidemiologically necessary suppression measures and the civil liberties of citizens. in japan we recently saw an increase of cases after almost stopping the spread. this might be due to a more carefree behaviour of the people or a simple result of increased virus testing. since the asian countries are ahead of the european countries europe should learn from asia how to manage such an outbreak. given the lack of antiviral therapy or vaccine, the following measures should be implemented during the "dance" phase: 1. large scale pcr-testing to identify and quarantine infected patients and contacts. 2. quantifying sars-cov-2 transmission using epidemic control with digital real-time contact tracing. 3. serosurveillance of the population to figure out the people who have passed infection and acquired immunity. 4. maintaining social distancing and hygiene rules 5. prohibit all major events and maintaining travel restrictions across national and international borders. 6. wearing of surgical masks or even self-made face masks is mandatory since they prevent transmission of human coronaviruses and influenza viruses from symptomatic individuals. 7. introduction of body temperature scans as an additional measure for personal protection during everyday activities. 8. protect all health-and elderly care workers with ppe including n95 /ffp3 masks. 9. travel entry ban for persons from covid-19 risk regions or, alternatively, quarantining those persons upon entry. 10. re-implementation of regional lockdowns in case of endemic outbreak of sars-cov-2. for any lockdown, it is helpful to predict the time point at which no further new infections will occur by using normalized case number curves. upon reaching the plateau day, a residual time-period of about 2-3 weeks must be fixed for safe release. depicting normalized curves as seen in fig. 3 also indicates compliance of the population on the governmental recommendations. following those rules, a safe dance around the infection curve is possible to keep the population at a reduced infection rate in order to get the economy back to work and revitalise social and cultural life. if there is a pandemic with a new pathogen of unknown lethality and mutation rate, a hammer and dance suppression strategy should always be preferred over the strategy of herd immunity to dramatically reduce the evolutionary potential for pathogens. in the above-mentioned article from tomas pueyo a list of measures of varying effectiveness and cost is given. the decision-makers in each country must determine which weapon arsenal or, to put it less martial, which dancing shoes are best suited to permanently limit the spread of the virus. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding on the origin and continuing evolution of sars-cov-2. national science review innovative screening tests for covid-19 in south korea how we should respond to the coronavirus sars-cov-2 outbreak a decade after sars: strategies for controlling emerging coronaviruses summary of probable sars cases with onset of illness from 1 rapidly increasing cumulative incidence of coronavirus disease (covid-19) in the european union/european economic area and the united kingdom first cases of coronavirus disease 2019 (covid-19) in france: surveillance, investigations and control measures potential scenarios for the progression of a covid-19 epidemic in the european union and the european economic area influenza-associated pneumonia as reference to assess seriousness of coronavirus disease (covid-19) transmission of 2019-ncov infection from an asymptomatic contact in germany covid-19 national emergency response center e, case management team kcfdc, prevention. contact transmission of covid-19 in south korea: novel investigation techniques for tracing contacts response to covid-19 in taiwan: big data analytics, new technology, and proactive testing the preventive strategies of community hospital in the battle of fighting pandemic covid-19 in taiwan wearing face masks in public during the influenza season may reflect other positive hygiene practices in japan respiratory virus shedding in exhaled breath and efficacy of face masks endothelial cell infection and endotheliitis in covid-19. the lancet contrast enhanced ultrasonography (ceus) to detect abdominal microcirculatory disorders in severe cases of covid-19 infection: first experience large-vessel stroke as a presenting feature of covid-19 in the young the hammer and the dance key: cord-254148-wc762p6v authors: prell, tino; siebecker, frank; lorrain, michael; eggers, carsten; lorenzl, stefan; klucken, jochen; warnecke, tobias; buhmann, carsten; tönges, lars; ehret, reinhard; wellach, ingmar; wolz, martin title: recommendations for standards of network care for patients with parkinson’s disease in germany date: 2020-05-13 journal: j clin med doi: 10.3390/jcm9051455 sha: doc_id: 254148 cord_uid: wc762p6v although our understanding of parkinson’s disease (pd) has improved and effective treatments are available, caring for people with pd remains a challenge. the large heterogeneity in terms of motor symptoms, nonmotor symptoms, and disease progression makes tailored individual therapy and individual timing of treatment necessary. on the other hand, only limited resources are available for a growing number of patients, and the high quality of treatment cannot be guaranteed across the board. at this point, networks can help to make better use of resources and improve care. the working group pd networks and integrated care, part of the german parkinson society, is entrusted to convene clinicians, therapists, nurses, researchers, and patients to promote the development of pd networks. this article summarizes the work carried out by the working group pd networks and integrated care in the development of standards of network care for patients with pd in germany. of evidence-based treatment guidelines, the selection of motivated practitioners, regular training, commitment to compliance with the guidelines, patient-centered treatment, and transparent outcome quality [9, 12] . the core element of patient care within a network should be the implementation of a standardized treatment pathway. this defines the best possible sequence of treatment steps on the basis of guidelines and medical expertise. if possible, all patients within a network should be treated with specialized neurologists, registered neurologists, and gps working in a collaborative manner. there should be flowing boundaries to allow equal, individual care concepts based on medical necessity. in our opinion, the establishment of such a treatment pathway must be individually oriented in each network to the corresponding network structures, regional characteristics, and resources available in each case, making simple transferability between different networks impossible. nevertheless, core elements will certainly be available in different networks. this review, therefore, provides an overview of evidence-based recommendations for the network care of patients with pd within the framework of a multisectoral, multiprofessional setting. the authors met in cologne in 2019 for a roundtable discussion and to organize the foundation of the working group pd networks and integrated care, part of the deutsche gesellschaft für parkinson und bewegungsstörungen (dpg). the working group has the following aims: synchronization of supply networks in germany, development of minimum standards, development of joint research projects, further development of nursing staff qualifications, and development of qualification standards for therapy groups. the dpg working group pursues the goal of improving patient care in close cooperation with other physicians, therapists, and patient support groups. the roundtable discussion was sponsored by dpg (travel costs). following introductions and stated aims, various points of interest (existing german network structures and aims, communication strategies, standards of network care, etc.) were discussed. no formal votes were taken at the meeting. the discussions identified general points of agreement. to give recommendations for a standard of network care, one has to acknowledge the existing care paths for patients with pd in germany. typically, the initial symptoms are not identified as parkinsonism or pd-specific symptoms by the patients themselves. instead, they usually approach their gp with motor or even more important and frequent nonmotor complaints (e.g., obstipation, pain and depression). occasionally, physiotherapists treating back pain or degenerative joint symptoms realize that these are the first signs of motor symptoms (e.g., rigidity) related to pd and that the patient should be referred for a pd diagnostic workup. patients who live in rural areas are more likely to have their gp identify symptoms as being related to pd (13) . however, the gp model in germany is not as rigid as in other countries, and many citizens not only have a primary care physician but can also consult specialists (e.g., internal medicine, orthopaedists, etc.), depending on their prior health care contacts and requirements throughout their lifetime. parkinsonism refers to a clinical presentation characterized by the presence of bradykinesia plus rest tremor or rigidity [13] . bradykinesia is a generalized slowing of movements and repetitive motion fatigue. it may present as hypomimia ("masked face"), hypophonia, worsening of fine-motor tasks, micrographia, difficulty turning in bed, or reduced arm swing with side difference. additionally, distinct changes of gait and balance, like short steps, shuffling gait, and uncertainty when turning around are common. rigidity is the resistance that can be assessed clinically by passively flexing and extending a patient's limb. typically, the patient complains about stiffness and pain, which often manifests as shoulder or back pain. while kinetic and postural tremors may occur, the rest tremor is the most common type of tremor in early pd. patients with these clinical signs should be referred to a neurologist for further diagnosis, because, in germany, the time between the appearance of the first symptoms and the diagnosis is significantly longer when patients with these symptoms see their gp [5] . patients with symptoms that may be related to pd should be asked about pd-typical nonmotor signs such as sensory symptoms (loss of smell, pain), depressed mood, rapid eye movement-sleep behavior disturbance, periodic limb movement disorder, or constipation, which frequently occur years before motor signs are realized. thus, one recommendation for standard of care in the initial phase of the disease course is physician awareness of the first signs of pd (which could be achieved with better information and secondary prevention standards in the network) and early referral of patients to a movement disorder specialist (which could be achieved by specific disease management programmes). the movement disorder specialist should be a neurologist with many years of experience caring for patients with pd. in germany, there are specific recommendations for patient referral in this context that can guide decisions in the outpatient setting [6] . in 2015, the official international parkinson and movement disorder society (mds) clinical diagnostic criteria for pd were proposed [13] . the benchmark for these criteria is an expert clinical diagnosis. however, the criteria can be easily applied by clinicians with less expertise in pd diagnosis [14] . in the mds criteria, motor symptoms remain the core feature of the disease, defined as bradykinesia plus rest tremor or rigidity (explicit instructions for defining these symptoms are given). after consideration of absolute exclusion criteria (which rule out pd), red flags, and supportive criteria, the diagnosis of clinically established pd or probable pd can be made, or pd can be ruled out. besides anamnesis, clinical assessment with cerebral imaging (cranial mr imaging [mri] is preferred) should be performed to exclude symptomatic causes of pd symptoms. in case of divergent clinical and mri-based diagnoses, the clinical assessment should take precedence. however, a differentiated approach is required for the numerous subsequent apparatus and drug tests. the evidence shows that levodopa and apomorphine tests are not as meaningful as standard levodopa therapy in differentiating between established pd and atypical parkinson's syndrome. a negative test does not rule out a response to longer-lasting levodopa treatment. this suggests that levodopa and apomorphine tests should not be routinely used in differential diagnosis but may be valuable in specific clinical situations [15] . a reduction in olfactory capacity is a sensitive but not specific indicator of pd. therefore, standardized olfactory tests are only recommended in combination with other diagnostic procedures for the diagnosis of pd. striatal dopamine active transporter-single photon emission computed tomography (spect) imaging should be used early in the course of the disease to detect a nigrostriatal deficit in clinically unexplained parkinsonism or tremor syndrome [15] . in contrast, the postsynaptic (i 123-iodobenzamide) spect should not be used for the differential diagnosis of established parkinson's syndrome (syn. idiopathic parkinson syndrome) to differentiate atypical neurodegenerative disease variants. the myocardial 123 mibg-spect can be used to distinguish multiple system atrophy from pd [15] . in addition, functional brain imaging with positron emission tomography is a valuable diagnostic tool for the differential diagnosis of idiopathic parkinson syndrom and atypical parkinsonism [16, 17] . for clinical neurological confirmation of the diagnosis and therapy control, the patient should be examined after 3 months, and thereafter according to clinical need but at least once a year [15]. because even proven experts have to revise the diagnosis of an ips during the course of the disease, the diagnosis should be reviewed at regular intervals. with the rising availability of electronic patient records, another recommendation is that a standard set of information should be generated and stored in the record of each patient with pd after the results of the first diagnostic tests. this information should be available for the patient and his or her health care provider team. referral to a movement disorder specialist is important to improve the accuracy of diagnosis, for case selection and to provide guidance in terms of specialized device-aided therapies, namely, dbs, levodopa/carbidopa intestinal gel (lcig) and apomorphine. consultation from the medical staff of a specialized center may improve motor function and the quality of life in patients in advanced pd stages [18, 19] . patients with the following constellations and symptoms should be referred to a movement disorder specialist even if the disease duration is <4 years [20] [21] [22] : referral to a movement disorder specialist should also be considered for the patient to have access to the most innovative treatment and clinical research options. a substantial number of patients are highly interested in contributing to research, the opportunities for which are typically limited to regional neurologists. recommendations for these patient referrals in the outpatient setting in germany have been proposed [6] . some health care insurance systems reimburse treatment of patients with pd in specialized units. a well established and frequently used multiprofessional inpatient treatment concept in germany is pd multimodal complex treatment (pd-mct). prerequisites for patients taking part in mct are documented physician diagnosis of pd, a constant anti-parkinsonian drug titration, and the application of activating therapies (at least 7.5 h/week). it involves physicians, physiotherapists, occupational therapists, speech therapists, and other specialists for the optimization of pd treatment [3] and usually lasts 7 to 21 days. this therapy programme has been shown to be effective, with a reduction of motor symptoms and nms [23, 24] . richter et al. [3] performed an analysis of 55,141 inpatients with pd who were integrated into this mct from 2010-2016. they found that a large majority of patients with pd need to leave their residence county for an inpatient stay in a specialized pd unit. this limited access to multimodal therapy programmes means that patients sometimes have to travel long distances to receive specialized therapy [3] . there are no generally valid definitions of which patients should be treated within the complex programme and which should not. in view of the heterogeneity, it is difficult to make binding statements about this. a prerequisite should be that the motor or nmss can no longer be satisfactorily treated by outpatient therapy. another prerequisite should be that patients are dealing with limitations in their activities of daily life and have a reduced quality of life. this can be the case, for example, with side effects under oral therapy, motor deterioration, or the high burden of nms. other typical indications for inpatient treatment would be the discontinuation of dbs or the initiation and optimization of therapy with lcig or apomorphine. however, as the disease progresses and progressive limitations in mobility and cognition are observed, the benefits of inpatient treatment must be weighed against the increasing risk of delirium. overall, clinical experience shows a substantial benefit of pd-mct for a large number of patients. the preselection process could ideally be managed by network structures and players. additionally, the positive effect achieved by intense medical and nonmedical intervention should be maintained after release by immediate intensified ambulatory intervention and home-training concepts in order for the patients to benefit from the positive experience. this would be an important incentive for the patient to take part in pd-mct. for patients with pd who need to adapt to complex medication schemes, drug pumps, or dbs devices, a classical outpatient or inpatient setting is not appropriate to sufficiently address clinical problems, while in a neurologist's office or even in a movement center, outpatient clinic time and staff capacities are limited and the results of changes in medication or stimulation of the dbs device can only be monitored in the next (often late) consultation. an in-house stay is associated with an artificial environment that does not reflect the individual's everyday life demands and is less suited for patients with dementia who often cannot cope with an altered environment. furthermore, many patients with pd decline hospitalization for personal reasons such as job issues or having to care for other family members. for these patients, at the border between inpatient and outpatient care and the need for sophisticated treatment strategies, the new comprehensive, individual, and interdisciplinary concept of a pd day clinic has proven to be effective [25] . in the meantime, in germany, several university clinics with a pd focus have established this or a similar pd day clinic concept to close the gap in pd care that have been found to be a transnational issue [26] [27] [28] . the concepts and standards of qualified pd day clinics have been certified recently by the tüv and the german parkinson patient society [29] . in general, a neurologist should be responsible for long-term medical care of patients with pd, and movement disorder specialists should be involved when there is a special issue. however, for various reasons, this is not always possible. neurologists may not be available in rural areas, and even for patients in nursing homes, access to specialized neurological treatment is often limited. this is an important issue, because the number of patients in long-term care facilities will rise sharply in the coming decades [30] . for patients with pd, the interaction between the gps and neurologists is essential. pd networks can make a decisive contribution to ensuring high-quality care of these patient groups. medical treatment is not the only option to control the motor symptoms and nms during the course of the disease. other nonmedical treatment options from other specialists are frequently necessary to improve functional status, performance of daily activities, and quality of life. these specialists include, among others, physiotherapists, occupational therapists, speech therapists, pd nurse specialists, and social workers [31] . specific recommendations for physiotherapists, physicians, and patients with pd were published in the european physiotherapy guidelines for parkinson's disease [32] . health professionals must have sufficient pd-specific knowledge and expertise [33] . physiotherapy has a positive impact on functional activities involving gait, transfers, and balance [32, 34] . the occupational therapist focuses on enabling performance and engagement in meaningful activities [35] . home-based, individualized occupational therapy can improve the self-perceived performance of daily activities in patients with pd [36] . timely referral to physiotherapy, and occupational therapy is recommended because difficulties in daily activities can occur in every disease stage. given the high prevalence of dysphagia and dysarthria during the course of the disease [37] , speech-language therapy, including swallowing techniques, is frequently necessary for patients with pd. a collaborative approach between these disciplines should focus on complementary and different aspects. therapists have to be aware of each other's expertise, and effective and timely communication is essential [35] . pd networks are promising tools to share information about diagnostic results, current treatment goals, and plans. in addition, there are many different nonphysician pd specialists for inpatient and outpatient care, such as pd nurse specialists or parkinson assistants (passs). their different roles and functions are described in another paper in this issue. depending on the location (inpatient or outpatient), the focus of their tasks can be different. these specialists are often familiar with aspects of case management; medication adherence; provision of information, education, psychosocial support, and coping skills; and caregiver support [38] . patients with pd should have 1) regular access to clinical monitoring and adjustment of medication in consultation with the treating physician; 2) regular contact with caregivers, including home visits, as appropriate; and 3) access to reliable sources of information on clinical and social issues affecting patients with pd and their caregivers/families. these functions could be provided by pd nurse specialists or a pass. the positive therapeutical effects of pd nurse specialists are currently evaluated for their health economic impact [39] . in particular, patients with advanced pd may benefit early from palliative care. doctors and nursing staff can provide information about the final phase so that the family can take advantage of adequate care options. palliative care should be aligned with patient priorities and complement other treatments. therefore, advanced care planning might also increase knowledge about end of life issues. generally, it should start early in the course of the disease. it can be started when particular symptoms occur (pain, dyspnoea, dysphagia, and aspiration) or at the very end of life [40, 41] . besides general markers of advanced disease (frequent infections and hospitalizations, malnutrition, etc.), the palliative performance scale can be used to measure the functional status of a patient and to determine the eligibility for enrolment in a palliative care programme [41, 42] . dysphagia with symptomatic aspiration might be taken as a clear indicator when palliative care should begin, because it also involves a discussion about life-prolonging therapies such as tube feeding. figure 1 provides an overview of common players and structures in a local supply network. information on clinical and social issues affecting patients with pd and their caregivers/families. these functions could be provided by pd nurse specialists or a pass. the positive therapeutical effects of pd nurse specialists are currently evaluated for their health economic impact [39] . in particular, patients with advanced pd may benefit early from palliative care. doctors and nursing staff can provide information about the final phase so that the family can take advantage of adequate care options. palliative care should be aligned with patient priorities and complement other treatments. therefore, advanced care planning might also increase knowledge about end of life issues. generally, it should start early in the course of the disease. it can be started when particular symptoms occur (pain, dyspnoea, dysphagia, and aspiration) or at the very end of life [40, 41] . besides general markers of advanced disease (frequent infections and hospitalizations, malnutrition, etc.), the palliative performance scale can be used to measure the functional status of a patient and to determine the eligibility for enrolment in a palliative care programme [41, 42] . dysphagia with symptomatic aspiration might be taken as a clear indicator when palliative care should begin, because it also involves a discussion about life-prolonging therapies such as tube feeding. figure 1 provides an overview of common players and structures in a local supply network. the therapist network (outpatient) directly surrounding the patient and his or her environment is not only linked to the patient, but therapists are also linked to each other. this results in mutual inter-relationships and a flow of information between all professional groups involved (not only between the directly neighbouring ones). a supraregional supply network in the form of clinics and centers is connected to this ''micro-network''. here, exchange and cooperation results. different stationary and semistationary care options are offered and supplemented with, for example, telemedical services (e.g., medical video observation and sensor-based motion analysis). the therapist network (outpatient) directly surrounding the patient and his or her environment is not only linked to the patient, but therapists are also linked to each other. this results in mutual inter-relationships and a flow of information between all professional groups involved (not only between the directly neighbouring ones). a supraregional supply network in the form of clinics and centers is connected to this ''micro-network". here, exchange and cooperation results. different stationary and semistationary care options are offered and supplemented with, for example, telemedical services (e.g., medical video observation and sensor-based motion analysis). self-management means having knowledge, skills, and confidence to manage daily tasks when living with a chronic disorder such as pd. it includes the concepts of self-management tasks (medical, role, and emotional management) and self-management skills (problem solving, decision-making, resource utilization, the formation of a patient-provider partnership, action-planning, and self-tailoring) [43] . patients with pd should be able to monitor progress and problems and to set, communicate, and harmonize their individual therapeutic goals with all members of the health care provider team. in addition, required information for the individual aspects of the disease symptoms, treatments, and side effects/risks should be tailored to the patient requirements and transferred adequately to the patient. health care providers involved in the care of patients with pd can positively influence self-management skills with distinct approaches that mainly focus on education and support. self-management in pd may, therefore, contribute to slower disease progression, reduced complications, and lowered costs [44] . however, self-management support interventions for patients with pd vary in content, structure, and intensity, and little is known about which existing self-management support programmes are most effective. as indicated by a recent overview of self-management support programmes for patients with pd, clinicians should ensure that the key components of education, goal setting, and guided problem solving are included. moreover, adding these skills to the rehabilitation process and including caregivers and peer support systems seems promising [44] . as mentioned above, pd requires close interaction between different care partners in order to provide the best possible care for the patient. rural location, nursing home residence, and the presence of physical or cognitive impairment are common reasons for limited access to specialized pd health care [45] . a pd network can improve access to specialized health care and manage the distribution of resources, tasks, and responsibilities. by doing so, pd networks can help to avoid unnecessary hospitalization and reduce costs [8] . different methods exist to bring pd-specific knowledge and care to the patients in a pd network structure. in this context, telemedicine has shown promising effects for the management of pd. this includes synchronous methods (videoconferencing) and asynchronous methods (e.g., e-mail, smartphone assessments, remote monitoring, and wearable devices) [10, 11, 46] . telemedicine has the potential to allow pd-specific efficient care to be delivered to more patients and more regularly than a traditional model of care [47] . from the patient's view, telemedicine has the advantages of access to specialists, convenience, and time savings [48] . at present, it is applied in several clinical settings due to sanctions imposed for infection prophylaxis in the current sars-cov-2 pandemic, and it is seen to be a suitable tool with which to give advice and treat patients with pd. it also can be used to support outpatient palliative care teams with special neurological knowledge when the patient chooses to die at home [49] . since 2019, the remuneration of video consultation hours has been based on the insured, basic, or consultation flat rate in germany. nevertheless, telemedicine is still limited by patients' limited access to high-speed internet and usability issues (especially in elderly patients) [46] . nevertheless, with the new digital health act (''digitale-versorgung-gesetz" (dvg)), reimbursement for video-based home telemedicine support has begun in germany, and now, home telemedicine needs to be integrated into pd health care workflows. the german health care system is struggling with the issues of separation of care sectors (e.g., outpatient vs. inpatient care) and considerable differences in the provision of care in urban and rural areas. in order to optimize the specialized care of patients with pd in germany, the current care structures must be changed. this can be achieved by establishing pd networks, which act as a link between outpatient and inpatient treatment as well as between patients, caregivers, gps, nonspecialized neurologists, movement disorder specialists, and other therapists. this is a promising way to ensure that a stage-appropriate and patient-specific therapy for pd can be initiated promptly and maintained permanently in accordance with the current guidelines. additionally, new e-health processes might overcome current barriers and limited access to specialized health care and provide both patients and health care professionals with the potential for future seamless care, a strong interaction between health care partners, and involvement of patients and caregivers. interestingly, many patients with pd are using digital media tools and smartphones and thus have access to digital technology [50] . furthermore, the recently released digital health act (dvg) will enable patient-centered technologies as digital health care applications for better support of trans-sectoral pd health care. especially against the background 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patientenperspektive auf die versorgungssituation im krankheitsbild morbus parkinson in deutschland-eine querschnittserhebung the promise of telemedicine for chronic neurological disorders: the example of parkinson's disease the promise of telemedicine for movement disorders: an interdisciplinary approach patient views on telemedicine for parkinson disease telemedicine in palliative care: implementation of new technologies to overcome structural challenges in the care of neurological patients the use of digital technology and media in german parkinson's disease patients effects of community based nurses specialising in parkinson's disease on health outcome and costs: randomized controlled trial this article is an open access article distributed under the terms and conditions of the creative commons attribution acknowledgments: travel costs for experts meeting by dpg. tino prell has received bmbf research grant, and honoraria for presentations/lectures abbvie gmbh, ucb pharma gmbh, desitin gmbh, licher mt gmbh, and bayer ag deutschland. frank siebecker reports no conflict of interest. michael lorrain has received honoraria and compensation for consultancy and lecturing from abbvie, afi, bayer, bial, biogen, desitin, merck, nordrheinische akademie, teva, ucb, and zambon. carsten eggers received payments as a consultant for abbvie inc. ce received honoraria as a speaker from abbvie inc., daiichi sankyo inc., bayer vital inc. ce received payments as a consultant for abbvie inc. and philyra inc. stefan lorenzl reports no conflict of interest. jochen klucken reports institutional research grants from bavarian research foundation; emerging field initiative, fau, eit-health, eit-digital, eu (h2020), german research foundation (dfg), and bmbf, and industry-sponsored institutional iits and grants from teva gmbh, licher mt gmbh, astrum it gmbh, and alpha-telemed ag. he is coemployed by the university hospital erlangen, germany, fraunhofer institute for integrated circuits e.v., germany, and the medical valley digital health application center gmbh, bamberg, germany. he works on advisory boards in the field of healthcare technologies and digital health of different associations of medical professionals, industries, and political authorities. he holds shares of portabiles healthcare technologies gmbh, portabiles gmbh, alpha-telemed ag, and received compensation and honoraria from serving on scientific advisory boards for lichermt gmbh, abbvie gmbh, ucb pharma gmbh; he has lectured at ucb pharma gmbh, teva pharma gmbh, licher mt gmbh, desitin gmbh, abbvie gmbh, solvay pharmaceuticals, bial deutschland gmbh; celgene gmbh, lundbeck-foundation. dr. klucken has a patent related to gait assessments pending. tobias warnecke has received honoraria from abbvie (lecture fees, consultant). abbvie acts as coinitiator of the parkinsonnetwork muensterland+ (pnm+) and is cocontractor of the university hospital of muenster. carsten buhmann has received fees as speaker and/or advisor from abbvie, bial, desitin, grünenthal, licher, novartis, tad pharma, ucb, and zambon. lars tönges has received travel funding and/or speaker honoraria from abbvie, bayer, bial, desitin, ge, ucb, and zambon, and consulted for abbvie, bayer, bial, desitin, ucb, and zambon, in the last 3 years. reinhard ehret reports no conflict of interests. ingmar wellach has received honoraria an compensation for consultancy and lecturing from abbvie gmbh, ucb pharma gmbh, desitin gmbh, bial deutschland gmbh, zambon deutschland gmbh, fagron gmbh & co. kg, grünenthal gmbh, and bayer ag deutschland. martin wolz has received honoraria for presentations/lectures from zambon, valeant, desitin, teva, ucb pharma, abbvie, bial, licher, and daiichi sankyo. key: cord-269559-gvvnvcfo authors: kergaßner, andreas; burkhardt, christian; lippold, dorothee; kergaßner, matthias; pflug, lukas; budday, dominik; steinmann, paul; budday, silvia title: memory-based meso-scale modeling of covid-19: county-resolved timelines in germany date: 2020-08-03 journal: comput mech doi: 10.1007/s00466-020-01883-5 sha: doc_id: 269559 cord_uid: gvvnvcfo the covid-19 pandemic has led to an unprecedented world-wide effort to gather data, model, and understand the viral spread. entire societies and economies are desperate to recover and get back to normality. however, to this end accurate models are of essence that capture both the viral spread and the courses of disease in space and time at reasonable resolution. here, we combine a spatially resolved county-level infection model for germany with a memory-based integro-differential approach capable of directly including medical data on the course of disease, which is not possible when using traditional sir-type models. we calibrate our model with data on cumulative detected infections and deaths from the robert-koch institute and demonstrate how the model can be used to obtain countyor even city-level estimates on the number of new infections, hospitality rates and demands on intensive care units. we believe that the present work may help guide decision makers to locally fine-tune their expedient response to potential new outbreaks in the near future. the covid-19 pandemic continues to hold our way of life on this planet in a tight grip. over the whole world, we have now reached more than 10 million infections [1] . while new infections still rise at alarming pace in the united states, brazil, or india, most other asian and european countries that were hit much earlier by the pandemic seem to have succeeded in reducing the number of new daily cases. this success can largely be attributed to fast and locally tailored political measures that introduced severe travel restrictions [2] [3] [4] [5] [6] and curtailed public life. initially, the measures were met by a largely understanding general public. however, the partial necessity of police enforcement and increasing protest against contact restrictions, locally even encouraged by politicians [7] , demonstrate rising anger, fear, or even mental health problems due to the current situation [8, 9] . thus, it is critical to carefully reopen the economy and reestablish public life, while avoiding a relapse and a potential collapse of the health-care system, which may entail much stricter measures again. to reach this goal, however, decisions must be made quickly and often locally at county level, based on reliable data and trustworthy predictions. clearly, accurate models are of essence to capture the disease dynamics at exactly this spatial meso-scale, to predict the number of new infections per day or the number of patients that may require intensive care. here, we focus on the situation in germany, where county-resolved daily and cumulative infection cases are reliably reported by the robert-koch institute (rki, [10] ). we combine two previous modeling advances [11, 12] into a locally resolved, history-type model that captures the spatiotemporal evolution of the pandemic in germany. we use a generalization of typically known sir-type compartment models that allows for a much better representation of the courses of disease [12] . while becoming infected is well represented by a simple ordinary differential equation (ode), the remaining course of disease is captured rather restrictively by these ode-based, sir-type models [13] . based on the integro-differential model introduced by kermack and mckendrick already in 1927 [14] and recently reintroduced by [12, 13] , we model the spatial spread of covid-19 in the following way: for all s < 0, t > 0 and x ∈ ω with ω ⊂ r 2 open denoting the considered spatial region. the normalized initial history datum is given by ) denotes the normalized number of susceptibles. the weight γ i ∈ l 1 ((0, ∞); r ≥0 )) with γ i l 1 ((0,∞)) = 1 describes the evolution of infectiousness, where γ i (τ ) defines the infectiousness of an individual at τ days after the infection event. the interaction term β ∈ l ∞ ((0, ∞) × ω 2 ; r ≥0 ) denotes the interaction between the infectious and the susceptible population. the considered balance law is of nonlocal-history type. nonlocality as well as history in balance laws are receiving increasing attention to model real world phenomena. they provide a more detailed way to model evolution and can be seen as the mesoscopic link between purely macroscopic and fully microscopic models. in the considered application, the microscopic equivalent-agent models [15] [16] [17] -can be interpreted as a measure valued solution to the proposed model. the classically used compartment models (sir, seir) [18] have been widely used to model the viral spread. their recently revealed relationship to hamiltonian mechanics is quite insightful [19] , demonstrating that they constitute a mere simplification of the here considered integrodifferential equations. in terms of the spatial resolutionwhich of course can also be modeled in the compartment models [20, 21] -the classical sir model can be seen as the singular limit of the interaction term, i.e. β(·, * , ) → b(·)δ( * − ) for a given b ∈ l ∞ ((0, ∞); r ≥0 ). the models are generalized with respect to the evolution of infectiousness of infected individuals. the considered model can represent-based on medical data-any course of infectious-ness, in contrast to, e.g., an assumed exponential decay in the widely used sir model. as introduced in [11] , we discretize our spatial domain ω, germany, at county-level (or even city-level), where current containment rules are steadily evaluated and adapted in case local infection numbers rise up again. our county-interaction network is adapted from the global epidemic and mobility (gleam) model [22, 23] , focusing on mid-and shortrange interactions motivated by the severely restricted air travel [24, 25] . taken together, this spatially resolved integrodifferential model allows us to accurately analyze and predict disease dynamics at its various stages and the effect of local measures. to model the spread of the disease in a discretized spatial setting, we consider a finite partition of the domain ω, i.e. where n denotes the number of counties or cities in germany, depending on the spatial resolution. we obtain the following memory type vector-valued initial value probleṁ is the vector-valued normalized initial history datum. we introduce • to denote the transformation of a vector • into a quadratic diagonal matrix, where the entries along the diagonal equal those of the vector. the time-dependent, vector-valued function s ∈ w 1,∞ ((0, ∞); [0, 1] n ) denotes the normalized number of susceptibles. the matrix-valued function b ∈ l ∞ ((0, ∞); r n ×n ≥0 ) denotes the infection rates and interaction between the considered regions ω k with k ∈ {1, . . . , n }. the existence and uniqueness of a solution of the proposed integro-differential equations-continuous as well as discretized in space-is proven e.g. in [12] for all γ i for which there exists > 0 s.t. γ i | (0, ) ≡ 0. this is a rather natural condition, since the incubation time-the period during which the infected are not yet infectious-is positive. based on the history of s, other quantities and subgroups can be determined directly from including medical data on the various courses and infectiousness levels of the disease via corresponding integration weights: we distinguish between the states infectious γ i , symptomatic γ s , tested and quarantined γ q , hospitalized γ h , in intensive care γ icu , recovered γ r and deceased γ d . following the contribution of [ disease progression in our model: (a) light symptoms, recovering without hospitalization, 95% share; (b) hospitalization, recovering without intensive care, 4% share; (c) patients in intensive care and recovering, 0.4% share; (d) patients in intensive care that eventually die, 0.6% share. figure 1 depicts the four different courses of the disease as represented in our model, including their corresponding state transitions and infectiousness levels. note that only the weighted sum γ i = 4 i=1 share i · γ i ,i is necessary for the solution of eq. (1), but the individual contributions allow for detailed descriptions of disease progression from medical data and corresponding post processing. we normalize the integral over γ i such that it represents the probability of infection. we further assume that patients in courses (b) to (d) will be tested positive and are thereby considered in reported infection numbers. the ratio of total versus detected infections is defined as dark ratio ω, thereby representing the factor of unknown cases. since the dark ratio is not necessarily constant in space, this is taken into account by locally scaling the function γ q that represents the detected and quarantined state of course (a) to the appropriate value. since the dark ratio seems to closely correlate with testing capacities [11] , we introduce federal-state-wise dark ratios ω j , assembled in the vector ω, that vary only over states with j ∈ {1, . . . , 16}, due to locally differing behavior patterns and, in particular, political measures to reduce social contacts, the infection rates vary in space and time. based on our previous findings in [11] , we introduce federal-state-wise initial infection rates β j0 with j ∈ {1, . . . , 16}, and two reduction factors β red 1 and β red 2 representing the major restrictions of 1) cancelling large events and 2) contact restrictions. those model parameters are calibrated using data reported by the rki, as described in sect. 2.3. since the shut-down measures were introduced at slightly different times t j1 and t j2 in the different federal states of germany, we model the time-dependent reduction of infection rates via piece-wise constant functionsβ j1 (t) andβ j2 (t) to obtain the overall infection rates in each state (2) to model cross-county interactions, we adapt the gleam short-and mid-range mobility network [22, 23] as introduced in [11] and capture cross-county infections by where β k are time-dependent infection rates, c k are the crosscounty infection weights, n k is the number of inhabitants in the largest city of county k, n max = 3e6 corresponds to the number of inhabitants in germany's largest city berlin, and r kl is the distance between counties k and l. importantly, β k and c k are identical for all counties within one federal state, such that eq. 3 introduces 16 additional model parameters c j with j ∈ {1, . . . , 16} which need to be calibrated based on reported data in the literature (see sect. 2.3). the parameters for the gleam model are taken from [22] and given in table 1 . figure 2b displays the mobility network across germany [11] . note that both the county-internal as well as the cross-county infections contribute to the basic reproduction number in our spatial model. for the distinction of individual counties, we use the municipal directory (gemeindeverzeichnis) from the german federal statistical office (statistisches bundesamt) [27] , which delivers area, shape and population data. we consider city-wise population data, which is accumulated over the entire county or corresponding spatial domain for the respective model. the center of population of each county serves as its spatial coordinates. the detailed description of the courses of disease allows for elaborate post-processing of the solution to evaluate any described quantity. generally, evaluation differs for cumulative and current quantities. the number of cumulative discovered infections q or the number of deceased d are evaluated by double integrals such as current quantities like infectious i(t), those with symptoms, hospitalized or icu patients follow from expressions such as from data the number of positively tested people on day zero is known, but the integro-differential equation model requires initial values for the infected at each spatial node as well as an initial history as a starting point for the integration. initially, we assume exponential growth in all counties described by the ansatz with ν = 0.345 from fitting an exponential function to rki data on cumulative covid-19 cases in all of germany from march 2 to march 6. the initial estimated number of infected ε at time t = 0 in each county can be calculated by combining eq. (4), the number of initially reported cases q 0 , eq. (6) and the time derivativeṡ. from the result and eq. (6), the initial history can be estimated. the high-resolution network model brings with it the challenge for spatially consistent initial conditions. however, most counties did not yet have any known cases on the very first day, limiting the possibility of simply scaling overall initial infections per county by the dark ratio. thus, for the county-based model we selected the distribution of initial infections according to data for march 16 [10] , linearly scaling down the overall number of infections to the number reported on our starting date march 2. to approach our spatially resolved county-model, we followed a cascade optimization strategy. data analysis and preliminary simulations had shown that we require federalstate-dependent dark ratios ω j and infection rates β j0 , j ∈ {1, . . . , 16} [11] . figure 2a illustrates the estimates for the state-wise dark ratios ω j , which we obtain by assuming a germany-wide identical mortality of μ = 6‰ [26] and fitting to the individually reported death tolls, with ω ≈ 6.5. using state-wise identified dark ratios ω j , we first used a coupled system of 16 nodes connecting each federal state to obtain a germany-wide average β and reduction factors β red 1 and β red 2 by fitting the cumulative data for germany via the following objective function the residual r 1 is minimized using a particle swarm optimization (pso) algorithm described in detail in sect. 2.4 with weights w i = 1/(t end − t start )/max(q rki ), w d = 1/max(d rki ) and w s = 0.1/max(q rki ). we then considered state-wise data to fit β j , j ∈ {1, . . . , 16}, while keeping c = 1, leading to the distribution over ger-many depicted in fig. 2c . we fit the cumulative number of confirmed infections reported by the rki [10] for the time period from march 2 until april 25 with the cumulative number of detected infections q as defined in eq. (4), normalized by the maximum cumulative number of reported infections from the rki. this is the time period during which the various shutdown measures were in place without any noticeable relaxation. on top of that, we include the change-rate of infections on our last day april 25 into the residual vector with the weights the weights are state-wise normalized to balance the contribution of heavily and less affected states. the residual r 2 is again minimized using the pso algorithm. finally, we increased the resolution to full county level, amounting to a coupled system of 401 nodes. to re-balance the changed influence of the larger network, we iteratively fitted statewise cross-county weights c j for ∈ {1, . . . , 16} to match the state-wise cumulative infections of the 16 node state-wise model (q sw ) with the accumulated numbers from the 401 node county-based model (q cw ) on the last day of the fit. we used a damped gradient-descent like algorithm to update c j at iteration i + 1 following the rule empirically, we obtained converged cross-county weights within 30 iterations with a limited step size δc max = 0.25 and a damping exponent ζ = 1.5. the final state-wise distribution of optimized cross-county weights c j is displayed in fig. 2d. particle swarms are distributed optimization schemes that treat each realization of the d optimization variables as particles with a position x i and a velocity v i in a d-dimensional bounded search space. particles are initialized with a uniformly random position within the boundaries of the search space and zero initial speed. for the iteration i > 0 the following set of equations describes the behaviour of any particle: the velocity v i+1 is a linear combination of three quantities. the previous velocity v i weighted with the constant factor a results in an inert motion property. the term p i loc − x i represents a force that pulls the particle towards its local attractor p i loc , which is the best position this specific particle has visited so far. multiplication with the constant weight b loc controls the influence of this quantity. in addition to that the randomized diagonal matrix r i loc with values between 0 and 1 enables optimization in varying directions. the global attractor p i glob represents the best position any particle has visited so far and works analogously to the local attractor with the factor b glob . we chose established values for a, b loc and b glob as summarized in table 2 , used a total of 300 particles and followed the 'nearest' strategy when particles cross boundaries of the search space during optimization [29] . to prevent overly fast convergence to a visited attractor without broad coverage of the search space, we employed a so-called ring topology neighborhood, such that the global attractor of a particle corresponds only to the best local attractor of its two neighbors below and above. this way, good positions are slowly propagated through the whole swarm, allowing for enhanced exploration of the search space, which well balances run-time efficiency and identification of the true global optimum. to validate the model, we evaluated the temporal correlation between model predictions and rki data by computing the pearson correlation coefficient r p , the coefficient of determination r 2 = r 2 p and the corresponding p-value to assess statistical significance via the function [r p , p] = figure 3 shows how the optimized spatially resolved memorybased model with 401 network nodes representing each county of germany well reproduces the cumulative confirmed cases in each of its federal states from march 2 until april 25. for cumulative infection data reported by the rki [10] , we find astonishing and statistically significant (all p < 1e − 12) agreement on the temporal evolution. the only state with an r 2 < 0.98 is bremen-a city-state with overall very low infection numbers and a population of less than 700.000. here our quasi-continuum modeling approach and the underlying exponential growth seem to approach their validity limit, and stochastic effects start to prevail. although only the last data point of reported deaths was considered for parameter identification, the model captures the temporal evolution of covid-19 related deaths in each state of germany with remarkable accuracy (all r 2 > 0.91). here, we observe least agreement in the city-state hamburg. in general, the model better captures the evolution in higher-populated states, with overall more infections and death tolls. we note that our fitting procedure only operates on state-based information. to further validate our model, we compare county-wise cumulative infection numbers q as reported by the rki and our model (fig. 4 left and figure 5 shows how the model informs on the temporal evolution of cumulative confirmed cases, with more detailed resolution on the subgroups of symptomatic, infectious, and hospitalized, patients in the icu, as well as the dead. a first kink in the infectious group is clearly visible at the beginning of march due to the cancellation of major events, which then drops significantly when contact restrictions become effective shortly afterwards. figure 6 shows the model predicted spatial distribution at county resolution of infectious, symptomatic, hospitalized, and patients in intensive care, following from the individual disease courses in fig. 1 . we consider a period from early march, where the exponential growth of the disease started in germany, until early june under the assumption that the contact reduction factors stay in place. in early march, most of the infected were at an early stage of the disease, i.e., most of them were infectious but did not have disease specific symptoms yet (on average, the first symptoms appear on the fifth day after the infection event [26] ). this explains the delay in symptomatic infections clearly visible in fig. 5 . in our model, we assume that most of the symptomatic voluntarily quarantine themselves and no longer infect others, implying that the infectiousness decreases when people move to the symptomatic group (cf. fig. 1 ). the infectious state ends at the latest when the symptomatic have been tested positive for the virus and are quarantined. the symptomatic state of covid-19 lasts approximately nine days on average [26] , explaining why the symptomatic group is about double in size compared to the infectious group in fig. 5 . figure 6 also shows the delay in covid-19 cases that need inpatient treatment or even intensive care. as reported in [26] , infected are typically hospitalized for nine days after the infection event at a probability of 4.5%. as this is encoded in the courses of disease, the snapshot on march 2 reports hardly any hospitalized patients. according to [26] , patients finally, we show how our model can be adapted to locally increase resolution to individual city-or community level. fig. 7 spatial distribution of the infectious (i) on april 2 at county level for all of germany (top) and with locally increased resolution to community-level (bottom). the non-densified part of the domain is greyed out for the sake of better visual contrast. zoomed regions show county-and community resolution for counties erlangen, fürth, nürnberg and their rural surroundings. note that the proposed macroscopic model reaches its validity limit for very low daily new infections within one subregion figure 7 shows the germany-wide county-level simulation (top), with a zoom into the metropolitan area of nürnberg, erlangen and fürth and its surrounding counties. increasing the resolution within this domain to community level (bottom) but maintaining county-level for the rest of germany leads to a network of 464 nodes. the zoom-in clearly shows that county infections are dominated by their largest cities, following the three purple areas that represent nürnberg, fürth and erlangen from bottom to top, underpinning our formulation of the cross-county infection terms (cf. eq. (3)). surrounding communities suffer much less infections due to their much smaller populations. gray areas correspond to rural public space not assigned to a specific community [27] . we have presented a memory-based network model to predict the spatio-temporal outbreak dynamics of the covid-19 pandemic in germany. the model considers the effects of political measures, the cancellation of major events and contact restrictions, and the different possible courses of the disease, which is not possible when using traditional sirtype models. it well represents the evolution of confirmed cases and deaths reported by the rki from march 2 until april 25. we have then used the model to predict the further developments until june and have provided estimates for the county-wise required capacity of the local health care system, i.e. the number of patients that require hospitalization and even intensive care. finally, we have demonstrated that the model can be refined to predict the interaction and local outbreak dynamics at community level. by now, medical data on observed disease progression at most stages during a covid-19 infection is abundantly available and continues to improve. our versatile integrodifferential approach directly integrates these data into the model and can easily be extended, corroborating its superiority over standard sir-type models. in general, the model can thus handle an arbitrary number of courses of the disease. similarly, it may expand to consider region-dependent demographics or varying capacities and quality of treatment of the health-care system. while the model can serve as a valuable tool to assess the effects of new super spreader events-which may occur any time-on the distribution of cases in germany, it reaches its validity limit when the number of infections becomes small. to additionally capture this even smaller scale, a coupling to individual agent-based models [15] [16] [17] may be beneficial. covid-19 dashboard by the center for 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beispielszenarien der sars-cov-2-epidemie 2020 in deutschland the particle swarm-explosion, stability, and convergence in a multidimensional complex space particle swarm optimization in highdimensional bounded search spaces publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements open access funding provided by projekt deal. we cordially thank sarah nistler for tedious data collection and the entire covid-19 modeling group at fau for valuable discussions and feedback on this work.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecomm ons.org/licenses/by/4.0/. key: cord-259562-e1htl489 authors: petzold, moritz bruno; bendau, antonia; plag, jens; pyrkosch, lena; mascarell maricic, lea; betzler, felix; rogoll, janina; große, julia; ströhle, andreas title: risk, resilience, psychological distress, and anxiety at the beginning of the covid‐19 pandemic in germany date: 2020-07-07 journal: brain behav doi: 10.1002/brb3.1745 sha: doc_id: 259562 cord_uid: e1htl489 background: the current covid‐19 pandemic comes with multiple psychological stressors due to health‐related, social, economic, and individual consequences and may cause psychological distress. the aim of this study was to screen the population in germany for negative impact on mental health in the current covid‐19 pandemic and to analyze possible risk and protective factors. methods: a total of 6,509 people took part in an online survey in germany from 27 march to 6 april. the questionnaire included demographic information and ascertained psychological distress, anxiety and depressive symptoms, and risk and protective factors. results: in our sample, over 50% expressed suffering from anxiety and psychological distress regarding the covid‐19 pandemic. participants spent several hours per day thinking about covid‐19 (m = 4.45). psychological and social determinants showed stronger associations with anxiety regarding covid‐19 than experiences with the disease. conclusions: the current covid‐19 pandemic does cause psychological distress, anxiety, and depression for large proportions of the general population. strategies such as maintaining a healthy lifestyle and social contacts, acceptance of anxiety and negative emotions, fostering self‐efficacy, and information on where to get medical treatment if needed, seem of help, while substance abuse and suppression of anxiety and negative emotions seem to be associated with more psychological burden. the new virus sars-cov-2 has now rapidly spread to nearly all countries over the world, and the world health organization (who) declared an international pandemic in march 2020 (ghebreyesus, 2020) . the pandemic comes with a large number of potential stressors that might cause psychological distress and mental health burden (inter-agency standing committee, 2020). potential stressors related to the virus might be the fear of an infection with covid-19 and the consequences for oneself or loved ones. the taken measures that aim to slow down the spreading of the virus also come with lots of stressors such as social isolation, economic consequences, and uncertainty about the future (inter-agency standing committee, 2020). therefore, an increase in psychological distress and negative consequences for the mental health of large populations worldwide can be assumed. in a rapid developing situation with a pandemic of a scale that was not known in the last 50 years, substantial research on the psychological consequences of the pandemic is lacking. first studies provide evidence regarding psychological distress in the context of the covid-19 pandemic. an online survey in the general population in china showed that more than half of the participants rated the psychological impact of the events as moderate-to-severe and 16.5% reported depressive and 28.8% anxiety symptoms of moderate-to-severe intensity during the initial stage of the pandemic. these proportions seemed to be relatively stable-a second survey 4 weeks later showed no significant reduction in those symptoms (wang, pan, wan, tan, xu, mcintyre, et al., 2020) . another study from china showed a lower prevalence of symptoms of psychological distress in chinese workforce during the covid-19 outbreak tan, hao, et al., 2020) , and particularly, individuals with preexisting (mental) health issues seem to suffer from psychological strain in the context of the pandemic . studies that focused on the psychological consequences of previous epidemics or pandemics showed that these were associated with substantial psychological distress and mental health problems, for example, during the ebola epidemic 2014 (greenberg, wessely, & wykes, 2015; mohammed et al., 2015) or the sars outbreak in 2003 (maunder et al., 2006) . the first case in germany was detected in january 2020 (bayrisches staatsministerium für gesundheit und pflege, 2020), and case numbers have been rising afterward (see figure 1 ). in parallel, stepwise more rules appeared to inhibit a further exponential growth of the infection numbers, for example, the closure of all educational, cultural and gastronomical institutions, and a reduction in retail and service sectors (bundesgesundheitsministerium, 2020) . since 23 march, throughout germany, more rigorous national rules became effective, including further closures of institutions and restrictions of physical contact and staying outside. to our knowledge, there is no published research on factors of psychological distress in the general population in germany during the current pandemic. hence, the aim of the present study was to assess psychological distress, anxiety, and depression with regard to the covid-19 pandemic and to analyze possible risk and protective factors. this is a cross-sectional observational study using a convenience sample of the general population in germany via online survey, approved by the ethics committee of charité universitätsmedizin berlin (ea1/071/20) and registered on clinicaltrials.gov (nct04331106). to survey the psychological dimension of the covid-19 pandemic, an online self-report questionnaire via sosci survey was used. data collection started 27 march 2020, when in germany, 42,288 cases of infection and 253 deaths attributed to covid-19 were reported (robert koch institut, 2020) . the end of the first wave of data collection was 10 days later: 6 april 2020, when in germany 95,391 cases and 1,434 deaths were reported (robert koch institut, 2020 the charité. completing the entire survey required 10-15 min. the present paper only examines cross-sectional data of the first wave. further longitudinal measurements will be carried out. all participants gave informed consent prior to participation. figure 1 shows the covid-19 situation in germany during recruitment period regarding cases of infection, death, and recovery. except the minimum age of 18 years, residence in germany, and the ability to complete the questionnaire in german, there were no other inclusion or exclusion criteria. the online questionnaire contained demographic information and the experiences with the virus (e.g., being in quarantine, tested or diagnosed for the coronavirus). additionally, the subjective risk of being infected within the next month was rated from 0% to 100% and the daily average amount of hours spent thinking about covid-19 was recorded. to screen for general anxiety and depressive symptoms, the ultra-brief screening scale of the patient health questionnaire-4 (phq-4) (löwe et al., 2010) was used. the intensity of four items describing major anxiety/depressive symptoms was rated on a 4-point scale from 0 ("not at all") to 3 ("nearly every day"). the phq-4 can be examined as a total score or be divided into an anxiety (gad-2) and a depression subscale (phq-2). to assess selected aspects of anxiety regarding covid-19, nine items were included (e.g., the fear of being infected and the fear of social or economic consequences). all statements were rated on a 6-point likert scale, ranging from 1 ("not true at all") to 6 ("totally true"). additionally, a modified version of the validated dsm-5 severity-measure-for-specific-phobia-adult-scale (beesdo-baum et al., 2012) was used to ascertain the extent of anxiety symptoms caused by the pandemic. the scale consists of 10 items, rated on a 5-point likert scale from 0 ("never") to 4 ("all the time"). the questionnaire inquired eight items regarding protective factors in dealing with the pandemic (e.g., self-efficacy in general, social self-efficacy) and five items targeting risk factors (e.g., suppression, substance use). protective and risk factors were adapted from the recommendations on coping with psychological distress in the pandemic of the inter-agency standing committee (iasc) of the united nations (un) (inter-agency standing committee, 2020). items were rated on a 6-point likert scale. all questions were administered in german. the questionnaire consisted of eight pages. we included only participants who completed at least page 4 (n = 6,509). 93.6% of the participants (n = 5,721) completed all pages. average percentage of missing data on item level was 2.1% (range: 0.0-7.1). missing data were handled by casewise deletion. all analyses were carried out using ibm spss statistics version 25. significance level was set to .05 (two-tailed). for the analysis, descriptive statistics, pearson's and spearman's correlations, and t tests for independent samples were used. 70.1% of the participants were female (n = 4,563), 29.0% male (n = 1,887), and 0.9% identified as diverse (n = 59). mean age was 36.2 years (sd = 11.65, range 18-99). 37.6% reported to have children (n = 37.6%). 15.1% had a secondary school degree (n = 985), 32.4% had a higher education entrance qualification (n = 2,109), and 50.0% had a university degree (n = 3,254). 16.7% of the participants reported to work in a medical context (n = 1,084). 10.7% of the participants suffered from a severe physical illness (n = 695). the participants lived in a household with 2.54 persons on average (including themselves). figure 2 shows the experiences of the participants with covid-19. about one third of the participants knew someone diagnosed with covid-19 or already suspected themselves to be infected. about 7% were currently under quarantine, and <5% had been tested for covid-19. about 1% of the sample had been diagnosed with covid-19. average rating of the risk of being infected with covid-19 within the next month was 38.3% (sd = 25.26, range: 0-100). most participants rated the risk with 50% (21.8%, n = 1,422). the lowest 25% of the sample ranked it as 20.0% or lower. median of risk perception was 40.0%. the highest 25% ranked the risk at least as 50%. average rating of the risk of being infected with influenza ("flu") was 18.2% on average, the participants thought about covid-19 for 4.45 hr/ day (sd = 3.80, range from 0 to 24). 25% of the participants thought <2 hr, while 25% thought 6 hr or more per day about covid-19. 10% reported to think more than 10 hr/day about covid-19. women where to get medical treatment showed significant negative correlations ranging from r = −.07 to r = −.24. the overall score of the modified specific-phobia scale was 10.15 (sd = 6.95), with women showing significantly higher scores than men (m = 10.67, sd = 6.94 vs. m = 8.88, sd = 6.78; p > .001). the participants showed an average phq-4 score of 4.15 (sd = 3.19, range 0-12). 25% of the participants showed a score of at least 6, while 10% of them showed a score of at least 9. women showed a significantly higher phq-4 score (indicating more depressive and anxious symptomatology) than men (m = 4.4 vs. m = 3.5). the participants showed an average phq-2 score of 2.11 (sd = 1.70, range 0-6). 25% of the sample showed a score of at least 3 and 10% a score of at least 5. the average gad-2 score was 2.03 (sd = 1.76, range 0-6). 25% of the participants showed a score of at least 3, while 10% showed a score of at least 5. in this study, we wanted to explore how the current covid-19 pandemic is connected to a psychological burden, especially to upcoming anxiety, among the general population in germany. first, we found that the participants spend a tremendous amount second, we found that the risk perception of getting infected with covid-19 in the next 4 weeks was very high. these data show that as expected, the fear of becoming infected with covid-19 is very prevalent in the general population. even in a time where the prevalence of covid-19 infections seems difficult to estimate, the risk rating of being infected within the next 4 weeks seems to be higher than the expected number of infections in 4 weeks. an infection probability of 40% within the next 4 weeks (the median) would mean over 30 million of infected people in germany by beginning of may which seems rather unlikely when the current development is taken into account (robert koch institut, 2020). our (tham, ibrahim, hunt, kapur, & gooding, 2020 ). in the current situation, fears regarding the covid-19 pandemic have to be seen as normal consequences in an exceptional situation rather than as pathologic reactions (petzold, plag, & ströhle, 2020a , 2020b outbreak , where more than half of the participants reported a moderate-to-severe psychological impact of the covid-19 pandemic on themselves, while about 17% of reported moderate-to-severe depressive symptoms and nearly 30% reported moderate-to-severe anxiety symptoms. interestingly, personal experiences with covid-19 were not strongly connected to covid-19 anxiety. this could mean that psychological and social determinants may have a larger influence on anxiety in that early phase of the pandemic than immediate experiences with this virus itself. this is undermined by our finding that self-efficacy (meaning a person's believe in his or her own ability to master situations or show a certain behavior) showed essential significant negative correlations with covid-19 anxiety. low self-efficacy has been shown to be connected with higher anxiety (bandura, 1988; muris, 2002) . our results make the assumption reasonable that self-efficacy could be a protective factor also against pandemic-driven anxiety and future longitudinal studies should test this assumption. the result that working in a medical context is associated with more anxiety regarding the covid-19 pandemic is in line with findings from a recent study from hospitals in singapore and india that showed high proportions of physical and psychological strain in healthcare workers . a further comparison of different professions in the healthcare sector would be interesting-as for example in a study in singapore nonmedical healthcare workers (e.g., pharmacists, technicians) reported more psychological strain than medical personnel tan, hao, et al., 2020) . these results are of a high practical value as they empirically underpin the recommendations on the reduction of psychological distress in the current pandemic that are given by international or in our sample, the average phq-4 score was with a mean of 4.15 higher than the phq-4 score that has been reported by previous research in the general population of 1.76 (löwe et al., 2010) . with all given precautions, this could show that in the current situation there is an increase in depressive and anxiety symptoms in the german general population. due to the nature of the study, this cannot be interpreted as a robust and reliable research result and should be merely seen as an empirical fundament to build hypotheses in this direction. if elevated levels of anxiety and depression turn out reliable and robust in other studies and especially in the longitudinal course, appropriate interventions should be established to reduce psychological strain-for example, cognitive behavioral therapy . in a first longitudinal study from china (wang, pan, wan, tan, xu, mcintyre, et al., 2020) , a statistically significant but not clinically relevant reduction in ptsd symptoms as a result of the covid-19 pandemic was found from end of january to end of february 2020. at the same time, there were no significant changes regarding anxiety, depression, and stress. furthermore, the study identified protective factors such as confidence in doctors and satisfaction with health information, risk perception and outcome expectation (perceived survival likelihood), and personal precautionary measures (wang, pan, wan, tan, xu, mcintyre, et al., 2020) . in our sample, women showed higher scores of covid-19 anxiety, more time of thinking about covid-19 per day, as well as more depressive symptoms than men. this is in line with the results of other studies regarding the psychosocial distress caused by the covid-19 pandemic (qiu et al., 2020; . up to now, it is not possible to draw conclusions if this is something specific to the covid-19 pandemic as higher values of anxiety and depression are reported in women in general (salk, hyde, & abramson, 2017) . our study represents the first study that assesses psychological distress, anxiety, and depression as well as risk and protective factors in the current covid-19 pandemic in germany. we started recruitment quite early so we assessed our participants still in a situation where case numbers were rising exponentially and media coverage was really large. this allows to study the psychological consequences at an early stage of the pandemic and lays a good basis for further longitudinal follow-ups. with a sample size of over 6,000 participants, our sample is large enough to detect even small effects. our sample was fully registered and approved by the local ethics committee. nevertheless, there are some limitations. we recruited our sample as convenience sample mainly through social media. this might have led to a sample bias. people who are familiar with or have easy access to social media might have been more likely to participate in our study, which might have led to a rather young sample. furthermore, people who show higher levels of psychological distress and anxiety might be more likely to take part in a study like ours. this could have led to an overestimation of these factors in our sample. this strategy of recruitment does reduce the generalizability of our results which is shown by several differences between the demographics in our sample and the general population in germany. the sample shows in comparison with the general population a much higher gender imbalance, a lower average age, and a higher percentage of persons working in a medical context (bundesinstitut für bevölkerungsforschung, 2020). our study is a cross-sectional examination and does not allow any causal interferences. our questionnaire was rather short, using simple scales, not all of them were validated. therefore, all of the study results in general should rather be interpreted as first hints, which might be helpful for further studies as well as to empirically underpin existing recommendations on the reduction in psychological distress in the pandemic. our results suggest that in this early phase of the covid-19 pandemic with low percentages of diagnosed cases in our study population, we can already observe its fundamental impact on anxiety and in the pandemic such as a healthy lifestyle, social support, acceptance of negative emotions, and avoidance of suppression and substance abuse is supported by our data. the authors declare that there is no conflict of interest. the peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.1745. the data that support the findings of this study are available from the corresponding author upon reasonable request. moritz bruno petzold https://orcid.org/0000-0002-7801-1434 antonia bendau https://orcid.org/0000-0002-3789-6205 self-efficacy conception of anxiety bestätigter coronavirus-fall in bayern -infektionsschutzmaßnahmen laufen psychometric properties of the dimensional anxiety scales for dsm-v in an unselected sample of german treatment seeking patients retrieved from www.bunde 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note on addressing mental health and psychosocial aspects of covid-19 outbreak-version 1.1. retrieved from https://inter agenc ystan dingc ommit tee.org/ syste m/files/ 2020-03/mhpss %20cov id19%20bri efing %20not e%202%20mar ch%202020-engli sh.pdf. international federation of red cross and red crescent societies (2020). mental health and psychosocial support for staff, volunteers and communities in an outbreak of novel coronavirus a 4-item measure of depression and anxiety: validation and standardization of the patient health questionnaire-4 (phq-4) in the general population long-term psychological and occupational effects of providing hospital healthcare during sars outbreak an evaluation of psychological distress and social support of survivors and contacts of ebola virus disease infection and their relatives in lagos, nigeria: a cross sectional study-2014 relationships between self-efficacy and symptoms of anxiety disorders and depression in a normal adolescent sample covid-19-pandemie: psychische belastungen können reduziert werden dealing with psychological distress by healthcare professionals during the covid-19 pandemia a nationwide survey of psychological distress among chinese people in the covid-19 epidemic: implications and policy recommendations covid-19: fallzahlen in deutschland und weltweit gender differences in depression in representative national samples: meta-analyses of diagnoses and symptoms is returning to work during the covid-19 pandemic stressful? a study on immediate mental health status and psychoneuroimmunity prevention measures of chinese workforce examining the mechanisms by which adverse life events affect having a history of self-harm, and the protective effect of social support when worries make you sick: a review of perseverative cognition, the default stress response and somatic health immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china a longitudinal study on the mental health of general population during the covid-19 epidemic in china reflecting on rumination: consequences, causes, mechanisms and treatment of rumination longitudinal associations between rumination and depressive symptoms in a probability sample of adults mental health considerations during covid-19 outbreak key: cord-287548-3wv9xcxh authors: plümper, thomas; neumayer, eric title: the pandemic predominantly hits poor neighbourhoods? sars-cov-2 infections and covid-19 fatalities in german districts date: 2020-08-20 journal: eur j public health doi: 10.1093/eurpub/ckaa168 sha: doc_id: 287548 cord_uid: 3wv9xcxh background: reports from the uk and the usa suggest that covid-19 predominantly affects poorer neighbourhoods. this article paints a more complex picture by distinguishing between a first and second phase of the pandemic. the initial spread of infections and its correlation with socio-economic factors depends on how the virus first entered a country. the second phase of the pandemic begins when individuals start taking precautionary measures and governments implement lockdowns. in this phase the spread of the virus depends on the ability of individuals to socially distance themselves, which is to some extent socially stratified. methods: we analyse the geographical distribution of known cumulative cases and fatalities per capita in an ecological analysis across local districts in germany distinguishing between the first and the second phase of the pandemic. results: in germany, the virus first entered via individuals returning from skiing in the alps and other international travel. in this first phase we find a positive association between the wealth of a district and infection rates and a negative association with indicators of social deprivation. during the second phase and controlling for path dependency, districts with a higher share of university-educated employees record fewer new infections and deaths and richer districts record fewer deaths, districts with a higher unemployment rate record more deaths. conclusion: the social stratification of covid-19 changes substantively across the two phases of the pandemic in germany. only in the second phase and controlling for temporal dependence does covid-19 predominantly hit poorer districts. in germany, the virus happened to be spread initially via individuals returning from ski holidays in the alps and, to a much lesser extent, through business and other travellers from china, italy and other hotspots, which meant that the majority of infected people in the beginning were relatively young and well-off. 9, 11 once the virus had reached germany, the subsequent spread of infections was facilitated by super-spreader social events such as a carnival session in gangelt, a small town in the district of heinsberg, a beer festival in the small city of mitterteich, district of tirschenreuth, and a wine event in bretzfeld, hohenlohekreis. these super-spreader events create local cluster effects if the social event is mainly attended by locals. in fact, even two months after the above events took place, these were still the districts with the highest number of known infections per 100,000 citizens in germany. figure 1 maps cumulative known sars-cov-2 cases, normalized by population, in german districts on 13 april. even at a first glance we see that the rate of infection declines from south to north and from west to east. even within the western part of germany, regions in which a greater share of the population is catholic also have a higher incidence, which may be correlated to spreader events such as carnival that is much more popular in predominantly catholic regions. 12, 13 the north-south divide appears to be stronger than the east-west divide. this may be down in part to the greater ease by which southern germans can reach by car what turned out to be virus hotspots in ski resorts in northern italy and austria. insert figure 1 about here once the existence and dangers of the pandemic have become public knowledge, people and governments implement precautionary measures and the spread of the virus slows down. 14, 15 at the same time, the geographical pattern of infections slowly changes. for a virus to spread, social interaction between an infected and an uninfected person is required. since the number of new infections remains strongly influenced by the number of active infections in a district, the pattern that has evolved during phase 1 will not disappear quickly. thus, hotspots remain hotspots for some time. but not forever. figure do not affect all people in the same way. 16 the ability to reduce social interactions and to 'stay home' is not distributed evenly in a society. 17, 18 the spread of the virus in phase 2 is shaped by the extent to which individuals manage to reduce their social contacts. in general, white collar activities can be moved to a home office, while other workers still need to commute to their workplace and work if their employer does not lock down the workplace. poorer people find social distancing more challenging than richer people, having less access to resources to shield them from the economically damaging effect of the lockdown. regardless of how and where the virus had spread first in the initial phase of the pandemic, in phase 2 the virus is likely to become a poor man's disease. in fact, we find that in the second phase of the pandemic, poorer and more socially deprived districts start to have higher than average covid-19 mortality rates. the transition from phase 1 to phase 2 is a smooth process rather than a hard cut, as this depends on when people start consciously changing their behaviour and some do so earlier than others. still, a definite break comes with the lockdown. the first german states to go into lockdown were bavaria and the saarland. their curfew begun on 21 march; one day later the whole of germany followed. hartl et al. 19 ideally, we would test our first prediction with data on cases from late march or early april, since it takes roughly a week from the implementation to the effectiveness of policy measures on infection rates. unfortunately, the first date at which we were able to capture the full distribution of confirmed infections and deaths across all german districts is 13 april, with data sourced from the website of the robert koch institute. whilst clearly introducing measurement error as overlapping with the second phase of the pandemic, the strong path dependency of any pandemic means that the cumulative number of infections on 13 april will be sufficiently strongly correlated with the cumulative number of infections around 30 march, which would have been the ideal date. since it takes more time for people to die from covid-19, 13 april may represent close to the ideal end period for phase 1 for our analysis of fatalities. to study our second prediction, we take as our second dependent variable new infections and fatalities that happened in the second period between 14 april and 19 may. these cases occurred after people had time to adjust to the by now fully known risks and the lockdown had been imposed. a major relaxation of the lockdown took place on 19 may such that one can take 19 may as the end of phase 2 of the pandemic. we divide cases and fatalities by a district's population size in 10,000 people. consequently, the dependent variables in our regressions represent cumulative cases or fatalities per capita and cumulative new infections or new fatalities per capita. we estimate our regression models with ordinary least squares and robust standard errors. as our measure of wealth of a district we include the average income subject to income tax in thousands of euro. we also control for the share of the workforce that is universityeducated. this variable is a proxy for the share of the population that can work from a home office and is correlated at r = 0.85 with an index of working from home potential calculated by alipour et al. 20 to measure social deprivation we include the unemployment rate. average taxable income is highly negatively correlated with the unemployment rate at r = -0.58, which is why we include average taxable income and the unemployment rate only in separate regressions. as two proxy variables to account for the way in which the virus first entered germany and spread initially we include the latitude location of a district and the share of its population that is catholic. the former accounts for the ease by which residents could drive to the alps for ski tourism, whilst the latter accounts for the greater popularity of carnival as potential super-spreader events in predominantly catholic districts. 12 in addition, we include dummy variables for whether a district is predominantly urban and is geographically in an extremely remote location. the virus spreads more easily in more densely populated urban habitats 21,22 and while extreme remoteness is often seen as a costly locational disadvantage, 23, 24 it partly protects the local population from infections as there will be less exchange with people from the outside. all data for the explanatory variables are sourced from regional databases of the german statistical offices. table 1 reports results for average taxable income as the central socio-economic explanatory variables, table 2 does the same for the unemployment rate. in the first phase, average taxable income is positively associated with cumulative cases measured on 13 april at the district level. model 1 suggests that a district that has an average income of 10,000 euros higher than the mean income of german districts has 6.3 [95% c.i.: 3.0 to 9.6] additional cases per 10,000 people relative to the district mean. this is a substantively important effect given that the average number of known cumulative cases of german districts on april 13 stood at 14.9 with a standard deviation of 12.3. in phase 2 we regress the cumulative number of known infections between 14 april and 19 may on the same set of variables. during this period, the mean of cumulative new infections per 10,000 people is 6.3 [s.d. = 5.7] . in this period the association between cumulative cases and average taxable income of a district becomes negative but is not statistically significant (model 2). our results also suggest that mortality rates are lower in richer and therefore higher in poorer districts in phase 2 (model 4). taxable income thus shows a negative association with cumulative cases in phase 1 but not in phase 2, demonstrating that the pandemic increasingly affects poorer districts too even if, as in germany, the pandemic started in richer districts. likewise, average income has no systematic association with cumulative deaths in phase 1 but becomes negatively associated with deaths in phase 2. the opposite pattern to what we find for taxable income holds for the unemployment rate (table 2) . districts with a higher unemployment rate reported lower cumulative cases in phase 1 (model 5) and higher cumulative deaths in phase 2 (model 8). hence, regardless of the socio-economic indicator we use, we find that in phase 2 the pandemic increasingly affects poorer and more socially deprived districts too in terms of cumulative infections and actually affects them more in terms of cumulative deaths. insert table 2 about here there are thus interesting differences between our analysis of infection rates and mortality rates. in phase 1, the population of poorer and more socially deprived districts is less likely to get infected with sars-cov-2 than the population in richer and less deprived districts but there are no statistically significant mortality differences between these districts. in phase 2 and controlling for path dependency, the population of poorer and more socially deprived districts is at least equally likely to get infected, but the probability to die from covid-19 is statistically significantly higher. in germany at least, covid-19 increasingly becomes a disease of the poor after lockdown -arguably, because the rich find it easier to follow the rules of social distancing, a result that is consistent with harris. 6 we studied the relationship between socio-economic factors and the covid-19 pandemic in germany, distinguishing between two phases and analysing both infections and fatalities. we have shown that the population of poorer districts is not necessarily more likely to get infected with sars-cov-2. in germany during the first phase of the pandemic, poorer districts and districts with a higher unemployment rate had fewer infection rates. due to the inherent limitations of an ecological study, our analysis at the district level cannot conclusively identify the causal mechanisms. yet, it seems likely that the distribution of the virus during the first phase of the pandemic in germany has been largely influenced by ski tourism. districts geographically closer to the alps are relatively wealthy and have little social deprivation by german standards. as a consequence, the pandemic started in germany predominantly as a rich man's disease. in this initial phase, mortality rates in poorer and more socially deprived districts were not higher though poorer and more socially deprived people tend to have more co-morbidities, which increase covid-19 mortality. 25 since lockdown, however, and controlling for the strong path dependency in the spread of the disease, poorer and more socially deprived districts no longer report lower infection rates and deaths become increasingly concentrated in these districts. the gap in infection rates between richer and poorer districts closes and a gap in mortality rates begins to open with poorer districts now having higher than average mortality rates. the same applies if we employ the unemployment rate as a measure of social deprivation. covid-19 is slowly becoming a poor man's disease. an ecological analysis cannot trace the causal mechanism but it is very likely that more people in richer districts as well as in districts with a higher share of university educated employees could work from home and afford to behave in a socially distanced way than people in poorer and more socially deprived districts. 26 this is entirely consistent with studies from other countries showing a higher mortality rate among individuals with lower socio-economic status, with the higher prevalence of co-morbidities in such individuals one of the likely causal mechanisms. 25 the recent emergence of hotspots in slaughterhouses in the districts of gütersloh and oldenburg indicate that the pandemic has reached the very poor: temporary migrant workers from bulgaria and romania. the subtle difference in results between the 'infections model' and the 'deaths model' is particularly interesting. these results lend indirect empirical support to previous findings suggesting that the case fatality rate, that is, the number of deaths per known infected people, is higher in poorer districts. 27 sorci et al. 28 have used a very different research design to ours, regressing the case fatality rate on a battery of explanatory variables including some socioeconomic factors, whereas our estimates have the population fatality rate as the dependent variable. for their sample they find that higher than average per capita income is weakly associated with lower than average fatality rate. our results are consistent with their findings in both phases: in phase 1 poorer and more socially deprived districts combine a low infection rate with an average death rate, in phase 2 poorer and more socially deprived districts combine an average infection rate with a higher than average death rate. we suspect that this finding results from the higher prevalence of comorbidities in relatively poor districts in germany and with variations in the ability to follow social distancing rules. covid-19 magnifies the effect of behavioural differences on health outcomes, but does not in itself discriminate between rich and poor. all viruses spread through social interactions and we should not be surprised that pandemics crystallize the socio-economic determinants of social interactions and the socio-economic constraints on the ability to follow social distancing rules. none declared. no specific funding was received. the replication data and do-file will be made available on dataverse.org upon publication. • initially, whether covid-19 predominantly affects poorer or richer neighbourhoods depends on how the virus first entered a society. • in germany, the virus mainly entered via tourists returning from ski holidays in the alps and accordingly wealthier districts initially recorded higher and more socially deprived districts recorded lower covid-19 infection rates during the first phase of the pandemic in which the virus could spread largely unhampered by social distancing measures. • lockdown policies have enormous public health benefits controlling the pandemic but also exert a strong effect on the social stratification of covid-19 because the ability to socially distance oneself from others now determines the individual risk of an infection and at the district level covid-19 increasingly becomes a disease of poorer and more socially deprived districts. • controlling for the path dependency of infections, wealthier districts now record lower and more socially deprived districts record higher covid-19 mortality rates during the second phase of the pandemic in which lockdown was in place. covid-19 exacerbating inequalities in the us poverty kills people: after coronavirus we can no longer ignore it. the guardian 5 may who is more susceptible to covid-19 infection and mortality in the states? the subways seeded the massive coronavirus epidemic in new york city. nber working paper 27021 revealing the unequal burden of covid-19 by income, race/ethnicity, and household crowding: us county vs. zip code analyses how coronavirus -a 'rich man's disease' -infected the poor a pandemic in times of global tourism: superspreading and exportation of covid-19 cases from a ski area in austria a hundred days into the coronavirus disease (covid-19) pandemic carnival and citizenship. the politics of carnival culture in the prussian rhineland spreading the disease. the role of culture effective containment explains subexponential growth in recent confirmed covid-19 cases in china a simplified model for expected development of the sars-cov-2 (corona) spread in germany and us after social distancing health versus wealth: on the distributional effects of controlling a pandemic nber working paper 27046 the differential impact of covid-19 across demographic groups: evidence from nyc, unp the determinants of the differential exposure to covid-19 in new york city and their evolution over time, unp measuring the impact of the german public shutdown on the spread of covid19. covid economics, vetted and real-time papers germany's capacity to work from home. discussion paper 13152. bonn: institute for labour economics 2020. and infection risk in rural ecuador rural america and coronavirus epidemic: challenges and solutions the costs of remoteness: evidence from german division and reunification multilevel determinants of breast cancer survival: association with geographic remoteness and area-level socioeconomic disadvantage. breast cancer research and treatment opensafely: factors associated with covid-19 death in 17 million patients my home is my castle -the benefits of working from home during a pandemic crisis: evidence from germany estimating the global infection fatality rate of covid-19 note: 95% confidence interval in parentheses. *** p<0.01, ** p<0.05, * p<0 key: cord-276363-m8di6dpt authors: holm, majbrit v.; blank, patricia r.; szucs, thomas d. title: influenza vaccination coverage rates in europe – covering five consecutive seasons (2001–2006) in five countries date: 2008-06-28 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2008.00036.x sha: doc_id: 276363 cord_uid: m8di6dpt objective to understand potential drivers and barriers to influenza vaccination in the general population. methods 47 982 household surveys were conducted in five european countries between 2001 and 2006. results overall influenza vaccination coverage increased over the years and reached 26·2% in 2005/06. among the elderly ≥65 years, the rate increased significantly to 67·8% (2005/06). the most common reason for being vaccinated over the 5 years was the perception of influenza as a serious illness, which people want to avoid. the main reason for not getting vaccinated among those never previously vaccinated was feeling that they were unlikely to catch influenza. a recommendation by the family physician was the most encouraging factor for vaccination. the severity of influenza and the efficacy of vaccination are well documented in the medical literature. 1,2 eradication of influenza is impossible but continuous immunization of the population can minimize the impact of the disease. 3 in addition to providing substantial health benefits, vaccination may also be associated with significant economic benefits, not only among the elderly but also among healthy working adults and children. despite this knowledge and ongoing efforts by policy-makers, physicians and other healthcare providers, influenza vaccination rates in the five european countries surveyed remain limited, with the additional effect that manufacturing capacity may be too low for producing a sufficient amount of an appropriate monovalent vaccine when a pandemic occurs. 3 the who states that the risk of a new pandemic is at its highest level since the last pandemic in 1968. 4 this situation might influence the immunization coverage rates in the population. published literature evaluating vaccination coverage rates in europe shows that importance placed on influenza vaccination varies greatly between countries. 3 two recent studies covering several european countries have been published. 5, 6 this report is an update of the earlier work by szucs and muller. we now have data available for five consecutive influenza seasons which allows us to go beyond the usual cross-sectional approach to analyzing vaccination rates. the main focus of this paper is on high-risk group coverage. a second objective is to understand the determinants for being or not being vaccinated, and to describe the populations' opinions regarding influenza and vaccination. in this context, we examine whether the threat of avian influenza had an impact on recent changes in vaccination coverage in the different countries. this survey is an ongoing assessment of influenza coverage rates in france, great britain, italy, spain, and germany. during [7] [8] [9] [10] [11] four target groups were specified. 1. individuals aged ‡65 years 2. individuals who suffer from a chronic illness 3. individuals who work in the medical field 4. combined group of individuals aged ‡65 years or who suffer from a chronic illness or who work in the medical field. for example, in germany the group of chronic illness sufferers is defined according to the german standing commission on immunization, as children, adolescents and adults suffering from chronic diseases of respiratory organs, chronic cardiovascular or liver diseases, as well as nephropathies and diabetes, or other metabolic disorders. in our study, people suffering from heart diseases, pulmonary diseases, diabetes, or other chronic illnesses were included in the chronic illness group. the survey questions have been published previously. 6 the questions covered reasons to get vaccinated this winter, reasons for not getting vaccinated against influenza, and options that would encourage persons to get vaccinated against influenza. for the 2005 ⁄ 06 survey, questions on influenza pandemics and avian influenza were added. the survey populations were representative of the adult population from age 14 years (germany, italy, spain); from age 15 (france), or from age 16 (great britain). in spain, persons above age 75 were not covered. sample weights were applied to correct for small deviations from the applicable age and gender quota and the annual datasets were pooled. statistical evaluation used spss ò version 13 for windows. bivariate associations of categorical variables were assessed using the chi-squared test. a chi-squared test for trend was used to assess time trends. in the case of continuous variables, differences of means were tested using oneway anova. for all statistical tests, two-sided p = 0ae05 was used as the level of statistical significance. ninety-five percent confidence intervals (ci) were reported as appropriate. due to the descriptive nature of these data, no correction for multiple testing was made. covariates identified as predictors of influenza vaccination in univariate analysis were considered as candidates for multivariable analysis. logistic regression was used to identify independent correlates of the outcome of interest, i.e. vaccination coverage. the overall sample consisted of 47 982 persons. in table 1 an overview of the sample is given for the year 2005 ⁄ 06 only. in an earlier publication, similar data can be found for the years 2002 ⁄ 03 and 2003 ⁄ 04. 6 spain was expected to show a lower number of people over 65 years of age, as the survey covered only persons £75 years old. the reason for the great deviation in the number of chronic ill persons in germany compared to other countries remains unclear as there is no difference in the way the question was asked in the five countries. the overall vaccination coverage across countries, based on an average of the country samples, decreased from 22ae5% (95% ci: 21ae6-23ae4%) in season 2001 ⁄ 02 to 21ae3% (95% ci: 20ae5-22ae1%) in season 2002 ⁄ 03. thereafter, it increased to 23ae4% (95% ci: 22ae6-24ae2%) in season 2003 ⁄ 04, to 23ae6% (95% ci: 22ae7-27ae1%) in season 2004 ⁄ 05, and to 26ae2% (95% ci: 25ae3-27ae1%) in season 2005 ⁄ 06 ( figure 2 ). the increase between season 2004 ⁄ 05 and season 2005 ⁄ 06 was statistically significant (p < 0ae001). this was mainly due to significant increases in immunization uptake in germany and italy, where the coverage increased to 32ae5% and 24ae1%, respectively, in 2005 ⁄ 06. adjusting the overall vaccination rate in europe (weighting the population sizes) resulted in an average vaccination rate of 26ae8% in season 2005 ⁄ 06. vaccination rates were highly age-dependent. older age was associated with higher vaccination rates. in season 2005 ⁄ 06 the immunization uptake across all countries was holm et al. ª 2008 the authors higher for all age groups compared to the previous seasons ( figure 1 ). across all five seasons, vaccination rates appeared to be associated with gender in great britain, italy, and spain. in great britain a higher vaccination rate was observed in women, whereas in italy and spain, the majority of the vaccinated were men (details not shown). in the year 2005 ⁄ 06, 39ae0% of the respondents expressed the intention to get vaccinated against influenza in the coming winter of 2006 ⁄ 07. over the years, the proportion of those expressing such an intention was on average 36%, about 13% higher than the actual vaccination rate. the gap was highest in germany (between 15% and 20% over the years) and almost non-existent in italy in season 2005 ⁄ 06. the overall vaccination coverage rate in persons aged ‡65 increased over time ( figure 2 ). the increase between season 2004 ⁄ 05 and season 2005 ⁄ 06 was statistically significant (p < 0ae001). coverage in the elderly was highest in great britain (79%) and lowest in germany and italy (63ae4%). it was significantly different from the population under 65 years of age. since season 2003 ⁄ 04, data on health status in terms of chronic illness were collected. persons with a chronic illness showed significantly higher vaccination coverage than those not suffering from a chronic disease (figure 2) . the highest coverage among the chronically ill persons was found in great britain (66ae4%) and the lowest in france (51ae8%). working in the medical field did not seem to be a driving factor for vaccination as the vaccination coverage rate in this sub-population was not significantly different from the rest of the sample (figure 2) , at the unadjusted level. however, adjustment for age and other covariates revealed the presence of an association ( table 2 ). for persons in the combined target group a significant difference in coverage was found compared to the non-target group population. the vaccination rate in this group increased over the years and the increase in season 2005 ⁄ 06 was significantly different from the previous season. the coverage rate in the combined target group was highest in great britain (60%) and lowest in germany (49%). however, this result could be influenced by the observed difference in the proportion of chronically ill respondents in germany (table 1) . table 2 shows unadjusted odds ratios for the target groups for the year 2005 ⁄ 06. odds ratios across all seasons did not greatly differ from the 2005 ⁄ 06 results. adjusted odds ratios were investigated in logistic regression models. the adjustment took into account gender, age over 65 years, work in medical field, and chronic illness. for years where data on chronic illness were not available, data were only adjusted for the remaining covariates. the odds ratios for the combined target group were only adjusted for age. multivariate adjustment showed significantly higher vaccination rates for healthcare workers in great britain (or: 1ae8, 95% ci: 1ae5-2ae2), france (or: 1ae7, 95% ci: 1ae4-2ae1), italy (or: 1ae4, 95% ci: 1ae1-1ae9), and spain (or: 2ae1, 95% ci: 1ae6-3ae6). the impact of chronic illness on the vaccination rate was significantly lower after multivariate adjustment, mainly due to taking into account the effect of age (germany or: 2ae3, 95% ci: 2ae0; 2ae6, italy or: 5ae0, 95% ci: 4ae2; 6ae0, france or: 3ae4, 95% ci: 2ae7; 4ae2 and spain or: 3ae3, 95% ci: 2ae8; 4ae0). all other odds ratios were not substantially changed by multivariate adjustment (details not shown). for those who reported to have been vaccinated in season 2005 ⁄ 06, the most frequently stated reasons were that influenza is a serious illness that people want to avoid and that they have received a recommendation from their family physician or nurse (table 3 ). in france the most commonly stated reason for vaccination was that the vaccine is provided free. over the 5-year period, the ranking of the cause for getting vaccinated did not change substantially. the proportion of respondents whose decision to get vaccinated was influenced by the recent attention given to avian influenza or a possible influenza pandemic varied from 13% in germany to 1ae3% in spain. it was 8ae6% in great britain, 4ae1% in italy, and 2ae0% in france. across all countries, the persons who gave the threat of avian influenza as a reason for vaccination were not found to be statistically different from other vaccinated persons (table 3) . only the proportion of those vaccinated for first time was statistically higher among the group influenced by the attention given to avian influenza (p < 0ae001). for those of the total survey population who had never been vaccinated, the reasons for not being vaccinated varied across countries over the 5 years of observation. overall, the most frequently stated reasons were no expectation of catching influenza, not having considered vaccination, and absence of a family physician's recommendation ( table 3) . the level of knowledge about influenza and the vaccine among the general population was similar across countries. seventy-nine percent of the respondents agreed with the statement that you can catch influenza even if you are vaccinated against it. sixty-eight percent agreed with the statement that if you catch influenza after having had the vaccine, the infection is less severe. fifty-eight percent said that it is important to get the influenza vaccine each year and 52% agreed that the side effects associated with the vaccine (fever, headache...) are acceptable. most of the participants did not agree with the following statements: the vaccine is not useful if you are in good health and if you have the vaccine, you will not catch influenza. a recommendation by the family physician or nurse, and receiving more information regarding the vaccine being efficacious and well tolerated were regarded as the most important factors that might encourage vaccination (table 3) . data on this question were not available for france. the vaccination coverage rate in the total sample is currently 26ae2% (season 2005 ⁄ 06). a statistically significant increment of 2ae5% was observed between season 2004 ⁄ 05 and season 2005 ⁄ 06. this was mainly due to significant increases in immunization uptake in germany and italy. in germany reimbursement of vaccination for all age groups has been implemented in several federal states to encourage vaccination rates. 12 this may explain the high coverage rates in germany. a sub-analysis of the data obtained in great britain showed that wales reached a vaccination rate of 33ae3% in season 2005 ⁄ 06, higher than the german coverage rate (32ae5%). the immunization rates in the defined highrisk target groups were also increased in season 2005 ⁄ 06. in particular, higher age and suffering from chronic illness were important predictors of vaccination. in the elderly ‡65 years, the lowest coverage was observed in germany and italy and the highest in great britain. in great britain or, odds ratio; ci, confidence interval; p-value, pearson chi-squared. *reference category. holm et al. ª 2008 the authors general practitioners are encouraged to recommend vaccination to eligible high-risk patients, which may have contributed to the high vaccination rates in the target groups. 9 in spain, vaccination coverage increased in those aged ‡60 after the age threshold of vaccination recommendations was reduced to age 60 in some communities. as healthcare workers are in contact with patients, it is critical for this group to be vaccinated in order to reduce transmission of the disease. additionally, they play an important role in the communication and motivation of the public to get vaccinated. however, the vaccination rate among healthcare workers remained low compared to the other high-risk groups. a recent published literature review identified low coverage rates in this professional group to be especially a problem in europe, 13 with vaccination rates between 12% and 25%. 5 our observation of coverage rates increasing from 19% to 25% over the years is consistent with earlier findings. considering the entire population, 39% of the 2005 ⁄ 06 respondents expressed the intention to get vaccinated in season 2006 ⁄ 07. the gap between those who intended to get vaccinated and those who actually received vaccination was stable over the years, at 13% on average. there was, however, substantial variation between countries. the persistence of this gap indicates the potential to increase vaccination coverage rates in europe. however, realizing this potential, activating the correct drivers, and dealing with the barriers to vaccination remains a challenge. only italy seems to have been able to diminish the gap. in season 2003 ⁄ 04 the italian vaccination campaign was intensified by the ministry of health due to the increased focus on severe acute respiratory syndrome. 14 a realistic vaccination coverage rate target in europe could be set at the level of vaccination intentions (39%). in the general population, the characteristics of those who gave the attention to avian influenza -a possible influenza pandemic as a reason for vaccination -were not found to be statistically different from the rest of the vacci-nated group. nonsignificant trends hinted at a larger proportion of women and a slightly lower mean age of this relatively small subgroup. the majority of persons influenced by the attention given to avian influenza were those who were vaccinated for the first time. in the general population, a recommendation from the family physician is the most important encouragement for vaccination. this confirms findings from several previous studies. 5, 15, 16 it was also stated that more information on the vaccines regarding tolerability and efficacy would motivate persons to get vaccinated. we did not cover the vaccination rates of schoolchildren in our article. however, high vaccination coverage in children and subsequent positive external effects will be difficult to achieve at least in some countries. this reason makes high vaccination rates in the risk populations even more important. telephone surveys are an appropriate method to investigate influenza vaccination uptake at the population level. telephone interviews have been used on several occasions to study vaccination rates in europe. 6, 17, 18 the main advantage of telephone interviews is a potentially high response rate obtained in an affordable and fast manner. the selection process based on random dialing of telephone numbers has been shown to be of high quality. 19 in france, the questionnaire was a self-administered mail survey. in mailed questionnaires, there is a high risk of respondents omitting questions and of a low return rate. on the other hand, mailed questionnaires are an even more affordable option for large-scale surveys. 19 the limitations of the present data collection are described in greater detail in an earlier publication. 6 an increasing problem is the use of wireless telephones. in the usa people with landlines had a higher odds (1ae27) of being vaccinated than those with only access to wireless telephones. 20 if this is believed to be similar in europe, we might have slightly overestimated the vaccination rate. the different methodological approach used in france may have affected the reliability of the comparison across countries. this is supported by the fact that the french gave different reasons for and against vaccination compared to the other countries. the who considers the current influenza pandemic risk to be at its highest level since the last pandemic. 4 hence, efforts should be made at all national and international levels to increase vaccination coverage according to the who objectives (i.e. 50% vaccination coverage to be reached in the elderly in 2006 and 75% in 2010). 21 among elderly ‡65 years, a vaccination rate of 50% or higher was reached in all the countries studied. so far, only great britain has reached the 2010 target of 75% with an immunization rate of 79% in season 2005 ⁄ 06. the existence of national targets may provide a partial explanation for this success. 22 vaccines for preventing influenza in the elderly individual and community impact of influenza control of influenza. public health policies influenza vaccination in europe: an inventory of strategies to reach target populations and optimise vaccination uptake influenza vaccination coverage rates in five european countries-a population-based cross-sectional analysis of two consecutive influenza seasons rki-ratgeber infektionskrankheiten -merkblä tter fü r ä rztezielgruppen der impfung the influenza immunisation programme [www document protocole de mise en place de la chimio-prophylaxie dans une collectivité de personnes à risque lors d'une é pidè mie de grippe, en pé eriode de circulation du virus grippal influenza coverages in spain and vaccination-related factors in the subgroup aged 50-64 years zahlt meine kasse fü r die grippeschutzimpfung? influenza vaccination of healthcare workers: a literature review of attitudes and beliefs seasons determinants of adult influenza and pneumonia immunization rates cross-sectional study on influenza vaccination influenza vaccination coverage in elderly people influenza vaccination coverage and reasons to refrain among high-risk persons in four european countries health measurement scales. a practical guide to their development and use telephone coverage and health survey estimates: evaluating the need for concern about wireless substitution prevention and control of influenza pandemics and annual epidemics, 56th wha, 10th plenary meeting department of health. summary of flu immunisation policykey points about flu immunisation policy in england this study was made possible by an unrestricted research grant from aventis-pasteur msd, lyon, france. we thank the geig for making the french data available for analysis. furthermore, we would like to thank bertrand verwee, christine pilet from sanofi pasteur, and matthias schwenkglenks from the european center of pharmaceutical medicine, basel, switzerland, for their comments on the study and on the data analyses. key: cord-339735-6964ktxr authors: empl, michael t.; kammeyer, patricia; ulrich, reiner; joseph, jan f.; parr, maria k.; willenberg, ina; schebb, nils h.; baumgärtner, wolfgang; röhrdanz, elke; steffen, christian; steinberg, pablo title: the influence of chronic l-carnitine supplementation on the formation of preneoplastic and atherosclerotic lesions in the colon and aorta of male f344 rats date: 2014-08-28 journal: arch toxicol doi: 10.1007/s00204-014-1341-4 sha: doc_id: 339735 cord_uid: 6964ktxr l-carnitine, a key component of fatty acid oxidation, is nowadays being extensively used as a nutritional supplement with allegedly “fat burning” and performance-enhancing properties, although to date there are no conclusive data supporting these claims. furthermore, there is an inverse relationship between exogenous supplementation and bioavailability, i.e., fairly high oral doses are not fully absorbed and thus a significant amount of carnitine remains in the gut. human and rat enterobacteria can degrade unabsorbed l-carnitine to trimethylamine or trimethylamine-n-oxide, which, under certain conditions, may be transformed to the known carcinogen n-nitrosodimethylamine. recent findings indicate that trimethylamine-n-oxide might also be involved in the development of atherosclerotic lesions. we therefore investigated whether a 1-year administration of different l-carnitine concentrations (0, 1, 2 and 5 g/l) via drinking water leads to an increased incidence of preneoplastic lesions (so-called aberrant crypt foci) in the colon of fischer 344 rats as well as to the appearance of atherosclerotic lesions in the aorta of these animals. no significant difference between the test groups regarding the formation of lesions in the colon and aorta of the rats was observed, suggesting that, under the given experimental conditions, l-carnitine up to a concentration of 5 g/l in the drinking water does not have adverse effects on the gastrointestinal and vascular system of fischer 344 rats. electronic supplementary material: the online version of this article (doi:10.1007/s00204-014-1341-4) contains supplementary material, which is available to authorized users. l-carnitine is a key component of the so-called carnitine shuttle, a multienzyme transport system that is required to transfer activated long-chain fatty acids (acyl-coas) into the mitochondrial matrix, where they are degraded via β-oxidation (violante et al. 2013) . in the course of this process, l-carnitine is conjugated to acyl-coas by carnitine palmitoyltransferase 1 (cpt1) yielding acylcarnitines, which are then transported to the inner mitochondrial compartment by the carnitine acylcarnitine translocase (cact) in exchange for free carnitine (houten and wanders 2010) . thereafter, carnitine palmitoyltransferase 2 (cpt2) retransforms acylcarnitines to acyl-coa esters, which are then degraded to acyl-coa subunits, thus generating substrates for the citric acid cycle and reducing equivalents for the electron transport chain (houten and wanders 2010) . although l-carnitine is present in plants, the main nutritional sources for humans are foodstuffs of animal origin (mitchell 1978) . depending on dietary habits, daily intake from food sources ranges from <0.16 to 2.4 mg/kg body weight (bw), the bioavailability being generally lower in humans that regularly consume a diet high in l-carnitine (e.g., abundant consumption of red meat) and even lower when high amounts of this compound are supplemented exogenously (harper et al. 1988; rebouche 2004; sahajwalla et al. 1995) . the body concentration of l-carnitine is tightly regulated by an equilibrium between endogenous synthesis (from lysine and methionine), renal reabsorption, and dietary l-carnitine supply, the latter especially influencing the renal clearance rate (evans and fornasini 2003; jeukendrup et al. 1998; rebouche 2004 ). therefore, oral or intravenous dosages above a certain basal or physiological "threshold" level lead to an increased l-carnitine elimination and diminished uptake (evans and fornasini 2003; rebouche and seim 1998) . although the mechanisms of the intestinal absorption of l-carnitine have not yet been fully elucidated, they most likely involve carrier-mediated transport as well as passive diffusion, the latter being the more important intake route for non-dietary (i.e., high) carnitine concentrations (evans and fornasini 2003; li et al. 1992) . active renal reabsorption, intestinal uptake and tissue distribution of l-carnitine are mediated by so-called carnitine/organic cation transporters (octn), two of them (octn1 and octn2) having been identified in humans (tamai 2013) . rare mutations in the slc22a5 gene encoding for octn2 lead to systemic carnitine shortage and consequently to the development of a primary carnitine deficiency (pcd), which in most cases manifests itself clinically in form of a hypoketotic hypoglycemic encephalopathy as well as disorders of the heart and skeletal muscle (erguven et al. 2007; lahjouji et al. 2001) . in contrast, the clinically less severe secondary carnitine deficiency (scd) is caused by organ (e.g., kidney or liver) or metabolic disorders (e.g., impaired fatty acid metabolism), carnitine malabsorption, malnutrition as well as pharmacological treatment (erguven et al. 2007; flanagan et al. 2010) . the treatment of these disorders, especially in the case of pcd, consists in the daily supplementation of l-carnitine in doses (100-400 mg/kg bw or 990 mg 2-3 times/day) adapted to the patients' plasma level (bain et al. 2006; longo et al. 2006) . since the early 1980s, l-carnitine is also being extensively advertised and used by athletes, bodybuilders or even obese individuals as a nutritional supplement with allegedly "fat burning" and performance-enhancing properties (jeukendrup et al. 1998 ), although to date there are no conclusive data supporting these claims neither in rats (eder 2000; melton et al. 2005; saldanha aoki et al. 2004 ) nor in humans (barnett et al. 1994; brass 2000; cerretelli and marconi 1990; grunewald and bailey 1993; jeukendrup and randell 2011; jeukendrup et al. 1998; vukovich et al. 1994) . taking into account the inverse relationship between exogenous supplementation and bioavailability, one must conclude that, when fairly high oral doses are given, a significant amount of l-carnitine would remain in the gut. the unabsorbed compound can be partially degraded to trimethylamine (tma) or γ-butyrobetaine by enterobacteria in the gut lumen of rats and, to a greater extent, humans (koeth et al. 2013; rebouche and chenard 1991; rebouche et al. 1984; zhang et al. 1999) . after absorption, tma is oxidized to trimethylamine-n-oxide (tmno) by hepatic flavin monooxygenases (baker and chaykin 1962; bennett et al. 2013; koeth et al. 2013 ): in addition to that, and following l-carnitine supplementation, tmno can be directly produced in the gut by the gastrointestinal microbiota (koeth et al. 2013) . in an acidic environment and in the presence of nitrite ions, i.e., under conditions which prevail in the upper gastrointestinal tract, the known carcinogen n-nitrosodimethylamine (ndma) can be formed from these amine precursors (bain et al. 2005; lijinsky et al. 1972; loh et al. 2011; tricker and preussmann 1991) . additionally, bacterial metabolism can also lead to ndma formation from amines such as tma and dimethylamine (maduagwu and bassir 1979) . consumption of l-carnitine in doses that are not completely absorbed might thus enhance bacterial ndma production in the colon and consequently contribute to colorectal tumor formation, as has been shown by knekt et al. (1999) and loh et al. (2011) for dietary ndma. therefore, we investigated whether a chronic administration of different l-carnitine concentrations via drinking water leads to an increased number of aberrant crypt foci (acf), which are considered preneoplastic lesions associated with colorectal cancer formation (bird 1995) , in the colon of male fischer 344 rats. as a recent study by koeth et al. (2013) showed that tmno resulting from l-carnitine supplementation promotes atherosclerosis in mice, we additionally examined its influence on the occurrence of atherosclerotic lesions in the aorta of the above-mentioned rats. eighty male fischer 344 ducrl rats (f344 rats) were purchased at 5-6 weeks of age (100-120 g bw) from charles river (sulzfeld, germany) and housed in type iv polycarbonate cages (ehret, emmendingen, germany). the cages were placed in airflow cabinets (uni protect; ehret) operated in a positive pressure mode (50 pa) and providing a temperature of 21-23 °c, a relative humidity of 50-60 %, a maximum light intensity of 45 lux, 15-20 air shifts per hour as well as a 12/12 h day and night cycle. the bedding consisted of poplar granules (lignocel ® select; jrs, rosenberg, germany), which were changed once a week, and the diet was a standard pelleted rodent maintenance diet (cat. nr. 1324; see online resource 1 "specifications of the animal feed" for details on feed composition) purchased from altromin (lage, germany). the animals in each cage had access to tunnel housing made of red polycarbonate (bioscape, castrop-rauxel, germany) and certified carcinogen and toxicant-free aspen rods (abedd ® lab&vet service, vienna, austria) as enrichment. experimental design and procedure upon arrival, littermates were randomly assigned to one of four test groups consisting of 20 animals each and housed pairwise in each cage to minimize distress during the whole experimental period of 58 weeks. animals in the control group (group 1) received drinking (tap) water without any supplementation. water for groups 2, 3 and 4 was supplemented with 1, 2 or 5 g l-carnitine/l for 52 weeks, respectively. l-carnitine (carnipure™; lonza, basel, switzerland) with a purity of 99.5-99.9 % was purchased from denk ingredients (munich, germany). because of a sialodacryoadenitis virus (sdav) infection (see results), the l-carnitine treatment was started in week 6 upon arrival after an acclimatization and recovery period of 5 weeks. to avoid microbial contamination, drinking water was autoclaved before carnitine supplementation and changed twice a week. in the course of water changes, water consumption was recorded, while the weight of the animals was assessed once a week. moreover, the stability of l-carnitine in the water was assessed by liquid chromatography/mass spectrometry (lc-ms) for a period of 7 days under experimental conditions (see online resource 1 "assessment of l-carnitine stability" for details). after the 52-week administration period, individual rats were anaesthetized by co 2 (6 l/min flush) and decapitated. blood was immediately collected for further analysis and the gastrointestinal tract entirely removed and processed as previously described (nicken et al. 2012) . briefly, the colon was removed, washed with phosphate buffered saline (pbs) and opened longitudinally. thereafter, the tissues were fixed in formalin (roti ® -histofix 4 %; carl roth, karlsruhe, germany), stained with methylene blue solution (0.1 % w/v in pbs) and acf formation was assessed using a stereomicroscope (szx16; olympus, hamburg, germany). additionally, the kidneys, liver and spleen were removed and weighed. for histopathologic examination, heart, thoracic aorta and liver were fixed in 10 % neutral buffered formalin. organ trimming was performed in accordance with the registry of industrial toxicology animal-data (rita) and north american control animal database (nacad) guidelines for organ sampling and trimming in rats and mice (morawietz et al. 2004; ruehl-fehlert et al. 2003) , followed by embedding in paraffin wax, sectioning at 2 µm thickness, and staining with hematoxylin and eosin (he). micrographs of representative lesions were obtained using an olympus bx51 microscope equipped with a dp72 12.8 megapixel digital color camera and cellsens standard v. 1.7.1 software (olympus corp., tokyo, japan). figures were further processed with adobe ® photoshop ® v. 7.0 (adobe systems, inc., san jose, ca, usa), thereby adjusting contrast and brightness, if necessary. assessment of the ndma concentration in the urine of the experimental animals urine was collected in the next-to-last week of the study by housing 16 animals (4 from each group) individually in metabolic cages (tecniplast, hohenpeißenberg, germany) for 24 h. gathered urine samples (5-10 ml) were stored in 50-ml tubes (greiner bio-one, frickenhausen, germany) protected from light at −80 °c until analysis. sample extraction was performed using supelclean coconut charcoal spe tubes (sigma-aldrich, schnelldorf, germany) based on the u.s. environmental protection agency method 521 for the detection of nitrosamines in drinking water (munch and bassett 2004) . d 6 -ndma (restek, bad homburg, germany) was added to the urine samples as internal standard prior to sample preparation. the nitrosamines were eluted from the spe tubes using methylene chloride (vwr, leuven, belgium) and concentrated under a gentle stream of nitrogen at 35 °c to a volume of approximately 0.5 ml. an aliquot of 5 µl was injected into the gc-ms. as residue-free urine was not available, the method validation was performed by using synthetic human urine (synthetic urine, nussdorf, germany) spiked with ndma (restek). the samples were analyzed on an agilent 7890a gas chromatograph (waldbronn, germany) coupled to an agilent 5975c mass selective detector (msd) applying the following parameters: column: agilent db-wax (polyethylene glycol, 30 m; 0.25 mm i.d.; 0.5 µm film thickness); carrier gas: helium, 1.9 ml/min, constant flow; oven temperature program: 1 min 35 °c, +20 °c/min, 1 min 200 °c, backflush 3 min 200 °c; programmed temperature vaporization on an agilent multimode inlet: inlet temperature program: 0.06 min 37 °c, +600 °c/min, 5 min 240 °c; vent flow 100 ml/min at 0.34 bar; ionization: 70 ev, ei, sim mode (m/z: 80, 74, 42). statistical analysis of the data was performed with prism v. 6.04 (graphpad software, inc., la jolla, ca, usa). the shapiro-wilk normality test was used to assess probability distribution of the datasets. normally distributed sets were subjected to a one-way analysis of variance (anova) followed by tukey's post hoc test, while non-normally distributed data or data with too few independent experiments to perform a shapiro-wilk test (analysis of ndma levels in rat urine) were subjected to a kruskal-wallis test followed by dunn's post hoc comparison. the relationship between the frequencies of pathohistological findings and l-carnitine treatment was analyzed by means of pearson's chi-squared test. statistical significance was considered if p ≤ 0.05. in the first week upon arrival, the animals showed signs of an sdav infection. sdav is a relatively common and rat-specific coronavirus with high morbidity and very lowto-no mortality (gaillard and clifford 2000; jacoby and gaertner 2006) . the infection was relatively silent, the most prominent clinical symptom being sneezing followed by red-colored nasal discharge. in groups 1, 2 and 3, one animal had to be euthanized before the completion of the study. all other animals completed the study in good general condition. no statistically significant differences were observed between the groups regarding the final body weight, all animals weighing ~410 g at the end of the study (table 1 , "final bw"). similarly, the final weight of the various organs sampled from the different animals did not differ across the groups in a significant manner (table 1 , "kidney, liver and spleen weight"). interestingly, the rats in the highest dose group (group 4) drank significantly more water than the animals in groups 1 (control) and 2 (lowest dose; table 1 , "water uptake"). based on average water consumption of 18.3 ml/ rat/day (table 1 , "water uptake") and an average body weight of 260 g/rat (table 1; regarding acf formation and acf multiplicity (crypts/acf), there was no statistically significant difference between the four groups tested (tables 2, 3) histopathological alterations in the aorta were only seen in one animal of the control group, showing mild focal degenerative changes in the media accompanied by a mild infiltration of macrophages and mineralization ( fig. 1a ; table 3 ). histopathological examination of the heart revealed a mild multifocal chronic lymphohistiocytic myocarditis with myocardial degeneration and fibrosis in about 50 % of the rats, but no statistically significant difference between the groups was observed ( fig. 1b; table 3 ). moreover, all animals displayed a variable degree of bile duct hyperplasia (fig. 1c) , and a high number of animals showed a mild-to-moderate multifocal acute to subacute table 1 physiological data of the experimental animals where applicable, values are shown as mean ± standard deviation * weight of one kidney ▲ calculated on the basis of the mean of the weekly water consumption of two animals/cage a kruskal-wallis test followed by dunn's post hoc analysis b one-way anova followed by tukey's post hoc analysis α significantly different (p < 0.01) when compared to group 1 β significantly different (p < 0.001) when compared to group 2 group 1 (0 g/l) group 2 (1 g/l) group 3 (2 g/l) group 4 (5 g/l) number of animals 19 19 19 20 starting body weight (g) a 105.1 ± 7.9 102.4 ± 7.0 101.9 ± 6.9 101.6 ± 6.9 final body weight (g) a 410.5 ± 19.6 408.5 ± 14.6 413.2 ± 14.1 414.5 ± 18.3 kidney weight (g)* ,a 1.27 ± 0.08 1.26 ± 0.07 1.28 ± 0.1 1.30 ± 0.09 liver weight (g) b 11.4 ± 1.0 11.4 ± 0.9 11.6 ± 0.8 11.9 ± 0.9 spleen weight (g) b 0.82 ± 0.1 0.83 ± 0.08 0.84 ± 0.06 0.84 ± 0.08 water uptake (ml/day/animal) ▲,a 18.1 ± 1.6 18.1 ± 2.1 18.3 ± 1.2 18.8 ± 1.8 α,β table 3 pathohistological findings in the aorta, heart and liver of the animals all pathohistological findings analyzed with pearson's chi-squared test a number of animals with the mentioned alteration/total number of animals analyzed, frequency (%) in parentheses group 1 (0 g/l) group 2 (1 g/l) group 3 (2 g/l) group 4 (5 g/l) (100) 20/20 (100) fig. 1 a aorta of a control animal: mild focal degenerative changes in the media accompanied by a mild infiltration of macrophages and mineralization (arrowheads; l lumen). b heart of an animal of group 4: mild multifocal chronic lymphohistiocytic myocarditis with myocardial degeneration and fibrosis. c liver of a control ani-mal: mild bile duct hyperplasia, associated with mild lymphohistiocytic infiltration. d liver of a control animal: focal suppurative and necrotizing hepatitis. he. scale bars a, b, d 100 µm, scale bar c 50 µm suppurative and necrotizing hepatitis (fig. 1d) , without statistically significant differences between the groups (table 3) . except for a hepatocellular carcinoma in one animal of group 1, no tumors were observed within the livers of the remaining animals. no significant differences regarding the ndma content in the urine of the animals was observed between the test groups (fig. 2) . interestingly, animals receiving 2 or 5 g/l l-carnitine (groups 3 and 4) seem to excrete the lowest amount of ndma, median concentrations reaching 281.9 ng/ml and 267.6 ng/ml, respectively (group 1: 314.2 ng/ml; group 2: 348.8 ng/ml). however, it has to be noted that the differences are not statistically significant and that the amount of urinary ndma strongly varies between the animals tested in each group (fig. 2 ). the final mean body weights of the rats recorded during the course of this study correspond to weights measured in untreated f344 rats of the same age (solleveld et al. 1984) . in contrast, the water uptake of the rats in this study was somewhat reduced when compared to the water uptake of laboratory rats in general (hofstetter et al. 2006 ). although rats administered 5 g/l l-carnitine drank significantly more water than animals in groups 1 and 2, this finding can be considered as biologically irrelevant, and it is questionable whether it is actually related to l-carnitine supplementation. f344 rats have extensively been used in longterm (i.e., 2 years-long) carcinogenicity studies (dinse et al. 2010; solleveld et al. 1984) and are characterized by an extremely low spontaneous incidence rate (0.1-0.6 %) of neoplasms of the small and large intestine (haseman et al. 1998) , rendering them particularly useful for the investigation of putative colon carcinogens. however, acfs seem to spontaneously develop in the colon of f344 rats with a fairly high incidence (40-60 %), even in animals killed at an earlier age than those in the present study (furukawa et al. 2002; tanakamaru et al. 2001) . with an overall frequency of 7.7 % and no statistically significant difference in the number of acfs/animal between the testing groups, acf formation in this study is considered to be spontaneous and not related to l-carnitine supplementation. in addition, the data obtained in the course of this study show that the administration of different l-carnitine dosages does not lead to increased amounts of ndma being excreted via the urine when compared to untreated animals, a fact which might furthermore explain the low acf incidence. even though there was no difference between the test groups, it should be noted that a "basal" ndma level (376.2 ± 169.8 ng/ml on average) was nevertheless observable in the urine of the animals in group 1. this might be the result of its endogenous formation or of a contamination of unknown etiology (kraft et al. 1981; tricker and preussmann 1991; vermeer et al. 1998) . regarding the possible influence of the sdav infection on the outcome of the study at hand, it should be mentioned that the repair processes in the affected tissues (respiratory tract, eye, salivary and lacrimal glands) begin 5-7 days post infection and in the case of the salivary and lacrimal glands are completed after about 21 days (jacoby and gaertner 2006; percy et al. 1988 ). since sdav infection occurred at the very beginning of the study and the animals had 3-4 weeks to recover, we do not suspect any major effect of the virus on the outcome of this study, especially since sdav is not known to interfere with colon tumorigenesis or heart-related pathologies (jacoby and gaertner 2006) . the latter statement as well as the fact that immunocompetent animals develop immunity and recover relatively fast with barely any sequelae (jacoby and gaertner 2006) led to the decision, not to kill the entire colony and to continue the experiment. particularly, male rats develop a plethora of pathologic changes with increasing age, the most common being spontaneous tumors, chronic nephropathy and lesions of the cardiovascular system (coleman et al. 1977; king and russell 2006) . the pathologic findings related to the cardiovascular system described in this study clearly fall in this category. focal myocardial degeneration in conjunction with inflammation (lymphohistiocytic infiltrations) followed by myocardial fibrosis have been described in aged f344 rats as well as other rat strains in incidences comparable to the ones reported herein, especially when the animals were fed ad libitum (blankenship and skaggs 2013; coleman et al. 1977; goodman et al. 1979; hall et al. 1992; keenan et al. 1995a, b; maeda et al. 1985) . in this context, the left ventricular papillary muscle, which was the primarily affected fig. 2 ndma concentrations in the urine of the experimental animals collected in the next-to-last week of the study. shown is the median as well as ndma levels of each individual animal/group (mean of three measurements/sample). the dataset was subjected to a kruskal-wallis test followed by dunn's post hoc comparison location in numerous animals (data not shown), is reported to be a preferred site for these lesions (percy and barthold 2008) . additionally, aging rats commonly exhibit hyperplastic bile ducts (king and russell 2006) . according to coleman et al. (1977) , 75 % of approximately 12-month old f344 rats show bile duct hyperplasia, a finding which contrasts the incidence of 100 % reported in this study, although, still in accordance with the same source, similar incidences were reported in significantly older animals (>18 months). in contrast, control f344 rats used in 2-year carcinogenicity studies of the u.s. national institutes of health carcinogenesis testing program only marginally suffered from bile duct hyperplasia, 24.5 % of male and 12.5 % of female f344 rats being affected (goodman et al. 1979) . the frequency of multifocal suppurative and necrotizing liver lesions observed in this study (68.4-90.0 %) was distinctly above the relatively low incidence of spontaneous necrotizing processes previously described in aged male f344 rats (approx. 6,9 %; hall et al. 1992 ). although we were unable to identify characteristic viral, bacterial, mycotic or parasitic structures within the liver lesions employing routine and special histological staining methods (gram stain, periodic acid schiff-reaction, and groccott silver stain; data not shown), we cannot rule out the possibility that the animals were infected with agents such as clostridium piliforme (tyzzer's disease), salmonella spp. or corynebacterium kutscheri (percy and barthold 2008; thoolen et al. 2010) . however, since all test groups were equally affected, this finding is clearly not related to the l-carnitine treatment. additionally, it should be considered that these lesions were mainly of mild, subclinical grade and acute in character and therefore might have developed only in the last days (or the last week) of the study, thus rendering an influence on acf formation or the onset of cardiovascular lesions unlikely. no definitive (sub-)intimal atherosclerotic lesions were observed in the aortas of the animals. the degenerative changes observed in the aorta of one control rat were interpreted as an incidental finding of unknown etiology and are in agreement with the previously reported rare spontaneous lesions described in old f344 rats (king and russell 2006) . since this finding concerns only one animal in the control group, a carnitine-related cause can be excluded. additionally, it should be noted that rats, except for specially bred strains, generally do not develop atherosclerosis (king and russell 2006; moghadasian 2002) . this fact together with differences in the composition of the gut microbiota, the metabolism of l-carnitine as well as the amount (1.3 g/l) of l-carnitine given to the apolipoprotein e-knockout mice might explain the divergent results of the study at hand to that conducted by koeth et al. (2013) regarding the formation of atherosclerotic lesions. in conclusion, this study provides evidence that the daily administration of l-carnitine in concentrations of 70.4, 140.8 and 351.9 mg/kg bw/day for 1 year does not lead to an adverse effect in the colon or cardiovascular system of male f344 rats. trimethylamine: metabolic, pharmacokinetic and safety aspects disposition and metabolite kinetics of oral l-carnitine in humans the biosynthesis of trimethylamine-n-oxide effect of l-carnitine supplementation on muscle and blood carnitine content and lactate accumulation during high-intensity sprint cycling trimethylamine-n-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation role of aberrant crypt foci in understanding the pathogenesis of colon cancer findings in historical control harlan rcchan™: wist rats from 4-, 13-, 26-week studies supplemental carnitine and exercise guidance for industry-estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers l-carnitine supplementation in humans. the effects on physical performance pathological changes during aging in barrier-reared fischer 344 male rats comparison of ntp historical control tumor incidence rates in female harlan sprague dawley and fischer 344/n rats l-carnitine supplementation and lipid metabolism of rats fed a hyperlipidaemic diet a case of early diagnosed carnitine deficiency presenting with respiratory symptoms pharmacokinetics of l-carnitine role of carnitine in disease spontaneous development of aberrant crypt foci in f344 rats common diseases. in: krinke gj (ed) the laboratory rat neoplastic and nonneoplastic lesions in aging f344 rats commercially marketed supplements for bodybuilding athletes histopathologic observations in weanling b6c3f1 mice and f344/n rats and their adult parental strains pharmacokinetics of intravenous and oral bolus doses of l-carnitine in healthy subjects spontaneous neoplasm incidences in fischer 344 rats and b6c3f1 mice in two-year carcinogenicity studies: a national toxicology program update the laboratory rat a general introduction to the biochemistry of mitochondrial fatty acid beta-oxidation viral disease fat burners: nutrition supplements that increase fat metabolism fat metabolism during exercise: a review-part iii: effects of nutritional interventions diet, overfeeding, and moderate dietary restriction in control sprague-dawley rats: ii. effects on age-related proliferative and degenerative lesions diet, overfeeding, and moderate dietary restriction in control sprague-dawley rats: i. effects on spontaneous neoplasms metabolic, traumatic, and miscellaneous diseases risk of colorectal and other gastro-intestinal cancers after exposure to nitrate, nitrite and n-nitroso compounds: a follow-up study intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis urinary excretion of dimethylnitrosamine: a quantitative relationship between dose and urinary excretion carnitine transport by organic cation transporters and systemic carnitine deficiency the effect of enteral carnitine administration in humans nitrosation of tertiary amines and some biologic implications n-nitroso compounds and cancer incidence: the european prospective investigation into cancer and nutrition (epic)-norfolk study disorders of carnitine transport and the carnitine cycle microbial nitrosamine formation in palm wine: in vitro n-nitrosation by cell suspensions nutritional influences on aging of fischer 344 rats: ii. pathology l-carnitine supplementation does not promote weight loss in ovariectomized rats despite endurance exercise carnitine metabolism in human subjects. i. normal metabolism experimental atherosclerosis: a historical overview revised guides for organ sampling and trimming in rats and mice-part 3: a joint publication of the rita and nacad groups version 1.0; document # epa/600/r-05/054) determination of nitrosamines in drinking water by solid phase extraction and capillary column gas chromatography with large volume injection and chemical ionization tandem mass spectrometry influence of a fatrich diet, folic acid supplementation and a human-relevant concentration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine on the induction of preneoplastic lesions in the rat colon rat pathology of laboratory rodents and rabbits depletion of salivary gland epidermal growth factor by sialodacryoadenitis virus infection in the wistar rat dose translation from animal to human studies revisited kinetics, pharmacokinetics, and regulation of l-carnitine and acetyl-l-carnitine metabolism metabolic fate of dietary carnitine in human adults: identification and quantification of urinary and fecal metabolites carnitine metabolism and its regulation in microorganisms and mammals l-carnitine dissimilation in the gastrointestinal tract of the rat revised guides for organ sampling and trimming in rats and mice-part 1: a joint publication of the rita and nacad groups multiple-dose pharmacokinetics and bioequivalence of l-carnitine 330-mg tablet versus 1-g chewable tablet versus enteral solution in healthy adult male volunteers carnitine supplementation fails to maximize fat mass loss induced by endurance training in rats natural history of body weight gain, survival, and neoplasia in the f344 rat pharmacological and pathophysiological roles of carnitine/organic cation transporters (octns: slc22a4, slc22a5 and slc22a21) essential similarities between spontaneous and meiqxpromoted aberrant crypt foci in the f344 rat colon proliferative and nonproliferative lesions of the rat and mouse hepatobiliary system carcinogenic n-nitrosamines in the diet: occurrence, formation, mechanisms and carcinogenic potential volatile n-nitrosamine formation after intake of nitrate at the adi level in combination with an amine-rich diet peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient carnitine supplementation: effect on muscle carnitine and glycogen content during exercise dietary precursors of trimethylamine in man: a pilot study the authors wish to thank (in alphabetical order) judith bigalk and nicole brauer for excellent technical assistance and dr. laura c. bartel, maria d. brauneis, janine döhring, julia hausmann, anne von keutz, dr. petra nicken, bettina seeger and shan wang for valuable help in taking care of the experimental animals. the authors would also like to acknowledge the financial support of the german federal institute for drugs and medical devices (bonn, germany). key: cord-239527-69bxbhjh authors: montag, felix; sagimuldina, alina; schnitzer, monika title: are temporary value-added tax reductions passed on to consumers? evidence from germany's stimulus date: 2020-08-19 journal: nan doi: nan sha: doc_id: 239527 cord_uid: 69bxbhjh this paper provides the first estimates of the pass-through rate of the ongoing temporary value-added tax (vat) reduction, which is part of the german fiscal response to covid-19. using a unique dataset containing the universe of price changes at fuel stations in germany and france in june and july 2020, we employ a difference-in-differences strategy and find that pass-through is fast and substantial but remains incomplete for all fuel types. furthermore, we find a high degree of heterogeneity between the pass-through estimates for different fuel types. our results are consistent with the interpretation that pass-through rates are higher for customer groups who are more likely to exert competitive pressure by shopping for lower prices. our results have important implications for the effectiveness of the stimulus measure and the cost-effective design of unconventional fiscal policy. the drastic economic downturn accompanying the covid-19 pandemic was met by an unprecedented fiscal response of the german government. on 3 june 2020, a stimulus package worth 130 billion euro was announced. to the general surprise of the public, it included a reduction of the standard value-added tax (vat) rate from 19 to 16 percent and of the reduced rate from 7 to 5 percent for the second half of 2020, at an estimated cost of 20 billion euro or 0.6 percent of gdp. the aim of this fiscal policy is to temporarily reduce prices and stimulate consumption through inflation expectations. for this to work, however, it is crucial that firms pass on the vat reduction to consumers. our paper provides the first estimates of the pass-through rate of the temporary vat reduction for a major sector of the economy. we estimate the pass-through rate for diesel and gasoline using a unique dataset containing the universe of price changes at fuel stations in germany and france in june and july 2020 and employing a differencein-differences strategy. we find that pass-through is incomplete for all types of fuels and very heterogeneous across fuel types. this variation of pass-through rates is consistent with the interpretation that customers of different fuel types are differentially likely to shop for lower prices, resulting in differing competitive pressure. whilst fuel stations pass on most of the vat rate reduction to diesel customers who on average drive more than twice as many kilometers per year than drivers of gasoline driven cars, pass-through rates for gasoline are much lower. studying the effect of the temporary vat rate reduction in the context of the fuel market is particularly interesting for two reasons. first, it is a market where granular data is available in real-time, which allows us to evaluate the effect while it is happening. second, it is a market where price adjustments are costless and in fact happen frequently. thus, despite the temporary nature of the vat rate change, price adjustment costs cannot be held accountable for imperfect pass-through. this paper makes two contributions to the literature. first, our results have implications for the effective design of unconventional fiscal policy. feldstein (2002) proposed that stimulating inflation in an environment where monetary policy is ineffective could be done by targeting household expectations directly. this type of policy was later coined unconventional fiscal policy by d' acunto et al. (2018) . a temporary reduction in the vat rate is only likely to affect household expectations, however, if the reduction is passed on to consumers and they therefore expect prices to be lower temporarily. by analyzing the pass-through rate of the temporary vat reduction, we shed light on a necessary condition for this type of unconventional fiscal policy to be effective. furthermore, we show that by targeting competitive markets where consumers are likely to search for lower prices, policymakers can make unconventional fiscal policy more cost-effective. et al. (2020b) predict that the 2020 temporary vat reduction will successfully increase inflation expectations and expenditure. benzarti, carloni, et al. (forthcoming) show that pass-through is often asymmetric and that prices respond twice as much to vat increases as to decreases. furthermore, whilst the 2007 vat increase was permanent, the current vat change only lasts for six months. conclusions from the 2007 permanent vat increase therefore are not necessarily informative about the 2020 temporary vat reduction. second, we are the first to provide estimates of the average pass-through rate of an unanticipated vat rate change in a major sector of the economy using high-frequency, establishment-level price data. our unique dataset allows us to observe all price changes for around 23, 000 fuel stations across germany and france before and after the temporary vat rate reduction. fuel stations in germany are treated after the 1 july, whereas fuel stations in france are unaffected by the german policy change. thus, we can employ a difference-in-differences strategy, using french fuel stations as control group. previous empirical studies on tax pass-through use aggregate price indices, finding mixed results. they include evidence for under-shifting (e.g. benzarti and carloni, 2019) , full passthrough (e.g. benedek et al., 2019) or over-shifting (e.g. besley and rosen, 1999) . notable exceptions using firm-level price data are kosonen (2015) , studying the effect of a vat reduction for hairdressers in finland, and büttner and madzharova (forthcoming) , who estimate the effect of a large number of vat increases and decreases in the european union (eu) using product-level monthly sales data for home appliances in a panel model across a large number of european countries over time. like kosonen (2015) , we focus on the effect of one particular policy change on one particular market. in contrast to kosonen (2015) , using a control market in a different country allows us to avoid potential general equilibrium effects affecting the control group, which might lead to an over-or under-estimation of the pass-through rate, as noted by benedek et al. (2019) . finally, a feature of high-frequency price data in a market with many price adjustments is that it allows us to trace out the evolution of pass-through rates over time. to estimate the average pass-through rate of the vat reduction, we use a differencein-differences strategy, where we compare daily prices of the three main fuel types sold at fuel stations in germany and france before and after the policy change. 1 supply shocks, in particular fluctuations in the price of crude oil, should affect germany and france similarly and are thus eliminated by time fixed effects. we also account for regional differences in demand over time by controlling for changes in mobility, using regional data from the google covid-19 community mobility report. we find that the pass-through rate for diesel is 83 percent, whereas it is 61 percent for e10 and 40 percent for e5. 2 this translates into price decreases of 2 percent for diesel, 1.5 percent for e5 and 1 percent for e10. at the same time, retail margins for diesel only increased by 0.7 percent, whilst retail margins for e5 and e10 increased by between 10 and 12 percent. 3 our results show that pass-through of the vat rate reduction is fast and substantial but remains incomplete. whilst prices decrease for consumers, margins also substantially increase for sellers. furthermore, there is a substantial difference in pass-through rates 1 this dataset has previously been used by montag and winter (2020) to analyze the effect of price transparency. 2 e5 and e10 are the two main types of gasoline sold in germany, which differ in how much bioethanol they contain. 3 our measure of retail margins only subtracts taxes, duties and the price of crude oil at the port of rotterdam from fuel prices. it includes fuel station and refinery margins, as well as different cost types. the percentage change in retail margins due to the vat change is therefore an underestimate of the actual percentage change in retail margins. between fuel types. since stations sell all three types of fuel, unobserved station characteristics cannot explain these differences. instead, differences in competitive pressure due to different propensities of customer groups to shop for lower prices are consistent with the observed effects. whereas fuel stations pass on more than half of the vat rate decrease to frequent drivers and professional drivers, usually driving diesel cars, as well as to price sensitive gasoline customers who buy the cheaper e10, they pass on less than half of the tax rate reduction to customers of e5 suggesting that they are less responsive to price differentials. the remainder of the paper is structured as follows: section 2 describes the industry, section 3 gives an overview of the data and presents descriptive evidence, section 4 discusses the empirical strategy, section 5 presents the estimation results and section 6 concludes. in 2019, total revenues from retail fuel sales were worth 92 billion euro or approximately 3 percent of german gdp. in addition to its standalone value to the economy, this market has large externalities on the rest of the economy. fuel prices are a key determinant of travel costs, commuting costs and, more broadly, the cost of personal transportation. the first important distinction to make within fuels for passenger vehicles is between diesel and gasoline. 4 in germany, diesel has a volume share of 44 percent of fuel for passenger vehicles with combustion engines and gasoline accounts for the remaining 56 percent. 5 substituting between these two types of fuel is very costly, both on the demand and supply side. 6 within gasoline, there is differentiation according to the octane rating and the share of ethanol. standard gasoline (commonly referred to as super) has an octane rating of 95. it has a volume share of 95.4 percent of the gasoline market. 7 some high-perfomance vehicles will require gasoline with an octane rating of 98 (commonly referred to as super plus), which has a 4.6 percent volume share within gasoline. at the same time, there is no added benefit of fueling super plus if the vehicle can process super. since the price of super plus is always considerably higher, there is no demandside substitution from super to super plus either. as fuel stations do not report prices of super plus to the market transparency unit in germany, it is not part of our analysis. within super, there is a final distinction according to the share of ethanol. standard gasoline has a 5 percent share of ethanol and is thus commonly referred to as e5. in 2011, a new type of gasoline was introduced with a 10 percent ethanol share, referred to as e10. the aim of increasing the share of ethanol is to reduce greenhouse gas emissions and decrease the amount of fossil fuel used in transportation. although e5 and e10 are not taxed differently, e10 is usually around 4 eurocent cheaper than e5. this is partly driven by the relative prices of crude oil and ethanol on the world market and partly by a minimum quota of biofuels that need to be sold by fuel stations. after the introduction of e10 in 2011, there was controversy about whether biofuels damage the engine. although biofuels can pose a significant threat to the engine of a vehicle that is not certified to be compatible with e10, around 90 percent of gasoline-run vehicles, including all vehicles produced after 2012, are compatible with e10. 8 according to the german automobile association, e10 is around 1.5 percent less efficient than e5. 9 all fuel stations in germany are required to sell both types of fuel. nevertheless, in 2019 e5 still had a volume share of 85.6 percent within super and e10 only of 14.4 percent. overall, many motorists who could buy less expensive e10 choose not to do so and buy e5 instead. reasons for this could include preferences or a lack of information, which point towards a lower price sensitivity of e5 customers compared to e10 customers. two further observations can be made about the difference between drivers of gaso-line and diesel passenger vehicles. whereas only 32 percent of registered passenger vehicles in germany have a diesel engine, compared to 66 percent that run on gasoline, frequent drivers tend to use diesel cars. 10 on average, gasoline passenger vehicles drive 10, 800 kilometers, whereas diesel passenger vehicles drive 19, 500 kilometers per year. 11 the largest share of the fuel price consists of taxes. a lump-sum energy tax of 0.6545 euro per liter is levied on gasoline (0.4704 euro per liter for diesel). 12 in addition, there is a 19 percent value-added tax which is levied on the net price of diesel and gasoline, including the energy tax. this value-added tax is temporarily reduced to 16 percent between july and december 2020. to be reported by stations to a government agency, which makes this data available to researchers. 16 furthermore, we add data on the daily price of crude oil, the principal input product for diesel and gasoline, at the port of rotterdam. finally, we use data on daily regional mobility patterns from the covid-19 community mobility report provided by google. our analysis starts on 15 june 2020 and currently goes until 31 july 2020. the 15 june is a natural starting point, as it marks the beginning of the european commission's re-open eu plan and is the date on which france and germany lifted many of their travel restrictions and re-opened their borders. 17 as long as the temporary vat rate reduction remains in place, we plan to extend our period of analysis and estimate how the pass-through rates evolve. using the data on price changes, we construct daily weighted average prices. table 1 shows that the price level is generally higher in france than in germany. gross prices in france increase by around 2 eurocent between the pre-and post-vat cut periods. in germany, gross prices remain constant for e5 and e10 and decrease by 1 eurocent for diesel. at the same time, the increase in the net price in germany is between 2 and 3 eurocent, depending on the fuel type, which is larger than in france and thus suggests that the vat reduction was not completely passed on to consumers. 16 see https://www.prix-carburants.gouv.fr/rubrique/opendata/. 17 see https://reopen.europa.eu/en/. notes: "pre-vat cut" and "post-vat cut" refer to fuel stations in germany and france before and after the reduction of the vat rate, respectively. the pre-vat phase goes from 15 june until 31 june 2020. the post-vat phase starts on 1 july 2020. we also calculate retail margins by subtracting taxes, duties and the share of the price of crude oil that goes into the production of diesel and gasoline, respectively. 18 although these retail margins still contain different cost types, such as the cost of refining or transportation costs, the main source of input cost variation, the price of crude oil, is eliminated. the latter, we aim to capture local changes in the propensity to use a car for professional activities. both of these variables are measured as the percentage change of activities compared to the median value for the corresponding day of the week during the five-week period 3 january to 6 february 2020. the data is disaggregated for 96 sub-regions in france and 16 regions in germany. we use the geolocation of each fuel station to match the measures of local mobility to each station. table 1 shows that mobility patterns in france and germany are similar. whereas visits to retail and recreational facilities were around 10 percent lower in the second half of june compared to the baseline beginning of the year, in july, the number of such visits returned close to their pre-pandemic levels. at the same time, in both countries visits to workplaces were around 15 percent lower in the second half of june compared to the baseline and 20 percent lower in july. this is likely because many people go on vacation in july. it also indicates that overall trends in both countries are very similar. we begin by estimating the average effect of the vat reduction on fuel prices and retail margins in germany. to do this, we compare the evolution of prices and retail margins at fuel stations in germany to stations in france, before and after the decline in the value-added tax. for the three main types of fuel, we focus on two main outcomes: daily fuel prices and retail margins. to causally estimate the effect of the temporary reduction in the vat rate on fuel prices and retail margins, we use a difference-in-differences strategy, and compare stations in germany and france, before and after the reduction in the vat rate. specifically, we estimate the following regression: where y it is the logarithm of the price or retail margin of gasoline or diesel at a fuel station i at date t, and v at it is a dummy variable that equals one for stations affected by the vat reduction at date t. these are fuel stations in germany from 1 july 2020 onwards. x it is a vector of controls, which includes regional mobility data for retail and recreational purposes, and mobility to work. µ i and γ t correspond to fuel station and date fixed effects, respectively. to causally identify the effect of the vat reduction on fuel prices and retail margins, two main assumptions must be satisfied. first, there should be no transitory shocks that would differentially affect fuel stations in germany and france before and after the reduction in vat, other than the policy change itself. second, there should be no spillover effects from the vat reduction in germany onto the fuel market in france. station fixed effects control for any time-invariant differences between fuel stations in france and germany, and date fixed effects capture the transitory shocks, such as fluctuations in the price of crude oil, that identically affect french and german stations. the two countries are similar in their geographic location, size, and wealth. since in our analysis we also focus on a relatively narrow window around the reform, this should alleviate concerns on transitory shocks differentially affecting french and german fuel stations. one might still suspect that certain transitory shocks could confound our empirical strategy. we now discuss the most obvious candidates. on the demand side, public and school holidays in france and germany are highly correlated. travel restrictions put in place due to covid-19 were lifted simultaneously in the two countries. starting from 15 june 2020, residents of the schengen area and the united kingdom could freely cross the territories of france and germany again. most holidaymakers within europe typically travel across several countries in the eu, and as france and germany are both popular travel destinations in close geographic proximity, demand shocks likely hit fuel stations in the two countries in a similar way. in addition, we directly account for demand-related shocks by including regional information on the daily mobility to work and to retail and recreational places as control variables into our empirical specification. way. due to their geographic proximity, the fuel stations in france and germany procure most of their crude oil from similar sources. the two countries are also members of the european single market, which implies harmonized border checks, common customs policy, and identical regulatory procedures on the movement of goods within the eu. finally, no major reforms were implemented in france during our analysis period. in general, there are no fuel price-setting regulations in germany and france, and both countries have mandatory disclosure of fuel prices, which reaffirms our choice of france as a suitable control group. this section presents the effects of the temporary vat reduction on fuel prices and retail margins. we estimate these effects for the three main fuel types: e5, e10, and diesel. table 2 shows the results of estimating the regression model presented in equation 1 using the logarithm of price as an outcome variable. the coefficients in columns (1) to (3) correspond to the effect of the temporary vat rate reduction on e5, e10 and diesel prices without mobility controls. columns (4) to (6) show the effects on prices when we control for mobility. the results in columns (1) to (3) show that the reduction in the vat led to a decline in prices of all fuel products, which is statistically significant at the 1 percent level and economically significant. the average price for e5 decreases by 1.05 percent after the vat reduction, whilst average prices for e10 and diesel decrease by 1.55 and 2.12 percent, respectively. including mobility controls does not significantly change the estimates. reassuringly, however, mobility for retail, recreational, and work purposes positively correlates with fuel prices. when controlling for regional differences in mobility, average prices for e5, e10 and diesel are estimated to decline by 1.01, 1.53 and 2.08 percent, respectively. next, we estimate the pass-through rates of the vat change. under full passthrough, we expect prices for each fuel product to decrease by about 2.52 percent. 20 an estimated decline of 2.08 percent in diesel prices is therefore relatively close to full pass-through. around 83 percent of the vat reduction is passed on to consumers who refuel with diesel. for e10, the pass-through rate is 61 percent. finally, we estimate that 40 percent of the vat decline is passed on to consumers of e5. for all fuel products, pass-through of the vat reduction is fast and relatively high, but incomplete. to show the economic importance of the pass-through rate, we illustrate the actual price development, as well as estimated counterfactual prices under full and zero passthrough until 31 july 2020. figure 1 after the vat reduction, the observed price of e5 is at around 1.27 to 1.28 euro per liter, which is closer to the price that we predict under zero pass-through than to that under full pass-through. the observed e5 price is on average 2 eurocent higher than the counterfactual were the retailers to fully pass on the reduction in the vat rate to consumers. figure 2 shows the vat incidence for e10. the interpretation of the lines is equivalent to figure 1 . in the post-vat reduction period the observed e10 price declines to around 1.23 to 1.25 eurocent per liter. in the full pass-through case, we predict e10 prices to be on average 1 eurocent lower, which is a smaller difference compared to e5. figure 3 presents an analogous graph of value-added tax incidence for diesel prices. 20 with a decrease in the vat rate from 19 percent before the reform to 16 percent after the reform, this is 1.16−1.19 1.19 * 100 ≈ −2.52%. notes: columns (1)-(3) present estimates without mobility control variables on e5, e10, and diesel log prices, respectively. columns (4)-(6) present estimates on e5, e10, and diesel log prices from estimation with mobility controls. all columns use data from 15 june to 31 july 2020. standard errors clustered at the fuel station level in parentheses. * p < 0.10, * * p < 0.05, * * * p < 0.01 among the three fuel products, consumers who fuel with diesel experience the highest pass-through. the observed diesel price in germany, captured by the solid red line, is relatively close to the price that we predict under the full pass-through scenario. on average, consumers pocket about 2.3 eurocent per liter of diesel from the vat reduction. as we would expect, the ranking of pass-through rates corresponds to the ranking of customer groups with respect to their likelihood of shopping for lower prices. as described in section 2, consumers buying e5 are those least likely to search for lower prices. most of these consumers can already save in a similar order of magnitude by switching to e10, but choose not to do so. the incentive for fuel stations to pass on the vat rate reduction to these consumers is therefore also the lowest. in contrast, diesel customers are on average much more likely to be frequent drivers and are therefore much more likely to search for lower prices. fuel stations therefore experience more competitive pressure for diesel customers and are hence more inclined to pass on the vat rate reduction to these customers. table 3 presents the results of estimating the regression model presented in equation 1 using the logarithm of retail margins as an outcome variable. the coefficients in columns (1) to (3) correspond to the effect of the vat rate reduction on e5, e10 and diesel margins without controlling for mobility. columns (4) to (6) show the effects on margins when mobility controls are included. the results in columns (1) to (3) show that the reduction in the vat led to an increase in retail margins for all fuel products, which is statistically significant at the 1 percent level and economically significant. the average retail margin for e5 increases by 11.34 percent after the vat reduction, whilst the average retail margin for e10 and diesel increases by 10.59 and 0.46 percent, respectively. including mobility controls does not significantly change the estimates. after controlling for regional differences in mobility, average retail margins for e5, e10 and diesel increase by 11.68, 10.83 and 0.70 percent, respectively. notes: columns (1)-(3) present estimates without mobility control variables on e5, e10, and diesel log retail margins, respectively. columns (4)-(6) present estimates on e5, e10, and diesel log retail margins from estimation with mobility controls. all columns use data from 15 june to 31 july 2020. standard errors clustered at the fuel station level in parentheses. * p < 0.10, * * p < 0.05, * * * p < 0.01 these findings are in line with the estimated pass-through rates. for diesel, where most of the vat reduction is passed through to consumers, there is only a very modest increase in retail margins. instead for gasoline, particularly for e5, less than half of the vat rate reduction is passed on to consumers and thus the temporary decrease in the vat rate led to an increase in retail margins by 10 to 12 percent. note, that for our measure of retail margins we simply subtract taxes and duties, as well as the share of the crude oil price attributable to the production of diesel and gasoline, from the gross price. the retail margins therefore include the refinery margin, as well as the station margin and different cost types, such as the cost of refining or the cost of transportation. since this means that our estimated retail margin is an overestimate of the actual retail margin, the estimated percentage increase in margins due to the vat rate reduction can be taken as a lower bound. thus, the vat rate reduction likely led to an increase in retail margins of much more than 10 percent. figure 4 illustrates the evolution of retail margins for e5, as well as the counterfactual under full and zero pass-through. the solid red line corresponds to the observed evolution of retail margins at german fuel stations between 15 june and 31 july 2020. in addition, we plot margins predicted under zero and full pass-through scenarios with shortand long-dashed lines, respectively. for e5, the observed evolution of retail margins is closer to the zero pass-through case than to full pass-through. figures 5 and 6 present analogous graphs of the evolution of retail margins under full and zero pass-through for e10 and diesel. in contrast to e5, the observed margins of e10 and diesel are closer to the full pass-through scenario than to zero pass-through. in this paper, we show that, so far, pass-through of the temporary vat rate reduction, as part of the german fiscal covid-19 response, is fast and substantial but remains incomplete for all fuel types. furthermore, we find a high degree of heterogeneity between the pass-through estimates for different fuel types. since the same stations are selling the different types of fuels and the supply structure is very similar, this leaves differences in competitive pressure from different customer groups as a candidate explanation for the differences in the pass-through rates. in particular, the more likely customers of a particular fuel type are to shop for lower prices the higher is the pass-through rate for this fuel type. although fuel markets are not the prime target of unconventional fiscal policy, the mechanisms behind whether and to what extent firms pass on taxes to consumers are the same as for other markets. studying how fuel stations pass on the temporary vat reduction is thus a worthwhile exercise and informs us about market-wide pass-through. a key result is that demand characteristics and competitive pressure play a crucial role in how a temporary tax reduction is passed on to consumers. a key takeaway for policymakers is that by targeting competitive markets with high pass-through rates they can increase the cost-effectiveness of unconventional fiscal policy. in ongoing research we investigate these determinants of pass-through rates in more detail. we construct the price panel and compute retail margins at fuel stations in france and germany as follows. for each fuel station in our data set, we observe a fuel price every time it is changed along with a precise time and date stamp of a change. on average, fuel stations in germany change fuel prices 15 times a day, whereas there is typically one price change a day at french fuel stations. based on the distribution of price changes at german fuel stations, we construct hourly fuel prices from 6 am until 10 pm for each day between 15 june -31 july, 2020. for france, since fuel prices do not change frequently over a day we keep a fuel price at 5 pm for our empirical analysis. for german fuel stations, we compute daily weighted average price from the hourly distribution of price changes that we observe. to construct the weights, we use the data on hourly fueling patterns reported in a representative survey among drivers by the german federal ministry of economic affairs. figure 7 shows shares of motorists in germany who fuel at a given time period during a day. we further re-weight the hourly shares to produce weights for the hours between 6 am and 10 pm. to compute retail margins, we adjust fuel prices to taxes and duties in france and germany, and subtract a fuel share of the price of crude oil, which is a major input cost. in germany, taxes and duties consist of the value-added tax, a lump-sum energy tax, and a fee for oil storage. before the vat reduction, the value-added tax was at the rate of 19%, and starting from 1 july 2020 it is temporarily reduced to 16%. a lump-sum energy tax is at 0.6545 euro per liter for e5 and e10 gasoline, and at 0.4704 euro per liter for diesel. a fee for oil storage is at 0.27 euro per liter for e5 and e10, and at 0.30 euro per liter for diesel. 21 in france, the value-added tax rate is at 20%, with the exception of corsica island, 21 see https://www.avd.de/kraftstoff/staatlicher-anteil-an-den-krafstoffkosten/. varieties of vat pass through who really benefits from consumption tax cuts? evidence from a large vat reform in france what goes up may not come down: asymmetric incidence of value added taxes sales taxes and prices: an empirical analysis unit sales and price effects of preannounced consumption tax reforms: micro-level evidence from european vat unconventional fiscal policy managing households' expectations with unconventional policies unconventional fiscal policy to exit the covid-19 crisis a role for discretionary fiscal policy in a low interest rate environment more and cheaper haircuts after vat cut? on the efficiency and incidence of service sector consumption taxes price transparency against market power assuming that among the other products only jet fuel is of value, we split the price of a barrel into the cost of producing gasoline, diesel, and jet fuel to compute a share of the brent price that corresponds to a particular fuel product. around 54% of the brent oil price per barrel corresponds to the production of 19 gallons of gasoline, and around 34% -to the production of 12 gallons of diesel, which we further transform into the input key: cord-344553-uya1j94u authors: bodova, k.; boza, v.; brejova, b.; kollar, r.; mikusova, k.; vinar, t. title: time-adjusted analysis shows weak associations between bcg vaccination policy and covid-19 disease progression date: 2020-05-06 journal: nan doi: 10.1101/2020.05.01.20087809 sha: doc_id: 344553 cord_uid: uya1j94u in this study, we ascertain the associations between bcg vaccination policies and progression of covid-19 through analysis of various time-adjusted indicators either directly extracted from the incidence and death reports, or estimated as parameters of disease progression models. we observe weak correlation between bcg vaccination status and indicators related to disease reproduction characteristics. we did not find any associations with case fatality rates (cfr), but the differences in cfr estimates are at present likely dominated by differences in testing and case reporting between countries. the reports on a possible use of the well-established and widely used bcg vaccine as a protection against covid-19 (de vrieze, 2020) raised a lot of interest and media coverage. currently, four clinical trials have been designed to evaluate the potential of bcg for protection against the sars-cov-2 infection in health-care workers (bonten, 2020; khattab, 2020; curtis, 2020; cirillo and dinardo, 2020) . these studies are driven by the so called non-specific effects of bcg vaccine on viral infections, observed in animal models, as well as in humans, although the molecular basis of this phenomenon is not completely understood (moorlag et al., 2019) . the associations between bcg vaccination policy and covid-19 disease progression have also been a subject to controversy in data analysis, with some studies claiming significant effects on the number of cases and case fatality rates (miller et al., 2020; berg et al., 2020) , while others criticizing weaknesses of those studies and claiming no statistically significant differences (szigeti et al., 2020; hensel et al., 2020; fukui et al., 2020; singh, 2020) . while correcting for many covariate factors (such as population size, population age distribution, etc.), most of these studies, however, failed to correct for the differences in time progression of the epidemics in each country. covid-19 epidemic usually starts from relatively few imported cases and spreads quickly through exponential growth with high reproduction numbers. at unchecked growth rates, a significant percentage of the country population would be infected before the disease would subside. however, this growth rate only continues until effective measures, such as lockouts or social distancing policies, are introduced, changing the dynamics of the epidemics substantially, with infection rates rarely reaching a significant percentage of the whole population in the first wave flaxman et al. (2020) . in this study, we have estimated a variety of indicators characteristic for different stages of covid-19 epidemics, also adjusting for time since the beginning of the epidemics in each country, and found that several key indicators show weak, but statistically significant, associations with bcg vaccination status. figure 1 : comparison of estimated reproduction numbers r100 (left) and r100+10 (right) between countries with and without the universal bcg vaccination policy. to compare the covid-19 disease progression between countries with recent universal bcg vaccination policy and those without, several parameters derived from the case and death reports in each country were selected. the parameters reflect early-stage disease spread characteristics (when they are likely not yet affected by social distancing policies), early-stage case fatality rates (before potential effects from overwhelmed health care system), and progression of the disease after the changes characteristic for social distancing policies take effect. estimates of early stage r are lower in countries with recent bcg vaccination policies. the reproduction number r, the average number of secondary cases of disease caused by a single infected individual, has been estimated using epiestim package (cori et al., 2013) , based on 7-day windows, the first estimate starting on the day when cumulative number of 100 reported cases have been reached (r100), the second estimate starting on 10th day afterwards (r100+10). in many countries, this time period would not reflect the effects of social distancing policies, but would also somewhat avoid the initial period when the case reporting is likely to be unreliable. in both cases, the countries with recent bcg vaccination policies show lower r estimates ( figure 1 ) and these shifts were statistically significant (mann whitney u-test, p = 0.04 for r100 and p = 0.006 for r100+10). we have also examined the number of days between 10 and 100 reported cases (c10), 100 and 1000 reported cases (c100), 10 and 100 reported deaths (d10), and 100 and 1000 reported deaths (d100). these time periods reflect r in various early stages of the epidemic, longer periods meaning slower spread of the disease. note, that c10 numbers are likely unreliable (due to initial problems in establishing testing and reporting policies in each country), and there are only few countries that reached 1000 reported deaths before our data set cutoff. also note that if we assume a constant case fatality rate within a specific time period (typically 6-10 days) and a specific country, and also assume exponential growth in cases within this time period, the numbers d10 and d100 do not actually reflect the death rate, but instead only depend on the underlying value of r. death reports are likely more accurate than case reports, which are much more affected by testing and reporting policies in each country. on average, all of these time periods are slightly longer in countries with recent universal bcg policies, with statistically significant results for d10 (mann-whitney u-test, p = 0.02). no differences in case fatality rates. we have estimated case fatality rates on days when 100 and 1000 cumulative deaths were first reached in each country (cfr100 and cfr1000 respectively), and also used cmmid methodology (nishiura et al., 2009; russel et al., 2020) to correct for estimation of active cases (ccfr100 and ccfr1000) . while some small shifts were observed between countries with and without recent universal bcg vaccination policies (see supplementary material), these shifts are not statistically significant. significant differences in the coefficients of the vazquez model. one of the difficulties in modelling and predicting the extent of the coronavirus spreading in a population is the divergence of the observed data (the number of confirmed active cases in individual countries) from the trends expected from the traditional sir type models. ziff and ziff (2020) have recently observed that deaths in china do not follow the typical epidemiological curve and instead of an exponential growth they follow a combined polynomial growth with exponential decay (pged). polynomial growth has been also confirmed for multiple other countries (merrin, 2020) and even though the initial spread in many countries is approximately exponential, it is followed by a steady polynomial growth and in a longer run by an exponential decay (komarova and wodarz, 2020) . for a possible explanation of the transition from exponential to polynomial growth, it is natural to look into self-imposed or government-imposed social distancing measures. these measures transform the structure of virus transmitting contact networks in a population, possibly to small-world network structures or even fractal networks. in contrast to small-world networks, social networks under standard conditions contain a significant fraction of nodes with high number of connections (that correspond to potential superspreaders). interestingly, polynomial growth of the number of infections in time in well connected scale-free networks emerges naturally as a consequence of infection initially reaching the highly connected nodes and their neighbors, while their isolation or recovery significantly reduces the interconnectivity of the residual network (szabó, 2020) . theoretical study of the infection spread in scale-free networks by vazquez (2006) leads to an explicit formula for the number of infected individuals in time in a form of pged. the formula contains three key parameters: p -the coefficient of the polynomial growth (not necessary an integer), τ -the rate of decay of the exponential tail (1/τ is an analogue to the rate of removal of individuals from the infected class to inactive recovered class in the traditional sir-type models), and a -the constant prefactor (scaling the total population). based on the value of these parameters, it is straightforward to determine nmax, the number of infected at the peak of the epidemic, which is independent of the choice of the reference time for the start of the infection. these parameters were obtained by the best fit on the linear scale to the data in each of the considered countries/regions. interestingly, we have found that the parameters τ and nmax significantly differ between countries split into two groups-with and without recent universal bcg vaccination policies ( figure 2 ). the τ parameter shifts to the higher values, signifying higher recovery rate in the countries with recent universal bcg vaccination policies (mann-whitney u-test p = 0.04). in addition, these countries have generally lower numbers of infected cases at the peak of the epidemic (nmax) corrected for underreporting (mann-whitney u-test p = 0.002). east and west germany. the case of germany is interesting, since the country has been split into east and west germany in 1949 and reunited in 1990. in east germany, the policies regarding bcg vaccination followed eastern bloc practices, with universal vaccination policy in place between 1951 and 1998. in west germany, the vaccination has been introduced in 1961, but in 1975 it was discontinued in favor of vaccinating high risk groups only. [the information has been reconstructed from the notes in bcg atlas, however we were not able to confirm this from other sources.] in the present crisis, the whole germany follows similar practices in case reporting and treatment of the disease. interestingly, east germany exhibits much lower estimates of r than west germany at the corresponding phases of the epidemic (r100 = 2.8, r100+10 = 1.55 in east germany vs. r100 = 3.14, r100+10 = 2.76 in west germany; see also figure 3 ). also, the death rate from covid-19 seems to be significantly lower in east germany, even when correcting for differences in age distribution (table 1 ). while some of the previous studies have observed associations between bcg vaccination policy and spread of covid-19 (miller et al., 2020; berg et al., 2020) , others criticized their work and showed 3 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 6, 2020. (2020). most of these studies have used indicators that were quite straightforward, such as the number of reported cases per million inhabitants on a particular date. here, we have instead chosen a variety of indicators that reflect characteristics of various phases of the epidemics in each country, and moreover, these indicators were implicitly or explicitly adjusted according to the time from the beginning of the epidemic in each country. in fact, we hypothesize that such time adjustment is one of the key factors in such an analysis considering what we know about the spread of covid-19. in our data, we have observed several statistically significant associations, and we conclude that there is an association between bcg vaccination policy and spread of covid-19. however, whether this association is causal or is merely an observed correlation due to some other common factor, is impossible to say. moreover, most observed shifts in various coefficients are rather small and while the universal bcg vaccination policy may have had a positive impact in some of the countries, the observed impact clearly cannot replace effective policies such as lockdowns and social distancing measures which currently constitute the most effective weapon against the epidemic. at best, the existence of universal bcg vaccination policy may have provided a few days time for governments to effectively institute such policies. one of the interesting observations is that we did not find any correlation between bcg vaccination policy and cfr. while this may suggest a hypothesis that bcg vaccination may help to limit spread, but may not be effective against difficult progression of the disease in susceptible individuals, we would 4 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 6, 2020. reproduction numbers r were estimated using seven day windows using smoothed incidence numbers. be careful to draw such conclusions. this is because the estimates of cfr are clearly unreliable at this point of time, with many countries showing cfr estimates well over 10%. likely, huge differences between countries do not reflect real differences in outcomes of the disease, but rather discrepancies in the amount and effectiveness of testing, with many light or asymptomatic cases remaining undetected. in fact, such a conclusion is partly supported by the evidence from east/west germany, where we can assume consistent reporting of cases and outcomes, and where differences in cfr seem to be consistent with historical differences in bcg vaccination policies, even after correcting for differences in the age distribution of the population. obtaining case and death reports. the information on reported cases, deaths, and recoveries related to covid-19 assembled by john hopkins university center for system science and engineering (dong et al., 2020) has been downloaded from humanitarian data exchange (humanitarian data exchange, 2020) on april 14, 2020. the data set covers reports from 266 countries from january 22, 2020 until april 13, 2020. for further analysis, only 41 countries with at least 100 reported cumulative deaths have been retained. we also used the data set for germany maintained by robert koch institute, containing reported cases, deaths, and recoveries split geographically and into age groups; the data set was downloaded through arcgis (robert koch-institut and bundesamt für kartographie und geodäsie, 2020). for our analysis, the data were split geographically into east germany (brandeburg, mecklenburg-vorpommern, sachsen, sachsen-anhalt, thüringen, and berlin) and west germany (schleswig-holstein, hamburg, niedersachsen, bremen, nordrhein-westfalen, hessen, rheinland-pfalz, baden-württemberg, bayern, and saarland). bcg status of individual countries. for countries included in the study, we have assembled information from the bcg world atlas (zwerling et al., 2011) and from the who-unicef estimates of bcg coverage (world health organization, 2020) (see supplementary materials). based on this information, the countries were divided into positive bcg status (the countries with current universal bcg vaccination policy and countries with past universal policies discontinued after 1990 or with recent reports of high vaccination coverage from who) and negative bcg status (the countries without universal bcg 5 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 6, 2020. . vaccination policy and those that discontinued universal bcg policies and did not satisfy the above conditions). estimation and extraction of indicators. the indicators were extracted from the time series data sets using simple scripts, as outlined in the results (see supplementary material for tables). all of the indicators are computed in time that is relative to a particular milestone, i.e. reaching a particular cumulative number of case reports or death reports. in this way, compared indicators are synchronized at a particular stage of the epidemic. since the number of cases and deaths is highly dependent on the stage of the epidemic, using such synchronized indicators is a key in our analysis. case fatality rate indicators cfr100 and cfr1000 were computed on the days when the cumulative number of reported deaths surpassed 100 and 1000 respectively; the cumulative number of deaths was divided by the cumulative number of reported cases 7 days prior to that date. as alternative indicators for case fatality rates, denoted as ccfr100 and ccfr1000, we have used methodology established by the centre for the mathematical modelling of infectious diseases (nishiura et al., 2009; russel et al., 2020) , systematically compensating for confirmation-to-death delay using lognormal distribution with mean delay of 13 days and a standard deviation of 12.7 days (linton et al., 2020) . regardless of the method, the main problem with cfr indicators is inconsistent reporting on the number of cases in different countries, as this depends highly on testing strategy, reporting methodology, as well as testing capacities of individual countries. thus, cfr estimates are likely dominated by these factors. we are not aware of any simple method that could overcome this problem at this point of time. note that indicators d10 (time from 10 death reports to 100 death reports) and d100 (time from 100 death reports to 1000 death reports), even though based on the numbers of reported deaths, are unlikely to reflect cfr, but instead simply serve as more stable estimates reflecting the underlying reproductive number r. this is because if we assume exponential growth phase and a constant cfr over this period of time, the cfr coefficient will cancel out in the computation of the expected number of days to reach 10-fold increase in the number of deaths. indicators r100 and r100+10 were computed using epiestim r package (cori et al., 2013) . this method is based on bayesian inference, modelling new infections as a poisson process with rate governed by the instantaneous reproduction number and the number and total infectiousness of infected individuals at the current time interval. the instantaneous reproduction number has a gamma-distributed prior and during the inference is assumed to be constant within each seven-day sliding window to yield an estimate at the end of the window. the infectiousness is approximated by the distribution of the serial interval, which is defined as the time between the onset of symptoms of a case and the onset of symptoms of secondary cases infected by the primary case. following previous work (churches, 2020) , we have set the distribution of serial intervals as a discrete gamma distribution with mean of 5 days and standard deviation of 3.4 days. here, we concentrated on monitoring early stages of the epidemic in each country, when such simple exponential growth model is relatively accurate representation of the spread of the disease. moreover, the estimated values are used mostly in the non-parametric mann-whitney test, which only considers their relative ordering, not exact values. to avoid initial uncertainty in the reproductive number estimates due to small numbers of case reports, and to adjust for the differences in the start date of epidemics in each country, the seven-day interval for the first estimate (r100) starts on the day when 100 cases have been reported and the second estimate (r100+10) is taken 10 days later. the case incidence numbers have been smoothed over a window of 7 days in order to account for differences in testing procedures on different days of the week (i.e. no or little testing over the weekend in many countries). such smoothing will not affect the parameters of exponential growth models. it has been verified that confidence intervals at chosen points of time are not unproportionally large. application of vazquez model. the number of infected individuals in the vazquez model (vazquez, 2006; ziff and ziff, 2020) has the form where a, p, and τ are parameters and t = 1 (units are days) corresponds to the first day of an infection. in practice, the available data does not report the number of infected individuals in the population due to 6 . cc-by 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 6, 2020. . limited testing availability and potential testing errors. therefore we use the total number of active cases (confirmed -recovered -deaths) as a proxy for the total number of infected individuals. in countries with sufficient testing, we assume that the identified active cases represent a constant fraction of the total active cases and the formula for n(t) differs only in the constant factor a. we used a nonlinear least squares method to infer the parameters in the above relationship from the data. however, instead of directly fitting the parameters a, p, and τ , we used an equivalent formulation n(t) = nmax · t tmax p · e p(1−t/tmax) , with parameters nmax (the maximal number of active cases during the infection), p (the power of the polynomial growth term), and tmax = p * τ (the time when the peak is reached). for consistency, we have truncated the data to reduce the impact of testing irregularities during the initial onset of epidemic. therefore we start the data from the day when a certain number of active cases n a was reached. the threshold n a was chosen in proportion to the population in the country to reduce effects of randomness in reporting and to account for the spreading potential. italy served as the reference with a threshold of 200 cases (threshold chosen was always at least 10). mandated bacillus calmette-guérin (bcg) vaccination predicts flattened curves for the spread of covid-19 reducing health care workers absenteeism in covid-19 pandemic through bcg vaccine (bcg-corona) covid-19 epidemiology with r. r views, an r community blog edited by rstudio bcg vaccine for health care workers as defense against covid 19 (badas) a new framework and software to estimate time-varying reproduction numbers during epidemics bcg vaccination to protect healthcare workers against covid-19 (brace) can a century-old tb vaccine steel the immune system against the new coronavirus? science an interactive web-based dashboard to track covid-19 in real time report 13: estimating the number of infections and the impact of non-pharmaceutical interventions on covid-19 in 11 european countries does tb vaccination reduce covid-19 infection?: no evidence from a regression discontinuity analysis exercising caution in correlating covid-19 incidence and mortality rates with bcg vaccination policies due to variable rates of sars cov-2 testing johns hopkins university novel coronavirus (covid-19) cases data application of bcg vaccine for immune-prophylaxis among egyptian healthcare workers during the pandemic of covid-19 patterns of the covid19 epidemic spread around the world: exponential vs power laws incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data differences in power-law growth over time and indicators of covid-19 pandemic progression worldwide correlation between universal bcg vaccination policy and reduced morbidity and mortality for covid-19: an epidemiological study non-specific effects of bcg vaccine on viral infections early epidemiological assessment of the virulence of emerging infectious diseases: a case study of an influenza pandemic csv mit den aktuellen covid-19 infektionen pro tag (zeitreihe) using a delay-adjusted case fatality ratio to estimate under-reporting. available at the centre for mathematical modelling of infectious diseases repository bcg vaccines may not reduce covid-19 mortality rates propagation and mitigation of epidemics in a scale-free network bcg protects against covid-19? a word of caution polynomial growth in branching processes with diverging reproductive number who-unicef estimates of bcg coverage fractal kinetics of covid-19 pandemic the bcg world atlas: a database of global bcg vaccination policies and practices key: cord-288721-3bv3aak6 authors: schneider, annika; kurz, sandra; manske, katrin; janas, marianne; heikenwälder, mathias; misgeld, thomas; aichler, michaela; weissmann, sebastian felix; zischka, hans; knolle, percy; wohlleber, dirk title: single organelle analysis to characterize mitochondrial function and crosstalk during viral infection date: 2019-06-11 journal: sci rep doi: 10.1038/s41598-019-44922-9 sha: doc_id: 288721 cord_uid: 3bv3aak6 mitochondria are key for cellular metabolism and signalling processes during viral infection. we report a methodology to analyse mitochondrial properties at the single-organelle level during viral infection using a recombinant adenovirus coding for a mitochondrial tracer protein for tagging and detection by multispectral flow cytometry. resolution at the level of tagged individual mitochondria revealed changes in mitochondrial size, membrane potential and displayed a fragile phenotype during viral infection of cells. thus, single-organelle and multi-parameter resolution allows to explore altered energy metabolism and antiviral defence by tagged mitochondria selectively in virus-infected cells and will be instrumental to identify viral immune escape and to develop and monitor novel mitochondrial-targeted therapies. mitochondria are crucial for cellular energy metabolism, critically involved in the coordination of signalling processes within cells and orchestrate induction of apoptotic cell death 1, 2 . besides this, cell-autonomous defence mechanisms during viral infection link innate immune sensing of infection and inflammation at the level of mitochondria 3, 4 . the research in the recent years has expanded our knowledge about the different roles of mitochondria. for the different functions mitochondrial shape and motility, but also size, are important and are highly dynamic processes 5 . mitochondrial shape and size are continuously changed during the dynamics of mitochondrial fusion and fission and mitochondrial turnover is controlled by mitophagy 5 . viruses modify the host cell to create an ideal ambience, which includes metabolic support for viral gene expression and replication. such modifications of cellular metabolism and structure of viruses can also affect mitochondria. there are more and more reports about viruses known to influence mitochondrial dynamics. viruses known to enhance mitochondrial fission are hepatitis b virus (hbv), hepatitis c virus (hcv) and epstein-barr virus [6] [7] [8] [9] . viruses, which interfere with or enhance mitophagy are hbv, hcv and measles virus [6] [7] [8] 10 . sars coronavirus is reported to enhance the fusion of mitochondria 11 . but the influence of viral infection on mitochondrial membrane potential and stress response has not been addressed in detail because of methodological constraints. so far, analysis of mitochondria and their functions relied mostly on bulk analysis of mitochondrial populations analysed ex vivo. in infected tissues where both, infected and non-infected cells are simultaneously present, it is very difficult to discriminate between mitochondria from infected versus healthy non-infected cells. this may be achieved by serial tissue sections analysed by electron microscopy, where viral particles could be visualized. however, this is a very time demanding process yielding results with little statistical power. we therefore aimed to develop a technology, where high numbers of single mitochondria and their function can be analysed in the context of viral infection in order to characterize changes induced by viral infection. we chose the liver, and more specifically hepatocytes, as viral infection increases size and mitochondrial fragility of liver mitochondria. we first aimed to determine the influence of viral infection of the liver on the size of liver mitochondria by flow cytometry. to that end, we established a reference curve using polystyrene microparticles with defined sizes (0.88 µm, 1,34 µm and 3 µm). forward scatter analysis of these polystyrene microparticles revealed clear demarcation of the differently sized microparticles and a direct linear correlation of forward scatter results with microparticle size (r 2 = 0.99) ( fig. 2a) , consistent with earlier reports that forward scatter measurements directly correlate with microparticle size down to 0.5 µm 20, 21 . the flow cytometric analysis revealed that mitochondria isolated from healthy non-infected liver ranged in size from 0.8 µm up to 1.4 µm (fig. 2b ) assuming that isolated mitochondria are spherical in morphology, which is indicated by electron microscopy (see fig. 1b ). since mitochondria from hepatocytes are much larger than those from non-parenchymal liver cells or immune cells, we assume that mitochondria ≥0.8 µm in size are derived from hepatocytes. mitochondria purified from virus-infected livers had a slightly higher mean size compared to healthy liver (1.04 ± 0.06 µm compared to 0.97 ± 0.04 µm, respectively) and ranged in size from 0.8 µm up to 3 µm (fig. 2b) . infection with recombinant replication-deficient adenoviruses is a well-established preclinical model system to study hepatotropic infections [22] [23] [24] . however, to confirm the results we repeated the experiments by infection with wildtype replication-competent lymphocytic choriomeningitis virus (lcmv). also after lcmv-infection, we detected an increase of mitochondrial size confirming the results obtained after adenoviral infection (supp. fig. 1a ). in order to investigate whether innate immunity generated during viral infection, was responsible for this increase in mitochondrial size, we induced a type i interferon response by application of poly i:c 25 . flow cytometric analysis of mitochondria isolated after poly i:c application did not reveal any differences in their size compared to the control groups suggesting other mechanisms (supp. fig. 1b) . the exact determination of the size of single mitochondria now opened the possibility to use this information for further analysis. next, we evaluated mitochondrial functionality by determining the mitochondrial membrane potential using the potentiometric dilc 1 (5) fluorescence dye. dose titration experiments of the dilc 1 (5) dye demonstrated a dose-dependent increase in fluorescence intensity in purified mitochondria (supp. fig. 1c ). upon addition of the electron chain uncoupling agent cccp, we found a profound reduction in dilc 1 (5) fluorescence (fig. 2c ) demonstrating that flow cytometric determination of changes in dilc 1 (5) fluorescence reflected mitochondrial membrane potential. by flow cytometric analysis we observed a significant decrease in the mitochondrial membrane potential of mitochondria isolated from virus-infected vs. healthy livers after either adenoviral or lcmv infection compared to healthy controls ( fig. 2d and supp. fig. 1d ). in contrast, we did not detect changes in the membrane potential after innate immune stimulation by poly i:c (supp. fig. 1e ). yet, the size of mitochondria may influence dilc 1 (5) signal intensity. indeed, we found a direct correlation between mitochondrial size and dilc 1 (5) staining ( fig. 2e and supp. fig. 1f ) suggesting that larger mitochondria purified from virus-infected livers should show higher dilc 1 (5) fluorescence intensity. we therefore compared mitochondria with the same size isolated from healthy or virus-infected livers. such direct comparison demonstrated that mitochondria of the same size from healthy vs. virus-infected livers showed a remarkable decrease in the membrane potential of mitochondria from infected livers (fig. 2f ) and suggested that viral infection caused changes in mitochondrial functionality. mitochondria also function to take up calcium from the cytosol and thereby coordinate cellular function 26 , which can also serve as a stress test. when challenged with high concentrations of calcium (100 µm), mitochondria isolated from virus-infected livers are much more fragile shown by time-dependent loss of membrane potential and change of their morphology indicated by decrease in side-scatter (fig. 2f ). this accurately detects mitochondrial swelling after loss of membrane potential following ca 2+ challenge which is also detected by bulk analysis with a classical stress test by adding ca 2+ and detection of loss of membrane potential by rh123-fluorescence and swelling by measuring optical density at 540 nm (supp. fig. 1g ) 18 . consistent with the loss of membrane potential and changes in side-scatter signals, we detected loss of mitochondrial integrity after www.nature.com/scientificreports www.nature.com/scientificreports/ calcium challenge. number of viable mitochondria detected per second by flow-cytometry declined after calcium challenge, consistent with loss of mitochondrial integrity, and did so much faster in samples from virus-infected livers (fig. 2f ). comparing mitochondria with different sizes, it became evident that larger mitochondria are more fragile and disappeared more rapidly after ca 2+ -challenge (fig. 2f ). taken together, here we detected an increase in size and a decrease in membrane potential as well as mitochondrial fragility of liver mitochondria after viral infection. however, since both, non-infected as well as infected hepatocytes are present in livers after adenoviral infection (see fig. 1b ), current protocols for isolation and analysis yield a mixture of mitochondria derived from healthy as well as infected hepatocytes. this makes it necessary to develop a methodology, by which mitochondria from healthy and virus-infected hepatocytes can be separated in order to characterize changes in mitochondrial function specifically in virus-infected cells. we generated a recombinant adenovirus expressing the fluorescent protein dsred fused to a mitochondrial localization sequence (ad-cmv-mitorl) that accumulates and selectively labels mitochondria within infected cells (supp. fig. 2 ). we combined this mitochondrial labelling in infected cells with a multispectral flow cytometric single organelle measurement of isolated mitochondria. upon infection of hepatocytes with ad-cmv-mitorl in vitro we detected mito-dsred-fluorescence in mitochondria using confocal microscopy (fig. 3a) . since ad-cmv-mitorl also codes for luciferase, we detected in vivo bioluminescence of the liver after infection, thus www.nature.com/scientificreports www.nature.com/scientificreports/ confirming successful infection of hepatocytes in vivo (fig. 3b ). this allowed us to test whether mitochondria from ad-cmv-mitorl-infected hepatocytes (mito-dsred + mitochondria) could be distinguished from those of non-infected hepatocytes (mito-dsred − mitochondria) within the same liver. after density-gradient purification, mitochondria isolated from virus-infected livers were counterstained with mitotracker green and analysed by flow cytometry allowing discrimination of mito-dsred + mitochondria from mito-dsred − mitochondria from the same liver (fig. 3c ). mito-dsred + mitochondria from virus-infected hepatocytes had a mean size of 1.19 ± 0.06 µm as compared to mito-dsred − mitochondria from healthy hepatocytes with a mean size of 0.96 ± 0.01 µm (fig. 3d ). this confirmed the results obtained from mitochondria isolated from non-infected livers and further demonstrated a more pronounced size difference when mitochondria from virus-infected could be distinguished at the single organelle level from those of healthy hepatocytes. this was most likely related to a relative underestimation of size for mitochondria from virus-infected livers due to contamination with mitochondria from non-infected cells that are smaller than hepatocyte mitochondria. yet, we cannot formally exclude that mito-dsred localizing to mitochondria after infection with ad-cmv-mitorl may have contributed to the size difference. the almost identical forward scatter results and size of mito-dsred − mitochondria compared to mitochondria isolated from non-infected livers (fig. 3d) indicated that there was no influence of viral infection in neighbouring hepatocytes on mitochondrial size after isolation. differences in size of mitochondria may have also an influence on other parameters detected by flow cytometry and we therefore systematically measured mitochondrial autofluorescence from 450 to 800 nm using a spectral flow cytometer. as expected, we detected increased fluorescence at 590 nm in mito-dsred + mitochondria, where the maximum of dsred fluorescence emission (590-650 nm) is expected 27 . interestingly, we detected higher autofluorescence signals between 500 and 550 nm as well as above 650 nm in mito-dsred + mitochondria (fig. 3e) . as the strength of autofluorescence may be influenced by the size of mitochondria, we analysed autofluorescence signals against the size of isolated mitochondria (fig. 3f ). we found that autofluorescence intensity between 430 and 550 nm directly correlated with mitochondrial size, which may explain the higher autofluorescence observed in larger mito-dsred + mitochondria. together, these data demonstrate the usefulness of single-organelle analysis by flow cytometry in combination with in vivo mitochondrial labelling in virus-infected hepatocytes to exactly determine physical parameters such as size or autofluorescence. mitochondrial crosstalk enables changes in membrane potential. since discrimination of mitochondria isolated from virus-infected compared to non-infected hepatocytes was reliably achieved using flow cytometry, we proceeded to test for changes in mitochondrial functionality upon infection. we assumed that the difference in membrane potential detected between mitochondria isolated from virus-infected livers compared to non-infected livers (see fig. 2 ) has previously been underestimated, and that our method would allow to more specifically discriminate mitochondria from virus-infected hepatocytes compared to non-infected hepatocytes. we determined whether mito-dsred + differed from mito-dsred − mitochondria with respect to dilc 1 (5) fluorescence intensity. to our surprise, we found that the dilc 1 (5) signal was similar for all sizes of mito-dsred + compared to mito-dsred − mitochondria (fig. 4a ). since dilc 1 (5) fluorescence was homogenous in all mitochondria isolated from ad-cmv-gol infected livers (see fig. 1 ), although they consisted of a mixture of mitochondria from infected and non-infected hepatocytes, we wondered whether there was an exchange of molecules between mitochondria. therefore, we mixed dilc 1 (5)-labelled mitochondria with non-labelled mitochondria and by time-dependent flow cytometric analysis found that dilc 1 (5) fluorescence decreased in pre-labelled and increased in un-labelled mitochondria reaching an equilibrium of intermediate fluorescence intensity within 30 seconds (fig. 4b) . however, mito-dsred was not exchanged between mitochondria, because we found clearly distinct dsred staining of mitochondria isolated from ad-cmv-mitorl-infected livers, and mito-dsred − mitochondria showed the same absent dsred fluorescence intensity as mitochondria isolated from non-infected livers. in order to further evaluate mitochondrial functionality, we challenged mitochondria with ca 2+ as stress test and performed time kinetic measurements of dilc 1 (5) fluorescence and side-scatter of mito-dsred + and mito-dsred − mitochondria isolated from ad-cmv-mitorl infected livers. remarkably, the differences in mitochondrial characteristics observed when comparing mitochondria isolated from infected livers to mitochondria from non-infected livers (see fig. 2f ) where not present any more when comparing mito-dsred + to mito-dsred − mitochondria originating from the same liver. in fact, loss of dilc 1 (5) fluorescence, decrease in side scatter and mitochondrial events were the same for mito-dsred + mitochondria as compared to mito-dsred − mitochondria (fig. 4c) . when in direct physical contact with mito-dsred + mitochondria, also mito-dsred − mitochondria showed the same fragility as mitochondria from virus-infected hepatocytes. there, was still a small difference in the large mitochondrial group after calcium stimulation and flow cytometric analysis of the ssc and dilc 1 (5) which could be explained by the fact that 5 to 10 minutes after calcium stimulation the number of events was drastically reduced. only approximately 10% from the initial number of events are still detectable (shown by number of events/s). because of the statistical variation the conclusions at later time points has to be taken with caution. interestingly, also mixing of dilc 1 (5) labelled mitochondria isolated from either ad-cmv-golor lcmv-infected with those from healthy uninfected livers yielded in rapid loss of mitochondrial membrane potential to that measured in mitochondria from infected livers (fig. 4d and supp. fig. 2 ) taken together these data demonstrate that mitochondria which are in close physical proximity exchange information leading to changes in mitochondrial membrane potential but not in mitochondrial size. here, we describe the influence of viral infection on the phenotype and function of mitochondria employing a new methodology combining spectral flow cytometry with virus-encoded markers to simultaneously evaluate multiple mitochondrial parameters at the level of single organelles. most studies involve confocal microscopy to detect mitochondria, which is also available in an automated manner to quantify large datasets of mitochondria 28 . www.nature.com/scientificreports www.nature.com/scientificreports/ while most of these microscopic studies are performed in cell cultures to explore mitochondrial dynamics at the level of single cells, there are only few reports specifically detecting tagged mitochondria in tissues for ex vivo or in vivo analysis 29, 30 . since in vivo microscopic analysis of mitochondria requires a complex experimental setup, is rather time consuming and does not allow for analysis of large numbers of mitochondria, we aimed to establish a methodology to evaluate mitochondria directly ex vivo following isolation from virus-infected tissue. so far, most of available methods analyse properties of mitochondria ex vivo at the level of mitochondrial populations, www.nature.com/scientificreports www.nature.com/scientificreports/ such as extracellular flux analysis, western blot analysis, calcium uptake or swelling assays. beyond visualization by microscopy, flow cytometry has emerged as technology to characterize mitochondria 18, 31, 32 . however, mitochondria isolated from virus-infected tissues can be derived from both, virus-infected cells as well as healthy cells, which may skew the experimental results. we therefore generated recombinant adenoviruses containing a mito-dsred expression cassette to selectively label mitochondria of infected cells. fusion of a fluorescent marker to mitochondrial target sequences has previously been reported to reliably and specifically label mitochondria as shown by confocal microscopy 33, 34 . we combined virus-encoded mito-dsred labelling of mitochondria to separate mitochondria of virus-infected cells from those originating from healthy cells, with the power of multi-parameter analysis by spectral flow cytometry. using this methodology, we provide evidence that mitochondria can be reliably separated from virus-infected cells and that viral infection led to an increase in size as well as a decrease of mitochondrial membrane potential. such changes in biophysical and functional properties of mitochondria were not triggered by innate immunity following recognition of infection through microbe-associated pattern recognition receptors indicating other reasons for these changes, which still have to be defined. time kinetic measurements of single mitochondria by flow cytometry further allowed us to detect a previously unknown mitochondrial cross-talk that involves rapid exchange of small molecules like the potentiometric dye dilc 1 (5) . such exchange of molecules among mitochondria required physical contact, occurred within seconds and did not include mitochondrial matrix-embedded proteins. this indicates a dynamic regulation of mitochondrial properties by cell autonomous mechanisms that require further investigation. taken together, the combination of mitochondrial labelling through mito-dsred together with single organelle analysis using spectral flow cytometry is ideally suited to further unravel biophysical and functional properties of mitochondria as well as mechanisms and consequences of mitochondrial interconnectivity in virus-infected cells. given the important role of mitochondria in cellular metabolism, anti-viral defence, cell signalling and cell death, the multiparametric analysis of single mitochondria opens new avenues to explore these complex mitochondrial functions in more detail in virus-infected cells. mice. c57bl/6 j mice were purchased from charles river (sulzfeld, germany). mice were maintained under specific pathogen-free (spf) conditions in the central animal facility of the klinikum rechts der isar, in accordance with the guidelines of the federation of laboratory animal science association. animal experiments were approved by the animal care commission of bavaria. male mice between the ages of 6-10 weeks were used. the expression cassette for cloning into recombinant adenovirus consists of the genes for the fluorescent protein dsred linked to a mitochondrial targeting sequence and cbg99-luciferase separated by p2a linker sites from the porcine teschovirus 1 followed by a bgh poly(a) signal. gene expression was driven by the ubiquitous minimal cmv-promoter (ad-cmv-mitorl). ad-cmv-gol generation has been reported before 23 . recombinant second generation serotype 5 adenoviruses were generated using the gateway ® technology from thermofisher as described before 23 . briefly, expression cassettes with cmv promotor, dsred linked to the mitochondrial targeting site, cbg99-luciferase and the bgh poly(a) signal were synthesized (eurofins genomics, germany) and cloned into gateway ® pentr ™ 11 dual selection vector (thermofisher scientific, germany). recombination of pentr ™ with expression cassette into pad/pl-dest ™ gateway ® vector (thermofisher scientific, germany) was performed in vitro via the lr clonase ® enzyme mix (thermofisher scientific, germany). the obtained pad/pl-dest ™ with expression cassette was linearized using the paci restriction enzyme and the resulting adenoviral dna was transfected with lipofectamine 2000 (thermofisher scientific, germany) into hek293 cells (crl-1573 ™ ; atcc, usa). cell debris and supernatant were harvested when complete detachment of the cells occurred. this suspension was freeze/thawed, centrifuged and used for further infection of hek293 cells. cells from several cell culture dishes were harvested and resuspended in tris-buffer and freeze/thawed three times. cell debris was removed by centrifugation and supernatant purified by a two-step cscl gradient ultracentrifugation. the band containing adenovirus was harvested and dialyzed. virus titer was determined via adenovirus hexon titration. hek293 cells were infected with serial dilutions of purified adenovirus. after 35 to 40 hours, cells were fixed with methanol, and virus infected cells were stained with anti-hexon antibody (anti-hexon 2297hrp, acris, germany) and detected via dab (dako, usa). the infected cells were counted and the titer was calculated. bioluminescence imaging. imaging of luciferase expression in infected mice was monitored by ivis lumina lt-series iii instrument (perkinelmer las, germany). five minutes before measurement mice have been anesthetized with 2.5% isofluran and treated intraperitoneally with 100 mg/kg bodyweight d-luciferin-k-salt (pjk gmbh, germany). isolation of mitochondria from murine liver tissue. heparin/nacl (300 u/150 µl) was injected i.p. into the mouse 5 minutes prior to preparation. mice were sacrificed and livers were perfused via portal vein for 1 minute with pbs to remove blood. liver was removed and weighed, and the liver was rinsed with isolation buffer www.nature.com/scientificreports www.nature.com/scientificreports/ (220 mm mannitol, 80 mm sucrose, 10 mm hepes, 1 mm edta, ph 7.4). the whole isolation procedure was performed on ice and in ice-cold isolation buffer. the tissue was rinsed with 1 ml isolation buffer and cut with a blunt end scissor into small pieces. the liver fragments were resuspended in 1 ml isolation buffer supplemented with 0.5% bsa and protease inhibitor (protease inhibitor cocktail, edta-free, roche, switzerland) per 0.1 gram of weighted organ and homogenized in a potter-elvehjem with 3 strokes at 800 rpm. the homogenate was transferred to cooled 50 ml falcon and centrifuged at 600 x g for 10 minutes to remove nuclei, intact cells and cellular debris. the supernatant was transferred to a glass tube and centrifuged at 4000 x g for 10 minutes to sediment mitochondria. the received crude pellet was gently dislodged with a glass pestle from the side of the glass tube. mitochondrial purification by density gradient centrifugation. mitochondria were purified as previously described 35, 36 . in brief, a discontinuous percoll density gradient was used for mitochondrial purification. crude mitochondria were resuspended in ip-buffer (300 mm sucrose, 5 mm tes, 0.2 mm egta, ph 6.9), loaded on a percoll density gradient (60%, 30% and 18% diluted in ipp buffer: 300 mm sucrose, 10 mm tes, 0.2 mm egta, 0.1% w/v bsa, ph 7.2) and separated at 9000 × g for 10 minutes. the phase containing mitochondria between 60% and 30% percoll-layer was recovered with a glass pipette and transferred to a 30 ml glass tube, resuspended in 15 ml ip-buffer and centrifuged for further 10 minutes at 9000 × g. the pellet was washed again in 10 ml ip-buffer and centrifuged at 9000 × g for 10 minutes to get rid of remaining percoll. the supernatant was removed and mitochondrial pellet was dislodged from the side of the glass tube. the received mitochondria were resuspended in 100 µl ip-buffer and kept on ice. determination of protein concentration. the protein content in the mitochondrial preparations was determined using the dc tm protein assay kit (bio rad laboratories, germany). the assay was performed according to the manufacturer´s protocol. four different bsa-dilutions reaching from 0.25 mg/ml to 1.5 mg/ml in ip-buffer were used as standards. the optical density was measured at 750 nm with a multiplate reader (infinitem100 pro, tecan, germany). determining mitochondria by flow cytometry. mitochondria were diluted to 10 µg protein per µl in ice-cold mitochondrial staining buffer msb (0.2 m saccharose, 10 mm mops-tris, 5 mm succinate, 1 mm phosphoric acid, 10 µm egta). the different mitochondrial probes were diluted in msb, mixed with the mitochondrial dilution in a 1:1 ratio and incubated at room temperature for 20 minutes. the cell permeable carbocyanine-based mitotracker green probe (mtg, 200 nm), which contains a mildly thiol-reactive chloromethyl moiety, was used to selectively stain all undamaged mitochondria regardless of the membrane potential. dilc1(5) (100 nm), a cationic carbocyanine dye, was used to measure the membrane potential of isolated mitochondria. mitochondria were pelleted at 9000 x g for 2 minutes and washed once in ice cold pbs. mitochondrial pellet was resuspended in msb to a final concentration of 10 µg/µl and stored on ice for analysis. immediately before analysis, samples were diluted in ice-cold and filtered pbs to the final analysis concentration of 0.05 µg/µl. samples were analysed using the spectral cell analyzer sp6800 (sony biotechnology inc, japan). the sample flow rate was set to record about 1500 events per second. as mitochondrial uncoupling by the protonophore cccp is well known to dissipate mitochondrial membrane potential (mmp), 5 µm cccp (sigma-aldrich, st. louis, missouri, usa) was used as a positive control for membrane potential dependence of diic 1 (5) (biotium, hayward, usa). the mitochondrial permeability transition (mpt), a process characterized by a large increase of permeability of the inner mitochondrial membrane (imm), leading to an influx of solutes with a molecular weight less than 1.5 kda and water into the mitochondrion, is a ca 2+ -induced process. the influx of solutes and water leads to swelling of mitochondria. in mpt-measurements 100 µm ca 2+ in msb was added to induce swelling and samples were analyzed immediately after administration and every following 5 minutes for 45 minutes in total. cyclosporina (sigma-aldrich, st. louis, missouri, usa) inhibiting mpt and thereby reversing the effect of ca 2+ , was added at a concentration of 5 µm. mitochondrial size was determined using polystyrene particle size standard beads (flow cytometry grade, spherotech) in three sizes: 0.88 μm, 1.34 μm and 3 μm. beads of each size were separated via ultrasound, vortexed and 20000 beads/size were added per ml filtered pbs. immediately before analysis, mitochondria were diluted in bead mixture to the final analysis concentration of 0,05 µg/ml. data were analysed using flowjo software (version 10, flowjo, oregon, usa). western-blot. 30 µg of protein per sample was loaded onto 4-20% mini-protean ® tgx stain-free ™ precast gels (bio rad laboratories, münchen) and separation was performed within a gel chamber filled with 1x sds electrophoresis buffer at 100 v for 1 to 2 hours. after separation, proteins were blotted using the trans-blot ® turbo ™ mini pvdf transfer packs (bio rad laboratories, germany). proteins were transferred onto membranes at 2.5 a for 30 minutes using the trans-blot turbo ™ (bio rad laboratories, germany). membranes were blocked with 10% milk in tbs-t (tbs + 0.1% tween-20) for 1 hour at room temperature, washed three times with tbs-t and incubated with primary antibodies in 5% bsa in tbs-t overnight at 4 °c. the membranes were washed three times with tbs-t and incubated for 4 hours at room temperature with hrp-coupled secondary antibodies in 10% milk powder in tbs-t. blots were washed three times and developed using cheluminate-hrp picodetect (applichem gmbh, germany), which was evenly distributed on the membrane. the luminescence was detected for up to 20 minutes using the imaging-system chemidoc tm xrs (bio rad laboratories, germany). to visualize www.nature.com/scientificreports www.nature.com/scientificreports/ several proteins on the same blot, primary and secondary antibodies were removed by incubating membranes for 45 minutes at 50 °c in stripping buffer containing ß-mercaptoethanol. subsequently membranes were washed three times with tbs-t and incubated as previously described with primary and secondary antibodies. following primary antibodies were used: adenine nucleotide translocator (ant) (santa cruz biotechnology usa), cytochrome-c-oxidase (cox iv), cytochrome-c (cyt-c), glyceraldehyde 3-phosphate dehydrogenase (gapdh), glucose-regulated-protein 78 (grp78), histon 2b (h2b), voltage dependent anion channel (vdac) (all cell signaling technology, usa), lysosome-associated membrane protein 2 (lamp2) (thermo fisher scientific, usa), peroxisomal membrane protein 70 (pmp70) (origene technologies, usa), following secondary antibodies were used: rabbit anti-goat hrp (santa cruz biotechnology, usa), mouse anti-rabbit hrp, goat anti-mouse hrp (jackson immunoresearch, uk). histology. mouse livers were fixed for 48 hours in 4% paraformaldehyde. dehydrated livers (leica asp300s, germany) were embedded in paraffin. serial 2 µm-thin sections were prepared with a rotary microtome (hm355s, thermofisher scientific, usa) and subjected to histological and immune-histochemical analysis. hematoxylin-eosin (he) staining was performed on deparaffinized sections with eosin and mayer's haemalaun according to standard protocol. immunohistochemistry was performed using a bondmax rxm system (leica, wetzlar, germany, all reagents from leica) with primary antibody against egfp (a-11122, diluted 1:500 in antibody diluent, invitrogen, thermofisher scientific, usa). slides were deparaffinized, pre-treated with epitope retrieval solution 1 for 30 minutes. bound antibody was detected with a polymer refine detection kit without post primary reagent and visualized with dab as a dark brown precipitate. counterstaining was done with hematoxyline. electron microscopy. tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 m sodium cacodylate buffer ph 7.4 (science services, munich, germany), postfixed in 2% aqueous osmium tetraoxide 37 , dehydrated in gradual ethanol (30-100%) and propylene oxide, embedded in epon (merck, darmstadt, germany) and cured for 48 hours at 60 °c. semithin sections were cut and stained with toluidine blue. ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (zeiss libra 120 plus, carl zeiss nts gmbh, oberkochen, germany). pictures were acquired using a slow scan ccd-camera and item software (olympus soft imaging solutions, münster, germany). statistics. student's t tests were calculated using graphpad prism software. significance was set at p < 0.05 and denoted as *p < 0.05, **p < 0.01, ***p < 0.001 and ***p < 0.0001. all results are expressed as the mean ± standard deviation (sd). the data within this manuscript are available from the corresponding author upon reasonable request. mitochondrial control of cellular life, stress, and death mitochondrial signaling pathways: a receiver/integrator organelle mechanisms of mavs regulation at the mitochondrial membrane identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 mitochondria: dynamic organelles in disease, aging, and development hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis hepatitis c virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy epstein-barr virus latent membrane protein-2a alters mitochondrial dynamics promoting cellular migration mediated by notch signaling pathway mitophagy enhances oncolytic measles virus replication by mitigating ddx58/rig-i-like receptor signaling sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/ traf3/traf6 signalosome living in the liver: hepatic infections correlated morphometric and biochemical studies on the liver cell. i. morphometric model, stereologic methods, and normal morphometric data for rat liver bioluminescence imaging allows measuring cd8 t cell function in the liver isolation of mitochondria from cultured cells and liver tissue biopsies for molecular and biochemical analyses analysis of mitochondria by flow cytometry real-time flow cytometry analysis of permeability transition in isolated mitochondria flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death measurement of mitochondrial mass by flow cytometry during oxidative cell sizing: a light scattering photometer for rapid volume determination overcoming limitations of microparticle measurement by flow cytometry tnf-induced target cell killing by ctl activated through cross-presentation outcome of anti-viral immunity in the liver is shaped by the level of antigen expressed in infected hepatocytes perforin inhibition protects from lethal endothelial damage during fulminant viral hepatitis reduced type i interferon production by dendritic cells and weakened antiviral immunity in patients with wiskott-aldrich syndrome protein deficiency calcium uptake mechanisms of mitochondria biochemistry, mutagenesis, and oligomerization of dsred, a red fluorescent protein from coral deep analysis of mitochondria and cell health using machine learning multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo in vivo imaging of disease-related mitochondrial dynamics in a vertebrate model system flow cytometry of isolated mitochondria during development and under some pathological conditions why to compare absolute numbers of mitochondria analysis of mitochondrial dynamics and functions using imaging approaches strategies for imaging mitophagy in high-resolution and high-throughput a semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies progressive stages of mitochondrial destruction caused by cell toxic bile salts a chrome-osmium fixative for electron microscopy this work was funded by the deutsche forschungsgemeinschaft (dfg, german research foundation) -projektnummer 272983813 -trr 179 to d.w and p.k. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-44922-9.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-315064-2mgv9j6n authors: escher, felicitas; pietsch, heiko; aleshcheva, ganna; bock, thomas; baumeier, christian; elsaesser, albrecht; wenzel, philip; hamm, christian; westenfeld, ralph; schultheiss, maximilian; gross, ulrich; morawietz, lars; schultheiss, heinz‐peter title: detection of viral sars‐cov‐2 genomes and histopathological changes in endomyocardial biopsies date: 2020-06-12 journal: esc heart fail doi: 10.1002/ehf2.12805 sha: doc_id: 315064 cord_uid: 2mgv9j6n aims: since december 2019, the novel coronavirus sars‐cov‐2 has spread rapidly throughout china and keeps the world in suspense. cardiovascular complications with myocarditis and embolism due to covid‐19 have been reported. sars‐cov‐2 genome detection in the heart muscle has not been demonstrated so far, and the underlying pathophysiological mechanisms remain to be investigated. methods and results: endomyocardial biopsies (embs) of 104 patients (mean age: 57.90 ± 16.37 years; left ventricular ejection fraction: 33.7 ± 14.6%, sex: n = 79 male/25 female) with suspected myocarditis or unexplained heart failure were analysed. emb analysis included histology, immunohistochemistry, and detection of sars‐cov‐2 genomes by real‐time reverse transcription polymerase chain reaction in the ikdt berlin, germany. among 104 embs investigated, five were confirmed with sars‐cov‐2 infected by reverse real‐time transcriptase polymerase chain reaction. we describe patients of different history of symptoms and time duration. additionally, we investigated histopathological changes in myocardial tissue showing that the inflammatory process in embs seemed to permeate vascular wall leading to small arterial obliteration and damage. conclusions: this is the first report that established the evidence of sars‐cov‐2 genomes detection in embs. in these patients, myocardial injury ischaemia may play a role, which could explain the ubiquitous troponin increases. emb‐based identification of the cause of myocardial injury may contribute to explain the different evolution of complicated sars‐cov‐2‐infection and to design future specific and personalized treatment strategies. in december 2019, a novel coronavirus with potential zoonotic origin, named severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was identified as the causative agent of a cluster of suspicious pneumonia cases in wuhan, hubei, china. the incredible fast worldwide spread of the coronavirus disease 2019 (covid-19) prompt the world health organization (who) to declare covid-19 as a pandemic on 11 march 2020. 1 more than 1 776 867 confirmed cases of covid-19 and more than 111 828 fatalities in 185 countries have been attributed to sars-cov-2 as of 14 april 2020 (https://who.sprinklr.com/). molecular tests (real-time reverse transcriptase polymerase chain reaction, rt-qpcr) were generally used to confirm the clinical diagnosis of covid-19. a recent report showed that sars-cov-2 could be detected in different types of clinical specimens such as broncho alveolar lavage, sputum, nasal swabs, feces, blood, and urine. 2 the ubiquitous distribution of the main viral entry receptor angiotensin converting enzyme 2 (ace2) for sars-cov-2 entry into the target cells led to the hypothesis of the involvement of other potential target organs for sars-cov-2 besides the respiratory tract, for example, the heart, the liver, the brain, the pancreas, or the kidneys. 3 infection with the sars-cov-2 is associated with systemic illness by hyper-inflammation. 4 cardiovascular complications with embolism due to covid-19 have been reported recently. [5] [6] [7] [8] acute myocardial injury associated with covid-19 manifested as an increase of high-sensitivity cardiac troponin levels. 1 however, direct sars-cov-2 rna in the heart muscle has not been demonstrated so far. the damage caused by sars-cov-2 to the cardiovascular system and the underlying mechanisms remain to be investigated. accordingly, we prospectively analysed endomyocardial biopsies (embs) from a cohort of 104 samples of patients with suspected myocarditis or unexplained heart disease for the presence of sars-cov-2 rna by rt-qpcr and hints for histopathological injury. up to 8 embs each of 104 patients [mean age: 57.90 ± 16.37 years; left ventricular ejection fraction (lvef): 33.7 ± 14.6%, sex: n = 79 male/25 female] with suspected myocarditis or unexplained heart failure were analysed between 3 february and 26 march 2020 in german clinical centres in accordance with sars-cov2 spread in germany. embs were routinely taken from left ventricle. in 60.4% a hypertrophy was seen according possible due to a cardiac oedema. coronary artery disease was excluded angiographically in all patients prior to emb. the suspected diagnosis had been made by clinicians. embs were send for further diagnosis to the laboratory ikdt (institute for cardiac diagnostic and therapy berlin, germany). analysis included histology, immunohistochemistry, and molecular virology. following emb extraction, samples were transferred to formalin for histological analyses and to rnalater ™ solution (thermo fisher scientific, waltham, ma, usa) for immunohistological and molecular analyses. dna was extracted by puregene core kit a (qiagen, hilden, germany) according to manufacturer's instructions. total rna from one emb was isolated using trizol reagent ™ (thermo fisher scientific, waltham, ma, usa), solubilized in depc-h 2 o, and treated with dnase (peqlab, erlangen, germany) to remove any traces of dna followed by reverse transcription with high-capacity cdna reverse transcription kit (thermo fisher scientific, waltham, ma, usa). random hexamer primers (5 μm) were used in addition to specific primers targeting the e-gene of sars-cov2 (0.2 μm each). dna and cdna concentrations were measured by pcr-based quantifiler ™ human dna quantification kit (thermo fisher scientific, waltham, ma, usa) or by expression of housekeeping gene hprt, respectively. detection of other cardiotropic viruses (enteroviruses, adenoviruses, human herpesvirus 6, epstein-barr virus, parvovirus b19) using (rt-) qpcr or nested pcr was applied as described elsewhere. 9,10 commercially available rt-qpcr kits targeting the e-gene and rdrp-gene (tib molbiol, roche diagnostics, germany) and the assay n2 (n-gene) published by the cdc were chosen to initially screen samples for presence of sars-cov2 genomes. as aforementioned assays were proven to be the most robust, rdrp-gene assay was used for confirmation of results. 11 all rt-qpcr assays were performed using taqman universal pcr mastermix (thermo fisher scientific, waltham, ma, usa) in a 20 μl reaction mix consisting of 2× pcr buffer including enzyme mix, primers, and probes concentrations as recommended by manufacturers and 21.5 μl and 5 μl of cdna. thermal cycling was carried out as recommended by manufacturers on either abi quantstudio 12k flex or biorad cfx96 thermal cyclers. in brief, real-time rt-pcr was performed with 45 cycles using 1.5 μl of cdna. however, for validation of our pcr results additional pcr runs were performed with 5 μl of cdna not altering the results obtained by the 1.5 μl approach. synthetic in vitro rna of e-gene and rdrp-gene assays were diluted 1:10 prior to cdna synthesis and plasmid positive control of n-gene assay was diluted 1:104 prior to rt-qpcr to account for an expected low yield in total rna extracted from emb samples. histology was developed from formalin-fixed tissue by haematoxylin & eosin (he); azan, and periodic acid-schiff (pas) staining in light microscopy. for immunohistological evaluation, specimens were rnalater fixed, embedded in tissue tec (slee, mainz, germany) and immediately snap-frozen in methyl butane which had been cooled in liquid nitrogen and then stored at à80°c until processing. embedded specimens were cut into cryosections placed on 10% poly-l-lysine-precoated slides. myocardial inflammation was diagnosed by cd3 + tlymphocytes/mm 2 (dako, glostrup, denmark), cd11a + /lfa-1 + lymphocytes/mm 2 (immuno tools, friesoythe, germany), cd11b + /mac-1 + macrophages/mm 2 (immuno-tools, friesoythe, germany), cd45r0 + t memory cells (dako, glostrup, denmark), perforin + cytotoxic cells/mm 2 (bd bioscience, san jose, california). in addition, we stained intercellular adhesion molecules and mhc class ii cell surface receptor (cd54/icam-1 and hladr, immunotools, friesoythe, germany). staining were quantified by digital image analysis. 12 approval was not required. endomyocardial biopsy results of total patient cohort are summarized in table 1 . out of the 104 emb samples, five patients were positive for sars-cov-2 e-gene specific sequences indicating the first description of sars-cov-2 presents in a case series. besides latent infection with parvovirus b19, no other viral pathogens were detectable in sars-cov-2 positive samples. based on the clinical history, the clinicians expressed a suspicion of a previous covid-19 infection, but they were not tested with throat swab sample during admission to the hospital. the clinical courses of the five patients were different and showed highly acute to mild forms. patient 1 was a 48-year-old male with newly diagnosed heart failure and significantly reduced systolic function (ef 22%). suspected diagnosis was acute myocarditis. he described sudden onset of high-grade fever and dyspnoea within a few days. in addition, he suffered from thrombi and embolia. he reported a prior vacation in tyrol, austria. this patient showed a highly acute status was admitted to the intensive care unit (icu) and due to severe infection. the diagnosis of a small-vessel vasculitis was established, and cyclophosphamide and additional steroids were initiated. the patient recovered adequately. after receiving emb results, immunosuppressive treatment was stopped immediately. patient 2 was a 62-year-old male with mildly reduced ef (40%) and moderate lv-hypertrophy, and without respiratory infect. this patient had a new cardiac impairment of lv function since january 2020. the cause was unknown, so a possible myocarditis was assumed. with the exception of cardiac symptoms, this patient had a mild course and did not need to be monitored by icu. patient 3 was a 60-year-old female with heart failure symptoms but preserved ef (60%) with pronounced lvhypertrophy. initially, she was admitted to the icu with severe acute respiratory syndrome. blood tests revealed elevated levels of markers of myocyte injury (see table 2 ), which remained positive during the first days of her hospitalization. after respiratory improvement the emb was carried out 4 weeks after onset of syndromes. in this interesting case, the cardiac symptoms occurred with a pronounced relapse after the initial event. patient 4 was a 36-year-old male with a significantly reduced systolic function (ef 25%) with a history of mild respiratory infect 3 weeks ago. the clinical course developed without complications and icu surveillance. during hospitalization, the levels of troponin decreased laboratory values on day 15 were in reference range, and he recovered during this time. patient 5 was a 39-year-old male with heart failure symptoms but preserved ef with suspected diagnosis of acute myocarditis. the patient had a history of upper airway infection with headache and fever up to 4 weeks before admission. he suffered from shortness of breath, t-wave inversions in the anterolateral leads in ecg, elevated cardiac troponin i, and cardiac magnetic resonance imaging compatible with myocarditis. the course of this patient was acute and required icu treatment. in patients 2-5, treatment strategies were not modified after receiving the result of sars-cov-2 rt-qpcr in emb. they were treated symptomatically, in part with initiation of guideline-directed medication for heart failure. patient characteristics and emb results are summarized in table 2 . sars-cov-2 loads determined in the embs were low (ct values: 36.66 ± 1.99) corresponding to less than 100 to 500 viral copies/reaction. viral loads were determined from the internal sars-cov-2 positive control with a ct value of 32.73 ± 1.12 corresponding to approximately 10e + 4 copies/reaction while the ct values of sars-cov-2 negative samples were below 40 cycles and thus below detection limit. results from rt-qpcr are shown in table 3 . histological assessment of embs revealed an active myocarditis according to the dallas criteria in patient 1 13,14 ( figure 1a) . histological analysis could also show necrosis of myocytes and interstitial tissue and granulation tissue in the periphery of necrosis of the type observed after an infarction ( figure 1a) . immunohistochemical emb analysis confirmed pronounced intramyocardial inflammation. analysis of immune cell infiltrates of sars-cov-2 genome positive embs showed elevated number of t-cells, macrophages, lymphocytes, and t-memory cells (cd45r0) in four of the five patients ( figure 1a-c, e, f) . moreover, all sars-cov-2 patients exhibited an elevated number of cell adhesion molecules (cd54/icam-1). patient 2 showed inflammatory response on limit values. we could show that the inflammatory process in cardiac tissue seemed to permeate vascular wall. the inflammatory process was leading to arterial obliteration and damage (figure 1b-d) . the final mechanism of tissue damage in consequence of vascular obliteration appears to be similar to systemic forms of vasculitis leading to ischaemia. the neighbouring myocardium displayed vacuoles in myocytes as a sign of restricted metabolism. perivascular fibrosis with variation of fibre densities could be seen in cases 2 to 5 (not shown in figures). this phenomenon indicated relicts of previous damage. in this study, we established for the first time the evidence of sars-cov-2 genome detection in 5 of 104 embs of patients with suspected myocarditis or unexplained heart failure. after the first cases describing pneumonia of unknown origin in wuhan, china, sars-cov-2 rapidly spread worldwide with critical challenges for the public health and medical communities. cardiovascular involvement in covid-19 seems to be a notable complication. first single case reports could show viral particles in interstitial cytopathic macrophages and their surroundings in emb of a severe covid-19 shock patient by electron microscopic analyses. whether direct myocardial injury due to viral involvement or the effect of systemic inflammation appear to be the most common mechanisms responsible for cardiogenic shock situation needs to be further investigated. 19 in the analytic stage, real-time rt-pcr assays remain the molecular test of choice for the aetiologic diagnosis of sars-cov-2 infection. specificity of e-gene and rdrp-gene assays tested with clinical respiratory samples and sars-cov and mers-cov did not result in cross-reactivity and false positive results. high sensitivity of both assays as indicated by low pcr limit of detection for purified rna could also be confirmed for rna spiked into and extracted from swab samples. 15 however, direct sars-cov-2 rna detection in the myocardium has not been demonstrated so far. herein, we demonstrated by rt-qpcr that sars-cov-2 genomes is present in different cases. in this study, we described series of different histories of cardiovascular patients admitted to the hospital. one main clinical finding is that cardiac involvement with positive sars-cov-2 genomes in embs can either occur acutely or with latency after onset of symptoms of infection. based on the results of currently published research, it seems important to discuss the manifestations and characteristics of myocardial damage induced by covid-19. 16 herewith, we validated the direct cardiac involvement associated with intramyocardial inflammation in patients with sars-cov-2 genome positivity in embs. in patient 1, we could show an active myocarditis and in patient 5 a borderline-myocarditis according the dallas criteria. in the remaining patients, an inflammatory cardiomyopathy was determined. recent literature data have shown that cardiac troponin i concentration is increased in all patients with sars-cov-2 infection, and values exceeding the 99th percentile in the upper reference limit can be observed in 8-12% of positive cases. 17 moreover, patients with covid-19 are known to be at higher risk of acute pulmonary embolism, and elevated d-dimer levels on admission are predictive of adverse outcomes for patients with covid-19. 5 the first vascular sign has been referred to as 'vascular thickening' or 'vascular congestion' in the lung. bai et al. 18 reported vascular thickening to be significantly associated with covid-19 compared with non-covid-19 pneumonia (59% vs. 22%, p < 0.001). the physiopathologic mechanisms behind these changes remain unclear, but their role in diagnosis and possible future treatment strategies is substantial. in this regard, a very recent report showed that pericytes demonstrating high ace-2 expression might act as target cells for sars-cov-2, while pericyte injury can result in endothelial cell dysfunction. 22 recent reports showed that besides pericyctes ace-2 is expressed to different levels also in cardiomyocytes, endothelial cells, fibroblasts, and leucocytes. 23 however, ace-2 expression does not argue for permissive infection of a respective target cell by sars-cov-2. on the other hand, recent reports have demonstrated that sars-cov-2 genomes could be detected besides airway epithelium cells also in the intestinal enterocytes, spleen, liver, kidney, and heart. 2, 24 in addition, recent histologically post-mortem analyses in covid-10 positive patients revealed lymphocytic endotheliitis in different organs with evidence of direct viral infection, indicating endothelial dysfunction as a possible principle determination of microvascular dysfunction by shifting the vascular equilibrium towards more vasoconstriction with subsequent organ ischaemia and inflammation. 21 although nearly all organs seemed to be affected by covid-19, we currently do not know in-depth details about the organ-specific infection by sars-cov-2. in this regard, tavazzi and coworkers have shown recently in their case description using electron microscopy on embs of a patient with covid-19 in cardiogenic shock that sars-cov-2 particles could be localized to interstitial macrophages and their surroundings but not in cardiomyocytes. 19 as to whether this observation is due to a transient viraemia or infected macrophage migration from the lung has to be evaluated. our finding of sars-cov-2 genome detection in embs of patients suffering from myocarditis/inflammatory cardiomyopathy cannot rule out or confirm the infection of cardiac cells but revealed incremental insights into organ-specific infection of sars-cov-2 using possibly macrophage migration as a shuttle from the lung to the heart. in this study, we investigated histopathological changes in myocardial tissue in the series of sars-cov-2 positive embs. in line with the recently published study, we could show that the inflammatory process in embs seemed to permeate vascular wall leading to small arterial obliteration. the final mechanism of tissue damage in consequence of vascular obliteration appears to be similar to systemic forms of vasculitis. we therefore hypothesize that in these patients, myocardial injury ischaemia may play a role, which could explain the ubiquitous troponin increases. as a result, this ischaemia could trigger possible cardiac arrhythmias. a limitation of this study is that we did not had enough material for sars-cov-2 genome in depth analysis to certainly exclude cross reaction with other corona virus strains due to the limited material available by the embs. however, the high sensitivity and specificity of the used pcr systems to detect solely sars-cov-2 genomes have been demonstrated recently. 15 another limitation is that we cannot completely exclude that the detection of sars-cov-2 genomes in the heart might result from contamination of circulating blood. unfortunately, we have no blood samples to the corresponding embs on hand to analyse this aspect. however, sars-cov-2 load in blood seemed to be low in comparison with other clinical types of specimens. 2 nevertheless, sars cov-2 can potentially bind to its cellular ace2 receptor in heart tissue cells and can therefore be detected in the heart muscle. in this regard, a recent report showed that pericytes in the heart demonstrating high ace-2 expression might act as target cells for sars-cov-2 while pericyte injury can result in endothelial cell dysfunction. 20, 22 if sars cov-2 can replicate in these target cells of the heart, this has to be investigated in subsequent analysis. the low detection rate and low viral loads of sars-cov-2 genomes may be due to the limited number, size, and quantity of embs. heart tissue cells (e.g. pericytes) are not the main target cells of sars-cov-2 while specimens of the main target the lung of infected patients are easier and in larger quantity to obtain than embs and may contribute to the low detection rate in embs. however, we showed that sars-cov-2 is detectable in the heart muscle but can only speculate about the clinical relevance of sars-cov-2 infection of the heart. as to whether sars-cov-2 infection may induce myocarditis is questionable, however, may trigger an ongoing progress to myocarditis of other reason. in conclusion, in this study, we could show for the first-time evidence of sars-cov-2 genome detection in 5 of 104 patients with suspected myocarditis or unexplained heart failure with different history of symptoms and time duration. in addition to inflammation and consequential damage, one possible histopathological mechanism may be vascular involvement with arterial obliteration which can lead to ischaemia. a possible sars-cov-2 infection should therefore be considered in patients with acute unexplained heart failure or new cardiac arrhythmias. we believe that recognition by the scientific community of myocarditis as a possible complication associated with covid-19 may be helpful for strict monitoring of affected patients. emb-based identification of the cause of myocardial injury may contribute to explain the different evolution of complicated sars-cov-2-infection and to design future specific treatment strategies. an antiviral therapy is not yet available. based on our histopathological results, possible anticoagulant/antiaggregation therapy should be investigated. clinical features of patients infected with 2019 novel coronavirus in detection of sars-cov-2 in different types of clinical specimens preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan covid-19 and the cardiovascular system cardiac involvement in a patient with coronavirus disease 2019 (covid-19) cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) therapeutic implications of a combined diagnostic workup including endomyocardial biopsy in an all-comer population of patients with heart failure: a retrospective analysis viral persistence in the myocardium is associated with progressive cardiac dysfunction large, stable, contemporary interspecies recombination events in circulating human herpes simplex viruses dilated cardiomyopathy myocarditis: the dallas criteria european society of cardiology working group on myocardial and pericardial diseases. current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr potential effects of coronaviruses on the cardiovascular system: a review cardiac troponin i in patients with coronavirus disease 2019 (covid-19): evidence from a meta-analysis performance of radiologists in differentiating covid-19 from viral pneumonia on chest ct sepe sars-cov-2 genomes in endomyocardial biopsies 7 myocardial localization of coronavirus in covid-19 cardiogenic shock. myocardial localization of coronavirus in covid-19 cardiogenic shock the ace2 expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov-2 endothelial cell infection and endotheliitis in covid-19 what is a pericyte? cell type-specific expression of the putative sars-cov-2 receptor ace2 in human hearts sars-cov-2 productively infects human gut enterocytes all nurses, clinicians, and infectious diseases specialists working hard in this difficult period are greatly acknowledged for their efforts and daily care for patients suffering from sars-cov-2 infection. this work was done within a profit grant of the investitionsbank berlin (profit no. 10169028, berlin, germany). for their excellent technical assistance, we thank k. winter, c. seifert, s. ochmann, c. liebig, and k. errami (ikdt berlin, germany). none declared. key: cord-252244-y5w9hjy8 authors: loeffler-wirth, h.; schmidt, m.; binder, h. title: covid-19 trajectories: monitoring pandemic in the worldwide context date: 2020-06-05 journal: nan doi: 10.1101/2020.06.04.20120725 sha: doc_id: 252244 cord_uid: y5w9hjy8 background: covid-19 pandemic is developing worldwide with common dynamics but also with partly marked differences between regions and countries. they are not completely understood, but presumably, provide one clue to find ways to mitigate epidemics until exit strategies to its eradication become available. method: we provide a monitoring tool available at www.izbi.de. it enables inspection of the dynamic state of the epidemic in 187 countries using trajectories. they visualize transmission and removal rates of the epidemic and this way bridge epi-curve tracking with modelling approaches. results: examples were provided which characterize state of epidemic in different regions of the world in terms of fast and slow growing and decaying regimes and estimate associated rate factors. basic spread of the disease associates with transmission between two individuals every two-three days on the average. non-pharmaceutical interventions decrease this value to up to ten days where complete lock down measures are required to stop the epidemic. comparison of trajectories revealed marked differences between the countries regarding efficiency of measures taken against the epidemic. trajectories also reveal marked country-specific dynamics of recovery and death rates. conclusions: the results presented refer to the pandemic state in may 2020 and can serve as working instruction for timely monitoring using the interactive monitoring tool as a sort of seismometer for the evaluation of the state of epidemic, e.g., the possible effect of measures taken in both, lock-down and lock-up directions. comparison of trajectories between countries and regions will support developing hypotheses and models to better understand regional differences of dynamics of covid-19. coronavirus disease arrived in 187 countries with 5.5 mio infections and more than 300,000 deaths worldwide so far (25 th may 2020). the disease affects almost all spheres of life, especially public health, economics and well-being. present situation and near future lasting from months to one-two years (in worst-case more, in best-case less) will require coexistence with the virus until effective pharmaceutical countermeasures (medication, vaccine) are available and applicable [1] . this coexistence requires adjustment of a balance between a controllable low level of infections and maximum-possible levels of public life and economics. controlling the infection requires feedback loops sensitive to early and robust indications of secondary outbreak waves. this includes permanent surveillance of epidemiologic and medical indicators by testing programs, monitoring of case numbers and symptoms and forecasting methods on one hand, and suited 'no-pharmacological intervention' (npis) strategies on the other hand, to held the case numbers low (ideally further decreasing) to prevent secondary outbreaks. various 'number-tracker' tools are active (e.g., [2] [3] [4] [5] ). they mostly plot case numbers (infected, recovered, death) over time, usually on a country-by-country (or region-by-region) basis. as an illustration, we show the number of current infections and of covid-19 related deaths as a function of time in selected countries ( figure 1 ). these 'epi-curves' reveal how the epidemic was expanding in time and space from china (end of 2019 and january 2020) via other asian countries (south korea, iran) and western europe towards eastern europe, amerika, and other parts of the world (february to april 2020). they also reveal different phases of epidemic, namely, an initial 'take-off stage', an 'exponential growing stage' followed by 'slowed growth', 'turning into a decline' and 'decline' [5] . charting the outbreak day by day in each country and comparing them, e.g. by setting an arbitrary starting threshold of, for example, 100 infections, illustrates the succession of events as a global story [2] . for a straightforward evaluation simple measures are typically used such as doubling time of cases, reproduction numbers (mean number of people infected by a typical case) or the number of new infections per 100,000 citizens in a certain region, which all provide a limited snapshot-view with pro's and con's in different states of epidemic. as a next, more elaborated level, standard epidemic models provide a theoretically well-founded description of dynamics of disease incidence in terms of rate constants for transmission and recovery of covid-19 and detailed infection-transmission 'serial interval' functions. different models, mostly assuming a series of diseases states such as the 'susceptible-infected-removed' (sir) types (see below) have been used to describe 'epi-curves' of selected countries and regions under consideration of i) spatial heterogeneous outbreak and transmission scenarios, and ii) the effect of npis [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] . in case of the latter, models have been applied not only in retro-perspective but also to forecast epidemic in dependence on measures taken. because of still limited knowledge about disease mechanisms and detailed data about its spread in the population forecasting either provides short-term extrapolations or hypothetical predictions of possible future scenarios as the result of different model assumptions. we here provide the covid-19 viewer, a monitoring tool which aims at bridging the temporal 'epicurve' and the modelling levels. our monitoring substitute the time-coordinate used in the epi-curves by infected cases (cumulative or current ones). the obtained trajectories then enable to visually estimate the dynamic state of epidemic in terms of simple shape characteristics such as slope, parallel shifts or turning points with direct relations to transmission and removal rates of the disease. comparative analysis between trajectories of different countries enables to judge different scenarios of npis, population size, and social factors. daily actualized data and interactive web-functionalities enable monitoring pandemic based on newest data. our trajectory-approach is complemented by a series of simple model calculation which visualize the obtained trajectories for comparison with real ones. the paper is organized as follows: in the results section we introduce and illustrate the different trajectories and plots available in the monitoring tool by showing examples from different countries of the world, which are thought to serve as worked examples referring to the actual state of pandemic in the second half of may 2020. the majority of plots shown in the publication were directly taken from the web-tool. the interested reader thus can actualize the data and/or chose countries of interest for similar views. we address the effect of nips in europe, the spread of epidemic in germany and compare mortalities between selected countries. the materials and methods section shortly explains the major functionalities. details of the methods, model simulations and fits as well as supplementary figures were provided in the supplement (appendix). figure 1 : covid-19 cases (left plot: currently infected, right plot: died individuals) in different countries as a function of date. the '100-cases per country' threshold is crossed between end of february and end of march for the countries shown (except china). the time courses reflect growing (e.g., us, rus, nl), slightly decaying (e.g., i, d, tk), strongly decaying (e.g., ch, rok) regimes of epidemic or indications of bi-or multiphasic growth (e.g. am, ir). the courses of the dead toll as a function of time reflect country-specific percentages of covid-19 victims. the plots were generated in the corona-viewer on a daily actualization-basis as described in the text. the trajectory-monitoring tool ('covid-19 viewer') was programmed as web application using the rpackage 'shiny' [22] . it processes the number of newly infected and of removed (sum of recovered and died) individuals from 187 countries (and of diamond princess cruise liner with 712 cases) as provided by the corona virus resource center of johns hopkins medical university ('world data': https://systems.jhu.edu/research/public-health/ncov/) and from robert-koch-institut ('germancountry' data: https://www.rki.de/de/content/infaz/n/neuartiges_coronavirus/fallzahlen.html). data are daily updated. the tool is available via the websites of izbi (www.izbi.de) and the leipzig health atlas (https://www.health-atlas.de/models/28). the 'covid-19 viewer' is an interactive tool to monitor the development of the pandemic in 187 countries and in the 16 german states using simple and intuitive plots ( figure 2 , appendix i). the tool is interactive and enables the user to select different presentations of data. the so-called 'rise-fall' trajectory was chosen as 'standard visualization'. it shows the newly confirmed covid-19 cases per country and per day (averaged over the past 7-days) as a function of accumulated total cases per country in double-logarithmic scale. the 'rise-fall' trajectory typically divides into a 'rising' exponential growth part reflecting growth of epidemic and a 'falling' decay regime due to counter measures and/or progressive immunization in the population. it allows estimating transmission and removal rates and reproduction numbers (appendix i). the time range can be chosen and, as an illustration, pressing the 'start animation' button generates a movie of the dynamics of epidemic in the selected countries in terms of progressing rise-fall trajectories. the user can chose 'custom' trajectories to combine different numbers (infected or removed cases, deaths, daily or cumulative counts, figure 1 ) along the coordinate axes for alternative views (use the hoover window for curve assignment and details such as date, numbers). trajectories can be generated for all countries, groups of countries (use left-handed table for selection) or single countries. german states can be selected by choosing 'germany-state codes'. in addition to the standard plot, conventional time series plots show the different numbers (infected, removed, recovered, died as cumulative or per-day) as a function of date. the viewer offers standard browsing functionalities (zooming in and out, image download). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint figure 2 : covid-19 viewer: screenshot with major functionalities indicated (above) and example plots (below). the sir (susceptible-infected -removed) model provides a simple, adequate and straightforward interpretation of the data (see figure 3 for illustration and appendix ii). it describes the disease as a sequence of three states, s (susceptible), i (infected) and r (removed), where infection proceeds via interactions between s and i individuals. recovered individuals are assumed to get immunized. the respective numbers were reported by census systems, which can differ between countries, e.g. by counting only hospitalized individuals, counting died covid-19 positive cases as not covid-19 caused and/or referring to different test-frequencies. all case numbers must therefore be understood as 'visible', i.e. reported ones. the rise-fall trajectories enables classification of the type of the growth and identification of the epidemic threshold (no growth). custom trajectories allow to estimate time-dependent sir model parameters such as the effective transmission and removal rate factors, c e (t) and k(t), respectively. time courses of the rate factors were extracted from the local slopes of the trajectories (appendix i and ii). the ratio of the rate factors estimates the effective reproduction number r e (t) defined as the number of individuals who get contaminated by one infected person on the average. the timedependent rate factors depend, in addition to the intrinsic properties of covid-19 on a series of external factors such as public health measures (non-pharmaceutic interventions, npis) to slow down transmission of epidemics (affecting c e ) and effective medical services after infection (affecting k). in addition to the estimation of sir parameters as described above, we performed least-squared fits of the trajectories where the daily numbers of newly infected and removed cases were calculated as a function of the cumulative number as predicted by the sir model (appendix ii). the fits provide estimates of n max , the maximum cumulative number of infected cases, and of the rate constants. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint monitoring the state of epidemic using 'rise-fall' trajectories the 'rise-fall' trajectory plots the newly confirmed covid-19 cases (averaged over a running 7 day windows) as a function of accumulated total cases per country in double-logarithmic scale. the 'select all' function shows the trajectories of all countries considered (figure 4a ). overall, these doublelogarithmic trajectories reveal two basic features: an initially linear increase with a slope of unity indicates exponential growth of epidemic. this linear regime is followed for many countries by a downwards turn which indicates slowing down of growth owing to npis 'locking down' infections and/or possibly also to progressing immunization of the population in later phases of epidemic leading to the depletion of the reservoir of susceptible individuals and/or other factors. the 'rise-fall' plots use the cumulative number of cases n as a robust measure of progressing epidemic in a population. naturally, it is larger for countries with larger population sizes providing a larger overall reservoir for covid-19 infections compared with smaller countries. shape of the 'rise-fall' trajectories are however virtually independent of country size. the trajectories thus reflect intrinsic properties of epidemic in terms of its transmission and removal potential. the two sets of trajectories shown in the left and right part of figure 4a refer to situation at april 17th 2020 and about six weeks later, respectively. for most countries, among them france, italy, spain and germany, the trajectories turn into falling courses during this time and/or the falling parts further drop and intersects the '0.01 -slope' line referring to a more than tenfold reduction of the transmission rate of epidemic (see below). these trends thus indicate decay of pandemic after the npis taken in most of countries. on the other hand, brazil and russia emerged to the countries with most cumulative cases after usa, with still growing case numbers. most western european countries of larger and medium size reached the decaying part in the first week of april 2020 (except sweden and great britain) roughly two-three weeks after npis were taken in these countries. countries from different parts of the world such as austria, iceland, south korea, australia, new zealand and china reached low levels of new infections as indicated by strong vertical decays. larger countries (e.g. russia, india, brazil, pakistan) were in the rising part. some countries show a two-phasic growth as indicated by the parallel right shift of linear regions in their growing part (e.g. sweden, denmark, iran, ukraine, armenia) indicating that fast exponential growths are followed by slower phases due to reduced transmission rates (see below). singapore and japan show relatively slow growing phases with reduced rates and late turns into falling regimes while south korea's turn is very sharp presumably because of the 'crash down' measures taken there. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. hence, the 'rise-fall' trajectories illustrate the current state of the epidemic and its developmental course with country-wise resolution. they enable monitoring the state in terms of differences and similarities between the countries and geographic regions revealing specifics and commons of epidemic spread: (i) a unique linear slope of most of the trajectories in the intermediate abscissa range is indicative for exponential growth in early phases of the outbreak of the pandemic (low level of immunity in the population). the nearly identical position of these lines refers to covid-19 typical pandemic spread rate and maximum basic reproduction numbers r 0 (appendix ii). (ii) parallel, downshifted lines suggest still exponential growth, however with reduced rates reflecting reduced effective reproduction numbers 1 < r e < r 0 . in these countries (e.g., sweden, iran), the epidemic is not stopped. (iii) the 'flattening' of slope and downwards curvature seen, e.g. for most european countries such as italy, spain or germany reflects slowing down growth owing to efficiency of npis and, possibly to a minor degree of progressive and significant immunization in the population. (iv) the sharp, virtually vertical drop of trajectories reflects the stop of epidemic observed, e.g. for china and south korea, and after may 1st for new zealand, australia, and also part of european countries. (v) the different qualitative features of the trajectories are virtually independent of (population) size of the countries. 'smaller' countries like island, cyprus, armenia or georgia show overall similar features such as linear rise, parallel shifts (armenia), a maximum and steep falling parts (e.g., island). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint the 'rise-fall' trajectory uses cumulative cases n along the abscissa as a robust measure of the extent of the epidemic. this number doesn't consider the degree of recovery and thus it doesn't reflect the current amount of infected cases (i). custom trajectories make use of the independent number of removed cases (r) reported and plot cumulative, current and differential (per day) numbers in different combinations (appendix ii). using the current number of infected cases (i= n -r) as x-axis one sees whether the extent of infection increases or it decays. while the rise-fall trajectory, ï��n-vs-n, tends asymptotically towards a maximum cumulative number of infections (n max ) for each country, which reached the falling regime, the ï��n-vs-i trajectory turns from a growing i into a decaying branch at i max , the maximum number of infected individuals. these trajectories turn in clock-wise directions for most countries meaning that the rate factor of transmission of epidemic, c e (t), strongly decays (appendix i and ii). for example, austria and japan show full turns while the turns of sweden and usa remain incomplete leading to less pronounced decays of the respective c e (t)-courses (figure 5a, b) . in contrast, the ï��r-vs-i trajectories turn typically in counter-clockwise direction referring to an increase of the removal rate factor as explicitly seen in the respective k(t) plots. the ratio of the effective transmission and of the removal rates then estimates the effective reproduction number as a function of time, r e (t) (figure 5b ). the trajectories of the countries selected for illustration reflect different types of trends such as strong and straight repression and stop of epidemic via reduction of transmission in austria, reduced growth but still expanding epidemic in usa and sweden or indications of a second wave of expanding epidemic in iran. here, the respective trajectories and plots of rate factors and of r e (t) show different aspects of the dynamic of the epidemic. for example, s and j are characterized by relatively low levels of rate factors compared with a and ir, a difference seen also in the parallel shifts of the respective trajectories. in appendix ii we find analogous differences between western and south european countries (e, f, i, figure s 3) compared with middle european ones (d, a, ch), which suggest differences in the spread mechanism of covid-19 and, possibly, also in the recovery dynamics. the removal rate obtained depends on the time-delay between infection and recovery, which is neglected our simple trajectory-approach (see also epicurves in figure 5a ). 'cumulative balance' and 'current case' custom trajectories complete the visualization options: lower levels of transmission and removal rates associate with such trajectories running closer to the diagonal. overall, the trajectories enable tracing an epidemic in terms of case numbers reported directly be the census agencies of the respective countries. derived numbers such as the rate factors and reproduction numbers 'translate' these numbers into features more directly describing the dynamics of the epidemic. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint effective reproduction numbers as shown in figure 5b provide suited summary measures of the case numbers with a well-defined epidemiological meaning. their value defines the transmission potential in the population in terms of the mean number of individuals who are infected by one infectious person on the average. for the comparison of all or a selection of the countries available, the monitoring tool generates a ranked boxplot of their actual reproduction numbers. presently, the epidemic is not stopped in roughly 50% of all countries because their r e is still larger than unity ( figure 6 ). the tool also generates the respective plot for r e -values obtained two and four weeks before. at the latter date about 70% of countries show r e > 1, which demonstrates the presently decaying trend. time courses of a selection of countries illustrate different types of decays which eventually relate to the type of npis taken. for example, early, consequent eradication of epidemic in island and croatia result in fast and steep decays. slower but monotonous decays were observed in russia, spain and portugal. also wave-like changes before the final decay (japan, singapore) or even worsening of situation (armenia, sweden until middle of may) were found. presently (may, 25 th ) sweden shows the highest reproduction numbers among all countries studied. note, that r e is a relative measure considering daily changes and current numbers of infections and recoveries (appendix i), meaning that restricted outbreak clusters affecting only relatively small numbers of individuals suggest spread of epidemic in a larger, not affected population. hence, a combination of charcteristic numbers should be used to characterize dynamic of epidemic, namely transmission rate factor (or doubling time of cases) more at the beginning, effective reproduction numbers more in the phase of vast spread of epidemic and absolute numbers of new cases in the phase of mitigation and near eradication. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint next, we asked how the npis taken in middle and western europe and scandinavia in the first three weeks of march 2020 affected the dynamics of the epidemic. the rise-fall trajectories of countries selected from [9] reveal that they now are mostly in the falling regime however with modifications such as parallel downwards shifts, wave-like decays and even lacking decays as already discussed above (figure 7fehler! verweisquelle konnte nicht gefunden werden.a) . in figure 7fehler ! verweisquelle konnte nicht gefunden werden.b we re-plot the trajectories separately for each country together with marks indicating which measure was taken when along the trajectories. in most cases, trajectories start turning downwards about two weeks after a complete lockdown in the respective country. before this, one often finds slowing down of the exponential growth as indicated by small differences compared with the trajectory of us referring to exponential growth. in norway, denmark, and also sweden one observes a relatively strong first slowing down as indicated by the parallel downwards shift of the trajectories which roughly refers to a reduction of the transmission rate constant by about 30 % (figure 7d ). sweden, without complete lockdown measures, but also great britain show weakest decay of the trajectories and largest values of the effective reproduction numbers r e > 1 in contrast to all other countries except belgium (figure 7c ). comparison of the reproduction numbers two and four weeks earlier indicates consistent high values in britain and sweden and also a delayed decay in italy, spain and france, the european countries, which were heavily hit by covid-19 in february and march. the time courses of the reproduction numbers r e (t) respond nearly immediately on the measures in many cases showing, at least, small drops in support of a recent study [9] which assumes that the reproductive number -a measure of transmissionimmediately responds to interventions being implemented ( figure 7d , the first measure and complete lockdown were indicated). consistent decays to values r e < 1 after about two weeks were seen in scandinavia (except sweden) and austria, switzerland and germany while in belgium, france, italy and spain the decays last roughly four weeks until they fall below the epidemic threshold. in sweden and great britain, virtually unchanged levels of r e above the et were observed. a recent model analysis of the effect of npis in germany applies a sir model with changed rate constants at so-called 'change points' which are assumed to take place when measures were applied [23] . we found a decay of the transmission rate during the time when measures were applied which drops overall by 60 -70% in rough agreement with [23] (figure 7d , right part). interestingly, japan showed a similar resonse to npis as the european countries, namely a slow, but instantaneous growth of epidemic turned into the falling regime at the beginningf may ( figure 5 ), two-three weeks after npi measures were intensified at april 10 th . overall, our simple analysis reveals that npis were followed by drops of the reproduction number mainly due to a decay of the transmission rate factor and by halt of epidemic after two to four weeks after complete lock down. sweden (and partly gb) shows also a drop of r e and the transmission rate which however overall are insufficient until end of may to stop epidemic. both, the time-course of reproduction number and the rise-fall trajectory are sensitive to detect the slowing down and the halt of epidemic. the available country-wise numbers used do not allow to analyse the observed effect assuming heterogeneous effects of npi on different subpopulations which, in principle, could explain steps and wavelike changes in the courses of the trajectories as an alternative to alterations of the rate factors in a homogeneous population assumed here. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint figure 7 : the effect of non-pharmaceutical interventions across ten european countries selected in analogy with [9] . the trajectory of us is shown for comparison. a) rise-fall trajectories of selected countries mostly decay thus indicating marked decrease of epidemic in most cases. b.) country-by-country plots of the rise-fall trajectories together with marks assigning the nips (dates and assignments were taken from [9] ) show that the trajectories turn downwards about two weeks after lockdown in most cases (the grey box refers to the 'two weeks after the last measure' date). exceptions are sweden (no complete lockdown) and united kingdom. the two green boxes indicate the data obtained at march 10 th (mostly before measures) and may 1th. c) the effective reproduction numbers are still clearly above the critical value of r e =1 for sweden and great britain. italy and france show the strongest decay of re over the last 4 weeks. d) courses of the effective reproduction number as a function of time indicate a marked drop of re(t) immediately after the lock down in most countries. also the first measure taken is indicated. for germany all 5 measures were indicated together with the courses of the rate factors. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint countries across the world differ in many factors related to covid-19 epidemic such as the particular npi measures, social behaviour, family structure and education systems with differing school rules, population densities, urban structure, transport system and also age distribution. the heterogeneity of these factors is assumed to be smaller inside each of the countries than between them. for germany, the covid-19 viewer provides 'rise-fall' trajectories for all sixteen german states, which cover population sizes between about 0.7 mio (bremen) up to 18 mio inhabitants (nordrhein-westfalen). they include three city-states (hamburg, bremen, berlin), while the other states are 'area'-states include countryside regions and towns of different sizes. the trajectories overall express very similar courses of the epidemic across germany (figure 8a) , which suggest relatively similar dynamics of the epidemic in different parts of the country and, particularly, that germany-wide npis 'locked-down' epidemics in the different states in a similar way. analysis of the maximum cumulative number of infected individuals, n max , using fits of the 'rise-fall' trajectories however reveals considerable differences especially between the south and west of germany and its east and north (figure 8b ). in bavaria, which is located in the south of germany, roughly eight-times more people are infected on relative scale than in mecklenburg-vorpommern located in the north-east. in general, 'area' states from the west and south of germany were more affected by epidemic than states in the east and north. this difference associates with an earlier outbreak in the former states with higher amounts of infected individuals (figure 8c ). npis were taken germany-wide at the same time between 9 th and 23 th of march, which suggests that delayed measures with respect to the outbreak will increase the burden of infections. in summary, germany-wide the trajectories reflect similar dynamics of epidemic where however earlier outbreaks especially in the west and south and in larger cities gives rise to increased numbers of infected persons, possibly because of the delay of npi. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint figure 8 : covid-19 in germany: a) 'rise-fall' trajectories across german states: the trajectories of german states resemble that of whole germany indicating similar dynamics of covid-19 across germany. trajectory of us is shown for comparison. b) the relative maximum cumulative number of infected cases (per 1,000 residents of the respective states divides clearly into states from west and south germany and states in the east and north of germany. city states (be, hh, hb) are found in the former group. c) epi-curves (cumulative case numbers as a function of time) reveal that epidemic arrived earlier in western and southern states mostly by a few days compared with the eastern and northern ones. the curve of the city state bremen (hb) slightly differs from that of the other ones. mortality is an important endpoint of covid-19 epidemic related to a series of factors such as the intrinsic severity of the virus [24] in first instance, but also age, sex, genetic and immunological predisposition [25] , disease history and also co-morbidities of the patients [26] , as well as the effectivity of medical measures such as icu services [27] , the capacity of health care systems and also socio-economic factors. censing of deaths, e.g. by counting covid-19 positively tested deaths as covid-19 caused or not, is another factor affecting the reported numbers. so far we subsumed the numbers of death cases together with recovered individuals as removed ones. separate counting shows that, overall, the dead toll of covid-19 ranges from less than 1% up to more than 10% of counted infections, depending on country and time, when the data were registered (see below). the 'custom trajectory' page provides 'mortality trajectories' in terms of cumulative death cases versus cumulative infections . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint (alternatively one can choose daily cases). constant percentages refer to parallel diagonal lines as indicated ('iso-percentage' lines). for illustration we selected groups of countries in figure 9 for comparison with part of the 'rise-fall' trajectories in figure 4b . larger-size west-european countries (great britain, france, spain, italy) all show similar mortality trajectories referring to about 10% of the (visible) infected individuals. mortality of germany and austria is smaller (about 4%), possibly due to the smaller mean age of infected persons at the beginning of epidemic. the respective mortalitytrajectories however slowly grow in direction of the level of the other european countries with increasing number of infections. note that the slopes of the trajectories of the latter countries (e.g. france, italy, spain) is slightly steeper than that of the iso-percentage lines which indicates slowly growing mortalities across in these countries. presently, mortality in europe is largest in sweden, belgium, netherlands and great britain with further increasing trends. relative small mortality is found in russia and belarus possibly caused by governmental-control about covid-19 related death-census. mortalities in ukraine and estonia and in east asia (china, japan) are comparable with mortality in us, where the latter asian countries and also south korea show an increasing trend of mortality. in south america, one finds higher mortalities than in us with further increasing trends. overall, comparison of the mortality trajectories reveals systematic differences and trends, which need further analysis for interpretation. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint we here presented the 'covid-19 trajectory viewer', which generates a series of trajectories and plots based on public available covid19 data. it enables the comparison between epidemic development with country-wise resolution worldwide. trajectories are based on two types of counts, namely the number of infected and of removed (recovered and died) individuals. plots use either these counts directly, their cumulative values or increments per day and combine them in different ways, which allows to inspect the actual state of the epidemic from different perspectives. in addition, the monitoring tool enables calculation and visualization of derived parameters, namely the effective transmission and recovery rate factors and the effective reproduction number. they estimate the transmission and removal 'power' as basic characteristics showing whether epidemic growths or declines. changes of these parameters during epidemic development reflect different factors affecting the dynamic of epidemic, namely (i) the possible consequences npis, (ii) eventually growing immunity due to decaying numbers of susceptible individuals, and, (iii) also differences in the methods of counting and reporting data between different countries. our monitoring metric is sensitive for subtle alterations of the dynamics of the epidemic making it suitable to estimate the effectivity of npis and to serve as 'seismometer' for secondary outbreaks to early indicate such events ( figure 10 ). three possible future pandemic scenarios for covid-19 dynamics have been suggested based on previos influenca courses [1] , firstly, 'peaks and valleys' where the first big wave in spring 2020 is followed by repetetive smaller waves with geographic specifics depending on local npis; secondly, the 'fall peak' suggesting a large secondary peak in fall, winter 2020; and, third, a 'slow burn' of ongoing transmission and case occurrence, but without a clear wave pattern, again with geographic variations affected by the degree of mitigation measures in place in various areas. one or none of them, or even all three in parallel in different countries will be possible, where trajectories and rate factor curves will provide an instrument to distinguish the different scenarios. thereby, one has to keep in mind that these are data on visible, symptomatic covid19 cases. unsymptomatic cases remain usually undetected and can exceed the number of symptomatic ones considerably. a recent publication shows that more than 80% of all positively tested covid-19 cases on a cruise liner did not show any symptoms, raising questions about the true prevalence of "silent" infections [28] with possible consequences for the immunization dynamics in a population. moreover, our simple monitoring does not explicitly consider heterogeneities of the spread of the epidemic in a population (e.g. cities versus countryside, elderly versus younger, hospitalized versus non-hospitalized, symptomatic versus asymptomatic, highly exposed professions versus less exposed ones, etc.). such effects are hidden in the data and can be considered in terms of the trajectory approach by using more detailed data, e.g. by stratifying populations geographically, with respect to professions, age, symptoms etc. and/or by applying more elaborated models. our visualization in terms of trajectories and derived rate factors and their interpretation is based on the simple sir model dividing the visible population into three types of individuals. such three-state models have been widely and successfully used in many areas of sciences to describe different kinds of dynamics, ranging from elementary reaction kinetics in chemistry to photo-physics, molecular transformations in biology and many other fields. the basic assumption behind the sir model is the mass action law, claiming that changes of the population of a state directly relates to its population number. the different trajectories visualize this relationship by plotting changes of newly infected or . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint removed individuals as functions of the number of cumulative or currently infected individuals. the double logarithmic scaling of the axes accounts for the fact that the solution of the ordinary differential equations behind the sir model predicts exponential dynamics in important limiting regimes such as the early or late outbreak limits, which in turn, suggest linear courses of the trajectories. this way the obtained trajectories reflect a virtually common maximum transmission rate in the exponential growth phase in many countries suggesting that each infected individual infects another one every two-three days (figure 10a ). the initial growth is followed by down-steps and parallel shifted lines indicative for exponential growth with reduced transmission rate (e.g., transfer of infection between two individuals every five days). downturns of different sharpness indicate markedly reduced spread dynamics, and also halt of the epidemic in terms of falling courses if transmission frequency reaches a level of more than one per ten days. the close temporal relatedness between slowing down of the transmission dynamics and the dates when measures of the npi-type were taken suggests causal relations and shows that an associated 'falling' regime can be monitored using the trajectory approach. the npi result in dropping transmission rates and reproduction numbers where the steepness of decay in europe is larger for countries such as austria and germany, which were hit by the main infection wave a few weeks later than italy, france and spain showing slower decays. early nips on a relative time scale with respect to growth dynamics obviously facilitate faster slowing down afterwards. so-called 'complete lockdown' measures seems to be an essential measure for stopping epidemic despite considerable differences between countries, e.g. in handling go-out restrictions ('ausgangssperre', relatively moderate rules in germany versus strong ones in italy, spain and france). the swedish model seems to fail regarding transmission dynamics conceding further expanding epidemic and high death toll (figure 10a ). our trajectories show that lowering a of transmission rates by more than 50-70% compared with its maximum, intrinsic value, is required to stop epidemic and to turn it into the decaying regime. joint plotting of trajectories using the covid-19 viewer shows that at present majority of east asian (china, south korea, singapore) and european countries are in the falling regime, while most american countries are in the exponential growth phase. epidemic seems virtually eradicated in the island states new zealand, iceland but also other small countries such as croatia in a similar way as observed at 'diamond princess' cruise liner held under isolation (figure 10b ). it shows that isolation in combination with strong npis effectively stop epidemic. on the contrary, slowing down but still exponential growth are seen in other small and relatively isolated countries such as armenia (surrounded by mountains and closed borders to part of neighbouring countries) reflecting inefficiency of measures taken. wavelike up and down as seen for iran indicate repeated waves of growing epidemic (figure 10c ). new outbreak clusters become evident as spiked upturns in the falling regime as indicated presently for south korea (figure 10d ). the removal rate factor is a second, important characteristic of covid-19 dynamics, which additively composes of recovery and death rates, where the former number is dominating. removal rates can differ by a factor of two-to-ten between different countries (e.g. germany and austria versus spain and france, figure s 3) by unknown reasons. possible explanations are specifics of the recovery process due to healthcare measures applied and/or epidemiological factors such as age-and/or health-risk of the respective populations. also counting criteria of recovered individuals are another, possibly more relevant factor, which can differ between countries. often census agencies apply recovery counting algorithms (e.g. by assuming recovery two weeks after infection if no other information is available in germany) presumably biasing estimation of removal rate factors. moreover, also counting of deaths is census-dependent. on the other hand, the initially low but afterwards increasing mortality rates in austria and germany can be rationalized by the increasing age of infected individuals (disease was initially spread in communities of younger persons). thus, comparing trajectories supports detection . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20120725 doi: medrxiv preprint of differences of recovery and mortality rates between countries for subsequent analysis of the possible reasons. figure 10 : example trajectories indicating different dynamic regimes of covid-19: a) basic rise and falling regimes refer to transmission intervals of 3-4 and more than 10 days, respectively. they were observed in european countries under complete lock down such as austria. incomplete lock down as applied in sweden only slowed down spread of epidemic. it associates with roughly two times more infections and a more than four-fold deathtoll. b) eradication of epidemic can be expected in island states (iceland, new zealand) and other relatively small countries (e.g. croatia) showing disappearance of new cases two to three months after the outbreak (see epicurves in the insertion). another example of eradication is covid-19 spread at the princess diamond cruise liner with about 760 infections. c) wave-like up and downs of epidemic were observed in armenia and iran. the trajectories transform into wave-like oscillations of the effective reproduction number (insertion). d) a new spike of cases is seen in the trajectory of south korea. the trajectory for us is shown for comparison. covid-19 pandemic develops in different phases around the world ranging from exponential growth to decaying regimes and even eradication from region to region and from country to country. it is characterized by high dynamics, which necessitate prompt monitoring to evaluate the outcome of npi measures in either, 'lockdown' or 'lock up' direction to indicate improvement or worsening in terms of suited metrics such as increasing or decreasing numbers of cases, rate factors or reproduction numbers. the covid-19 viewer provides this information in the worldwide context on a daily actualized basis. we understand our report as a worked example reflecting aspects of the pandemic in may 2020, which supports future monitoring using the covid-19 viewer as a sort of working instruction. many aspects of the covid-19 pandemic are not completely understood. this includes dark figures of infections, detailed spreading mechanisms and associated socio-economic, politic and health factors. here more studies reasoning differences between regions and countries are required. the trajectory approach complements epi-curve reporting by bridging the gap to modelling methods. inspection and comparison of the trajectories and of the time courses of rate factors extracted are expected to inspire development of substantiated hypotheses and elaboration of improved models to better understand mechanisms of epidemic spread and decay and theirs specific in different countries and regions. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . covid-19: the cidrap viewpoint: part 1: the future of the covid-19 pandemic: lessons learned from pandemic influenza coronavirus pandemic (covid-19) coronavirus tracked: the latest figures as countries fight to contain the pandemic the covid tracking project mitigation and herd immunity strategy for covid-19 is likely to fail a first study on the impact of current and future control measures on the spread of covid-19 in germany projecting the spread of covid19 for germany imperial college covid-19 response team estimate of the development of the epidemic reproduction number rt from coronavirus sars-cov-2 case data and implications for political measures based on prognostics quantifying the effect of quarantine control in covid-19 infectious spread using machine learning covid-19 spread: reproduction of data and prediction using a sir model on euclidean network sequential data assimilation of the stochastic seir epidemic model for regional covid-19 dynamics effective containment explains subexponential growth in recent confirmed covid-19 cases in china estimating effects of physical distancing on the covid-19 pandemic using an urban mobility index a time-dependent sir model for covid-19 with undetectable infected persons. eprint arxiv viola priesemann: inferring change points in the covid-19 spreading reveals the effectiveness of interventions autocatalytic model for covid-19 progression in a country using phenomenological models for forecasting the extended sir prediction of the epidemics trend of covid-19 in italy and compared with hunan evaluation of the secondary transmission pattern and epidemic prediction of covid-19 in the four metropolitan areas of china shiny: web application framework for r. r package version inferring change points in the spread of covid-19 reveals the effectiveness of interventions sars-cov-2 (covid-19) by the numbers a global effort to define the human genetics of protective immunity to sars-cov-2 infection factors associated with hospitalization and critical illness among 4,103 patients with covid-19 disease features of 16,749 hospitalised uk patients with covid-19 using the isaric who clinical characterisation protocol covid-19: in the footsteps of ernest shackleton estimating epidemic exponential growth rate and basic reproduction number the disease-induced herd immunity level for covid-19 is substantially lower than the classical herd immunity level estimating individual and household reproduction numbers in an emerging epidemic time-dependent sir model for covid-19 with undetectable infected persons. arxivorg 2020 key: cord-270948-qfsjtflv authors: klosterhalfen, stephanie; kotz, daniel; kuntz, benjamin; zeiher, johannes; starker, anne title: waterpipe use among adolescents in germany: prevalence, associated consumer characteristics, and trends (german health interview and examination survey for children and adolescents, kiggs) date: 2020-10-22 journal: int j environ res public health doi: 10.3390/ijerph17217740 sha: doc_id: 270948 cord_uid: qfsjtflv waterpipe (wp) use is popular among youth worldwide, but epidemiological data from germany are scarce. we aimed to describe prevalence rates of wp use (current, last 12 months, ever) and analysed correlates and trends among 11to 17-year-olds in germany. analyses were based on data from the “german health interview and examination survey for children and adolescents” study during 2014–2017 (n = 6599). changes in wp use prevalence compared with 2009–2012 were used to describe trends. associations with sociodemographic characteristics and cigarette smoking were assessed with multivariable logistic regression models. prevalence of current wp use among adolescents was 8.5% (95% confidence interval (ci) = 7.5–9.6), use in the last 12 months was 19.7% (95% ci = 18.3–21.2), and ever use was 25.8% (95% ci = 24.2–27.5). high prevalence rates were particularly found among 16–17-year-olds. during 2009–2012, these prevalence rates were 9.0%, 18.5%, and 26.1%, respectively. wp use was associated with older age, male sex, migration background, lower educational level, and current smoking status. among current wp users, 66.2% (95% ci = 60.0–71.9) identified themselves as non-smokers, and 38.1% (95% ci = 32.5–44.0) had used wp ≥ three times in the last month. wp consumption is popular among german youth, and prevalence rates have not changed over time. specific prevention strategies to reduce harmful wp consumption among youth should be implemented. in recent decades, there has been a worldwide increase in the prevalence of waterpipe (wp) use among young people. historically, the popularity of wps spread from india, across continents, until its consumption became accepted in the western world as an alternative form of tobacco smoking. regular consumption of wps by broad sections of the population is a phenomenon that was not observed prior to the end of the 20th century [1] [2] [3] . although the name (hookah, shisha, narghile, argileh, boory, goza, or hubble bubble), size, and design of wps vary from region to region, they all function in the same way. the characteristic of this time-consuming (average duration of 47 min) method of tobacco smoking is that the smoke passes through water before being inhaled into the lungs once cooled [4] . figure 1 shows the required components of a wp. to fill the tobacco head, a special, mostly sweet and flavored wp tobacco called maassal can be used, as well as alternative tobacco-free products such as steam stones [5] . to heat the tobacco, wp charcoal (or alternatively, an electronic heat source [6] ) is used. see: figure 1 . components of a waterpipe. int. j. environ. res. public health 2020, 17, x 2 of 16 through water before being inhaled into the lungs once cooled [4] . figure 1 shows the required components of a wp. to fill the tobacco head, a special, mostly sweet and flavored wp tobacco called maassal can be used, as well as alternative tobacco-free products such as steam stones [5] . to heat the tobacco, wp charcoal (or alternatively, an electronic heat source [6] ) is used. see: figure 1 . components of a waterpipe. wp use differs from conventional cigarette use not only with respect to the length of a smoking session; wp tobacco tastes often sweeter due to the added flavors, and the inhalation of cooled smoke seems less irritating to the mucosae and lungs. in addition, wps can be smoked as a group, e.g., at a party, and can therefore create a social experience [7] . these aspects provide insight into why wp use is popular among adolescents, why many wp wp use differs from conventional cigarette use not only with respect to the length of a smoking session; wp tobacco tastes often sweeter due to the added flavors, and the inhalation of cooled smoke seems less irritating to the mucosae and lungs. in addition, wps can be smoked as a group, e.g., at a party, and can therefore create a social experience [7] . these aspects provide insight into why wp use is popular among adolescents, why many wp users do not perceive themselves as "conventional smokers" [8] , and why some users underestimate the health risks of wp consumption [9] [10] [11] [12] . first experiences with the consumption of tobacco typically take place during the period of experimentation during adolescence. the most frequent first tobacco product tried by young people is the cigarette (followed by cigar, smokeless tobacco, and wp) [13] . during this period, adolescents are at special risk of developing dependency, and the risk of early deterioration of health increases [14] . different cultural and socioeconomic backgrounds as well as use of other tobacco products can be determinants regarding the consumption of wp by adolescents [15] [16] [17] . the aromatic taste of wp tobacco (e.g., apple, cherry, melon) appeals to young people and can be associated with a more pleasant, longer smoking experience which leads to increased nicotine exposure and dependence potential [18] [19] [20] . furthermore, the consumption of wp is associated with other harmful health effects similar to those associated with cigarette smoking [21] . in addition to the increased risk of carbon monoxide poisoning, which can result from combustion of the wp charcoal [22] , smoking wp can cause acute to chronic impairment [23] , negative impacts on executive brain function, or carcinogenic changes in various organs including the lungs and cardiovascular system [24] [25] [26] . sharing a wp among different people can also increase the risk of transmission and infection with bacterial or viral diseases [27] , which is particularly relevant during times of acute pandemic such as the present global novel coronavirus disease (covid-19) pandemic. the number of shisha bars (almost 6000) and the consumption of wp tobacco have risen in germany [28] . the increasing number of wp cafés can influence societal acceptance, and these serve as a place of social exchange for adolescents, just like pubs in former generations [12, 29] . in germany, there are legislative measures at the both state and the federal level to regulate wp consumption (bundesnichtraucherschutzgesetz ("federal non-smoker protection act"), jugendschutzgesetz ("youth protection act"), tabakerzeugnisgesetz ("tobacco products act"), nichtraucherschutzgesetz ("non-smoker protection act")). the german tobacco products act regulates ingredients, emission levels and information requirements for tobacco and related products. in 2016, the ingredients of wp tobacco changed (% content of glycerin). the youth protection act regulates the distribution of tobacco products. in 2007, the age limit for the consumption of tobacco products in public has been raised from 16 to 18 years. it is not permitted to sell tobacco products to minors. children and adolescents under the age of 18 are not allowed to smoke in publicly accessible rooms in places open to the public and otherwise in public places. these measures were accompanied by a tobacco prevention program. purchase of wp tobacco and accessories or the entry to a shisha bar are not permitted to people under 18 years of age. apart from regional studies, there are only a few population-based studies on the prevalence of wp consumption among adolescents in germany. the german health interview and examination survey for children and adolescents (kiggs) study and studies of the federal centre for health education (bzga) such as the drug affinity study have collected data on awareness about and use of wp, differentiated according to migration background, frequency of consumption, and combined consumption of tobacco cigarettes, wps, e-products, and tobacco heaters [16, 30] . national and international study findings indicate that male adolescents or youth with a migration background use wp more often than girls or people without a migration background [3, 16, 30] . regarding socioeconomic or educational factors, there seems to be a relationship between wp use and lower educational levels in germany, whereas international studies have reported opposite findings [3, 16, 30] . a study by the german health insurance dak ("dak-präventionsradar") has collected prevalence figures of wp consumption among school children [31] . prevalence rates of 6-14% for current and 22-44% for ever use of wps are reported for adolescents in germany under 18 years of age [16, 27, [30] [31] [32] . regarding international prevalence rates, current wp consumption varies widely, from 2.2% in romania to 36.9% in lebanon [15, 33] . several studies from the united states (us) reported increasing rates of wp use among 11-to 18-year-olds between 2009 and 2017 [34] . smoking a wp is a common form of tobacco use among adolescents in the us [12] . however, little is currently known about the factors associated with wp use. the influence of a one-or both-sided migration background, the socioeconomic status (ses) of the family, and sex, have not yet specifically been investigated in germany. data are also missing on the percentage of wp users who perceive themselves as smokers or non-smokers. this is an important issue, which can influence the perception of health risks of wp tobacco consumption and the creation of prevention programs. we, therefore, aimed to evaluate wp use and associated factors among german adolescents. more specifically, based on data of the second wave of the german health interview and examination survey for children and adolescents (kiggs wave 2), in the present study, we aimed to (i) investigate the prevalence of wp consumption among 11-to 17-year-old boys and girls; (ii) describe the frequency of wp use and the self-assessed smoking status; (iii) examine the associations between sociodemographic factors, smoking status and wp consumption among adolescents; and (iv) to monitor trends between the previous and the current wave of the kiggs study. due to a large study sample, the kiggs study-in contrast to other population-wide studies conducted in germany-allows the surveillance of prevalence figures more detailed (e.g., one-or both-sided migration backgrounds, survey of 11-year-olds, survey 12-month prevalence) and to include statements on self-assessed smoking status. these data can help in the identification of different risk profiles to develop targeted group-specific and gender-sensitive prevention strategies. the kiggs study is part of health monitoring conducted by the robert koch institute (rki) on behalf of the federal ministry of health in germany. kiggs focusses on health status, health behavior, living conditions, protective and risk factors, and healthcare among children, adolescents, and young adults living in germany. cross-sectional data have been collected at three time points: the kiggs baseline study (2003) (2004) (2005) (2006) , kiggs wave 1 (2009-2012) and kiggs wave 2 (2014-2017). the response rate (according to aapor response rate 2) of kiggs wave 2 was 40.1% in total [35] . a multi-step approach was used to include people with a migration background in kiggs wave 2. the share of children and adolescents of non-german nationality in kiggs wave 2 corresponds to the population figures from the federal statistical office [36] . the concept, methodology, and analyses of kiggs are described in detail elsewhere [35, [37] [38] [39] . comparable to the kiggs baseline study, respondents for kiggs wave 2 were selected randomly based on the population registers of 167 representative german municipalities and cities (two steps sampling process). the study population of kiggs wave 1 consists of re-invited participants from the baseline study supplemented by newly invited children aged 0-6 years. kiggs wave 2 (like kiggs baseline study) was comprised of an interview and examination part, whereas kiggs wave 1 was conducted as a telephone interview survey [37] [38] [39] . to achieve an optimal number of respondents and sample composition, a variety of measures were applied (e.g., phone calls or home visits) [35, 36] , resulting in a total of 15,023 respondents aged 0-17 years. the analyses of wp consumption were restricted to data from 11-to 17-year-old respondents (n = 6599), collected using a written questionnaire. to identify trends in comparison with the previous wave, the results from wave 1 were compared with the currently collected prevalence rates from wave 2. the study was approved by the ethics committee of hannover medical school (no. 2275-2014). the prevalence of wp use was assessed with the question "have you ever smoked a waterpipe or shisha?" respondents who affirmed having used a wp were defined as "ever wp user" and were further asked "have you smoked a waterpipe or shisha in the last 12 months?" (yes defined as "last-12-month wp user") and "if you think about the last 30 days, on how many days did you smoke a waterpipe or shisha?" (response options: ≥1 day, defined as "current user" or "none in the past 30 days"). regarding the frequency of use during the past month, we classified responses according to one, two, or ≥three times. to determine the ses of the family, an index was generated based on information of the parents' level of education, occupational status, and income (equivalized disposable income). thus, respondents were classified as belonging to a family with "low", "medium", or "high" ses [40] . school type was surveyed by asking the parents "which type of school does your child go to?", with nine response options: "primary school", "secondary school", "middle school", "school with secondary and middle educational program", "integrated comprehensive school", "academic secondary school", "technical secondary school", "special school", and "other". due to its federal structure, there is no uniform school system in germany. as some federal states now have a two-tier school system, we categorize for the following analyses, two groups for secondary school: "secondary/middle/comprehensive school" and "technical/academic secondary school". young people who no longer attended school were assigned to the corresponding category based on the highest level of education they achieved [41] . to assess migration background, all respondents were asked about their own and their parents' country of origin: "in which country were you born?" and "in which country were your parents born?" a one-sided migration background meant that one parent was not born in germany or had no german citizenship; a both-sided migration background meant that the child himself/herself migrated to germany and had at least one parent who was not born in germany or both parents were born abroad [42] . to assess smoking status, respondents were asked "do you currently smoke?", with the following response options: "no", "daily", "several times a week", "once a week", or "less than once a week". all respondents who answered in the affirmative were defined as a "current smoker". the data collected for kiggs wave 2 are available from the rki research data center (https: //www.rki.de/en/content/health_monitoring/public_use_files/public_use_file_node.html). the descriptive analyses of wp use patterns (current, last 12 months, ever) stratified by sociodemographic characteristics and smoking status are presented, differentiated for female and male respondents, as percentages with 95% confidence intervals (cis). weighting with regard to age, sex, federal state, german citizenship, and the child's parents' level of education was applied to ensure representative data for children and adolescents living in germany. comparison of current prevalence figures and those obtained between 2009 and 2012 was based on descriptive statistics and is presented as percentage with 95% ci. three multivariable logistic regression models were applied to explore associations between different wp use patterns and sociodemographic characteristics and smoking status for girls and boys: model i = current wp use vs. never wp use, model ii = last 12 month wp use vs. never wp use and model iii = ever wp use vs. never wp use. respondents with missing data were excluded from the regression analyses. data were analyzed using stata 15.1 (stata corp., college station, tx, usa). stata's survey procedures were applied to account for the clustered sampling design. the prevalence of current wp use among 11-to 17-year-old adolescents in germany was 8.5% (95% ci = 7.5-9.6; n = 446) in the period 2014-2017. almost every fifth adolescent had used wp within the last 12 months (19.7%, 95% ci = 18.3-21.2; n = 1101), and 25.8% (95% ci = 24.2-27.5; n = 1415) were ever wp users (weighted data). table 1 presents prevalence of different wp use patterns, sociodemographic characteristics, and smoking status, stratified by sex (weighted data, missing data regarding ses (n = 145), education (n = 710), migration background (n = 33), and current smoking status (n = 852)). the pattern of missing values showed a higher amount of missing values among boys with migration background, boys with lower ses and lower education level, and among girls with lower ses and multivariable analyses showed that the odds of missing values are especially high among boys with a both-sided migration background (data not shown). boys were more likely than girls to report current (10.6% vs. 6.3%), last 12-month (22.1% vs. 17.3%), and ever (28.1% vs. 23.4%) wp use. respondents with a migration background and current smokers reported using wp more often than those without a migration background or non-smokers (current, last 12 month, and ever wp use). the results of the three multivariable regression analyses regarding sociodemographic characteristics, current smoking status, and wp consumption are presented in table 2 . the adjusted odds ratios (ors) of current vs. never wp use (model i) were higher in adolescents with older age and current smokers (girls: or = 1.97, 95% ci = 1.69-2.29 and or = 48.27, 95% ci = 24.12-96.59; boys: or = 2.20, ci = 1.92-2.52 and or = 67.57, 95% ci = 18.02-253.32). concerning migration background, we found that boys with a one-sided migration background used wp more often than boys without a migration background (or = 3.03, 95% ci = 1.36-6.77). we found similar associations when comparing wp use in the last 12 months vs. never wp use (model ii). in addition, girls with a both-sided migration background showed a lower or for wp use than girls without a migration background (or = 0.38, 95% ci = 0.22-0.65), and girls with a lower educational level showed a higher or for wp use than girls with higher educational levels (or = 1.82, 95% ci = 1.32-2.51). we also found the above-mentioned associations when comparing ever vs. never wp use (model iii), except that the adjusted or was also higher in girls from a family with low ses compared with girls belonging to a family with high ses (or = 1.66, 95% ci = 1.02-2.71). the numerator for the calculation refers to the total number in the corresponding series (e.g., 50.6% of 17-year-old girls report wp ever use). bold printed indicates the prevalence for the respective group. defined as using wp in the last 30 days. • one-sided indicates children and adolescents having one parent not born in germany or without german citizenship; two-sided indicates children and adolescents who themselves migrated to germany and have at least one parent who was not born in germany, and children and adolescents whose parents were both born in a country other than germany or non-german nationals. * socioeconomic status generated as a household characteristic based on parental levels of education, occupational status, and income. german equivalents to school types: secondary school = hauptschule; middle school = realschule; comprehensive school = gesamtschule; technical secondary school = fachoberschule (fos); academic secondary school = gymnasium. 1 † all listed covariates were included in models i-iii. data are presented as odds ratio (or) 95% confidence interval (ci). * p < 0.05; ** p < 0.01; *** p < 0.001. > age was treated as a continuous variable in the regression analyses. • one-sided indicated children having one parent not born in germany or without german citizenship; both-sided indicates children who themselves migrated to germany and have at least one parent who was not born in germany and children and adolescents whose parents were both born in a country other than germany or non-german citizens. defined as using wp in the past 30 days. ‡ socioeconomic status generated as a household characteristic based on parental levels of education, occupational status, and income. german equival ents to school types: secondary school = hauptschule; middle school = realschule; comprehensive school = gesamtschule; technical secondary school = fachoberschule (fos); academic secondary school = gymnasium. the prevalence rates of wp use in wave 2 (2014-2017) were similar to those identified earlier in wave 1 (2009 wave 1 ( -2012 , as shown in figure 2 . among german 11-to 17-year olds surveyed in the period 2014-2017, 8 .5% reported being current wp users and about 26% reported being ever users. the use of wp seems to be most common in the age group of 16-17-year-olds. a considerable proportion (62%) of current wp users had smoked a wp twice or more in the last month. only one-third of wp users considered themselves smokers. we found positive associations of wp use with older age, male sex, and current smoking status. regarding the associations between wp consumption and education level or migration background, an inverse relationship was observed for both genders in some analyses. as shown in table 2 , the association between lower educational level and the use of wp was more pronounced among girls, whereas the association between the migration background and the use of wp is found primarily among boys. the prevalence rates did not differ much from those obtained during 2009-2012. the prevalence rates found in the present study are highly congruent with data collected in 2015 by the bzga [43] . the two nationwide surveys yielded comparable prevalence rates for both current use (kiggs wave 2 (11-to 17-year-olds): 8.5% vs. drug affinity study (12-to 17-year-olds): 8.9%) and ever use (25.8% vs. 27.3%). the prevalence identified in the european school survey project on alcohol and other drugs (espad) in austria was strikingly higher: in 2019, 21% of 14-to 17-year-olds reported current and 51% reported ever wp use [44] . a possible reason for the difference may be the difference in age groups, as prevalence increases with age. this may also explain the comparatively high prevalence rates for young people (14-to 16-year-olds) living in the german state of bavaria (current wp use: 20.1%; ever use: 48.9%) who also participated in the espad study in 2015 [45] . comparing the prevalence rates reported in germany with those in the us (2009-2017), nationally representative estimates indicate lower prevalence figures (current use among high school students (grades 9 to 12): 4.8%, ever use: 14.3%); however, representative state-wide estimates showed comparable figures (current use: 11.6%; ever use: 22.5%) [34] . within the present study we aimed to explore the frequency of current wp consumption and self-assessed smoking status. most current wp users reported a wp use frequency of no more than twice in the past 30 days. this consumption pattern is also seen in previous studies [32, 46] . reasons for the difference in consumer behavior, for example, in comparison with (daily) cigarette consumption, could be owing to the inflexibility of the stationary tobacco use method and its time-consuming nature. most current wp users identified themselves as non-smokers. thus, wp consumption is not perceived as smoking, a result which has been also reported elsewhere [8] . the results of the kiggs study showed variation in wp use according to sex, age, migration background, and current smoking status. we found higher ors for current and ever use among respondents who were male, older, and who had a one-sided migration background (boys). these findings are in line with prior national and international studies [30, 33, 34, 47] . migration background is a known correlate of wp use described in previous kiggs waves and other studies. whereas in the first wave of the german health survey for children and adolescents (kiggs wave 1, 2009 wave 1, --2012 [48] , boys with a both-sided migration background were found to use wps more often (current and ever) than those without a migration background, we found counterintuitively low prevalence for wp use with a both-sided migration background but high prevalence with a one-sided migration background among boys in kiggs wave 2. hence, we speculate that the particular high amount of missing values among boys with a both-sided migration background might explain their low prevalence of wp use. the case of girls was reversed; we found an association of a both-sided migration background and lower ors for current and ever wp use. similar results have been described for smoking adults (over 18 years) in germany [49] . our findings also point out that young people who regard themselves as current smokers were up to 68 times more likely to use wp than non-smokers. associations regarding this kind of dual use have been reported in other studies [33, 47] . concerning the trend in prevalence figures, our study found stable figures over time. for 12to 17-year-old boys, the bzga reports similar trends in the figures for current wp use. first, these figures decreased from 2007 (16.3%) to 2011 (9.8%), but then remained at this level until 2015. for 12to 17-year-old girls, a similar trend can be seen over time. the prevalence figures for the current use of wp ranged from 7.4% (2011) to 6.4% (2015) [50] . the present study entails the following limitations. owing to the cross-sectional design, it was not possible to make conclusive statements about causality with respect to the results. responses given in kiggs wave 2 are self-reported data, which are always associated with biases. respondents may remember of the corresponding answer categories inaccurately (recall bias) or may give socially acceptable answers (social desirability bias). as there are different terminologies for wp, the use of pictures within the questionnaire would probably have been preferable to ensure that all respondents have the same understanding of the tobacco product. to be able to assess the health risks arising from the consumption of wp tobacco, the ingredients of wp tobacco play an integral part. unfortunately, the composition of wp tobacco or the number of puffs during a session could not be investigated in this study. over the course of the kiggs study, there has been a change in methods: kiggs wave 2 was conducted using self-report questionnaires and kiggs wave 1 using telephone interviews, which are more susceptible to socially desirable response behavior [51] . as with all surveys, the possibility of bias owing to selective non-participation also exists. it is assumed that people who participate in a health study also have greater health awareness and therefore differ from the general population in terms of smoking behavior (selection bias) systematic identification of patterns of missing items was not feasible, but could help to interpret results more accurately in further studies. the described selection effects were partially corrected by weighting. thus, the observed results may be generalizable to 11-to 17-year-olds in germany, which is a strength of the kiggs study. furthermore, it was possible to identify different wp consumption patterns (current, in the last 12 months, and ever), the frequency of wp consumption, and the combined consumption of tobacco cigarettes and wps, as well as the association of wp use with sociodemographic characteristics and cigarette smoking status. older age, male sex, migration background, lower educational level, as well as current cigarette smoking were found to be associated with wp use among german adolescents. wp consumption is popular among adolescents but does not seem to have increased substantially in recent years. continuous monitoring of trends in prevalence and use behavior is important to yield an evidence basis for developing targeted group-specific and gender-sensitive prevention approaches within public health prevention strategies. in addition to preventive programs within schools, it would be also useful to provide information about the health hazards and addiction of wp use in sports clubs or on preferred social networks (e.g., youtube, facebook, twitter) visited by young people. a targeted gender-specific approach could also be made here. the law for the protection of youth, which has been adapted since 2016 and prohibits the sale of wps by mail order to minors, is an important step to reduce the illegal sale to minors. a consistent and frequent age control in shisha bars should continue to be carried out by public authorities. information campaigns (also for parents) may help to decrease the private use of wps. the ban on marketing tobacco with characteristic flavors (e.g., menthol) implemented in germany by § 5 of the tobacco products act in 2020 is an important step to prevent young people from consuming flavored wp tobacco. a further ban on the advertising of tobacco products or combined warnings (consisting of pictures and text) are planned for wp tobacco in germany from may 2024, which will help to increase the awareness about health hazards connected to wp use. further research should explore why many adolescent wp users do not see themselves as smokers, that is, the beliefs and motives that underlie this. more research is needed on the consumption patterns (e.g., number of puffs, duration of a wp session) and on the type of wp use (e.g., types of wp tobacco, use of charcoal). moreover, the association between the consumption of wp and other substances, e.g., cigarettes, should be investigated more in detail. we thank analisa avila, els, of edanz group (https://en-author-services.edanzgroup.com/ac) for editing a draft of this manuscript. the authors declare no conflict of interest. advisory note: water-pipe tobacco smoking: health effects, research needs and recommended actions by regulators estimating the beginning of the waterpipe epidemic in syria the global epidemiology of waterpipe smoking tobacco smoking using a waterpipe: a re-emerging strain in a global epidemic waterpipes. facts about smoking tobacco-free waterpipes can also be a health hazard self-assessment of adolescents regarding water pipe consumption health effects of waterpipe tobacco use: getting the public health message just right adolescents' perceptions of health risks, social risks, and 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wave 2 has been completed socioeconomic status and subjective social status measurement in kiggs wave 2 trends in educational inequalities in smoking among adolescents in germany: evidence from four population-based studies health interview and examination survey for children and adolescents the drug affinity of young people in the federal republic of germany 2015. smoking, alcohol consumption and use of illegal drugs: current prevalence and trends survey of 9th and 10th grade students in bavaria expert panel on waterpipe assessment in epidemiological, studies. consensus statement on assessment of waterpipe smoking in epidemiological studies waterpipe tobacco smoking trends among middle and high school students in the united states from smoke on the water distribution and patterns of tobacco consumption in germany the decline in cigarette consumption by adolescents and young adults in germany and the increasing importance of hookahs, e-cigarettes and e-shishas empirische sozialforschung key: cord-345834-l2e5v39s authors: anacleto, m.a.; brito, f.a.; de queiroz, a.r.; passos, e.; santos, j.r.l. title: diffusive process under lifshitz scaling and pandemic scenarios date: 2020-08-20 journal: physica a doi: 10.1016/j.physa.2020.125092 sha: doc_id: 345834 cord_uid: l2e5v39s we here propose to model active and cumulative cases data from covid-19 by a continuous effective model based on a modified diffusion equation under lifshitz scaling with a dynamic diffusion coefficient. the proposed model is rich enough to capture different aspects of a complex virus diffusion as humanity has been recently facing. the model being continuous it is bound to be solved analytically and/or numerically. so, we investigate two possible models where the diffusion coefficient associated with possible types of contamination are captured by some specific profiles. the active cases curves here derived were able to successfully describe the pandemic behavior of germany and spain. moreover, we also predict some scenarios for the evolution of covid-19 in brazil. furthermore, we depicted the cumulative cases curves of covid-19, reproducing the spreading of the pandemic between the cities of são paulo and são josé dos campos, brazil. the scenarios also unveil how the lockdown measures can flatten the contamination curves. we can find the best profile of the diffusion coefficient that better fit the real data of pandemic. in december 2019, the world started to face a new type of severe pneumonia which appeared in wuhan, china. only two months later the international committee on taxonomy of viruses named the virus responsible for these pneumonia cases as severe acute respiratory syndrome coronavirus 2 or sars-cov-2, whose disease was popularly known as coronavirus disease 2019, or simply covid-19 [1] . such disease was classified as a public health emergency of international concern at the end of january 2020, by the world health organization. up to now, sars-cov-2 has spread all over the world, presenting more than 3.5 million of cases, taken approximately 247107 lives, and has a new epicenter in the united states of america which has reported almost one-third of the total amount of cases [2] . few places in the world were able to fully control the pandemic of covid-19. in europe, for instance, italy and spain were for a long time the world's two worst-hit countries by the covid-19. now, the numbers of active cases in these two countries are slowly decreasing and they are facing the final stage of the pandemic. one of the best countries in europe to adopt measures against covid-19 so far is germany, who is also entering in the controllable phase of the pandemic. behind the success of germany are measures of social distance or lockdown procedures, and a large number of people tested for sars-cov-2 [3] . after spreading in asia and europe, now covid-19 is a challenge for the usa, as well as for low and middle-income countries, such as brazil. there is a serious concern on the international scientific community about the behavior of sars-cov-2 in such countries, since they face other severe problems such as poverty, food security, economic growth, besides other diseases like human immunodeficiency virus, tuberculosis, and malaria [4] . so far, several scientific works and reports based on numerical simulations have been published, lighting the evolution of the pandemic in different countries and reflecting the actions as well as the strategies of each country to mitigate the effects of covid-19. some of these studies can be found in references [4] [5] [6] [7] [8] . among recent works on this subject, we also highlight an interesting proposal of an age-structured model presented by canabarro et al. [9] , a model based on hospital infrastructure developed by pacheco et al. [10] , and a study to predict covid-19 peaks around the world based on active cases curves, introduced by tsallis et al. [11] . in this work we intend to collaborate with the current investigations by proposing a model which can describe the evolution of the pandemic through the solutions of a modified version of the diffusion equation. the standard diffusion equation describes the macroscopic behavior due the effect of many micro-particle bodies, as it is observed in a brownian motion, for instance [12] . an interesting modification of this equation was proposed in the seminal paper of petr horava [13] , in his studies about quantum gravity, where he extended the definition of spectral dimension to theories on smooth spacetimes in anisotropic or lifshitz scaling. in our investigation, we introduce a new version of the diffusion equation inspired by horava's work, and we use it to fit real active cases data of covid-19 from germany, spain and brazil. the diffusion equation with the lifshitz scalling and the equation of motion for the diffusion coefficient are going to be introduced in section ii. in section iii we are going to show different solutions for the diffusion equation which can be used to fit the evolution of covid-19. the availability of our models are carefully discussed in section iv. the spreading of the pandemic between two different cities is modeled in section v. then, we present our final remarks and perspectives in section vi. the first issue to describe a pandemic evolution consists in choosing an appropriate diffusion process. the complexity of a virus transmission such as sars-cov-2, demands a diffusion process characterized by a probability density ρ = ρ(x, τ ; x , τ ; σ) measuring the diffusion from a time τ to a time τ , and from a space coordinate x to x at a diffusion time σ. notice that σ and τ are two different types of time, τ would be understood as the standard time variation of the pandemic, while σ would control the collective response to the pandemic, such as social distance measures, for instance. besides, as each country can adopt several strategies to mitigate the pandemic effects, it is expected that the diffusion process would account for different degrees of anisotropy. a general continuous diffusion equation that attends such criteria was introduced by horava in his seminal work [13] , whose form is here τ is the so-called euclidean time and z is the lifshitz critical exponent, which measures the anisotropic scaling of a given model [13, 14] . the lifshitz critical exponent is essential to determine the spectral dimension, which can be applied to several geometric objects presenting fractal behavior [13] . the relative sign (−1) z+1 concerns the requirement of ellipticity of the diffusion operator valid for integer z, but the results can be analytically continued for any positive real z [13, 14] . an extra relevant ingredient to proper modeling a pandemic spread is a diffusion coefficient, which can account for the transmission rate of the virus. therefore, this discussion suggests that a pandemic scenario is governed by the following anisotropic diffusion equation where φ(τ ) is a dynamic diffusion coefficient. we are going to show that a proper balance between φ and z yields to distributions that can fit real pandemic data. let us also constrain the diffusion coefficient φ(τ ) with the standard lagrangian where the negative kinetic part stands for the euclidean time. therefore, the equation of motion for the diffusion coefficient is such that by integrating the previous equation once, we find the first-order differential equation where we considered the function w is known as superpotential in analogy with the bosonic sector of a super symmetric field theory. the diffusion equation (2) has the general solution where ρ 0 stands for the initial probability density subject to the pandemic (or to the diffusion process). in this work, we consider ρ as the probability density of active cases of covid-19 to make a parallel between our model and real pandemic data. the number of active cases of covid-19 is defined as follows [3] active cases = total cases − total deaths − recovered , therefore, it represents the current number of patients detected and confirmed as infected with sars-cov-2. the number of active cases is also a relevant metric for public health and primary care functions, as it allows measuring capacity versus hospitalization needs. such a number is used to plot the distributions of active cases of covid-19 for different countries as we see in [3] , and it was used to predict the pandemic peaks in different countries as one can see in [11] . before starting to make numerical integrals of eq. (7) to depict the active cases curves, let us comment on the dimensionality of the model. a (spatial) bi-dimensional model seems most natural to discuss the diffusion process in the population. this usually is captured by the bi-dimensional networks with persons being nodes and relation being links [15] . moreover, one particular feature of the transmission of this virus is that it occurs by contact between persons [1] . this feature is captured by a nearest-neighbor interaction between the nodes. it can be argued that one can project down this nearest-neighbor interaction bi-dimensional network into a long-range interaction chain (one-dimensional chain) with appropriate boundary conditions. thus in the passing to the continuum model we can in a reasonable approximation consider a d = 1 model with x ranging from −∞ to +∞. for the numerical integration in the reciprocal space, we can take therefore −∞ ≤ ω ≤ ∞, and −∞ ≤ k ≤ ∞. moreover, we choose evaluate ρ at x = x indicating that the probability density is measured in the same spatial location after the evolution of the diffusion process. the simplest model that we can analyze consist in where the diffusion coefficient is normalized. this first model will enable us to observe the influence of the critical exponent and of the diffusion time in our curves. the fig. 1 shows four different behaviors for the solution of the diffusion equation. the diffusion time and the critical exponent z are competing parameters. particularly in the top left curve, with a same diffusion time large values of z ≥ 1 makes the gaussian flattened, whereas bottom right for z < 1 the gaussian tends to blow up even for sufficiently large diffusion time. the best scenario in the sense of flattening the gaussian curve seems to be the increasing of both diffusion time and critical exponent z (top right). the features of σ presented in the previous scenarios yield us to indeed understand the diffusion time as equivalent to a lockdown period. in this work we adopted the lockdown definition introduced by flaxman et al. [5] , which means a scenario where regulations and laws regard strict social interaction. these regulations/laws include the banning of any non-essential public gatherings, closure of educational, public and cultural institutions, and ordering people to stay at home apart from exercise or essential tasks. in this example we are going to consider the following superpotential where λ and α are real constants. such superpotential leads us to which is known as λ φ 6 potential, and it is depicted in the left panel of fig. 2 . the minima or vacua values of v correspond to φ v = 0, and φ v = ± √ 2 α. the region between these different minima is called a topological sector, and it mediates the transmission rate in our pandemic model. this potential was applied in subjects like higher-order phase transitions in ginzburg-landau theory [16, 17] , modeling domain walls in ferroelastic transitions [18] , and in new physics beyond the standard model of particles at high energies scales [19, 20] . the previous definition of w yields to the first-order differential equation whose analytic solutions are exhibiting anti-kink and kink-like profiles as one can see in figure 2 . once φ(τ ) can be interpreted as the transmission rate of the virus, in fig. 2 , we observe that for τ τ 1 the virus is not transmitted, however its transmission increases until an approximately constant rate (φ v = √ 2 α) as τ gets bigger than τ 1 . the applicability of such a model is going to be carefully discussed later, in section iv. as a next example, let us deal with a more complex model for the diffusion coefficient. such a model is derived from this potential is known as double sine-gordon model and it is applied to study ultra short optical pulses in he 3 [21, 22] , in decaying of false vacuum and phase transitions in field theory [22, 23] . the left panel of fig. 3 shows part of the periodic form of potential v (14). there we can see one topological sector between vacua φ v = ± n π/(2 κ). an interesting feature about this model is that the parameter β can deform this potential at φ = 2 π n/κ. such deformation is responsible for the double (anti)kink profiles observed in the right panel of fig. 3 , and it is also related to the variation of the transmission rate of our pandemic model. the previous potential yields to the first-order differential equation which is satisfied by the analytic solutions for s = ±1, and n = 0, ±1, ±2, ... . the behavior of these analytic solutions v can be appreciated in fig. there it is shown that φ(τ ) has three different regimes of transmission rate, the first one for τ τ 1 where φ v = (2 n − 1) π/(2 κ), the second regime occurs when τ ≈ τ 1 and φ ≈ π n/κ, and the third one appears for τ τ 1 and φ v = (2 n+1) π/(2 κ). these regimes can lead us to distributions with different waves of contagious, as we are going to show below. in the next section we are going to analyze the viability of model iii and compare the numerical curves of ρ derived from it with those obtained through model ii. as it is known, the active cases data can present a high level of uncertainty once it depends on the number of the tests performed by each country and also the countries' transparency in reporting the tests. the data set used to depict the graphics of this section were taken up to may 02, 2020. therefore, some discrepancies between our predictions and the pandemic evolution are expected. to depict active cases curves that could j o u r n a l p r e -p r o o f journal pre-proof reproduce the existent data and able to predict the behavior of the pandemic, we decided first to test our model against a now well-established data set, and for that, we choose data from germany. nowadays germany is the 6th leading country in the world in numbers of covid-19 cases, accumulating a total of 164967 cases up to may 02 [24] . moreover, germany is the third leading country in the total number of tests, reporting a total amount of 2547052 tests for sars-cov-2, which corresponds to 30400 tests per million of population (considering the data up to may 02) [24] . therefore, to constraint some free parameters in our model, we use the data from germany as guidance. despite this procedure, we still have other free parameters to represent the particular features of each country's strategy to deal with the pandemic. we present the features of our models against real active cases data from germany, brazil, and spain below. in this first scenario we shown in figs. 4, and 5, the active cases curves for germany integrated from model ii and iii, respectively. there, the black solid curves are the numerical solutions generated from eq. (7), which best fitted the real data depicted in blue dots. the parameters constrained in the fitting process were ρ 0 = 10 7 (for fig 4) , ρ 0 = 10 5 (for fig. 5) , and time scaling t = 2 τ representing the number of days of pandemic. moreover, we also used σ = 28 (for figs. 4, and 5), referring to the number of lockdown days in germany [25] . we also depicted the active cases data from germany (blue dots), since february 15, 2020 (day 1), until may 02, 2020 (day 78) [24] . the left panels of figs. 4, and 5 predict that the pandemic of covid-19 would be fully controlled in germany after day 102 of infection (or after may 26, 2020), when the number of active cases is less than 1000 people. besides, the active cases curves unveil that the kink and the double anti-kink solutions, eqs. (13) , and (16) deform the standard gaussian curve. by comparing the right panels of figs. 4, and 5, we realize that the double anti-kink solution yields us to a better fitting of real data, reproducing a change in the decreasing of the number of active cases at t ≈ 60 day. the graphics of figs. 6, and 7 shown the currently infected people by sars-cov-2 in spain. at the present moment, spain reported an amount of 245567 cases of covid-19, which makes it the second leading country in the world pandemic rank [26] . the active cases data for spain are depicted as blue dots in figs. 6, and 7, and to plot our numerical curves we considered σ = 43 lockdown days [25] . we can observe that the black solid curves are in good agreement with the pandemic data, and they predict that the active cases of covid-19 would be fully controlled in spain after day 120 (june 11, 2020), where the number of infected people is less than 1000. moreover, in this scenario the kink and the double anti-kink like solutions eqs. (13) , and (16), strongly deform the standard gaussian curve. as in the case of germany, the double anti-kink curve leads us to a better fitting of real data, reproducing a second wave of contagious after day 50. the pandemic of covid-19 starts in brazil eleven days after spreads in europe. despite this few difference in time, the evolution of the contamination in brazil was deeply different from germany and spain, as we can see in the graphics of figs. 8, and 9. the numerical solutions presented in figs. 8, and 9 were depicted using the same values for ρ 0 and t from germany curves and the same value for z from spain curves. moreover, we derived two possible scenarios of lockdown measures, the black solid curves show a model with σ = 30 lockdown days while the red solid curves were depicted with σ = 90 lockdown days. furthermore, we also included the active cases data from brazil as blue dots [27] . the real data from brazil reveal an abrupt change of contamination which started on day 48 (april 13, 2020) and developed to a new increasing rate after day 56 (april 26, 2020). along this period, besides the pandemic, brazil has been facing a political crisis, which would explain the behavior of the covid-19 infection here observed. another problem with brazil's data is its high degree of uncertainty. up to may 02, 2020, brazil reported a total of 97100 cases of covid-19, figuring at the 9th leading country in the world pandemic rank [3] . however, brazil has performed only 339552 tests of covid-19, representing a total of 1597 per million of people [3] . for these reasons, it is challenging to make any prediction about the evolution of the pandemic in brazil. although, our curves seem to be in good agreement with the active cases data so far, and we also can realize that a long time social distance flatten the curves of the active cases. the peaks of the two active cases curves from figs. 8, and 9 have considerable differences about 115000, and 141000 cases, respectively. moreover, the peaks are predicted to happen on days 92 (may 27, 2020), and 86 (may 21, 2020) for black and red solid curves from fig. 8 , and on days 95 (may 30, 2020), and 110 (june 04, 2020) for black and red curves from fig. 9 . it is relevant to mention, that up to may 02, 2020, brazil can attend in maximum 32703 patients with needs for icu [28] .the solutions from fig. 8 predict that the pandemic in brazil would not be fully controlled earlier than day 131 (june 22, 2020). in the longer predicted scenario, observed in the red solid curve of fig. 9 , the pandemic would be fully controlled after day 198 (august 28, 2020), when the number of active cases is less than 1000. we also realize that the double anti-kink like solutions are able to fit better the real data than the single kink-like ones, reproducing the increasing in the number of active cases after day 48. . the peaks of black and red curves are approximately 268000 and 153000 cases, respectively. we also depicted the active cases data from brazil (blue dots), since february 26 (day 1), 2020 until may 02, 2020 (day 67) [27] . as it is known the primary mechanism used by covid-19 to spread is through person-to-person contact. consequently, big cities favor the spreading of the virus to small centers, once they facilitate the mixing of people from different areas. moreover, their strategical positions close to airports and crossed by state roads, make such cities susceptible to rapidly spread the virus to innermost regions. this phenomenon of advancing of covid-19 into the countryside happened in several places in the world, such as around new york city and close to several metropolises from brazil. it was also modeled in several regions of brazil using a markov chain approach as one can see in the work of costa et al. [29] . in this section, we adapted our diffusion equation to describe the dissemination of the virus around different cities. in order to reproduce such a scenario, we rewrite our diffusion equation as (17) where n is the number of the cities, and j i (σ) are free sources of the diffusion process. the previous equations present the following general analytic solutions and the free sources should obey the conservation constraint the simplest definition for the sources is to work with j i (σ) = constant. in this application we are going to understand such sources as proportional to the basic reproductive rate at the beginning of the pandemic, commonly known as r 0 [30] . the basic reproductive rate is a non-dimensional quantity which measures the secondary cases of contamination produced by one case introduced in susceptible populations [31] . such a rate is a key ingredient in several mathematical models to describe pandemic scenarios, and any attempt to estimate its value is a real challenge. consequently, by working with an equivalence between j i and r 0 , our model suggests that the basic reproductive rate is changed between different cities as the pandemic evolves in a given region. to exemplify our methodology, let us consider two cities from brazil which experienced the phenomenon of the spreading of sars-cov-2 to innermost regions. the cities here considered are são paulo, and são josé dos campos. these two cities have about 12 million and 721 thousand of inhabitants, respectively, and they are approximately 100 km distant apart. they are experiencing the so-called yellow-phase of the reopening plan designed by the state of são paulo government, where people have access to public places such as parks, restaurants, and cultural events with limited capacity [32] . the pandemic started in são paulo at february 25 (day 1), and up to august 02, 2020 it accumulated more than 200000 cases with more than 9600 fatalities [33] . the first case of covid-19 in são josé dos campos was reported in march 18, 2020 and the city has been registered more than 17000 cases, besides 204 deaths up to august 02, 2020 [33] . moreover, the most recent basic reproductive rate estimated for the state of são paulo in the beginning of the pandemic is r 0 = 9.24 [34] . in such an j o u r n a l p r e -p r o o f application, the best model to fit the cumulative cases data from the two cities was model ii, therefore, the asymptotic behavior of ρ in eq. (18) is going to be proportional to the product j i φ v , corresponding to a kink like profile for the density distribution. so, in the following application, we choose to test our model against the cumulative cases data. then, let us consider n = 2 as the number of the cities, and whereρ 0 is a proportionality constant and r 0 is the basic reproductive rate. taking the previous ingredients into eq. (18) yield us to depict the graphics presented in fig. 10 . there, we consider labels i = 1, and i = 2 to describe são paulo and são josé dos campos, respectively, and we worked with σ = 6 lockdown days, corresponding to the anticipation of holidays in the city of são paulo [35] . such anticipation of holidays was planned to increase the social distancing, attempting to reduce the spreading of covid-19. we can observe that our model successfully reproduces the evolution of the pandemic in theses two cities if we consider z = 50, which is the same value used to fit spain and brazil's active cases curves in the previous section. moreover, the fact that j 2 = −r 0 means that the basic reproduction rate was passing from são paulo to são josé dos campos between february 25 and march 18, as the pandemic spreading evolves. x = x , ρ 0 = 10 2 , α = 2.90 × 10 3 , s = 1, λ = 0.72 × 10 −1 , z = 50, τ = 50, τ 1 = 69, j 1 = 9.24,ρ 0 = 1, and σ = 6 (black solid curve). we also depicted the cumulative cases data from the city of são paulo (blue dots), since february 25 (day 1), 2020 until august 02, 2020 (day 160) [33] . in the right panel we present the cumulative cases solutions for model ii with t = 2τ (time in days), d = 1, x = x , ρ 0 = 10, α = −117.38, s = 1, λ = 0.82 × 10 −1 , z = 50, τ = 85, τ 1 = 77.5, j 2 = −9.24,ρ 0 = 1, and σ = 6 (red solid curve). besides, we depicted the cumulative cases data from the city of são josé dos campos (blue dots), since march 18 (day 1), 2020 until august 02, 2020 (day 137) [33] . in this work we introduced a modified version of the diffusion equation, mediated by a lifshitz scaling together with the diffusion coefficient φ(τ ). the diffusion time σ is analogous to the so-called fictitious time in stochastic quantization theories [36] . we were able to find an analytic solution for this diffusion equation, and to use the standard gaussian curves to interpret σ as the lockdown time, if such an equation is applied to model pandemic cases. therefore, we investigate two possible models with φ(τ ) having (anti)kink, and double (anti)kink-like profiles. these models were used to fit real active cases data of covid-19 from three different countries (germany, spain, and brazil). we successfully depicted the active cases curves for germany and spain up to may 02, 2020, and use them to constraint some of our free parameters to generate curves for brazil. then, we predicted four scenarios for the advance of the pandemic in brazil, based on 30 and 90 lockdown days. these scenarios alerted for a potential escalation of the pandemic in brazil if no lockdown measure is taken. moreover, the solution which best fitted the active cases data up to may 02, 2020, is the red curve from fig. 9 , where it was considered 90 lockdown days. such a solution predicted that the pandemic would be fully controlled after day 198 (august 28, 2020). from the previous analyses we are able to observe how crucial the lockdown measures are to flatten the active cases curves and to control the pandemic spread, corroborating with the remarks from [9] . we also applied our model in a new phase of the pandemic, where the virus is moving towards innermost regions of the countries. in this application we considered several diffusion processes interacting through free sources. each one of these diffusion processes corresponds to the spreading of sars-cov-2 in a given city. as a simplest case to model, we worked with constant sources and understood them as proportional to the so-called basic reproductive rate at the beginning of the pandemic (r 0 ). in order to exemplify our model we built the cumulative cases curves of two cities from brazil -são paulo and são josé dos campos, and compare them with real data. our results unveil that this pandemic model can be successfully applied in the context of coupled spreading of covid-19. it is relevant to point that the lifshitz scaling exponent was essential to depict the numerical curves here studied. moreover, we also verified that the double (anti)kink-like profiles were able to fit better the real active cases data than the single (anti)kink-like solutions. consequently, we can conjecture that multiple anti(kink)-like models, like those introduced in [37] , would improve the level of precision of our active cases curves, and would enable us to model multiple pandemic phases. another interesting perspective consists in investigate solutions for the diffusion equation derived from models with multiple lump like solutions, such as those presented in [38] . moreover, the methodology here adopted can be applied to other pandemics, as well as to other covid-19 data set like new daily cases, for instance. covid-19 dashboard the potential impact of the covid-19 epidemic on hiv, tb and malaria in low-and middle-income countries report 13 -estimating the number of infections and the impact of non-pharmaceutical interventions on covid-19 in 11 european countries contacts in context: large-scale setting-specific social mixing matrices from the bbc pandemic project expected impact of covid-19 outbreak in a major metropolitan area in brazil data-driven study of the covid-19 pandemic via age-structured modelling and prediction of the health system failure in brazil amid diverse intervention strategies coronavirus disease 2019 (covid-19) dynamics considering the influence of hospital infrastructure double sine gordon model, in solitons, topics in current physics germany: active coronavirus cases what are the lockdown measures across europe? spain: active coronavirus cases brazil: active coronavirus cases ministério da saúde covid-19 info metapopulation modeling of covid-19 advancing into the countryside: an analysis of mitigation strategies for brazil evaluating reduction in covid-19 cases by isolation and protective measures in são paulo state, brazil, and scenarios of release stochastic quatization we inform the credit statements about the preparation of this manuscript bellow • m.a. anacleto conceptualization, writing -review & editing, and brito conceptualization, investigation, writing -original draft, writing -review & editing, resources, supervision, and project administration de queiroz conceptualization, investigation, writing -original draft, writing -review & editing, and project administration passos conceptualization, writing -review & editing, and project administration we would like to thank cnpq, capes and pronex/cnpq & paraiba state research foundation (grants no. 165/2018 and 0015/2019), for partial financial support. maa, fab, ep and jrls acknowledge support from cnpq (grant nos. 306962/2018-7, 312104/2018-9, 304852/2017-1 and 420479/2018-0, respectively). the authors also would like to thank the anonymous referees for thoughtful comments which undoubtedly raised the quality of this work. key: cord-257940-12nf27j4 authors: schwendicke, falk; krasowski, aleksander; gomez rossi, jesus; paris, sebastian; kuhlmey, adelheid; meyer-lückel, hendrik; krois, joachim title: dental service utilization in the very old: an insurance database analysis from northeast germany date: 2020-09-30 journal: clin oral investig doi: 10.1007/s00784-020-03591-z sha: doc_id: 257940 cord_uid: 12nf27j4 objectives: we assessed dental service utilization in very old germans. methods: a comprehensive sample of 404,610 very old (≥ 75 years), insured at a large statutory insurer (allgemeine ortskrankenkasse nordost, active in the federal states berlin, brandenburg, mecklenburg-western pomerania), was followed over 6 years (2012–2017). our outcome was the utilization of dental services, in total (any utilization) and in five subgroups: (1) examinations and associated assessment or advice, (2) restorations, (3) surgery, (4) prevention, (5) outreach care. association of utilization with (1) sex, (2) age, (3) region, (4) social hardship status, (5) icd-10 diagnoses, and (6) german modified diagnosis-related groups (gm-drgs) was explored. results: the mean (sd) age of the sample was 81.9 (5.4) years. the utilization of any dental service was 73%; utilization was highest for examinations (68%), followed by prevention (44%), surgery (33%), restorations (32%), and outreach care (13%). utilization decreased with age for nearly all services except outreach care. service utilization was significantly higher in berlin and most cities compared with rural municipalities, and in individuals with common, less severe, and short-term conditions compared with life-threatening and long-term conditions. in multi-variable analysis, social hardship status (or: 1.14; 95% ci: 1.12-1.16), federal state (brandenburg 0.85; 0.84–0.87; mecklenburg-western pomerania: 0.80; 0.78–0.82), and age significantly affected utilization (0.95; 0.95–0.95/year), together with a range of co-morbidities according to icd-10 and drg. conclusions: social, demographic, regional, and general health aspects were associated with the utilization of dental services in very old germans. policies to maintain access to services up to high age are needed. clinical significance: the utilization of dental services in the very old in northeast germany showed significant disparities within populations. policies to allow service utilization for sick, economically disadvantaged, rural and very old populations are required. these may include incentives for outreach servicing, treatment-fee increases for specific populations, or referral schemes between general medical practitioners and dentists. electronic supplementary material: the online version of this article (10.1007/s00784-020-03591-z) contains supplementary material, which is available to authorized users. for decades, interventions to improve dental health have been focused on children and adolescents, with widely acknowledged success in many high-income countries. while adults and older individuals also benefitted from a general improvement in oral health, showing a reduced number of restored or missing teeth [1, 2] , data on the resulting treatment needs in these populations are scarce. especially for the very old, defined as those aged 75 years or older, there is very limited knowledge on their needs for and utilization of dental service. this group of very old, notably, is the only growing one in many high-income countries, with remarkably complex oral health dynamics: retaining an increasing number of teeth up to such high age, this group is, oftentimes suddenly, affected by general health deterioration, impacting on the capability for oral self-care as well as the physical abilities to utilize in-office dental care [3] [4] [5] [6] . in a previous study and building on claims data, we found a disparate utilization of prosthetic services in the very old, with those aged 85 years or older, those living rural, and those with severe general health conditions utilizing prosthetic services, by large, to a lower degree than younger, urban living and only limitedly sick seniors [7] . the only service the former group used more often was maintenance of existing prosthetics. notably, claims data come with a range of possible limitations, e.g., selection bias, confounding bias, or misclassification bias. however, employing claims data allows to investigate groups which are otherwise hard to represent, e.g., the very old, the sick, and the rural living ones. claims data also come with robust sample sizes and represent everyday care. they also suffer from limited risks of recollection or reporting bias and have a high generalizability for their respective healthcare setting [8, 9] . in the present study, we used claims data from a large health insurance in northeast germany to assess dental service utilization in the very old. we hypothesized that utilization differed according to age, general health, socioeconomic status, and place of living. for reasons of comparability, the design and conduct of this study largely aligns with that of a previous publication on prosthetic treatment patterns in the same population [7] . the investigated cohort was evaluated based on routinely collected claims data from a statutory (public) health insurance in germany. individuals aged 75 years or older from one large insurer, the aok nordost, were followed over 6 years (2012 to 2017). the aok nordost is a regional branch of the allgemeine ortskrankenkasse (aok), acting mainly in the northeast of germany in the federal states of berlin, brandenburg, and mecklenburg-western pomerania. our reporting follows the record statement [10] . the aok nordost insures around 1.8 million individuals from the described three federal states. insured individuals may, however, also move into other areas of germany, which is why for our geographic analyses only individuals living in these federal states between 2012 and 2017 were included. the area of interest encompasses the german capital, berlin, and two rural states, brandenburg and mecklenburg-western pomerania, with only few larger cities (> 70,000 inhabitants). all three states are considered economically weak in comparison with other parts of germany. data for this study were claims data, including claims from 1 january 2012 to 31 december 2017. data were routinely collected and provided under ethical approval in a pseudonymized form using a data protection cleared platform via the scientific institute of the aok nordost, the gewino. a comprehensive sample of very old, aged 75 years or above, insured with the aok nordost in 2012, was drawn and followed over 6 years. no further eligibility criteria were defined. variable ascertainment was only possible via insurance base data and claims data. the database had been curated for plausibility at gewino and once more by the study team. no formal sample size estimation was performed given this being a comprehensive sample. our outcome was the relative utilization (in % of the population) of dental services. within the statutory german insurance, dental services are provided on a fee-per-item basis using fee items catalogs of the statutory or private german insurance [11, 12] . the vast majority (88%) of patients are statutorily insured. for the statutory insurance, all items are drawn from the fee item catalog bewertungsmaßstab (bema), which contains a large range of granular items comprising (1) examinations, assessment and advice, radiographic evaluations etc. (examinations); (2) restorative dentistry (restorations), note that within german insurance coding, crowns are not subsumed under "restorations" and hence there is no overlap between this service group and our previous analysis on prosthetic dentistry; (3) oral surgery and medicine (surgery); (4) prevention (for adults, the only preventive measure available until 2015 was removal of calculus; in 2015, further fee items (focusing on oral hygiene measurement, and oral hygiene plan, denture cleaning, and fluoride application) were introduced but these were not available for the present analysis); and (5) outreach care. further items include, for example, periodontal treatment, prosthetic therapies, and adjunct measures. we here report on any utilization in bema (min 1 item claimed/year) as well as stratified along the item blocks 1-5. as this is the first detailed analysis on dental service utilization in the very old in northeast germany, we provide largely descriptive analyses. the utilization of dental services was assessed according to following independent variables: (1) sex (male/female); (2) age (in years) in each year of follow-up; (3) region, we used municipalities as regional units, mainly as on a lower (more granular) spatial level only few individuals were retained in some areas. municipalities included the capital berlin (with over 3.5 million inhabitants), medium-sized cities (70,000-200,000 inhabitants), and rural areas. further analyses were performed on federal state level; (4) social hardship status (income < 1246 euro/month per capita in 2019); (5) icd-10 diagnoses, derived from outpatient diagnostic data; (6) inpatient hospital diagnoses and treatments, derived from german modified diagnosis-related groups (gm-drgs). the gm-drgs classify diseases in groups of similar pathogenesis, characteristics, and treatment complexity, and are mainly used for reimbursement reasons. only the 25 most frequently recorded icd-10 and gm-drg codes were used. the data used for this study were provided by the gewino using a data protection approved storage and analysis platform after cleaning and consistency controls. data were pseudonymized and included individuals' age, sex, social hardship status, spatial code of their place of living (allowing classification into municipalities), all bema items claimed per year as well as icd-10 codes and gm-drgs for each year, among further variables. comparability of data between different years and data consistency was given. a comprehensive sample had been used, and neither participants nor providers were aware that the collected claims data will be used for routine data analyses later on. the data collection is not prone to selection and detection bias. however, given this being claims data from only one insurance, the overall population of very old germans differs and data may be affected by biases associated with claims data, as laid out above and in the discussion. no further measures against these biases could be taken. the statistical analysis was performed on a sample (n = 404,610) of the database provided by aok nordost. the only inclusion criterion was that an individual had to be insured in the year 2012 and had to be aged 75 years or above at this point. for the descriptive analysis of utilization of dental services, we considered an individual to have consumed a particular service if at least once during the period 2012 to 2017 the provision of such a service was claimed. descriptive statistics of age groups were computed based on the age distribution in 2012. an individual was assigned to having a social hardship if the individual was assigned to this status at least once during the period 2012 to 2017. for geographical analysis, we excluded all individuals that relocated from one of the federal states (berlin, brandenburg, and mecklenburg-western pomerania) to another federal state, thereby decreasing the sample size to 390,044. however, we did not correct for relocations within the three federal states during the observational period. for each particular outpatient diagnosis (icd-10 codes) and inpatient hospital diagnosis and treatment (gm-drgs), we summed up all claims and ranked them from most to least frequent. we then selected the 25 most frequent diagnoses each (in total 50) and computed for each of them the number of individuals that were assigned to having a diagnosis, respectively, treatment, during 2012 to 2017. we applied logistic regression, a method to model a binary outcome variable as a linear combination of predictor variables. the response variable was the utilization of any type of dental services claimed by an individual at least once in the year 2012. as predictor variables we included age, sex, being deceased, social hardship status, federal state (note that we allowed the category "other" for relocated individuals), and the described outpatient and inpatient hospital diagnosis variables, all of them referring to the year 2012. all analyses, modeling, and visualization were performed using python (version 3.7, available at http://www.python.org) and auxiliary modules from its scientific computing ecosystem. overall, 404,610 very old (75 years or older) individuals were followed over a period of up to 6 years (173,733 of these did not survive follow-up). the mean (sd, median, min, max) age of the sample was 81.9 (5.4, 81, 75, 109) years. the population comprised significantly more females than males and those aged 75-84 years old than those aged 85 years or older. about one-third lived in berlin, and the other two-thirds in the more rural brandenburg and mecklenburg-western pomerania. social hardship status was claimed by nearly half of the population at least once during the follow-up period (table 1 ). our sample was overall more female and much older and claimed far more hardship status than the national average. the utilization of any dental service was 73%; utilization was highest for examinations (68%), followed by prevention (44%), surgery (33%), and restorative (32%) and outreach care (13%). utilization decreased with age for nearly all services except outreach care (fig. 1) . utilization of restorations, surgery, and prevention decreased by 75-80% (in relative terms, e.g., from 36% to 6% for restorations) between age 75 and 95 years; the decrease after age 95 years was limited. a slightly less pronounced, but nevertheless consistent, decrease was found for examinations. in contrast, outreach care increased and was, at age 95 years or above, the main service (together with examinations, which one would assume is the minimum consequence of outreach care). utilization was further different between regions ( table 1 , fig. 2 ). utilization of any dental service was generally higher in cities than rural areas, and highest in berlin and three other urban municipalities (rostock, potsdam, schwerin). utilization further differed geographically according to specific services. utilization of restorations was nearly 50% increased in certain cities and one rural southwestern municipality compared with most other rural areas. surgical services were provided more often in berlin and the south as well as cities in general; a similar pattern was observed for preventive services. for outreach care, no such strict pattern was observed; certain cities as well as a stretch of municipalities along the coastline showed higher utilization. utilization of any dental service was assessed according to icd-10 codes (table 2) . utilization was higher for the majority of codes, e.g., for eye conditions (e.g., presbyopia, cataract, astigmatism), gonarthrosis, cox-arthrosis, benign hypertension, anti-coagulants therapy, varicose, prostate hyperplasia, osteoporosis, hyperlipidemia and hypercholesterinemia and unspecified chronic pain. a similar pattern was found for most specific services. notably, individuals with dementia showed a similar utilization with regard to any services, but mainly received examinations, not restorative or surgical care. the same was found for patients with urinary incontinence. for outreach care, an opposite pattern was observed, with higher utilization by those with dementia and incontinence, and lower utilization by those with eye conditions, for example ( table 2) . we further assessed the utilization of any dental service stratified according to different gm-drgs (table 3) . utilization of any service was higher in participants hospitalized for non-severe gastrointestinal ulcerations, non-severe arrhythmia, bronchitis, non-severe hypertension, syncope, non-severe renal insufficiency, and non-complicated cardial diagnostics or eye operations. utilization was lower in patients with severe gastrointestinal ulcerations as well as severe heart insufficiency. these trends of higher or lower utilization were similar for other services, except outreach care, where a different pattern emerged: utilization was higher in patients with non-severe but also severe ulcerations, paraplegia/tetraplegia, non-severe hypertension, infections, head or skin injuries, joint operations, apoplexy, and geriatric rehabilitation. it was lower in patients with bronchitis (table 3) . in multi-variable analysis, social hardship status (or: 1.14; 95% ci: 1.12-1.16), federal state (brandenburg 0.85; 0.84-0.87; mecklenburg-western pomerania: 0.80; 0.78-0.82) and age significantly affected utilization (0.95; 0.95-0.95/year), together with a range of co-morbidities according to icd-10 and gm-dgrs (table 4, table s1 ). pseudo-r 2 indicated that the model generally had limited explanatory power (r 2 = 0.15). understanding dental service utilization in specific populations and groups may allow to increase access to the right services for every individual, thereby improving health and services' efficiency and equitability [13] . the present study tried to evaluate how factors driving services' needs (age, sex, general health) and access on patient level (income and financial means, place of living) and system level (physical and organizational) impact on utilization [14, 15] . we hypothesized that the utilization of dental services in the very old was associated with an individual's age, general health status, place of living, and social status. moreover, we assumed to find service-specific disparities. we confirm these hypotheses; social, demographic, regional, and general health aspects were associated with the utilization of dental services in very old germans. a number of aspects should be discussed. first, utilization in this specific group was comparably high; in general, dental service utilization in germany is higher than that in most other countries, likely due to the setup of the service provision, with most services being available at no costs at all to the patient [16]. moreover, regular consumption of dental services is incentivized using a bonus scheme, with patients getting a discount on their out-of-pocket expenses for prosthetic services in case they can demonstrate a history of regular yearly checkups. such incentive will be especially efficacious in old individuals, who either have or expect to have prosthetic services with higher likelihood than younger ones. we also found only minimal changes in the age-specific utilization over the 6-year period; that is, seniors of similar age did not show considerably increased utilizations in 2012 compared with 2017, for example. the only detectable increase occurred between 2012 and 2013, most likely associated with a general policy shift in dental healthcare in germany (an entry fee existing until 2012, with patients paying 10 euro to the practice-which passed it on to the insurer-whenever entering the practice for the first time in a quarter of a year had been abolished in 2013). these findings of rather constant utilization over the first half of the last decade as well as the increase in utilization of dental services from 2012 and 2013 are in line with previous research [17, 18] . our findings are in so far relevant, as a number of major policy shifts targeting the very old requiring care assistance have been introduced between 2013 and 2015, the effects of which our analysis did not capture (so far). this might be as we only included individuals aged 75 years or older in 2012 and followed them for 6 years (i.e., those entering this group later on were not included), but also as we did not focus on those requiring care assistance, i.e., probably "diluted" their relevance in our analysis. it would be relevant to re-assess this cohort, expanding it to individuals aged 75 years or older in 2018 and focusing on only those receiving care assistance. we find a drastic and only limitedly service-specific decrease in utilization with age; individuals aged 85 years, for example, consumed only a fraction of services compared with those age 75 years. notably, from age 95 years onwards utilization was fairly stable, indicating a possible "survivor" effect. the only exception from these observations was outreach care, as discussed below. age is associated with an increasing prevalence of chronic and severe diseases or hospitalization [19] . in line with our previous analysis on prosthetic services, such severe general health conditions (e.g., severe gastrointestinal ulcerations as well as severe heart insufficiency) were found to significantly decrease utilization. notably, for most other (especially icd-10 coded) conditions, the overall utilization was unaffected. this might be as icd-10 codes were derived from ambulatory assessments, where individuals need to attend their general practitioners and hence show some kind of mobility and self-capability. moreover, individuals with dementia (and incontinency) showed reduced utilization of therapeutic services (but not examinations). this might be as these individuals do not accept more intense (and time consuming) care for treatment. we further assessed the impact of social hardship status on utilization. such status is a proxy for low income. it has been found associated with increased utilization of prosthetic services, as individuals with this status usually pay very low or no additional fee at all for any prosthetic service; that is, financial utilization barriers for this type of dental treatment are very low or absent [7] . for the present analysis, hardship status was used only as a social marker, as the analyzed dental services (examination, restorations, surgery, prevention, outreach care) are coming at no costs for all statutorily insured individuals, regardless of their age. [29] this is a remarkable difference of the german compared with many other healthcare systems, where retirement oftentimes means loss of professionally supported health insurance [20] [21] [22] and a subsequent collapse of service utilization [23] . it is noteworthy that utilization for those with hardship status was found significantly increased in multivariable analysis (in bivariate analyses this was less clear, indicating possible confounding by age, place of living, or health status, for example). as those with low social status are also likely to show the poorest oral and general health [24], it is highly relevant to find them to consume services more often, too. it is beyond this study to elucidate the reasons underlying this utilization. notably, though, existing public policies to support healthcare utilization in vulnerable groups in germany, e.g., those with chronic diseases [25] , do not capture those with economic constraints and poor oral health, i.e. cannot be at the heart of this association. independently of the found increased utilization, policy makers may want to revisit such policies and to strengthen dental service utilization for the very old, the very sick, and the very poor. we also found an association between utilization and place of living [13] . such association has been assumed to be grounded in rural areas being underserviced due to workforce shortages while urban areas suffer from provider clustering and associated supply-side-induced demand [26, 27] . we confirm such ruralurban disparities for any service utilization in the very old. the two rural federal states in our study, brandenburg and mecklenburg-western pomerania, show much lower dentist densities than berlin [28] , possibly explaining our findings. notably, utilization in the whole population (not only the very old) has been found to follow the opposite pattern, with higher utilization in the two rural states than in berlin [17] . hence, the observed inequalities seem to be moderated by age: older individuals seem to seek care more often, but are not able to physically access it in rural areas, while younger individuals could access it more easily in urban areas, but are not seeking care. we want to highlight that our analyses on smaller spatial level (municipalities) showed a more nuanced picture, with some rural areas showing high utilization of specific (but not all) services. we are so far unable to entangle possible reasons underlying this observation, which may be grounded in local dentist densities (some municipalities show surprisingly high densities) or a locally increased proportion of dentists with specific contractual agreements with care homes (thereby increasing access to care for the very old). more in-depth analyses seem warranted to first confirm and then explain such peculiar patterns, as they may allow to identify local best practices which could be translated to regional or national level. we identified service-specific utilization patterns not only across regions, as described, but also age. our findings of a generally decreasing utilization between age 75 years and 95 years have been identified before, with utilization of dental therapeutic services decreasing by around 50% along this age span in a national sample [17] . in the national sample, restorative care was provided far more often than surgical care, while we found restorative care being consumed to a similar degree like surgical care. this might be as our sample was generally older and also represented a different target population (see below). we assume that these two factors drive a treatment concept where maintaining teeth (using restorative care) is deprioritized while achieving an overall pain free status (by removing teeth, for example) is getting more important (and usually also being the only available option). notably, prevention (which was only calculus removal in the present study) continued to be provided up to high age (albeit to a lower intensity). the only service where utilization was increasing with age was outreach care, while this seemed to allow for only very limited provision of therapies. it is relevant to understand the drivers behind treatment patterns in outreach care, and it may not be sufficient to only incentivize outreach visits, but also support outreach management or referral concepts for those requiring more complex care. in light of the covid-19 pandemic and the near-global shutdown of any dental visits (except for emergencies) to care homes (also in germany), outreach care is likely to be re-evaluated with regard to its benefits and risks. overall, our study calls for a range of possible policy and research initiatives: first, healthcare policy and decision makers should install incentives to provide services to the high needs elderly population. this may come by increasing single treatment fees for this group, or more generally by making outreach services more attractive. the latter may be realized by increasing fees once more or trialing and allowing different kinds of servicing, e.g., involving task delegation to assistance personnel. outreach care should further be provided not only to individuals in long-term care centers (nursing homes) but also to those residing at home (which is the vast majority of elderly). similarly, referral schemes between general medical practitioners and dentists may be helpful to identify high-risk individuals; mandatory follow-ups after such referrals may make sure that sick and remote older individuals (who seldom proactively seek care) are not plainly overlooked by standard dental healthcare. integrated service models (for example oral and dental hygiene enforcement for patients at risk for pneumonia) should further be strengthened. dental research, on the other hand, is called to action to develop applicable concepts fig. 2 regionally specific utilization of dental services, stratified in services blocks, in northeast germany. relative (in %) any utilization and specific service utilization is shown. larger cities with an increased or decreased utilization compared with the surrounding municipalities are further highlighted by arrows table 2 utilization of dental services according to international disease classification (icd-10, german modification) codes by the very old in northeast germany. any and specific service utilization (in %) is shown icd-10-gm *categories z00-z99 are intended for cases in which facts are indicated as "diagnoses" or "problems" which cannot be classified as disease, injury or external cause under categories a00-y89 **this chapter includes (subjective and objective) symptoms, abnormal results of clinical or other investigations, and inaccurately identified conditions for which there is no classifiable diagnosis elsewhere table 3 utilization of dental services according to german modified diagnosis-related groups (gm-drgs encompassing effective management of dental diseases at optimal infection and transmission control measures. right now, servicing is at a minimal level due to fears of infection and it can be expected that infection control will remain a highly relevant topic in this vulnerable population even when covid-19 is finally brought behind us. moreover, dental research should develop and evaluate the described complex care models involving delegation or cooperation. a number of initiatives are currently underway in germany in this direction (e.g., https://innovationsfonds.g-ba.de). further, primary and secondary prevention models in this group should be enhanced; currently prevention concepts in the elderly are by large identical with those in younger individuals. policy makers may want to revisit such age-group-specific prevention concepts when they are available. generally, we see a great need to emphasize prevention in this group (based on our findings, prevention was near-absent for the very old in the northeast). dentists and dental bodies may want to actively participate in such research and also the implementation of possible policies, especially considering that with the very old, there is a growing group with high needs who can truly benefit from dental care. this study has a number of strengths and limitations. first, this is one of few longitudinal studies evaluating dental service utilization in very old individuals. our cohort involved over 400,000 individuals from three federal states spanning an area of similar size as austria or the netherlands and belgium combined. second, we evaluated a range of demographic, social, general health, and regional factors, some of which (drgs, icd-10) have not routinely been employed when evaluating dental healthcare. third, and as a limitation discussed above, claims data suffer from a range of biases. provided and claimed treatment cannot be equated with needs or morbidity. exploring causality is only limitedly possible, and within the present (largely descriptive) analyses, this was also not within our scope (the available longitudinal data may permit some more in-depth analyses in the future). any identified bivariate association may suffer from confounding bias, and even the performed multivariable analysis showed only limited explanatory value, likely as further relevant factors (e.g., medication, care status) were not available and accounted for, or as available factors (e.g., social hardship status, place of living) came with very limited granularity. fourth, individuals insured by aok nordost are not fully representative for other individuals from the same target area or even the whole of germany: more affluent people are often not statutorily insured (there is a minimum income level defined as entry barrier into private insurances in germany). this may affect the individual's health status and his or her utilization behavior (reflecting health literacy, but also specific incentives set by insurers towards seeking or avoiding care) as well as the number and type of services provided by the dentists (as services are remunerated differently). the northeast of germany is overproportionally old and, as mentioned, economically comparably weak (notably, there is a significant economic disparity within the northeast, too, which our data reflect on). the rural parts of the northeast suffered from emigration to other areas of germany especially after the reunification, while berlin experienced an over-proportional immigration in the 1960s from aboard as well as the last 20 years from within germany. these specifics will impact service utilization but may not be found to this degree in other areas of germany. future studies on the present dataset may explore them in detail, if possible, to better understand what impact on utilization they have. in conclusion, and within the limitations of this study, social, demographic, regional, and general health aspects were associated with the utilization of dental services in very old germans. we identified consistent and considerable disparities in utilization between populations. policies to allow service utilization also for the sick, economically disadvantaged, rural, and very old should be developed, tested, and employed. competing interests the authors declare that they have no competing interests. ethical approval and informed consent all experiments were carried out in accordance with relevant guidelines and regulations. data collection was ethically approved by the ethics committee of the aok nordost. no informed consent was required for this study given that data were pseudonymized. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. global burden of severe tooth loss: a systematic review and meta-analysis trends in caries experience in the permanent dentition in germany 1997-2014, and projection to 2030: morbidity shifts in an aging society our current geriatric population: demographic and oral health care utilization ageing, dental caries and periodontal diseases elder's oral health crisis. the journal of evidence-based dental practice zahnverlust und prothetische versorgung prosthetic treatment patterns in the very old: an insurance database analysis from germany the limitations of using insurance data for research misclassification in administrative claims data: quantifying the impact on treatment effect estimates the reporting of studies conducted using observational routinely-collected health data (record) statement befundbezogene festzuschüsse als innovatives steuerungsinstrument in der zahnmedizin defining and targeting health care access barriers societal and individual determinants of medical care utilization in the united states barriers to and enablers of older adults' use of dental services can we predict usage of dental services? an analysis from germany dental visits among older u.s. adults, 1999: the roles of dentition status and cost disparity in dental coverage among older adult populations: a comparative analysis across selected european countries and the usa dental care utilization and retirement oral health conditions of community-dwelling cognitively intact elderly persons with disabilities versorgungsprävalenzen bei älteren senioren mit pflegebedarf bekanntmachung eines beschlusses des gemeinsamen bundesausschusses über eine änderung der chroniker-richtlinie accessibility of general practitioners and selected specialist physicians by car and by public transport in a rural region of germany vertragszahnärztlichen versorgung von pflegebedürftigen und menschen mit behinderungen, kzbv/bzäk zahnarztdichte in deutschland nach bundesländern im jahr der wirtschaft: trend zu festsitzenden versorgungen hält an acknowledgments we thank the gewino for providing access to the data within.author contributions the study was conceived by fs. fs, akr, and jk planned the analyses. fs, akr, and jk performed the analyses. all authors interpreted the data. fs wrote the manuscript. all authors read and approved the manuscript.funding open access funding enabled and organized by projekt deal. this study was funded by the bundesministerium für bildung und forschung (bmbf tailohr, az 01gy1802).data availability data used in this study cannot be made available by the authors given data protection rules, but may be requested at the gewino. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-289285-aof7xy13 authors: michaelis, martin; geiler, janina; naczk, patrizia; sithisarn, patchima; leutz, anke; doerr, hans wilhelm; cinatl, jindrich title: glycyrrhizin exerts antioxidative effects in h5n1 influenza a virus-infected cells and inhibits virus replication and pro-inflammatory gene expression date: 2011-05-17 journal: plos one doi: 10.1371/journal.pone.0019705 sha: doc_id: 289285 cord_uid: aof7xy13 glycyrrhizin is known to exert antiviral and anti-inflammatory effects. here, the effects of an approved parenteral glycyrrhizin preparation (stronger neo-minophafen c) were investigated on highly pathogenic influenza a h5n1 virus replication, h5n1-induced apoptosis, and h5n1-induced pro-inflammatory responses in lung epithelial (a549) cells. therapeutic glycyrrhizin concentrations substantially inhibited h5n1-induced expression of the pro-inflammatory molecules cxcl10, interleukin 6, ccl2, and ccl5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with h5n1 replication and h5n1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). glycyrrhizin also diminished monocyte migration towards supernatants of h5n1-infected a549 cells. the mechanism by which glycyrrhizin interferes with h5n1 replication and h5n1-induced pro-inflammatory gene expression includes inhibition of h5n1-induced formation of reactive oxygen species and (in turn) reduced activation of nfκb, jnk, and p38, redox-sensitive signalling events known to be relevant for influenza a virus replication. therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of h5n1 disease. highly pathogenic h5n1 influenza a viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . at least for the first wave of an h5n1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . therefore, antiviral therapy for influenza a viruses including highly pathogenic h5n1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] . the neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza m2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . however, the use of both drug classes is limited by the emergence of resistant virus strains. in seasonal influenza strains, the majority of h3n2 viruses and a great proportion of h1n1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . moreover, a drastic increase in oseltamivir-resistant h1n1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . preliminary data from the united states predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating h1n1 strains to be oseltamivir resistant [14] . h5n1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza a virus strains and treatment-resistant h5n1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . more-over, parenteral agents for the treatment of seriously ill patients are missing. glycyrrhizin, a triterpene saponine, is a constituent of licorice root. it has been found to interfere with replication and/or cytopathogenic effect (cpe) induction of many viruses including respiratory viruses such as respiratory syncytial virus, sars coronavirus, hiv, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . the severity of human h5n1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . delayed antiviral plus immunomodulator treatment reduced h5n1-induced mortality in mice [31] . therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of h5n1. also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza a virus replication and virus-induced pro-inflammatory responses [32] [33] [34] . stronger neo-minophagen c (snmc) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in japan for the treatment of chronic hepatic diseases and is marketed in japan, china, korea, taiwan, indonesia, india, and mongolia. here, we investigated the influence of snmc on h5n1 replication, on h5n1-induced cytokine expression, on h5n1-induced cellular oxidative stress, and on critical h5n1-induced cellular signalling events in human pneumocytes (a549 cell line). glycyrrhizin (stronger neo minophagen c) was obtained from minophagen pharmaceuticals co., ltd. (tokyo, japan). the influenza strain a/vietnam/1203/04 (h5n1) was received from the who influenza centre (national institute for medical research, london, uk). the h5n1 influenza strain a/thailand/ 1(kan-1)/04 was obtained from prof. pilaipan puthavathana (mahidol university, bangkok, thailand). virus stocks were prepared by infecting vero cells (african green monkey kidney; atcc, manassas, va) and aliquots were stored at 280uc. virus titres were determined as 50% tissue culture infectious dose (tcid 50 /ml) in confluent vero cells in 96-well microtiter plates. a549 cells (human lung carcinoma; atcc: ccl-185, obtained from lgc standards gmbh, wesel, germany) were grown at 37uc in minimal essential medium (mem) supplemented with 10% fbs, 100 iu/ml of penicillin and 100 mg/ml streptomycin. human monocytes were isolated from buffy coats of healthy donors, obtained from institute of transfusion medicine and immune haematology, german red cross blood donor center, johann wolfgang goethe-university, frankfurt am main. after centrifugation on ficoll (biocoll)-hypaque density gradient (biochrom ag, berlin, germany), mononuclear cells were collected from the interface and washed with pbs. then, monocytes were isolated using magnetically labeled cd14 microbeads (miltenyi biotec gmbh, bergisch gladbach, germany) following the manufacturer's instructions. monocytes were cultivated in imdm supplemented with 10% pooled human serum, 100 iu/ml of penicillin, and 100 mg/ml streptomycin. the cellular viability was assessed on confluent cell layers with celltiter-gloh luminescent cell viability assay (promega gmbh, mannheim, germany) according to the manufacturers' protocol. cell viability was expressed as percentage of non-treated control. to determine intracellular np localisation, h5n1-infected a549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, mallinckrodt baker b.v., deventer, the netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in pbs) directed against the influenza a virus nucleoprotein (np) (millipore, molsheim, france). an alexa fluor 488 goat anti-mouse igg (h&l) (invitrogen, eugene, oregon, usa) was used (1 h incubation, 1:1000 in pbs) as secondary antibody. nuclei were stained using 49,6-diamidino-2phenylindole (dapi) (sigma-aldrich chemie gmbh, munich, germany). fluorescence was visualised using olympus ix 1 fluorescence microscope (olympus, planegg, germany). for flow cytometric analysis, the same antibodies were used. the cytopathogenic effect (cpe) reduction assay was performed as described before [34] . confluent a549 cell monolayers grown in 96-well microtitre plates were infected with influenza a strains at the indicated multiplicities of infection (mois). after a one hour adsorption period, cells were washed to remove non-detached virus. the virus-induced cpe was recorded at 24 h post infection (p.i.). unless otherwise stated, a549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. for time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus. total rna was isolated from cell cultures using tri reagent (sigma-aldrich, munich, germany). real time pcr for h5 was performed using described methods [35] . the following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-fam-tca aca gtg gcg agt tcc cta gca-tamra. the fraction of cells with fractional dna content (''sub-g1'' cell subpopulation) indicates cytotoxicity. sub-g1 cells are considered to be dead (usually apoptotic) cells. cells were fixed with 70% ethanol for two hours at 220uc. the cellular dna was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (facscalibur, bd biosciences, heidelberg, germany). caspase activation was measured using the caspase-glo 8, 9, or 3/7 assays (promega, mannheim, germany) following the manufacturer's instructions. cell culture supernatants were collected and frozen at 280uc. cytokines/chemokines were quantified by specific elisa duo sets (r&d systems gmbh, wiesbaden, germany) following the manufacturer's instructions. nfkb activity was investigated in h5n1 (moi 0.01)-infected cells by quantification of the nfkb subunits rel a (p65) and nfkb1 (p50) from nuclear extracts using the transam tm transcription factor dna-binding elisas (active motif, rixensart, belgium). nuclear extract were prepared using the nuclear extract kit (active motif, carlsbad, ca, usa) following the manufacturer's instruction. cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; bd biosciences, heidelberg, germany). monocytes (1610 6 in 100 ml of imdm with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. after a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with pbs containing 0.3% tritron x-100. then, nuclei were stained with 49,6-diamidino-2phenylindole (dapi). the upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with pbs. the number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an olympus ix 1 fluorescence microscope (olympus, planegg, germany). cells were lysed in triton x-sample buffer and separated by sds-page. nuclear extract were prepared using the nuclear extract kit (active motif, carlsbad, ca, usa) following the manufacturer's instruction. proteins were detected using specific antibodies against bactin (sigma-aldrich chemie gmbh, munich, germany), jnk, phosphorylated jnk, p38, or phosphorylated p38, (all purchased from new england biolabs gmbh, frankfurt am main, germany) and were visualised by enhanced chemiluminescence using a commercially available kit (amersham, freiburg, germany). reactive oxygen species (ros) were detected using the image-it live green reactive oxygen species kit (molecular probes, distributed by invitrogen, karlsruhe, germany). two groups were compared by t-test. more groups were compared by anova with subsequent student-newman-keuls test. the a549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type ii pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. if not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect a549 cell viability (data not shown) but clearly decreased cpe formation in a549 cells infected with the h5n1 influenza strain a/thailand/1(kan-1)/04 at mois of 0.01, 0.1 or 1 ( figure 1a ). similar results were obtained in a549 cells infected with strain a/vietnam/1203/04 (h5n1) (suppl. figure 1a) . staining of a549 cells for influenza a nucleoprotein 24 h after infection with strain h5n1 a/thailand/1(kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza a nucleoprotein positive cells ( figure 1b) . to examine the influence of glycyrrhizin on virus progeny, a549 cells were infected with the h5n1 influenza strain a/ thailand/1(kan-1)/04 at moi 0.01 or moi 1 and infectious virus titres were determined 24 h post infection ( figure 1c ). while glycyrrhizin in concentrations up to 50 mg/ml did not affect h5n1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (moi 0.01: 13-fold reduction, moi 1: 10-fold reduction). next, influence of glycyrrhizin on h5n1 replication was confirmed by the detection of viral (h5) rna using quantitative pcr. only glycyrrhizin concentrations $100 mg/ml significantly reduced figure 1b) or h5n1 a/vietnam/1203/04-infected (suppl. figure 1c ) a549 cells (moi 0.01) 24 h post infection. time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( figure 1d ). addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect h5n1 replication. for investigation of h5n1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: cxcl10 (also known as interferon-cinducible protein 10, ip-10), interleukin 6 (il6), interleukin 8, (il8; also known as cxcl8), ccl2 (also known as monocyte chemoattractant protein 1, mcp-1), and ccl5 (also known as rantes). a549 cells were infected with h5n1 a/thailand/ 1(kan-1)/04 or h5n1 a/vietnam/1203/04 at moi 0.01, 0.1, or 1. glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. cytokine expression was detected 24 h post infection by elisa. glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in h5n1-infected cells in a dose-dependent manner (figure 2, figure 3a ). effects were more pronounced at lower mois. notably, expression of all cytokines except il8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml figure 3a ) although these glycyrrhizin concentrations had no effect on h5n1 replication in a549 cells (figure 1, figure s1 ). cytokine expression by influenza a virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza a virus pathogenicity [5, 39] . in a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of h5n1 a/thailand/1(kan-1)/04 (moi 0.1)-infected a549 cells through 8 mm filters. monocyte migration towards supernatants of h5n1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. treatment of h5n1infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( figure 3b ). influenza viruses including h5n1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( figure 4a-c) . glycyrrhizin concentrations $100 mg/ml inhibited h5n1 a/thailand/1(kan-1)/04 (moi 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in a549 cells as determined 24 h post infection ( figure 4a-c) . lower glycyrrhizin concentrations did not affect h5n1-induced apoptosis. the detection of cells in sub-g1 phase resulted in similar findings ( figure 4d ). substances that inhibit h5n1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of rnp complexes [34, 42] . in accordance, glycyrrhizin also interfered with nuclear export rnp at moi 1 ( figure s2 ). similar results were obtained in moi 0.01 h5n1 a/thailand/1(kan-1)/04infected cells ( figure s3 ). influence of glycyrrhizin on h5n1-induced activation of nuclear factor kb (nfkb), p38, and on h5n1-induced cellular reactive oxygen species (ros) formation activation of nfkb, p38, and jnk have been associated with influenza a virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . while glycyrrhizin did not influence nfkb activity in non-infected a549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited nfkb activation in h5n1-infected cells ( figure 5a ). moreover, glycyrrhizin inhibited h5n1-induced phosphorylation of the mapks p38 and jnk ( figure 5b ). in addition to their roles during influenza a virus replication and virus-induced cytokine/chemokine expression, nfkb, p38, and jnk are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . antioxidants had been already found to interfere with influenza a virus-induced signalling through nfkb, p38, and jnk, with influenza a virus replication, and with influenza a virus-induced pro-inflammatory gene expression [32] [33] [34] . since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with h5n1-induced ros formation. indeed glycyrrhizin exerted clear antioxidative effects in h5n1 (moi 0.01)-infected cells ( figure 5c ) causing significant reduction of ros formation already at a concentration of 25 mg/ml ( figure 5d ). here, we show that glycyrrhizin inhibits the replication of highly pathogenic h5n1 influenza a virus, h5n1-induced apoptosis, and h5n1-induced expression of pro-inflammatory cytokines in lung-derived a549 cells. after intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . therefore, the glycyrrhizin concentrations found to interfere with h5n1 replication and h5n1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. notably, although higher glycyrrhizin concentrations were needed to interfere with sars coronavirus replication [22] than with h5n1 replication, beneficial results were reported in glycyrrhizin (snmc)-treated sars patients in comparison to sars patients who did not receive glycyrrhizin [23] . notably, investigation of different glycyrrhizin derivatives against sars coronavirus led to the identification of compounds with enhanced antiviral activity [53] . therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs. experimental results suggested that glycyrrhizin might be able to affect seasonal influenza a virus disease by antiviral and immunomodulatory effects [26, 27] . mice were prevented from lethal h2n2 infection by glycyrrhizin although no influence on virus replication was detected. the mechanism was suggested to be induction of interferon-c in t-cells by glycyrrhizin [54] . moreover, glycyrrhizin was shown to influence seasonal influenza a virus replication through interaction with the cell membrane [25, 28] . however, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. since glycyrrhizin addition during the adsorption period did not influence h5n1 replication in our experiments it appears not likely that membrane effects contribute to anti-h5n1 effects detected here in lower concentrations. our results rather suggest that glycyrrhizin interferes with h5n1-induced oxidative stress. influenza a virus (including h5n1) infection induces ros formation. antioxidants were found to inhibit influenza a virus replication and influenza a virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . here, glycyrrhizin interfered with h5n1-induced activation of nfkb, p38, and jnk representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza a virus replication and influenza a virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . glycyrrhizin 50 mg/ml significantly reduced h5n1-induced activation of nfkb. in addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with h5n1-induced ros formation and with phosphorylation of the redox-sensitive mapks p38 and jnk. in our model, activation of p38 appears to be critical for h5n1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant n-acetyl-cysteine (nac) [34] . interestingly and in contrast to glycyrrhizin, nac failed to inhibit h5n1 replication or h5n1-induced cytokine/chemokine expression in therapeutically relevant concentrations. glycyrrhizin diminished h5n1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with h5n1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. therefore, h5n1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ros formation than h5n1 replication. indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . in a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with h5n1-induced pro-inflammatory gene expression but not with h5n1 replication in human monocyte-derived macrophages [57] . in addition, other immunomodulatory treatment regimens that did not influence h5n1 replication reduced mortality in h5n1-infected mice [31, 58] . therefore, glycyrrhizin represents a potential additional treatment option that interfers with both h5n1 replication and h5n1induced expression of pro-inflammatory cytokines in lung cells. interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic cd8 + t-lymphocytes. global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper nk cell cytolytic activity [60] . in this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] . in conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation snmc) interfere with highly pathogenic h5n1 influenza a virus replication and h5n1-induced proinflammatory gene expression at least in part through interference with h5n1-induced ros formation and in turn reduced activation of p38, jnk, and nfkb in lung cells. since we used the clinical formulation snmc effects of other ingredients like glycin or cystein cannot be excluded. vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza a virus pandemic [61] . anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of h5n1-caused disease. the threat of avian influenza a (h5n1). part i: epidemiologic concerns and virulence determinants the threat of avian influenza a (h5n1): part ii: clues to pathogenicity and pathology the threat of avian influenza a (h5n1). part iii: antiviral therapy the threat of avian influenza a (h5n1). part iv: development of vaccines pathogenesis of emerging avian influenza viruses in mammals and the host innate immune response of chickens and men: avian influenza in humans vaccines for pandemic influenza. the history of our current vaccines, their limitations and the requirements to deal with a pandemic threat the pandemic influenza vaccine challenge antiviral drugs for the control of pandemic influenza virus surveillance of resistance to adamantanes among influenza a(h3n2) and a(h1n1) viruses isolated worldwide high prevalence of amantadine-resistance influenza a (h3n2) in six prefectures, japan, in the 2005-2006 season increased incidence of adamantane-resistant influenza a(h1n1) and a(h3n2) viruses during the 2006-2007 influenza season in japan epidemiologic study of influenza infection in okinawa, japan, from 2001 to 2007: changing patterns of seasonality and prevalence of amantadine-resistant influenza a virus infections with oseltamivir-resistant influenza a(h1n1) virus in the united states oseltamivirresistant influenza viruses a (h1n1), norway, 2007-08 use of oseltamivir in 12 european countries between 2002 and 2007-lack of association with the appearance of oseltamivir-resistant influenza a(h1n1) viruses global transmission of oseltamivir-resistant influenza distribution of amantadine-resistant h5n1 avian influenza variants in asia reduced sensitivity of influenza a (h5n1) to oseltamivir h5n1 transmission and disease: observations from the frontlines human infection with highly pathogenic avian influenza virus (h5n1) in northern vietnam glycyrrhizin, an active component of liquorice roots, and replication of 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infection antiviral activity of glycyrrhizic acid derivatives against sars-coronavirus glycyrrhizin, an active component of licorice roots, reduces morbidity and mortality of mice infected with lethal doses of influenza virus acetylsalicylic acid (asa) blocks influenza virus propagation via its nf-kappabinhibiting activity global impact of influenza virus on cellular pathways is mediated by both replication-dependent and -independent events glycyrrhizin inhibits highly pathogenic h5n1 influenza a virus-induced proinflammatory cytokine and chemokine expression in human macrophages tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection inhibition of the cytokine response does not protect against lethal h5n1 influenza infection a novel immunomodulatory mechanism of ribavirin in suppressing natural killer cell function confronting the next influenza pandemic with antiinflammatory and immunomodulatory agents: why they are needed and how they might work the authors thank mrs. kerstin euler, mrs. gesa meincke, and mrs. christina matreux for technical support. key: cord-292380-ulsejzqt authors: iwanejko, jakub; wojaczyńska, elżbieta; turlej, eliza; maciejewska, magdalena; wietrzyk, joanna title: octahydroquinoxalin-2(1h)-one-based aminophosphonic acids and their derivatives—biological activity towards cancer cells date: 2020-05-22 journal: materials (basel) doi: 10.3390/ma13102393 sha: doc_id: 292380 cord_uid: ulsejzqt in the search for new antitumor agents, aminophosphonic acids and their derivatives based on octahydroquinoxalin-2(1h)-one scaffold were obtained and their cytotoxic properties and a mechanism of action were evaluated. phosphonic acid and phosphonate moieties increased the antiproliferative activity in comparison to phenolic mannich bases previously reported. most of the obtained compounds revealed a strong antiproliferative effect against leukemia cell line (mv-4-11) with simultaneous low cytotoxicity against normal cell line (mouse fibroblasts-balb/3t3). the most active compound was diphenyl-[(1r,6r)-3-oxo-2,5-diazabicyclo[4.4.0]dec-4-yl]phosphonate. preliminary evaluation of the mechanism of action showed the proapoptotic effect associated with caspase 3/7 induction. at present, our attention is focused on the covid-19 pandemic and there is a tendency to neglect the civilization diseases which however remain the main reason for mortality worldwide. in particular, recently published data have shown that cancers are some of the leading causes of death in poland, with prostate cancer (almost 20% of patients) and lung cancer as the most common in the male population. the third cause of male mortality is colorectal (colon and rectum) cancer. in the female population, lung cancer dominates, followed by breast and colorectal cancer. among young poles, leukemias, lymphomas and brain cancers predominate and account for about 56% of cases [1] . anticancer drug design is a challenging field with a continuous demand for new, selective and non-toxic agents for treatment [2] . among various classes of compounds, aminophosphonic acids and their derivatives meet these requirements. due to their similarities to α-amino acids and a wide range of applications, α-aminophosphonic acids are continuously gaining importance in organic synthesis. so far, a number of applications such as antiviral [3, 4] or cytotoxic agents [5, 6] , enzyme inhibitors [7, 8] immune system activators [9] or antibacterial activities [10] have attracted considerable attention. the importance of these acids and their derivatives in the search for new pharmaceutical uses has been extensively discussed in numerous review articles [11] [12] [13] [14] . the α-aminophosphonic acid and its derivatives pose a crucial role in a variety of biological activities (figure 1 ), including cytotoxic properties. ampa (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor antagonist [15] ; (b) antimicrobial aminophosphonate [16] ; (c) anti-hiv agent [17] ; (d) cytotoxic agent against human cervical carcinoma [18] . aminophosphonates bearing an n-heterocyclic fragment have received particular attention over the last years among other potential anticancer agents [19] [20] [21] [22] . α-amino acids and their esters are considered versatile pharmacophores, known for antitumor activity [23] . the group of nikalje successfully coupled indole-2,3-diones with α-aminophosphonates, which led to new selective, antiproliferative compounds, analogues of commercially available drugs, orantinib and sunitinib [24] . in another noteworthy example, huang and co-workers proved that the incorporation of an aminophosphonate moiety to irinotecan, a chemotherapeutic agent, increased the cytotoxicity against certain cancer cell lines [25] . the fact of their negligible mammalian toxicity [12] testifies to the usefulness of these compounds in drug discovery research. a convenient route to this class of compounds is the pudovik reaction-a nucleophilic addition of dialkyl phosphites to imines [26] . another approach, kabachnik-fields one-pot three-component protocol, requires an amine, a carbonyl compound and an alkyl phosphite, however, it fails with electron-deficient amines [27] . most of the reported reactions are base-or acid-catalyzed, and noncatalyzed procedures remain scarce [28] . a recent review covers the reactions and synthetic methods for aminophosphonates [29] . in our previous research, we reported on the synthesis and antiproliferative action of novel phenolic adducts of bicyclic imine 1 (scheme 1) [30] . two phenolic mannich bases were found to be comparatively active to cisplatin with a noticeable increase of selectivity against cancer cell lines (one of them shown on the scheme). the tested compounds exhibit a resemblance to bioactive diketopiperazines in their bicyclic fragments. our new goal was to synthesize phosphorus analogs of these structures and evaluate their cytotoxicity against cancer cell lines. among a variety of synthetic methods for heterocyclic aminophosphonates formation [31] , our group has focused on a convenient nucleophilic addition of dialkyl phosphites to a cyclic imine. bearing in mind the remarkable precedents of improving the efficacy of cytotoxicity against cancer cell lines by insertion of aminophosphonate moiety and the results of our previous research, we directed our examinations toward the evaluation of antiproliferative properties of the phosphonic derivatives of octahydroquinoxalin-2(1h)-one. [15] ; (b) antimicrobial aminophosphonate [16] ; (c) anti-hiv agent [17] ; (d) cytotoxic agent against human cervical carcinoma [18] . aminophosphonates bearing an n-heterocyclic fragment have received particular attention over the last years among other potential anticancer agents [19] [20] [21] [22] . α-amino acids and their esters are considered versatile pharmacophores, known for antitumor activity [23] . the group of nikalje successfully coupled indole-2,3-diones with α-aminophosphonates, which led to new selective, antiproliferative compounds, analogues of commercially available drugs, orantinib and sunitinib [24] . in another noteworthy example, huang and co-workers proved that the incorporation of an aminophosphonate moiety to irinotecan, a chemotherapeutic agent, increased the cytotoxicity against certain cancer cell lines [25] . the fact of their negligible mammalian toxicity [12] testifies to the usefulness of these compounds in drug discovery research. a convenient route to this class of compounds is the pudovik reaction-a nucleophilic addition of dialkyl phosphites to imines [26] . another approach, kabachnik-fields one-pot three-component protocol, requires an amine, a carbonyl compound and an alkyl phosphite, however, it fails with electron-deficient amines [27] . most of the reported reactions are base-or acid-catalyzed, and non-catalyzed procedures remain scarce [28] . a recent review covers the reactions and synthetic methods for aminophosphonates [29] . in our previous research, we reported on the synthesis and antiproliferative action of novel phenolic adducts of bicyclic imine 1 (scheme 1) [30] . two phenolic mannich bases were found to be comparatively active to cisplatin with a noticeable increase of selectivity against cancer cell lines (one of them shown on the scheme). the tested compounds exhibit a resemblance to bioactive diketopiperazines in their bicyclic fragments. our new goal was to synthesize phosphorus analogs of these structures and evaluate their cytotoxicity against cancer cell lines. among a variety of synthetic methods for heterocyclic aminophosphonates formation [31] , our group has focused on a convenient nucleophilic addition of dialkyl phosphites to a cyclic imine. bearing in mind the remarkable precedents of improving the efficacy of cytotoxicity against cancer cell lines by insertion of aminophosphonate moiety and the results of our previous research, we directed our examinations toward the evaluation of antiproliferative properties of the phosphonic derivatives of octahydroquinoxalin-2(1h)-one. in our studies of the aminophosphonic acids and aminophosphonates mv4-11 (b phenotypic myelomonocytic) cell line carrying translocation t(4;11) was used. mv4-11 corresponds to aml m5b (according to the previously used french-american-british (fab) classification based on the morphology features of the cells). such an aml subtype is characterized by a tendency to occupy the gums, lymph nodes and skin [32] . mv4-11 cell line growth in suspension has about 50 h doubling time that makes it a good and a sensitive model for searching for new synthesized compounds against leukemia cells. all the reagents and solvents were purchased from commercial suppliers and used without further purification. melting points were carried out on the apotec ® schmelzpunktbestimmer melting point apparatus and are uncorrected. 1 h, 13 c and 31 p nmr spectra were collected on jeol 400yh and bruker avance ii 600 instruments. nmr spectra were recorded in cdcl3, unless specified otherwise. the temperature of the samples was 298 k. fourier-transform infrared spectra were measured using the perkin elmer 2000 ftir spectrometer. the principle peaks and their assignments are listed in table 1 . the high-resolution mass spectra (hrms) measurements were performed using the waters lct premier xe tof instrument. optical rotations were measured at ambient temperature on optical activity ltd. model aa-5 automatic polarimeter; [α] d values are given in 10 −1 deg cm 2 g −1 . column chromatography was performed on silica gel 60 (particle size 0.063-0.200 mm). thin-layer chromatography was conducted with the merck silica gel 60 pre-coated plates (f254) and visualized with uv light and/or iodine vapors. (1r,6r)-3-oxo-2,5-diazabicyclo [4.4 .0]dec-4-ene (1). typical procedure (1r,2r)-trans-diaminocyclohexane (4.00 mmol, 456 mg, 2.00 equiv) was dissolved in 2-proh (8 ml). to the stirred solution ethyl glyoxylate solution (50% solution in toluene, 2.00 mmol, 0.420 ml, 1.00 equiv) was added and the mixture was stirred for 24 h at room temperature (293 k). the solvent was removed in vacuo and the product was purified by silica gel column chromatography (eluent: in our studies of the aminophosphonic acids and aminophosphonates mv4-11 (b phenotypic myelomonocytic) cell line carrying translocation t(4;11) was used. mv4-11 corresponds to aml m5b (according to the previously used french-american-british (fab) classification based on the morphology features of the cells). such an aml subtype is characterized by a tendency to occupy the gums, lymph nodes and skin [32] . mv4-11 cell line growth in suspension has about 50 h doubling time that makes it a good and a sensitive model for searching for new synthesized compounds against leukemia cells. all the reagents and solvents were purchased from commercial suppliers and used without further purification. melting points were carried out on the apotec ® schmelzpunktbestimmer melting point apparatus and are uncorrected. 1 h, 13 c and 31 p nmr spectra were collected on jeol 400yh and bruker avance ii 600 instruments. nmr spectra were recorded in cdcl 3, unless specified otherwise. the temperature of the samples was 298 k. fourier-transform infrared spectra were measured using the perkin elmer 2000 ftir spectrometer. the principle peaks and their assignments are listed in table 1 . the high-resolution mass spectra (hrms) measurements were performed using the waters lct premier xe tof instrument. optical rotations were measured at ambient temperature on optical activity ltd. model aa-5 automatic polarimeter; [α] d values are given in 10 −1 deg cm 2 g −1 . column chromatography was performed on silica gel 60 (particle size 0.063-0.200 mm). thin-layer chromatography was conducted with the merck silica gel 60 pre-coated plates (f 254 ) and visualized with uv light and/or iodine vapors. [(1r,6r)-3-oxo-2,5-diazabicyclo [4.4 .0]dec-4-yl]-phosphonic acid (3a). procedure: the imine 1 (1.00 mmol, 152 mg, 1.00 equiv) was dissolved in ch 2 cl 2 (15 ml) followed by the addition of the tris (trimethylsilyl) phosphite (1.00 mmol, 0.334 ml, 1.00 equiv). the reaction was kept at ambient temperature for 24 h, with magnetic stirring. the solvent was removed under reduced pressure and the residue dissolved in methanol (15 ml) followed by stirring overnight at ambient temperature. methanol was removed under reduced pressure and the acid was separated by crystallization (anhydrous etoh/et 2 o 1:5 v/v) which led to the product as a colorless solid. the ftir analysis was conducted to additionally confirm the structures of the products. in the spectrum of imine 1, the strong band at 1622 cm −1 has been assigned to the double-bonded imino group [33] . this stretching vibration was not present in the other products which confirmed the addition of phosphorus nucleophiles. moreover, these compounds exhibited signals in the range of 1416-1453 cm −1 (c-n), characteristic for secondary cyclic amines, which corresponds to the spectra of aminophosphonates known in the literature [34] . absorption in the region of 3100-3400 cm −1 has been attributed to the n-h stretching from the lactam group. the differences are observed for acids 3a and 3b, due to the possible intermolecular hydrogen bond formation. the p=o stretch was found at 1166-1349 cm −1 , in accordance to the literature [35] . the other essential signals between 909 and 1122 cm −1 were assigned to p-o and p-ar bonds as identified in tusek-bozic's work [36] . . balb/3t3 cell line was cultured in dmem (gibco, scotland, uk) supplemented with 2 mm l-glutamine and 5% fbs). all the culture media contained antibiotics: 100 u/ml penicillin (polfa tarchomin sa, warsaw, poland) and 100 µg/ml streptomycin (sigma-aldrich chemie gmbh, steinheim, germany)). all the cell lines were cultured in a humid atmosphere at 37 • c and in 5% co 2 . twenty four hours before adding the tested compounds, each of the cell lines was seeded in 96-well plastic plates (sarstedt, numbrecht, germany) in an appropriate medium at a density (10 4 cells/well), except a549 cell line (0.25 × 10 4 /well), and mcf7 cell line: (0.75 × 10 4 /well). the selected cell lines were exposed to each of the tested chemical compounds at four different concentrations in the range of 100 to 0.1 µg/ml for 72 h. as a reference, cisplatin (teva pharmaceuticals, poland) was used, and dmso (sigma-aldrich chemie gmbh, steinheim, germany) served as a solvent control at concentrations corresponding to these present in the dilutions of the tested compounds. for adherent cells, a sulforhodamine b assay (srb), and for leukemic-an mtt assay was performed. after 72 h of incubation, cells were fixed in situ by gently adding of 50 µl per well of ice-cold 50% tca (trichloroacetic acid, poch, gliwice, poland) and were incubated at 4 • c for one hour. afterwards, wells were washed five times with water and 50 µl of 0.4% solution of srb (sulforhodamine b, sigma-aldrich chemie gmbh, steinheim, germany) in 1% acetic acid (poch, gliwice, poland) was added to each well and plates were again incubated at rt for 30 min. the unbound dye was removed by washing plates five times with 1% acetic acid, while stained cells were treated with 10 mm tris (tris base, sigma-aldrich, chemie gmbh, steinheim, germany). the absorbance in each well was read using the elisa plate reader (biotek synergy h4, swindon, uk) equipped with gen5 software at the 540 nm wavelength [37] . the percentage of proliferation inhibition of leukemia cells by the tested compounds was determined by an mtt assay. briefly, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (sigma-aldrich, chemie gmbh, steinheim, germany) was added to each well and plates were left for 4 h at 37 • c. then, plates were centrifuged for 5 min, at 88× g, at 4 • c, the supernatant was thrown out and 200 µl of dmso per well (poch, gliwice, poland) was added. the plates were left in rt for 10 min and the absorbance in each well was read using the elisa plate reader (biotek synergy h4, swindon, uk), equipped with gen5 software at the 570 nm wavelength [38] . the results are presented as mean ic 50 values (the concentration of the compound, that inhibits cell proliferation by 50%) ± standard deviation. ic 50 values were assessed by the prolab-3 system based on cheburator 0.4, software developed by nevozhay [39] . at each concentration, chemical compounds were tested in triplicates in a single experiment. each experiment was repeated at least three times independently. for the cell cycle analysis, mv4-11 cell line was used. the cells were seeded in 24-well plastic plates (sarstedt, darmstadt, germany) at a density of 0.25 × 10 6 cells/1 ml. next, after 24 h, the tested compounds at the final concentration ic 50 and 2 × ic 50 , were added in a volume of 1 ml to the cells. the cells were exposed to the tested compounds for 48 h. next, the cells growing in suspension were collected, counted with the trypan blue solution (sigma-aldrich chemie gmbh, steinheim, germany), centrifuged for 5 min at +4 • c, 5 min, at 324× g, resuspended in 1 ml of 70% ice-cold ethanol (poch, gliwice, poland) and frozen at −20 • c for at least 24 h. after that, the cells were transferred to 5 ml propylene tubes (dedicated for flow cytometry analysis), washed in pbs (iiet, pas, wroclaw, poland) and centrifuged (+4 • c, 10 min, 324× g). then, the rnase solution (in pbs, 8 µg/ml) (life technologies, carlsbad, ca, usa) was added (500 µl for 0.5 × 10 6 cells) and the cells were incubated for 60 min at 37 • c with gentle mixing. after that, the cells were placed on ice, a propidium iodide (pi) solution (sigma-aldrich chemie gmbh, steinheim, germany) (in concentration 0.1 mg/ml) was added to the cells for 30 min. next, the flow cytometry analysis of the cell cycle was performed using bd lsr ii fortessa (becton dickinson, san jose, ca, usa), equipped with facs diva version 6.1. software (bd). the analysis of the obtained results was performed using flowing software version 2.5.1 developed by perttu terho. for each sample, the percentage of cells in each cell cycle phase was determined. each experiment was performed three times independently. the mv-4-11 cell line was seeded at the density of 0.25 × 10 6 cells/ml (total 1 × 10 6 cells) in a culture medium on 24-well plastic plates (sarstedt, numbrecht, germany) and was exposed to the chemical compounds at the concentration of ic 50 and 2 × ic 50 for 48 h. as a solvent control, dmso (poch, gliwice, poland) was used at a concentration corresponding to the highest concentration of compounds. after 48 h of incubation, the cells were collected, washed in pbs (324 g, 10 min, 4 • c) and counted. the cells (0.5 × 10 6 /ml) were diluted in a 0.5 ml annexin binding buffer (10 mm hepes/naoh; 140 mm nacl, 2.5 mm cacl 2 : iiet pas, wrocław, poland), diluted in distilled water at a ratio of 1:4. 5 µl of annexin v conjugated with apc (bd bioscience, san jose, ca, usa) was added to each 195 µl of cell suspension. after 15 min of incubation at room temperature in the dark, and pbs washing, the pi solution at 0.5 mg/ml (sigma-aldrich gmbh chemie, steinheim, germany) was added to the samples. the data were processed using bd lsrii fortessa, equipped with diva 6.1 software. the data were analyzed using flowing software version 2.5.1., developed by perttu terho and described as: double negative (live), double-positive (late apoptotic), annexin v positive-propidine iodine negative (early apoptotic)-and annexin v negative-propidine iodine positive (necrotic). each experiment was repeated 4-5 times. the mv-4-11 cells were seeded at the density of 0.25 × 10 6 cells/ml in culture medium on 24-well plastic plates (sarstedt, numbrecht, germany). after a 48 h exposition to the compounds at the concentration of ic 50 and 2 × ic 50 and incubated for 48 h, cells were collected and washed in pbs. as a solvent control, dmso was used at the concentration corresponding to the highest concentration of the compounds. camptothecin (sigma-aldrich gmbh chemie, steinheim, germany) was used as a positive control. the appropriate volume of lysis buffer ph 7.5 (50 mm hepes, 10% sucrose, 150 mm nacl, 1% triton x-100) (iiet, pas, wroclaw, poland) with the addition of 1% of dtt (dl-dithiotreithol, sigma-aldrich gmbh chemie steinheim, germany) was prepared. the reaction buffer ph 7.5 (20 mm hepes, 10% sucrose, 100 mm nacl) iiet pas, wrocław, poland with the dtt addition and with 10 µm of caspase-3 substrate (ac-devd-amc, cayman chemicals, ann arbor, mi, usa) was prepared and warmed to 37 • c before using. after incubation time the cells were centrifuged (324× g, 10 min, 4 • c), 50 µl of the lysis buffer was added to each sample and the probes were left at 4 • c for 30 min. then 40 µl of lysates were added to white 96-well plates (perkin-elmer, waltham, ma, usa) in triplicate, 160 µl of a pre-warmed reaction buffer was added to each well and the fluorescence was read out using a fluorescence plate reader (biotek synergy h4, swindon, uk) equipped with gen5 software the measurements were performed at 355 nm and 460 nm for 2 h every 10 min at 37 • c. at the same time, an mtt test was performed in order to normalize results. based on the results obtained, an mfi (mean fluorescence intensity) vs. reaction time curve was plotted and the vmax (reaction rate) values were determined. after normalization, the values obtained for the tested samples were compared to the control to assess how many times caspase-3 activity in tested probes is higher/lower than in control. each experiment was conducted at least 4-5 times. the mv-4-11 cell line was seeded at the density of 0.25 × 10 6 cells/ml in a culture medium on 24-well plates (sarstedt, numbrecht, germany). the cells were exposed to the compounds at the concentration of ic 50 and 2 × ic 50 for 48 h. then, the cells were collected, washed in pbs and counted in a trypan blue solution. the collections of 0.2 × 10 6 cells/sample were centrifuged (300× g, 5 min, room temperature) and pellets were suspended in a jc-1 solution (cayman chemicals, ann arbor, mi, usa) in a warm culture medium (final concentration 2.5 µg/ml). after 10 min of incubation at 37 • c in the dark, cells were centrifuged (300× g, 5 min, room temperature) and pellets were suspended in 200 µl of pbs (iiet, pas, wrocław, poland). as a solvent control, dmso was used at the concentration corresponding to the highest concentration of the compounds. valinomycin (sigma-aldrich gmbh chemie, steinheim, germany) was used as a positive control. the results were read using bd lsrii fortessa (becton dickinson, san jose, ca, usa), equipped with facs diva 6.1. software and were analyzed with flowing software 2.5.1, developed by perttu terho in dot plots presenting jc-1 monomers to aggregates. the mv-4-11 cells were seeded at a density of 0.25 × 10 6 cells/ml in culture medium on 24-well plates (sarstedt, numbrecht, germany). the cells were exposed to the compounds at the concentration of ic 50 and 2 × ic 50 and incubated for 48 h. as a solvent control, dmso was used at a concentration corresponding to the highest concentration of the compounds. tamoxifen (sigma-aldrich gmbh chemie, steinheim, germany) was used as a positive control. after 48 h of incubation, the cells were collected and washed in pbs (324 g, 10 min, 4 • c). for this purpose, 96-well black plates were used (perkin elmer, walthman, ma, usa). 100 µl of propidine iodine (pi) (final concentration: 10 µg/ml) was added to each sample, except the probes intended for background measurement. after 2 min of room temperature incubation, the cells were centrifuged (400× g, 5 min, room temperature), pellets were suspended in 100 µl of pbs (iiet pas, wrocław, poland) and again centrifuged. next 100 µl of 0.05 mmol/l dansyl cadaverine (sigma aldrich gmbh chemie, steinheim, germany) was added to each sample and the samples were incubated at 37 • c for 10 min. next, the cells were centrifuged and washed with pbs. finally, the obtained pellet was suspended in 300 µl of pbs and transferred into the appropriate wells. each sample was made in triplicate. autophagic vacuole staining intensity was detected at the excitation wavelength of 335 nm and emission of 512 nm, and the degree of cell death at excitation wavelength 536 nm and emission of 617 nm. each experiment was conducted at least 4-5 times. the mv-4-11 cell line was seeded at the density of 0.25 × 10 6 cells/ml in a culture medium on 24-well plastic plates (sarstedt, germany) to the final volume of 2 ml. the cells were exposed to the compounds at the concentration of ic 50 and 2ic 50 and incubated for 48 h. as a solvent control, dmso was used at the concentration corresponding to the highest concentration of the compounds. after 48 h of incubation, the cells were collected, washed in pbs (324 g, 5 min, room temperature) and counted. the collection of 0.2 × 10 6 cells were stained with 10 µg/ml of acridine orange (ao) (sigma aldrich gmbh chemie, steinheim, germany) for 20 min at 37 • c, washed two times with pbs and read using bd lsr ii fortessa (becton dickinson, san jose, ca, usa), equipped with facs diva 6.1. software and were analyzed with flowing software 2.5.1, developed by perttu terho. each experiment was performed at least 4 times. the presented compounds were synthesized using the previously published methods [40, 41] . a cyclic imine 1, derived from optically pure trans-(r,r)-1,2-diaminocyclohexane was reacted with h-phosphonates or phosphine oxides to give compounds 2a-f with good yields as mixtures of epimers (table 2) . a single epimer 2g of p-taddol derivative (taddol = α,α,α ,α -tetraaryl-2,2-disubstituted 1,3-dioxolane-4,5-dimethanol) was obtained by crystallization from mixture 2f. nmr spectra of products 1 and 2a-g are shown in the supplementary materials (figures s1-s8 ). the presented compounds were synthesized using the previously published methods [40, 41] . a cyclic imine 1, derived from optically pure trans-(r,r)-1,2-diaminocyclohexane was reacted with hphosphonates or phosphine oxides to give compounds 2a-f with good yields as mixtures of epimers (table 2) . a single epimer 2g of p-taddol derivative (taddol = α,α,α′,α′-tetraaryl-2,2disubstituted 1,3-dioxolane-4,5-dimethanol) was obtained by crystallization from mixture 2f. nmr spectra of products 1 and 2a-g are shown in the supplementary materials (figures s1-s8 ). in a modified protocol, we obtained aminophosphonic acids 3a and 3b (scheme 2). an addition of tris(trimethylsilyl)phosphite and further methanolysis yielded product 3a (supplementary materials, figure s9 ). when a ketimine (x = ph) was used in the reaction, an additive of bromotrimethylsilane was required for the reaction to complete. an activation of c=n bond was necessary, since c-substituted imines are less reactive. the diastereoselectivity of the reactions was improved, especially in the case when a sterically hindered phenyl ketimine was used. the dr values provided in table 2 represent the compositions of the epimeric mixtures used in further studies and were determined by 31 p nmr and confirmed by 1 h nmr spectroscopy. in the case of compound 3b (supplementary materials, figure s10 ), only traces of the second epimer were detected at the level of in a modified protocol, we obtained aminophosphonic acids 3a and 3b (scheme 2). an addition of tris(trimethylsilyl)phosphite and further methanolysis yielded product 3a (supplementary materials, figure s9 ). when a ketimine (x = ph) was used in the reaction, an additive of bromotrimethylsilane was required for the reaction to complete. an activation of c=n bond was necessary, since c-substituted imines are less reactive. the diastereoselectivity of the reactions was improved, especially in the case when a sterically hindered phenyl ketimine was used. the dr values provided in table 2 represent the compositions of the epimeric mixtures used in further studies and were determined by 31 p nmr and confirmed by 1 h nmr spectroscopy. in the case of compound 3b (supplementary materials, figure s10 ), only traces of the second epimer were detected at the level of the accuracy of nmr technique (ca. 2%). in a modified protocol, we obtained aminophosphonic acids 3a and 3b (scheme 2). an addition of tris(trimethylsilyl)phosphite and further methanolysis yielded product 3a (supplementary materials, figure s9 ). when a ketimine (x = ph) was used in the reaction, an additive of bromotrimethylsilane was required for the reaction to complete. an activation of c=n bond was necessary, since c-substituted imines are less reactive. the diastereoselectivity of the reactions was improved, especially in the case when a sterically hindered phenyl ketimine was used. the dr values provided in table 2 represent the compositions of the epimeric mixtures used in further studies and were determined by 31 p nmr and confirmed by 1 h nmr spectroscopy. in the case of compound 3b (supplementary materials, figure s10 ), only traces of the second epimer were detected at the level of the accuracy of nmr technique (ca. 2%). all compounds were evaluated according to their antiproliferative activity towards human acute myeloid leukemia (aml-m5b) cell line (mv4-11) and three adenocarcinoma cell lines of different origin: lung (a549), colorectal (lovo) and breast (mcf-7). the results were compared with those obtained on the normal murine fibroblasts cell line (balb/3t3). among the modified compounds, only derivatives 2f and 2g (similarly to 1) exert antiproliferative activity against all the cancer cell lines tested, however in contrast to compound 1 their activity towards balb/3t3 cells was visibly lower as compared to the cancer cells (table 3) . on the other hand, 2b and 3a were most active towards leukemia cells, though they were not toxic neither for solid tumors cell lines nor murine fibroblasts. a preliminary sar (structure-activity relationship) analysis reveals a few conclusions about structural features that result in the desired activity. among the tested phosphonates, phenyl derivative 2b performed better than benzyl (2c), methyl (2a) and taddol esters (2f). however, the latter derivative bearing a substituent introducing a big steric hindrance was found to be more versatile and acted on all of the investigated cell lines. comparison of diastereomeric mixture (2f) and isolated single isomer (2g) reveals no significant differences between epimers with opposite configurations of the stereogenic center c-4. therefore, the separation of phosphonate diastereomers for biological tests seems unnecessary. remarkably, h-phosphinate 2d was practically inactive, while phosphine oxide 2e exhibited a moderate activity in comparison to the majority of the tested phosphonates. this indicates the directions of further modifications, which should be focused on esters of aminophosphonic acids. the acid itself (3a) exhibited high cytotoxicity, but only toward leukemia cells; comparison of compounds 3a and 3b suggests that complete substitution of a stereogenic center decreases the antiproliferative activity. compounds 2b, 2e and 2f were selected for further studies. for evaluation of the impact of the selected compounds on cell cycle distribution, we analyzed the percentage of cells in each of the cell divisions upon incubation with selected compounds. analyzing cell cycle distribution (figure 2a-d) , we could only observe a decrease of cells in the s phase after 48 h incubation with 2e. in parallel, the tendency to increase the percentage of cells in g0/g1 phase was observed. cisplatin used as a control of the test increased cells percentage in the g2m phase. analysis of dead cells (subg1) showed a significant increase in dead cells caused by 2f used in higher concentrations. next, we decided to analyze apoptotic and necrotic cells using annexin v/pi staining ( figure 2e-g) , as well as the activity of caspase 3/7 in the treated cells ( figure 2h ). compound 2b increased the level of early apoptotic cells with an increase of caspase 3/7 activity. compound 2f increased the percentage of necrotic cells (but also the tendency to increase the level of apoptotic cells: early and late, was observed) and also increased the activity of caspase 3/7. in the case of compound 2e, an increased percentage of late apoptotic cells was accompanied by an increase of caspase 3/7 activity. the drop of the percentage of cells with high mitochondrial membrane potential (∆ψ) was observed in mv4-11 cells incubated with compound 2f and 2e ( figure 2i ). for labeling autophagic vacuoles, two techniques with dansyl cadaverin and with acridine orange were used. in both methods, compound 2f increased the level of acidic autophagic vacuoles ( figure 2j,k) . increased the activity of caspase 3/7. in the case of compound 2e, an increased percentage of late apoptotic cells was accompanied by an increase of caspase 3/7 activity. the drop of the percentage of cells with high mitochondrial membrane potential (δψ) was observed in mv4-11 cells incubated with compound 2f and 2e ( figure 2i ). for labeling autophagic vacuoles, two techniques with dansyl cadaverin and with acridine orange were used. in both methods, compound 2f increased the level of acidic autophagic vacuoles ( figure 2j ,k). in this study, we prepared aminophosphonic acids and their derivatives based on octahydroquinoxalin-2(1h)-one scaffold via pudovik reaction. the syntheses proceeded efficiently giving stable products in case of all types of h-phosphonates, phosphine oxides and phosphite used. since no significant differences of antiproliferative activities for a diastereomeric mixture (2f) and a single epimer (2g) were observed, further studies were conducted for mixtures of both stereoisomers of each compound. compound 2b, which was found to be the most active in proliferation inhibition of the mv4-11 cells, induces apoptosis of these cells with an increased caspase 3/7 activity. compound 2f, which inhibited the proliferation of all neoplastic cell lines, tends to decrease the percentage of cells in the g2m phase and increases the percentage of dead cells, including cells undergoing necrosis. this compound increases the activity of caspases 3/7 and reduces the mitochondrial potential of cells. a long-lasting drop or rise of ∆ψ from control levels may induce a loss of cell viability. the higher is the level of intracellular atp, the more stable are the ∆ψ values, making atp a compound buffering mitochondrial ∆ψ [42] . however, compound 2f can also enhance autophagy. this phenomenon may not be beneficial from the point of view of cancer therapies, although there are different opinions, as well as it may depend on specific mechanisms of action of studied compounds [43] . derivative 2e reduces the percentage of cells in the s phase of the cell cycle, increases the percentage of cells in late apoptosis and necrotic cells, increases caspase 3/7 activity, and reduces mitochondrial potential, so it works as a pro-apoptotic agent. a similar mechanism of action was reported by huang's group in the paper on the synthesis of potential anticancer candidates for the use in the therapy of ovarian cancer cells [25] . the tested compounds, with the emphasis put on 2b, 2e and 2f, could be used as promising scaffolds in further antiproliferative drug design. supplementary materials: the following are available online at http://www.mdpi.com/1996-1944/13/10/2393/s1, figure s1 . 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an effective route to new bicyclic compounds: aminophosphonates, enamines and imines aminophosphonates and aminophosphonic acids with tetrasubstituted stereogenic center: diastereoselective synthesis from cyclic ketimines mitochondrial membrane potential the roles of autophagy in cancer funding: this research received no external funding. the authors declare no conflict of interest. key: cord-264526-bxpzo2xu authors: aydin, malik; naumova, ella a.; paulsen, friedrich; zhang, wenli; gopon, felix; theis, christian; lutz, sören; ehrke-schulz, eric; arnold, wolfgang h.; wirth, stefan; ehrhardt, anja title: house dust mite exposure causes increased susceptibility of nasal epithelial cells to adenovirus infection date: 2020-10-11 journal: viruses doi: 10.3390/v12101151 sha: doc_id: 264526 cord_uid: bxpzo2xu adenovirus (adv) infections in the respiratory tract may cause asthma exacerbation and allergic predisposition, and the house dust mite (hdm) may aggravate virus-induced asthma exacerbations. however, the underlying mechanisms of whether and how adv affects asthmatic patients remains unclear. to address this question, we investigated nasal epithelial cells (naepcs) derived from a pediatric exacerbation study cohort for experimental analyses. we analyzed twenty-one different green-fluorescent proteinand luciferase-tagged adv types in submerged 2d and organotypic 3d cell culture models. transduction experiments revealed robust transduction of adv type 5 (adv5) in naepcs, which was associated with an increased uptake of adv5 in the presence of hdm. in healthy and asthmatic naepcs exposed to hdm before infection, we observed a timeand dose-dependent increase of adv5 uptake associated with upregulation of entry receptors for adv5. furthermore, electron microscopic and histologic analyses of 3d cell cultures revealed an impairment of the respiratory cilia after hdm exposition. this ex vivo pilot study shows the impact of adv infection and hdm exposition in a primary cell culture model for asthma. currently, more than 100 human adenoviruses (adv) (http://hadvwg.gmu.edu/) have been identified. they have been phylogenetically divided into seven species (a to g) based on hemagglutination features, oncogenic potential in rodents, dna homology, and genome organization [1, 2] . adenoviruses are non-enveloped viruses, which contain a double-stranded dna viral genome of an approximate length of 26-46 kbp [3] . the capsid consists of 252 capsomeres, and the virus shape is icosahedral with 240 hexon-, 12 penton-, and fiber proteins including shaft and knob [4] . for adv cell entry, several cellular receptors have been described, including the coxsackie andadenovirus receptor (car), cd46, sialic acid, desmoglein-2 (dsg-2), and heparan sulfate proteoglycan (hspg) [4] . adv are known as pathogens, but they have also been explored as viral vectors in gene therapeutic applications. in clinics, human adv have become increasingly important in recent years. they cause different clinical symptoms in a wide range of diseases, e.g., pneumonia, conjunctivitis, gastroenteritis, or myocarditis [5] [6] [7] [8] . threatened groups include children younger than five years of age or immune-deficient patients after transplantation. in addition, adv have also been causatively associated with pneumonia outbreaks in us-military bases [9] . several adv can be isolated from patients with lung infections [8] , and here we addressed the question of whether this is associated with asthma exacerbation. there is strong evidence that asthma exacerbations are associated with virus-mediated upper and/or lower respiratory infections [10] , and therefore there is a broad interest in studying the role of viruses in asthma pathogenesis and exacerbation [11] [12] [13] [14] . it was described that predominantly rhinovirus (rv), and other viruses, particularly adenoviruses (adv), cytomegalovirus, bocavirus, coronavirus, herpes simplex virus, influenza virus, parainfluenza virus, respiratory syncytial virus, or enteroviruses, may be involved in asthma development [15] . in addition to viruses, house dust mite (hdm) as a major allergen is strongly associated with asthma and presents an important risk factor for virus-induced asthma exacerbation [12, 13] . although various studies have investigated the molecular roles of some respiratory viruses in allergic pathways [14, 15] , the relationship between adv infection and hdm sensitization in asthma exacerbation has not been sufficiently analyzed. here, we aimed at analyzing this relationship in primary nasal epithelial cells (naepcs) as an ex vivo cell culture model, to better study allergies and the immunology of asthma [16] [17] [18] . to analyze adv infection in the context of hdm sensitization, we utilized primary nasal naepcs derived from our pediatric exacerbation study cohort in submerged 2d and organotypic 3d cell culture models. for this approach, we applied twenty-one previously described green-fluorescent protein (gfp)-and luciferase-tagged adv types [19] , encompassing adv of all seven species. moreover, we performed electron microscopic and histologic analyses of 3d cell cultures to study the impairment of respiratory cilia after hdm exposure. we found that hdm exposure may increase adv5 infection in vitro, and that major adv surface receptors may play a role. we established a pediatric exacerbation study network in two children's hospitals in germany. participating study centers were wuppertal (witten/herdecke university, germany) and niederberg/velbert (teaching hospital of the university hospital essen, germany). pediatric subjects with a chronic bronchitis/wheeze (3 months to ≤5 years of age) or asthma (>6 years to 17 years of age) suffering from acute exacerbation episodes were recruited. in addition, healthy controls (age range >3 months to 65 years of age) were recruited for comparison. a detailed study description and protocol is currently in preparation for publication. for the experimental approach used here, only naepcs from asthmatics and healthy donors were used (n = 3 per each group as biologic with each n = 3 technical replicates). for the control experiments, cells from healthy donors were compared with cells from asthmatic subjects (treated and untreated). only asthmatics with an hdm sensitization (released ige levels in serum, positive immunocap ® results for dermatophagoides pteronyssinus, cap class >3) were chosen for this work. furthermore, only healthy donors without any positive sensitization to dermatophagoides pteronyssinus were selected. clinically, asthmatic patients showed an immunologic reaction to dermatophagoides pteryonossinus, and the healthy donors did not present any allergic reactions. for this prospective study, all analyzed biomaterials and data involving human participants were collected in concordance with the ethical standards and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. ethics approval was obtained from the local ethics committees (witten/herdecke university (158/2017) and medical chamber (ärztekammer nordrhein, nr 2019312), germany). the study was retrospectively assigned the human study at german clinical trials register (drks) with the registration number drks00015738. all study cohort relevant data analyses were pseudonymously performed. all participated subjects or their legal custodians/parents provided a written informed consent. this article does not contain any animal studies. the naepcs were obtained by performing a nasal brushing (cytobrush eswab ® copan italia) from both nostrils, resuspended in warm begm ® medium (purchased from lonza, basel, switzerland), and shaken for 30 s. approximately, 150,000-250,000 cells per nasal brushing procedure (including both nostrils) were collected, the total number was approximately 1 × 10 6 cells at passage 2 (p2). to eliminate possible contamination with erythrocytes and fibroblasts, lysing procedures were performed before experimental set-up. after centrifugation at 350× g for 8 min, the cell number was calculated using a neubauer counting chamber. cells were incubated in collagen i pre-coated t75 flasks (greiner bio-one, austria) for up to a maximum of two passages (p2) at 37 • c and 5% co 2 atmosphere. at p2, the cells were seeded using an organotypic 3d air liquid interface (ali) cell culturing technique adapted to the instructions of stemcell tm technologies (https://www.stemcell.com/). for this, 25,000 cells were seeded in collagen i pre-coated transwells using pneumacult tm ali proliferation medium added to the basal and apical chambers, and the medium was changed every single day. at day 4, the medium in both chambers were removed, and the cells received pneumacult tm ali differentiation medium at the basal chamber. the medium was changed every 2 to 3 days assuring airlifting for up to 4 weeks until the pseudostratified morphology was reached, which was confirmed by ciliary beats and production of mucus (pneumacult tm ali and differentiation medium were purchased from stemcell tm technologies, canada). naepcs were characterized through flow cytometry using cd45-apc (miltenyi biotec, germany), cd326 (epcam)-pe (miltenyi biotec, germany), and anti-cytokeratin-fitc (miltenyi biotec, germany), followed by fixation and permeabilization using inside stain kit (miltenyi biotec, germany) according to manufacturer's instructions. the selected antibodies for the characterization of naepcs were adapted in a previously published study [20] . the adv receptors, cd46-apc (miltenyi biotec, germany), cxadr/car-pe, and dsg-2-pe (affymetrix, thermo fisher scientific) were chosen for flow cytometry analyses. the specimens were fixed with 0.1 m cacodylate buffer containing 2.5% glutaraldehyde, 2% polyvinylpyrrolidone, and 75 mm nano 2 for 30 min. at 4 • c. samples were washed in 0.1 m cacodylate buffer without glutaraldehyde and subsequently incubated in a solution containing 2% arginine-hcl, glycine, sucrose, and sodium glutamate for 18 h at room temperature (rt). the specimens were rinsed in distilled water, followed by immersion in a mixture of each 2% tannic acid and guanidine-hcl for 6 h at rt. the samples were rinsed again in 0.1 m cacodylate buffer and incubated in a 1% oso 4 solution for 30 min at rt. after three rinsing steps with 0.1 m cacodylate buffer, the specimens were dehydrated, dried in liquid co 2 , and finally examined with a zeiss sigma sem (zeiss, oberkochen, germany) scanning electron microscope using 2 kv acceleration voltage after sputtering with gold palladium. as detectors, the in-lens and se detectors were used. the specimens were processed for tem according to a previously published protocol [21] . in brief, samples were fixed in ito's fixative (2.5% glutaraldehyde, 2.5% paraformaldehyde, and 0.3% picric acid) dissolved in phosphate buffered saline (pbs) (ph = 7.3) and embedded in epon. semi-thin sagittal sections of 1 µm were cut with a microtome (ultracut e; reichert jung, vienna, austria) and subsequently stained with toluidine blue. sections were viewed with an epifluorescence microscope (aristoplan; ernst leitz, wetzlar, germany) and photographed (keyence biorevo bz9000 microscope). ultrathin sections were stained with uranyl acetate and lead citrate and viewed with a transmission electron microscope (em109; carl zeiss meditec gmbh, oberkochen, germany). the naepcs were seeded in collagen coated 96 wells (day 0). at day 1, the culture medium was changed. for exposition experiments, dermatophagoides pteronyssinus (citeq biologics, netherlands, product code: 02.01.64) was used in different concentrations (1 µg/ml, 10 µg/ml, and 100 µg/ml) for two time points. the first time point included an exposition duration of 24 h. the read-out parameters will be presented in the course of the manuscript. after 24 h exposition with dermatophagoides pteronyssinus, the supernatant was removed, fresh begm ® cell culture medium was added, and naepcs were incubated for 24 h. after this incubation time, a second exposition with dermatophagoides pteronyssinus was performed for 24 h, and after that, the read-out parameters were measured (see below). these two time points were presented as 24 h and 72 h throughout the manuscript. in addition, to easily follow the manuscript, the term hdm was used for dermatophagoides pteronyssinus throughout the manuscript. we used different recombinant adv types deleted for the early gene region e3, which was replaced by a transgene expression cassette encoding the reporter genes luciferase and gfp [19] . in brief, recombinant viruses were amplified in permissive cell lines and purified using cesium-chloride gradients as described before [19] . we explored n = 21 different adv types derived from different species to transduce naepcs. twenty-four hours before transduction, we seeded 1 × 10 4 naepcs per well in collagen i pre-coated 96 wells plates. gfp and luciferase expressing adv types were added to each well using 50 viral particles per cell (vpc). twenty-four hours post transduction of naepcs, we determined luciferase activity within the transduced cells using the nano-glo ® luciferase assay system from promega. to perform the luciferase assay, we removed 100 µl supernatant, added nano-glo ® luciferase assay substrate and nano-glo ® luciferase assay buffer (dilution: 1:50) to cells, and incubated for up to 10 min at 37 • c and 5% co 2 until cells were detached. subsequently, luciferase expression levels were quantified using a tecan elisa reader. statistical analyses were performed using graphpad prism version 8.3.0 for windows, graphpad software, la jolla california usa, www.graphpad.com. data were presented as mean and standard error of the mean (sem) or as absolute values with percentages or fold changes (n = 3 to 5). comparisons between two groups were performed with unpaired/paired, two-tailed, and t-tests. comparisons between more than two groups were performed with one-way-anova and holm-sidak's multiple comparison posttest. the significance levels were set at * p < 0.05, ** p < 0.01, and *** p < 0.001. to prove the purity of the cultured cells derived from our pediatric exacerbation study cohort, flow cytometric analyses were performed for each culture. as presented in figure 1a , the isolated cells were cd45 neg epcam pos pan-cytokeratin pos . in addition, the morphology of the cultured cells was analyzed through raster electron microscopy (rem) as well as histology, which revealed the purity of the cultures (figure 1b,c) . to address the question whether naepcs were submissive to adv infection, we transduced cultured naepcs in a monolayer with 21 different luciferase and gfp expressing adv types ( figure 2 ) at 50 vpc. twenty-four hours post-transduction, the luciferase activity of infected cells was determined. permeabilization, anti-cytokeratin-fitc was used for intracellular staining. (b) after passage 2 (p2), the cells were seeded for organotypic 3d air-liquid interface cultures. nasal mucus secretion and ciliary beats were observed through light microscopy after 6 to 8 weeks of culturing. final specimens were then processed for raster electron microscopic imaging. (c) histologic analyses confirmed the morphology of the nasal epithelial cell population (10x magnification). to address the question whether naepcs were submissive to adv infection, we transduced cultured naepcs in a monolayer with 21 different luciferase and gfp expressing adv types ( figure 2 ) at 50 vpc. twenty-four hours post-transduction, the luciferase activity of infected cells was determined. as shown in figure 3 , adv type 5, followed by 9, 21, 3, and 35, showed the highest transduction rates in naepcs if directly compared to other adv types. therefore, adv5, as a common respiratory virus and the most analyzed virus in terms of gene therapeutic approaches, was then used for further analyses. to determine the infections rates of adv5 in naepcs, we transduced naepcs with adv5 using different vpc and measured the luciferase expression level and visualized gfp pos cells through immunofluorescence microscopy. twenty-five hours post-transduction, there was a clear correlation as shown in figure 3 , adv type 5, followed by 9, 21, 3, and 35, showed the highest transduction rates in naepcs if directly compared to other adv types. therefore, adv5, as a common respiratory virus and the most analyzed virus in terms of gene therapeutic approaches, was then used for further analyses. to determine the infections rates of adv5 in naepcs, we transduced naepcs with adv5 using different vpc and measured the luciferase expression level and visualized gfp pos cells through immunofluorescence microscopy. twenty-five hours post-transduction, there was a clear correlation between the virus dose, the luciferase expression levels, and the gfp + -expressing cell numbers. a cytopathic effect of infected cells associated with adenovirus infection was not observed (figure 4a,b) . it is assumed that patients with allergies and asthma suffer from increased virus infections [22] . therefore, we studied the virus transduction efficiency of adv5 in hdm-provoked naepcs. different concentrations of hdm (1 µg/ml, 10 µg/ml, and 100 µg/ml) were used to provoke naepcs ex vivo, and the schematic outline of this experiment is shown in figure 5 . between the virus dose, the luciferase expression levels, and the gfp + -expressing cell numbers. a cytopathic effect of infected cells associated with adenovirus infection was not observed (figure 4a,b) . between the virus dose, the luciferase expression levels, and the gfp + -expressing cell numbers. a cytopathic effect of infected cells associated with adenovirus infection was not observed (figure 4a,b) . it is assumed that patients with allergies and asthma suffer from increased virus infections [22] . therefore, we studied the virus transduction efficiency of adv5 in hdm-provoked naepcs. different concentrations of hdm (1 μg/ml, 10 μg/ml, and 100 μg/ml) were used to provoke naepcs ex vivo, and the schematic outline of this experiment is shown in figure 5 . during the nasal brushing procedure, we collected the cells in cell culture medium, centrifuged at 350x g for 8 min, and washed with pbs. the cell pellet was resuspended in cell culture medium, and the cells were seeded in collagen i pre-coated t75 flasks. after passage 2 (p2) was reached, naepcs were collected and seeded in collagen i pre-coated 96 well plates. different hdm concentrations were used (1 μl/ml, 10 μg/ml, 100 μg/ml). the transduction concentration of adv5 was set at 10 virus particle per cell (vpc). this figure was generated using biorender.com luciferase assays were performed one and three days post-transduction correlating with adv transduciton rates. there was a trend of increased adv transduction in healthy control cells and in cells derived from asthmatics. especially on day 3 after hdm exposure, we observed an enhanced adv mediated luciferase activity in hdm-provoked naepcs from asthmatics, compared to naepcs derived from healthy cells ( figure 6 ). during the nasal brushing procedure, we collected the cells in cell culture medium, centrifuged at 350× g for 8 min, and washed with pbs. the cell pellet was resuspended in cell culture medium, and the cells were seeded in collagen i pre-coated t75 flasks. after passage 2 (p2) was reached, naepcs were collected and seeded in collagen i pre-coated 96 well plates. different hdm concentrations were used (1 µl/ml, 10 µg/ml, 100 µg/ml). the transduction concentration of adv5 was set at 10 virus particle per cell (vpc). this figure was generated using biorender.com luciferase assays were performed one and three days post-transduction correlating with adv transduciton rates. there was a trend of increased adv transduction in healthy control cells and in cells derived from asthmatics. especially on day 3 after hdm exposure, we observed an enhanced adv mediated luciferase activity in hdm-provoked naepcs from asthmatics, compared to naepcs derived from healthy cells ( figure 6 ). to shed light on the underlying mechanism for enhanced adv5 uptake, major adv receptor expression levels, in particular, car, cd46, and dsg-2, were characterized on naepcs through flow cytometry. in comparison to hdm-provoked naepcs, we observed a significant increase of car and cd46 expression levels on naepcs of asthmatics as well as on naepcs of healthy donors. thus, we speculate that an increased adv receptor expression level may explain the enhanced adv5 transduction efficiency of naepcs after hdm exposition (figure 7a,b) . in contrast, there was no figure 6 . the effect of hdm stimulation and adv5 infection on naepcs. naepcs were stimulated at different time points (day 1 and day 3) with different hdm concentrations (1 µg/ml, 10 µg/ml, and 100 µg/ml), and the cells were subsequently transduced with adv5 at 10 virus particle numbers per cell (vpc). twenty-four hours post-transduction, luciferase assays were performed. we observed an increased adv5 transduction efficiency in pre-stimulated naepcs with hdm, particularly at day 3 in asthmatic specimens. this was in contrast to samples of healthy controls. values were normalized and presented as fold change given as mean and standard error of mean (sem). to shed light on the underlying mechanism for enhanced adv5 uptake, major adv receptor expression levels, in particular, car, cd46, and dsg-2, were characterized on naepcs through flow cytometry. in comparison to hdm-provoked naepcs, we observed a significant increase of car and cd46 expression levels on naepcs of asthmatics as well as on naepcs of healthy donors. thus, we speculate that an increased adv receptor expression level may explain the enhanced adv5 transduction efficiency of naepcs after hdm exposition (figure 7a,b) . in contrast, there was no significant difference in the expression level of dsg-2 on hdm-treated naepcs, compared to healthy naepcs controls (figure 7c ). the mean-pe values for car were set as absolute numbers. as shown, the asthmatic group had significant differences at car expression levels after hdm stimulation, particularly at day 3 compared to day 1. (b) the mean-apc values for cd46 were set as absolute numbers. as shown, the healthy control group showed significant differences between time points and concentration levels, compared to the asthmatic group (c) desmoglein-2-pe was not significantly different in terms of time points and concentration levels in healthy controls and asthmatics. to analyze whether hdm influences the barrier function on naepcs that may lead to enhanced adv transduction, we provoked 3d naepcs cultures with hdm and performed raster-em on days 1 (d1) and day 3 (d3). raster-em scan provided interesting insights into the irritated epithelium after high dosage of hdm, in particular at d3 (figure 8a ). measuring the lengths and thickness of the ciliary, there was no statistical differences for the different groups (figure 8b) . histologic analyses confirmed an increased mucus production and an irritated basal layer after hdm treatment (figure 8c ). furthermore, we observed a different tight junction conformation in organotypic 3d naepcs cultures of asthmatics compared to healthy controls. the tight junction conformation in untreated asthmatic samples was tightly packed and was presented in a higher number than the untreated control samples (figure 8d,e) . here, we characterized the effects of hdm exposition on adv infection in an ex vivo cell culture models of naepcs of exacerbated pediatric asthmatics and healthy controls from our pediatric exacerbation study. with this experimental approach, we successfully characterized the effects of hdm exposition on human adv infection in naepcs ex vivo. using a library of 21 luciferase-and gfp-tagged advs, we analyzed adv infection rates in naecps in the presence of hdm as a common allergen. we found that adv luciferase expression levels were significantly increased 3 days after hdm exposure in asthmatics, compared to healthy controls, hinting towards the hypothesis that replication of adv may be influenced by hdm stimulation. however, this hypothesis needs to be further analyzed by performing adv genome replication studies in naecps. it was described previously that virus infection can synergize with allergens in the induction of asthma exacerbations. the first study describing the interaction between rv infection and hdm exposure was performed by bossios et al. (2008) [23] . they observed an increase in cytokine levels in immortalized human bronchial epithelial cell line when rv and hdm were applied or when cells were provoked with hdm before rv infection [23] . interestingly, akbarshahi et al. (2018) found a similar effect by showing that hdm exposure affected the antiviral response provoked by virus infections [12] . furthermore, golebski et al. (2014) described a relationship between hdm and poly i:c stimulation in terms of gene and protein expression [24] . the results of our study broaden the preceding findings, as we have observed an increased uptake of adv5 after hdm stimulation potentially mediated by enhanced major adv receptor expression. our results represent the first in vitro evidence that hdm stimulation may increase susceptibility to adv infections. adenovirus receptors, including car, cd46, or dsg-2, are required for uptake of adv into the respective target cell. adv5 mostly interacts with car, whereas cd46 and dsg-2 receptors are used by adv3, 7, 14, and others. car is localized on the basolateral and apical surface of the epithelial cells. lung epithelial cells are interconnected by tight junctions. by this formation, the basolateraly-located car receptors may protect the cell from the interaction with adv [25] [26] [27] . the flow cytometric analyses of hdm-provoked naepcs showed higher levels of car on naepcs. in contrast to the work of excoffon et al., our data do not include the analyses of a car localization shift but include the adenoviral susceptibility of hdm-provoked cells [27] . we speculate that an upregulation of adv receptors upon hdm inhalation may lead to increased susceptibility to adv infection in patients with allergic rhinitis or asthma exacerbation in vivo. however, to support this hypothesis, additional in vivo experiments are needed. we furthermore noticed a low but measurable increase of cd46 receptor expression levels on naepcs after hdm exposition. contrary to our results, tsai and colleagues (2018) observed decreased cd46 levels and increased apoptosis in primary nasal mucosa samples from adult mild asthmatics when cultured with hdm extracts [28] . naepcs gained increased attention as a model to study asthma development, to study immune responses during virus infection in asthma patients, and to define biomarkers specific for asthma and virus infections [29] [30] [31] . their innate immune reactions such as toll-like receptor pathways, secretion of il-33, or thymic stromal lymphoprotein boost adaptive immune responses and start a broad range of stimulation processes, e.g., activation of naïve t cells [18] . by using naepcs from healthy and allergic subjects, vroling et al. showed that both groups had differences in the chemokine, growth factor, and transcription factor levels [16, 17] . thus, naecps represent a valuable alternative as an ex vivo model, compared to tracheal or bronchial epithelium of the small airways. this further supports the use of naecps in our study to explore the relationship between asthma development, hdm exposure, and adv infections. future studies should analyze immune responses in these cells after hdm exposure and adv infection. note that the used set-up of the present study was completely based on a primary human biomaterial database. this is in contrast to other published studies, which exemplarily used cancer cell lines to correlate their experimental findings with clinical observations. in summary, this work provides novel insights into the mechanism of adenoviruses on the airway epithelium of asthmatics. to the best of our knowledge, this is the first ex vivo study presenting the impact of hdm sensitization on adv infection in an in vitro exposition model. our study may open new paths for the interaction between allergens and virus infections, and may provide a basis for further potential gene therapeutic approaches in treatment of childhood asthma exacerbation. this work analyzed the interaction of the hdm exposition in the presence of adv infection in an in vitro model based on naepcs. we demonstrate that a pre-stimulation with hdm may induce an increased adv infection rate at least partially mediated by increased car receptor expression. moreover, electron microscopy and histologic imaging revealed an effect on the cilia in organotypic 3d cell cultures when exposed to hdm that may be 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studies we thank the center for biomedical education and research, school of life sciences (zbaf), and its members for their intellectual input during seminars and lectures of the zbaf ph.d. program. we thank manuela besser for the technical preparation of few 3d cultures for histologic analyses. we appreciate the support of susanne haussmann from the witten/herdecke university, germany; elke kretschmar from the friedrich-alexander-university erlangen, germany; and the colleagues, including nurses, physicians, and technicians of the emergency room of the children's hospital of the helios university medical hospital of the witten/herdecke university, germany. in addition, we thank the children and parents who participated in this study. finally, figure 5 was created with biorender.com. funding: this research received funding from the internal research promotion of the faculty of health at witten/herdecke university, germany (iff 2019-13). the funding organizations had no role in the design and conduction of the study; sample collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. the authors have declared that they have no competing interests. this work has not been published before, and it is not under consideration for publication elsewhere. the manuscript has been approved for publication by all co-authors. key: cord-329900-lq91rb8c authors: seiffert, moritz; brunner, fabian j.; remmel, marko; thomalla, götz; marschall, ursula; l’hoest, helmut; acar, laura; debus, eike s.; blankenberg, stefan; gerloff, christian; behrendt, christian-alexander title: temporal trends in the presentation of cardiovascular and cerebrovascular emergencies during the covid-19 pandemic in germany: an analysis of health insurance claims date: 2020-08-04 journal: clin res cardiol doi: 10.1007/s00392-020-01723-9 sha: doc_id: 329900 cord_uid: lq91rb8c aims: the first reports of declining hospital admissions for major cardiovascular emergencies during the covid-19 pandemic attracted public attention. however, systematic evidence on this subject is sparse. we aimed to investigate the rate of emergent hospital admissions, subsequent invasive treatments and comorbidities during the covid-19 pandemic in germany. methods and results: this was a retrospective analysis of health insurance claims data from the second largest insurance fund in germany, barmer. patients hospitalized for acute myocardial infarction, acute limb ischemia, aortic rupture, stroke or transient ischemic attack (tia) between january 1, 2019, and may 31, 2020, were included. admission rates per 100,000 insured, invasive treatments and comorbidities were compared from january–may 2019 (pre-covid) to january–may 2020 (covid). a total of 115,720 hospitalizations were included in the current analysis (51.3% females, mean age 72.9 years). monthly admission rates declined from 78.6/100,000 insured (pre-covid) to 70.6/100,000 (covid). the lowest admission rate was observed in april 2020 (61.6/100,000). administration rates for st-segment elevation myocardial infarction (7.3–6.6), non-st-segment elevation myocardial infarction (16.8–14.6), acute limb ischemia (5.1–4.6), stroke (35.0–32.5) and tia (13.7–11.9) decreased from pre-covid to covid. baseline comorbidities and the percentage of these patients treated with interventional or open-surgical procedures remained similar over time across all entities. in-hospital mortality in hospitalizations for stroke increased from pre-covid to covid (8.5–9.8%). conclusions: admission rates for cardiovascular and cerebrovascular emergencies declined during the pandemic in germany, while patients’ comorbidities and treatment allocations remained unchanged. further investigation is warranted to identify underlying reasons and potential implications on patients’ outcomes. graphic abstract: [image: see text] electronic supplementary material: the online version of this article (10.1007/s00392-020-01723-9) contains supplementary material, which is available to authorized users. since its outbreak in wuhan, hubei province, china, in december 2019, the novel sars coronavirus (sars-cov-2) has spread rapidly, causing an outbreak of acute and severe respiratory illness worldwide [1] . the first disease outbreak news from the world health organization (who) had been issued january 5, 2020. after a rapid spread of sars-cov-2 in italy [2] and other european regions, several countries issued strict infection control measures. elective in-hospital procedures were widely cancelled or postponed to provide additional capacities for the treatment of covid-patients in germany, starting in march 2020 [3, 4] . while an association between acute viral respiratory disease and subsequent cardiovascular events had been described for other diseases (e.g., influenza) [5, 6] , several centers reported a decline in hospital admissions for acute cardiovascular and cerebrovascular emergencies during the covid pandemic [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . since acute cardiovascular and cerebrovascular diseases remain leading causes for morbidity and mortality, further investigation of this concerning trend is warranted to identify potential implications for both health-care professionals and regulators. this analysis sought to determine trends in admission rates, invasive treatments, and comorbidities of inpatients treated for cardiovascular and cerebrovascular emergencies during the sars-cov-2 pandemic in germany. this was a retrospective analysis of routinely collected health insurance claims data. based on their primary or admission diagnosis, we included all patients with inpatient treatment between january 1, 2019, and may 31, 2020, for cardiovascular and cerebrovascular emergencies. these included (1a) st-segment elevation myocardial infarction (stemi), (1b) non-st-segment elevation myocardial infarction (nstemi), (2) acute limb ischemia, (3) acute aortic rupture, (4a) acute stroke and (4b) transient ischemic attack (tia) (for detailed coding see supplemental table 1 ). data collected during the pandemic (covid: january through may 2020) were compared to a control period in the previous year (pre-covid: january through may 2019). the primary study end point was the absolute number of monthly hospitalizations and the corresponding admission rate per 100,000 insured inhabitants. secondary end points included the absolute number and proportion of invasive procedures provided to these patients (for detailed coding see supplemental table 1) , as well as all-cause mortality during the hospital stay and the proportion of relevant comorbidities and socio-demographic variables. the longitudinal data of germany's second-largest insurance fund, barmer, includes the outpatient and inpatient medical care provided to up to 9.4 million (from 2008 to 2020) german citizens (13.2% of germany's population) involving more than 24 million hospitalizations between january 1, 2008, and may 31, 2020. the barmer cohort is similar to western european countries and has been widely used for research projects before [20, 21] . a regular random sample validation of internal and external validity is performed by the medical service of the health funds (mdk) in germany, and various peer-reviewed validation studies have been published before [22, 23] . the diagnoses and comorbidities routinely collected in health insurance claims data follow the commonly accepted international standard for reporting diseases and health conditions using world health organization (who) international classification of diseases in its 10th revision of the german modification (icd-10-gm) and operations and procedures codes (ops) as a german adaptation of the international classification of procedures in medicine (icpm) by who. the primary diagnosis of the hospital case was used to discriminate between the cardiovascular or cerebrovascular emergencies. in approximately 0.6% of the most current hospitalizations (in may 2020), the admission diagnosis was utilized on a supplementary basis if no primary diagnosis was available. in addition to age (in years) and gender (dichotomized), we used the following icd-10 codes to identify diabetes (e10*, e11*, e12*, e13*, e14*), hypertension (i10*, i11*, i12*, i13*, i14*, i15*), heart failure (i50*), atrial fibrillation (i48*), chronic ischemic heart disease (i25*), obesity (e66*), chronic renal disease (n18*), chronic obstructive pulmonary disease (copd, j44*), and cancer (c00-97*) as comorbidities among the study sample. all-cause mortality was provided during the index hospital stay. we summarized the baseline characteristics of the patients with means for age and with percentages and 95% confidence interval (ci) for discrete variables. a comparison of 95% ci and chi square test was used to test for differences. for table 1 , we compared symmetric samples from january 01, 2019, to may 31, 2019 (pre-covid), vs. january 01, 2020, to may 31, 2020 (covid), to adjust for seasonal effects. missing data (0.6%) were handled by exclusion. as additional sensitivity analysis, we compared the study variables using shorter time periods (e.g., march 2019 vs. march 2020, april 2019 vs. april 2020, may 2019 vs. may 2020). besides, we determined the admission rates using either both primary and admission diagnosis vs. only primary diagnosis (only reimbursed cases). data processing was performed with software sas version 9.04 (sas institute, north carolina, usa) and spss version 25 (ibm corporation, new york, usa), visualization was performed with software adobe illustrator version 24.1.2 (adobe, california, usa). we identified 115,720 hospitalizations (mean age 72.9 years, 51.3% females, 95% ci 51.0% to 51.6%) for cardiovascular or cerebrovascular emergencies between january 1, 2019, and may 31, 2020 (monthly mean: 6,807 patients) ( table 1) . among all hospitalizations, a total of 8,202 deaths occurred. the monthly hospital admission rate for cardiovascular or cerebrovascular emergencies declined from a maximum of 83.8 per 100,000 in january 2019 to a minimum of 61.6 per 100,000 in april 2020, which increased to 67.1 per 100,000 in may 2020 (mean: 75.2 per 100,000). comparing pre-covid to covid time frames, overall monthly admission rates declined from 78.6 per 100,000 to 70.6 per 100,000. this was observed across all strata ( fig. 1) : admission rates per 100,000 during covid compared to pre-covid decreased for stemi (7.3 vs. 6.6, − 12.2% percentage points, p.p.), nstemi (16.8 vs. 14.6, − 15.2% p.p.), acute limb ischemia (5.1 vs. 4.6, − 12.4% p.p.), stroke (35.0 vs. 32.5, − 8.9% p.p.), and tia (13.7 vs. 11.9, − 14.6% p.p.). no relevant differences were observed for aortic ruptures (0.6 vs. 0.5) ( fig. 1 and table 1 ). the lowest admission rates were noted in april 2020 (61.6 per 100,000) with declines in stemi (− 16.3%), nstemi (− 21.5%), acute limb ischemia (− 22.5%), stroke (− 17.8%), and tia (− 30.2%) compared to the respective rates in april 2019. for may 2020, slowly recovering admission rates were observed for nstemi, stemi, ali, stroke, and tia compared to april 2020 (fig. 1 ). the admission rates of nstemi and stemi and the corresponding numbers of daily sars-cov-2 infections in germany from january through may 2020 are depicted in fig. 1a . comorbidities, cardiovascular risk profiles and sociodemographic variables were similar among patients admitted during covid compared to pre-covid eras (table 1) . besides patients admitted for stroke (8.5-9 .8%), the inhospital mortality was similar among patients admitted during covid compared to pre-covid eras (table 1) . table 1 admission rates and baseline characteristics and comorbidities of pre-covid vs. covid groups (january to may 2019 vs. january to may 2020) proportions for comorbidities are presented as % with 95% confidence interval in parentheses # statistically significant differences (p < 0.05). n.s. the percentage of patients admitted for cardiovascular or cerebrovascular emergencies, who underwent interventional or open-surgical procedures during the hospital stay, were similar between pre-covid and covid periods for stemi (84.7-86.3%), nstemi (58.0-60.5%), acute limb ischemia (81.9-82.8%), aortic rupture (51.5-56.7%), stroke (18.4-19.1%), and tia (2.1-2.2%) (fig. 2 and table 1 ). all sensitivity analyses were confirmative (not shown). this analysis of a large dataset of routinely collected health insurance claims demonstrated a marked decrease in hospital admission rates for several cardiovascular and cerebrovascular emergencies during the covid-19 pandemic in germany. these patients' comorbidities and the percentage of them, who were treated invasively, remained unchanged. a concerning decline of more than 40% in hospital admissions for acute coronary syndromes after the covid-19 outbreak had been reported in smaller series of severely affected regions [9, 11-14, 18, 24] . we observed reductions of 12.2% in patients presenting with stemi and 15.2% with nstemi in a large representative sample of the german population during a longer phase of observation (january through may 2020). the maximum decline of nstemi presentations was detected in april 2020 (− 21.5% compared to 2019), followed by a slow recovery in may 2020, essentially mirroring sars-cov-2 infection rates in germany. these observations were of particular interest asopposed to other countries-germany was not affected as strongly by the covid-19 pandemic and a significant limitations of health-care resources did not become evident. this may explain why we found a milder decline compared to regions heavily affected by the disease outbreak [25] and was in line with reports from other less-affected areas reporting reductions of approximately 25% in acute cases. interestingly, admissions for several cardiovascular and cerebrovascular emergencies started to increase again in may, suggesting a potential recovery after a significant drop in new daily covid-19 cases and a liberalization of public restrictions in germany. similar trends were observed for patients with acute cerebrovascular and peripheral vascular diseases. using surrogate markers for the quantity of care, a large recent analysis reported a decrease of 39% in march 2020 in patients presenting with acute ischemic stroke to us hospitals [8] . the results of the current study confirmed declines in strokes and tia during the pandemic in germany, albeit less severe. consistent with these observations, we detected a marked decline in the admission rates for acute limb ischemia during the pandemic. whether this translates into increased disease severity and worse outcomes as others have suggested [26] will need to be investigated. additional information on age, gender, and comorbidities revealed patient populations to be similar before and during the pandemic, arguing against the notion that milder affected patients might particularly refrain from seeking medical care, thus shifting the severity of diseases. likewise, treatment allocation of the patients admitted remained unchanged. furthermore, in the current study, patients presenting to the emergency departments with acute cardiovascular or cerebrovascular disease had a similar chance to receive interventional or open-surgical therapy before and during the pandemic in germany. this seems to be an indicator for an ongoing high-level medical therapy during this time period. since rapid diagnosis and treatment of acute coronary syndromes are important to avoid further complications [27, 28] , recently published results from italy are alarming reporting a delay in coronary revascularization and increasing rates of case fatality and major complications in patients suffering from acute myocardial infarction during covid-19 [14] . to guide the management of acute coronary syndromes and cardiovascular disease during the covid-19 pandemic and to contain collateral damage, the european association of percutaneous cardiovascular interventions and german cardiac society recently issued comprehensive expert documents [29, 30] . the root causes for the observed decline in acute admissions for cardiovascular and cerebrovascular diseases remain unclear and may be multifactorial: avoidance of medical care due to patient-based anxiety and fear of contagion during the pandemic may play an important role. however, an attitude toward increased deferrals of less urgent cases by health-care personnel and lifestyle changes among other confounders during the time of extensive social restrictions need to be evaluated as well. patient information and public education will be of paramount importance to contain collateral damages caused by delayed medical treatment for acute cardiovascular and cerebrovascular diseases. most previous reports on this topic used either surveys [31], selective multi-center data, or surrogate parameters (e.g., use of imaging software) [8] to quantity patient care. population-based data investigating emergency admissions in this setting validly remain scarce. nevertheless, the following limitations merit consideration. first, health insurance claims data were not primarily collected for research purposes and retrospective observational studies are unsuitable to prove underlying causal relationships. however, data from health insurance claims, as used in this analysis, may help to identify potential implications for both healthcare professionals and regulators at an early stage. second, while longitudinal data were available between 2008 and today, pandemic measures in germany began in february 2020 with no relevant follow-up available. therefore, future analyses investigating patients' outcomes are required. this is particularly important for mortality, albeit differentiating covid-19-related mortality from mortality due to other causes in the light of covid infection will remain complex from insurance claims data. third, admission diagnoses were used for current analyses in a small share of cases in may 2020 (0.6%) if the main diagnosis was not yet submitted. however, comorbidities were only analyzed for individuals with complete datasets available to avoid classification bias. last, the barmer sample included a relevant proportion of the entire population in germany. a selection bias appeared unlikely and the barmer cohort was comparable to other european countries in terms of comorbidities, age, and gender. in contrast to clinical registries or administrative data, the barmer cohort was less affected by selection bias and includes all age groups, all hospitals, and all medical specialties. however, spatial analyses aiming for regional differences were not feasible in the existing dataset. in this large-scale retrospective analysis of health insurance claims, we observed a marked decrease of in-hospital admission rates for acute cardiovascular and cerebrovascular emergencies including myocardial infarction, acute limb ischemia, stroke, and transient ischemic attack during the covid-19 pandemic in germany. no changes were seen in these patients' comorbidities and treatment allocations. the underlying root cause for these developments and subsequent implications on patient outcomes warrant further investigation. public education will be important to contain collateral damage caused by delayed treatment for acute cardiovascular and cerebrovascular diseases. ted work. dr. gerloff reports personal fees from amgen, bayer vital, bristol-myers squibb, boehringer ingelheim, sanofi aventis, abbott, and prediction biosciences outside the submitted work. dr. behrendt and dr. debus report grants from the german federal joint committee and grants from german stifterverband outside the submitted work. the other authors declare no conflicts of interest. ethics approval several review boards have determined that using anonymized data from claims or national statistics retrospectively is not human subject research because de-identified datasets were used. consent to participate all analyses were following the european union general data privacy regulation (eu-gdpr), considering the theoretical concept of k-anonymity. thus, patient informed consent was not obtained for this retrospective secondary data analysis [31] . availability of data and material (data transparency) the data will be shared on reasonable request to the corresponding author. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. a novel coronavirus outbreak of global health concern pattern of vascular disease in lombardy, italy, during the first month of the covid-19 outbreak cardiovascular disease and surgery amid covid-19 pandemic the global impact of covid-19 on vascular surgical services role of acute infection in triggering acute coronary syndromes acute myocardial infarction after laboratory-confirmed influenza infection 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time-series analysis in a tertiary greek general hospital where have the stemis gone during covid-19 lockdown? an increased severity of peripheral arterial disease in the covid-19 era esc guidelines for the management of acute myocardial infarction in patients presenting with st-segment elevation: the task force for the management of acute myocardial infarction in patients presenting with st-segment elevation of the european society of cardiology (esc) accf/aha guideline for the management of st-elevation myocardial infarction: a report of the american college of cardiology foundation eapci position statement on invasive management of acute coronary syndromes during the covid-19 pandemic coronavirus disease 2019 (covid-19) and its implications for cardiovascular care: expert document from the german cardiac society and the world heart federation acknowledgment open access funding provided by projekt deal. key: cord-337339-0vkigjv2 authors: osterrieder, nikolaus; bertzbach, luca d.; dietert, kristina; abdelgawad, azza; vladimirova, daria; kunec, dusan; hoffmann, donata; beer, martin; gruber, achim d.; trimpert, jakob title: age-dependent progression of sars-cov-2 infection in syrian hamsters date: 2020-07-20 journal: viruses doi: 10.3390/v12070779 sha: doc_id: 337339 cord_uid: 0vkigjv2 in late 2019, an outbreak of a severe respiratory disease caused by an emerging coronavirus, sars-cov-2, resulted in high morbidity and mortality in infected humans. complete understanding of covid-19, the multi-faceted disease caused by sars-cov-2, requires suitable small animal models, as does the development and evaluation of vaccines and antivirals. since age-dependent differences of covid-19 were identified in humans, we compared the course of sars-cov-2 infection in young and aged syrian hamsters. we show that virus replication in the upper and lower respiratory tract was independent of the age of the animals. however, older hamsters exhibited more pronounced and consistent weight loss. in situ hybridization in the lungs identified viral rna in bronchial epithelium, alveolar epithelial cells type i and ii, and macrophages. histopathology revealed clear age-dependent differences, with young hamsters launching earlier and stronger immune cell influx than aged hamsters. the latter developed conspicuous alveolar and perivascular edema, indicating vascular leakage. in contrast, we observed rapid lung recovery at day 14 after infection only in young hamsters. we propose that comparative assessment in young versus aged hamsters of sars-cov-2 vaccines and treatments may yield valuable information, as this small-animal model appears to mirror age-dependent differences in human patients. emerging coronaviruses have caused serious global public health concerns in the past two decades and cause infections that lead to severe respiratory and occasionally systemic disease [1] . these include severe acute respiratory syndrome (sars)-cov as well as middle east respiratory syndrome (mers)-cov, both of which resulted in high morbidity and mortality in infected humans [2, 3] . similar to other emerging cov, the novel sars-cov-2 likely arose from an ancestor in bats and amplified in a yet unknown animal reservoir before making its jump into the human population [4] . sars-cov-2 has pushed global health systems to the brink of breakdown. the remarkably fast and unexpected spread of sars-cov-2 can be attributed to efficient replication in the upper respiratory tract and robust human-to-human transmission. while covid-19 is primarily a respiratory syndrome, it can induce quite variable clinical signs including fatigue, headache, and gastrointestinal symptoms, which can in severe cases result in fatality [5] . it has become evident that differences in the type and severity of sars-cov-2-induced disease and its sequelae seem to be strongly correlated with the age of patients and exacerbated by pre-existing medical conditions (e.g., chronic obstructive pulmonary disease, heart disease, diabetes, obesity) [1, [6] [7] [8] . the availability of reliable animal models is of critical importance for pathogenesis studies as well as the development and preclinical evaluation of vaccines and therapeutics [2, 9, 10] . for sars-cov-2, the susceptibility of several animal species was predicted by in silico analysis based on comparisons of the entry receptor for sars-cov and sars-cov-2, angiotensin converting enzyme 2 (ace2). these predictions are of relevance because the ace2 sequence is deemed an important factor governing susceptibility [11] . more specifically, the interaction of the viral spike (s) glycoprotein receptor binding domain with its ace2 counterpart was examined [12, 13] , and in some cases confirmed in vivo [9] . productive sars-cov-2 infection was shown in non-human primates, which developed respiratory disease recapitulating moderate disease as observed in humans [14] [15] [16] [17] . mice are not naturally susceptible to sars-cov-2, but mouse-adapted virus strains have been developed and used in balb/c mice [18, 19] . moreover, transgenic mice expressing human ace2 represent a lethal sars-cov-2 infection model resulting in significant weight loss and permitting robust virus replication in the respiratory tract including the lungs [20] . ferrets have provided valuable data in the case of sars-cov [21, 22] , and two studies describe the infection of ferrets with sars-cov-2 and successful transmission to in-contact animals without clinical signs [23, 24] . first and preliminary studies also focused on the assessment of a syrian hamster model that had previously been used successfully in sars and mers research [21, 22, 25, 26] . it was suggested that hamsters are highly susceptible, although they were reported to show no or only moderate respiratory signs and body weight losses. however, it is important to note that only young male hamsters of 4 to 5 weeks of age were used in these studies [27, 28] . we sought to explore age-related differences in the course of sars-cov-2 infection in syrian hamsters and to establish a small-animal model that resembles the more severe sars-cov-2-infection observed particularly in elderly patients. we conducted all animal work in compliance with relevant national and international guidelines for care and humane use of animals. the animal use protocol for the experiments reported here was approved by the landesamt für gesundheit und soziales in berlin, germany (approval number 0086/20; approved on 30.04.2020). virus stocks were prepared from a previously published sars-cov-2 isolate (betacov/germany/bavpat1/2020) [29] , which was kindly provided by drs. daniela niemeyer und christian drosten, charité berlin, germany. the isolate, referred to as sars-cov-2 münchen (sars-cov-2m) [30] , was handled under the appropriate safety precautions in a bsl-3 facility (freie universität berlin, institut für virologie) and propagated on vero e6 cells (atcc crl-1586) in minimal essential medium (mem; pan biotech, aidenbach, germany) supplemented with 10% fetal bovine serum (pan biotech), 100 iu/ml penicillin g and 100 µg/ml streptomycin (carl roth, karlsruhe, germany). thirty-six 6-or 32-to-34-week-old female and male syrian hamsters (mesocricetus auratus; breed rjhan:aura, janvier labs, saint-berthevin, france) were kept in individually ventilated cages (ivcs; tecniplast, buguggiate, italy) in an approved bsl-3 facility. ivcs were equipped with enrichment (carfil, oud-turnhout, belgium). all animals had unrestricted access to food and water and were allowed to acclimate to the conditions for seven days prior to infection. cage temperatures and relative humidities were recorded daily and ranged from 22-24 • c and 40-55%, respectively. the 6-week-old hamsters were randomly distributed into two groups: mock (n = 12, 6-week-old) and young infected (n = 12, 6-week-old). the third group represents the aged infected hamsters (n = 12, 32-34-week-old). iptt-300 transponders (biomedic data systems, seaford, de, usa) were subcutaneously implanted into all hamsters 2 days prior to infection to allow the identification and monitoring of body temperatures. animals were mock-infected with 60 µl medium from uninfected vero e6 cells or infected with 1 × 105 pfu sars-cov-2m in 60 µl by intranasal instillation. for transponder implantation, hamsters were sedated with butorphanol (2.5 mg/kg; cp-pharma, burgdorf, germany) and midazolam (2 mg/kg; braun, melsungen, germany). for infections, hamsters were sedated with ketamine (25 mg/kg; serumwerk bernburg, bernburg, germany) and midazolam (2 mg/kg; braun). on 2, 3, and 5 days post-infection (dpi), three randomly assigned hamsters of each group were euthanized by exsanguination under medetomidine (0.15 mg/kg; pharma-partner, hamburg, germany), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg) anesthesia [31] . blood, nasal washes, bucco-laryngeal swabs, lungs (left and right), kidneys, spleens, duodenums, and blood sera were collected for (histo)pathological examinations and/or virus titrations, rt-qpcr, and serological examination. during the 14-day experiment, body temperatures, body weights, and clinical signs of all animals were monitored twice daily. animals that had a body weight loss of more than 10% weight over a 72 h period were euthanized in compliance with the animal use protocol. such humane termination applies to the two hamsters euthanized 7 dpi. for histopathology and in situ hybridization (ish), the left lung lobe was carefully removed, immersion-fixed in formalin, ph 7.0, for 48 h, embedded in paraffin, and cut in 2 µm sections. for histopathology, slides were stained with hematoxylin and eosin (he) after dewaxing in xylene and rehydration in decreasing ethanol concentrations. lung sections were microscopically evaluated in a blinded fashion by a board-certified veterinary pathologist to assess the character and severity of pathologic lesions using lung-specific inflammation scoring parameters as described for other lung infection models before [32] . three different scores were used that included the following parameters: (1) lung inflammation score including severity of (i) interstitial pneumonia (ii) bronchitis, (iii) epithelial necrosis of bronchi and alveoli, and (iv) hyperplasia of type ii-alveolar epithelial cells; (2) immune cell infiltration score taking into account the presence of (i) neutrophils, (ii) macrophages, and (iii) lymphocytes in the lungs as well as (iv) perivascular lymphocytic cuffing; and (3) edema score including (i) alveolar edema and (ii) perivascular edema. ish was performed as reported previously [33] using the viewrna™ ish tissue assay kit (invitrogen by thermo fisher scientific, darmstadt, germany) following the manufacturer's instructions with minor adjustments. probes for the detection of n gene rna of sars-cov-2 (ncbi database nc_045512.2, nucleotides 28,274 to 9533, assay id: vpnkrhm) and the mouse housekeeping gene eukaryotic translation elongation factor-1α (ef1a; assay id: vb1-14428-vt, affymetrix, inc., santa clara, ca, usa), which shares 95% sequence identity with the syrian hamster orthologue, were designed. lung sections (2 µm thickness) on adhesive glass slides were dewaxed in xylol and dehydrated in ethanol. tissues were incubated at 95 • c for 10 min with subsequent protease digestion for 20 min. sections were fixed with 4% paraformaldehyde in phosphate-buffered saline (alfa aesar, thermo fisher, kandel, germany) and hybridized with the probes. amplifier and label probe hybridizations were performed according to the manufacturer's instructions using fast red as the chromogen, followed by counterstaining with hematoxylin for 45 s, washing in tap water for 5 min, and mounting with roti ® -mount fluor-care dapi (4, 6-diaminidino-2-phenylindole; carl roth). for negative and morphologically intact controls, lungs from uninfected hamsters of each group (n = 4) were included. in addition, an irrelevant probe for the detection of pneumolysin was used as a negative control for unspecific reactions. he-stained and ish slides were analyzed and images were taken using an olympus bx41 microscope with a dp80 microscope digital camera and the cellsens™ imaging software, version 1.18 (olympus corporation, münster, germany). for the display of overviews of whole lung lobe sections, slides were automatically digitized using the aperio cs2 slide scanner (leica biosystems imaging inc., vista, ca, usa), and image files were generated using the image scope software (leica biosystems imaging inc.). the percentages of lung tissues affected by inflammation were determined histologically by an experienced board certified experimental veterinary pathologist (k.d.) as described previously [34] . lung inflammation scores were determined as (0) absent, (1) minimal, (2) mild, (3) moderate, or (4) severe, and quantified as described previously [35] . immune cell influx scores and edema scores were rated from (0) absent to (1) sporadic, (2) mild, (3) moderate, or (4) severe. ish signals were digitally quantified on scanned whole slides of each animal using the aperio positive pixel count algorithm (leica biosystems imaging inc.) with minor adjustments. specifically, a positivity score encompassing the number of positive pixels in relation to the total number of pixels per tissue scan as well as the total intensity of positive pixels were determined ( figure s3 ). for an assessment of virus titers from 25 mg of lung tissue, tissue homogenates were serially diluted and plated on vero e6 cells in 12-well cell culture plates (sarstedt, nümbrecht, germany). at 3 dpi, cells were fixed in 4% formalin, stained with 0.1% crystal violet (in 25% methanol), and plaques were counted. rna was extracted from nasal washes and tracheal swabs with the rtp dna/rna virus mini kit (stratec, birkenfeld, germany) according to the manufacturer's instructions. the innuprep virus dna/rna kit (analytic jena, jena, germany) was used for rna extractions from tissue samples. viral rna was quantified using a one-step rt qpcr reaction with the neb luna universal probe one-step rt-qpcr (new england biolabs, ipswitch, ma, usa) and the 2019-ncov rt-qpcr primers and probe (e_sarbeco) [36] on a steponeplus realtime pcr system (thermo fisher scientific, waltham, ma, usa) according to the manufacturer's instructions. viral rna copies were then normalized to cellular rpl18 as previously described [37] . all primers and probes are listed in table s2 . standard curves for absolute quantification were generated from serial dilutions of sars-cov-2 rna obtained from a full-length virus genome cloned as a bacterial artificial chromosome and propagated in e. coli or from serial dilutions of the purified hamster rpl-18 pcr product. the latter was generated by pcr using rpl-18 qpcr-primers (table s2 ) and a cdna template obtained from hamster lung tissue. in lung tissue, viral rna copies were calculated per 1×10 5 hamster rpl-18 transcripts. for serum neutralization assays, 50 µl of medium containing 103.3 tissue culture infectious doses 50 (tcid 50 ) of sars-cov-2m were mixed with 50 µl of diluted serum. each sample was tested in triplicate. after 1 h incubation at 37 • c, the mixture was transferred to confluent vero e6 cells in a 96-well plate (sarstedt, nümbrecht, germany). viral replication was assessed after 3 days by the detection of cytopathic effects. statistical analyses were performed using graph-pad prism v8 (graphpad software inc., san diego, ca, usa). the statistical details of all analyzed experiments can be found in the respective figure legend. for our experiments, we used a total of 36 female and male syrian hamsters (mesocricetus auratus), which were either 6 (n = 24) or 32 to 34 weeks old (n = 12). hamsters were kept in individually ventilated cages (ivcs) and randomly assigned to three groups: mock (n = 12, 6-week-old), young infected (n = 12, 6-week-old) and aged infected (n = 12, 32-to 34-week-old). animals were mock-infected with supernatants of cell culture medium taken from uninfected vero e6 cells or infected with 1 × 10 5 plaque-forming units of sars-cov-2 münchen (sars-cov-2m; betacov/germany/ bavpat1/2020) [30] . during the 14-day experiment, body temperatures, body weights, and clinical signs were recorded daily. animals were euthanized and sampled at different time points after infection to assess virus titers in various organs and to examine pathological changes in the lungs (figures 1 and 2) . each group (n = 3 for 2, 3, and 5 dpi, n = 1 for 7 dpi and n = 2 for 14 dpi; bar = 0.5 cm) (b) affected areas of inflammation were identified histologically for each lung and compared between the groups. (c) lung inflammation score taking into account (i) severity of pulmonary inflammation; (ii) bronchitis (iii) bronchial and alveolar necrosis; iv) hyperplasia of alveolar epithelial cells type ii. (d) immune cell influx score taking into account the infiltration of lung tissue with (i) neutrophils; (ii) macrophages; (iii) lymphocytes; (iv) perivascular lymphocytic cuffing; and (e) edema score including (i) alveolar and (ii) perivascular edema. scores and parameters in c to e were graded as absent (0), minimal (1), mild (2), moderate (3), or severe (4) as described [35] . (f) time-dependent distribution of sars-cov-2 rna signals in young and adult hamsters representative of each group as detected by in situ hybridization (group sizes as in a; for digital image analysis of the differences, see figure s3 ; bar = 0.5 cm). first, we observed age-dependent sars-cov-2-induced body weight losses, with more pronounced weight reductions in aged compared to young hamsters ( figure 1a-c) . mean body weight losses peaked at 6 to 7 days post-infection (dpi), with partial recovery until 14 dpi in both infected groups. there were no differences in body temperatures between the infected groups or between infected and mock-infected animals ( figure 1d ). next, we determined viral titers and sars-cov-2 rna copy numbers in various tissues by rt-qpcr [36, 37] and performed virus titrations from lung homogenates using vero e6 cells ( figure 1e -h). results were similar between age groups and confirmed high viral loads in respiratory samples at early time points after infection, but a relatively rapid clearance of infection. while we did not identify any other sex-specific differences, it seems important to note that the rt-qpcr of blood samples revealed viremia in two male individuals from both age groups with viral rna copy numbers of >10 5 at 5 and 7 dpi, respectively ( figure 1i ). in these individuals, we also detected relatively high levels of viral rna in the spleen, kidneys, and duodenum, indicating a systemic spread of sars-cov-2 in some cases (table s1 ). to further investigate the potential dissemination of infection, we tested the aforementioned organs from all animals sacrificed at 5 dpi and found them to be either negative for sars-cov-2 rna or to contain only low levels of viral rna (table s1 ). viral loads in bucco-laryngeal swabs and nasal washes appeared to be a reliable surrogate of the presence or absence of virus in the lungs in both age groups. the average loads ranged between 10 4 and 10 7 copies, respectively, at early times after infection, indicating that these sampling techniques can be used to monitor sars-cov-2 replication in syrian hamsters ( figure 1g,h) . at 14 dpi, hamsters had mounted a humoral immune response as evidenced by relatively high titers of neutralizing antibodies. it is worth noting that antibody titers appear to be higher in young when compared to aged hamsters in the animals tested (table s1) . histopathology revealed clear age-dependent differences, with young hamsters launching an earlier and stronger immune cell influx into the lungs associated with a faster recovery than their aged counterparts (figure 2a-e) . at 2 dpi, young hamsters developed a marked necro-suppurative bronchointerstitial pneumonia with strong alveolar and interstitial influx of neutrophils and macrophages as well as perivascular lymphocytic cuffing, which was much milder or absent in the aged group ( figure s1a, right panel) . in contrast, only aged animals developed pronounced alveolar and perivascular edema indicating vascular leakage at 3 dpi ( figure 2e and figure s2c) . a more diffuse, severe bronchointerstitial pneumonia was similarly present in both groups at 5 dpi with an onset of tissue regeneration, including hyperplasia of the bronchial epithelium ( figure s1c , arrowhead) and type ii alveolar epithelial cells. only at the 5 dpi time point was the arterial and venous endothelium of animals in both groups swollen and vacuolated, with necrotic endothelial cells separated from the underlying basement membrane by the presence of subendothelial lymphocytes and neutrophils ( figure s2c, left) , which was consistent with what has been described as endothelialitis in human sars-cov-2 infection [38] . at 7 dpi, recovery as indicated by the marked hyperplasia of bronchial epithelial cells and type ii alveolar epithelial cells were seen in both groups ( figure s2a ). interestingly, lung tissues had almost recovered in young hamsters at day 14, while the aged animals still had persistent tissue damage and active inflammation ( figure s2b ). from 2 dpi onwards, sars-cov-2 rna was detected by in situ hybridization in bronchial epithelial cells, debris in the bronchial lumen, alveolar epithelial cells type i and type ii, as well as macrophages in both groups, again with clear age-dependent differences over time ( figure 2f , figures s2d and s3) . young animals had high amounts of viral rna in numerous bronchial epithelial cells and within the bronchial lumen that was accompanied by marked spreading through the lung parenchyma on 2 and 3 dpi. in contrast, aged animals had less virus rna present in the bronchi. we detected only a scattered pattern of infected bronchial epithelial cells and sporadic areas of parenchymal infection at 2 and 3 dpi. at 5 dpi, viral rna was undetectable in the bronchi of young hamsters, and only small infected areas containing low levels of rna with a patchy distribution were detected. it is noteworthy that aged animals, at the same time after infection, had increased numbers of infected areas with a similarly patchy distribution throughout the lungs as well as copious amounts of viral rna associated with cellular debris in the bronchial lumen. using this technique, no viral rna was detected at 14 dpi in either group. in summary, our study examined the suitability of a small animal model to study sars-cov-2 infections and expands on determining the role that age might play in the differences with respect to disease progression. intranasal infection of syrian hamsters resulted in weight loss and robust virus replication in the upper and lower respiratory tract. a limitation of this study is the lack of an age-matched mock group for the older hamsters, which we are unable to include for legal reasons as we are bound under german law to follow the 3r principle. we further demonstrate that aged 32-to 34-week-old hamsters experienced higher and more consistent weight loss after intranasal infection, while body temperatures and virus replication in upper airways and lungs were similar between both age groups. furthermore, we show that, using in situ hybridization, viral rna was detectable in bronchial epithelial cells, type i and type ii alveolar epithelial cells, and macrophages. all these cell types are potential targets of sars-cov-2 in human lung tissue; hence, the infection of hamsters of different ages seems to closely reflect what has been reported for human patients [28, 39] . in contrast to sars-cov-2 titers, histopathological changes differed markedly between young and aged syrian hamsters over time: younger animals launched more severe reactions at early time points after infection, while lesions and inflammation in the lungs became more pronounced and widespread at later time points in the elderly. notably, age-related differences of sars-cov-2 infections have also been observed in non-human primates [17] , and while this manuscript was under revision, in hamsters [40] . the study by imai et al. also describes infections of syrian hamsters of different age groups, and they also observed more robust body weight losses in older hamsters [40] . based on the data presented here, we propose that comparative preclinical assessments of sars-cov-2 vaccines and other treatment options in young versus aged hamsters may yield valuable and relevant results, as this small animal model appears to mimic age-dependent differences in humans. the development of a humoral immune response emphasizes that hamsters are likely suitable for vaccination trials. our observations also confirm that body weight loss is a readily quantifiable parameter that has proven very useful to measure disease severity in sars-cov-2 infection of syrian hamsters. this makes the difference in body weight loss between age groups with more consistent losses in aged hamsters only more important as it provides an objective way to judge clinical efficacy of antiviral therapy or vaccination. association between age and 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real-time rt-pcr validation of assays to monitor immune responses in the syrian golden hamster (mesocricetus auratus) tropism, replication competence, and innate immune responses of the coronavirus sars-cov-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures syrian hamsters as a small animal model for sars-cov-2 infection and countermeasure development this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors acknowledge the excellent technical assistance by ann reum, annett neubert, and simon dökel. we would like to thank carfil inc. for their generous support of our animal husbandry. the authors declare no conflict of interest. key: cord-342160-snfm62js authors: panait, luciana cătălina; stock, graham; globokar, majda; balzer, jörg; groth, bernhard; mihalca, andrei daniel; pantchev, nikola title: first report of cytauxzoon sp. infection in germany: organism description and molecular confirmation in a domestic cat date: 2020-07-17 journal: parasitol res doi: 10.1007/s00436-020-06811-3 sha: doc_id: 342160 cord_uid: snfm62js cytauxzoonosis is described as an emerging tick-borne disease of domestic and wild felids caused by protozoans of the genus cytauxzoon. while in the americas the condition is described as a fatal disease, in europe, reports on the clinical expression of the infection are scarce. this study describes the first case of cytauxzoon sp. infection in germany, in a domestic cat. a 6-year-old male domestic cat living in saarlouis (saarland) was presented with anorexia, lethargy and weight loss. the cat had an outdoor lifestyle and had not travelled abroad. serum clinical chemistry analysis revealed azotaemia with markedly increased symmetric dimethylarginine, hypercreatinemia, hyperphosphatemia and hypoalbuminemia. moreover, a mild non-regenerative anaemia was present. approximately 1 year prior to these findings, the domestic cat was diagnosed with a feline immunodeficiency virus (fiv) infection. these results pointed toward a decreased glomerular filtration rate, presumably as a result of kidney dysfunction. round to oval signet ring–shaped intraerythrocytic organisms, morphologically suggestive for a piroplasm, were revealed during blood smear evaluation with a degree of parasitaemia of 33.0%. pcr analyses and sequencing of a region of the 18s rrna gene confirmed the presence of a cytauxzoon sp. infection, with 99–100% nucleotide sequence identity with previously published cytauxzoon sp. isolates. as this is the first molecularly confirmed cytauxzoon sp. infection in a domestic cat in germany, these findings suggest that cytauxzoonosis should be considered as a differential diagnosis in cases of anaemia in outdoor domestic cats, particularly in areas where wild felid populations are present. in recent years, cytauxzoon felis, a tick-borne pathogen of felids endemic in north america, is gaining clinical importance. domestic cats infected with c. felis generally develop a peracute and highly fatal disease . furthermore, other closely related piroplasms infecting domestic and wild felids have been reported. cytauxzoon manul was described in naturally infected pallas's cats (otocolobus section editor: leonhard schnittger manul) from mongolia (ketz-riley et al. 2003; reichard et al. 2005) . in europe, an unnamed species of cytauxzoon was reported not only in domestic cats from spain (criado-fornelio et al. 2004; díaz-regañón et al. 2017) , france (criado-fornelio et al. 2009; legroux et al. 2017) , italy (carli et al. 2012 (carli et al. , 2014 , portugal (alho et al. 2016) and switzerland (nentwig et al. 2018 ) but also in wild cats (felis silvestris) in spain (barandika et al. 2016; león et al. 2017) , romania (gallusová et al. 2016) , italy (veronesi et al. 2016) and bosnia and herzegovina (hodžić et al. 2018) , iberian lynx (lynx pardinus) in spain (luaces et al. 2005; millán et al. 2007 millán et al. , 2009 meli et al. 2009; garcía-bocanegra et al. 2010) and eurasian lynx (lynx lynx) in romania (gallusová et al. 2016) . studies on cytauxzoon sp. in europe show different infection rates depending on countries and host species involved. while the infection rate in domestic cats is ranging from 0.8% in france (criado-fornelio et al. 2009 ) to 23% in italy (carli et al. 2012) , in wild felids, the assessed prevalence is generally higher than 50% in romania (gallusová et al. 2016) , bosnia and herzegovina (hodžić et al. 2018) and spain (barandika et al. 2016; león et al. 2017) . the european isolates of cytauxzoon seem to be less virulent than c. felis, and the clinical manifestations appear to be associated with immune-mediated diseases or secondary infections (díaz-regañón et al. 2017; nentwig et al. 2018) . however, reports of cytauxzoon sp. infections in europe are scarce and most of them refer to subclinical cases. nonspecific clinical signs such as lethargy, anorexia, anaemia, fever, weight loss, tachycardia, tachypnoea, diarrhoea and vomiting have also been described (carli et al. 2012 (carli et al. , 2014 alho et al. 2016; legroux et al. 2017) . the present paper reports the first molecular confirmation of cytauxzoon sp. infection in germany and provides a detailed clinical picture of the disease in association with feline immunodeficiency virus (fiv) infection. in march 2017, a 6-year-old male domestic cat was referred to a veterinary facility in saarlouis (49.31°n, 6.75°e), a small city located in saarland (south-western germany), with anorexia and lethargy. the cat had an outdoor lifestyle and had never travelled abroad. according to the owner, the patient had refused to eat in the last 3 days and lost approximately 1.5 kg in the previous few weeks. episodes of vomiting could not be excluded due to the outdoor lifestyle of the cat. the patient received supportive therapy for 5 days with maropitant citrate (cerenia®, 1 mg/kg, sc), metamizole (novalgin®; 35 mg/kg, im), amoxicillin (betamox® long acting 150 mg/ml, 15 mg/kg, sc) and fluids (lactated ringer's solution, 15 ml/kg, sc) . despite the therapy, the cat was euthanized on the 5th day, due to the worsening of the clinical state, being presented in a lateral decubitus with severe lethargy and ataxia. nevertheless, the owner elected against necropsy. no information regarding the history of tick infestation or previous antiparasitic treatments was available. in march 2017, a complete blood count and serum clinical biochemical analysis (including the assessment of liver, kidney and pancreatic parameters) were performed at idexx reference laboratory in ludwigsburg, germany (table 1) . symmetric dimethylarginine (sdma) was measured using a commercially available high-throughput immunoassay (idexx sdma test, idexx laboratories inc.; ernst et al. 2018) . no urine examination was performed. approximately 1 year prior to these investigations (january 2016), a blood sample from the same patient was sent to idexx laboratories for similar haematologic and clinical biochemical analysis ( table 1 ). at that time, the blood sample was also tested for feline leukaemia virus (felv) antigen (p27; petchek felv, idexx laboratories inc.), as well as for antibodies against fiv (p24/gp40; petchek plus anti-fiv, idexx laboratories inc.) and feline coronavirus (fcov) (fcov elisa cat, afosa, germany). in march 2017, may-grünwald-giemsa-stained blood smears were prepared from peripheral blood and were evaluated for the presence of blood pathogens. the degree of parasitaemia was estimated by counting the number of infected erythrocytes per 2000 erythrocytes with an olympus bx61 microscope at × 1000 magnification. the measurement of the well-defined organisms (with a characteristic shape, n = 100) was assessed with a dp72 camera and cell^f software (olympus corporation, japan). total nucleic acids were extracted using the qiaamp dna blood biorobot mdx kit (qiagen, hilden, germany) following the manufacturer's instructions. a polymerase chain reaction (pcr) assay for piroplasmida targeting the 18s rrna gene was performed according to carret et al. (1999) and katargina et al. (2011) . furthermore, a nested and a conventional pcr protocol were used for the amplification of a part of the 18s rrna gene of the phylum apicomplexa using primer pairs bth-1f/bth-1r, gf2/gr2 and 7549/7548, respectively, following previously published reaction procedures and protocols (reichard et al. 2005; hrazdilová et al. 2019) . pcr products were visualized by electrophoresis in a 2% agarose gel. after purification, the amplicons were submitted for sequencing on both strands (macrogen, the netherlands). consensus sequences were compared with sequences available in genbank™ dataset by basic local alignment search tool (blast) analysis. in march 2017, the haematological analyses revealed a z o t a e m i a w i t h m a r k e d l y i n c r e a s e d s d m a , hypercreatinemia, as well as hyperphosphatemia and hypoalbuminemia. furthermore, a mild non-regenerative anaemia was present. abnormal laboratory findings are shown in table 1 . the results indicated decreased glomerular filtration rate that suggested the presence of kidney disease. urinalysis was not performed to assess specific gravity, protein concentration or sediment. in january 2016, the haematological and clinical chemistry variables had been within normal limits. felv and fcov tests were negative, but a positive fiv antibody test was obtained. blood smear examination in march 2017 revealed morphologically inconspicuous leukocytes, and erythrocytes displayed no significant anisocytosis or polychromasia. blood smear evaluation revealed round to oval signet ringshaped organisms inside the erythrocytes, with a light blue cytoplasm and a blue nucleus usually located in an eccentric position (fig. 1) . one to four merozoites were observed within individual red blood cells. the mean length of the well-defined forms was 1.2 ± 0.2 μm, and the measured width was 1.0 ± 0.2 μm. the intensity of parasitaemia was estimated at 33.0%. the intracellular parasites were morphologically suggestive for a piroplasm. moreover, the typical signet ring-shaped organisms could be clearly differentiated from feline haemotropic mycoplasma due to their evident large nuclear area . microscopic examination of a blood smear in january 2016 had revealed no blood cell morphologic abnormalities and no microorganisms. the presence of cytauxzoon sp. dna was confirmed by positive pcr results in all three protocols and subsequent sequencing. the longest consensus sequence available (1010 bp) was submitted to genbank™ under the accession number mn629916. blast analysis of the sequenced 18s rrna gene region revealed 99.9% nucleotide sequence identity with a genbank™ sequence of cytauxzoon sp. (legroux et al. 2017; nentwig et al. 2018 ). the study reported herein describes the clinical picture, laboratory findings and diagnostic procedures of a cytauxzoon sp. clinical infection in a domestic cat from germany. a previous piroplasmid fatal infection was described in a captive bengal tiger from a zoo in germany, when parasitic inclusions resembling cytauxzoon spp. were visualized in histological sections of various tissues, the largest number being found in blood vessels of lymph nodes and spleen. even though there was no evidence of tick infestation of the tiger, three bobcats (lynx rufus) originating from an american zoo were incriminated as a possible source of the infection (jakob and wesemeier 1996) . the cat patient in the current report was also diagnosed with a fiv infection, being presented in a critical condition a few days following clinical examination due to suspected kidney disease. since clinical cases of canine babesiosis and theilerioses are associated with acute or chronic nephropathy or glomerulonephritis (camacho-garcía 2006; solano-gallego et al. 2016), the renal disturbances observed in this patient could have been attributed to the piroplasmid infection to fiv or both. moreover, in c. felis infection, several cases of concomitant kidney disorders have been described in domestic and wild felids (meier and moore 2000; peixoto et al. 2007; lewis et al. 2012) . previous studies have also confirmed that fiv infection can induce the accumulation of immune complexes in renal tissue; therefore, a causative relationship between fiv infection and glomerulonephritis has been posited (reinacher and frese 1991; poli et al. 1995; harley and langston 2012; hosie et al. 2009 ). in the light of this information and the laboratory results (hypoalbuminemia pointing toward a renal loss of protein, increased creatinine and phosphate values as well as a very high sdma value), one could postulate that the patient had glomerular disease due t o i m m u n e -c o m p l e x d e p o s i t i o n i n t e r m s o f a membranoproliferative glomerulonephritis. other potential causes of hypalbuminaemia in this patient would include decreased production, other causes of loss (i.e. blood loss and protein-losing enteropathy) or haemodilution. in cases of renal injury, sdma serum concentration increases earlier than creatinine, remaining elevated also in cases of chronic kidney fig. 1 may-grünwald-giemsa stained peripheral blood smear (× 1000). cytauxzoon sp. appears as single or paired signet ring-shaped organisms (arrows) within erythrocytes. a high parasitaemia can be observed disease (on average with 40% reduction of glomerular filtration rate, compared with up to 75% reduction needed to increase the creatinine value) (relford et al. 2016) . regarding the pathogenesis of fiv infection, cats remain subclinically infected for several years until functional immunodeficiency (by increasing the susceptibility to secondary infections and neoplasia) and/or immune-mediated diseases will eventually translate into clinical manifestation around 4 to 6 years of age or older (hosie et al. 2009 ). in the present case, the cat did not display any noticeable clinical signs at 5 years of age, approximately 1 year before merozoites resembling cytauxzoon sp. were detected in high numbers in the blood smear. therefore, it can be presumed that, as a result of immunosuppression caused by fiv, the piroplasm contributed as an opportunistic factor triggering the clinical picture. furthermore, an increased pathogenicity in association with immunosuppression induced by concurrent diseases has been previously suggested in cytauxzoon sp. infection (carli et al. 2012; díaz-regañón et al. 2017 ). however, the clinical role of cytauxzoon sp. in domestic cats without immunosuppression and coinfections remains debatable, as the information about pathogenesis and clinical involvement is limited. although some cases of clinical illness and fatal outcome have been described (alho et al. 2016; legroux et al. 2017; nentwig et al. 2018) , the majority of cytauxzoon sp. infected cats were apparently healthy, with only few animals showing mild anaemia (carli et al. 2012 (carli et al. , 2014 díaz-regañón et al. 2017; nentwig et al. 2018) . microscopic examination of the blood smear revealed a degree of parasitaemia of 33%. other clinical studies showed lower levels of parasitaemia (luaces et al. 2005; carli et al. 2012 carli et al. , 2014 legroux et al. 2017) , while similar percentages of piroplasminfected erythrocytes were observed in the blood smear of three cats with cytauxzoonosis from switzerland (nentwig et al. 2018) . as the molecular confirmation of cytauxzoon sp. infection was established post-mortem, no specific anti-piroplasm treatment was applied. although, in c. felis infection, the recommended therapeutic protocol consists of atovaquone and azithromycin (cohn et al. 2011) , the optimal therapy for cytauxzoon sp. infection remains unknown due to the lack of controlled clinical studies. in a recently published case report, three 2-month-old kittens infected with cytauxzoon sp. were treated with a combination of atovaquone and azithromycin, with a putative success (nentwig et al. 2018) . also, previously published cytauxzoon sp. infections in domestic cats have been medicated with various antiprotozoal drugs, including imidocarb dipropionate and doxycycline (carli et al. 2012 (carli et al. , 2014 alho et al. 2016; legroux et al. 2017) . clinical studies in europe reported that a single imidocarb dipropionate administration was not successful in treating cytauxzoonosis, although the addition of doxycycline cleared parasitaemia in another patient (carli et al. 2014; legroux et al. 2017 ). based on the sequencing results performed on the 18s rrna gene amplification product, cytauxzoon sp. detected in the present case revealed a high homology (99-100%) with cytauxzoon sp. reported in european domestic felids (legroux et al. 2017; nentwig et al. 2018 ) and with cytauxzoon manul (99.8%) described from pallas's cats from mongolia (reichard et al. 2005) . in contrast, the sequence displayed a more distant identity of 95% to c. felis 18s rrna partial sequences deposited in genbank®. a significant association between the detection of cytauxzoon sp. dna in european domestic cats and outdoor lifestyle has been reported, particularly in rural areas (carli et al. 2012; díaz-regañón et al. 2017) . in agreement with these reports, the cat from the present study showed an outdoor lifestyle, which entails a higher risk of infection due to exposure to tick vectors and wildlife reservoirs. for c. felis infections of domestic cats, the american bobcat (l. rufus) is assumed to act as the main reservoir (kier et al. 1982) and amblyomma americanum and dermacentor variabilis are the confirmed tick vectors (blouin et al. 1984; reichard et al. 1991) . however, for the european isolates of cytauxzoon sp., little is known about vectors and routes of transmission. in europe, high prevalences of cytauxzoon sp. infection were found in felis silvestris in romania (gallusová et al. 2016) , italy (veronesi et al. 2016) , bosnia and herzegovina (hodžić et al. 2018) and spain (barandika et al. 2016; león et al. 2017) . moreover, germany has one of the biggest european populations of f. silvestris, which is concentrated in the low altitude mountain areas (balzer et al. 2018) . studies on wild animal distribution suggest that saarlouis city is surrounded by an abundant wild cat population (steyer et al. 2016) . consequently, considering that they might live in close proximity to urban and rural areas and crossbreed with domestic cats, it is possible that wild cats play a central role in the transmission of cytauxzoon sp. in west germany as well as in other european countries. ixodes ricinus is the most commonly found tick in germany (petney et al. 2012) , and it has been hypothesised to be a possible vector for cytauxzoon sp. (gallusová et al. 2016) . this study describes, to the best of our knowledge, the first case of molecularly confirmed cytauxzoon sp. infection in germany. this case provides a new geographical record of cytauxzoon sp. infection in domestic cats in central europe and describes the clinical and laboratory findings in association with fiv infection. results advocate that cytauxzoonosis should be considered as a differential diagnosis in cases of anaemia in domestic cats with an outdoor lifestyle, particularly in areas where populations of wild felids are present. additional studies are required to identify the possible arthropod vectors which may be involved in the transmission and to clarify the relationship between cytauxzoon sp. infection in cats and the presented clinical signs. code availability not applicable. data availability all data generated or analysed during this study are included in this published article. conflict of interest the authors declare that they have no conflict 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silvestris) first detection of cytauxzoon spp. infection in european wildcats (felis silvestris silvestris) of italy publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-254169-sjoiv70c authors: nakano, katsuyuki title: future risk of dengue fever to workforce and industry through global supply chain date: 2017-03-16 journal: mitig adapt strateg glob chang doi: 10.1007/s11027-017-9741-4 sha: doc_id: 254169 cord_uid: sjoiv70c the primary vector of the dengue fever virus, the aedes aegypti mosquito, is distributed across the tropical and sub-tropical latitudes; however, the area at risk of infection has been expanding steadily. this study aimed to identify the industries most vulnerable to the effects of dengue fever by 2030. the assessment was done by considering the international supply chain, with aspects such as the labor intensity, and the relevant geographical and socioeconomic aspects being taken into account. in addition, multi-regional input-output tables were employed to analyze the ripple effects of productivity losses resulting from workers contracting the disease. the results indicate that more than 10% of the workers involved in the supply chain of all the major industries in the united states (usa), china, japan, and germany could be considered at risk of contracting dengue fever by 2030. moreover, the risk was even higher in india and brazil, namely, more than 70%. the effect of widespread dengue fever infection could influence industrial activities severely, not only in the regions most at risk (india and brazil) but also in the other regions (usa, japan, and germany). labor-intensive industries, such as agriculture, fisheries, and the distribution sector are particularly at risk and will have to consider appropriate contingency measures. it is recommended that the downstream side of the supply chain, the industries in the usa, japan, and germany, supports the introduction of worker’s health management system against the infectious disease into their business partners. this study employed limited data and only estimated the possible effects of the disease by 2030. further comprehensive analysis is required with more data modeled for the future to verify and enhance the reliability of the present results. dengue fever is a systemic viral infection transmitted to humans by various types of mosquito, such as the aedes aegypti. after infection, the symptoms typically appear after an incubation period of 3 to 7 days (simmons et al. 2012 ). in the initial febrile phase, the patients suffer from high temperature (≥38.5°c), headaches, vomiting, myalgia, and joint pain, often accompanied by a transient macular rash. this phase lasts for 3 to 7 days, after which most patients recover without any complications (simmons et al. 2012) . however, in a small number of cases, the condition of the patient deteriorates and dengue hemorrhagic fever could develop. this condition could be fatal without proper medical care. in december 2015, the first dengue vaccine was licensed; however, currently, it is not available widely and treatment therefore remains supportive (pitisuttithum and bouckenooghe 2016; world health organization 2016) . at present, the dengue virus is found across the tropical and sub-tropical latitudes (simmons et al. 2012) , and the annual rate of infection is estimated at 390 million (95% credible interval 284-528), of which 96 million (95% credible interval 67-136) of those infected develop symptoms at different levels of severity (bhatt et al. 2013) . the increase in the incidence of dengue fever has been ascribed to the changing human ecology, demography, and globalization, and probably climate change (world health organization 2012). for example, for the first time in metropolitan france, two cases of autochthonous dengue fever were diagnosed in september 2010 (la ruche et al. 2010) . furthermore, after 70 years with no confirmed autochthonous cases of dengue fever in japan, 19 cases were reported in tokyo during august and september 2014 (kutsuna et al. 2015) . it has been predicted that climate change would contribute to an expansion at the fringes of the current distribution of dengue fever, with 4.39 billion people being at risk of contracting the disease by 2030 (hales et al. 2014) . the mortality rate resulting from the disease can be reduced to almost zero by implementing timely and appropriate clinical management (world health organization 2012). for example, suaya et al. (2009) conducted a survey of 1695 patients, with no fatalities occurring. although dengue fever is normally not a critical illness, the more severe cases of infection do need a specialized treatment. on average, ambulatory and hospitalized patients lose 6.6 and 9.9 days of work, respectively. absence from work often results in economic losses (suaya et al. 2009 ), and the estimated total annual cost (i.e., direct and indirect medical costs) associated with the disease amounted to us$2.1 billion in the americas for the period 2000 -2007 (shepard et al. 2011 . it has been calculated that these costs were mostly related to productivity losses induced by non-fatal dengue fever cases, whereas the deaths resulting from the disease accounted for only a small proportion (2.6%) of the total costs (shepard et al. 2011) . such productivity losses appeared as stagnation of the production activities and increased production costs. as industries are often interlinked locally and internationally, a mishap occurring in one industry could easily affect the other industries. for example, the floods suffered in thailand in 2011 damaged not only the thai economy but also that of other asian countries (haraguchi and lall 2015) . furthermore, the progress of climate change is expected to have a detrimental effect on industrial activities. in view of the above, a consideration of the future risks to the supply chain and the introduction of countermeasures to sustain industrial activities are required. life cycle assessment (lca) (iso 2006) has been employed to assess the environmental effects throughout the product supply chain. this method enables the quantification of environmental effects such as carbon dioxide (co 2 ) emissions and the consumption of resources. a number of databases have been developed to support lca, e.g., ecoinvent (wernet et al. 2016) and gabi (think step 2015) . in addition, multi-regional input-output tables (mrios) such as exiobase (wood et al. 2015) and eora ) have been developed to assess the environmental effects of the international supply chain of industrial activities. the quantification of the consumption of scarce resources by the supply chain (wiedmann et al. 2013) has indicated that the resource supply could be at risk. nakatani et al. (2015) assessed the risk of water shortages, whereas norris et al. (2014) employed input-output tables to assess the social risks associated with supply chains. nakano (2015a) extended the lca method to evaluate the effects of environmental change on the product supply chain, and evaluated the effects of climate change on the international supply chain of japan (nakano 2015b) . santos et al. (2013) analyzed the economic losses and inoperability that could result from an influenza epidemic by employing the input-output tables of the united states (usa). industries and governments have limited capacity to introduce adaptive actions to risks, and such studies support the introduction of efficient countermeasures by identifying the weak points in the system. however, no studies evaluating the effects of infectious diseases on all industrial activities through the international supply chain have been conducted before, to my knowledge. to establish an effective adaptive plan, improved understanding is needed of the effects of climate change on humans (ebi et al. 2006 ). therefore, this study aims to propose a method to evaluate the effects of an infectious disease on industrial activities by utilizing a method that indicates the priorities for considering countermeasures. the study focused on the dengue virus because predictions indicate an increasing risk of infection owing to climate change and the vaccine not being available widely. widespread infection of workers and/or their families by a virus such as dengue and the consequent absences from work would result in inevitable productivity losses. moreover, it is clear that labor-intensive industries are relatively more at risk to such losses. in addition, geographical aspects have to be considered, as the virus-bearing mosquitos favor areas that have high temperatures and water available. furthermore, socioeconomic aspects are important, as developed countries have a greater capacity to provide appropriate treatment smoothly. therefore, the labor intensity of an industry and the geographical and socioeconomic aspects should be taken into account to identify regions where the risk of dengue virus infection is high. the intergovernmental panel on climate change (ipcc) has indicated that risk results from the interaction of hazard, vulnerability, and exposure (ipcc 2014). the life cycle assessment framework for adaptive planning (lca-ap) to climate change adopted this concept and is able to evaluate the potential climatic effects on industries throughout the supply chain (nakano 2015a) . this method quantifies inputs (e.g., water and workforce) to industrial activities that could be affected by climatic factors. such inventory analysis results indicate the risk of exposure to the infection (nakano 2015a) . in lca-ap, the inventory analysis results are adjusted by accounting for the geographical aspect (hazard) and the socioeconomic aspect (vulnerability). furthermore, lca-ap is able to utilize the wellestablished lca database. lca-ap was therefore used in this study to evaluate the risk to the industry of dengue infection through the international supply chain. the potential effects of dengue infection were calculated by multiplying the inventory analysis result and a factor accounting for the country-specific climate hazard, as well as the socioeconomic vulnerability characteristics, as follows: where ci is the category indicator (potential impact from dengue), exp is the inventory analysis result (expressing exposure), haz is the hazard of dengue, and vul is the vulnerability. figure 1 shows the assessment procedure. the population at risk of dengue per value of production of each industrial sector (direct effect) was calculated from the portion of the population at risk of dengue infection in 2030 owing to climate and socioeconomic change, and the number of workers per value of production of each industrial sector. to quantify the indirect effect (upstream part of the supply chain), the mrio was used, which expresses international trade among the industrial sectors of each country. data below explains the detail of the assessment method. various methods have been proposed to predict the distribution of the risk of dengue infection in the future (messina et al. 2015) . in such calculations, the environmental change induced by climatic change, as well as other factors, such as socioeconomic change, have to be considered (messina et al. 2015) . hales et al. (2014) estimated the population at risk of dengue infection in 2030 by taking into account the future climate, population, and the gross domestic product (gdp) of each country and region. the gdp was used to evaluate the vulnerability of each country and region (hales et al. 2014) , with the study utilizing the data from these countries portion at risk of dengue under climate and socioeconomic change in 2030 (hales et al. 2014) number of workers per value of production of each industrial sector (wood et al. 2015) number of workers at risk of dengue per value of production of each industrial sector (direct impact) multi-regional input-output (mrio) tables (wood et al. 2015) number of workers at risk of dengue per value of production of each industrial sector (direct and indirect impact) (table 1) was used as f(haz, vul) in eq. (1). it was predicted that more than 90% of the population in southeast asia and latin america (tropical) would be at risk by 2030. in addition, a relatively high risk was indicated for south asia, the caribbean, and central sub-saharan africa. on the other hand, no risk was indicated for europe, whereas the highincome countries in asia pacific and north america had a small risk. input-output tables statistically express trade among all the industrial sectors in monetary value. analysis employing input-output tables is able to determine the environmental and wider sustainability effects of traded goods and services (hendrickson et al. 1998; wiedmann et al. 2011) . for example, the usa input-output tables were used to analyze the economic losses and inoperability caused by the 2009 h1n1 pandemic in the national capital region of the usa (santos et al. 2013) . mrios, such as exiobase (wood et al. 2015) , eora , and the global trade analysis project (gtap) (aguiar et al. 2016 ) have been developed to analyze the international supply chain. these mrios can quantify the monetary data, greenhouse gas emissions, and the labor force of each industrial sector in each country and region. in particular, the focus of exiobase (wood et al. 2015 ) is on environmentally relevant activities and the detail modeling of the industrial sectors that have more pronounced effects on the environment. in addition, eora has also been used for environmental analysis. the quality of eora is comparable with that of exiobase (geschke et al. 2014) . in this study, (wood et al. 2015 ) was adopted, comprising a matrix of 163 industrial sectors in 48 countries and regions. to quantify the involvement of workers throughout the supply chain (exp), the following equation was used: where w d is a diagonal matrix of direct workforce input (person/million euro (person/m eur)), i is an identity matrix (dimensionless), a is a technical coefficient matrix (dimensionless), k′ is a column vector expressing the final demand (m eur), and (i − a) is known as the leontief inverse matrix (miller and blair 2009 ). the leontief inverse matrix of exiobase, (i − a) −1 , is a square matrix of 7824 × 7824. to analyze the potential effect of each industry in each country per one monetary unit (1 m eur), the k′ of the target industry in the target country was set to 1 m eur and the others were set to 0. to assess the magnitude of the risk of dengue infection for one industry, the portion of workers at risk of contracting dengue infection (r impact ) was calculated by eq. (3), as follows: where ci is the category indicator (number of workers at risk/m eur) and exp is the inventory analysis result (number of workers/m eur). an industry for which a higher r impact is indicated has a higher potential risk relevant to the workers and the supply chain of the industry. such industries would be advised to establish countermeasures to dengue infection to safeguard their industry and supply chain. in addition, to clarify the dengue risk distribution in a supply chain, a portion of indirect influence (upper part of the supply chain) (r indirect ) is calculated by eq. (4): where ci indirect is the category indicator of the upstream part of the supply chain (number of workers at risk of contracting dengue fever/m eur). an industry indicating higher r indirect has a higher potential risk relevant to the workers in their upstream side of the supply chain. such an industry would be advised to establish relevant countermeasures to the risk beyond the border of the organization and/or the national border. the granularity of exiobase is 163 industries for 48 countries, enabling the calculation of exceptionally large results. the study intended to clarify major impacts of production losses to global economy due to disabled workforce; therefore, the focus was on the major industries in major countries. major countries, usa, china, japan, germany, india, and brazil, were selected by descending gross domestic product (gdp) values. the uk, france, and italy have larger gdps than brazil; however, these countries were excluded based on the similarity of their geographical and socioeconomical characteristics to germany. the selection of industry was based on the characteristics of industries such as classification of the industrial sector, economic scale, and labor intensity. from the primary sector of the economy, the fish product sector was selected for their highest labor intensity (wood et al. 2015) . from the primary and the tertiary sectors of economy, industries with higher labor intensities (fish products and hotels and restaurants) were selected. from the second sector of economy, industries with high supply values were selected from each segment (such as raw material). therefore, the major industries analyzed in this study include food products, fish products, textiles, plastics, chemicals, iron and steel, motor vehicles, construction, and hotels and restaurants. note that this study followed the exact definitions for each industrial sector name and boundary, as defined in exiobase (wood et al. 2015) . 3 results and discussion 3.1 proportion of workers at risk of contracting dengue infection table 2 shows the portion of workers at risk of contracting dengue infection in the supply chain (r impact ) of the countries and industrial sectors included in the analysis. as indicated by the table, more than 70% of the workers in all the major industries in india and brazil would be at risk in 2030. in the usa, 63% of workers in the fish product sector would be at risk, whereas in china, japan, and germany, the risk would be less than 50% for the workers in the major industries. however, more than 30% of the workers in the supply chain of the basic plastic and chemical industries in these countries would be at risk. moreover, more than 10% of the workers in all the major industries in all major countries would be at risk of contracting the virus; therefore, the activities of all the major industries could potentially be affected by the consequences of dengue virus infection. the portion of indirect effects (r indirect ) was calculated for the major industries in the major countries (table 3 ). the calculations indicated that 0% of the population would be at risk of contracting dengue fever in germany in 2030 (hales et al. 2014) ; therefore, the risk indicated in all the major industrial sectors in germany would be induced by indirect effects. parts of the usa and japan were classified as regions at risk of dengue infection; therefore, slight direct effects were observed for these countries. in contrast, the direct effects were significant in china, india, and brazil. in particular, in the construction sector in india and brazil, the textile sector, and the hotel and restaurant sector in brazil, more than half of the effects were induced by local industrial activities. however, in all the industries, indirect effects should not be ignored. the relation between the production value (billion euro (b eur)/year) and the effect (number of workers at risk of contracting dengue fever (person/m eur)) is illustrated in fig. 2 . an industrial sector located in the upper right side of the graph has higher production value and a large number of workers at risk of contracting dengue fever; therefore, this industry would be specifically advised to introduce countermeasures to manage the effects of the disease. note that the scales of the vertical and horizontal axes of fig. 2 are different among the countries to express the economic size and the effects relevant to each country. an extremely significant effect (342 person/m eur) was indicated for the fish product sector in the usa. similarly, significant effects were indicated for the fish product sector in the other countries, whereas the production values of this sector were small. in china, the textile, food product, and hotel and restaurant sectors showed relatively higher production values and effects. in japan, the motor vehicle sector had the largest production value, whereas the effect was small. in germany, the plastic sector showed a relatively higher production value and effect. however, the scale of the vertical axes of the graphs for both germany and japan was smaller by 1 digit in comparison with the other countries. no major industrial sectors in japan or germany were located in the upper right side of the graphs; therefore, no critical sectors were identified in either of these countries. in brazil and india, the construction sector showed relatively higher production values and effects. most of the effects were induced by the local construction industries (table 3 ) and, as this industry is labor intensive, appropriate countermeasures would have to be introduced in these two countries. the major emerging countries, such as china, brazil, and india showed significant effects relevant to the food product and hotel and restaurant sectors, whereas the production values were not significant ( table 4 ). the effects indicated for india (person/m eur) were relatively more significant for the major industries of the country in comparison with those of the other countries. this result is ascribed to india being located in southeast asia, which is more at risk of dengue fever infection, and to the labor intensity of the country being higher than in the developed countries. therefore, industries located in the bottom area in fig. 2 (e.g., the chemical, motor vehicle, and steel industries) would be advised to consider improving their health care systems to counteract the effects of dengue fever infection. in order to study the countermeasures for dengue fever infection in practice, an activity that has a greater effect in the supply chain has to be identified. therefore, the supply chains of nine major industrial sectors were analyzed for which relatively higher effects and production values had been indicated (fig. 3) . wood et al. (2015) the induced effects in the usa were found insignificant (less than 1%), whereas the effects of imports from asia-pacific and african countries were significant. the fish product sector in the usa purchased fish amounting to 474 m eur locally, 825 m eur from south american countries, 1 692 m eur from asia-pacific countries, 2 and 269 m eur from african countries 3 (wood et al. 2015) . these exporting countries have a higher percentage of population at risk of contracting dengue fever (table 1) . furthermore, the labor intensity of the fishing sector in the usa is 8.6 workers/m eur, whereas it is 2841 workers/m eur in the asia-pacific countries, and 444 workers/m eur in the african countries (wood et al. 2015) . the risk in the asia-pacific and african countries was significant, with 63% of workers classified as being at risk of contracting dengue fever (table 2 ). in view of these findings, it would be advisable for the fish product industry in the usa to promote risk management in their international supply chain. the textile sector in china (direct effect) contributed 13% of the effect, with 56% of the effects being induced locally in the country. the plant-based fiber sector accounted for 38% of the effects, namely, 10% in china, 15% in african countries, and 13% in asia-pacific countries. the plant-based fiber sector provides the main materials to the textile sector, such as cotton (gossypium spp) and jute (corchorus capsularis). the textile sector in china purchases 20,532 m eur from the chemical sector and 8497 m eur from the local plant-based fiber sector in china in 2007 (wood et al. 2015) . the purchase amount of the chemical sector exceeded that of the plant-based fiber sector; however, the labor intensity of the chemical sector (14.7 workers/m eur) was much lower than was that of the plant-based fiber sector (762 workers/m eur) (wood et al. 2015) , and the plant-based fiber sector was therefore identified as a significant sector. as the textile sector is labor intensive, it is important to strengthen the risk management in this sector. moreover, the plant-based fiber sector provides materials (e.g., cotton) to the textile sector; consequently, there is a need to reinforce the risk management for dengue fever infection. most of the effects (94%) were induced in china. the food product sector (direct effect) contributed 11% of the total, whereas the effects of indirect activities, such as the vegetable sector (17%), the cereal grain sector (13%), the wheat sector (12%), and the paddy rice sector (11%), contributed more than 50%. the economic scale of the food product sector in china is large (225 b eur) (wood et al. 2015) , and the proportion of workers in the supply chain at risk of contracting dengue fever is significant (27%, as shown in table 2 ); therefore, it is important to consider appropriate countermeasures to dengue fever infection. 1 south american countries except brazil and mexico 2 asia pacific countries except japan, china, south korea, india, taiwan, indonesia, and australia 3 african countries except south africa (d) hotels and restaurants in japan although the direct effect was not actually 0%, as the risk of dengue fever infection does exist in japan, most of the indicated effects were induced abroad. food imports from the asia-pacific countries had significant effects, namely, 23% from the fish product sector and 10% from the vegetable sector. the hotel and restaurant sector in japan purchased 8252 m eur from the local food product sector, 7750 m eur from the local beverage sector, and 1041 m eur from the fish product sector in the asia-pacific countries (wood et al. 2015) . although the value of the products purchased from the asia-pacific countries was smaller than was that of the local purchases, there was significant risk (18%) of the asia-pacific workers in the supply chain contracting the dengue virus (table 2) . japan imports various foods from asian countries, such as shrimp (e.g., penaeus indicus) from southeast asia, and appropriate risk management relevant to these foods would have to be considered. no workers in germany were predicted at risk of contracting dengue fever, the domestic effects were zero, and all the effects indicated were induced in asia, africa, and other countries. a number of sectors, such as the vegetable sector in asia-pacific countries (2%) and the crop sector in the african countries (2%) contributed to the effects. no significant industry contributed to the result. as regards the direct transactional value relevant to the motor vehicle sector in germany, the value of the fabricated metal product sector in germany was 12,424 m eur and that of the rubber and plastic product sector was 7606 m eur. in contrast, the value of both the vegetable sector in the asia-pacific countries and the crop sector in the african countries was less than 1 m eur (wood et al. 2015) . the supply chain of the motor vehicle sector is complex and includes agricultural activities. for example, vegetable oil is a raw material of chemicals, such as surfactants, and the starch produced by the crop sector is a component of the surface-sizing agents used in the paper manufacturing process. the motor vehicle sector in germany purchased 147 m eur from the paper and paper product sector (wood et al. 2015) . the absolute amount of these agricultural inputs to the motor vehicle sector is small; however, these agricultural industries indicated relatively higher impacts. significantly, 23% of the workers in the supply chain were classified at risk of contracting dengue fever (table 2) . therefore, the motor vehicle sector in germany would be advised to analyze the components for which agricultural products from asia and africa are used in order to have appropriate contingency measures in place. similar to the motor vehicle sector, no effects were predicted for the plastic industry in germany. relevant to the international supply chain, no sectors showed a clear and significant contribution to the result; however, the wholesale sector (5%) and the vegetable sector in the asia-pacific countries (4%) were indicated in the result. labor intensity in these countries is high, and 38% of workers in the supply chain were at risk of contracting dengue fever (table 2 ). similar to the motor vehicle sector in germany, it is recommended that the components be examined that use agricultural products from asia and africa. in india, 96% of the effects are derived locally, including the direct effect of the iron and steel industry (26%). the service sectors, such as the retail trade sector (16%) and the wholesale trade sector (5%) contributed to the result in the supply chain. in addition, the effects of raw material acquisition activities, relevant to the coal and lignite sector (10%) and the iron ore sector (2%), were detected in the result. as shown in table 2 , 71% of workers were at risk of contracting dengue fever; therefore, proactive risk management is required in this sector and its supply chain. direct effects accounted for 57% of the effects in the construction sector in india. the construction sector is labor intensive (223 workers/m eur) compared with other sectors in india, such as the plastic sector (30 workers/m eur) and the paper sector (93 workers/m eur) (wood et al. 2015) . the sectors providing materials to the construction sector, such as the wood product sector (5%) and the forestry sector (3%) had significant effects on the supply chain. in addition, the service sectors, such as the retail trade sector (6%) and the land transport sector (3%) contributed to the result. the effects induced locally in india contributed 97% to the total effects indicated for the country. the production value of this sector is large (135 b eur) (wood et al. 2015) , and the effects per monetary unit are significant (297 workers/m eur). consequently, this sector should consider countermeasures to the consequences of dengue fever infection. the domestic effect accounted for 83% of the total, including a direct effect of 27%. in the supply chain, service sectors, such as the retail trade sector (8%) and the wholesale trade sector (6%), and agricultural sectors, such as the sugar cane sector (11%) and the forestry sector (2%), were indicated in the results. the chemical sector in brazil purchased 2.8 b eur from the local sugar cane and beet sector and 2.7 b eur from the local naphtha sector (wood et al. 2015) . sugar cane has been used to produce ethanol as an alternative source of energy for motor vehicles in brazil. the international supply chain contributed 17% to the effects; however, no significant effect was indicated from any sector in brazil. however, the chemical sector would be advised to enhance risk management in their own sector and their supply chain, especially the sugar cane industry and the trade sectors. this study indicated that the effects of dengue fever could influence other countries through the international supply chain. the dengue infection data in 2030 (hales et al. 2014 ) considered current-level countermeasures according to gdp. therefore, the results indicated that further introduction of countermeasures are required to deduce the risk. the regions where dengue fever is not prevalent, such as the usa, japan, and germany, should consider appropriate countermeasures, as a percentage of workers involved in the supply chain of these countries have been classified as at risk of contracting the disease. in particular, industries that are reliant on labor (e.g., agriculture and the distribution industry) would have to consider adaptation measures carefully. worldwide, various adaptation actions have been introduced (lesnikowski et al. 2013) . bowen et al. (2014) suggested that reducing the health risks induced by climate change, cross-sectoral partnerships should be improved and influential organizations in the development of mitigation and adaptation measures should be identified. the downstream side of the supply chain has buying power, whereas the adaptive activities of the upstream side are influenced by the attitude of costumers (berkhout et al. 2006; fleming et al. 2014) . therefore, the downstream side of the supply chain, such as industries in the usa, japan, and germany, could contribute to promoting a health management system in industries at risk of dengue fever infection. the organization for economic co-operation and development (oecd) guidelines for multinational enterprises (oecd 2011) recommend avoiding bthe foreseeable environmental, health, and safety-related impacts associated with the processes, goods, and services of the enterprise over their full life cycle.^for example, ajinomoto incorporated company advises their suppliers to provide a regular healthcare management to their workers (ajinomoto 2013) . these guidelines and activities could lead to support introduction of countermeasures to infectious disease to business partners and reduce business risk. however, a number of enterprises still do not implement such activities. therefore, it is recommended that international initiatives, such as the united nations global compact (united nations 2017), clearly advice companies to support introduction of worker's health management systems to their business partners. these activities reduce business risks (godfrey et al. 2009 ); therefore, i recommend that investors positively evaluate such risk management activities. a comprehensive and systematic framework is needed to study a policy option for the formulation of robust, pro-development climatic measures and effective health management (chalabi and kovats 2014) in order to counteract the effects of widespread diseases. however, this study focused only on the risk of dengue fever infection, with other diseases (e.g., malaria) and disasters (e.g., extreme weather conditions) not being taken into account. furthermore, the results of this study include uncertainty. this is ascribed to the study having to model the predicted population at risk of dengue fever infection in 2030 on the actual industrial structure and international supply chain in 2007. the situation in 2007 was adopted because of the difficulty of predicting the future economic situation. however, as the textile industry has been relocating to a country where labor cost is lower, the industrial structure and the international supply chain have been changing. therefore, the future economic situation has to be considered in order to increase the reliability of the study. in this study, only the supply side was evaluated, although, typically, infectious diseases would affect the demand side as well. for example, the pandemic of severe acute respiratory syndrome (sars) in hong kong, from november 2002 to august 2003, significantly affected local consumption and the travel industry (siu and wong 2004) . therefore, future study would have to consider the effects of infectious diseases on the demand side as well. the primary vector of dengue fever, the urban-adapted a. aegypti mosquito has been distributed across the tropical and sub-tropical latitudes (simmons et al. 2012 ), but the total area at risk of dengue infection has been expanding (hales et al. 2014) . the study clarified the effects of dengue fever infection on various global industries by projections for 2030, taking into account the international supply chain. the results indicated that more than 10% of workers involved in the supply chain of all the major industries in the usa, china, japan, and germany would be at risk of contracting dengue fever by 2030, whereas more than 70% of the workers in india and brazil would be at risk by that time. the effects of dengue fever could influence industrial activities in the regions at risk (e.g., india and brazil) and in the other regions (e.g., usa, japan, and germany) through the supply chain. in particular, industries that are highly labor intensive (agriculture, fishery, and the distribution industry) would be advised to consider adequate adaptation measures. it is recommended that the downstream side of the supply chain, such as industries in the usa, japan, and germany, identify processes at risk of infectious disease in the supply chain where worker's health management systems should be introduced. this study indicated that implementation of such systems would reduce business risks and improve shareholder value (godfrey et al. 2009 ); therefore, i recommend that such interventions to upstream side of the supply chain be included in investment criteria. as the study focused only on the effects of dengue fever in 2030, with limited data being available, comprehensive analysis with data modeled for the future could 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footprint analysis dengue vaccine research world health organization (2012) global strategy for dengue prevention and control 2012-2020. world health organiszation conflict of interest the author declares that there is no conflict of interest. key: cord-348495-pa6iqc83 authors: perrotta, d.; grow, a.; rampazzo, f.; cimentada, j.; del fava, e.; gil-clavel, s.; zagheni, e. title: behaviors and attitudes in response to the covid-19 pandemic: insights from a cross-national facebook survey date: 2020-05-15 journal: nan doi: 10.1101/2020.05.09.20096388 sha: doc_id: 348495 cord_uid: pa6iqc83 in the absence of medical treatment and vaccination, the mitigation and containment of the ongoing covid-19 pandemic relies on behavioral changes. timely data on attitudes and behaviors are thus necessary to develop optimal intervention strategies and to assess the consequences of the pandemic for different demographic groups. we developed a rapid response monitoring system via a continuously run online survey (the "covid-19 health behavior survey") across eight countries (belgium, france, germany, italy, the netherlands, spain, the united kingdom, the united states). the survey was specifically designed to collect key information on people's health status, behaviors, close social contacts, and attitudes in response to the covid-19 pandemic. we developed an innovative approach to recruit participants via targeted facebook advertisement campaigns in order to generate balanced samples for post-stratification. in this paper, we present results for the period from march 13-april 19, 2020. we estimate important differences by sex: women show a substantially higher perception of threat along with a lower level of confidence in the health system. this is paralleled by sex-specific behaviors, with women more likely to adopt a wide range of preventive behaviors. we thus expect behavior to serve as a protective factor for women. our findings also show a higher level of awareness and concern among older respondents, in line with the evidence that the elderly are at highest risk of severe complications following infection from covid-19. while across all the samples respondents were less concerned for themselves than for their country or for the world, we also observed substantial temporal and spatial heterogeneity in terms of confidence in institutions and responses to non-pharmaceutical interventions. the ongoing coronavirus disease 2019 (covid-19) outbreak started in wuhan city, china, in december 2019 and quickly spread globally, soon reaching pandemic proportions [1] . by mid-april 2020, the virus had already caused over 1.6 million cases and over 100,000 deaths worldwide [2] , placing a substantial burden on national healthcare systems and posing unprecedented challenges for governments and societies. as yet, governmental responses to mitigate the coronavirus epidemic have varied considerably across countries. non-pharmaceutical interventions, specifically intended to reduce sustained local transmission by reducing contact rates in the general population, have so far ranged from moderate containment measures, such as school closures and cancellations of public gatherings, to drastic measures, such as travel bans and nationwide lockdowns [3] . in western democracies, individual behaviors, rather than governmental actions, are potentially crucial to control the spread of covid-19 [4] . human behavior is in fact a key factor in shaping the course of epidemics [5] . individuals may spontaneously modify their behaviors and adopt preventive measures in response to an epidemic when mortality or the perception of risk is high, and this may in turn change the epidemic itself by reducing the likelihood of transmission and infection [6, 7, 8] . however, a key problem is a lack of data to assess people's behavior and reactions to epidemics. decision-making and the evaluation of non-pharmaceutical interventions require specific, reliable, and timely data not only about infections, but also about human behavior. especially in the ongoing covid-19 pandemic, where medical treatment and vaccination are still only remote options, mitigation and containment mainly rely on massive and rapid adoption of preventive measures [9] . understanding how the members of different demographic groups perceive the risk, and consequently adopt specific behaviors in response to it, is therefore key to measure the effectiveness of non-pharmaceutical interventions, design more realistic epidemic models, and enable public health agencies to develop optimal control policies to contain the spread of covid-19. we seek to narrow this data gap by monitoring individual behaviors and attitudes in response to the covid-19 pandemic in multiple countries. in march 2020, we launched a crossnational online survey, called the "covid-19 health behavior survey" (chbs), to collect timely data on people's health status, behaviors, close social contacts, and attitudes related to covid-19. we recruit respondents through advertisement campaigns on facebook, that we created via the facebook advertising manager (fam). this novel approach to recruiting respondents allows us to combine the flexibility of online surveys for rapid data collection, with the controlled environment of targeted advertisement. this makes it possible to recruit a balanced sample across demographic groups, that is approximately representative of the general population, after applying appropriate post-stratification weights [10, 11, 12] . other similar online initiatives have emerged recently [13, 14, 15, 16] , but to the best of our knowledge, this is the first cross-national study addressing multiple key factors, ranging from individual behaviors and attitudes to health-related indicators to social contact patterns. moreover, our sampling approach and continued data collection allows us to compare people's behaviors across countries that are at different stages of the covid-19 pandemic, and to assess changes in behaviors after pivotal events, such as nationwide lockdowns. in this paper, we present first results based on survey data collected over the period march 13 to april 19, 2020 in belgium, france, germany, italy, the netherlands, spain, the united kingdom, and the united states. over this period, a total of 66,266 participants completed the questionnaire. our goal in this paper is to provide insights into the relation between participants' demographic characteristics and (i) the threat they perceive covid-19 to pose to various levels of society, (ii) the confidence they have in the preparedness of different national and international organizations to handle the current crisis, and (iii) the behavioral measures (preventive measures and social distancing measures) they have taken to protect themselves from the coronavirus. from a public health perspective, this information is key to understand the behaviors and attitudes of specific demographic groups in different countries and help guide the decision-making process to design adequate policies to contain the spread of covid-19. in the following sections, we outline our methodological approach and discuss the innovative aspects of participant recruitment through facebook advertising campaigns, as well as the statistical adjustments needed to approximate a sample representative of the general population. then we describe the main features of our sample and present results of the first analyses regarding behaviors and attitudes in response to covid-19. we close with a discussion and an outlook for the next steps in our broader project. the chbs is designed to collect information on respondents' health behaviors and attitudes related to covid-19. participation in the survey is anonymous and voluntary. respondents can stop participating at any time and can skip questions they feel uncomfortable answering. the questionnaire consists of four topical sections: (i) socio-demographic indicators (age, sex, country of birth, country of residence, level of education, household size and composition); (ii) health indicators (underlying medical conditions, flu vaccination status, pregnancy, symptoms experienced in the previous seven days); (iii) opinions and behaviors (perceived threat from covid-19, level of trust in institutions, level of confidence in sources of information, preventive measures taken, disruptions to daily routine); (iv) social contact data, i.e. the number of interactions that respondents had the day before participating in the survey in different settings (at home, at school, at work, or in other locations). to facilitate validation and warrant comparability with existing surveys, we included standard questions from several sources, such as the european social survey (ess) [17] regarding socio-demographic characteristics, ipsos [18] regarding opinions on the coronavirus outbreak, and the polymod project [19] regarding social contacts. note that we ask respondents about their behavior and attitudes related to the coronavirus outbreak only if they indicated that they were aware of it. in more detail, we asked respondents how much, if at all, they had seen, read or heard about the coronavirus outbreak, with the answer options "a great deal", "a fair amount", "not very much", "nothing at all", and "prefer not to answer". respondents who indicated that they knew nothing at all, or that they preferred not to answer, were not asked any further questions related to the outbreak. we created the questionnaire first in english, and then translated it into the different official languages of the countries in our study, with support from professional translators. we considered country-level differences when adjusting the questionnaire for different countries, where applicable (e.g. differences in the educational system). in the online implementation, the questionnaire is available in both english and the national language(s) of the respective country in which respondents are located. the questionnaire was implemented in the online survey tool limesurvey (version 3.22.8+20030) and hosted by the society for scientific data processing (gwdg). the full english questionnaire (as used in the united states) is reported in appendix a. the link to the questionnaire is distributed through advertisement campaigns that we created via the fam. facebook is currently the largest social media platform, with 2.45 billion monthly active users worldwide as of september 2019 [20] . in the united states, about 69% of adults used facebook in 2019 [21] , with similar penetration rates in europe, ranging from 56% in germany to 92% in denmark [22] . the fam enables advertisers to create advertising campaigns that can be targeted at specific user groups, as defined by their demographic characteristics (e.g. sex and age) and a set of characteristics that facebook infers from their behavior on the network (e.g. interests). an increasing number of studies explore the use of facebook in demographic and health research to recruit participants for online surveys [23, 24, 25] . two main advantages of this approach are rooted in facebook's wide reach and the possibility to directly target members of different demographic groups. two concerns that are often raised are that in online samples self-selection might lead to bias in results, and that online sub-populations may not be representative of the general population. however, there is increasing evidence that samples obtained from facebook do not significantly differ from samples obtained from more traditional recruitment and sampling techniques in central demographic and psychometric characteristics, especially if post-stratification weights are applied adequately [10, 12, 26, 27] . we created one advertising campaign per country and stratified each campaign by sex (male and female), age group (18-24, 25-44, 45-64 , and 65+ years), and region of residence (largely following the nuts classification in europe and the census regions in us; see table s4 in the supplementary material), resulting in 24 to 56 strata per country, further stratified using six different ad images. figure s1 in section 1 of the supplementary material illustrates the structure of our facebook advertising campaigns in the united states. note that we aggregated the different regions of residence into larger macro-regions, to keep the number of strata in facebook manageable (see table s4 for the exact mapping). we launched the campaigns on march 13, 2020, in italy, the united kingdom, and the united states. we added germany and france on march 17, spain on march 19, the netherlands on april 1, and belgium on april 4, 2020. in the period 21-26 march, we were unable to recruit a significant number of participants due to technical issues with the fam which prevented the delivery of our advertisements. we select participants for our analysis in three steps. first, we include only participants who reported that they lived in the country that the respective advertising campaign and countryspecific questionnaire targeted, and who reported their sex, age, and region of residence (the central variables in our post-stratification weighting approach, see details below). second, when analysing responses to a given question, we exclude respondents who chose the options "don't know" or "prefer not to answer". in the analysis reported here, this particularly affects the question about awareness of the coronavirus outbreak (see section 2.1); however, as table s2 in the supplementary material shows, the share of respondents to whom this applies is less than 1% across countries. third, given that in calendar week 12 we were only able to collect a small number of completed questionnaires in spain (less than 100), we excluded these data from our analysis as the sample size would render our analysis unreliable for this period. note that all period references consider local time zones across countries and regions. after participant selection, we apply post-stratification weights to the final data set in order to correct for potential issues with non-representativeness in our sample. we use a standard procedure in survey research, in which appropriate weights are computed based on population information from more traditional data sources (e.g. census data). here we use population data from eurostat (2019) [28] and the us census (2018) [29] . specifically, for each stratum i (given by each combination of sex, age, and macro-region) in each country, we compute the fraction p i andp i of, respectively, the true population counts n i and the sample countsn i , compared to the total population i n i and the total sample size ini . the weights w i are then defined as w i = p i /p i , thus giving less weight to groups which are over-represented (w i < 1) and more weight to groups which are under-represented (w i > 1) in the sample. we provide more details about our approach to post-stratification in section 3 of the supplementary material. in our analysis, we focus on (i) perception of threat from covid-19, (ii) confidence in the preparedness of different national and international organizations to respond to this threat, and (iii) behavioral measures taken to protect oneself from the virus. all our analyses are based on the weighted sample, whereas the reported sample sizes refer to the unweighted sample. we asked respondents to rate the threat they perceived covid-19 to pose for different levels of society (i.e. to themselves, their family, their local community, their country, and the world) on a 5-point likert-type scale (1 = very low threat, 5 = very high threat), including the options "don't know" and "prefer not to answer", which are treated as missing values (table s3 in the supplementary material reports the corresponding sample size for each item). for comparison, we asked respondents to answer the same questions also for the seasonal flu. we normalized respondents' answers to each item to the range 0-1, meaning that values around 0.5 correspond to moderate perceived threat, whereas 0 and 1 correspond to low and high perceived threat, respectively. in a similar way, we asked respondents to rate the confidence they had in the preparedness and ability of different organizations to effectively deal with the covid-19 pandemic (i.e. doctors and healthcare professionals in their community, hospitals in their local area, health care services in their country, the world health organization, their local government, and their national government) on a 4-point likert-type scale (1 = not confident at all, 4 = very confident), also including the options "don't know" and "prefer not to answer", which are treated as missing values (see table s3 in the supplementary material). we normalized answers to the range 0-1, and aggregated responses related to the local health system (doctors and healthcare professionals in respondents' community and hospitals in their local area) by averaging them across items within respondents. finally, we asked respondents which measures, if any, they had taken to protect themselves from the coronavirus. for this, we showed a list of actions, from which they can choose all that apply. this list includes preventive measures (e.g. washing hand more often), measures of social distancing (e.g. avoided social events), measures of reduced mobility (e.g. avoided public transportation), panic buying (e.g. stockpiling of food), and potential discriminatory actions (e.g. avoided eating in asian restaurants). see the questionnaire in the appendix a for the full list of actions. in the analysis, we consider the shares of participants who reported having adopted specific behaviors in response to covid-19, including: (i) the stockpiling of food and/or medicine; (ii) the use of a face mask; (iii) the increased use of hand sanitizer; (iv) the increased washing of hands; (v) social distancing (if participants selected at least one of the following: avoided shaking hands, avoided social activities, and avoided crowded places); and (vi) the reduced use of transportation (if participants selected at least one of the following: avoided travelling by public transportation, and avoided travelling by taxi). in our analyses, we used non-parametric tests for median comparisons (wilcoxon test to compare two groups and kruskall-wallis test to compare three or more groups) and considered p-values of less than 0.05 to be significant. data analysis was performed with the programming language python (version 3.7). . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. . https://doi.org/10.1101/2020.05.09.20096388 doi: medrxiv preprint 3 results a total of 66,266 participants completed the survey in belgium (n=5,520), france (n=6,216), germany (n=11,030), italy (n=9,310), the netherlands (n=4,711), spain (n=7,145), the united kingdom (n=8,412), and the united states (n=13,922) in the period between march 13, 2020 (calendar week 11) and april 19, 2020 (calendar week 16). as table 1 shows, participation by week was high in all countries, with a median number of 1,610 participants per week in belgium, 1,490 in france, 1,646 in germany, 1,810 in italy, 1,743 in the netherlands, 1,842 in spain, 1,114 in the united kingdom, and 2,498 in the united states. table 1 also shows the demographic characteristics of the participants in each country, based on the unweighted sample. the sex ratio is somewhat skewed towards females compared to the overall population, ranging from 63% female in germany to 71% female in france. in terms of age, older respondents tend to be over-represented, with a median age of 48 years (iqr 31-62) in belgium, 45 years (iqr 29-61) in france, 40 years (iqr 27-56) in germany, 39 years (iqr 26-56) in italy, 55 years (iqr 38-64) in the netherlands, 49 years (iqr 36-60) in spain, 56 years (iqr 41-65) in the united kingdom, and 55 years (iqr 37-65) in the united states. when it comes to education, there is some variation across countries. more specifically, in belgium (46%), france (71%), spain (59%), the united kingdom (46%), and the united states (60%) most respondents attained university-level education, whereas in germany (62%), italy (50%), and the netherlands (58%) most respondents attained secondary-level education. after applying post-stratification weights, the bias described above is reduced and the sample approximates the shares reported in nationally representative surveys in terms of sex, age, and educational attainment, as shown in figure s3 in the supplementary material. for more details, see section 3 of the supplementary material. the perception of threat: higher for the elderly and for women, while everyone is less concerned for oneself than for the country as a whole in all countries, there is significant variation in threat perceptions across levels of society (p < 0.001). in particular, the perception of threat increases sharply from the personal sphere (oneself and the family) to more distal contexts, i.e. the local community, the country, and, ultimately, the world 1 . considering specifically the perceived threat to oneself and to the world, the latter is on average 32% greater, whereas this difference ranges from 24% in italy to 37% in the united states. apart from these variations at the country level, the threat perception posed by covid-19 is both age-and sex-specific. as shown in figure 1b, 7 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. . overall, the perceived threat increases with age, with few notable exceptions, including the perceived threat to the family in germany, the netherlands, spain, and the united states, the perceived threat to the local community in the netherlands, spain, and the united states, and the perceived threat to the country and to the world in the united states (all p > 0.06). importantly, as figure 1c shows, the perceived threat is significantly higher among women than among men 2 . the development of threat perceptions over time shows different temporal patterns across countries, as can be seen in figure 1d . in particular, there is significant variation over time in germany (p < 0.001), and the united states (p < 0.001). in germany the perceived threat shows a negative trend, with the median value compared to that of week 12 decreasing by about 4% in week 13, 9% in week 14, 15% in week 15, and 18% in week 16. in the united states the trend changes over time, with the median value compared to that of week 11 increasing by about 20% in week 12, 28% in week 13, and 31% in week 14, but then dropping to being only 17% higher in week 15, and 15% higher in week 16. in france, italy, and the united kingdom, the temporal pattern is more mixed, with significant variation over time across levels of society (p < 0.02), except for the perceived threat to oneself in france (p = 0.5), italy (p = 0.6), and 2 all p < 0.01, except in spain for the perceived threat to oneself (p = 0.06), to the family (p = 0.2), and to the local community (p = 0.05) 8 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. finally, figure 2 compares the threat perception for seasonal influenza (panel a) with that for covid-19 (panel b). the perceived threat posed by covid-19 is significantly higher than the perceived threat posed by influenza (all p < 0.001). in more detail, the perceived threat to oneself is on average 51% higher (ranging from 41% in germany to 61% in belgium), the threat to the family is 46% higher (40% in the netherlands to 51% in the united states), the threat to the local community is 44% higher (33% in france to 54% in the united kingdom), the threat to the country is 64% higher (51% in germany to 71% in belgium), and the threat to the world is 55% higher (47% in italy to 62% in belgium). more details about the perceived threat posed by influenza can be found in section 5 of the supplementary material. landscape, men have higher confidence in local and national health systems in all countries, there is significant variation across organizations (all p < 0.001). first, respondents' confidence in the national health system tends to be lower than their confidence in the local health system 3 . considering the median values, respondents' confidence in the national health system is about 13% lower than their confidence in the local health system in belgium, 19% lower in france, 6% lower in germany, 6% lower in 3 all p < 0.007, except italy (p = 0.2) and the united kingdom (p = 0.9) . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. . the netherlands, 2% lower in spain, and 14% lower in the united states. second, respondents' confidence in local and national governments differs substantially in all countries (p < 0.04). in particular, their confidence in the national government is about 3% lower than their confidence in the local government in germany, it is 26% lower in france, 12% lower in spain, and 31% lower in the united states. by contrast, it is about 1% higher in belgium, 7% higher in italy, 6% higher in the netherlands, and 8% higher in the united kingdom. apart from this variation, several other patterns stand out in the level of confidence by age group and sex. as shown in figure 3b , overall the elderly tend to be more confident in the preparedness of the various organizations, with the exception of the who, in which young adults aged between 18 and 24 years show instead greater confidence. additionally, the level of confidence is sex-specific across organizations, as can be seen in figure 3c . male respondents are more confident in the local or national health systems 4 , whereas female respondents are more confident in the who and in the local government 5 . as for the national government, instead, confidence is higher among female respondents in germany (p < 0.001), and spain (p = 0.01), but it is higher among male respondents in the united states (p < 0.001). figure 3d shows the development of the level of confidence over time. similarly to the perceived threat shown in figure 1d , the temporal patterns vary across countries. in particular, 4 all p < 0.02, except for the netherlands, spain, and the united kingdom 5 as for the who, all p < 0.002, except for belgium, france, and italy, while for the local government, all p < 0.02, except belgium, italy, and the united states . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. . there is significant variation across weeks in germany (p < 0.01), italy (p < 0.03), the united kingdom (p < 0.01), and the united states (p < 0.01). while in germany and in the united kingdom, the trend is positive with the median value in week 16 being about 7% and 21% higher compared to the first week, on the contrary, in italy this trend is negative with the median value in week 16 being about 12% lower than in week 11. in the united states, instead, the temporal pattern is more variable, with the level of confidence in the health systems increasing, whereas the level of confidence in the different levels of government diverges. it is higher for the local government (about 8% higher in week 16 than in week 11), while for the national government the trend changes over time, with the median value compared to that of week 11 decreasing by about 3% in week 12, 17% in week 13, and 12% in week 14, but then increasing to being only 7% lower in week 15, and 5% lower in week 16. on the other hand, the temporal pattern in france shows significant variation, except for the local health system (p = 0.09), whereas there is no significant variation in the level of confidence over time in belgium (all p > 0.07), the netherlands (all p > 0.08), and spain (all p > 0.5). moreover, looking at the level of confidence in the who separately, this consistently shows a negative trend in france (about 14% lower in week 16 compared to week 13), germany (about 13% lower in week 16 compared to week 12), italy (about 12% lower in week 16 compared to week 11), the united kingdom (about 7% lower in week 16 compared to week 11), and the united states (about 24% lower in week 16 compared to week 11). figure 4 shows the self-reported behaviors broken down by country (panel a), age group (panel b), sex (panel c), and week (panel d). as shown in figure 4a , the least frequently reported behavior is the stockpiling of food and/or medicine, ranging from about 18% of respondents (iqr [16] [17] [18] [19] [20] [21] in the netherlands to about 31% (iqr 24-33) in germany. secondly, the share of participants who reported wearing a face mask ranges from about 6% (iqr 4-9) in the netherlands to about 57% (iqr 54-61) in italy. as for hand hygiene, the share of participants who increased the use of hand sanitizer ranges from about 49% (iqr 46-54) in germany to about 70% (iqr 68-77) in the united states, while the share of participants who increased washing their hands ranges from about 89% (iqr 85-91) in germany to about 94% (iqr 91-96) in spain. finally, the most frequently reported behaviors are, respectively, increased social distancing, which ranges from about 93% (iqr 90-95) in the united kingdom to about 98% (iqr 97-99) in italy, and the reduced use of transportation, which ranges from about 72% (iqr 68-75) in the united states to about 82% (iqr 78-85) in france. moreover, the share of participants adopting specific behaviors related to covid-19 shows a variable pattern across age groups in all countries, as shown in figure 4b . in particular, significant variations in the age distribution is observed in respondents who stockpiled food and/or medicine, increased the use of hand sanitizer, and reduced the use of transportation 6 . however, there is no significant variation in the age distribution in terms of respondents who engaged in social distancing, increased hand washing, and worn a face mask 7 . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. . fig. 4 . proportions of participants who reported having adopted specific behaviors in response to covid-19 broken down by country (a), age group (b), sex (c), and week (d). behaviors include the stockpiling of food and/or medicine, wearing a face mask, increased use of hand sanitizer, increased hand washing, increased social distancing, and reduced use of public transportation. bar charts show median values and 95%ci as errors. weighted sample. as shown in figure 4c , behaviors related to covid-19 are sex-specific, with female respondents showing the highest adoption rates for specific behaviors 8 . the development of behaviors over time shows different temporal patterns between countries, as can be seen in figure 4d . in particular, the use of a face mask substantially increased over time (all p < 0.001, except belgium, and the netherlands), as well as hand hygiene in germany, italy, the united kingdom, and the united states (p < 0.04), and the reduced use of transportation in the united kingdom (p < 0.001), and the united states (p < 0.001). social distancing has increased sharply in the united kingdom (p < 0.001), and in the united states (p < 0.001), whereas it has decreased in germany (p = 0.001), reflecting different stages of the epidemic and different policies. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020 understanding how different demographic groups perceive the risk of covid-19, and thus adopt specific behaviors in response to it, is key to enable public health agencies to develop optimal intervention strategies to contain the spread of the disease. in this paper, we have presented insights from survey data collected through a cross-national online survey, the covid-19 health behavior survey (chbs). the survey is ongoing, and here we presented results based on data collected during the period march 13-april 19, 2020 in belgium, france, germany, italy, the netherlands, spain, the united kingdom, and the united states. in this closing section, we summarize the main findings and provide our interpretation in light of the current evidence on the covid-19 pandemic. first, we found that the perception of the threat that covid-19 poses was on average highest in italy, followed by the united kingdom, spain, belgium, france, the united states, the netherlands, and germany. conversely, respondents' confidence in the preparedness of local and national organizations to deal with covid-19 was on average highest in the netherlands, followed by italy, germany, spain, the united kingdom, the united states, belgium, and france. in particular, italy was the first most affected country in europe in terms of numbers of cases and deaths, as well as the first country in europe to implement a nationwide lockdown on march 11, 2020 . this may explain the high threat perceived by the population in this country, and, together with the high confidence in the different health systems and different levels of government, the willingness to adopt preventive behaviors and adhere to social distancing measures. after italy, nationwide lockdowns were implemented also in spain (march 14), france (march 17), belgium (march 18), the netherlands (march 24), and the united kingdom (march 24) to slow the progression of the virus and to prevent overloading the healthcare system [30] . in the united states, instead, restrictive measures were implemented at the state level, starting in california on march 19, 2020 . notably, regarding the united kingdom and the united states, our data collected before and after lockdown measures were implemented (considering the united states as a whole) allow to observe temporal variation in the self-reported behaviors and attitudes: the perceived threat has increased in the population, along with the adoption of social distancing measures. in the case of the united kingdom, after the lockdown was implemented, the level of confidence in the health systems and different levels of government sharply increased, possibly reflecting discontent in the population about previously announced strategies. by contrast, the results for germany are more difficult to interpret. in germany, somewhat less restrictive measures were implemented on march 22, including school closures, cancellations of public gatherings, and the encouragement of social distancing. however, in contrast to the united kingdom and the united states, for which we observe a change in the temporal trends only after the implementation of non-pharmaceutical interventions, we find for germany that the share of respondents who had adopted social distancing measures was already high before such measures were implemented, and did not change much after this point. furthermore, compared to other countries, the level of confidence in the health systems and different levels of government in germany was high from the beginning of our observation period, and has further increased since then, whereas the perceived threat of covid-19 has decreased over time. this might be interpreted as a case of spontaneous bottom-up behavioral changes emerging from the population, following high trust in decisions and preparedness of the government. also, of the european countries considered in this study, germany had the third highest number of cases (about 140,000), but placed only sixth in terms of deaths (about 4,000) as of april 19, 2020 [31] , which might explain the lower perceived risk perception in the 13 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 15, 2020. . second, we observe a clear pattern in threat perceptions regarding different levels of society, sharply increasing from moderate threat for the personal sphere (threat to oneself and the family) to very high threat for more distal contexts (i.e. the local community, the country, and the world). yet, even though the perception of threat to oneself among our respondents was comparatively low, we found that a high share of them had increased their hand hygiene. this insight renders it uncertain as to what extent behavior can be straightforwardly linked to perceptions of personal threat. furthermore, we found that the perceived threat posed by covid-19 is significantly higher than the perceived threat posed by seasonal influenza. one likely explanation for this is that although seasonal influenza causes regular annual epidemics worldwide [32] , the novelty and uncertainty that surround covid-19 leads risk perception to be substantially higher. third, apart from variation at the country level, we also found sex-and age-specific differences. looking at the age component, our findings suggest that younger people perceive the threat to themselves lower than older people. this is in line with the evidence that older adults are at highest risk of severe complications following infection from covid-19 [33] . by contrast, the age structure in the perceived threat to the family is less pronounced, which suggests that respondents were concerned about their family members, regardless of their own age and the perceived threat to themselves. fourth, we also found sex-specific patterns in the data. specifically, female respondents perceived the threat that covid-19 poses substantially higher, reported a lower level of confidence in the health system, and were more willing to adopt protective behaviors. since the case fatality rate for covid-19 is substantially higher for men [34] , we might expect that men are more concerned about it. our results demonstrate that this is not necessarily true, and fact may have to be considered in the design of future communication campaigns. we gained these insights by using a novel approach to collecting health behavior data in times of a pandemic. we employed facebook advertising campaigns to continuously recruit a large number of participants for our survey over a long period of time. this approach allows us to target specific demographic groups in a comparative, cross-national approach, and to collect balanced samples to which post-stratification methods can be applied. these advantages notwithstanding, our approach also has some limitations. first, online surveys potentially suffer from bias due to self-selection and non-representativeness of the sample. in the case of facebook, there is increasing evidence that samples obtained from this social media network are not significantly different in central demographic and psychometric characteristics from samples obtained by more traditional recruitment and sampling techniques [12] . furthermore, by applying post-stratification weighting, which is a standard procedure in survey research, we can correct for non-representativeness in observable characteristics (but not necessarily for self-selection based on unobservable characteristics), at least at the level of the entire sample. ideally, in our cross-temporal comparisons, we would apply this approach at the level of the week, to warrant complete comparability of observations over time, but issues of data sparsity complicate this approach. we do not expect this to strongly affect our results, but it should be kept in mind that our weekly results might suffer from somewhat larger bias than our aggregate results. second, our data collection started at different time points across countries, and also pertains to different points in the trajectory of the pandemic across countries. this also encompasses differences in the implementation of non-pharmaceutical interventions ordered by local and national governments, and needs to be kept in mind when comparing and interpreting our results across countries. 14 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 15, 2020. . third, the data presented here have the form of repeated cross sections, which enables us to assess changes in the population samples over time, but does not allow us to assess changes within individuals. we are planning to address some of the limitations in the future in the following way. first, we aim to expand our post-stratification weighting scheme, by applying multilevel poststratification, which will enable us to achieve greater consistency among differently sized strata and greater precision in the estimates for population subsets, such as the weekly estimates presented here. second, we aim to carry out a follow-up survey among participants who agreed to provide their email address for this. this panel perspective offers a unique possibility to understand how the covid-19 pandemic affects the population in the long run and to assess the impact of loosening the lockdown measures on social contact patterns and health behaviors in a cross-national perspective. to conclude, our work reduces the gap in human behavioral data, by providing timely and accurate data on individual behaviors and attitudes across countries. our work also illustrates how social media networks, like facebook, together with appropriate survey designs and statistical methods, offer an innovative and powerful tool for rapid and continuous data collection to monitor trends in behaviors relevant for mitigation strategies of covid-19. taken together, the insights gained from our survey data are particularly relevant for policy makers and help design appropriate public health strategies and communication campaigns, and to design realistic epidemic models, which can account not only for the spatio-temporal spread of the infection, but also for accurate data on individual human behaviors. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 15, 2020. . https://doi.org/10.1101/2020.05.09.20096388 doi: medrxiv preprint . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 15, 2020. . https://doi.org/10.1101/2020.05.09.20096388 doi: medrxiv preprint . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 15, 2020. . https://doi.org/10.1101/2020.05.09.20096388 doi: medrxiv preprint world health organization world health organization estimating the number of infections and the impact of non-pharmaceutical interventions on covid-19 in european countries: technical description update how will country-based mitigation measures influence the course of the covid-19 epidemic? capturing human behaviour non-pharmaceutical interventions for pandemic influenza, national and community measures behavioural change models for infectious disease transmission: a systematic review towards a data-driven characterization of behavioral changes induced by the seasonal flu how behavioural science data helps mitigate the covid-19 crisis what's to like? facebook as a tool for survey data collection demographic research with non-representative internet data traditional versus facebook-based surveys: evaluation of biases in self-reported demographic and psychometric information are people excessively pessimistic about the risk of coronavirus infection? evaluating covid-19 public health messaging in italy: self-reported compliance and growing mental health concerns social psychological measurements of covid-19: coronavirus perceived threat, government response, impacts, and experiences questionnaires data for good: new tools to help health researchers track and combat covid-19 public opinion on the coronavirus outbreak: a multi-country poll from ipsos social contacts and mixing patterns relevant to the spread of infectious diseases facebook reports third quarter 2019 results social media use 2018: demographics and statistics measuring labour mobility and migration using big data: exploring the potential of social-media data for measuring eu mobility flows and stocks of eu movers online surveys and digital demography in the developing world: facebook users in kenya migrant sampling using facebook advertisements: a case study of polish migrants in four european countries broad reach and targeted recruitment using facebook for an online survey of young adult substance use quota sampling using facebook advertisements not by the book: facebook as a sampling frame eurostat regional yearbook annual estimates of the resident population by sex, age, race, and hispanic origin for the united states and states covid-19 pandemic in europe coronavirus source data world health organization people who are at higher risk: older adults covid-19 weekly surveillance report we would like to thank all the participants who took the time to voluntarily complete our survey, and the staff and colleagues of the max planck institute for demographic research who contributed to the realization of this project, in particular k. this study was funded through the support of the max planck institute for demographic research, which is part of the max planck society. this study was conducted in agreement with the data protection regulations valid in germany. informed consent was obtained from all participants, enabling the collection, storage, and processing of their answers. ethical approval for the study was obtained from the ethics council of the max planck society. all authors designed the questionnaire and collected the data. dp conceived the project idea, devised the idea for the manuscript, analyzed the data, and wrote the manuscript. ag developed the strategy and technical implementation for data collection and the recruitment of survey participants, and wrote the manuscript. fr supported the strategy development and the technical implementation of the data collection, and wrote the manuscript. jc and edf designed the post-stratification weighting scheme. dp, ag led the project and the implementation of the online survey. all authors provided input and edited and reviewed the manuscript. the authors declare that they have no competing interests. key: cord-011794-ejoufvvj authors: binder, florian; reiche, sven; roman-sosa, gleyder; saathoff, marion; ryll, rené; trimpert, jakob; kunec, dusan; höper, dirk; ulrich, rainer g. title: isolation and characterization of new puumala orthohantavirus strains from germany date: 2020-04-23 journal: virus genes doi: 10.1007/s11262-020-01755-3 sha: doc_id: 11794 cord_uid: ejoufvvj orthohantaviruses are re-emerging rodent-borne pathogens distributed all over the world. here, we report the isolation of a puumala orthohantavirus (puuv) strain from bank voles caught in a highly endemic region around the city osnabrück, north-west germany. coding and non-coding sequences of all three segments (s, m, and l) were determined from original lung tissue, after isolation and after additional passaging in veroe6 cells and a bank vole-derived kidney cell line. different single amino acid substitutions were observed in the rna-dependent rna polymerase (rdrp) of the two stable puuv isolates. the puuv strain from veroe6 cells showed a lower titer when propagated on bank vole cells compared to veroe6 cells. additionally, glycoprotein precursor (gpc)-derived virus-like particles of a german puuv sequence allowed the generation of monoclonal antibodies that allowed the reliable detection of the isolated puuv strain in the immunofluorescence assay. in conclusion, this is the first isolation of a puuv strain from central europe and the generation of glycoprotein-specific monoclonal antibodies for this puuv isolate. the obtained virus isolate and gpc-specific antibodies are instrumental tools for future reservoir host studies. electronic supplementary material: the online version of this article (10.1007/s11262-020-01755-3) contains supplementary material, which is available to authorized users. puumala orthohantavirus (puuv) is the most important hantavirus in europe [1] . it causes the majority of human hantavirus infections and hemorrhagic fever with renal syndrome (hfrs) cases [2] . in central and western europe hantavirus outbreaks occur in two to five year intervals and are driven by massive increase of the bank vole (myodes glareolus) population, the reservoir of this orthohantavirus species [3] . human hantavirus disease is notifiable in germany since 2001 and the majority of recorded cases is mainly due to puuv infections in southern and western parts of germany, whereas dobrava-belgrade orthohantavirus (dobv) with the striped edited by detlev h. kruger. the online version of this article (https ://doi.org/10.1007/s1126 2-020-01755 -3) contains supplementary material, which is available to authorized users. field mouse as reservoir causes infections in the northeastern part of germany [3] . the characterization of the pathogenicity and identification of virulence markers are highly dependent on adequate puuv isolates. currently, the number of puuv isolates is very limited and does not represent the real diversity of puuv strains in europe. in particular, no central european puuv isolate exists [4] . the majority of puuv isolates, and hantaviruses in general, was obtained based on passaging in reservoir animals or veroe6 cells and is highly adapted [5] [6] [7] . previous investigations indicated that veroe6 cell adaptation of puuv kazan strain results in the inability of the adapted strain to infect the bank vole reservoir [8] . the recent development of bank vole-derived primary or permanent cell lines may allow the isolation of reservoir-adapted puuv strains [9] [10] [11] [12] . hantavirus proteins are usually detected in infected cells by monoclonal antibodies. nucleocapsid (n) protein-specific monoclonal antibodies have been developed against a large range of hantaviruses [13] [14] [15] . in contrast, the number of glycoprotein precursor (gpc), as well as gc-and gn-specific monoclonal antibodies is rather low [16] [17] [18] . the majority of these antibodies were raised by infection of bank voles or immunization with recombinant n protein or heterologous virus-like particles (vlps). the generation of envelope protein-specific monoclonal antibodies with reactivity to virus proteins in infected cells is highly dependent on structural constraints [19] . autologous vlps represent a useful tool to generate highly efficient immune responses against a variety of viruses and for the generation of monoclonal antibodies in particular [20] . puuv strain astrup [21] gpc-derived vlps were generated in this study as previously described for maporal orthohantavirus [22] . lower saxony, north-west germany, and district osnabrück in particular, is a well-known endemic region for puuv infections [23, 24] . this endemic region was also again heavily affected by the hantavirus outbreak year 2019 [25] . here, we aimed to isolate a central european puuv strain from bank voles in the district of osnabrück using standard veroe6 cells and the recently established carpathian lineage bank volederived kidney cell line (mgn-2-r [10] ). complete genome determination by shot-gun and hybrid-capture-mediated highthroughput sequencing (hts) was used to follow the potential adaptation of the puuv isolates in veroe6 and reservoir cell lines. finally, the reactivity of the isolates was determined with novel monoclonal antibodies raised against puuv gpc vlps. bank voles were trapped in spring 2019 in the puuv endemic region around osnabrück following a standard snap trapping protocol [25, 26] . in the field, a small piece of lung was taken for virus isolation and rt-qpcr analysis. thereafter, carcasses were frozen, transported to the laboratory and completely dissected according to standard protocols. chest cavity lavage was collected by rinsing the chest cavity by 1 ml phosphate-buffered saline (pbs) and investigated for the presence of puuv-reactive antibodies. the presence of hantavirus rna was analyzed from lung tissue and were, in part, previously published in a surveillance study [25] . for virus isolation and further infection studies, veroe6 and bank vole kidney (mgn-2-r; [10] ) cells were used in parallel. virus titration was done on veroe6 cells only. mgn-2-r cells were grown in an equal mixture of hams' f12 and iscove's modified dulbecco's medium (imdm) + 10% fetal calf serum (fcs) and passaged two times per week at a 1:6 ratio. veroe6 cells were passaged twice a week in minimal essential medium (mem) + 10% fetal calf serum (fcs) and a split ratio of 1:4. for virus isolation, 1 × 10 5 mgn-2-r or veroe6 cells were seeded in 12.5 cm 2 flasks one day before rodent sampling in the field. the cells were carried to trapping sites in an isolation box with heat packs (around 33 °c constant for 2 days with outside temperature of 5-10 °c). after collecting voles from traps, a small incision in the chest area was made and a piece of lung (pea-sized) was taken and transferred into 1 ml dulbecco's modified eagle's medium (dmem) + 5% fcs + penicillin/streptomycin (ps) in a 5 ml safe lock tube. lung tissue material was homogenized in the field by grinding it through a fine metal grid against the tube wall. the homogenized tissue material was sterile filtered (0.45 µm) directly onto the cells resulting in approximately 500 µl tissue/medium suspension per 12.5 cm 2 flask. after 1-2 h incubation in the isolation box, 4 ml dmem + 5% fcs + ps was added. upon arrival in the laboratory flasks were incubated in a cell culture incubator at 37 °c and 5% co 2 for 10 days until first passage. in parallel, a pinhead-sized piece of lung was taken for rna isolation in 1 ml trizol (qia-gen, hilden, germany). after 10 days, trypsinized cells were resuspended in 2 ml dmem + 5% fcs + ps. for puuv rna screening, 325 µl of each cell suspension was taken for rna extraction and analyzed by rt-qpcr (see below). fresh veroe6 cells were resuspended in 2 ml dmem + 5% fcs + ps and 200 µl were mixed 1:1 with 200 µl of the inoculated cell suspension in a new 12.5 cm 2 flask. afterwards, 4 ml dmem + 5% fcs + ps were added and cells were incubated for 10 days until next passage. in parallel, one uninfected flask of veroe6 or mgn-2-r cells was passaged as a control. this procedure was continued until rt-qpcr-positive samples were detected. after first screening, only the flasks of the rt-qpcr-positive samples were further passaged. for detection of puuv nucleic acid, rna was extracted from homogenized lung tissue, or cell culture passages using qiazol lysis reagent (qiagen, hilden, germany) followed by a novel puuv s segment-specific rt-qpcr. for rt-qpcr, primers puuv-nss-s (5′-gwnata rcy cgy cat garc-3′) and puuv-nss-as (5′-art gct gac act gty tgt tg-3′) and the probe (5′-6-fam-crg tgg rrrt-gkacc crg atga-bhq-1-3′) were used. the pcr was done according to the quantitect probe one-step rt-qpcr mix (qiagen, hilden germany) protocol and contained 20 pmol/µl of each primer and 5 pmol/µl probe (eurofins, hamburg, germany). the following cycler protocol was used: 30 min of reverse transcription at 50 °c; 15 min initial denaturation at 95 °c; 45 cycles of 10 sec at 95 °c, 25 sec at 50 °c and 25 sec at 72 °c. for quantification of the number of rna copies/µl and sample, an in vitro transcribed rna was used. the in vitro transcription of a plasmid coding for nucleotides 83-355 of the s segment of a puuv strain from baden-wuerttemberg (binder et al., unpublished) was done according to the protocol of the manufacturer (riboprobe® in vitro transcription system t7, promega gmbh, mannheim, germany). the transcribed rna was serially diluted from 10 -2 to 10 -11 ng/ml with 700 rna copies/µl limit of detection (lod). initial tissue samples were screened for puuv rna and viral load as rna copies/µl was determined in triplicates for organs of isolated positive animals. rna from the cell culture adapted strains puuv sotkamo and tulv moravia were used as positive and negative control for the rt-qpcr, respectively. for metagenomics, we extracted rna from either a pinheadsized piece of lung tissue or 250 µl cell culture supernatant using 750 µl qiazol lysis reagent (qiagen, hilden, germany) in combination with rneasy mini kit (qiagen, hilden, germany). for generation of complete genomes of cell culture supernatants, a previously published workflow was used [27] . double-stranded, non-directional cdna libraries from lung tissue for sequencing on the illumina platform were prepared from total rna using the nebnext ultra ii rna library prep kit for illumina (new england biolabs, ipswich, ma, usa). per reaction, a total of 100 ng rna was used as an input. rna was fragmented for 8 min and final cdna libraries were amplified by 8 cycles of pcr to complete adapter ligation and to generate enough material for target sequence enrichment. a custom-made mybaits target capture array (arbor biosciences, ann arbor, mi, usa), containing biotinylated rna probes against all available puuv sequences deposited in ncbi genbank database (august, 2018), was employed to capture puuv-containing sequences from total cellular cdna sequencing libraries. the hybridization-based sequence enrichment (chemistry v3) was performed according to the manufacturer's instructions (arbor biosciences, ann arbor, mi, usa). the enriched cdna sequencing libraries were amplified with 14 pcr cycles to produce enough dna material for hts on the illumina platform. the enriched cdna libraries were quantified with the nebnext library quantification kit (new england biolabs, ipswich, ma, usa), pooled in equimolar amounts, and sequenced with a 600 cycle miseq reagent kit v3 (illumina, san diego, ca, usa) using paired-end sequencing (2 × 300 cycles) on a miseq sequencer (illumina, san diego, ca, usa). the resulting reads were trimmed and assembled against the known complete genome of strain astrup from the osnabrück region [21] with geneious r11.1.5 (https ://www.genei ous.com). for sequences lacking the 5′ and 3′ ends of the m segment, rna ligation was done using t4 rna ligase (thermo fisher scientific, waltham, ma, usa) and subsequent in vitro transcription with a first strand cdna synthesis kit (thermo fisher scientific, waltham, ma, usa). sequences were obtained by conventional dideoxy-chain termination sequencing after pcr with primers puuv os m2 fwd-5′ tga ggg caa tta tta tgt aa 3′ and puuv os m2 rev 5′ cca att gta tgt ggg cat tcc 3′. the obtained sequences were deposited at gen-bank, accession numbers mn639737-mn639763. phylogenetic trees were reconstructed with four novel and 18 published concatenated s, m, and l coding sequences or 202 partial s segment sequences of 365 nucleotides length. published sequences of other hantaviruses were obtained from genbank. analysis was performed by bayesian algorithms via mrbayes v.3.2.6 (https ://sourc eforg e.net/proje cts/ mrbay es/files /mrbay es/) on the cipres online portal [28] . a mixed nucleotide substitution matrix was specified in 4 independent runs of 10 7 generations. phylogenetic relations are shown as a maximum clade credibility phylogenetic tree with posterior probabilities for major nodes. for immunofluorescence assay (ifa), veroe6 and mgn-2-r cells were inoculated with 500 µl puuv osnabrück/v29 or puuv osnabrück/m43 supernatant in dmem + 5% fcs as described previously [10] . infected cells were fixed 10 days 1 3 post infection with a 1:1 mixture of acetone and methanol for 20 min at − 20 °c. after fixation cells were dried, re-hydrated with phosphate-buffered saline (pbs) and incubated with nucleocapsid (n) protein-specific antibody 5e11 [13] diluted 1:1000 in pbs for 1 h at room temperature (rt). a secondary anti-mouse alexa fluor 488 conjugated antibody (abcam, cambridge, uk) was used for detection of hantavirus proteins. nuclei were stained with 4′,6-diamidino-2-phenylindole (dapi, thermo fisher scientific). for titration studies of puuv, mgn-2-r and veroe6 cells were inoculated with 500 µl of the puuv osnabrück/v29 or puuv osnabrück/m43 virus isolate and passaged three times as described above. supernatants of both cell lines were collected after passage three and frozen at − 80 °c. subsequently, supernatants were serially diluted from 10 -1 to 10 -7 in dmem containing 5% fcs in a 96-well plate with three replicates each. a volume of 100 µl of each dilution was added to 24 h old cell monolayers of veroe6 cells in a 96-well plate. after incubation for 10 days, the virus titer was calculated using ifa for puuv n protein detection as described above. titers were calculated as 50% tissue culture infectious dose (tcid 50 )/ml by the spearman/kärber method [29] and mean titers of three experiments are given. titers after isolation (passage 3 of original lung tissue-derived sample) were used for comparison. for expression and generation of vlps in hek293 cells, a codon-optimized synthetic gene of the puuv gpc of the strain astrup [21] was purchased (geneart, regensburg, germany). the gene encoding the glycoproteins was pcr amplified using primer pair o grs 101/o grs 102 (aat-taaggt acc tcc aga ggc gac acc cgg aacc and aattattaag ctt tca ggg ctt gtg ttc ttt gg) and the pcr product and the acceptor vector phan-1 (roman-sosa, unpublished) were digested with the restriction endonucleases kpni and hindiii. the expression plasmid phan-2 was generated by standard molecular biology protocols. in this plasmid, the endogenous signal sequence of the puuv gn is substituted by the igg-light chain signal sequence and a double strep-tag with a glycine/serine-rich linker between the tags. then a permanently transfected hek293 cell line was generated upon transfection of the cells and selection in the presence of geneticin at 0.5 mg/ml. the vlps were affinity purified from the cell supernatants essentially as described [22] . recombinant vlps were used for five immunizations of four weeks apart of female balb/c mice. hybridoma cells producing monoclonal antibodies (mabs) were generated by standard fusion procedure [30, 31] and screened using a 2 µg/ml stock solution of vlps according to an in-house elisa protocol [32] and buffers without tween. resulting mabs were analyzed by ifa and western blot test for their reactivity to puuv osnabrück/v29, puuv sotkamo, puuv vranica and tulv moravia. veroe6 cells were infected with puuv osnabrück/v29, puuv sotkamo, puuv vranica or tulv moravia at moi 0.1 in dmem + 5% fcs. cells were harvested 10 (puuv osnabrück/v29, sotkamo) or 3 (puuv vranica, tulv moravia) days post infection in sds sample buffer (62.5 mm trishcl ph 6.8, 2% sds,10% glycerol, 6 m urea, 0.01% bromophenol blue, 0.01% phenol red) and proteins were separated by sds page, blotted onto polyvinylidenfluorid (pvdf) membranes. after blocking, the membranes were cut into strips and incubated over night with the antibodies 2e10 (1:1), 5f12 (1:1), 3b12 (1:200), 5b8 (1:1), 5h1 (1:1), 4g10 (1:100), 1b12 (1:2), 1g9 (1:100), 8g4 (1:50), 1h7 (1:1), 2h11 (1:5) or n protein-specific antibody 5e11 (1:1000, [13] , all diluted in pbs-tween 0.05%) at 4 °c. a horseradish peroxidase (hrp) labeled secondary goat anti-mouse igg antibody diluted 1:3000 in pbs-tween 0.05% (bio-rad, hercules, ca, usa) was used for detection of hantaviral proteins. a rabbit anti-β-tubulin antibody (abcam, cambridge, uk) was used as a loading control. investigation of chest cavity lavage samples from bank voles was done by igg elisa using recombinant puuv strain bawa n protein, as described earlier [32] . the monoclonal antibody 5e11 was used as a positive control [13] , chest cavity lavage of a igg elisa-and rt-pcr-negative bank vole was used as negative control. chest cavity lavage samples with an optical density (od) value below the lower cut-off value were considered as negative. positive and doubtful samples were retested a second time. when the od value of the elisa was in a range between the lower and upper cut-off value defined according to our standard protocol [32] , animals were considered doubtful. when the od value was above the upper cut-off value, the samples were considered as positive. rodent trapping at five sites from april 11th to 12th, 2019 in the osnabrück region resulted in the collection of 57 bank voles [25] . dissection on site and inoculation of veroe6 and bank vole mgn-2-r cells with homogenized lung samples resulted after three blind passages in four potential isolates that were detected by a novel puuv rt-qpcr (table s1 , fig. 1) . two of the potential candidates showed only low levels of puuv rna and were not able to consistently infect further passages (m52, m62). quantification by rt-qpcr analysis of different tissues from these four bank voles confirmed lung tissue for most of the samples as having the highest puuv rna load, although it was detected in almost all other tissues investigated (fig. s1 ). rt-qpcr investigation of lung tissues of all 57 bank voles resulted in the detection of hantavirus rna in 44 animals (tables 1, s1, [25] ). puuv rna-positive animals originated from all five trapping sites. serological analysis of chest cavity lavages detected puuv n protein reactive antibodies in 24 of 57 bank voles (tables 1, s1). five additional animals, positive for puuv rna, were found to be equivocal in our serological test. all 24 antibody-positive animals were also found to be puuv rna positive, indicating a high number of persistently infected voles. fifteen additional bank voles were only positive for puuv rna, but not for anti-puuv antibodies, indicating a high number of acutely infected animals in spring in this region (table 1) . interestingly three of the four potential isolates originated from seronegative bank voles (table s1 ). two isolates (osnabrück v29 and osnabrück m43) were obtained by passaging in veroe6 or mgn-2-r cells, which reached titers of almost 10 3 tcid 50 /ml ( fig. 2a and b , titer after isolation). shot-gun and hybrid-capture-mediated hts of both isolates resulted in the generation of complete genome sequences which are identical in sequence to the respective original strain in bank vole lung tissue except for one amino acid (aa) exchange each in the rna-dependent fig. 3 ). the genome organization of the novel puuv isolates indicated the typical sequence elements for puuv: the small (s) segment encodes an n protein of 433 aa residues and a putative nss protein of 90 aa in an + 1 overlapping reading frame, the medium (m) segment codes for the 1148 aa gpc and the large (l) segment for the rdrp of 2156 aa (see fig. 3 , genbank accession numbers: mn639737-mn639748). phylogenetic analysis of the concatenated s, m and l segment coding sequences grouped the novel isolates together with astrup prototype strain in sister relationship to puuv sequences from france (fig. 4a) . the phylogenetic analysis of a partial s segment sequence of the novel isolates and representative strains of all puuv clades and subclades from germany confirmed the close relationship of the new isolates to the osnabrück hills subclade (fig. 4b) . the puuv osnabrück m43 isolate was found to be contaminated by a bank vole reovirus; hts derived sequences of the passaged reovirus (genbank accession numbers: mn639755-mn639763) showed a strong similarity to a bank vole reovirus strain, but much lower similarity to a common vole reovirus [33] ). the non-reovirus contaminated isolate osnabrück v29 from veroe6 cells was found to have an insertion of 20 nucleotides in the 3′ non-coding region (ncr) when compared to the other isolate and the astrup reference sequence (fig. 3) . however, this insertion was also found in the original lung sample and therefore no cell culture-specific adaptations were observed in the ncrs of both virus isolates (fig. 3 ). figs. 1 and 2 ). this passaging resulted in no further mutations (genbank accession numbers: mn639749-mn639754). however, the virus isolate passaged in veroe6 cells is accompanied by an increase in the virus titer to 10 4 tcid 50 /ml (fig. 2) . in contrast, the passaging of the osnabrück v29 strain in mgn-2-r cells resulted in a decreased virus titer. as no cytopathic effect was observed, virus detection for titration in both cell lines was done by immunofluorescence assay using an n proteinspecific monoclonal antibody (fig. 2a) . eleven monoclonal antibodies were produced in this study by immunization of mice with puuv strain astrup gpcderived vlps. evaluation of the virus isolate osnabrück v29 using these monoclonal antibodies resulted in typical immunofluorescence patterns in the cytoplasm (fig. 5) . further analysis by western blot test using a lysate of isolate osnabrück v29 from veroe6 cells suggested that the majority of anti-gpc antibodies are directed against conformational epitopes; however, some recognize linear epitopes in gc or gn (table 2 ). subsequent evaluation of the reactivity of these monoclonal antibodies with other puuv strains and tulv strain moravia indicated some level of crossreactivity for some of them (table 2) . here, we describe the first isolation of a central european puuv strain. this strain of the central european lineage increases the available panel of puuv isolates: currently available isolates sotkamo, umea, vranica, and kazan, belong to the clades finnish, north scandinavian, most likely north scandinavian, and russian, respectively [34] . the puuv-like hokkaido virus strain kitahiyama128 originates from japan [12] . in our study, the isolation was based on an in-field dissection and inoculation of cells to prevent freeze/thaw cycles. the subsequent investigation of all 57 bank voles indicated that three of four isolates originated from anti-puuv-seronegative voles. this finding illustrates that a serological test in the field might be misleading in selection of samples for successful virus isolation. instead, an on-site molecular assay may enhance the chance for a successful virus isolation. nevertheless, the approach used here still indicates the challenges of hantavirus isolation; only four isolates were obtained from a total of 15 acutely infected bank voles. in addition, the determination of the complete genome sequences of two isolates including the ncrs expands our knowledge on the sequence diversity of puuv strains within the different regions of the genome. moreover, the hybrid-capture-based enrichment of puuv sequences allows a rapid determination of the complete genome and underlines the value of this workflow for hantavirus surveillance and molecular evolution studies [35] . a phylogenetic analysis of partial s segment nucleotide sequences confirmed the previously reported subclades of puuv in germany; the novel isolates belong to the subclade osnabrück hills within the central european clade. the position within the phylogenetic tree also confirms the local evolution pattern of puuv reported before [23, 36] . the observed high level of rt-qpcr-positive bank voles (44/57; 77%) confirms the district of osnabrück in spring 2019 as a hantavirus outbreak region [25] . the puuv rna detection rate was similarly high at all five trapping sites of bank voles. although 2019 was identified as a hantavirus outbreak year in germany, the distribution of notified human puuv cases was not as homogeneous as in previous outbreak years [25] . the passage of the puuv strains for isolation resulted in non-synonymous nucleotide exchanges in the l segment responsible for single amino acid exchanges in the rdrp (i3749m in m43 and d3963y in v29). the substituted amino acid residues are each very similar in their properties and, presumably, might not influence protein function. a more divergent adaptation at position s2053f has previously been observed for puuv strain kazan [8, 37] . although in this previous study nucleotide exchanges in the ncr of the s segment were observed [37] , here we did not find relevant mutations in this region after passaging in cell culture. the v29 strain showed an insertion in the 3′ ncr, but this insert was also found in the original lung material used for isolation. additionally, this sequence insert was found in another sequence from the same region (jn696358.1, [36] ). the isolate v29 was shown to replicate in veroe6 and a bank vole kidney cell line. the low titer in the bank vole mgn-2-r cell line might be due to the evolutionary lineage origin of this cell line (carpathian lineage); in central europe puuv is harbored by the western evolutionary lineage with spillover to the carpathian lineage in regions with sympatric occurrence of both [24] . in line with the assumption of an association of a puuv clade with an evolutionary bank vole lineage, the vranica puuv strain replicated in mgn-2-r cells, but not in bank vole kidney cells of another evolutionary lineage [9, 10] . interestingly, replication of puuv-like hokkaido virus in cells of its host, the gray red-backed vole, was comparable to puuv infection [12] . future investigations in cell lines and animals of different bank vole lineages are required to confirm this conclusion directly. the orthoreovirus contamination of one of the puuv isolates illustrates that bank voles may harbor additional reactivity of novel puuv gpc-specific monoclonal antibodies with hantavirus-infected veroe6 cells in immunofluorescence assay (ifa). antibodies were generated by immunization of balb/c mice with gpc-derived virus-like particles of puuv strain astrup. after screening and subcloning, monoclonal antibodies were tested in ifa. veroe6 cells were infected with puuv osnabrück v29 iso-late on coverslips and fixed for ifa after 10 days. the monoclonal antibodies were administered for 1 h at rt. detection of the specific antibody binding was done using an anti-mouse alexa fluor 488 conjugated antibody. after staining, coverslips were mounted on glass slides for imaging infectious agents that may influence the susceptibility to puuv infections or their outcome. of note, in bank voles several viruses have been detected, i.e., polyoma-, herpesand hepaciviruses [38] [39] [40] [41] , but also bacterial agents and endoparasites [42] [43] [44] . similarly, a hantavirus isolation approach was previously hampered by the coinfection by a striped field mouse adenovirus [45] . future investigations are needed to evaluate potential influences of coinfections in bank voles. it has been shown that hantavirus gn and gc form complex spike-shaped structures [46] that build conformational epitopes [17, 18] . therefore, we selected an immunization procedure using puuv-gpc-derived vlps, as the organization of the glycoproteins resembles the one of the virion. a panel of eleven monoclonal antibodies was produced here and all of them were reactive with the new puuv isolate in immunofluorescence assay. the staining pattern, which is reminiscent of the one of the secretory pathway organelles, i.e., the golgi apparatus and the endoplasmic reticulum, suggests that the epitopes recognized by these antibodies are already accessible during the maturation process of the proteins. interestingly, some of the monoclonal antibodies recognize linear epitopes as revealed by a western blot assay. although preliminary results suggest that the antibodies do not neutralize the virus when tested individually, synergistic effects with a protective effect cannot be ruled out yet as shown for anti-ebola virus monoclonal antibodies [47] . therefore, the novel antibodies represent a useful tool for further experimental, diagnostic, and therapeutic applications. in conclusion, the puuv isolate described here replicates in a bank vole cell line and its n and gpc proteins can be detected by specific monoclonal antibodies. therefore, this isolate will be useful for further studies on the virulence markers of central european puuv, its reservoir host association and the route of pathogenicity in the bank vole model. the novel gpc-specific monoclonal antibodies will enable future studies on virus entry and important domains for exposed immunogenic regions. funding florian binder acknowledges intramural funding by the friedrich-loeffler-institut. additional funding was provided by the bundesminsterium für bildung und forschung through the research network zoonotic infections (robopub consortium, fkz 01ki1721a, awarded to rgu; fkz 01ki1721h, awarded to laves) for trapping and rodent screening, the rapid project within the infect control veroe6 cells were inoculated with puumala virus (puuv) osnabrück/v29, puuv sotkamo, puuv vranica or tula virus (tulv) strain moravia. infected cells were fixed 10 (puuv osnabrück/v29, sotkamo) or 3 (puuv vranica, tulv moravia) days post infection for immunofluorescence assays or collected in sample buffer for western blot analysis. after fixation or western blot transfer, novel gpc-specific mabs 2e10, 5f12, 3b12, 5b8, 5h1, 4g10, 1b12, 1g9, 8g4, 1h7, and 2h11 were administered. gn-and gcreactive mabs were assigned where possible according to molecular weight of the immunoreactive bands in western blot analysis − negative; (+) weak reactivity; + positive; ++ strongly positive conflict of interest the authors declare that they have no competing interests. ethical approval all animals were handled according to the applicable institutional, national and international guidelines for the care and use of animals. bank vole trapping was conducted in line with the regular pest control of the laves veterinary task-force in lower saxony, germany (department of pest control, oldenburg) according to german federal law ( § 18, gesetz zur verhütung und bekämpfung von infektionskrankheiten beim menschen). the immunization of mice was done in line with the general immunization program of the friedrich-loeffler-institut (landesamt für landwirtschaft, lebensmittelsicherheit und fischerei, mecklenburg-vorpommern, permit: 28/17). open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. hantavirus infections hantaviruses-globally emerging pathogens weiss s (2019) molecular and epidemiological characteristics of human puumala and dobrava-belgrade hantavirus infections coding strategy of the s and m genomic segments of a hantavirus representing a new subtype of the puumala serotype isolation of the causative agent of hantavirus pulmonary syndrome propagation of nephropathia epidemica virus in cell culture isolation and characterization of puumala hantavirus from norway: evidence for a distinct phylogenetic sublineage cell culture adaptation of puumala hantavirus changes the infectivity for its natural reservoir, clethrionomys glareolus, and leads to accumulation of mutants with altered genomic rna s segment a new permanent cell line derived from the bank vole (myodes glareolus) as cell culture model for zoonotic viruses common vole (microtus arvalis) and bank vole (myodes glareolus) derived permanent cell lines differ in their susceptibility and replication kinetics of animal and zoonotic viruses more novel hantaviruses and diversifying reservoir hosts-time for development of reservoirderived cell culture models? viruses isolation of hokkaido virus, genus hantavirus, using a newly established cell line derived from the kidney of the grey red-backed vole (myodes rufocanus bedfordiae) characterization of monoclonal antibodies against hantavirus nucleocapsid protein and their use for immunohistochemistry on rodent and human samples sensitive detection of hantaviruses by biotin-streptavidin enhanced immunoassays based on bank vole monoclonal antibodies novel serological tools for detection of thottapalayam virus, a soricomorpha-borne hantavirus bank vole monoclonal antibodies against puumala virus envelope glycoproteins: identification of epitopes involved in neutralization the use of chimeric virus-like particles harbouring a segment of hantavirus gc glycoprotein to generate a broadly-reactive hantavirusspecific monoclonal antibody human recombinant neutralizing antibodies against hantaan virus g2 protein hantavirus gn and gc envelope glycoproteins: key structural units for virus cell entry and virus assembly virus-like particles: a versatile tool for basic and applied research on emerging and reemerging viruses. viral nanotechnologies complete genome of a puumala virus strain from central europe protocadherin-1 is essential for cell entry by new world hantaviruses spatiotemporal dynamics of puumala hantavirus associated with its rodent host myodes glareolus host-associated absence of human puumala virus infections in northern and eastern germany heterogeneous puumala orthohantavirus situation in endemic regions in germany in summer aphaea/ewda species card: voles and mouses a versatile sample processing workflow for metagenomic pathogen detection a restful api for access to phylogenetic tools via the cipres science gateway beitrag zur kollektiven behandlung pharmakologischer reihenversuche antigenic and cellular localisation analysis of the severe acute respiratory syndrome coronavirus nucleocapsid protein using monoclonal antibodies indirect elisa based on hendra and nipah virus proteins for the detection of henipavirus specific antibodies in pigs phylogenetic analysis of puumala virus subtype bavaria, characterization and diagnostic use of its recombinant nucleocapsid protein isolation and complete genome characterization of novel reassortant orthoreovirus from common vole (microtus arvalis) phylogeography of puumala orthohantavirus in europe secondary contact between diverged host lineages entails ecological speciation in a european hantavirus multiple synchronous outbreaks of puumala virus adaptation of puumala hantavirus to cell culture is associated with point mutations in the coding region of the l segment and in the noncoding regions of the s segment evidence for novel hepaciviruses in rodents identification of two novel members of the tentative genus wukipolyomavirus in wild rodents identification of novel rodent herpesviruses, including the first gammaherpesvirus of mus musculus molecular detection and characterization of the first cowpox virus isolate derived from a bank vole leptospira genomospecies and sequence type prevalence in small mammal populations in germany high prevalence of rickettsia helvetica in wild small mammal populations in germany. ticks and tick-borne diseases occurrence of gastrointestinal parasites in small mammals from germany. vector borne zoonotic dis a novel cardiotropic murine adenovirus representing a distinct species of mastadenoviruses molecular organization and dynamics of the fusion protein gc at the hantavirus surface cooperativity enables non-neutralizing antibodies to neutralize ebolavirus acknowledgements open access funding provided byprojekt deal. the authors would like to thank sönke röhrs for help with rodent trapping, stephan drewes for help with phylogenetic analysis and sven sander and patrick zitzow for excellent technical support with generation of monoclonal antibodies and sequencing of puuv isolates. the authors thank martin beer, klaus osterrieder and nicole tischler for constant support and helpful discussions.author contributions rgu and fb designed the study and wrote the manuscript. fb did virus isolation, infection studies, sequence analysis, phylogenetic analysis, and testing of monoclonal antibodies. sr and fb generated and screened the monoclonal antibodies. grs produced the vlps for immunization. ms and fb performed rodent trapping. dh, jt, and dk did the complete genome sequencing of puuv isolates. rr developed the puuv-specific rt-qpcr assay. all authors gave significant ideas for the presented work and were involved in writing and proof reading of the manuscript. key: cord-289555-1z4vbldd authors: mühldorfer, kristin; speck, stephanie; kurth, andreas; lesnik, rené; freuling, conrad; müller, thomas; kramer-schadt, stephanie; wibbelt, gudrun title: diseases and causes of death in european bats: dynamics in disease susceptibility and infection rates date: 2011-12-28 journal: plos one doi: 10.1371/journal.pone.0029773 sha: doc_id: 289555 cord_uid: 1z4vbldd background: bats receive increasing attention in infectious disease studies, because of their well recognized status as reservoir species for various infectious agents. this is even more important, as bats with their capability of long distance dispersal and complex social structures are unique in the way microbes could be spread by these mammalian species. nevertheless, infection studies in bats are predominantly limited to the identification of specific pathogens presenting a potential health threat to humans. but the impact of infectious agents on the individual host and their importance on bat mortality is largely unknown and has been neglected in most studies published to date. methodology/principal findings: between 2002 and 2009, 486 deceased bats of 19 european species (family vespertilionidae) were collected in different geographic regions in germany. most animals represented individual cases that have been incidentally found close to roosting sites or near human habitation in urban and urban-like environments. the bat carcasses were subjected to a post-mortem examination and investigated histo-pathologically, bacteriologically and virologically. trauma and disease represented the most important causes of death in these bats. comparative analysis of pathological findings and microbiological results show that microbial agents indeed have an impact on bats succumbing to infectious diseases, with fatal bacterial, viral and parasitic infections found in at least 12% of the bats investigated. conclusions/significance: our data demonstrate the importance of diseases and infectious agents as cause of death in european bat species. the clear seasonal and individual variations in disease prevalence and infection rates indicate that maternity colonies are more susceptible to infectious agents, underlining the possible important role of host physiology, immunity and roosting behavior as risk factors for infection of bats. bats are among the most successful and diverse mammals on earth. approximately 1230 chiropteran species are found on every continent except antarctica and inhabit a multitude of diverse ecological niches [1] . bats play essential roles in maintaining healthy ecosystems, as they act as plant pollinators, seed dispersers, and predators of populations of insects including harmful forest and agricultural pests [2] . most bat species are listed in the iucn red list of endangered species and almost half of these are considered threatened or near-threatened [3] . to estimate and prevent further population declines, research has been primarily focused on bat biology, ecology and behavior, while disease aspects were largely neglected [4] . in the last two decades, the importance of chiropteran species as potential vectors of significant viral diseases especially in regard to zoonoses has received growing attention. besides bat rabies that has been studied for more than half a century, extensive research efforts identified a large number of microbial agents [5] including important emerging zoonotic viruses detected in bats across the world [6] [7] [8] [9] [10] [11] [12] . however, most studies are limited to the identification of microorganisms detected and investigations regarding infectious diseases and causes of death in bats are sparse [13] [14] [15] [16] . in europe, research is predominantly focused on european bat lyssaviruses [17, 18] and coronaviruses [19, 20] , but first indications of bat-pathogenic bacteria [13, 14, [21] [22] [23] and novel viruses [24, 25] isolated from deceased bats in germany and great britain were found. in this study, we provide new data on infectious diseases in european bat species, considering factors likely to affect the susceptibility of bats to infectious agents including effects of seasonality, individual and species-specific heterogeneities, and possible intra-and inter-species transmission dynamics. all bat species in europe are strictly protected under the flora-fauna-habitat guidelines of the european union (http://ec.europa. eu/environment/nature/legislation/habitatsdirective/index_en.htm) (92/43/eec) and the agreement on the conservation of populations of european bats (www.eurobats.org) that prohibit invasive sampling of bats for research purposes. for the animals investigated in this study, carcasses of deceased bats found in germany were kindly provided by bat researchers and bat rehabilitation centers of different federal states. between 2002 and 2009, a total of 486 deceased bats of 19 european vespertilionid species (i.e., family vespertilionidae) were investigated (fig. 1a , [26] ). the bat carcasses originated from 6 different geographic regions in germany, i.e. berlin greater metropolitan area (n = 223), bavaria (n = 165), brandenburg (n = 38), lower saxony (n = 36), thuringia (n = 21), and baden-wuerttemberg (n = 3), and were collected by bat researchers and bat rehabilitation centers. most animals represented individual cases that were found dead, injured or moribund near human habitation. thus, the species composition in this study predominately reflected the urban and suburban bat fauna, which is characterized by a disproportionate abundance of a few bat species (fig. 1a , [27, 28] ). two groups of 2 and 21 adult noctules (nyctalus noctula), respectively, were collected from tree hibernacula destroyed during wood logging. a further group of 25 deceased adult n. noctula originated from a colony that was trapped in a rain pipe in december. nine dead juvenile pipistrellus pipistrellus were collected from a nursery roost. if bats died in care or had to be euthanized for animal welfare reasons, the carcasses were immediately stored at 220uc and were shipped to the leibniz institute for zoo and wildlife research, berlin, germany, for diagnostic investigations. of all carcasses examined histo-pathologically, about 90% were suitable for bacteriological investigation. a lesser extend (43%) was also examined for selected viral agents at the robert koch institute, berlin, germany. in addition, a brain sample of each animal was submitted to the friedrich-loeffler-institute, wusterhausen, germany, for rabies diagnosis. a full necropsy was performed on each bat and all macroscopic findings including ectoparasite infestation were recorded. for histo-pathological examination, small slices of multiple organ tissues (i.e., lung, liver, heart, kidney, adrenal gland, spleen, intestine, pancreas, brain, tongue, larynx, salivary gland and pectoral muscle) and tissues conspicuous for pathological changes were fixed in buffered 4% formalin, processed using standard methods and embedded in liquid paraffin. sections were cut at 2-5 mm and routinely stained with hematoxylin-eosin (he). in addition, special histological staining methods were used depending on microscopic findings, i.e. for the detection of bacteria (gram or giemsa staining), fungi (periodic acid schiff or grocott's gomori methenamine silver nitrate staining), iron (prussian blue stain), mineralization (von kossa staining), connective and collagen tissue (trichrome staining). details on pathological results are published elsewhere [26] . the causes of mortality were rigorously standardized with the primary cause of death identified for each bat as the most serious injury, disease or event subsequently fatal to the animal. to ensure independence of primary and contributing causes of death, the categorization was based on the severity of pathological findings. samples of lung, liver, heart and kidney, and tissues conspicuous for pathological changes (e.g. enlarged spleen) of 430 bats were plated onto columbia (5% sheep blood), chocolate, gassner, and macconkey agar (oxoid, germany) and were incubated at 37uc (chocolate agar 5% co 2 ) for 24-48 h. specific culture media and conditions for the isolation of yersinia, salmonella and anaerobic bacteria were used if appropriate. primary identification of bacterial strains was based on colony morphology, hemolysis, gram-staining, indol production, catalase and oxidase reaction. bacterial species identification was carried out using the relevant commercial api test system (biomérieux, germany). additional conventional biochemical tests [29, 30] were applied to confirm api test results where necessary. in case of ambiguous biochemical test results, 16s rdna gene analysis was performed for final identification [23] . salmonella isolates were characterized at the national reference laboratory for the analysis and testing of zoonoses (salmonella) at the federal institute for risk assessment, berlin, germany. identification and characterization of yersinia and pasteurella species have been reported earlier [22, 23] . homogenized organ tissue of lung, liver, heart, kidney, spleen, brain and salivary gland of 210 bats were pooled for each individual and used for rna/dna extraction and further molecular analysis by generic pcr assays detecting flavi[31] , hanta[32] , corona[33] , and influenza a-viruses [34] . also, pcr assays specific for 8 previously described herpesviruses [24] from european vespertilionid bats were used. for this purpose, rna/ dna was isolated using the nucleospinh rna ii kit (macherey-nagel, germany) and the nucleospinh tissue kit (macherey-nagel), respectively, according to the manufacturer's instructions. because of limitations in sample volume, for 180 out of the 210 bats pcr assays could only be applied for 4 different bat herpesviruses. internal controls were used for all pcr assays to test for inhibition. for confirmation, all retrieved fragments of bat herpesvirus-specific pcr assays were checked for sequence identity to previously published isolates [24] . for detection of lyssavirus antigen in brain tissue the fluorescent antibody test (fat) using a polyclonal antirabies conjugate (sifin, germany) was used [35] . fat-positive brain tissues were subject of virus isolation in murine neuroblastoma cell culture (na 42/13) using the rabies tissue culture infection test (rtcit) as described elsewhere [36] . lyssaviruses isolated in cell culture were characterized using both a panel of 10 anti-nucleocapsid monoclonal antibodies (mab) [37] and partial sequencing of a fragment of the nucleoprotein gene after rna extraction using trizol (invitrogen, germany) essentially as described [18] . genomic dna was extracted from organ homogenates using the nucleospinh tissue kit (macherey-nagel) according to manufacturer's recommendations. genetic identification of the bat species was performed by amplification and sequencing of a 241 bp fragment of the cytochrome b (cytb) gene [38] using primers fm up (59-ccc chc chc aya tya arc cmg art gat a -39) and fm down (59-tcr acd ggn tgy cct ccd att cat gtt a -39). in addition, for differentiation of the 2 distinct pipistrellus species, p. pipistrellus and p. pygmaeus, a rapid multiplex pcr assay was performed as described by kaňuch et al. the bat data were categorized in regard to different explanatory numeric and factor variables, e.g. bat species, sex and age class. the variable 'age class' ranked between 1 and 4 with increasing age (i.e. neonates, juveniles, subadults, and adults) and was used as numeric variable. for endoparasitic analysis, we defined a 3 level variable 'bat size' according to the body size of a certain bat species to reduce the degrees of freedom of the full model, i.e. large species (n. noctula, eptesicus serotinus, and vespertilio murinus), medium-sized species (e. nilssonii, plecotus auritus, myotis daubentonii, m. nattereri, and p. nathusii) and small species (p. pipistrellus, and m. mystacinus). to detect effects of seasonality, 4 different activity periods were specified according to the date of sampling, i.e. hibernation period (november to march), post-hibernation period (april/may), maternity period (june to august), and swarming period (september/october). as dependent binary variable for the respective models we either classified the mortality cause being disease or not (i.e. trauma), or the presence-absence of bacterial, ecto-and endoparasitic infections. we formulated 4 different hypotheses to test for individual and species-specific differences in disease susceptibility and infection rates: (a) disease-related mortality in bats is influenced by sex, age and species-specific differences, and degree of endoparasitic infection. (b) bacterial infection in bats is influenced by sex, age and species-specific differences, occurrence of traumatic injuries and cat predation. (c) ecto-or (d) endoparasitic infection in bats is affected by age, sex and species-specific differences. seasonal effects were not analyzed because of too many missing data points. because the long-term dataset was highly biased towards sampling procedure, preservation of bat carcasses and following diagnostic investigations, we split and filtered the full data into several subsets reflecting the different analyses (table 1) . all statistical analyses were performed using the r software v. 2.13.1 (r development core team 2011, vienna, austria). we used the chi-square test for given probabilities to evaluate significant differences in the sex ratio among bats of different species. for hypotheses a and b, we used a generalized linear mixed modeling approach (binomial glmm using function lmer in library lme4) with bat species included as random effect. this variable had not been significant as fixed effect (results not shown), but from other studies we can assume that there are speciesspecific differences in susceptibility of bats to certain infectious agents and therefore included it as random effect. we further used generalized linear models (glm with logit link and binomial error structure; for datasets with bat species .10 individuals) to test for individual and species-specific differences in parasite infection rates (hypotheses c and d). we created a full model for each hypothesis (a-d) to examine multiple and interaction effects of the specified variables. to select the final model variables, we used a stepwise backward algorithm (function stepaic in library mass) based on akaike's information criterion (aic) [40] . the daic of the final model was calculated relative to a random intercept model to demonstrate the effect size of the selected variables. results of the diagnostic analyses follow the full data splitting into several subsets (see section 'statistical analysis' in material and methods; table 1 ). all sampled bats belonged to 7 different genera (i.e. pipistrellus, nyctalus, myotis, eptesicus, plecotus, vespertilio, and barbastella) and 19 european vespertilionid species (fig. 1a) . three bat species, the common pipistrelle (p. pipistrellus, n = 138), the noctule bat (n. noctula, n = 92), and the serotine bat (e. serotinus, n = 53) constituted about 60% of all bat carcasses investigated in this study, whereas p. pygmaeus, nyctalus leisleri, myotis brandtii, m. bechsteinii, m. dasycneme, plecotus austriacus and barbastella barbastellus were represented in small numbers of 1 to 4 animals. the overall sex ratio was 1.5 males to 1 female with significant species-specific differences (fig. 1b) . animals in their first year of life (neonates, juveniles, and subadults) represented one third (32.5%, n = 158) of bat samples (fig. 1c) . overall, we were able to assign a cause of death to 70% (n = 304) of bats investigated in this study. two thirds of mortality were due to trauma (n = 145) or disease (n = 144), while almost 4% of bats had died of other non-infectious causes like pulmonary edema, dehydration and hypoglycemia (table 2 ). in 30% (n = 129) no significant pathological findings could be found. among the 145 traumatized bats, additional mild (n = 42), moderate (n = 28) and severe (n = 4) inflammatory organ changes were noted in one half (50.9%) of individuals, and 23% of the bats revealed bacterial (n = 19) and/or parasitic infections (n = 15) ( table 3) . of the 144 bats considered as dying of disease, fatal bacterial (n = 54), viral (n = 5) and parasitic infections (n = 2) were observed in 42%. besides, amniotic fluid aspiration was noted in a neonate noctule bat (n. noctula), and a juvenile common pipistrelle (p. pipistrellus) was euthanized because of severe forearm bone deformation. the remaining 81 bats (56.3%) revealed moderate to severe pathological changes of unknown etiology or unconfirmed bacterial or viral cause ( table 2 ). based on the glmm analysis, significant age-and sexdependent differences (daic = 23.13) were detected between the general causes of mortality, disease and trauma ( table 4 ). the disease presence in bat samples decreased continuously with increasing age. neonates and juveniles of both sexes were significantly more affected by disease than older age classes (table 4 ; fig. 2a) . we also found a significant trend in diseaseassociated mortality between the sexes, with adult females displaying higher disease prevalence (52.5%) than males (36.4%) ( table 4 ). no significant association was observed between a certain cause of mortality (i.e. disease or trauma) and severity of endoparasitic infection (daic = 0.75, result not shown). the seasonal distribution of disease-related mortality cases (fig. 2b ) described a trimodal pattern, with peaks in spring (april), summer (june to august) and winter (december). the proportion of traumatized individuals also increased obviously during the summer months up to and including the swarming period, but was low during the rest of the year. about 90% (n = 430) of bat samples were examined bacteriologically. among these, 42 different bacterial genera with more than 53 bacterial species were identified (table s1 ). predominant bacteria isolated were enterococcus faecalis (14.7%, n = 63), hafnia alvei (11.2%, n = 48), serratia liquefaciens (10%, n = 43), and pasteurella multocida (7.7%, n = 29). in 37% (n = 157) of bats no bacterial growth was observed at all. comparative bacteriologic and histo-pathologic analysis identified 22 different bacterial species that were clearly associated with pathological lesions and/or systemic infection, found in 17% (n = 73) of bats investigated bacteriologically ( table 5) . members of the families pasteurellaceae (above all p. multocida) (41.1%, n = 30), enterobacteriaceae (various bacterial species) (28.8%, n = 21), and streptococcaceae (above all enterococcus spp.) (21.9%, n = 16) were predominant bacteria associated with disease. more than half (54.8%, n = 40) of bacterial infections were observed in bats with traumatic injuries. the glmm analysis revealed low sex-and age-dependent differences in bacterial infection (daic = 1.97, result not shown). female bats (21.9%) and adults (21.6%) showed marginally higher prevalence of bacterial disease compared to males (18.3%) and to other age classes (15.6%), respectively. however, we found a strong influence of cat predation (daic = 16) associated with bacterial infection in bats (table 4 ). testing for human-pathogenic zoonotic viruses, no examined bat sample (0/210) was positive for influenza a virus, corona-, hanta-and flaviviruses, respectively. no inhibition of the pcr assays was notified. out of 486 bats tested for rabies virus infection, 2 serotine bats (e. serotinus) were positive for lyssavirus by fat and rtcit. the viruses were identified as european bat lyssavirus type 1 (eblv-1) sublineage a, both using mabs and sequencing. applying bat herpesvirus-specific pcr assays, 63 out of 210 bats proved to be infected with 7 of the previously described 8 bat herpesviruses ( table 6 ). the highest prevalence of 65% (24/37) was observed for bat gammaherpesvirus 6 (batghv6) in common pipistrelle bats (p. pipistrellus), followed by bat gammaherpesvirus 5 (batghv5, 42.1%) in nathusius' pipistrelle bats (p. nathusii) and bat gammaherpesvirus 4 (batghv4, 33.8%) in noctule bats (n. noctula). co-infection with different bat herpesviruses were recognized in 4 n. noctula (7.4%) infected with batghv3 and batghv4, and in one n. noctula (1.5%) infected with batghv4 and batghv5. batghv5 was not only detected in its initially specific host p. nathusii, but also in 3 other bat species, i.e. n. noctula, myotis myotis and m. mystacinus. although the prevalence of batghv3 (13.0%) and batghv4 (33.8%) differed significantly within its migrating host n. noctula, no difference was observed between the sexes. two juvenile n. noctula were found to be infected with batghv4. interestingly, for the sedentary bat species p. pipistrellus being infected with batghv6, a considerably higher prevalence was observed in 22 juvenile bats (72.7%) resulting in an overall prevalence of 65% also without difference between adult male and female bats. ectoparasites (mites, fleas, and ticks) were noted in 14% (n = 62) of bats, but a potential bias in ectoparasite numbers collected from dead animals in comparison to ectoparasite abundance on live animals has to be taken in account. female bats (17.1%) were slightly more infested by ectoparasites than males (14.7%), whereas in different age classes ectoparasite prevalence was almost balanced. the glm analysis revealed significant species-specific differences in ectoparasite infestation (daic = 14.58, table 4 ). most bat species revealed low ectoparasite prevalence (range 5.3-11.8%), while almost 43% (n = 20) of n. noctula were infested with mites and/or fleas (fig. 3a) . microscopic examination of organ tissues revealed endoparasitic infection in 29% (n = 124) of investigated bats, involving different protozoan (families eimeriidae and sarcocystidae) and helminth parasites (trematodes, cestodes, and nematodes). helminthes were predominantly found in the gastro-intestinal tract of the bats, while in some animals, granulomatous organ lesions were associated with larval migration of nematode species. based on the glm analysis, clear age-and species-specific differences (daic = 24.95) were observed between infected and non-infected bats ( table 4 ). the prevalence of endoparasitic infection in bat samples increased significantly with increasing age, whereas the increase in prevalence was more rapid between juveniles and subadults (8.5%) compared to the older age classes (4.5%). marginal differences were also observed between the sexes, with female bats showing slightly higher (30.4%) endoparasite prevalence than males (24.4%). regarding species-specific differences, large bats like n. noctula, e. serotinus and v. murinus revealed higher endoparasite prevalence compared to individuals of medium-sized or small vespertilionid species (table 4 ; fig. 3b ). this study was based on a passive surveillance sampling strategy that inherently influences the composition of bats sampled for diagnostic investigations [27] and might also effect the data presented on causes of death by ecological and anthropogenic impacts of urban landscapes [41] . trauma and disease represented the most important causes of mortality in deceased bats from germany, which differ from results of previous studies [13] [14] [15] where disease-related mortality often played a subordinate role. young bats and adult females were significantly more affected by disease, indicating that sex-and age-related disease prevalence in table 3 . pathological findings and bacterial, viral and parasitic infections specified for the general causes of mortality, trauma and disease. bats are strongly correlated with the maternal season. this assumption is further supported by the distinct increase of diseaserelated mortality from june to august, which corresponds to the maternity period of central european bat species. similar seasonal prevalence patterns in bats have also been described for parasitic (e.g. [42] [43] [44] [45] ) and viral infections (e.g. [19, 46, 47] ). in contrast, the increase of trauma-associated mortality cases from july to october resembles 4 major behavioral activity patterns of european bat species (i.e. weaning, mating, pre-hibernal fat storage, and migration) [48] and could therefore predispose bats to trauma. however, both seasonal peaks also coincide with time and locations where sick, injured or dead bats are more likely to be discovered as well as with the seasonal roosting behavior of bats adapted on urban habitats [27] . the additional seasonal peaks of disease-associated mortality corresponded to the post-hibernal and the early hibernal period of temperate zone bats. currently, there is a lack of knowledge of bat immunology. it is known for other mammalian species that hibernation reduces the innate and adaptive immune response; likewise an increasing risk of infection could be assumed for hibernating bats [49] . with the start of the hibernation season, large aggregations of bats originating from various colonies might enhance the risk of spreading infectious agents similar to maternity colonies. equally, the post-hibernal increase of disease-related mortality is suggestive for reduced immunity in association with prolonged fasting during hibernation. bacterial diseases have rarely been documented in bats. pasteurella spp., here identified in 7% of bats, were the predominant bacterial pathogens reported in european bats and infection appears to be strongly correlated with cat predation [13, 14, 23, 26] . in our study, bacterial infections were confirmed in 17% of bats investigated bacteriologically. most of these bacterial isolates represented opportunistic pathogens that usually do not harm the host unless the immune system is weakened [50] or preceding injury to natural host barriers (e.g. skin abrasion). primary bacterial pathogens like samonella enterica serovar typhimurium, s. enteritidis and yersinia pseudotuberculosis [22] were identified in almost 12% of affected bats. some of the bacterial species (e.g. burkholderia sp., cedecea davisae and clostridium sordellii) are newly described in bats. nevertheless, bacteriologic analyses can markedly be influenced by post-mortem bacterial invaders, freezing and storage of bat carcasses and the inability to detect certain bacteria by routine culture methods, resulting in some bacterial species that might have escaped detection. we found a strong association between cat predation and bacterial infection in bats as almost one half of bats (44%) caught by cats were affected by bacterial disease. various bacteria can be transmitted via cat bites [51] ; hence bats attacked by cats are likely to succumb to bacterial infection even if non-fatal injuries were present. this relation has been proven for p. multocida infections in european bat species [13, 14, 23, 26] . on the other hand, bats already debilitated by disease might easier fall prey to predators like cats. consequently, bats may also act as vectors for zoonotic pathogens, as domestic cats could pass these infectious agents on to humans. such cross-species transmission events from bats to domestic animals are well documented [9, 52] . for all tested human-pathogenic zoonotic viruses no infected bat could be detected in this study except lyssaviruses. bat rabies is the only bat transmitted zoonosis in europe that is known to have resulted in human cases [53] . unlike in other mammals table 4 . result of the final model variables corresponding to 4 different analyses: (a) disease-vs. trauma-related mortality, and presence-absence of (b) bacterial, (c) ecto-and (d) endoparasitic infection. where lyssaviruses ultimately cause lethal rabies, in bats nonlethal lyssavirus infections may also lead to the development of immunity [47] . with the detection of eblv-1 we confirm that this lyssavirus circulates among e. serotinus as previous studies showed [18] . in germany, bat rabies is also caused by eblv-2 and bokeloh bat lyssavirus (bblv) [54, 55] , but while the latter was recently isolated from m. nattereri, eblv-2 is associated with m. daubentonii and m. dasycneme [56] . the apparent absence of eblv-2 and bblv in the sampled bats is likely due to the fact that lyssavirus infections have a very low incidence in bat populations [18] and that the sample size was too limited, especially concerning the relevant species. there is a high prevalence for herpesviruses in different insectivorous bat species in germany (this study, [24] ). most of the previously described bat herpesviruses have been detected in low numbers in more than one bat species [24] . here, we observed a high species-specific prevalence among herpesvirusinfected bats, indicating that a certain type of european bat herpesvirus is primarily associated with a single bat species. this is supported by batghv6 and batghv7 that were again only identified in their initial hosts p. pipistrellus and p. auritus (both sedentary), respectively, underlining the typical strong speciesspecificity of mammalian herpesviruses. however, species' range overlap and close inter-species contacts in bat roosts may result in cross-species transmission and could explain the observed overcoming of the species barrier (this study batghv5, [24] ). interspecies transmission have also been discussed for other mammalian herpesviruses, i.e. bovine and equine herpesviruses (e.g. [57, 58] ). furthermore, for rna viruses (i.e. rabies virus) phylogenetic distance between different host species and overlap in geographic range have recently been demonstrated as strong predictors of host shifts and cross-species transmission in bats [59] . some of the bat species (i.e. n. noctula, p. pipistrellus, and p. nathusii) in this study appear to be more susceptible to herpesvirus infection. in n. noctula, 3 different gammaherpesviruses (batghv3, 4, 5) with significant prevalence differences were recognized. such type-specific differences in prevalence between the phylogenetically distant viruses batghv3 (13.0%) and batghv4 (33.8%) within one bat species indicates co-evolutionary virus-regulated mechanisms. parasite infestation in wildlife often occurs without clinical effects, but severe infection can reduce host fitness either in terms of survival or reproductive success [60] . most data on infection dynamics in bats came from parasite studies focusing on individual and seasonal variations in ectoparasite prevalence (e.g. [43] [44] [45] 61] ). host density, roost preference and movement pattern seem to be important factors explaining individual and speciesspecific parasite infestation rates in bats [43] [44] [45] . in european vespertilionid species, female-biased parasite loads are most likely associated with host physiology and differences in roosting behavior [42, 44] . we also found species-specific seasonal variations in ectoparasitic infestation, with n. noctula and m. daubentonii showing higher ectoparasite prevalence in spring and autumn compared to the breeding season (data not shown), which is in accordance with zahn and rupp [43] . additional findings of our parasite analyses are distinct variations in ecto-and endoparasite prevalence in relation to bat species. bats primarily roosting in trees or nest boxes were more frequently infested with ectoparasites like n. noctula (43%) and m. daubentonii (25%) compared to other species (range 5-12%) investigated in this study. high ectoparasite loads have generally been described in bats preferring enclosed roosts like burrows and cavities [61, 62] , suggesting that structural characteristics and the microclimate of roosting habitats influence ectoparasite survival and re-infection of bat hosts. this assumption is in accordance with results of pearce and o'shea [63] who found differences in ectoparasite prevalence and intensity in eptesicus fuscus in relation to environmental factors (i.e. temperature and humidity) of different roost sites. in contrast to these results, the endoparasite prevalence in european vespertilionid bats seems to be correlated with the body size of the bat species [26] . species-specific variations in diet and prey selection could possibly effect endoparasite prevalence in insectivorous bats [64] , as larger bats feed on insects of a wider size range including hard-bodied prey [65, 66] . this assumption is supported by the clear prevalence increase in subadult and adult bats compared to low endoparasite infection rates in juveniles primarily feeding on milk. a multitude of publications is restricted to pathogen presence or absence in different chiropteran species; here we demonstrate the impact of diseases and infectious agents on bats themselves. alongside to trauma-associated mortality and undefined mortality cases, disease aspects represented one third of mortality causes in 486 investigated bats of 19 european vespertilionid species. by comparing pathology and bacteriology results, we were able to detect 22 different bacterial species that were clearly associated with disease in bats. at least 12% of all bats had died due to bacterial, viral and parasitic infections. finally, we found clear seasonal and individual variations in disease prevalence and infection rates, indicating an increased susceptibility to infectious agents in female bats and juveniles during the maternity season. our data emphasize and provide the basis for disease related studies in bat species on population level to elucidate the complexity of the ecology of infectious agents and host species likewise. table s1 bacteria isolated from bats found in germany. 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bacterial pathogenesis bacteriologic analysis of infected dog and cat bites european bat lyssavirus transmission among cats human rabies due to lyssavirus infection of bat origin first isolation of eblv-2 in germany novel lyssavirus in natterer's bat bat rabies serological survey of herpesvirus infections in wild ruminants of france and belgium new hosts for equine herpesvirus 9 host phylogeny constrains cross-species emergence of rabies virus in bats behavioral adaptations to parasites: an ethological approach relationships between roost preferences, ectoparasite density, and grooming behaviour of neotropical bats roosting habits of bats affect their parasitism by bat flies (diptera: streblidae) ectoparasites in an urban population of big brown bats (eptesicus fuscus) in colorado when parasites become prey : ecological and epidemiological significance of eating parasites the implications of food hardness for diet in bats prey consumed by eight species of insectivorous bats from southern illinois the authors would like to thank berliner artenschutz team-bat-e.v., f. key: cord-310775-6d5vi2c5 authors: brinks, verena; ibert, oliver title: from corona virus to corona crisis: the value of an analytical and geographical understanding of crisis date: 2020-06-09 journal: tijdschr econ soc geogr doi: 10.1111/tesg.12428 sha: doc_id: 310775 cord_uid: 6d5vi2c5 the term ‘crisis’ is omnipresent. the current corona virus pandemic is perceived as the most recent example. however, the notion of crisis is increasingly deployed as a signifier of relevance, rather than as an analytical concept. moreover, human geography has so far little contributed to the interdisciplinary crisis research field which is fixated on the temporal aspects of crisis but neglects its spatiality. against this background, the first aim of the paper is to demonstrate the value of thinking about crisis analytically. therefore, we introduce theoretical knowledge developed within a recently emerging literature on crisis management. second, we demonstrate the relevance of including geographical thinking into crisis research more systematically. based on the tpsn‐framework by jessop et al., we illustrate spatial dimensions of the ‘corona crisis’, its perception and handling in germany. the empirical references are based on media reports. the spread of the coronavirus has turned into a crisis. this finding is hardly surprising and the majority of readers will agree. but when and how did it turn into a crisis? this question is much more difficult to answer, mainly due to the fact that the term 'crisis' is anything but easy to grasp. it is omnipresent and frequently used in very different contexts. the term is, for instance, used to signify the enhanced relevance of the respective research. as a consequence, the notion of crisis is mainly deployed intuitively, rather than analytically. similar to other disciplines, most geographical contributions related to crisis dynamics are driven by an empirical phenomenon, which is framed as being in crisis. in economic geography, the 'financial crisis' has received particular attention and geographers have made significant contributions by exploring the manifold spatial references of this global phenomenon (e.g. aalbers 2009; martin 2011) . within the geographic discipline, diagnoses of 'crisis' are often associated with neoliberalism and capitalism apparently producing manifold social, economic and political stresses (jones & ward 2002; larner 2011) . particularly, geographers in marxist tradition (most prominently represented by david harvey) deploy crises as an inherent and recurring feature of capitalism; or to cite harvey (2011, p. 11) : 'capital never solves its crisis tendencies' (emphasis in orig.). in addition, climate change, rising religious fundamentalism and newly emerging economic powers outside the traditional industrial centres pose challenges of truly global scope that invoke crises in all parts of the world (larner 2011) . this literature emphasises that we live in times of crises and doubtlessly provide important insights on the social, economic and political configurations that contribute to crisis diagnosis. however, so far little attention has been paid to specify the nature of 'crisis' itself as an exceptional and stressful human experience. if we accept the diagnosis that we live in times of crises, it becomes more important than ever to develop a more profound understanding of crisis as a particular context for action and a possibility for intervention. in this paper, we therefore propose a conceptual shift from the structural conditions that cause crises to an actor-centric approach focused on the practical consequences of crisis for individual and collective agency. we introduce theoretical knowledge developed within a recently emerging, interdisciplinary literature on crisis management. moreover, we illustrate this analytical understanding with reference to the recent 'corona crisis' to connect abstract ideas on the general characteristics of crises with empirical observations. the term crisis is ripe with temporal implications. these temporal aspects predominate in the crisis management literature. what lacks so far, however, is a systematic conceptualisation of the spatial aspects of crisis. while the crisis management literature does use spatial categories, such as epicentres, distance, scaling or territories, it lacks a systematic approach to integrate spatial imaginations into theories and practices of crisis management. social and economic geographers could thus contribute to the inter-disciplinary discourse by integrating the spatial dimension into the conceptualisation of crisis. in this paper, we set out to suggest a conceptualisation of the 'geography of crisis' informed by social and economic geography. the empirical material presented in this paper stems from different media sources. it has to be mentioned that this paper does not draw on an already fully-elaborated or finalised media analysis but is inevitably provisional and selective due to the highly dynamic development at the time this paper has been written. the analytical and conceptual thoughts presented here are based on current research on crises (brinks & ibert 2020) . within different research contexts, we analysed literature on crisis (management) and also benefited from empirical insights collected in interviews with crisis experts and own participation in interdisciplinary workshops on crisis and crisis management. the paper is subdivided into two main parts. first, the subsequent chapter introduces our definition of crisis based on literature from the interdisciplinary practice of crisis management and social scientific crisis research. second, we outline a geographical perspective on crisis, by exploring different dimensions of the spatiality of crisis. we use the tpsn approach as suggested by jessop et al. (2008) to systematise our observations. the paper concludes by highlighting the added value of a geographical approach. a crisis is related to, yet distinct from other terms, such as 'problem'. a problem denotes a gap between an observed condition and a desired condition (rittel & webber 1973) . such a gap is present in every crisis as well, for example, the gap between the fastgrowing numbers of people who became infected with the corona virus and the general desire that the population should be healthy. yet, such a gap is not a sufficient condition for a crisis. in order to talk of a crisis, a few more ingredients are necessary: uncertainty, urgency and threat (boin & 't hart 2007) . uncertainty denotes 'that we cannot predict or foresee what will happen when acting or not acting' (aspers 2018, p. 133) . in the corona case, uncertainty is caused by a lack of knowledge (e.g. about the ways of infections, dark figures of a-symptomatic cases), ambiguous signals (e.g. unspecific symptoms), a lack of viable means to counter the epidemic (e.g. the absence of an effective medicine and vaccination) and undetermined timeframes (e.g. when will a vaccination be available), to mention only few. the second ingredient, urgency, refers to the necessity to act, despite high degrees of uncertainty. in crisis, inactivity and non-decision are no options as they will only exacerbate the serious situation. yet, as acting has to take place under conditions of uncertainty, routines are no longer available and action has a strongly improvisational or experimental character (boin & rhinard 2008; milstein 2015) . the last ingredient to crisis is an existential threat of highly valued societal assets. the corona pandemic does not only threaten the health and lives of wide parts of the population, but also imperils economic interests and core institutions of the political order. due to the underlying fundamental uncertainty, 'the emotional response to crisis is not fear (such as fear from fire) but existential angst, which has no identifiable object that could offer a grip for a learnt response' (kornberger et al. 2020, p. 242) . in addition to these fundamental characteristics of crisis it is important to unpack the term a bit further. it is important to understand how crisis becomes enacted in practice. a crisis as an empirical observation cannot be deduced directly from the underlying societal conditions. rather similar, objectively measurable conditions (like unemployment rates, levels of distrust in political institutions) sometimes entail crisis diagnosis, and sometimes do not. sometimes relatively unimportant issues are treated as a crisis (the 'brent spar' controversy is a widely cited example of an escalating risk communication; löfstedt & renn 1997) , while even the most alarming scientific reports about climate change are not sufficient to mobilise a collective sense of urgency. what all crises share in common, thus, is not only a severe problem, but a shared perception of uncertainty, threat and urgency around that problem. the key importance of perceptions can also be found in the corona case. in our observation of the public discourse in germany, at the beginning of 2020 the government as many others in the western hemisphere looked at the early epicentre of the pandemic, the wuhan region in china, 'with a combination of fascination and fear' but without any sense of urgency or immediate threat until new information about corona infections in europe emerged (boin et al. 2020) . not earlier than 26 february 2020, we noticed a shift from 'corona epidemic' to 'corona crisis' in the german speaking debate for the first time in an article published in the online portal of the german newspaper der spiegel. two days earlier, in many parts of germany, carnival was celebrated on the streets -a mass meeting with thousands of people standing close to each other. from 16 march onwards, all public events of major size were prohibited, schools were closed across all federal states in germany and a few days later restaurants, production plants and retail shops followed. it was a matter of days, during which the publicly shared framing of the situation has changed fundamentally. typically, at some stage in the public 'framing contest' that takes place in advance of a crisis, the public opinion transcends an invisible 'tipping point' beyond which a problematic situation turns into a crisis (boin et al. 2009 ). however, this tipping point can only be noticed ex post, while it is impossible to determine it in advance. for most participants, thus, crisis comes unexpected. crisis is not only a matter of perception; it also unfolds performative qualities. here, performative means that the crisis diagnosis is not a mere description of the state of reality rather, a crisis diagnosis changes reality and therefore contributes to the enactment of crisis: 'if individuals (and the media) define a situation as a crisis, it is a crisis in its consequences' (rosenthal & kouzmin 1997, p. 286) . for decision-makers, once in place, the crisis immediately ascends the first place of the agenda. due to the performative qualities, the crisis unfolds its dynamics irrespective of subjective interpretations or experiences. for individual decision-makers, it is for instance no longer possible to ignore the crisis, or, if one tries, like donald trump did until the first weeks of march, it happens at immense political and economic costs. for professional crisis managers, the declaration of a crisis has very practical and robust consequences. they perceive a crisis as an effective 'coping structure' (term used in crisis management practice jargon) societies and organisations have to prioritise a certain topic and to mobilise resources to address a problem. crisis diagnoses emerge in multi-stakeholder constellations. some stakeholders even support the escalation of a crisis or reframe the crisis diagnosis in ways that exert pressure on organisations or states. crisis diagnoses thus are contested and the framing is subject to controversy in the public debate (boin et al. 2009 ). in contrast to the term 'catastrophe', the term crisis highlights that despite existential threats, it is not yet too late to prevent the disaster (boin & 't hart 2007) . in medicine, crisis marks the decisive phase in the course of an illness in which a positive or negative outcome is still possible (ricoeur 1988) . crisis, in other words, is strongly associated with the idea of an open future (kornberger et al. 2020 ) that can be created through individual or collective agency. in the current crisis, the first discussions emerge about the potential long-term structural effects of the corona crisis. for instance, visionaries from silicon valley highlight the enhanced possibilities to establish new practices of remote digital learning and work (thrun 2020) . at the same time, warning voices (sennett 2020 ) point at potentially problematic long-term effects of lockdown policies and the increased use of surveillance technologies on the human rights situation and vulnerable democratic institutions in weak democracies. when we think of or undertake research on crisis, we should also be aware of one additional observation. as mentioned above, decisions in crisis have to be made while the present is uncertain and the future is open (kornberger et al. 2020) . under such conditions, action does not take place within a given frame of meaning. rather, in crisis participants are forced to learn by interpreting the situation tentatively while acting on it. therefore, crises are usually perceived twice. in a first loop, participants encounter a critical turn in the course of events surprisingly. they experience an open-ended phase of chaos and escalation during which they struggle to regain control while action and sensemaking remain incompletely connected. in contrast to the abrupt beginning, the end of the acute crisis comes much more gradually. as a first step toward a (new) normality, after having responded to the challenges, participants eventually perceive a slowing down in the dynamic of escalation and try out new interpretations of the situation. however, against the background of the previously experienced uncertainty, participants tend to distrust this new stability. they remain unsure, how far their explanations will hold and whether or not the absence of another surprising turn is just a pause in the course of escalation or already a (re)turn to (new) normality. the ultimate end of the crisis, however, has to be 'declared' by decision-makers, which is another performative act. in a second loop, the course of events that led to the acute crisis is reconstructed ex post in the light of the newly established sense and certainty. as an interpretative act of sensemaking, the starting point and the end of the second loop are not fixed and can never be defined in advance. according to weick (1988, p. 306) , sensemaking is enacted since 'parts of what the explorer discovers retrospectively are consequences of his own making'. by the very process of acting in crisis, a rising stream of new information and experiences have to be included in the sensemaking process. thus, the second loop always starts after the first loop but usually at an early stage in the crisis course. in this second loop, the crisis is deliberately embedded in the classical phase model encompassing the phases of pre-crisis, acute crisis and post-crisis (e.g. fink 2002) , while the boundaries between the phases are still in motion. during sensemaking the considered timeframe is expanded both, into the past and the future. when reflecting on the pre-crisis phase, the focus is on weak warning signals that have been neglected beforehand or wrong decisions that contributed to an escalation of events. the post-crisis phase, in contrast, provides the (oftentimes missed) opportunity to learn from the crisis (birkland et al. 2009 ). once the crisis is overcome, time is ripe to reiterate the acute crisis several times in order to get a detailed understanding of the sequential order of actions. of course, the acute crisis itself cannot be repeated. yet, the pre-and post-crisis phases cannot emerge without the experiences made during the acute crisis. the awareness of these two loops of crisis experience is helpful to keep in mind for the corona crisis. while writing this paper, we are still witnessing the escalation of events and the tentative form of sensemaking while acting on the situation. yet, we can already discover first signs of time expansion. presently the public debate has already turned towards the past by discovering early warning signs that have previously been ignored. for example, a paper published in march 2019, in which the authors warned against (at that time) future outbreaks of a corona virus caused by cross-species transmission (fan et al. 2019) has received broader attention in the last weeks, that is one year after publication. in china and in italy suspicious accumulations of pneumonia cases attract the attention of epidemiologists aiming at reconstructing the outbreak. at about the same time, governments around the world start to plan for the future. they design graduated schemes back to normality, envisioning the possible ends of the crisis and speculating about new post-corona normalities. finally, the severity of a crisis is widely associated with its perceived scope. the scope of the crisis describes the degree to which the perceived escalation of problematic events can be contained within separable units of society. critical events are much more likely perceived as severe crises, the more they 'spill over' (bundy et al. 2017 ) existing boundaries. on the territorial level, 'transboundary crises' (boin & rhinard 2008) have an inter-regional or even an inter-national character. transboundary also denotes the overstepping of institutionalised boundaries, for example, the vertical sectoral responsibilities of political or administrative bodies (boin & rhinard 2008) . a true sense of crisis tends to emerge if multiple boundaries are overstepped and causes and effects of a crisis spill over from one compartment into the other. the scope of a crisis is also dependent on the sources of uncertainty. sometimes, these sources are clearly external, for instance, an earthquake or a cyber-attack. such external events can unfold disruptive qualities, yet they are usually easier to manage, as no decision-maker can be directly blamed for them. it thus seems sufficient to manage their negative consequences before returning back to old normality. more difficult are crises that are driven by internal sources. for instance, the structural crisis of a whole industry to a wide degree is caused by the insufficient strategic capabilities of the core decision-makers. here, the crisis is interpreted as a 'brutal audit' (orton & o'grady 2016 ) that unveils the lack of foresight and understanding of decision-makers. crises caused by internal factors enact a much higher degree of uncertainty, as any framing of the problem goes hand in hand with blaming of responsible persons or organisational units (boin et al. 2009 ). of course, in practice, it is difficult to clearly separate internal from external sources of uncertainty, as often critical external events raise the awareness of internal deficiencies. in the case of the corona pandemic, the crisis fulfils the character of a 'transboundary crisis' (boin & rhinard 2008) in an almost ideal-typical sense. the spread of the virus is no longer restricted to any geographically confined territory, vertical segments of society or particular societal layers. rather, within a few months, the virus is present almost everywhere on the globe, justifying the whoclassification as a 'pandemic'. further, it affects several societal systems, most crucially the health services, but beyond that also has severe spill-over effects to almost every economic sector, a wide range of institutions of political order and all parts of society. the tendency to transgress boundaries also makes the corona crisis particularly threatening. while the origin of the crisis is external to society, the corona pandemic can be seen as a brutal stress test that unveils internal dysfunctionalities in national health systems, social security programmes or value chains. even though the geographical dimensions of crisis are recognised by some crisis scholars, a systematic and theoretically-guided analysis of the spatiality of crisis has not yet been advanced in this field. such a systematic exploration, we argue, is a possible contribution of economic and social geography to social scientific crisis research. the agenda we suggest here is thus a bit different from previous geographical studies that use the term crisis prominently to signify they are dealing with severe problems within specific empirical fields, like, for instance, the bursting of financial bubbles in mortgage and real estate markets (e.g. aalbers 2009) or emergency practices in humanitarian aid (e.g. fredriksen 2014 ). we suggest the use of the tpsn framework (territory, place, scale, network), as developed by jessop et al. (2008) to explore the geography of crisis. according to gailing et al. (2019, p. 15 ) it provides a useful heuristic that can be flexibly applied to diverse empirical fields 'to allow for a synoptic perspective on this field'. at the same time, the authors also warn that tpsn should not be mistaken as a 'complete answer to everything' (gailing et al. 2019, p. 15) , as it lacks the necessary, field-specific theoretical terminology. hence, they argue that tpsn needs to be complemented with the respective theoretical terminology to unfold its full explanatory potential. for our agenda, the absence of theoretical assumptions in the tpsn-heuristic is an advantage. crisis, as we understand it, is not an 'empirical field' in the sense of gailing et al. (2019) , but rather a conceptual endeavour to advance a general understanding of practices and dynamics prevailing in situations of uncertainty, threat and urgency. in the following paragraph, we thus use theoretical claims from social scientific crisis research and combine it with spatial dimensions as suggested in the tpsn heuristic in order to delve deeper into the so far underdeveloped spatial aspects of crisis theory. in the following, some starting points for such an investigation are indicated by referring to the corona crisis as one illustrative empirical field (table 1) . even though crises increasingly cross territorial boundaries, the territorial dimension remains particularly important. the corona crisis produces countless cartographic visualisations documenting the spread of the pandemic. the number of infections announced by the johns hopkins university (2020) has become an internationally much-cited data source for tracing the dynamic development of the spread as well as regional differences worldwide. the total number of confirmed cases worldwide is presented on the national level. recently, a further map demonstrating the intensity of the outbreak in us counties has been launched by the university (johns hopkins university 2020). the territorial representation of the corona crisis is largely caused by the report system of public agencies which are bound to territorial units. likewise, many institutional crisis responses, such as the official declaration of an emergency situation, are bound to territories. however, territory affects crisis even beyond administrative responsibilities. the crossing of a territorial boundary, for instance, frequently cause shifts in the perception of crises as being more threatening (since the perceived distance to crisis declines) and escalating (fear of losing control). 'patient 1' as the first documented case in a certain territory is well reported as well as the first case of covid-19 outside of china on 13 january. manifold media reports refer to 'first cases' or 'first deaths' inside or outside a specific territorial unit. some places are more affected by crisis than others (see aalbers 2009 for the financial crisis). some crises culminate in a single epicentre. a school shooting creates such a mono-centric geography and 'place renewal' can be an adequate way for crisis recovery (wombacher et al. 2018 ). more typically, however, crises unfold complex, multi-local geographies. in the case of the corona pandemic, we can already identify several symbolically charged places. above all, the huanan seafood market in wuhan has been reported as the point of origin of the outbreak. related to that, the use of the term 'wuhan virus' by the us government can be conceived as a framing and blaming strategy jessop et al. 2008) . portrayal of outbreak according to territorial entities activation of territoriallybound resources 'first case' inside or outside a territory place emergence of places of crisis such as supermarkets 'epicentre' and 'superspreader' locations scale assignment of responsibility inter-national organisations such as the who network expert communities '#flattenthecurve' (boin et al. 2009 ) through spatial dissociation and association (ibert et al. 2019) . further places, such as the notorious après ski bars in ischgl in austria, or the football stadium in milan have become spots of investigation as potential 'super spreader' locations from where the virus disseminated across europe (merlot 2020) . surprisingly, supermarkets have emerged as relevant places of the corona crisis. as places of food provision, in times of lockdown these facilities have transformed rapidly into critical infrastructures equipped with additional safety precautions. in contrast, hospitals represent classical institutions of crisis response. yet, when becoming activated for this crisis, their regular safety standards needed to be adapted to the particular challenges of the corona pandemic. the notion of scale is closely related to spatial hierarchies (jessop et al. 2008) . it is a particularly important dimension in crises when it comes to negotiation of responsibility and coordination of action (which scale is the right one to (re)act on crises?). the corona crisis provides a vivid example here. in germany, for instance, the corona crisis induced a discussion of the federal constitution. where in other states, the national governments decided about the closing of retail stores, etc., the national government in germany is not authorised to decide about such measures since infection protection is situated at the federal state-level (bundesländer) (leitlein & schuler 2020) . moreover, the health authorities, which report about confirmed corona infections and are authorised to impose measures such as quarantine, are based on the level of administrative districts (landkreis) or district-free cities in germany. located at an inter-national scale, the who receives particular attention in these days. though not authorised to impose measures, the who has an important function in terms of policy recommendation. the who's declaration of the covid-19 outbreak being a 'pandemic' on 11 march can be interpreted as a means justifying considerable state interference with fundamental rights. the network perspective on crisis focuses on the relations between nodes (of every kind). it can be enriched by deploying the concept of 'relational proximity' (gertler 2008 ) and the function of medical experts in the corona crisis. medical professionals such as epidemiologists and virologists currently receive particular attention as policy advisors. they are embedded in trans-local professional communities. they share knowledge about the corona virus internationally, for instance, through rapid publication practices in academic journals (see for instance the lancet). members of these professional communities are characterised by relational proximity, which means that based on a shared repertoire of practices and similar expertise they are able to collaborate closely even across physical distance. another example of the network dimension are social media having an enormous relevance in the corona crisis in terms of establishing a common understanding of the situation and sharing (similar) experiences across distance. calls such as 'flatten the curve' or 'stay at home' went viral online and contributed to a shared perception of the corona crisis even when the locations and individual concerns with the corona virus are different. as jessop et al. (2008) argue, the empirical reality cannot be separated into the categories territory, place, scale and network. rather, the dimensions are interwoven in 'sociospatial relations'. similarly, gailing et al. (2019) find typical nexuses between several dimensions when studying empirical cases from the german energiewende. the following sub-sections aim at providing some examples of such interactions between spatial dimensions in the corona crisis -importantly, without any claim of completeness and admittedly presented in a rather sketchy and unsystematic fashion. at the present state, it would be an impossible endeavour to outline all spatial relations, too dynamic is the escalation in the course of events. therefore, we focus on three nexuses that can be detected in prominent public discourses to demonstrate the principle of our approach. network-place: topologies of interconnected places -as mentioned earlier, supermarkets have turned into strategic places in the fight against the pandemic across the globe. during the past few weeks, we witness a gradual reshaping of their physical setup and practices of staff to accommodate these places to the new requirements of 'social distancing' while maintaining a high turn-over of people. items from hospital environments, like surgical masks and gloves have been transferred to supermarkets in order to protect staff and clients. planes of acrylic glass have been fixed at checkout counters to minimise the physical contact between cashiers and customers and tapes attached on the floor remind shoppers to hold minimum distance. at the same time, familiar items, such as customer divider bars, loyalty cards or cash money have been banned from some supermarkets as they are now reinterpreted as potential carriers of the virus. however, it would be inaccurate to primarily conceive supermarkets as singular places. most supermarkets are not single-owned stores but rather branch stores belonging to chains of multi-national retail chains. of course, supermarkets are places, though places that belong to wider networks operated and orchestrated by grand retailers. supermarkets, in other words, are part of networks of practices (brown & duguid 2001) . the concrete local practices and settings are thus not idiosyncratic, but depend strongly on the affiliation to a certain retail chain. moreover, these practices might vary slightly from chain to chain while they are made similar from place to place through standards orchestrated through the respective networks. a similar topological perspective on crisis has been elaborated by fredriksen (2014) . the author focuses on emergency infrastructure which is used in different humanitarian crises. according to the author, emergency tents as material objects, which have constantly been developed further after crisis experiences, represent 'lessons learned' from different crises. moreover, since they are highly mobile and used at different sites affected by crisis, the places resemble one another and thus become nodes in a 'network topology' of crises (fredriksen 2014) . in a longer timeframe, experiences gained in supermarkets will most probably turn out to be extremely important for all kinds of retail stores. as soon as legislation will step by step relax the regulations on social distancing, the network of practice will most likely expand from the realm of supermarket(s) (chains) to other retailers, for instance in fashion retail or book stores. negotiation of crisis governance -the connection of scale and territory is obvious in crisis settings since public crisis response strategies are usually immediately connected to territorial units. scaling in the sense of deciding which level is the most effective one for coping with crises is a key question in crisis management (e.g. boin et al. 2005) . the different levels usually present territorial units where the smallest level is always fully integrated in the next larger level (municipal level, national scale, european scale, etc.). this leads to the key issue of coordination in crises. a certain threatening situation has to be assigned to a specific scale, responsible for crisis response. these responsibilities are usually determined beforehand. in the corona crisis, the formal assignment of authority in epidemic events (as mentioned, the federal states (bundesländer) are responsible instead of the national government in germany) is now critically eyed and political efforts have been started to change the respective law in order to allow the upscaling of competencies to the national level in such crises (waschinsky 2020). at the same time, local hubs of the outbreak are intensively investigated such as the district of heinsberg in the federal state of north rhine-westphalia in germany. authorities aim at deriving strategies for larger territorial areas, arguing that 'the district of heinsberg portrays the nationwide occurrence of infections in a nutshell' (ärzteblatt 2020) . however, in the corona crisis some of the limitations of thinking of crisis in territorial units and instruments of territorial scaling also come to light. even though the corona virus crosses geographical and territorial boundaries, the virus does not spread homogeneously in space. as in many other countries, the shutdown of public life in germany is a nationwide strategy (with variances across different federal states). this also means that more and less affected areas are treated the same way. when discussing potential strategies for the time after the shutdown these questions of territorial on a more general level, the corona crisis demonstrates the interplay of territory and scale by pointing to strategies, limitations and challenges of upscaling and downscaling processes (see also boin et al. 2005, on upscaling) . also fundamental differences occur between centralistic and federal states. determining the right scale, activation of respective structures when necessary and flexible adjustments in territorial scaling are central issues of crisis management. yet, as observed in other cases, the transgressive forces driving the corona crisis requires complex settings of multi-level governance that includes several scales and political sectors (bundy et al. 2017) . another interesting question is whether or not the scale of the crisis and the scale of crisis response always have to be congruent for most effective crisis management. territory-place-network: 'social distancing' policies -the spreading of the corona virus takes place from human to human being. without changes in the social behaviour, every infected person in average spreads the virus to 2-3 other people. therefore, most national authorities have enforced so-called 'social distancing' policies. the aim is to reduce the ratio of infection, in the ideal case below 1 (which means in the long run the epidemic will run out because then, statistically, each infected person infects less than one other person). a chain of infections can be interpreted as a network (kuebart & stabler, 2020) , with every infected person representing a node and every infection from person to person representing a tie. in the terminology of structural network analysis, decreasing infection rates lead to decreasing network connectivity. from a geographically informed perspective on proximity and distance, the term 'social distancing' is a bit misleading, as it suggests that social contacts should be avoided. in fact, rather on the contrary, social distancing encompasses a set of behavioural regulations that seek to allow social contacts, yet in a way that minimises physical proximity and thus promises to disrupt the chain of infections. 'social proximity' thus enables physical distancing since through grown and trusted relationships, familiar face-to-face interaction in physical co-presence can partly be substituted by online media and the like (boschma 2005) . social distancing policies do not only address interaction between people, they also include the spatial setting in which interaction occurs. the discourse on super-spreaders, for instance, focuses not only particular persons who spread the virus at disproportionally high rates, but almost always also includes particular types of places, where the infective encounters took place. hence, social distancing policies almost always are place sensitive and frequently entail the closure of the respective venues (night clubs, pubs, sports stadiums, concert halls, even playgrounds). finally, social distancing policies are enforced on a territorial level, most typically by the national states. however, different territorial approaches co-existed. while today most countries pursue social distancing policies, not all did so or did not from the very beginning. for instance, sweden, the netherlands and the uk preferred another approach of isolating only the most vulnerable individuals while the rest of the population can face the risk of infection in order to reach 'herd immunity' sooner rather than later. other countries, especially in asia, concentrated on infected persons and followed the strategy of preemptive mass-testing to identify infections early on and of isolating infected persons from the rest of the population. territorial differences in terms of crisis response are also known from other crises. regarding the h1n1 pandemic 2009 (better known as swine flu), baekkeskov and öberg (2017) analysed different vaccination policies of denmark and sweden, each supported by the dominant national expert opinions. while sweden followed the approach of vaccinating large parts of its population, denmark decided to recommend vaccination for risk groups only. both policies were supported by the majority of expert opinions reported in the respective national mass media. their findings emphasise that territorial differences in policy strategies are reflected by public discourses on the crisis in the territories. even though the general direction of social distancing policies is similar everywhere, there is much variation in detail between territories. for instance, italy and spain sought to decrease the amount of social contacts by imposing a lockdown, hence people are no longer allowed to leave their private homes apart from buying food or for health services. in germany, in contrast, authorities declared a prohibition of social interaction. german citizens are thus still allowed to leave their homes, as long as they follow the commandments of keeping a minimum distance to other citizens of 1.5 metres and seeking only the company of members who live in the same household or at most one other person. as a federal state, however, germany resembles a fragmented patchwork of territories with slightly different rules and approaches (see above). another set of interesting territorial differences in social distancing policies occur in the attribution of surgical masks in public spaces. in japan, for instance, 'mask-wearing since the 2000s … became a civic duty of those who sneeze and cough not to be a source infection, while for the healthy general public, mask-wearing embodies neoliberal ethics of being self-caring and self-responsible to one's health' (horii 2014) . while japan is the internationally most well-known example, similar practices can be observed in other countries, especially in east asia, as well. in the european context, by contrast, the same practice has been widely dismissed by public opinion until very recently. here, the wearing of surgical equipment is seen as part of a professional practice that is little useful when used inappropriately by laypersons and outside of the professional setting. therefore, surgical masks played a major role in some national policies in the east-asian context while they have been ignored in most western contexts. however, the perception of masks has shifted quickly recently, as austria exemplifies, whose government decreed at the beginning of april 2020 the duty of wearing a mask when entering a supermarket. in germany, the national government recommended the use of masks in the public space in mid-april. one by one, the federal states governments did not only take up this recommendation but, similar to austria, even tighten the rule by declaring the obligation of wearing masks in retail stores or in public transport. in this paper we set out to suggest a conceptually grounded notion of crisis and to explore its geography. the present corona crisis served as an illustrative empirical background to substantiate the analytical spatial dimensions with concrete examples. the term crisis, we suggest with references to contributions from crisis management and organisation studies, encompasses the elements of uncertainty, urgency and threat. crises are related to societal problems, yet cannot directly be deduced from them. rather, a crisis becomes only a crisis, if the situation is collectively perceived and declared as a crisis. moreover, crisis has performative qualities. a crisis diagnosis thus is not primarily a proper description of reality, but a creator of a new reality in which uncertainty, urgency and threat predominate, no matter if decision-makers like it or not. right because of the performative nature of crisis diagnoses, the discursive framing of the crisis is a highly contested issue in public debates. it takes place in complex, multi-stakeholder settings and different interests and worldviews are mobilised. some stakeholders might even be driven by a strategic interest in further escalating the situation (löfstedt & renn 1997) . while crisis management has spent considerable effort to theorise on the temporal aspects of crisis, reference to its spatial aspects remained sparse. against this background, we suggest that human geography can contribute to inter-disciplinary research on crisis by unpacking the geographical aspects systematically. we used the tpsn heuristic as suggested by jessop et al. (2008) to delve into the different dimensions of the spatiality of crisis: we explored its territorial dimension, its scalarity, place-based accounts and the relational spaces of networks. furthermore, the corona crisis served as a vivid example to illustrate that the tpsn approach is not primarily valuable to disentangle empirical observations and rearrange them along separate dimensions. rather on the contrary, we used the examples of the recently observable restructuring of supermarket spaces, of flexible re-scaling of crisis response policies and of social distancing policies to demonstrate that it seems much more promising to scrutinise the multiple forms of interaction and overlap of several spatial dimensions in the same empirical observation. what is the particular contribution of a conceptually informed, geographical understanding of crisis? we see at least three distinct qualities of such an approach: first, an emerging topic related to the corona crisis is regionally specific response strategies. here a geographically informed understanding of crisis has much to contribute to the debate. it could support approaches that seek to adapt policies to different regional characteristics (e.g. social distancing policies for urban or rural regions) or to regionally unequally affected areas (e.g. hotspots of the crisis vs. little or no affected areas). second, and related to the first point, systematically thinking about the geography of crisis can contribute a lot to the question of scalarity in crisis. the corona crisis (as many other crises) demonstrates the challenge of defining the scale of the crisis and respective crisis response strategies (which scale is the right one to (re)act on crises? how to choose the right scale? does the chosen scale necessarily have to match with a territorial unit?). in fact, due to its transboundary character it evades any single scale and instead calls for complex strategies of multilevel governance adapted to the institutional idiosyncrasies of different nation states. third, the corona crisis forces the rapid implementation of several new practices such as avoiding hand contact in supermarkets. thus, specific places transformed into critical localities, rapidly equipped with special safety infrastructure. the transformation of specific places is observable in our daily lives; however, it cannot fully be understood without references to other similar places. geography established an analytical understanding of the relations between mobility of practices (supermarket a and supermarket b) and context dependency of practices, enabling a more profound understanding of currently emerging crisis topologies. the corona crisis will certainly occupy us for a long time. a variety of studies and research projects will surely start in the near future (some already started) in order to reflect on specific aspects of the crisis. our aim in this paper was to closer investigate the notion of crisis and how a crisis diagnosis changes the present, as well as the view of the past and the future. crises unfold in time and space. the exact geography of a crisis, of course, depends on the empirical case. however, just as thinking about the temporality of crisis, the spatiality of crisis is worth investigating. we made one proposal by drawing on the tpsn framework (jessop et al. 2008 ) but possible approaches are far from exhausted. we argue for a stronger engagement with 'crisis' within human geography since its spatiality is so far kind of an empty space in crisis research. geographies of the financial crisis heinsberg-studie zur klärung von ansteckungswegen beginnt forms of uncertainty reduction: decision, valuation, and contest freezing deliberation through public expert advice disasters, lessons learned, and fantasy documents the corona crisis: a creeping crisis managing transboundary crises: what role for the european union? the crisis approach crisis exploitation: political and policy 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crisis: a pragmatist perspective cosmology episodes: a reconceptualization la crise: un phénomène spécifiquement moderne? revue de théologie at de philosophie 120 dilemmas in a general theory of planning crises and crisis management: toward comprehensive government decision-making available at <https:// www.tages spieg el.de/kultu r/richa rd-senne tt-zurcoron a-krise -wir-muess en-wachs am-sein/25657 932 bund will kompetenzen im kampf gegen das coronavirus an sich ziehen. handelsblatt enacted sensemaking in crisis situations the complexities of place in crisis renewal discourse: a case study of the sandy hook elementary school shooting the paper is based on ideas and thoughts, developed in the research project 'coping with crises in a resilient manner: the role of expert advice in the creation and use of "opportunities" in crisis situations', funded by the federal ministry of education and research in germany. warm thanks are due to our project team member tjorven harmsen for valuable inputs, conversations and discussions. we also wish to extend our gratitude to the leibniz research alliance "crises in a globalised world" that served as an inspiring platform for academic debate. key: cord-344431-2wq7msqz authors: holzinger, felix; oslislo, sarah; möckel, martin; schenk, liane; pigorsch, mareen; heintze, christoph title: self-referred walk-in patients in the emergency department – who and why? consultation determinants in a multicenter study of respiratory patients in berlin, germany date: 2020-09-10 journal: bmc health serv res doi: 10.1186/s12913-020-05689-2 sha: doc_id: 344431 cord_uid: 2wq7msqz background: emergency department (ed) consultations are on the rise, and frequently consultations by non-urgent patients have been held accountable. self-referred walk-in (srw) consulters supposedly represent a predominantly less urgent patient population. the emacross study aimed to explore consultation determinants and motives in srw patients with respiratory symptoms. methods: multicenter survey of adult ed patients with respiratory complaints in eight emergency departments in central berlin, germany. secondary hospital records data including diagnoses was additionally assessed. characteristics of srw and non-srw patients were compared. determinants of srw consultation were evaluated by binary logistic regression. consultation motives were analyzed descriptively. as a supplemental approach, network analysis (lasso-regularized mixed graphical model) was performed to explore connections between consultation determinants, consultation features and motives. results: between june 2017 and november 2018, n = 472 participants were included, the median age was 55 years (range 18–96), 53.2% of patients were male and n = 185 cases (39.2%) were srw consulters. the srw group showed lower proportions of potentially severe (pneumonia and respiratory failure, p < 0.001, χ(2) test) and chronic pulmonary conditions. determinants of srw consultation identified by logistic regression were younger age (p < 0.001), tertiary education (p = 0.032), being a first-generation migrant (p = 0.002) or tourist (p = 0.008), having no regular primary care provider (p = 0.036) and no chronic pulmonary illness (p = 0.017). the area under the curve (auc) for the model was 0.79. personal distress and access problems in ambulatory care were stated most frequently as consultation motives in the srw group; network analysis showed the scarcity of associations between demographic and medical srw determinants and motives triggering the actual decision to consult. conclusions: as to “who” consults, this study identified demographic and medical predictors of srw utilization. the said markers seem only remotely connected to “why” people decide for srw visits. to alleviate ed crowding by addressing frequent srw consultation motives, interventions focused on the ability for symptom self-assessment and at better-accessible alternative care seem sensible. trial registration: german clinical trials register (drks00011930); date: 2017/04/25. emergency department (ed) consultations are rising in many countries [1, 2] . a considerable proportion is managed on an outpatient basis [3] . ed utilization for non-urgent complaints which could alternatively be adequately managed by a general practitioner (gp)has become a much-discussed issue in the context of ed crowding [4, 5] . ed overburdening is supposed to contribute to a lack of care resources for actually critical patients, ultimately adding to adverse outcomes and even increased mortality [2] . in the discussion of non-urgent ed utilization, a notion frequently expressed especially by health professionals is an alleged misuse of emergency care structures by irresponsible consumers, but perceptions of patients may differ considerably [6] . understanding care demands as well as consultation patterns and triggers is thus of vital importance to allow for developing sensible solutions to the pressing problem of ed overuse: we need to better comprehend who these consulters are, what groups of society they belong to, and what they hope to gain by turning to the ed with certain complaints. this may greatly help in devising future care structures both demand-orientedas acceptance on the patient side is centraland resource-sparing. utilization motives of non-urgent ed patients have been evaluated in various settings [7, 8] . alleged contributing factors include perceived severity of symptoms, health-related anxiety, as well as considerations of convenience [3, 7, 9] . this is considered to be linked with organizational access barriers in primary care (pc) [6, 10] . many studies have included heterogeneous populations [7] with diverse consultation triggers, ranging from minor injuries to gastrointestinal or cardiorespiratory complaints [3, 11] . however, ed visits and how they come about may vary considerably depending on the nature of symptoms [12] . medical issues like thoracic pain or subjective dyspnea for example, although not always caused by serious disease, may be associated with greater worry, uncertaintyand thus subjective urgencythan e.g. acute musculoskeletal ailments or skin problems [3] , simply due to the not straightforward constellation. such less clear-cut situations, in which patients need to selfassess symptoms and then decide whether to visit an ed, constitute the most interesting cases when wanting to understand what drives utilization patterns. for this purpose, respiratory complaints constitute an ideal model, as they are very frequent consultation reasons in eds as well as in pc [3, 13, 14] and their underlying reasons encompass a wide spectrum, ranging from more serious (e.g. pneumonia) to non-serious (e.g. common cold) as well as acute and chronic conditions [15, 16] . concerning purportedly less urgent ed visits, means of arrival provide a first indicator: walk-in patients are presumably less severely ill than those arriving by ambulance [17] , as in the latter the necessity of ed treatment will usually have been either determined by a health care professional (e.g. referring physician), or the patient will have felt too severely afflicted to consider other transportation. among walk-in consulters, patients who decided to visit the ed on their own accord as self-referrals constitute the most interesting population for studying ed consultation reasons and associated factors [12, 18] . to gain a deeper understanding of ed utilization determinants in a population with an exemplary symptomatology, we aimed to comprehensively explore demographic and medical characteristics as well as consultation motives of self-referred walk-in ed patients presenting with respiratory symptoms. the multicenter mixed methods emacross (emergency and acute care for respiratory diseases beyond sectoral separation) study investigates characteristics, motives and health care utilization of patients with respiratory symptoms in a network of eight eds in the central district of berlin, germany (berlin-mitte). it is a subproject of emanet (emergency and acute medicine network for health care research), which focuses on acute care for a number of model conditions selected in the context of the ambulatory care sensitive conditions (acsc) concept [19] . emacross consists of a quantitative two-stage survey of respiratory ed patients, an evaluation of secondary hospital data, and a qualitative module [20] . this paper reports the results of the t0 survey and analysis of hospital records. the protocol was registered a priori in the german clinical trials register (drks00011930). the study was approved by the ethics committee of charité -universitätsmedizin berlin (ea1/361/16). participants were recruited in our network comprising the entirety of eds in the district, including two university medical centers, between 1st of june 2017 and 30th of november 2018. patients were assessed for eligibility at presentation based on symptoms reported to the triage officer. if inclusion criteria were met, written informed consent was obtained. recruitment was conducted regularly from monday to friday between 9 am and 5 pm and intermittently in the evenings and on weekends. the focus of recruitment was placed on regular physicians' office hours due to our interest in choosing the ed versus conceivable alternative care, ed selfreferral being not at all limited to out-of-hours periods [12] . the survey was conducted during waiting times or between investigations. patients of both sexes aged ≥18 years with respiratory symptoms (e.g. cough, dyspnea etc.) were included. an initially envisaged diagnosis-based enrollment [21] was abandoned as unfeasible after pilot testing due to the characteristics of ed care, definite diagnoses being available only late in the visits and outpatients frequently desiring to leave immediately after receiving their discharge letter. patients were excluded if unable (e.g. as to dementia or severity of acute condition) or unwilling to consent, or lacking adequate proficiency in one of the questionnaire languages (german, english, turkish, and arabic). recruitment was initially limited to outpatients. this proved problematic in the study workflow: patients had to be interviewed at a time when it was frequently undecided whether they would be ultimately admitted. in order to avoid having to exclude patients after completed interviews and thus losing valuable data, recruitment was extended to eventual inpatients as of october 2017; the study protocol was thus amended. the questionnaire assessed demographic and medical characteristics as well as consultation motives and health care utilization [21] . items were derived from established instruments where available and appropriate. assessment of health care utilization was based on the german health interview and examination survey for adults (degs) [22] , the phq4 questions were included as indicators of mental health [23] , general life satisfaction was measured with the short scale l-1 [24] , and education was assessed corresponding to the casmin classification [25] . other items were specifically developed for emanet. the final survey contained 43 questions (plus eventual sub-items). the german and english language versions of the questionnaire are available as additional file 1 and additional file 2. several pre-test rounds were carried out [26] . the questionnaire was tablet-based, data was entered by study nurses conducting face-to-face interviews. a few questions, e.g. for assessment of ed consultation motives, were posed openly and study personnel matched answers to a list of pre-formulated options. free text documentation was used in cases of no match to the list. concerning consultation motives, patients could thus freely relate their considerations; a combination of several reasons could potentially apply (multiresponse data). personnel received precise instructions, interpretation aids and repeated interviewer trainings. data was directly transferred to a secure database server. additionally, medical (e.g. triage, symptoms, diagnoses) and administrative (e.g. admission, discharge) data was extracted from hospital records via electronic case report form (ecrf). for quality assurance, random double entries of 5% of cases were performed and collated. three months after the baseline survey, a telephone or postal follow-up ensued to longitudinally assess health and utilization [21] . follow-up data is currently analyzed. target group: self-referred walk-in (srw) patients we delimited self-referred patients as cases in which no medical professional or institution was involved in the visit's initiation, namely participants not referred by a physician, hospital or department, or nursing home staff. walk-in cases were defined as patients reporting arrival by any means (e.g. by foot, car, public transport) other than emergency medical services (ems) or ambulance transport, and hospital records neither indicating such. srw patients were defined as cases with both characteristics, as compared to non-srw. patient characteristics data on medical and demographic characteristics was primarily derived from the t0 survey. most variables directly correspond to the respective survey questions. for some ordinal variables, categories were combined, e.g. in case of small subgroups or if otherwise deemed theoretically reasonable, e.g. a variable on previous frequency of similar symptoms, which was collapsed into "new symptoms" vs. "prior existence of comparable symptoms". ordinal variables with a substantial number of classes (e.g. 0-10 scales) were interpreted as continuous [27] . a summary variable for symptom-associated distress was created by combining scales assessing components of this construct (severity and associated threat) [28, 29] by calculating the average of the two scale values. the eight-level casmin education scale was collapsed into three levels of low, intermediate, and high educational attainment [30] . the "low" level comprised casmin levels 1a to 1c (primary + low secondary), the "intermediate" level 2a to 2c (intermediate + high secondary) and the "high" level 3a and 3b (tertiary education). for chronic pulmonary morbidity, hospital records and survey data were combined to enhance validity [31] . when cross-tabulating the dichotomous variables for both data sources (chronic pulmonary condition mentioned: yes/no), concordance was moderate at a cohen's kappa of 0.5 [32] , which is comparable to the literature [33, 34] . we considered a chronic pulmonary condition as likely present if this was either self-reported or a corresponding diagnosis documented. patients with two or more chronic conditions were defined as multimorbid [35] . consultation motives consultation motives were grouped into thematic summary categories based on pertinent classes of the framework of coster et al. [36] . categories were labeled as "distress", "access", "quality" and "convenience" (table 1) . "distress" encompasses all answers relating to symptom severity and anxiety, "access" covers issues of service-defined barriers to alternative care (e.g. appointment availability and office hours in pc), as well as situations of patients not knowing who to contact (e.g. visitors ignorant of local health care). "quality" summarizes expectations of better care in the hospital setting, and the "convenience" theme comprises patient-defined considerations regarding comfort and ease of ed access. ed consultation features and outcomes information on time of presentation, hospital admission, triage and diagnoses was available from hospital records. triage categories of the manchester triage system were combined in a binary variable delineating "high urgency" (levels 1, 2, and 3) and "low urgency" (levels 4 and 5) analogous to van der linden et al. [12] . concerning presentation time, we distinguished office-hours from out-of-hours based on usual opening times of gps' practices. "out-of-hours" was defined as 6.00 pm to 8.00 am on weekdays, plus all weekends. as german practices usually close on wednesday afternoons, an extended out-of-hours timeframe starting at 2.00 pm was defined for this day. patient characteristics as well as consultation motives were summarized descriptively. for logistic regression of srw determinants, a set of potential predictors and control variables was compiled, based on theoretical plausibility and the literature. the number of candidate predictors was limited by events per variable (epv) to avoid bias, a current recommendation being to aim for epv of 15 or higher [37] . we carried out univariate statistics and noted which variables showed significance at a relaxed level (p ≤ 0.25) [38] . however, non-significance did not result in immediately discarding a predictor. for non-significant variables, we carefully considered their potential importance for the model as e.g. control variables, in which case they were retained. this could also be the case if the variable had been identified as an important predictor in a previous study. a first multivariate model was constructed. we then checked the effects of discarding single predictors on the variable set and assessed fit and predictive accuracy of the candidate models to decide which variables to include in the final set. model fit was assessed by the hosmer-lemeshow test; cook's distance was used to investigate for influential outliers. classification was assessed by the area under the receiver operating characteristic (roc) curve. effect sizes are reported as odds ratios with 95% confidence intervals. we did not conduct an automated variable selection procedure like stepwise regression, as to avoid the risk of obtaining a biased model with falsely narrow confidence intervals and low p values [39, 40] . to overcome the problems of stepwise methods, several solutions to variable selection have been proposed, markedly focusing on expert knowledge and theory rather than strict significance thresholds, which was the approach chosen for our logistic regression analysis. alternatively, variable "because the situation felt threatening to me" "because my complaints were so severe" access 5 "because my gp's practice was closed" "because i could not get a timely appointment with my gp or specialist, although i tried to." "because i am just visiting this city" quality 7 "because diagnostic and therapeutic options are more comprehensive in the hospital" "because there are special experts in the hospital" "because the results of investigations are available more quickly" because the ed is always open and no appointment is necessary" "because the ed is closer to my home than a practice" note. question posed to the participants was "why did you decide to visit an emergency department with your current complaints?" selection by newer statistical techniques encompassing penalization and shrinkagelike ridge or lasso (least absolute shrinkage and selection operator) regressionwould be preferable to conventional automated selection methods in many constellations [37] . a lasso approach was part of our network analysis, which is outlined further down in this methods section. for group comparisons of categorical variables, the χ 2 test was used. the significance level for all analyses was set at 0.05. descriptive statistics and regression were performed in ibm spss version 25 and r (jasp 0.11.1 interface) [41] . for explorative investigation and visualization of patient characteristics in connection with consultation motives as well as ed consultation features and outcomes, a network analysis of complete case data was conducted. in such networks, variables are labeled as "nodes" and connections as "edges". analyses were performed with the r packages "mgm" [42] and "bootnet" [43] . the "mgm" package allows estimation of k-degree mixed graphical models (mgm) via regularized neighborhood regression; this method was suitable as the analysis included numerical as well as categorical variables. the mgm was estimated using lasso regularization, which sets very small parameter estimates to exactly zero and returns sparseand thus conservativenetwork models [43] . the lasso utilizes a tuning parameter to control the degree of regularization, which was selected by minimization of the extended bayesian information criterion (ebic) [44] . ebic penalizes solutions that involve more variables and more neighbors of nodes, with a hyperparameter γ determining the strength of the extra penalty on the number of neighbors [45] . this hyperparameter was set to 0.25 (default in "mgm") [42] . either of the estimates was required to be nonzero for an edge to be present (or-rule) [45, 46] . the network was plotted via the r package "qgraph" [47] . node placement is determined by the fruchterman-reingold algorithm which places nodes such that all the edges are of more or less equal length while aiming to avoid edges crossing. edge width is proportional to the edge-weight, green edges indicate positive relationships and red edges negative relationships [42] . to avoid edges without visual indication of a sign, multi-categorical variables (education, migration and travel) were binarized by combining categories as suggested by the preceding regression analysis. we assessed predictability of srw by connected nodes, defined as correct classification beyond the marginal [48] . accuracy of edge-weights was evaluated by nonparametric bootstrapping via "bootnet" [43] . a total of n = 472 cases were included, while n = 1121 initially screened patients had to be excluded. exclusion reasons and frequencies are shown in table 2 . details on recruitment monitoring and non-responder analysis in emanet have also been published elsewhere [49] . required data to determine srw vs. non-srw status was available for n = 463, of which 185 (40.0%) were classified as srw. for nine cases, necessary information for classification was missing. the frequencies of all combinations of the variables defining the target group are reported in table 3, while table 4 shows characteristics of the total cohort and srw vs. non-srw cases. the srw group was younger and included a greater proportion of females, migrants as well as tourists. srw patients also showed higher formal education status and less urgent triage, whereas the proportion of out-ofhours consultations was similar in both groups. morbidity in general and concerning chronic pulmonary conditions was higher in non-srw patients, while groups did not differ markedly regarding mental health. potential determinants of srw consultations were evaluated by logistic regression. results of the multivariate model are shown in table 5 . the hosmer-lemeshow test did not show significance (p = 0.969, χ 2 = 2.337, df = 8), thus supporting model fit. being a first-generation migrant or a tourist and having a high level of education (tertiary) were identified as predictive of srw in the multivariate analysis. higher age, having a chronic pulmonary condition and being regularly attached to a gp practice lowered the probability of an srw consultation. sex and out-of-hours were retained in the model as control variables. triage category was not shown as independently predictive in the multivariate model; the same applies to other variables evaluated during model building (previous utilization, multimorbidity, symptom-associated distress). roc curve analysis showed an auc (area under the curve) of 0.79 for the model. compared to non-srw, a considerably greater proportion of srw cases were managed as ed outpatients. concerning ed and hospital diagnoses, the non-srw group showed higher proportions of pneumonia and copd than srw patients did. respiratory failure was documented in 27.7% of non-srw cases, compared to only 7.0% in srw patients. in non-srw patients admitted to hospital, the proportion of respiratory failure diagnosed was also higher than in srw patients admitted (52.4%, vs. 34.2%, p = 0.046). srw patients had a higher share of upper airway condition diagnoses coded, as well as asthma. consultation outcomes are summarized in table 6 . in the framework of motive groups, "distress" was the main consultation reason voiced by both srw and non-srw patients, although by a greater proportion of the srw group. "access" constituted the second most important motive group, with a considerably greater share of srw patients relating such. the same applies, to a somewhat lesser extent, to "quality" and "convenience" (table 7) . inherent to their assessment as multi-response data, categories partially overlap, which is outlined for the srw group in table 7 . for example, about half of patients in the "access" and "quality" groups also related "distress" as additional consultation motive. as to better understand possible access problems in ambulatory care, a more detailed evaluation of the reasons reported by srw patients was conducted. unavailability of practices (out-of-hours, weekend, vacation) as well as difficulties in getting timely appointments were most prominently reported (35.2% and 32.4% of n = 71 participants in the "access" motive group). of the 25 cases stating that they could not reach their physician, eleven (44.0%) presented to the ed out-of-hours, 14 (56.0%) during regular office hours. patients were additionally asked whether they had tried to contact a doctor's practice prior to visiting the ed; the proportion reporting such was higher in the non-srw group than in srw patients (59.9% vs. 41.1%, p < 0.001). for srw patients, the access problems related suggest a substantial share of unsuccessful contact attempts. on the other hand, non-srw patients' better accomplishment in this regard will supposedly frequently have resulted in their eventually being referred to the ed by the ambulatory physician contacted. the estimated mgm network shows that demographic patient characteristics, morbidity and care attributes, consultation features and decision-making in ed utilization are complexly intertwined. in the regularized network ( fig. 1) , the srw node features the highest number of connections (edges) and is positively linked with the four consultation motive groups as well as with a high education level and being a first-generation migrant or tourist. negative edges are visible between srw and having contacted a practice prior to the ed visit, hospital admission, age, chronic pulmonary conditions and having a gp. predictability of srw in the network was 0.8, with an increase of 0.21 beyond the marginal probabilities. connections of srw in the network correspond to the results of the preceding regression of srw determinants, with non-zero edges estimated for all predictors identified in the logistic model. a flow plot (fig. 2) makes the direct connections between srw and its demographic and medical determinants and underlying motives more easily visible. notably, characteristics located in the second level (e.g. triage category, multimorbidity) did likewise not show statistical significance as srw predictors in the logistic model. among nodes with direct links to srw, it strikes that the demographic characteristics (green) and morbidity and care attributes (yellow) show more connections and interconnections than the motives (red). looking at the few connections between motives and other network nodes, the included edges appear fundamentally plausible, thus for example the edges between "motive: access" and "out-of-hours" and "practice contacted before ed visit". beyond this, motives seem comparably selfdetermined. except from a few tentative clues (e.g. edge between "migrant/tourist" and "motive convenience"), we cannot link motives to distinct patient groups or characteristics. predictability of the motive nodes in the network correspondingly does not exceed marginal probabilities (motives "access", "quality" and "convenience") or is minimal ("distress"). this is additionally suggestive of motives being mainly influenced by unknown factors not included in the network. as to auxiliary analyses, bootstrapping showed sizable confidence intervals around edge-weights, suggesting that many weights might not significantly differ; we thus refrained from interpretation of their order. we additionally explored the effects of adding hospital diagnoses to the network, categorized as "potentially more severe" (pneumonia, respiratory insufficiency), "potentially less severe" (upper airway conditions, rti, or r diagnoses only) and "chronic illness-related" (copd, asthma). however, predictability of srw did not improve by inclusion of diagnoses. the results suggest that the "road" ultimately leading to an srw visit starts with certain predisposing demographic traits and medical and care characteristics, like younger age, absence of chronic illness, migration background, and having no regular gp. corresponding findings of a higher tendency of the young to self-refer [12] and consult non-urgently [3, 7] have been described by others. the same applies to a higher ed utilization by migrant populations, which was reported for most european countries for which evidence was available in a systematic review by graetz et al. [51] . alleged reasons for greater migrant utilization encompass health status, cultural factors, as well as care structures in peoples' countries of origin, including possible experiences of poorquality pc [51] . in contrast, a population-based study from germany described a prevalent pc-based healthcare utilization pattern in first-generation migrants and linked this to lower socioeconomic and educational status [52] . in our cohort however, first-generation migrants were comparatively well-educated (tertiary education 47.0% vs. 23.7% in participants with no migration background). this might in part be an age effect (median 42 years, vs. 60 years in people with no migration background), but could also be influenced by the metropolitan setting, with some inner-city hospitals potentially attracting young and internationally mobile professionals not representative of a general migrant population. about 30% of the first-generation migrants in our study population reported to have lived in germany five years or less, which might support this notion. the countries of birth assessed in the survey do not offer any obvious further clues here, with 26.5% born in eu countries, followed by 14.7% middle east, 11.8% turkey and 10.8% latin america as largest subgroups. another possibility comes to mind in this context: the result of highly educated immigrants being overrepresented in the study could have been biased by participants' language skills, as interviews were conducted in german and english only. while the written questionnaire was additionally translated into turkish and as the network graph is force-directed, graphical spacing of two connected nodes does not reliably represent the magnitude of their association, thus barring spatial interpretations [50] . multi-categorical variables "migration and travel" and "education" were binarized to avoid unsigned edges. for migration, this meant categorization of participants not born in germany (first-generation migrants and tourists) vs. others; regarding education, dichotomization threshold was set between casmin categories for high education (=tertiary) vs. intermediate and lower education (=primary and secondary) arabic, these versions had to be answered in writing, requiring reading and writing skills proficient enough to answer a quite extensive set of questions. the lowerthreshold option of being interviewed was thus only available to people with a good working knowledge of german or english. this could have led to a selection bias towards better-educated people that could explain part of this result. the high proportion of people with academic education in our cohort corresponds to the berlin tertiary education rate, which distinctly exceeds the nationwide average [53] , a phenomenon underscoring the special circumstances of a big-city location. studies from other health care contexts have reported a higher tendency to self-refer or consult non-urgently in groups of lower socioeconomic status [54, 55] , and our discrepant results may be attributable to setting effects. concerning interactions between care sectors, the seemingly extenuating effect of having a gp on self-referrals and less urgent ed consultations is in line with other results [56, 57] . how do patientswhether they feature predisposing characteristics for srw or notreach their decision to consult further "down the road"? in contrast to previous studies not centering on a specific trigger symptom [6, 9, 12] , our results do not indicate an important role of convenience considerations. the relative preponderance of e.g. distress as a motive could be attributable to the nature of respiratory symptoms, their seriousness vs. benignancy being potentially more difficult to appraise for the patient than for e.g. an injury or rash, thus enhancing subjective urgency [3] . some patients appear to feel too severely ill to consider alternative care options, and thus are less likely to try to contact a practice, as suggested by the negative network edge between "practice contacted" and "motive: distress". concurrently, other studies have repeatedly described health concerns and medical necessity as important consultation motives in ed self-referrers [8, 58] . concerning access problems, the results are in line with previous studies having discussed the important role of pc availability as a determinant of ed utilization [9, 59, 60] . our data interestingly suggest that the said consultation motives are only sparingly connected with demographic and medical patient characteristics and cannot be attributed to distinct patient groups, so we cannot readily derive "why" from "who". motives and decision-making presumably depend on other factors. speculatively, distress for example might be influenced by individual experiences and personal susceptibility to health-related anxiety, and access problems may depend on the reachability of the individual patient's gp. however, this remains conjecture, and qualitative methods might constitute a more appropriate approach for studying the role of factors like personality traits, experiences or social environment. much has been written about non-urgent ed patients and inappropriate utilization. unlike others [11, 61] , we did not attempt to classify srw patients as appropriate or inappropriate (or urgent vs. non-urgent), as selecting reasonable criteria is controversial [7] . we would like to stress that we do not consider srw utilization as congruent with "non-appropriate": among the srw crowd, there are a non-negligible proportion of patients with hallmarks of medically serious situations, e.g. pneumonia or respiratory failure diagnoses. globally however, our study results show that srw patients are comparatively less severely ill and less likely to be hospitalized. while quantifying the extent of avoidable ed visits was not part of our research question, these observations suggest a substantial share. our results as well as the literature suggest that having a regular gp has a regularizing effect. measures to encourage pc attachment could supposedly advance bettertargeted utilization. beyond gp care, the demographic and medical predisposing traits identified inherently seem difficult to influence. thus, the prevalent problem areas of distress and access stand out as most promising gateways for health care interventions. the scope of patients' distress supposedly depends on the self-assessment of symptoms experienced [62] . besides personality traits, health literacy may affect the capacity for adequate interpretation of bodily sensations; corresponding deficits could contribute to patients seeing no alternative to an ed visit despite not being severely ill [63, 64] . a worthwhile avenue to explore in further studies might be provision of guidance for adequate "self-triage". evidence on corresponding online decision-support aids is currently controversial [65] , but some approaches seem promising [66] . the effectiveness of measures aimed at ameliorating pc access problems for reducing ed burden remains controversial. the current body of evidence suggests that additional offers like a co-location of gp posts and emergency departments are probably more promising than a simple expansion of regular gps' office hours [67] . a combination with information supporting utilization decisions could be worthwhile [68] , thus making the connection to the health literacy issue. however, this needs to be further substantiated scientifically, keeping in mind the question of cost-effectiveness of new care structures. in germany, the government recently has proposed a bill aimed at reforming emergency care structures to antagonize ed crowding [69] . this includes the establishment of special emergency centers at hospitals, where patients will be sent either to the ed or outpatient care structures, depending on severity of illness. these are planned to be operated jointly by the hospitals and the association of statutory health insurance physicians. international experiences with similar concepts appear promising: in the netherlands for example, emergency care access points (ecap) jointly created by eds and gp cooperatives have demonstrated considerable effects on reducing ed consultations [70] . the german plans also encompass fusing the currently separate call centers for ems and non-emergency out-ofhours doctors. ensuring adequate reimbursement for ems care provided on-site is also included in the reform, as currently ems transport is generally only paid for if patients are brought to hospital. the effects of this proposed package of measures on ed utilization remain to be seem in the coming years. this study provides a comprehensive insight into the determinants of srw consultations for respiratory complaints as well as underlying motives. to our knowledge, it is the first study to explore the complex connections of factors associated with ed utilization by a network method. network approaches and mixed graphical models have been increasingly applied in the context of clinical psychology and psychometrics [71, 72] , but their use for visualizing and studying complex relationships in health services research is novel. the network approach underscores the results of logistic regression by a different modeling method and offers additional insight into the interconnections of variables. however, we were quite careful regarding inference, bootstrapping having revealed limited edge weight stability. future studies with larger sample sizes might allow more robust estimations here. this limitation however does not apply to the same extent to the presence of edges, as observation of edges not set to zero in a regularized network already indicates that the edge is sufficiently strong to be included in the model [43] . several additional caveats apply. for once, potential selection bias must be considered. in an ed setting, not all patients may be similarly inclined to participate in a study, depending on factors like e.g. severity of illnessor language skills, as we have already discussed. on the other hand, the inclusion of all hospitals in the city district ensured access to a wide-ranging group of ed patients in a high-density urban area and serves to mitigate selection effects specific to single-center studies [21] . regarding representativeness, it must be noted that the deferred recruitment of inpatients induces an overrepresentation of less severe cases in our cohort, and certainly srw cases as well. however, while this influences the relative representation of utilizer groups in the study population, it does not affect interpretability of differences between groups. furthermore, the study's focus on respiratory complaints limits generalizability to unselected populations, even if the selected model symptom is frequent and includes a wide spectrum of underlying severe and non-severe constellations. studies considering all possible diagnoses though pose other problems, e.g. a need to differentiate between medical and surgical indications. beyond all this, we must emphasize that the study was conducted prior to the advent of covid-19, which has currently changed the implication of respiratory symptoms dramatically. concerning the data collection methods and tools used in our study, we would like to point out that many questionnaire items and scales were newly developed for this study on a theoretical basis, as we could not identify any validated tools (e.g. for assessing symptomassociated distress). thus, we cannot attest to the sensitivity and specificity of these scales. neither can we exclude that some questionnaire items might have been interpreted by study participants in a way not intended by us: when inquiring about consultation motives for example, patients might have felt prompted to justify their choice, rather than to just explain it. as to consultation motives, we would additionally like to stress that quantitative methods can only schematically assess decision-making processes and are ill suited to capture cognitive and emotional goings-on. qualitative studies have explored such issues in greater depth [6, 9] . concerning our study, the results of an ancillary gp interview module have been published [20] , a paper on the patient perspective is in preparation. as to the question of "who" consults in an srw manner, we identified demographic and medical determinants enhancing corresponding probabilities in respiratory ed patients. the young, well-educated, and pulmonary healthy as well as migrants must be mentioned here. having a regular gp reduces the chance of srw utilization. the said characteristics seem only barely connected to "why" people decide on srw visits. subjective distress and pc access problems play a pivotal role as consultation motives in the focused population, while convenience seems comparably inconsequential, thus tendentially confuting the notion of irresponsible utilization. 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network analysis: a brief overview and tutorial using network analysis to examine links between individual depressive symptoms, inflammatory markers, and covariates publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the patients taking part in our study.authors' contributions mm initiated the research network emanet, he also is principal investigator and speaker of the umbrella project. ls is co-speaker of emanet. fh and ch designed the subproject emacross, including quantitative and qualitative modules. fh, so and ch developed the study protocol including research questions and methods of evaluation. fh, so and mp analyzed and interpreted the data. fh drafted the manuscript for this paper, all co-authors read and critically revised the manuscript. fh drafted the final version, which all authors read and approved. all authors qualify as an author according to the icmje guidelines. this study is part of the project "emanet" and funded by the federal ministry of education and research (bmbf), grant number 01gy1604. the funding body had no role in the design and conduct of the study, data collection, analysis, and interpretation of the data, nor in the preparation, review and approval of the manuscript. open access funding provided by projekt deal. 4 charité -universitätsmedizin berlin, corporate member of freie universität berlin, humboldt-universität zu berlin, and berlin institute of health, institute of medical sociology and rehabilitation science, berlin, germany. 5 charité -universitätsmedizin berlin, corporate member of freie universität berlin, humboldt-universität zu berlin, and berlin institute of health, institute of biometry and clinical epidemiology, berlin, germany.received: 2 july 2020 accepted: 26 august 2020 the authors declare that they have no competing interests. key: cord-351732-wws6ring authors: sarteschi, christine m. title: sovereign citizens: a narrative review with implications of violence towards law enforcement date: 2020-09-24 journal: aggress violent behav doi: 10.1016/j.avb.2020.101509 sha: doc_id: 351732 cord_uid: wws6ring extremist movements are growing in the united states. one concerning extremist group is that of sovereign citizens. sovereign citizens have been labeled by the federal bureau of investigation as a terrorist threat. relative to other research about extremist groups, limited research exists about the sovereign citizen movement. the purpose of this article is to review all relevant literature concerning this movement, as it pertains to the threat posed to law enforcement, via descriptive research and to identify existing knowledge gaps. most empirical work, about sovereign citizens, thus far has focused on legal matters, mental health, radicalization, and postdiction of targeted violence. the work presented here serves as a foundation for future research concerning this group. the dhs definition is narrower and focuses only on sovereign citizen extremists which they define as "groups or individuals who facilitate or engage in acts of violence directed at public officials, financial institutions, and government facilities in support of their belief that the legitimacy of the u.s. citizenship should be rejected, almost all forms of established government, authority, and institutions are illegitimate and that they are immune from federal, state and local laws" (u.s. department of homeland security, 2014, p. 1). the dhs seems focused on sovereign citizens who engage in violence, as opposed to those who may believe in violence philosophically, but who have yet to engage in violence. this paper defines sovereign citizens in accordance with the fbi and the dhs. this paper also takes the position that any individual who is legally considered to be a citizen of the u.s. and who simultaneously believes that the u.s. is not a legitimate government, and as a result of that illegitimacy, believes themselves to be immune to all u.s. laws, is to be considered a sovereign citizen. these defined individuals may refer to themselves as something other than sovereign citizens and often do. one does not need to consider themselves a sovereign citizen to be a sovereign citizen. other names sovereigns may call themselves include: freeman on the land, flesh and blood human being, natural man, free person, sovereign man, layman, a natural person, a freedom of common-law citizen, and other names. the core belief of sovereign citizens, is their proclaiming the american government to be illegitimate. on the land," and a number of political candidates have made claims and have utilized tactics seemingly consistent with the sovereign citizen movement (baldino & lucas, 2019) . in june 2020, four members of a sovereign group "new westralia" broke into a historic courthouse and took up residence (hedley, 2020) . social media posts indicated that their goal was to establish themselves as the "proper governing body of law for western australia" (hedley, 2020, para. 7) . in another example involving australia, mark pytellek, a well-known sovereign citizen and his wife, were running a facebook group crusading against a new rule mandating flu vaccines for those who were visiting individuals in aged care homes (wilson, 2020) . the couple was selling an online service that charges a fee to add their name to a form letter titled "vaccine non consent document creator" to be sent to australian officials (wilson, 2020) . in addition, they are also encouraging users to pay $69.99 annually for access to a website called solutions empowerment that teaches sovereign rights and other secret strategies (wilson, 2020) . mr. pytellek has a record of legal trouble with authorities in australia. in a video from singapore, dated may 2020, there appears to be a sovereign citizen female yelling "i am not a person, i am sovereign" when confronted by police for refusing to wear a mask in public and for breaching safe social distancing measures, under covid-19 regulations (koay, 2020) . in russia, individuals calling themselves "citizens of the u.s.s.r." or "soviet citizens" are claiming they do not have to follow the laws of the russian federation nor do they recognize it as a legitimate legal body (luxmoore, 2019). much like the sovereign citizen movement in the u.s., many "soviet citizens" are learning how to supposedly evade laws via the internet. youtube, in particular, has channels with thousands of followers (luxmoore, 2019). one popular channel claims to offer "practical tips and advice for citizens of the ussr" in how j o u r n a l p r e -p r o o f journal pre-proof they can "legally avoid paying off debt" (luxmoore, 2019, para. 14). that video, as of may 2019, had 2.8 million views (luxmoore, 2019). germany's version of the sovereign citizen movement is known as reichsbürgers or the reich citizens movement. estimated to number approximately 19,000 individuals, across the country, they believe the laws of both imperial germany and nazi germany still apply (the sun, 2020) . they also print their own passports, refuse to pay taxes and engage in paper terrorism (gauvery herbert, 2020) . some are prone to violence. former mister germany, adrian ursache, a prominent member of the reich citizens movement, shot and killed a police officer in 2016. he had declared his property an independent state and when police tried to evict him from his "independent state" he attempted to kill them (the sun, 2020). two months later, one of urache's followers, wolfgang plan, shot at police as they attempted to remove his weapons. one officer died and two officers were injured during the incident. mr. plan was sentenced to life in prison. one exceptional reichsbürger case is that of peter pitzek. he refers to himself as the king of germany, and claims dominion over 1,300 subjects (gauvey herbert, 2020) . he was able to convince dozens of individuals to live on a compound in wittenberg, germany. they all gave him money, some their entire life savings. he amassed millions of dollars. he opened his own bank and was collecting deposits until german authorities shut it down. upon investigation, the authorities were only able to locate a small amount of money on his property. investigators concluded that the self-proclaimed king of germany had spent most of the money on himself, in the form of travel, cars and real estate. the rest, they estimated, had been laundered through a network of companies and then hidden in countries that lacked extradition treaties with j o u r n a l p r e -p r o o f journal pre-proof germany. he went to trial and was convicted. none of the money was ever recovered. after about a year in prison, an appeals court overturned his conviction. no longer incarcerated, he has since acquired new money-making ventures. more than 70 companies, for a fee, have incorporated in his kingdom. much like in australia, members of the far-right political establishment in germany have been adopting some of the language of the reichsbürger movement, suggesting that these ideas have gone mainstream. german authorities have been trying to crack down on the illegal activities of the reichsbürgers, but only with limited success (gauvey herbert, 2020). sovereign citizen-hood is spread via the internet, particularly on youtube. there are millions of videos on the topic. the sovereign citizen message is also spread through jails and prisons, typically via mailers and word of mouth. the marshall project described a sovereign citizen scheme operating in prisons. legitimate-sounding companies claim that the u.s. government is holding all prisoners for financial reasons and that for an initial fee of $4000, they will initiate a bond process that will ultimately earn a prisoner their freedom (thompson, 2019) . bond process, also known as straw man, redemption or accepted for value schemes, involve the use of fraudulent financial documents that appear legitimate (fbi, n.d.) . many unsuspecting victims find out, after the fact, that the bond process is simply an illegitimate money-making scheme. a number of high-profile individuals have attempted to use sovereign citizen strategies in courts, but none have been successful. one example is jared fogle, the former spokesperson for the subway sandwich restaurant chain, who declared himself a sovereign citizen in court filings. in one instance, he was attempting to correct an "error" regarding subject matter jurisdiction, essentially questioning the court's authority to render a verdict (bever, 2017) . mr. vogel pled guilty, in 2015, for receiving child pornography and for traveling and attempting to elicit sexual conduct with a minor. he was sentenced to 15 years in federal prison (bever, 2017) . all of his sovereign citizen arguments have been deemed frivolous and rejected by the court (evans, 2017) . some learn about the many supposed advantages of sovereign citizen-hood via a number of gurus peddling seminars, promising the ability to emancipate oneself from the federal government (pitcavage, 2012) . for instance, the new york times profiled sean david morton, a sovereign citizen who taught workshops claiming instant debt relief from mortgages, tax bills and student loans (powers, 2019). david wynn miller, another tax protester/sovereign citizen guru, and his adherents, claimed that the government cannot tax their income because they are individuals without citizenship and thus are sovereign unto themselves (united states v. kriemelmeyer, 2006) . mr. wynn miller went a step further than many other gurus and invented his own dialect called "in the truth." this dialect, he claimed, is based on mathematics, and is characterized by an overuse of prepositional phrases, hyphens, and colons and avoids the use of verbs, pronouns, adjectives, and adverbs (united states v. kriemelmeyer, 2006) . mr. wynn miller has since passed away but his tactics live on and are attempted by other sovereign citizens. syndrome (sids) and became angry at the government after they insisted upon an autopsy. he subsequently became involved with the dorean group, a company much like sean david morton's company, who offered seminars about debt relief and gaining freedom from government. mr. kane initially was a strong supporter of the dorean group, promoting their materials, but that changed after experiencing a number of legal problems. he eventually quit the group and started his own seminars. his seminars often included his advocating extreme violence, including the killing of internal revenue service (irs) agents. his violent rhetoric prompted the fbi to open an investigation. he increasingly has had more run-ins with law enforcement. his son joseph took a similar stance against the government. even as a young child, he had trouble with the law and was known to be disrespectful, problematic and defiant (carson, james, & o'neil, 2019) . actions. their results indicated that the trap-18 measure successfully distinguished between sovereign citizens who were violent from those who were not. the researchers found that individuals with higher trap-18 scores were two times more likely to be involved in committing an act of violence compared to those with lower scores. six warning behaviors (pathway, identification, novel aggression, energy burst, leakage, and last resort) and four distal behaviors (personal grievance, framed by ideology, greater creativity, and criminal violence) significantly postpredicted violence in their research (challacombe and lucas, 2019). for those unfamiliar with sovereign citizens, their behavior can seem odd, bizarre, confusing, and seemingly indicative of mental illness. this may explain why judges, after dealing with their disruptive behavior in the courtroom, frequently request mental health evaluations, to determine competency. in the literature, there are four studies to date, regarding the mental health of sovereign citizens and their fitness to stand trial. all of them seem to suggest that the vast majority of sovereign citizens are not mentally ill and are competent to stand trial. pytyck and chaimowitz (2013) took a case study approach, while analyzing two cases of sovereign citizens who were referred to the court for mental health assessment. one was a male and one was a female. in both cases, they refused to cooperate with officers before their arrests and were uncooperative in court, thus prompting their competency evaluations. both were deemed competent to stand trial. the authors argue that, in their view, the majority of sovereign citizens are not psychotic and are fit to stand trial. j o u r n a l p r e -p r o o f journal pre-proof parker (2014) conducted a retrospective study regarding competence among sovereign citizen defendants undergoing psychiatric evaluations. in this study, he reported the results of nine cases of individuals, identified as sovereign citizens, who were court-ordered to undergo evaluations of competence to stand trial. of the nine, three refused to participate in clinical interviews. their average age was approximately 39 years old, had at least a high school education. the majority were african-american and eight of the nine defendants were male. though two of the six had histories of mental health treatment, none showed significant cognitive deficits or had any history of psychosis. one defendant received a diagnosis of delusional disorder and another for depression. three received diagnoses of substance abuse disorders; one had no diagnosis. the defendant who received the delusional disorder diagnosis was deemed incompetent to stand trial, however, parker later came to believe that this defendant did not truly meet the criteria for delusional disorder and was competent to stand trial. in his view, those who adhere to the sovereign citizen belief system are mostly likely not delusional and are instead, exhibiting an extremist political philosophy. parker argues that being a sovereign citizen alone is not enough to warrant a mental health diagnosis or a judgment of incompetence (parker, 2014) . four studies available, thus far, support the notion that the majority of sovereign citizens are not diagnosably mentally ill or unfit to stand trial. sovereign citizens who are diagnosably ill and thus unfit to stand trial, are the exception rather than the rule. more research is needed to understand why sovereign citizens adopt such extremist beliefs. a good deal of information about sovereign citizens was found in legal review articles. four legal articles were identified through this review. perhaps one of the most cited articles, in u.s. legal opinions, was by loeser (2015) which provides a thorough examination of sovereign citizens as paper terrorists, the problems they cause in the courtroom and the danger they pose to public officials. loeser (2015) recommends a number of legal and educational solutions for stopping the sovereign citizen threat including deterrence through effective punishment, prefiling injunctions, procedural justice, online activism, and education about the history of government. likewise, the remaining articles (kalinowski, 2019) educate the reader about the nature and beliefs of sovereign citizens, in addition to examining the rationale for why sovereign citizens often wish to represent themselves in court (phillips, 2019). the most expansive and thorough article was that of netolitsky (2018) of the 30 cases, 40% were perpetrated by either antigovernment offenders or those who were sovereign citizens, though the researchers did not specify which of the 40% were sovereigns and which were merely labeled antigovernment. their analysis also revealed that the family members of sovereigns often share the same belief system, are typically armed and may be willing to use deadly violence against law enforcement (gruenewald et al., 2016) . in an effort to examine that threat more closely, open-source information was searched to capture instances when: 1) sovereign citizens attempted to harm, did harm or who killed leos; and 2) sovereign citizens who threatened to harm leos. the focus on all forms of violence directed at police, including those who make threats, was collected in an attempt to gain a fuller picture of the dangers faced by leos from sovereign citizens. sierra-arévaloa and nix (in press), for instance, found that between 79 and 86 percent of firearm assaults on police do not which curates and archives legal news and court cases. table 1 shows that of the 75 instances in which sovereign citizens attempted to harm or did harm leos, there were 27 leos killed by sovereign citizens between 1983 and july 2020. officers killed at traffic stops, accounted for eight of the 27 (30%) deaths. eight officers (30%) were killed during ambushes, six officers (22%) were killed during police standoffs, four officers' (15%) deaths occurred while doing routine checks or serving a warrant, and one officer (3%) was killed in a gun battle in a grocery store parking lot. insert table 1 here aside from the 27 deaths, there were an additional 65 leos who were wounded. many of the sites of the violence were traffic stops, ambushes, courthouses, parking lots, and others. examples of the violence, ranged from a police officer who had his thumb bitten off, to an officer shot and wounded at a gas station. in another incident, december, 2018, a sovereign j o u r n a l p r e -p r o o f journal pre-proof citizen was pulled over for failing to come to a stop at multiple stop signs. upon requesting her driver's license, the driver stated that she did not need a license because she was not "trafficking goods nor at commerce," a common claim among sovereign citizens (pennsylvania v. smith, 2020, para. 3). she did provide a self-created identification card and claimed to be an american national. after questioning the officer's authority, she was asked to exit her vehicle, a request that she refused (rellahan, 2020) . while attempting to forcibly remove her from the vehicle, an officer's arm became entangled with hers as she drove away, dragging the officer 10 to 15 feet (pennsylvania v. smith, 2020) . she then fled, driving at a high rate of speed, eventually being apprehended. after a three-day trial, she was convicted of aggravated assault (rellahan, 2020) . some of the cases involving sovereign citizens attacks on leos are quite unusual and elaborate. consider the case of gregory lee rodvelt. in september 2018, law enforcement arrived at the scene of his home in williams, oregon. according to the affidavit, the team soon learned that mr. rodvelt had booby-trapped the property (united states v. rodvelt, 2018) . upon arrival the officers approached a gate. they noticed a circular hot tub spa that was rigged in such a way that opening the gate would activate a mechanical trigger propelling the spa to roll towards the person opening the gate. observers described the apparatus akin to something from the indiana jones movie raiders of the lost ark. they also noticed another unusual apparatus on one of the overhead garage doors: a wooden mouse trap, rigged to discharge a shotgun blast if the garage door were to be lifted. law enforcement then moved forward to gain entry into the property. inside, they would find more booby-traps. upon entry, they were surprised by a wheelchair rolling towards them. it then exploded and fired a shotgun shell that hit an fbi agent j o u r n a l p r e -p r o o f journal pre-proof in the leg. the affidavit noted that mr. rodvelt had previously been arrested for domestic violence, dui, aggravated assault, and possession of explosives and destructive devices. he had no felony convictions at the time of his latest incident. in september of 2010, the local county sheriff and two employees of a petroleum company knocked on the door of mr. white (cnn wire staff, 2010) . they wanted to speak to him about an oil well on his property and inform him that the company owned mineral rights to his land (mustian, 2010) . mr. white had been blocking the company's access roads and leaving notes expressing his unhappiness with the company using certain chemicals to kill weeds (mustian, 2010) . it was his view that they had been poisoning his water (mustian, 2010) . upon seeing the deputy at his door, mr. white began shooting, hitting the deputy three times and also news reports indicate that mr. white had a volatile history. he was attracted to guns at a young age (mustian, 2010) . he liked to practice quick drawing, once even shooting himself in the leg (mustian, 2010) . he joined the military and after his service, worked with his brothers as a pipe layer (mustian, 2010) . eventually, he was hurt on the job and began living off the money he collected as part of a civil settlement from the injury (mustian, 2010) . newspaper accounts indicate that he had been arrested in 1993 and charged with kicking in a door (mustian, 2010) . it was also around that time that he began refusing to pay property taxes on his land. he complained to his family that drivers' licenses were "unconstitutional" and once pled guilty to driving without a license (mustian, 2010) . he was sued by the school district and other taxing entities. he responded by filing court documents containing various nonsensical antigovernment articles and a bumper sticker that read "fight organized crime, abolish the irs" (mustian, 2010) . he would also visit the district's clerk's office, distributing flyers printed by the "citizens crime commission" (mustian, 2010) . the flyers were meant to warn public officials about his contention that they were violating the law and would be standing trial for their crimes. the district's clerk's office hired a security guard and installed a panic button after mr. white explicitly threatened to kill all of their lawyers. (mustian, 2010) . in a 2010 interview, while in jail after his arrest, mr. white stated that he planned to retaliate against law enforcement via civil lawsuits and reiterated his hatred for law enforcement (mustian, 2010) . in december 2012, he was sentenced to life in prison plus 20 years (vanderlaan, 2012) . even in cases where no officer was harmed by sovereign citizens, deadly force was often intended. in march 2018, a vehicle in minnesota slid off the road due to poor weather conditions. troopers stopped to assist the two moorish sovereign citizen occupants and soon learned that the vehicle had been reported stolen. as one of the troopers began to handcuff the male suspect, the female suspect held a 9mm handgun to his head and pulled the trigger. the gun misfired and no bullet exited the chamber. the trooper acquired his weapon and shot the female suspect in the arm as she ran off. it was later learned she had an active arrest warrant for sex trafficking, along with a history of drug convictions (delong & sanchick, 2018). in an unusual case involving a "free citizen," (another name for a sovereign citizen), a man refused to leave a mall parking lot. the police determined that he had a revoked driver's license and a warrant for his arrest. the situation escalated when he refused to leave his vehicle. upon inspection, the police recovered "a device from the rv that was shaped like a pig and had '(expletive) the police' written on it. the pig held an energetic powder and appeared to be an explosive device" (venzer, 2019) . the man also specifically stated that he would kill multiple officers (venzer, 2019) . no one was harmed in the incident but the threat of harm explicitly directed at leos was ever present. such was the case with james michael tesi, a "natural living being," who referred to himself as tesi el of the moorish national republic (pitcavage, 2012). mr. tesi is thought to be the only known caucasian moor. he has a history of problematic interactions with police due to his belief in "sovereign freedom to travel." additionally, he did not think it necessary to follow other traffic laws. he violated seat belt laws, speed limit laws and refused to pay his traffic tickets (slater, 2016, p. 1) . in his latest confrontation with police (july 2011} court records indicate that mr. tesi was driving, when police attempted to pull him over for outstanding warrants (tesi v. texas, 2014) . mr. tesi swerved around the police vehicles and drove to his home and parked in his garage (tesi v. texas, 2014) . police approached mr. tesi who was still in his truck. mr. tesi kicked open his driver's side door, armed with a gun, and began firing at the officer. gunfire was exchanged and mr. tesi was shot in the leg. the police officer was not hit but could have easily been killed or seriously injured in the shootout. in another instance that could have been deadly for law enforcement, an officer pulled over sovereign citizen deadly violence against leos, has also occurred during the course of an ambush. in this research, table 1 (etters, 2015). the first deputy on the scene was chris smith who was shot immediately upon arrival (etters, 2015). another officer was shot but survived because of his bulletproof vest. people who knew mr. holley, reported that he had repeatedly spoken about his intention to kill as many leos as possible. he was known to be volatile, to hate the government and to read propaganda online (etters, 2015) . an investigation revealed that two weeks prior to the shooting, mr. holley had explicitly threatened to shoot leos if they came to his residence (rossman & portman, 2014) . the day before the shooting, his ex-girlfriend had called local authorities to report his threats (etters, 2015) . none of that information was shared with first responders (rossman & portman, 2014) . table 2 documents 19 instances in which sovereign citizens threatened to harm law enforcement. some sovereigns made threats in the course of an armed standoff or in elaborate plots designed to target law enforcement, some willing to die for their cause. david allen brutsche and devon campbell newman, for instance, plotted to kidnap a leo and hold their victim hostage in a makeshift jail. after the kidnapping, the duo planned to hold a trial and then "put a bullet in his head" (powers, 2015, para. 6) . "i'm willing to give my life for this" (powers, 2015, para. 1). undercover officers thwarted their plan before it could be carried out (valley, 2013) . insert table 2 here j o u r n a l p r e -p r o o f some sovereigns brazenly contact law enforcement and explicitly threaten them via a phone call, private social media messaging or by posting it on public social media accounts. such was the case for michael hall who was cited by the police for driving an unregistered and uninsured vehicle (labella, 2018) . he sent them private messages, putting the police "on notice" for their having violated his constitutional rights, writing that he will "travel freely upon any public road in any public highway i please until the day i die" and that he had "the right to kill a cop (labella, 2018, para 2 and 3). in another instance, mitchell taebel, a known sovereign citizen with a long-troubled history, was arrested after leading law enforcement on a high-speed chase (pohl, 2018) . two persons were severely injured, during the chase, when his vehicle slammed in to theirs. in a press conference, and on a number of other occasions, he stated, that the officers involved should have been killed and that he had the right to kill them under specific sections of the uniform crime code (pohl, 2018) . his actions are in line with many sovereign citizens who often cite the u.s. constitution or common law statues that they interpret as supporting their antigovernment and anti-authority agendas. this article sought to provide an overview of sovereign citizens, summarize the empirical research about the movement and to explore the threat they pose to law enforcement. to date, there are relatively few studies of sovereign citizens. this may be due to the fact that very few in addition, sovereign citizens are not a monothetic or ideologically pure group. their ideology oftentimes overlaps with other groups including militias, antigovernment groups, and patriot/white supremacy groups. others with overlapping philosophies include the black panther party, scientology, anti-vaccination, dooms day preppers, and other movements. this complicates counterterrorism efforts that have traditionally "focus[ed] on leadership decapitation and targeting organizational logistics and finances…" (hoffman & ware, 2020, para.30) . sovereign citizens, like some other groups, are decentralized and have "…no identifiable leaders, no existing organization and no infrastructure to disrupt" (hoffman & ware, 2020, para.30 ) thus making identification and prevention a formidable task. further complicating matters, is the lack of governmental authority to clarify issues of operational definition and ideology. without such guidance, accurate accounting, especially among those without access to classified intelligence, becomes a greater challenge. studies concerning the sovereign citizen movement thus far have largely focused on legal matters, mental health, radicalization, and postdiction of targeted violence. emerging evidence suggests that most sovereign citizens are not delusional or mentally ill. their behavior defies easy diagnostic categorization and requires further study. their beliefs, though atypical in nature, are largely not indicative of disordered thought. some sovereign citizens are motivated to commit violence for reasons that have yet to be fully explored. rahman, meloy, and bauer (2019) have applied the concept of extreme overvalued belief to lone-actor terrorism and individual acts of targeted violence. whether sovereign citizen ideology should be understood as an extreme, overvalued belief, will require more in-depth study. understanding what motivates sovereign citizens could reveal important insights for developing prevention strategies. exploring the movement's threat to law enforcement involved a collection of 94 instances in which sovereign citizens did harm police officers, attempted to harm them or threatened to do so. violence aimed at police often began with routine traffic stops, where sovereigns were accused of violating basic traffic laws or had outstanding warrants. these stops were sometimes made worse by sovereigns fleeing the scene, thus escalating the situation. the findings of this research are consistent with other research indicating that most deadly far-right attacks on law enforcement are triggered by routine traffic stops (grenewald, et al., 2016) . sovereign citizen deadly violence against leos also occurred during ambushes. though traffic stops are a large danger to leos, and often trigger sovereign citizen violence, the danger of an ambush is both real and deadly. the targeted attacks on leos, both those that were carried out successfully and those that were attempted but thwarted, highlight the predatory capacity of the sovereign citizen movement. invariably, the descriptive nature of this analysis has limitations. the sample size, involving attacks and threats on leos, is too small to draw satisfactory conclusions. in addition, the data were collected from media-based reports, which are subject to errors and can lack completeness (huff-corzine, mccutcheon, & corzine, 2014) . without access to classified government data, media access, of this type, is often the only available information. relatedly, despite a thorough and ongoing search, cases may have been missed. there may also have been cases that other researchers would have categorized differently. as mentioned above, some individuals were espousing sovereign citizen ideology but could have also been labeled as tax protestors or militias members, among others. other researchers might have omitted those seemingly blended, this research took a conservative approach and only included cases where definitive sovereign ties were clear. an example of a case that was omitted was martin winters, leader of a doomsday survivalist group. mr. winters was arrested for illegally obtaining assault rifles and building bombs (sullivan & ryan, 2014) . the fbi considered him an "extremely dangerous" individual who plotted to kill government agents, should they invade his property (masferrer, 2014) . one journalist labeled him a sovereign citizen but since his connection to the movement could not be independently verified, a decision was made to eliminate him from this sample of cases. despite its drawbacks, a descriptive approach can be valuable, especially given the limited empirical information available concerning sovereign citizens. even more limited is the information available to guide law enforcement agencies, who do interact directly with sovereign citizens, in the course of routine police work (grenewald, et al., 2016; smith, 2019) . given leo's regular contact with sovereign citizens, and the danger posed by sovereigns, the lack of mandated training programs, on this movement, is concerning (smith, 2019) . some law enforcement agencies offer optional training. such training should not be optional but instead mandatory and 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in tiny wa town as 'sovereign nation' attempts to overthrow government. the age the challenges of effective counterterrorism intelligence in the 2020s. law fare shooting for accuracy: comparing data sources on mass murder a legal response to the sovereign citizen movement we the people": rant by maskless s'porean woman in shunfu details emerge about man who shot chp officer on i-680 near alamo profiles of individual radicalization in the united states (pirus) man charged with sending police threatening messages from paper terrorists to cop killers: the sovereign citizen threat fbi arrests 'doomsday prepper the lone-actor terrorist and the trap-18 anatomy of a standoff. oa online moorish nationalist couple arrested again for trying to claim rights to a dc home: police overlap between black separatists and moorish sovereign citizen extremists the sovereign citizen movement and fitness to stand trial extreme overvalued belief and the legacy of carl wernicke immaculata student guilty in officer assault. daily local news 3 emergency dispatchers fired following deputy's shooting. tallahassee democrat a psychological and criminological analysis targeted violence against law enforcement officers. behavioral sciences and the law gun victimization in the line of duty: fatal and non-fatal firearm assaults on police officers in the united states sovereign citizen movement: an empirical study on the rise in activity, explanations of growth, and policy prescriptions (unpublished master's thesis) what you don't know about sovereign citizens can hurt you sovereign citizens movement state of minnesota v. blanshan. 11-cr-15-539. state of minnesota in court of appeals an examination of the american far right's anti-tax financial crimes doomsday prepper martin winters surrenders, held without bail. tampa bay times post-9/11 intel center now going after domestic terror. the daily best fbi abandons use of term 'black identity extremism 20-12-00096-cr. court of appeals 297 th district court of tarrant county apex court upholds former mister germany's prison sentence money-making schemes that ensnare prisoners and their families. the marshall project united states v. kriemelmeyer. 07-cr-052-c-01. united states district court for the western district of wisconsin united states v. rodvelt. 00202-cl. united states district court for the district of domestic violent extremists pose increased threat to government officials and law enforcement metro infiltrates sovereign citizens movement, uncovers plots to 'snatch,' execute officers white sentenced to life. oa online free citizen' with apparent pig-shaped, 'f-the police' explosive charged in maplewood standoff this couple are urging fellow anti-vaxxers to pay to have their name and address added to form letters key: cord-268094-ubz0q7e9 authors: curland, n.; gethöffer, f.; van neer, a.; ziegler, l.; heffels-redmann, u.; lierz, m.; baumgärtner, w.; wohlsein, p.; völker, i.; lapp, s.; bello, a.; pfankuche, v. m.; braune, s.; runge, m.; moss, a.; rautenschlein, s.; jung, a.; teske, l.; strube, c.; schulz, j.; bodewes, r.; osterhaus, a. d. m. e.; siebert, u. title: investigation into diseases in free-ranging ring-necked pheasants (phasianus colchicus) in northwestern germany during population decline with special reference to infectious pathogens date: 2018-02-06 journal: eur doi: 10.1007/s10344-018-1173-2 sha: doc_id: 268094 cord_uid: ubz0q7e9 the population of ring-necked pheasants (phasianus colchicus) is decreasing all over germany since the years 2008/2009. besides impacts of habitat changes caused by current rates of land conversion, climatic influences or predators, a contribution of infectious pathogens needs also to be considered. infectious and non-infectious diseases in free-living populations of ring-necked pheasants have been scarcely investigated so far. in the present study, carcasses of 258 deceased free-ranging pheasants of different age groups, predominantly adult pheasants, collected over a period of 4 years in the states of lower saxony, north rhine–westphalia and schleswig-holstein, were examined pathomorphologically, parasitologically, virologically and bacteriologically, with a focus set on infectious pathogens. a periocular and perinasal dermatitis of unknown origin was present in 62.3% of the pheasants. additional alterations included protozoal cysts in the skeletal musculature (19.0%), hepatitis (21.7%), enteritis (18.7%), gastritis (12.6%), and pneumonia (11.7%). in single cases, neoplasms (2.6%) and mycobacteriosis (1.7%) occurred. further findings included identification of coronaviral dna from trachea or caecal tonsils (16.8%), siadenoviral dna (7.6%), avian metapneumoviral rna (6.6%), and infectious bursal disease viral rna (3.7%). polymerase chain reaction (pcr) on herpesvirus, avian influenza virus (aiv), paramyxovirus type 1 (pmv-1), avian encephalomyelitis virus (aev), and chlamydia were negative. based on the present results, there is no indication of a specific pathogen as a sole cause for population decline in adult pheasants. however, an infectious disease can still not be completely excluded as it may only affect reproduction effectivity or a certain age group of pheasants (e.g., chicks) which were not presented in the study. the asian pheasant was introduced to europe by the romans (pennycott 2001) . meanwhile, phasianus colchicus (ph. c.) represents hybrids of different species (ph. c. torquatus and ph. c. mongolicus) in germany (glutz von blotzheim 1994) and is also called ring-necked pheasant because of its white neck feathers. considerable populations are limited to four federal states in germany, namely lower saxony, north rhine-westphalia, schleswig-holstein, and bavaria. the preferred pheasant habitat is wooded agriculture land with open fields for foraging and display but also with woodland edges and shrubs which provides protective cover (goldová et al. 2006) . reintroduction of reared wild or farmed pheasants is not as common in germany as in other countries, like the uk. therefore, population trends can be used as a realistic indicator for reproductive success and mortality rate. the ongoing decrease-indicated by hunting index and estimated population density (gethöffer et al. 2011 )-was the reason for this study assessing the prevalence of infectious and non-infectious diseases in the pheasant population. however, little is known about the actual occurrence and pathogenicity of pathogens among free-living populations of phasianus colchicus (aldous and alexander 2008) . published data mostly refer to birds held in captivity (bertran et al. 2009; pennycott 2001; welchman 2008; welchman et al. 2002) . as a member of the order galliformes, pheasants may also be infected by pathogens of poultry. concerning viral infections such as newcastle disease, infectious laryngotracheitis and infectious bronchitis which occur in game birds including pheasants, transmission is described as a result of bdirect or indirect contact with domestic birds or wild bird vectors ( aldous and alexander 2008) . like infectious bronchitis virus (ibv), the pheasant coronavirus (phcov) belongs to the group 3 of coronaviruses. cavanagh (2005) provides an overview about coronaviruses, including ibv and phcov, causing alterations in the respiratory tract and kidneys of pheasants (gough et al. 1996; lister et al. 1985; pennycott 2000; spackman and cameron 1983; welchman et al. 2002) . signs varied from sneezing, pale and swollen kidneys, visceral gout and high mortality rates (gough et al. 1996; pennycott 2000) , over effects on egg production (spackman and cameron 1983) , to sinusitis, airsacculitis and conjunctivitis, upper and lower respiratory tract diseases without signs of nephritis or effects on egg production . descriptions of coronavirus infections based on farm-raised pheasants. to the best of our knowledge, there are no data available from free-ranging pheasants. avian influenza viruses are divided into low pathogenic-and highly pathogenic avian influenza viruses (lpaiv and hpaiv) due to their pathogenic potential and into different subtypes by the antigenic domains of the haemagglutinin and the neuraminidase receptors. hpaiv have only been described for serotype groups h5 and h7 (iqbal et al. 2014) . since 2006, avian influenza virus has been detected several times in poultry and free-ranging birds of different species, mostly in galliformes and anseriformes. likewise, phasianidae are also susceptible. in a study on golden pheasants (chrysolophus pictus), most animals showed severe morbidity and mortality when artificially infected with hpaiv (van der goot et al. 2007 ). after infection with lpaiv pheasants shed virus for up to 45 days and most of the subtypes could be passed to contact pheasants (humberd et al. 2006) . avian metapneumovirus (ampv) is the causative agent of turkey rhinotracheitis and respiratory infections in other birds belonging to the order galliformes. main clinical signs in turkeys are related to the respiratory tract, as well as to the reproductive tract leading to loss in egg production and egg quality. in chickens, ampv-infection may often be mild or sub-clinical. because ampv-infection is often accompanied by other infectious agents of the respiratory tract such as ibv and different bacterial pathogens, it is not possible to identify the importance of this pathogen in respiratory diseases of pheasants yet (jones 1996) . serological proof of ampv infections in free-ranging pheasants in italy showed susceptibility of pheasants to this pathogen (catelli et al. 2001 ). in captive pheasants, an infection with avian encephalomyelitis virus has been described (welchman et al. 2009 ), but there is no evidence for the occurrence of this pathogen in free-living populations yet. furthermore, marble spleen disease caused by siadenovirus has already been described as a common disease in pheasants raised on farms but not yet in free-ranging individuals (fitzgerald et al. 1992; fitzgerald and reed 1989; pennycott 2000) . the disease occurs naturally in pheasants 3-8 months of age (swayne 2013) . characteristic pathomorphological lesions are splenomegaly associated with yellow and gray-white spots on the spleen's surface resulting in the characteristic marble-like look (orlic et al. 2003) , but normal appearance of spleen can also occur (pennycott 2000; pennycott 2001) . other typical gross lesions are oedematous congested lungs (fitzgerald and reed 1989; swayne 2013 ). data about the occurrence of infectious bursal disease in pheasants are controversial. in china, antibodies against infectious bursal disease virus (ibdv) were detected in 14 out of 40 samples of free-ranging pheasants (gu et al. 1998) . additionally, in a clinical trial about 20% of pheasants and guinea fowls experimentally infected with ibdv died (gu et al. 1998) . in another study, pheasants experimentally inoculated with serotype 1 viruses failed to induce any clinical signs (berg et al. 2001; ingrao et al. 2013 ). in the recent years, a flavivirus affecting game birds was emerging in central europe, especially in spain, the bagaza virus (bagv). this virus caused high mortality rates in red-legged partridges (alectoris rufa) and ring-necked pheasants (phasianus colchicus). in pheasants, bagv caused neurologic signs and was detected in the brain, while in red-legged partridges it showed pathomorphological changes even in other organs and could be detected in other tissues as well (gamino et al. 2012) . other flaviviruses have shown how fast conditions can change and new viruses emerge, like the usutu virus, which led to high mortality rates in eurasian blackbirds (turdus merula) (chvala et al. 2007 ) and the west nile virus (wnv), whose natural reservoir are birds (gamino and höfle 2013) . furthermore, diseases induced by different bacterial pathogens are common in captive pheasants. farm-raised pheasants experimentally infected with pasteurella multocida causing avian cholera showed a high mortality of 38% within the first 24 h. typical findings include haemorrhages in the subcutis and subserosa, hepato-splenomegaly with necrotic spots and fibrinonecrotic pneumonia, fibrinopurulent pleuritis and purulent airsacculitis. pheasants were also found to be highly susceptible (botzler 1991; petersen et al. 2001) . pheasants and domestic fowl appear to be very susceptible birds for avian tuberculosis caused by mycobacterium avium subsp. avium (prukner-radovcic et al. 1998) . avian tuberculosis, also termed avian mycobacteriosis, is a chronic disease characterized by granuloma formation in various organs (kul et al. 2005; moravkova et al. 2011 ). different mortality rates have been observed after infections with salmonella (s.), depending on the age of infected animals and serovar. for example, s. enterica serovar agona infection of farmed pheasants in japan, caused mortality rates ranging from 4 to 56% in different years and age groups. further pathomorphological findings are pericarditis and icteric liver (myoujin et al. 2003) . several mycoplasma (m.) species are commonly isolated from healthy pheasants and therefore do not seem to play a role as pathogens (e.g. m. glycophilum, m. pullorum, m. iners) (bradbury et al. 2001 ). however, m. gallisepticum has been described as a pathogen of severe respiratory disease associated with high mortality rates in captive pheasants. therefore, m. gallisepticum plays a significant role as pathogen of respiratory disease in pheasants (bencina et al. 2003; pennycott 2001; welchman 2008; welchman et al. 2002) . among parasitic infections, capillaria spp. (bencina et al. 2003; floristean et al. 2002; goldová et al. 2006) , syngamus trachea (gethings et al. 2015) , ascaridia spp. (pavlovic et al. 2003) , heterakis spp. (draycott et al. 2000; pavlovic et al. 2003) , and coccidia spp. (ruff 1999 ) may cause reduced fitness or increased mortality in captive pheasants and occur in free-ranging pheasants as well. for example, negative relationship between heterakis sp. and body condition was detected (villanua et al. 2006) . furthermore, heterakis gallinarum can transmit the pathogenic protozoan histomonas meleagridis, which is the agent causing bblackhead-disease^ (ruff et al. 1970) . as an example for indirect influence of parasites, eucoleus contortus seems to have no impact on the pheasants' condition, but these birds were preyed by foxes more than expected (millan et al. 2002) . moreover, an effect of the ectoparasite ixodes ricinus on survival and breeding success could be observed in britain, where tick infestations influenced breeding success and survival of female pheasants (hoodless et al. 2003) . the effect of infestation with chewing lice is an irritation and discomfort of their host, which the affected pheasants show by scratching their body (goldová et al. 2006) . the aim of the present study was to elucidate pathogens in free-ranging pheasants during the current population decline in northwestern germany using pathomorphological, virological, microbiological and parasitological investigations. for this study, pheasant carcasses collected between 2011 and 2014 originated from seven administrative districts in northwestern and northern lower saxony (n = 172; 66.7%) (cloppenburg, cuxhaven, grafschaft bentheim, emsland, osnabrück, stade, vechta). these districts are characterized by a high hunting bag of ring-necked pheasants within the last 40 years followed by a severe decline since 2008. additionally, samples obtained from pheasants from administrative districts of north rhine-westphalia (n = 60; 23.3%) (borken, coesfeld, hamm, krefeld, steinfurt, warendorf, wesel, euskirchen, gütersloh, recklinghausen, soest, viersen) and schleswig-holstein (n = 26; 10.1%) (dithmarschen, rendsburg-eckernförde) were collected from 2013 to 2014 and analyzed additionally. the pheasants, altogether 258 individuals, were found dead, sick, or obtained from hunting bags and mainly demonstrating abnormalities. the carcasses were sent by local hunters to the institute for terrestrial and aquatic wildlife research (itaw) in hannover and stored at − 20°c until post-mortem examination. pathomorphological, bacteriological, virological and parasitological analyses were conducted depending on the preservation of the carcass (table 1) . necropsies on all carcasses were conducted according to a standard procedure (siegmann et al. 2005 ) and samples of trachea, thyroid gland, lung, heart, liver, spleen, crop, proventriculus, gizzard, pancreas, small intestine, caecum, kidney, adrenal glands, infraorbital sinus, nasal cavity, gonads, cerebrum, cerebellum, spinal cord, peripheral nerve, thymus, skin, musculature, bones, bone marrow, caecal tonsils and bursa of fabricius were collected, if conservation status permitted. histopathological investigations were performed on 230 carcasses and in one additional case, only muscular tissue was examined. the nutritional status was scored as good, moderate, poor or cachectic. animals in a good body condition revealed a vast amount of fatty tissue within the thoracic and abdominal regions, whereas animals with a moderate body condition demonstrated reduced amounts of body fat tissue. animals in a poor body condition possessed only low amounts of fat reserves frequently associated with pectoral muscle atrophy. in contrast, cachectic animals lacked fat reserves and displayed a serous atrophy of the coronal myocardial fatty tissue. gender was determined during necropsy by sex dimorphism and gonads. the investigated pheasants consisted of 128 males (49.6%) and 118 females (45.7%), in 12 cases (4.7%) gender could not be determined because of autolysis or lacking parts of carcass. classification into age groups followed a correlation of different parameters: season, preliminary report, manifestation of sex dimorphism, body size and weight, and histopathological investigation of gonads (geriatric signs, active spermiogenesis, status of ovarian follicles). from hatching to sexual maturity, birds were referred to as juvenile, after this period, they were regarded as adults. thus, all pheasants were classified into adult (n = 213; 82.6%) or juvenile (n = 36; 14.0%), in nine cases age could not be classified (3.5%). for histopathology, tissue samples were fixed in 10% neutral-buffered formalin for 24 h and embedded in paraffin wax according to a standard laboratory procedure. tissue sections, cut at 4 μm, were stained with haematoxylin and eosin (he). depending on histological findings, selected additional sections were stained with periodic acid-schiff (pas) reaction, congo red, von kossa's, ziehl-neelsen's, gram and grocott's methenamine silver and heidenhain's azan trichrome stain according to standard laboratory protocols (welsch et al. 2010 ). for endoparasitic examinations, intestinal content of 225 pheasants was available for the combined sedimentationflotation method. the fecal sample was given in a tea strainer (mesh size 1 mm) and rinsed in a beaker with a jet of water. the filtrate containing helminth eggs and protozoan oocysts was allowed to sediment for 30 min. then the supernatant was decanted and the sediment was transferred into a 15-ml centrifuge tube, filled up with saturated zinc sulfate solution (znso 4 , specific gravity 1.30) and centrifuged at 450×g for 5 min. the liquid surface was transferred onto a slide with a wire eyelet and examined microscopically. if at least one egg or oocyst per intestinal content was detected, the sample was classified as bpositive^. a semiquantitative classification was applied using the following key: 1-5 eggs or oocysts were categorized as mild, 6-10 eggs or oocysts as moderate, 11-20 eggs or oocysts as severe and when more than 20 eggs or oocysts were detected, the sample was classified as enmasse. macroscopic evaluation of skin and plumage for ectoparasites was performed in 193 pheasants. in 2013, tissues samples from liver, heart and spleen, and on the basis of lesions in other organs as well, were investigated for bacterial growth using columbia sheep blood-agar (csb), and cystine lactose electrolyte deficient-agar (cled; both oxoid, wesel, germany). agar plates were incubated for 24 h at co 2 enriched atmosphere (5% co 2 ) (csb) or aerobically (cled) at 37°c and monitored for bacterial growth, repeated after 48 h. bacterial species identification was done from pure subcultures with biochemical methods using the commercial identification systems api® 20 e, api® 20 ne, api® staph and rapid id 32 strep (biomerieux, nuertingen, germany) and in-house methods. salmonella-specific enrichment and cultivation was done from heart, liver and spleen tissue following standard procedures of din en iso 6579, appendix d ((nal) 2007). in the year 2014, only organs from pheasants with pathomorphological changes were subjected to bacteriological examination. for molecular detection of chlamydia spp. dna was extracted from spleen samples (25 mg) with the nucleospin tissue kit™ (macherey-nagel, düren, germany) according to the manufacturer's instructions. the pcr was performed according to the standards of the national reference laboratory for chlamydiosis, friedrich-loeffler-institute, insel riems, germany, based on the original method (ehricht et al. 2006) . in five selected pheasants, investigations regarding mycoplasma spp. were performed. three adult pheasants with periocular dermatitis and two chicks, which belonged to one of these pheasants and died due to an unclear reason, were investigated. tissue samples of altered periocular skin (n = 5), trachea (n = 3) or tracheal swabs (n = 2) were cultured using sp4 liquid and agar media as described by bradbury et al. (miles and nicholas 1998) . the samples were immersed in sp4 broth before they were removed and stored at − 80°c until further investigations, respectively. a 10-fold dilution virology (next generation sequencing) 2 series up to 10 −2 was made and an aliquot of 50 μl was transferred onto agar plate. liquid and solid medium were incubated in an atmosphere of 5% co 2 for up to 10 days at 37°c, respectively. the agar plates were examined daily for mycoplasmal growth and the liquid broth was checked daily for colour changes indicating ph changes due to mycoplasma metabolism. in case of mycoplasmal growth, single colonies were taken for differentiation. for dna extraction from all tissue samples, swabs and the single colony subcultures the dneasy ® blood & tissue kit (qiagen, hilden, germany) was used according to the manufacturer's instructions diluted to 10 ng/μl. all samples were tested via mycoplasma-genusspecific pcr (vankuppeveld et al. 1992 ) and via mycoplasma gallisepticum specific pcr (hagen et al. 2004 ). mycoplasmagenus-specific pcr-products from single colony subcultures were sequenced by a commercial dna sequencing service (lgc genomics, berlin, germany). the bursa of fabricius was investigated for ibdv using rt-pcr (jackwood and sommer-wagner 2005; negash et al. 2012 ), a tracheal sample for ampv by rt-pcr (cavanagh et al. 1999) , and splenic tissue for siadenovirus by pcr (hess et al. 1999) as described before. samples of trachea and caecal tonsils were investigated for coronavirus by rt-pcr as described . the rt-pcr kit superscript® iii one-step rt-pcr system with platinum® taq dna polymerase (thermofischer scientific) was used for detection of ibdv and coronavirus, and the improm-ii™ reverse transcription system (promega) and the taq polymerase peqgold (peqlab biotechnologie gmbh, erlangen, germany vertrieben über vwr) for detection of ampv. rna was isolated from bursae, tracheae and caecal tonsils using peqgold trifast® reagent (peqlab, erlangen, germany) following the instructions of the manufacturer. dna was isolated from spleen samples with the innuprep dna mini kit (analytik jena, germany). furthermore, detection of paramyxovirus type i by qrt-pcr was performed as described before (fuller et al. 2010; teske et al. 2013 ), avian encephalomyelitis virus by rt-pcr (ottiger 2010) , and herpesvirus by pcr with taq polymerase (peqlab, erlangen) (pathogenesis corporation et al. 1996) . for detection of avian influenza virus by qrt-pcr virotype-influenza-a-rt-pcr-kits (qiagen co. (ldl)) were used, following the instructions of the manufacturer. frozen tissues of two pheasants were tested exemplary for the presence of viral sequences as a potential cause of histologically detected lesions by random pcr in combination with next-generation sequencing (viral metagenomics) as formerly described (van leeuwen et al. 2010 ). the investigation was performed on frozen brain and liver tissue of one pheasant with a lymphohistiocytic hepatitis and a periocular dermatitis and on periocular skin of another pheasant presenting with periocular dermatitis and conjunctivitis. obtained reads were analyzed by blastn and blastx analysis (schurch et al. 2014) . to possibly obtain more reads in positive case of a virus and check for its pathogenicity, the supernatant of homogenized tissue was subsequently inoculated into 11-day-old embryonated chicken eggs. after inoculation, eggs were harvested, embryos were culled and subsequently checked for the presence of macroscopic lesions. most pheasants were in good (n = 179; 69.4%), the remaining moderate (n = 40; 15.5%), poor (n = 4; 1.6%) or cachectic (n = 7; 2.7%) body condition. due to advanced autolysis and decomposition of some carcasses (n = 28; 10.9%), nutritional status was not assessed (fig. 1) . a poor nutritional status was documented in four cases. one juvenile pheasant suffered from a severe coccidiosis and was found positive for ampv-infection. another case showed a pyogranulomatous serositis and pneumonia and aeromonas hydrophila and e. coli were cultured from fibrin and renal tissue. in two cases, the cause for the poor nutritional remained unclear. in seven pheasants, cachexia was found. the causes included granulomatous-necrotising oophoritis caused by e. coli, neoplasia, granulomatous inflammations in multiple organs, severe gastritis, and severe capillaria spp. shedding, severe chronic tracheitis and severe hepatitis. in two cases, the cause remained undetermined. changes present in more than 10% of investigated birds (n = 230) are described in the following text, for less frequent alterations see table 2 . besides, 46.5% of the pathomorphological investigated pheasants demonstrated inflammatory lesions in multiple organs. ninety-nine out of 159 pheasants (62.3%) demonstrated inflammatory alterations in the dermis. mostly, lesions were found in the periocular dermis (n = 79; 79.8%), whereas the back part of the head (n = 46; 46.5%), abdomen (n = 30; 30.3%), skin adjacent to the cloaca (n = 12; 12.1%) or other localizations (n = 4; 4.0%) were affected less frequently, whereof cloaca and other locations were not investigated regularly. in 57 birds (57.6%), multiple skin locations were affected. non-purulent mostly perivascularly accentuated inflammations with different cellular compositions and gradual variable infiltrations of lymphocytes, plasma cells and macrophages were detected in 68 birds (68.7% of affected pheasants) (fig. 2) . suppurative inflammation occurred as pustular, focally suppurative, necro-suppurative and phlegmonous variants (n = 9; 9.1%). granulomatous (n = 7; 7.1%), ulcerative (n = 6; 6.1%) and necrotising (n = 1; 1.0%) forms were additionally observed. in some cases (n = 8; 8.1%), histological details were not discernable due to poor tissue preservation. in 34 affected pheasants (34.3%), dermatitis was mild or mild to moderate, the remaining 65 affected birds (65.7%) showed a moderate or severe dermatitis. inflammatory skin lesions were found in adult birds (n = 81, 64% of investigated adult birds) as well as in juvenile pheasants (n = 18, 50% of investigated juvenile birds) of both sexes (female: n = 51, 61% of investigated female birds; male: n = 47, 67% of investigated male birds; not determined: n = 1). the skin of a juvenile pheasant with severe perivascular infiltration is shown in fig. 2 . in 11.9% (n = 19) of investigated pheasants, dermal abrasions or lacerations were diagnosed, which probably were caused by trauma. stomach infiltration of the tunica mucosa with inflammatory cells were detected in 29 birds (12.6%). in most cases (n = 16; 7.0%) only the proventriculus, in 11 cases only the gizzard, and in two cases both parts of the organ were affected. infiltration consisted of variable compositions of plasma cells, lymphocytes and macrophages (n = 26). furthermore, single granulomatous inflammations (n = 3), one purulent gastritis and one necrotising proventriculitis were observed. intestine inspection of the intestine revealed enteritis in 43 pheasants (18.7%). catarrhal enteritis was supposed macroscopically in cases with foamy or liquid consistence and greenish to yellowish or brownish-beige colour of the intestinal content (n = 21). intestinal inflammation was observed histopathologically in 22 pheasants. the small intestine was affected in 9.1% (n = 21), both, small and large intestine, in 1.3% (n = 3) and caeca in 0.4% (n = 1). the character of the inflammation was mainly non-purulent with different compositions and gradual variations of infiltration by lymphocytes, plasma cells, eosinophilic granulocytes, macrophages (n = 17) and granulomatous (n = 1). additionally, in one case the caeca were affected by moderate fibrinous inflammation. liver in the liver various forms and severities of hepatitis and hepatocellular necrosis (n = 53, 23.0%) were present. inflammatory alterations consisted of infiltration of varying numbers of lymphocytes, plasma cells and macrophages in 25 pheasants (10.9%). necrosis and necrotising inflammations of the liver were found in 16 birds (7%). granulomatous and granulomatous to necrotizing hepatitis occurred in nine birds (3.9%). fractures of skeletal bones occurred in 28.3% (n = 65) of the investigated pheasants, and muscular lacerations were diagnosed in 10.8% (n = 25). a hyaline degeneration or atrophy of muscle fibers were observed in 57 pheasants (24.7%). muscle degeneration occurred mainly in traumatized birds (n = 28; 12.1%), but also pheasants which died in consequence of an inflammatory alteration of another organ system (n = 7; 3.0%), were shot (n = 6; 2.6%) or euthanized (n = 6; 2.6%) fig. 1 nutritional status of the investigated pheasants; birds were divided into shot or dead-found individuals revealed this alteration. myositis was detected in 10.0% (n = 23) of investigated pheasants. cell infiltration consisted mainly of variable compositions of plasma cells, lymphocytes and macrophages (n = 20; 8.7%). in the other cases, inflammatory character was necro-suppurative (n = 1; 0.4%), fibrinopurulent (n = 1; 0.4%) or not assessable (n = 1; 0.4%). pneumonia was diagnosed in 27 animals (11.7%). inflammatory alterations consisted of non-suppurative interstitial (n = 10), granulomatous to pyogranulomatous (n = 7), suppurative to necro-suppurative (n = 4), fibrino-purulent (n = 2) and necrotising (n = 1) forms. in two cases auto-and heterolysis prevented histological determination of the inflammatory character. a moderate and a mild to moderate lymphohistiocytic bronchitis was present in two pheasants, once associated with pneumonia. histopathological examination of the spleen revealed haemosiderosis in 40 birds (17.4%). in 82 (n = 225; 36.4%) examined pheasants, endoparasitic infections were detected by intestinal content examination (table 3) . coccidia oocysts were found in 52 (23.1%) individuals. furthermore, eggs of capillaria spp. (n = 32; 14.2%), ascaridia/heterakis spp. (n = 28; 12.4%), syngamus trachea (n = 10; 4.4%), trematodes (n = 4; 1.8%) and raillietina spp. (n = 1; 0.4%) were detected. in 30 pheasants, a combination of more than one endoparasitic species was found. most frequent was a combined shedding of coccidia oocysts with capillaria spp. eggs (n = 7; 3.1% of investigated pheasants), followed by a combination of coccidia oocysts with eggs of ascaridia/ the italic numbers only show percentage of effected birds. it is just a description of amount of positive birds 1 balt = bronchus-associated lymphoid tissue heterakis spp. (n = 6; 2.7% of investigated pheasants). other combinations varied greatly and same compositions only occurred in three pheasants or less and combined between two species up to four different endoparasites. additionally, histopathological examination of muscle tissue showed cysts with a typical appearance of sarcosporidia in 44 cases (19.0%). three randomly selected muscle samples were examined via genus-and in positive cases via speciesspecific pcr for atoxoplasma sp., isospora sp., eimeria sp., toxoplasma gondii, plasmodium sp., haemoproteus sp., leukozytozoon sp. and sarcocystis sp. in all three tested samples, genome of sarcocystis sp. was detected. the its-1 gene showed 87% similarity and the 18s rdna gene showed a 99% similarity to sarcocystis anasi. macroscopic evaluation revealed that 11 (5.7%) of the 193 examined pheasants were infested with feather-chewing lice (order phthiraptera, suborder ischnocera), and one pheasant (0.5%) showed infestation of a tick (ixodes ricinus). the results of the microbiological investigations are summarized in table 4 . no noticeable accumulation of a certain bacterial species was detected. mycoplasma (m.) spp. were detected in three out of five investigated birds via genus-specific pcr. all positively tested birds were adult, negatively tested birds were juvenile. m. spp. were detected in the trachea of three and additionally in the periorbital skin of one investigated birds, respectively. in one case, culturing was also successful for a trachea sample, but failed in the other cases due to bacterial contamination. the pcr product of the isolated single colony showed a 99% similarity to m. pullorum. m. gallisepticum was not detected. additionally, pcr to detect chlamydia spp. was negative in all 179 cases investigated. rna of avian influenza virus, avian encephalomyelitis virus, herpesvirus and paramyxovirus type 1 were not detected in any case (table 5 ). in eight out of 121 cases (6.6%) avian metapneumovirus (ampv)-specific genome fragments were found, in three cases associated with different alterations in the respiratory system. in two of these ampv-positive cases, coronavirus was additionally identified. the other five birds did not reveal inflammatory lesions in the respiratory tract. infectious bursal disease virus (ibdv) genome was detected in one out of 27 pheasants (3.7%) without corresponding alterations. pcr on siadenovirus gave a positive result in nine out of 119 cases (7.6%). in one of these pheasants, alterations in spleen (lymphatic depletion) and lung (pneumonia) were detected pathohistologically. coronavirus was detected by rt-pcr in 19 out of 113 birds (16.8%), four of which displayed morphological changes that have been described in pheasants naturally infected with coronavirus (nephritis (n = 1), pneumonia (n = 2), rhinitis (n = 2), sinusitis (n = 1)). most of the corona-rna positive birds died due to a trauma (n = 13), like the four individuals mentioned before. three pheasants died in consequence of an inflammation, another one because of asphyxia and the cause of death of the last two pheasants remains unclear. inflammatory changes, leading to death, were of different kind: one bird revealed multiple granulomatous inflammatory changes in different organs, another one had a moderate fibrinous typhlitis and the last one showed injuries with bacterial colonization and probably died due to a septicaemic shock. all these positive samples originate from different places of the sampling area and most of them revealed a good body condition (n = 15). no viral sequences were detected in the brain and liver of the first investigated pheasant, suffering from hepatitis and dermatitis. within the periocular skin of the second tested pheasant showing periocular dermatitis and conjunctivitis, one read was detected that was most closely related to reticuloendotheliosis virus strain ha1101 (89% identity on the amino acid level). after inoculation and harvesting, no macroscopic lesions were detected in the embryos. pathomorphological results of the necropsies showed different causes of death such as non-infectious causes (n = 145; 56.2%), different inflammations (n = 13; 5.0%), and cardiac and circulatory failure of unknown cause (n = 32; 12.4%). of the non-infectious causes, most were traumata (especially supposed road accident or predation) (n = 108; 41.9%). furthermore, 68 pheasants (26.4%) were shot during hunting and necropsied, in most cases due to abnormal clinical or morphological findings. a serious population decline in free-ranging pheasants in northwestern germany occurs since the years 2008/2009. the occurrence of pathogens and morphological changes were investigated in 258 pheasants collected between 2011 to 2014 in order to elucidate their role in the pheasant's deaths. this study was focused on a possible role of infectious diseases in the population decline during the recent years. according to sample size, morphological and aetiological findings, the study design was descriptive. the majority of investigated pheasants died due to non-infectious causes. especially, the high number of traumatized birds may result from the circumstances of sample collection. dead pheasants lying next to roads are found more often by humans than dead pheasants in the fields. however, different changes demonstrated even in the traumatized pheasants, provide hints to infectious pathogens, which are capable to play a role in the decline, although they seem not responsible for death in these cases. the investigated pheasant population deals with different inflammations of unknown causes. as mainly subadult and adult birds (82.6%) were investigated, the health of chicks in the german pheasant population is largely unknown. additionally, factors only affecting reproduction have to be considered into account. dermatitis was a frequent finding that has not been reported so far in pheasants or other free-ranging or captive birds yet. the type of inflammatory lesions differed between individuals and many birds showed more than one affected localization, but periocular skin was the primary location showing changes, but not obligatory. dermatitis of deeper parts of the skin without macroscopically detectable alterations was regularly diagnosed. both, carcasses and shot birds of all ages and both sexes were affected. the aetiology and the clinical relevance of this dermatitis remain unclear. due to the different characters and localizations of the inflammatory changes various causes are likely including mechanical impacts (e.g. scratching), ectoparasites as well as primary and secondary bacterial infections. in rowi (apteryx rowi), a cutaneous larva migrans of trichostrongylus species was detected as causal agent of dermatitis (gartrell et al. 2015) . another possible factor has been described in turkeys that were immunosuppressed by virus infections with subsequent impaired cutaneous antimicrobial barrier and increased susceptibility for opportunistic pathogens, like clostridial infections (huff et al. 2013; thachil et al. 2014) . a contribution of bacteria towards the inflammatory reaction in the tissues cannot be ruled out so far. additionally, an allergic reaction or an inflammatory response to toxins has to be considered (pass 1989) . further investigations on the cause, including pesticides, bacteria, bacterial toxins and immunosuppressive factors, should be performed. at this point, an influence on the pheasant's well-being cannot be verified, evaluated or excluded. inflammatory alterations in other organs occurred, but did not show a frequency or consistent pathological image, which could explain population decline. but taken into account the quantity of pheasants affected by more than one alteration (46.5%), the entirety of different inflammatory alterations in different organs may hint towards increased susceptibility towards diseases or a general weakening of the pheasants (fairbrother et al. 2004) . therefore, a more general effect of immunosuppressant or environmental factors seem likely, paving the way for a variety of opportunistic pathogens. even if it seems many adult individuals can still deal with these inflammatory changes, we do not know about influences on reproduction and offspring. the majority of investigated pheasants demonstrated a good body condition (69.4%). therefore, it seems food availability is still sufficient for adult individuals. however, a reduction of food availability can also influence reproduction afford (draycott et al. 2005; hoodless et al. 1999) . furthermore, pheasant chicks are insectivore in their first weeks of life, and only few juvenile pheasants were included in this study. accordingly, to evaluate food availability, more bhealthy looking^pheasants, in different seasons and different age groups should to be investigated. histopathological examination of skeletal musculature revealed sarcosporidia cysts without inflammatory reaction in 44 cases (19%). in farm-raised pheasants in slovakia microcysts of sarcocystis spp. were found in 30% of the samples (goldová et al. 2006) , in czech republic shot pheasant originating from one pheasantry were examined and 36.5% revealed muscular sarcocystosis (cerná and pecka 1984) . sarcosporidia have a two-host lifecycle with herbivores or omnivores as intermediate hosts and carnivores as definitive hosts. in the intermediate hosts, mature cysts are mostly found in striated muscle (olias et al. 2009 ). appearance of clinical signs or morphological changes are not described in many studies (barrows and 1977; box et al. 1984; dubey et al. 2010 ). an encephalitis, as recently described in pigeons infected with a hitherto unknown sarcocystis sp. (olias et al. 2009; olias et al. 2013) , was not observed in the investigated pheasants. if other inflammations occur or if the parasite is of clinical importance remains undetermined, although not likely. in the present study, 116 (51.6%) out of 225 pheasants displayed endoparasitic infections (tab. 4). on pheasant farms, a prevalence of endoparasitic infections of 82.5% in czech republic and 48.2% in slovakia is described (goldová et al. 2006) . a study on the occurrence of endoparasites in germany from 1999 to 2000 which includes mainly free-ranging pheasants showed an infestation of ecto-and endoparasites in 96.7% of pheasants (gassal and schmaschke 2006) . therefore, the results of our study show no excessive prevalence of endoparasites. most pathogenic species are roundworms (syngamus trachea, capillaria spp., heterakis isolonche, ascaridia spp.) and coccidia (eimeria spp.) which are common in wild and reared game birds and may reduce breeding success (cited according to (goldová et al. 2006) ) and were present in the investigated pheasants. according to literature (backhus 2000; dowell et al. 1983; gassal and schmaschke 2006; goldová et al. 2006; hillgarth and osborne 1991; hospes 1996) and based on our results (table 4) , an increase in parasite occurrence seems not to be present in the free-ranging pheasant population in northwestern germany. however, coccidia, for example, are known to have a more severe effect on small chicks, so the influence on this age group needs to be investigated. indeed, a parasitic infestation in weakened pheasants may be a supplementary factor for population decline. mycoplasma spp. were detected in three of five investigated individuals via pcr. in one case, m. pullorum was isolated from the trachea via culture. m. pullorum as well as m. glycophilum and m. gallinarum are regularly found in the respiratory tract of domestic pheasants and therefore do not seem to play a role as pathogen of respiratory disease (bradbury et al. 2001 ). however, the role of m. gallinaceum and m. iners is still discussed in literature . m. gallisepticum in contrast is a known pathogen of severe respiratory disease in pheasants, but was not detected in any of the investigated pheasants in this study. no increased occurrence of certain bacterial species was detected. however, as samples were frozen prior to the performed investigations, the results of the bacteriological examinations may be altered. nevertheless, a bacterial cause for the population decline seem to be unlikely, but cannot be ruled out. especially, acting as secondary pathogens bacteria may be a supplementary factor. the most surprising result was the detection of coronavirus rna in 16.8% of investigated carcasses that suggests a possible role of this pathogen. generally, chances to detect pathogens are usually low. therefore, it is speculated that this percentage reveals a high prevalence in wild pheasant population. however, sampling was done on carcasses found dead, which represents a preselection per se that raises the chance to detect virus. on the other side, carcasses were frozen prior to examination, which generally is sub-optimal and may decrease the chance to detect certain pathogens. generally freezing can affect degradation of rna and therefore reduce the prevalence. however, recent publications (ji et al. 2017 ) demonstrate that this effect is more in repeated freezing and thawing cycles. the effect of freezing cannot be fully excluded but as those samples were frozen only for a very limited time this effect is considered to be of minor relevance. in the investigated pheasants, only four of the coronavirus-rna positive birds showed pathomorphological signs in the respiratory tract or nephritis and most of the affected pheasants were killed by trauma. nevertheless, the localization of these affected birds all over the sampling area suggests a high distribution of this pathogen. more studies are needed to evaluate the prevalence and distribution of this pathogen, to approve this suspicion. in a verified case, this circumstance involves an influence on population development, because, besides respiratory signs or nephritis (cavanagh 2005; cavanagh et al. 2002; lister et al. 1985) , a more important consequence of coronavirus infection in pheasants is a negative influence on survival gough et al. 1996; pennycott 2000; pennycott 2001 ) and egg production (gough et al. 1996; spackman and cameron 1983; welchman et al. 2002) . mortality rates in captive pheasants range from 15% in breeding pheasants (gough et al. 1996) to 45% in juvenile pheasants (lister et al. 1985) . according to the coronavirus strain, death may occur with only few clinical signs apart from upper respiratory tract sneezing (gough et al. 1996) . in a wild pheasant population, a similar effect on survival and rearing success may lead to a decline of population, because wild bird populations depend on offspring survival, as described in the uk, where cause for population decline in gray partridges was ascribed to chick mortality (potts 1986) . serotyping is important to estimate the source of the detected coronavirus and its pathogenicity, to be able to determine subsequently the risk for free-ranging pheasant population (aldous and alexander 2008; cavanagh 2005) . because of high similarity and variability of strains, a differentiation is complicated (cavanagh 2005) . it was not possible to isolate replicating coronaviruses from positive samples in this study (data not shown). therefore, no further characterization of detected coronaviruses was possible yet. furthermore, the role of ampv (6.6%) and siadenoviruses (7.6%) has to be taken into account. avian metapneumovirus often occurs accompanied by other viruses, such as ibv (jones 1996) , and is discussed as a predisposition for other pathogens . siadenovirus, especially msdv, is reported to cause also predominantly respiratory clinical signs and often leads to peracute death (fitzgerald and reed 1989) . furthermore, immunosuppression may occur in infected pheasants (fitzgerald et al. 1992) . besides, this disease affects mainly pheasants from 3 to 8 months of age with mortality rates between 5 and 20% (fitzgerald and reed 1989) . therefore, a main consequence for influencing population development caused by these pathogens would be high mortality rates in offspring and juvenile pheasants. moreover, diseased free-ranging pheasants are an easy prey for predators, which may increase the effect on the population. further investigations should therefore focus on chick mortality. the one read, detected by next generation sequencing, was most closely related to rev, but it is unlikely that this virus plays a role in the observed lesions. most samples in this study originated from pheasants found dead, so a preselection was inherent. to evaluate results and provide a total prevalence, sample numbers should be enhanced and for example, every pheasant shot during hunting season without selection should be investigated. furthermore, to verify the spread of pathogens over the country, serological investigations of pheasants taken randomly out of the population are needed. results of this study point towards diseases like coronavirus infection, marble spleen disease and infectious bursitis disease, which affect especially young birds with high mortality rates. therefore, pheasant chicks should be included in further investigations, too. after all, a general factor decreasing health strength of the population should be considered. populations of free-ranging birds deal with multiple stress factors. for all farmland birds, the habitat is closely connected with human actions and influences, particularly agriculture with its influence on habitat and food availability, pesticides and fertilizer. furthermore, the weather, parasites and pathogens put stress on free-ranging pheasants. in this study, we present different pathomorphological changes detected 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romeis mikroskopische technik acknowledgements we thank the hunter's federation of lower saxony, north rhine-westphalia and schleswig-holstein for their support of the study. furthermore, special thanks to the laboratory personnel for the excellent technical assistance in the laboratory investigations. we would also like to thank the lower saxony ministry of food, agriculture and consumer protection, the state agency for nature, environment and consumer protection of north rhine-westphalia and the ministry of energy, agriculture, the environment and rural areas of schleswig holstein for their support of the study. this study was supported in part by the european union's horizon 2020 research and innovation program under grant agreement no. 643476 (compare). key: cord-345024-dtsi9qit authors: brauers, hanna; oei, pao-yu; walk, paula title: comparing coal phase-out pathways: the united kingdom’s and germany’s diverging transitions date: 2020-10-01 journal: environ innov soc transit doi: 10.1016/j.eist.2020.09.001 sha: doc_id: 345024 cord_uid: dtsi9qit political decisions and trends regarding coal use for electricity generation developed differently in the uk and germany, despite being subject to relatively similar climate protection targets and general political and economic conditions. the uk agreed on a coal phase-out by 2024. in germany, a law schedules a coal phase-out by 2038 at the latest. this paper investigates reasons for the different developments and aims to identify main hurdles and drivers of coal phase-outs by using the triple embeddedness framework. the comparative case study approach reveals that policy outcomes regarding coal consumption are deeply influenced by several actor groups, namely, coal companies, unions, environmental ngos, and the government. the most discussed aspects of a coal phase-out in both countries are energy security concerns, whether coal is mined domestically, (regional) economic dependence, as well as the relative power of actors with vested interests in coal consumption. to meet the paris agreement target of limiting global warming to at most 1.5 • c to 2 • c, coal consumption needs to be reduced drastically (unep, 2017, chap. 6; rockström et al., 2017) . the european union (eu) would have to cut its coal consumption to almost zero by 2030 to fulfil its already agreed upon climate protection commitments (rocha et al., 2016; climate analytics, 2017a) . some major eu coal producing and consuming countries have agreed on a coal phase-out, while others still plan further expansions in coal generation capacity. this paper aims to identify the main hurdles and drivers of coal phase-outs on a country-specific level. it contributes to the literature by investigating and comparing the current state regarding coal consumption for electricity generation of two (former) eu states, namely the united kingdom (uk) 1 and germany over the 1960 to 2019 era. both countries have a long history of coal production and consumption, being heavily dependent on coal for electricity supply. at the same time, they are (still) subject to the same eu climate and energy market regulations as well as the paris agreement (unfccc, 2015) , being required to reduce the amount of coal consumed. however, they are undertaking two contrasting strategies: namely a relatively rapid coal phase-out plan in the uk, compared to a strategy of conserving and delaying in germany. this paper aims to contribute to the literature by analysing why the developments in two major (formerly) coal producing and consuming countries are diverging so widely. it questions which actors and interests supported a continuation of coal's importance and the tef, a conceptual framework developed by geels (2014) , is part of the socio-technical transitions literature. industries suitable for this framework are reluctant to change, hold a high political influence, and are scale-intensive with many sunk investments, which is true for the coal sector. it recognises institutional change and includes strategic behaviour as well as the power of actors. by enabling the analysis of the co-evolution and the bi-directional relationships between an industry regime and its environments, it addresses shortcomings of previous methodologies (kungl and geels, 2018) . thereby, the framework refers to the situation of firms within an industry regime, which is itself embedded in two external environmentsthe socio-political and the techno-economic 3 environments. an industry regime is under selection pressures from its socio-political environment, where the criteria include, among others, legitimacy and social fitness, and the techno-economic environment, which demands economic competitiveness, efficiency, and financial performance. the tef acknowledges the ability of firms to respond to their environments and influence them through strategic actions. the responses of the coal regime (adaptation strategies) are both externally-oriented (toward the economic and the socio-political environment) and internally-oriented (toward changing the firm's set-up to fit better to the environments). hence, the framework includes bi-directional relationships and co-evolution of the regime and its environments (geels, 2014) . it can be used as a tool for analysing the destabilisation of industry regimes. in the analysis included actor groups are the firms of the incumbent coal regime, non-governmental organisations (ngos), governments, labour unions, civil society and competitors for coal (this selection is based on hess (2014) and turnheim and geels (2013) and the actors influence on coal transitions). additional background on the triple embeddedness framework is presented in the appendix. to analyse drivers and hurdles away from coal, two eu countries, where coal mining and using coal for electricity generation played or still plays a major role for the economy, were chosen. in 2015, the uk decided to phase out coal by 2025 (subsequently bringing the phase-out date forward to 2024 in 2020). in germany, the implementation of a phase-out plan is still under discussion; however, a coal phase-out by no later than 2038 is included in a draft law. the paper considers the 1960 to 2019 era, as the destabilisation of a regime is a long-term process and historic events can reveal broader societal and economic trends, creating path dependencies and lock-in effects (see also kungl and geels (2016) ). however, as most data (especially for east germany) is only available post-1989 and climate concerns were only perceived as more pressing after 2000, the paper focuses on 2000 to 2019. in addition, information from earlier periods is included within the analysis to provide context for both case studies. in germany, the installation of the so-called "coal commission" and its proposed phase-out plan is considered, but the implementation process of the phase out law is not analysed as it is still ongoing at the time of writing. due to the close connection of coal use for downstream electricity generation and upstream coal mining, both are included in the coal regime analysis. the paper focusses on the usage of coal in the electricity sector, as heat, until now, is of lesser importance for coal companies. 2 using the more commonly applied multi-level perspective (geels and schot, 2007) , would have diverted the attention to the niche part of the analysis. instead, we focus on the coal regime and incumbency, as well as the politics and power around a reduction in coal consumption. 3 in the original framework from 2014, the environment is called "economic" and not "techno-economic". data-collection is guided by the conceptual framework focusing on the relevant actors and contexts rather than on dependent and independent variables (kungl and geels, 2016, 2018) . our data collection is based on a triangulation of document analysis, regular visits to the german coal regions, and a series of workshops. the document analysis uses primary data from databases regarding coal production, consumption, employment, and share of gdp, etc. as well as secondary sources from scientific peer-reviewed journals, other articles, and books. additionally, we draw on a wide range of grey literature including daily newspaper articles, blogs, company press reports, annual reports, and various website information, written in english or german and referenced throughout the text. informal background interviews with regionally affected stakeholders, while visiting german coal regions 4 during three different research projects between 2012 and 2018, allows us to test and complement the acquired information. we study the german context to highlight resistance against the phase-out. a first draft of different socio-political and techno-economic aspects of the coal phase-out, including response strategies of the coal regime, allowed us to organize ten thematic workshops in germany between 2015 and 2019 to acquire additional information on specific aspects as well as the underlying narratives of affected stakeholders within the coal phaseout process. participants varied between 10-20 representatives from governmental bodies, the (conventional and renewable) energy industry, unions, academia, and civil society. each workshops focused on a different set of topics, either touching more socio-political (e.g. health concerns, climate and environmental regulation options) or techno-economic (e.g. number of job losses and possible replacements, technical replacement of coal with renewable energies, grid stability, affordability) aspects as well as the response strategies of the coal regime (e.g. modelling phase-out pathways, liability issues). we did not do fieldwork in the uk; instead ongoing exchanges with academic experts on the uk validated the quality of our case study findings. the triangulation of this gathered information was used to develop the tef, mapping socio-political and techno-economic aspects of the coal phase-out including response strategies of the coal regime for each country. in addition, intermediate results of the tef were regularly refined following presentations and discussions with stakeholders at five international academic conferences. the main aim of the paper is to provide an overall picture of the political economy of coal in both countries in a novel way. many of the single elements included in the tef are studied by other authors. our main contribution is to bring these results into the descriptive framework to better understand the complexities influencing the political economy of coal. additionally, new findings are generated by comparing the diverging developments of these two countries. fig. 1 provides a broad overview of coal mining and the total number of employees in coal mining (hard coal and lignite) in the uk and germany since 1958. it is apparent that coal's importance is in decline in both countriesbut at different speeds. despite the strong reduction in coal mining and employment since the 1960s, the share of electricity generated by coal is still 28% in germany in 2019 (bdew, 2020), whereas it constitutes only 2% in the uk (department for business, energy & industrial strategy, 2020c). 5 fig. 2 shows the development of the electricity mixes for the two countries. 6 it illustrates that the main substitutes for coal in the uk have been natural gas and renewables; in germany, renewable energy. this is also due to the fact that levelised costs of energy for renewables have fallen below the costs for conventional energy in both countries, especially due to falling capital costs and improving technologies (see for example for an analysis of energy prices in germany and the uk). the resulting strong increase of renewables has also resulted in new employment options for around 300,000 people for germany and slightly more than 100,000 for the uk (irena, 2019; oei et al., 2020a,b) . the uk is one of the few eu member states where coal played an important role in the energy sector, but which nevertheless announced a coal phase-out by 2024/2025 and is a founding member of the powering past coal alliance (department for business, energy & industrial strategy 2017). the role of coal has changed dramatically since the 1980s, especially in the last few years. while coal accounted for almost 80 percent of the uk's electricity generation at the beginning of the 1980s, it reached an all-time low in 2018, as shown in fig. 2 (department for business, energy & industrial strategy, 2020c). 7 coal was not needed at all to meet the uk's electricity requirements on 83 days in 2019 (evans, 2020) , and is even further reduced in 2020 due to reduced energy demand because of covid-19. coal production in the uk is almost eliminated with the closure of the last large deep mine in 2017. the influence of the uk's coal unions on political decisions has changed over time. margaret thatcher fought against the power of unions during the 1980s. reasons for this were the government's aspirations for power as well as the aim to liberalise the energy market and to increase competition (gouiffes, 2009; pollitt and haney, 2013) . after the end of the violent labour dispute (in 1985) , the union's influence had been reduced substantially (gouiffes, 2009; . 8 nevertheless, all major unions supported coal and lobbied against mine and power plant closures. following the strikes, hard coal production and employment continued to decline substantially (see also fig. 1 ), while overall coal consumption declined more slowly (department for business, energy & industrial strategy, 2020a). an accelerated coal phase-out process, steered by the government, started much later. in 2005, the socio-political environment in the uk observed a major shift in the perception of climate change, triggered by the 'big ask' campaign of the ngo friends of the earth (foe). an extensive media coverage of the campaign increased public and political awareness of climate change. the conservative party supported the 'big ask' campaign and adopted climate change accordingly as a major point of its strategy to modernise the party. within a month of this decision, 412 members of parliament (out of 646) signed the foe motion for a bill that would make emission reduction targets a law. the big three parties (conservatives, labour, and liberal democrats) started to compete by being greener than the others (carter and jacobs, 2014) . this made it more difficult for parties to openly support coal. starting in 2006, the cross-party 'green' competition created opportunities for politics prioritising the environment. another main influence on climate friendly political decisions were inter-departmental institutions, e.g. the office for climate change (with members from all the main departments related to greenhouse gas (ghg) emissions (energy, business, transport, treasury, etc.)) and the 5 the numbers for the electricity mix in germany in 2019 are preliminary. the numbers for the electricity mix in the uk in 2019 constitute the average of the first three quarters 2019. 6 for an overview of the longer term electricity trends in the uk and germany see fig. 5 and fig. 6 in the appendix. 7 since the opening of an interconnector with france in 1986, the uk has been a net electricity importer. at its peak in 2015, electricity imports were responsible for less than 0.9% of total primary energy supply and 6.6% of uk electricity generation (bolten, 2018) . ~95% of the electricity imports come from france and the netherlands (bolten, 2018) . as both countries have a lower electricity carbon intensity than the uk, the phase-out of coal in the uk electricity mix did not lead to substantial additional emissions elsewhere. 8 the strike resulted in more than 11,000 arrests, 7,000 wounded, 200 imprisoned and more than 8,000 convicted (gouiffes, 2009, 179) . committee on climate change. by bringing different interests together and by being more independent, they managed to create consensus around integrated approaches that undermined pure economic considerations that had previously dominated (carter and jacobs, 2014) . the uk can be considered as a "liberal market economy" with a preference for market-based and non-technology specific policy instruments like the carbon price floor (cpf) (hall and soskice, 2001) . the focus on cost efficiencyand not on e.g. supporting new entrantsalso explains the preference for large-scale technologies (see section 3.1.2). furthermore, the uk's 'liberal market economy' is characterized by "close-knit policy networks that are relatively open to incumbent industry actors but remain closed for outsiders and new entrants" (geels et al., 2016, 910) . as a mostly top-down policy style prevails, broad stakeholder engagement is limited . in general, there was a broad public consensus among civil society and ngos within the uk that tackling climate change was crucial (gillard, 2016; parkhill et al., 2013) . the declining role of coal combined with widely appreciated and available alternatives like local natural gas, nuclear energy, and renewable energy helped to generate public support for climate change policies. media coverage can shape public opinion and has also influenced the transition in the uk. isoaho and markard (2020) find in their discourse analysis of the guardian that incumbent actors first tried to legitimise coal until 2015. however, as they had already started to shift to alternatives, there was little resistance in public media when the government announced its coal phase-out pledge (see also antal and karhunmaa (2018) for a comparison of reporting of the guardian and the times on the german energy transition). in 2019, the government further increased its goals, deciding to target net-zero greenhouse gas emissions by 2050 (department for business, energy & industrial strategy, 2019b). several factors of the techno-economic environment facilitated the reduction of coal in the uk. compared to other countries (e.g. colombia, south africa or russia), coal resources were deeper in the ground and labour costs were higher, such that international imports were much cheaper than domestic mining. instead of subsidising coal mining like other countries, e.g. germany (see section 3.2.1), the uk started weaning itself off its dependence on coal mining. by taking the decision not to use public funds to support domestic mining in the 1980s, international competition led to a quick decline of domestic coal production and related employment. several policies introduced after 2006 constrained coal's business opportunities long before the final phase-out decision in 2015, especially the cpf, the renewables obligation (ro), the emissions performance standard (eps), as well as more in general the climate change act and the related carbon budgets. 9 the climate change act 10 of 2008, a main cornerstone of its climate policy, commits the uk to reducing greenhouse gas emissions by at least 80 percent by 2050 compared to 1990 levels, setting legally-binding carbon budgets. additionally, timetables for compliance with stricter eu pollution control regulations have required a response from all power plant operators and contributed to the closing decision of seven non-compliant and ageing power plants (~10 g w) between 2010 and 2015 (littlecott et al., 2018) . older power plants are mostly more polluting (in terms of co 2 and other emissions) and less efficient, which leads, next to higher amounts of pollution, to higher specific costs per mwh. in may 2020, the four still operating coal plants have reached an average lifespan of 43 years (power stations of the uk, 2020). the cpf and the eps, on the other hand, have restricted potential construction of new coal units (without carbon capture) (mendelevitch and oei, 2017) . renewable electricity policies, especially the renewables obligation, which required utilities to meet annual renewable electricity targets, incentivised incumbents to deploy a certain amount of renewable energies themselves, rather than enabling new market participants to enter the electricity market. entry barriers for new non-specialist market participants were high due to the complexity of the mechanism and related revenues were too uncertain for civil society actors (hall et al., 2016) . on the demand side, falling wholesale electricity prices, especially in the period after 2015, put pressure on the coal industry (littlecott et al., 2018) . however, the coal industry is subsidised by the government through various policies: a capacity market was introduced in 2014 and serves to guarantee idle power plants a steady income. other policies in 2017 included various tax benefits, inherited liabilities 11 related to coal mining, the supplementary balancing reserve 12 (2014-2017), and others. the inherited liabilities related to coal mining amounted to annual average subsidies of €48.6 million in the years 2006-2014. estimates for the annual average budgetary support for the supplementary balancing reserve are €94.3 million in 2016 (van der burg, 2017) . in addition, subsidies for renewables were cut back heavily in 2015 (johnstone et al., 2017) . renewable projects are, in most cases, smaller than conventional units and face problems in acquiring loans within a market based financial environment, such as the uk. this results in additional barriers for small scale renewable energy projects (especially with civil society ownership) to borrow funds from mostly centralised and internationalised private investment capital (hall et al., 2016) . this contributed to the fact that most installed renewable generation capacities in the uk are owned by firms that are already present in the energy market. 13 additionally, the slowing down of 9 the eps was part of the 2013 energy act and it sets a limit of 450gco 2 /kwh for new power plants of more than 50 mw. the carbon price floor was introduced in 2013 with £9/tco 2 ; the price of £18/t co 2 (~21€/t co 2 ) was frozen in 2015 until 2021 (house of commons, 2018). 10 climate change act 2008, chapter 27, parliament of the united kingdom. 11 the coal authority takes charge of inherited liabilities, for which coal-mine operators were not held responsible (van der burg, 2017) . 12 the supplementary balancing reserve puts generation capacity into a reserve. the reserve is kept outside the electricity market and can be used when there is a shortage in supply (van der berg 2017). 13 background talks with energy experts and see also https://www.gov.uk/government/publications/renewable-energy-planning-databasemonthly-extract. renewable energy investments increases the need to use natural gas as a replacement fuel for coal. so far, coal for electricity generation has been continuously replaced by cheaper electricity from both natural gas and renewables. however, in contrast to most eu countries, no renewable energy targets have been set for 2030 nor 2050 in the uk . this might further hinder renewable energy expansion and, therefore, increase the use of natural gas and potentially nuclear energy. 14 lockwood et al. (2019) find that large electricity generators have structural power in relation to decision makers. this enabled companies' ideas and related lobbying to influence the design of the capacity market policy and other subsidies in the uk. additionally, high hopes among all incumbents were placed on cc(t)s (carbon, capture, (transport), and storage) as a 'silver bullet' to allow for emission free coal combustion. the political decision to implement the eps and the cps, accelerating the coal phase-out might have been different, if it had been clear for all actors that carbon capture, (transport,) and storage (ccs or ccts) would not be available as a so-called 'clean coal' alternative, potentially leading to more resistance (littlecott et al., 2018) . 15 the coal industry used several framing techniques to influence public opinion and political decision makers. a main narrative was that without cheap coal, electricity prices would rise, which in turn would lower competitiveness of other british firms and hit households hard. the question of whether prices actually do increase because of climate policies or because the old power plants would have to go offline after ~50 years in operation anyways, has been avoided. other powerful frames repeatedly pushed into the public debate by the coal regime are job losses and blackouts. an example for this is a report by the british infrastructure group saying that coal power station closures would lead to a "sustained danger of intermittent blackouts for the foreseeable future". 16 the report was immediately refuted by several research institutes and ngos. 14 the government reduced its support for onshore wind and solar pv substantially, and signals on support for tidal power and biomass are unclear. nuclear power is struggling with opposition, the long planning and construction times, high costs, and ever-increasing problems with hinkley point c (the guardian (2019): hinkley point nuclear plant building costs rise by up to £2.9bn. https://www.theguardian.com/uk-news/2019/sep/25/ hinkley-point-nuclear-plant-to-run-29m-over-budget). 15 in 2009, a new regulation stated that no coal power station would get a permission without ccts (carter and jacobs, 2014) . this prevented new coal-fired power plants from being built, as ccts never became technologically available. 16 philpott, tim. 2016. 'electric shock: will the christmas lights go out next winter?' a british infrastructure group (big) report. https://www. theguardian.com/environment/2016/dec/19/campaigners-dismiss-christmas-electricity-blackout-report-as-laughable. fig. 3 summarises the triple embeddedness framework analysis' results for uk. the aging infrastructure, uneconomic mining, and climate policies led to the unfavourable (economic) conditions for coal in the electricity sector. when coal mining became uneconomic in the 1980s, state support was withdrawn, reducing the power of the unions. reducing the amount of domestic coal lowered further resistance to implement policies reducing coal's dominance in the electricity sector in subsequent years. the added focus on environmental protection and climate change by the government during the 2000s led to the implementation of crucial policies like the cpf and the eps. together with eu emission reduction targets, the coal industry's business was further weakened, and finally the coal phase-out by 2024/2025 announced. the eps prevents new coal-fired power plants (without carbon capture) from being built, the cpf made electricity generation by coal less competitive and air pollution regulations forced older power plants to be closed. the policies incentivised incumbents to change their strategy: invest in renewables and natural gas projects instead of further holding on to coal as their main business model. however, policies in the uk did not support the entrance of new (small-scale renewable) generators but instead continued support for the incumbent energy companies. for an in-depth analysis of parallel developments regarding nuclear power, see . the characteristics of coal in germany are similar to the uk: in both countries hard coal mining has been uneconomic for decades, coal infrastructure is mostly old and hard coal import dependence is rising . the consequences for the coal regime, however, have been very different due to a different political direction. the german government subsidised domestic mining from the 1950s onwards so that it could stay competitive with cheaper imported hard coal (matthes, 2017) . 17 these numerous direct and indirect subsidies continued until they were forbidden by european regulation in 2018 (gençsü et al., 2019). as a consequence, hard coal mining experienced a continuous 60 year controlled declinecompared to the abrupt collapse in the ukand ended in december 2018 . the consumption of hard coal stayed relatively constant over time, while power plants switched from domestic coal to imported hard coal. retired coal power plants were replaced with new units even after 2000 (pahle, 2010) . reasons for this were the underestimation of renewables and false hopes of operators to profit from the phase-out of nuclear energy. by the late 2010s, the share of hard coal within the electricity mix was cut in half, reaching 9 percent in 2019, resulting in very low utilisation rates (oei et al., 2020a,b) . the biggest drop in lignite production (and employment) already happened in the early 1990s following the reunification of germany as mines in eastern germany were adopted to western standards (e.g. higher environmental standards and higher labour productivity), enforcing several closures (stognief et al., 2019) . in 2020, lignite continues to be mined in three open cast mining regions in germany (oei et al., 2020a,b) . the adjacent power plants profit from relatively low operating costs and, therefore, contributed 19 percent of german electricity generation in 2019 (ag energiebilanzen, 2020). rising civil society pressure, as well as from the coal regions demanding financial support, pushed the government to introduce a 'commission on growth, structural change and employment' in 2018 -often also referred to as 'coal commission'. the commission included representatives of various social groups, such as unions, energy companies, industry, ngos, and residents of coal regions. it proposed a coal phase-out plan in january 2019 that foresees shutting down a total of 12.5 gw (27% of the active installed coal capacity at the end of 2017) of coal-fired power plants by 2022. all coal-fired electricity should be phased-out by 2035 or by 2038 the latest (bmwi, 2019). the planned instrument to phase-out hard coal is an auction mechanism. in late 2019, the government started negotiations with the lignite operators about the timing of decommissioning and the magnitude of compensations. in may 2020, more than one year after the commission presented its recommendations, it is still not implemented as a law, and criticized by many actors previously involved in the negotiations of the commission (oei et al., 2020b) . the following analysis focuses on historical hurdles that prevented an earlier coal phase-out in germany (compared to the uk). public opposition to coal began in the 1960s due to high-stack emissions causing acid rains and forest diebacks. until the 1980s, however, concerns and protests against coal remained a local issue. instead, the main focus of national protests by civil society and ngos was nuclear power, which was perceived as a more direct threat by the broad public. consequently, organizing anti-nuclear protests was the principal focus of environmental ngos like foe, greenpeace, and wwf (renn and marshall, 2016) . the german government slowed the decline of the coal industry since the 1950s, lowering the negative impact on firms, workers, and regions, but also prolonging the difficulties of reducing the dependence of coal until this date. for a detailed description of government policies to support the hard coal industry from the 1950s through 2018, see oei et al. (2019) . internationally, germany is known as a strong supporter for climate change action. the focus on climate change protection and careful steps toward a government planned coal phase-out increased in 2011. since then, germany's energy strategy has been called 'energiewende' (often translated as 17 cumulative subsidies between 1958 and 2008 of €295 billion supported hard coal, while lignite received a total of €57 billion (meyer et al., 2010 ). next to the capacity payments for lignite power plants, there were several other state subsidies still in place in 2017. these include royalty exemptions and reductions for resource extraction, support for the rehabilitation of mines, energy tax and electricity tax exemptions for power generation, and early retirement schemes for workers. for example, there were still €1,863 million subsidies every year only for coal mining in north rhine westphalia until 2014. there are interesting parallels to the uk, as there is a relatively newly introduced capacity reserve running in both countries until 2020, costing €230 million in germany and €138 million in the uk (van der burg, 2017). energy transition). it is based on the goals of a nuclear phase-out, the reduced consumption of fossil fuels, and an increase in energy efficiency (renn and marshall, 2016; . however, the government fails to achieve its domestic emission reduction targets due to continued coal consumption. germany aimed to reduce emissions by 40% in 2020 compared to 1990. in 2019, germany was on track to reduce emissions only by 33.2% (bmu, 2019). 18 germany can be characterized as "coordinated market economy" (hall and soskice, 2001) . this can result in close interactions between the government and powerful incumbents, as well as civil society organisations. while coal incumbents received continuous governmental support stognief et al., 2019) , germany also has a relatively strong and organized civil society with active cooperatives, citizens' groups, and a strong environmental tradition. therefore, civil society protests were already an important lever enabling the phase-out of nuclear power in germany . from 2011, the agreed upon nuclear phase-out relieved (human) resources of environmental actor groups. in parallel, concerns about coal intensified as knowledge about the impacts of climate change and human health impacts grew. tackling coal is more difficult for ngos, as it is not sufficient to simply address the risks of the energy technology (as in the nuclear case), but need to include also the potentially negative socio-economic aspects for coal workers and regions in their argumentation. rising international awareness of the climate crisis increased the pressure on the coal regime and the german government. consequently, ngos and activists managed to increase public attention, e.g. organizing a protest march in 2018 with more than 50,000 people into the hambach forest, which was planned to be cut down for the enlargement of an adjacent lignite mine . people living in the lignite mining regions have split opinions on a coal phase-out: one part of society fights against new mines to prevent their villages from being destroyed and to protect the environment. the other part lobbies to keep lignite as a main energy source in germany, mainly to protect their jobs and cultural heritage. both sides, however, believe that democratic processes have failed to make them actual stakeholders and part of the decisions shaping their energy futures (morton and müller, 2016) . the biggest unions in germany, representing workers of hard coal and lignite mines and power plants, are the ig bce and ver.di. both have a high influence on political decisions and were strong supporters of the gradual phase-out of coal in germany (compared to the uk) (renn and marshall, 2016) . the ig bce's position has been the most rigid, as they also represent workers in the energy intensive industries sector, which profits from low wholesale prices of electricity. before 2017, a coal phase-out before 2050 was characterised as impossible. any measure to reduce coal consumption was seen as direct attack on their represented workers (ig bce, 2016). ver.di also lobbied against tighter air pollution controls and in favour of capacity payments to keep the coal industry alive. their focus, however, put benefits and retraining programs for affected workers as the highest priority, rather than only trying to postpone the phase-out date (enervis, 2016). the german electricity market was liberalised in 1998, but competition remained limited resulting in continuously high market power of the four incumbent companies (rwe, enbw, e.on, and vattenfall). the renewable energy sources act (eeg), implemented in 2004, substantially changed the dynamics of the electricity market. feed-in-tariffs were available for all electricity market participants and were especially attractive to, and supportive of, new market participants. the german green industrial policies were more stable, financially certain, and less bureaucratic than the british policies hall et al., 2016) . the green growth discoursethat germany with its substantial manufacturing sector could profit financially from the energy transition, building wind turbines and solar modulesfurther pushed the transition . the eeg also resulted in a reduction of the market share of the formerly 'big four' electricity generating companies as well as to a shift within their production portfolio (renn and marshall, 2016; . in 2018, the domestic market share of the biggest five electricity generating companies ('big four' and leaga new company having bought all lignite assets from vattenfall) was reduced to 74% (bnetza and bundeskartellamt, 2019). this is comparable to the development of the wholesale electricity generation market share by the eight largest companies of the uk (edf, rwe, sse, drax, uniper, eex, scottishpower, and orsted) summing up to 72% (ofgem, 2019). in germany, big energy utilities face increasing re-municipalisation strategies as well as strong public support for nuclear phase-out and increasing renewables. in addition, many local small savings banks allocate capital to small and medium scale energy providers, like municipality owned stadtwerke (local public utilities), through an established framework of citizen investment to support regional development (hall et al., 2016 ). this regional financial support, paired with national renewable electricity policies of guaranteed feed-in tariffs, incentivised new small and community-based providers of renewable energies to enter the electricity market. continual market pressures on the coal business included overall decreasing energy demand due to the financial crisis, the rising market shares of renewables, and, consequently, decreasing wholesale electricity prices . in addition, the increasing price for allowances in the european emissions trading system, ~25 euro/per tonne of co 2 in 2019, in combination with shrinking gas prices made coal combustion increasingly uneconomic. consequently, older hard coal units observed greater variable costs than gas units, resulting in lower utilization rates. this is the principal reason underlying the decline of coal's share in the electricity mix since 2017 (sandbag, 2020; agora energiewende, 2020). 18 a paradox in germany is that despite big successes in growing wind, solar, and biomass capacities, greenhouse gas emissions remained relatively constant between 2011 and 2017 at around 900 million tonnes of co 2-eq . the target for 2030 is 543 million tonnes of co 2-eq (umweltbundesamt, 2017 (umweltbundesamt, , 2020b . new estimations indicate that germany might achieve its 2020 target due to the recession caused by the covid-19 pandemic. this short-term decrease, however, will be evened out by the uptake of the economy in the following years and, therefore, does not reflect the desired structural reduction. the main strategy of the incumbent coal firms was to lobby for coal friendly regulation and further (financial) state support: this is visible in their success at opposing the first attempt by the german government in 2015 to introduce a 'climate contribution' (klimaabgabe) (morton and müller, 2016) . the 'climate contribution' would have led, similar to a carbon tax, to closures of mostly older coal units (goodman, 2016) . instead, a so-called 'carbon reserve' mechanism was introduced: old lignite-fired power stations were paid compensation while providing only a very small, if any, contribution to germany's climate goals. "the defeat of the 'climate contribution' is one clear example of how the politics of coal can undermine the policy aims of the energy transition" (morton and müller, 2016, 10) . another example illustrating the coal industry's power over german politics is air pollution regulations. in 2017, germany lobbied with a small group of other (mostly eastern) eu countries against tighter eu air pollution rules 19 , which set stricter emission limits (best available techniques (bat) requirements) for large combustion plants as part of the industrial emissions directive (climate analytics, 2017b). nevertheless, a slim majority of european countries voted in favour of the stricter emission limits, taking effect for all eu member states in 2021 20 . however, in may 2020 germany is still not complying with european regulation not having transferred the new regulation into a corresponding law. the incumbent regime used several strategies to maintain the status quo of coal for as long as possible. one strategy was to misrepresent the effect of renewables on electricity prices for the general public. renewable feed-in-tariffs are mostly paid for by households and small industries, explicitly stated as such on all electricity bills. however, the increase of renewables actually lowered wholesale electricity prices -which especially benefited energy intensive industries. subsidies for conventional electricity, on the other hand, are directly paid for by the state budget and, thusly, are not clearly visible to consumers (lauber and jacobsson, 2016) . to save their business, electricity corporations additionally claimed that renewables threaten energy security because their fossil fuel plants would be rendered unprofitable and argued that renewables would make german industries uncompetitive by increasing energy costs. these claims were being made despite various studies showing that grid stability is not threatened by increasing amounts of renewables in the system and most energy intensive companies being freed entirely or at least partly from the eeg-surcharge (egerer et al., 2018) . the entanglement of rwe and municipal actors within the lignite region is a good example for the complexity of the coal regime in germany: traditionally, several city and regional governments in north-rhine westphalia are financially dependent on revenues of the rwe shares and have difficulties financing public services otherwise. municipal shareholders had little expertise (and belief) in renewables and consequently prevented a strategic reorientation of rwe away from coal, wanting to protect their regional coal interests . additionally, regional actors helped exert pressure on the national government to protect the overall coal regimeto safeguard local jobs and municipal services dependent upon the companies' financial success (oei, 2018) . rwe as well as the other members of the 'big four' in germany underestimated the fast growth of renewables and missed the opportunity to invest in non-fossil-fuel generation technologies. being used to large-scale projects, these companies did not want to participate in small-scale renewable projects and, additionally, they did not want to take some of their own profits from their conventional fleet away . consequently, medium-sized new actors formed, influencing and profiting from integrating renewable energy into the existing conventional wholesale power markets (wassermann et al., 2015) . this was strengthened through an overarching german trend toward re-municipalisation and the re-establishment of municipal utilities (stadtwerke). these served as key actors to promote structural change within the energy sector (berlo et al., 2017) . find that disruption in ownership in the electricity sector can help to support changing beliefs and practices. they distinguish between the "incumbent-led" energy transition in the uk and a "new-entrant-led disruptive" energy transition in germany. incumbents in the uk acted more strategically than incumbents in germany, saw change coming toward low carbon energy transition earlier, and deployed large-scale renewable energies themselves. nevertheless, incumbents in both germany and the uk lobbied for policies that supported their coal business activity. since 2015, germany's 'big four' have also started to diversify their strategies (though not necessarily their portfolio): vattenfall sold all its lignite assets and is promoting a target of carbon neutrality within a generation; enbw wants to align its strategy according to the paris agreement; e.on sold all of its electricity assets (to rwe and fortum); and rwe aims at carbon neutrality by 2040. furthermore, the bdew, the federal association of energy and water, which also represents the 'big four' together with most municipal utilities and renewable energy companies, was represented in the coal commission agreeing together with coal union representatives to the coal phase-out plan by 2035/38 (bmwi, 2019). fig. 4 summarises the tef analysis results for germany. this includes contextual information regarding the socio-political and economic environments as well as those forces (de-)stabilizing the coal regime. the long continuation of government subsidies for coal mining enabled the continuation of power generation and steel production with domestic energy sources. despite substantial pressure by international competition due to cheaper coal imports and climate protection measures, the political power of the coal regime limited changes to the status quo. climate change or air pollution concerns have not been strong enough to stop subsidies for the coal 19 dw (2018): environmental groups hit back as german coal companies try to sue eu. https://www.dw.com/en/environmental-groups-hit-backas-german-coal-companies-try-to-sue-eu/a-42801965. 20 european commission (2017): news release -commission to review permits of large combustion plants. july 31. http://ec.europa.eu/ environment/pdf/31_07_2017_news_en.pdf. industry or to implement policy instruments similar to those that forced closures in the uk. instead, concerns about high electricity prices, import independence, grid stability, as well as the implementation of the nuclear phase-out have resulted in a more gradual coal phase-out process. in 2010, shortly before germany decided to phase out nuclear energy, the share of coal and nuclear energy in gross electricity generation was still 64%. to replace both energy carriers posed a substantially larger challenge than replacing the 28% share of coal generation in the uk in 2010 (see fig. 2 ). thus, co 2 emissions were not reduced in a similar manner as in the uk. germany's example powerfully demonstrates that only incentivising and expanding renewable energies is not enough to diminish coal's importance. however, small scale renewable energy deployment and citizen ownership has created broad civil society support for the german energiewende, providing a valuable lesson for other countries and an important basis for a future coal phase-out. furthermore, prices for renewables have been brought down with the help of deployment in germany, which helped other countries in their renewable energy investments, including the uk (morris, 2016) . the aim of this paper is to analyse which actors and interests prevented or, in contrast, enabled a reduction of coal's importance. the comparative case study of the uk and germany employs the triple embeddedness framework. for both countries, it is apparent that socio-political and techno-economic environment pressures influence the coal regime and that powerful incumbents exist. another commonality for these two countries is that incumbents successfully prevented policies reducing their business opportunities for several decades while also securing financial support. common frames used to generate this support are claiming disadvantages in e.g. domestic competitiveness, energy security, import dependence, rising energy prices, black-outs, and unemployment. however, the decline of coal happened at different speeds and under different policy tools, influenced by varying contextual national factors, diversity of actor power within the coal regime, and those opposing coal. table 1 summarises the findings and highlights differences. in the uk, opposition of miners was already suppressed in the 1980s -not for climate reasons but other political reasons. having to import coal lowered opposition to reducing coal's importance in the power sector over the following decades. a driving force was ngo campaigns influencing public opinion on climate change and health aspects, which facilitated a competition between political parties for 'green' policies and the implementation of policy instruments like the carbon price floor and emission performance standards in the 2010s. this coincided with a point in time when, due to the age of coal-fired power plants, a decision between either major investments or a shutdown was necessary. furthermore, the decision for companies was comparatively easy as they were able to diversify into other domestic large-scale natural gas and renewables projects. in germany, since the 1950s, coal unions and influential coal companies slowed the decline of coal. thereby, reductions within hard coal mining and related employment happened in a linear manner until 2018 with the help of large governmental subsidies. the lignite sector, on the other hand, observed a rapid structural break in the early 1990s following german unification. a primary objective of east german mining was to create employment possibilitiesleading to 'inefficiencies' when having to compete with western mines optimized for capital return. after unification, this resulted in closures and layoffs as mining adapted to west european norms. our analysis shows that in germany the incumbent coal regime was able to uphold the status quo for a longer time than in the uk: in 2020, regional governments as well as municipalities that are shareholders of energy corporations in mining areas in germany are still supportive of coal due to ongoing employment concerns. additionally, energy intensive industries have built a coalition with the coal regime as they perceived it as a means for achieving lower wholesale electricity prices. coal mining in the uk, on the other hand, was reduced to a negligible amount since the 2000s, while germany still mined almost 200 million tonnes of coal. therefore, the uk was less dependent on jobs and regional income generation related to coal. germany's previous nuclear phase-out decision, following the 2011 fukushima accident, made the coal phase-out more difficult due to concerns about sufficient installed electricity generation capacities. this, however, also released more human and financial capacities for ngos to put pressure on the coal industry. phasing-out coal and nuclear energy simultaneously meant having to replace the majority of electricity generation capacities both for germany as a whole and for those companies owning the power plants. moreover, in contrast to the uk, germany could not increase domestic natural gas production and were dependent on comparatively expensive imported gas. most large electricity corporations in germany underestimated the potential for renewable energy and did not change their strategy to investing in renewables until much later. other main reasons for the earlier reduction in coal production and consumption include the relatively old infrastructure of coal-fired power plants in the uk, requiring major investments in refurbishment or new capacities, as well as the stronger opposition to any decision restricting coal by miners' unions in germany. these experiences show that different actor groups have significantly more influence on political decisions than others, contributing to the diverging decisions regarding coal in countries otherwise comparatively similar and subject to the same eu energy and climate regulations. accepting this influence can make policy sequencing approaches more attractive (introducing policies that face little initial resistance, to upscale stringency and introduce more controversial policies later) (pahle et al., 2017 ). this appears more feasible than simply targeting most efficient policy instruments that go against the interest of a regime with close ties to policymakers. we show that the diverging situations regarding coal arise due to a complex interplay of pressures by the socio-political and techno-economic environments as well as the response strategies of the incumbent regime. without efforts to limit the influence incumbents have on policy making, or at least balance it with other civil society actors, more ambitious energy transitions seem unlikely, or will at least be significantly slower. some overarching policy recommendations that can be drawn from these past experiences are the following: -support for large scale renewable energy investments can either come through a renewables obligation, such as in the uk, or through the entrance of new renewable energy actors, like in germany. -however, supporting renewable energies is not sufficient to enable a coal phase-out in line with (inter-)national climate targets. this is reflected in the struggle of the german government to implement the agreement of the coal commission against continuing opposition of incumbents. importantly, the achieved compromise is neither in line with the paris agreement nor germany's climate targets (oei et al., 2020b; kittel et al., 2020) . it is also slower than what citizens desire (rinscheid and wüstenhagen, 2019 ). -compatibility with the goal of ghg neutrality by 2050 needs to be included in transition planning. although the uk managed to achieve substantial ghg emission reductions with its shift from coal to natural gas, this will not be sufficient to achieve the upcoming more stringent climate targets for most countries. policies should be considered that prevent this next fossil fuel lock-in and instead create investments directly in renewables and energy storage as well as efficiency measures. -weakening the existing coal regime as well as showing them alternative business models enables change. margaret thatcher's political actions reduced the influence of the unions, but also resulted in other negative socio-economic consequences for mining regions still visible today. these impacts can be averted with targeted just transition policies, stopping coal use, and simultaneously providing social security for workers and new economic opportunities for dependent regions. -opportunities for change exist whenever larger investment decisions for plants or mines need to be taken. enforcing stringent climate and environmental regulation for new investments, as in the uk, can prevent stranded investments. missing such points in time can lead to ongoing legal debates regarding potential compensation payments, an ongoing discussion in germany. -phasing-out coal is not only about the replacement of coal with renewable energies within the energy system. for coal mining countries, like the uk and germany, the biggest challenge actually lies within the needed adjustments for the affected regional economies. past experiences show lessons of hardly managing (uk) or to passively delaying (germany) this process. current debates of the eu green deal try to reflect this by focusing on a "just transition" for all regions that will be affected by upcoming phase-out pathways. thereby, solutions strongly depend on regional contextual factors and must be adopted individually, as no single blueprint for a socially acceptable coal phase-out exists. we gratefully acknowledge support granted from the german ministry of education and research (bmbf) by financing the research group "coalexit" [grant number 01ln1704a] as part of the "global change" funding priority and the project "future of fossil fuels in the wake of greenhouse gas neutrality" [grant number 01la1810a] being part of the "economics of climate change" funding priority. the authors report no declarations of interest. firms-in-industries do "not only adapt to institutional pressures, but also respond strategically to shape them" (geels, 2014, 265) . aspects included from economic sociology highlight 'embeddedness', (cognitive, cultural and political). therefore, an important notion is "that economies and markets are underpinned by government regulations and institutions" (geels, 2014, 265) . another important contribution is also the recognition that "firms and industries use power and politics to shape formal institutions to their advantage" (geels, 2014, 265) and that "market elites and governmental elites often cooperate and that their voices are louder than those of labour unions, consumer groups and environmental groups" (geels, 2014, 265) , and that they can exert more political power than "ordinary citizens". the adaptation theory part acknowledges that firms act with deliberate and intentional strategies, rather than being mostly passive. both externally-and internally-oriented strategy schools are included. importantly, it is acknowledged that firms, in some contexts, have substantial scope to influence regulations and political environments. they use information strategies (e.g. setting up think tanks), financial incentives strategies (e.g. contributions to politicians and political parties), organize pressure strategies (e.g. creating industry associations, lobbying directly, confront using litigation, or threaten policymakers with layoffs), and discursive strategies (e.g. arguing that solutions are costly or technologically unfeasible). increasing pressures (from the economic and socio-political environment) and related performance problems can then incentivize actors to overcome lock-in mechanisms and tackle increasingly more foundational regime elements (geels, 2014) . the following two figures display long-term trends of electricity generation in the uk and germany. the effect of the miners' strikes in 1984 are clearly visible in the data for the uk. for germany, only data since its unification was available. bruttostromerzeugung in deutschland nach energieträger bruttostromerzeugung in deutschland ab 1990 nach energieträgern. arbeitsgemeinschaft energiebilanzen e die energiewende im stromsektor: stand der dinge 2019. rückblick auf die wesentlichen entwicklungen sowie ausblick auf 2020 the german energy transition in the british, finnish and hungarian news media bdew, 2020. bruttostromerzeugung in deutschland nach energieträger in den jahren die beendigung der energetischen nutzung von kohle in deutschland: ein überblick über zusammenhänge. herausforderungen und lösungsoptionen the incumbents' conservation strategies in the german energy regime as an impediment to remunicipalization-an analysis guided by the multi-level perspective projektionsbericht 2019 für deutschland gemäß verordnung (eu) nr. 525/2013. bundesumweltministerium commission on growth, structural change and employment -final report energy imports and exports. briefing paper number 4046. house of commons library the political economy of coal in poland: drivers and barriers for a shift away from fossil fuels cutting europe's lifelines to coal: tracking subsidies in 10 countries -united kingdom. overseas development institute policy brief explaining radical policy change: the case of climate change and energy policy under the british labour government 2006-10 integrating techno-economic, socio-technical and political perspectives on national energy transitions: a meta-theoretical framework a stress test for coal in europe under the paris agreement. scientific goalposts for a coordinated phase-out and divestment about 80% of eu and german, virtually all polish coal plants non-compliant with new eu 2021air pollution regulations moving beyond the heuristic of creative 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and the united kingdom: from regimes to democracies in sociotechnical transitions and discontinuities policy mixes for incumbency: exploring the destructive recreation of renewable energy, shale gas "fracking scenarios for coal-exit in germany -a model-based analysis and implications in the european context creative destruction or mere niche support? innovation policy mixes for sustainability transitions stewards or sticklers for change? incumbent energy providers and the politics of the german energy transition application and extension of a multi-dimensional framework. university of stuttgart institute for social sciences sequence and alignment of external pressures in industry destabilisation: understanding the downfall of incumbent utilities in the german energy transition the politics and economics of constructing, contesting and restricting socio-political space for renewables -the regime destabilization in energy transitions: the german debate on the future of coal insights from the uk coal phase out experience: report to chile decarbonization roundtable unpacking "regime resistance" in low-carbon transitions: the case of the british capacity market energy transition in germany: a case study on a policy-driven structural change of the energy system a just transition for coal miners? community identity and support from local policy actors the impact of policy measures on future power generation portfolio and infrastructure: a combined electricity and ccts investment and dispatch model (elco). energy syst. 1-30 staatliche förderungen der stein-und braunkohle im zeitraum 1950-2008. ö kologisch-soziale marktwirtschaft e.v. (fös). fös-studie im auftrag von greenpeace how germany helped bring down the costs of pv lusatia and the coal conundrum: the lived experience of the german energiewende energiewende 'made in germany' -low carbon electricity sector reform in the european context, 1st ed nrw im deutschen und europäischen kontext -energiewirtschaft, klimaziele und wirtschaftliche entwicklung, 129. politikberatung kompakt lessons from germany's hard coal mining phase-out: policies and transition from coal phase-out in germany -implications and policies for affected regions woran es beim kohleausstieg hakt und was zu tun ist. politikberatung kompakt, diw berlin wholesale electricity generation market shares by company largescale wind power in electricity markets with regular papers what stands in the way becomes the way sequencing in climate policy to ratchet up stringency over time transforming the uk energy system: public values, attitudes and acceptability -synthesis report dismantling a competitive electricity sector: the u.k.'s electricity market reform power stations of the uk, 2020. power stations of the uk coal, nuclear and renewable energy policies in germany: from the 1950s to the "energiewende germany's decision to phase out coal by 2038 lags behind citizens' timing preferences what does the paris climate agreement mean for finland and the european union? a roadmap for rapid decarbonization the politics of green transformations statistik der kohlenwirtschaft e.v, 2018b. datenangebot statistik der kohlenwirtschaft beschäftigte der braunkohlenindustrie in deutschland statistik der kohlenwirtschaft e.v, 2019b. steinkohle. statistik der kohlenwirtschaft approach to the distribution and orientation of power in socio-material change economic resilience of german lignite regions in transition regime destabilisation as the flipside of energy transitions: lessons from the history of the british coal industry (1913-1997) the destabilisation of existing regimes: confronting a multi-dimensional framework with a case study of the british coal industry (1913-1967) übersicht zur entwicklung der energiebedingten emissionen und brennstoffeinsätze in deutschland 1990-2015 -unter verwendung von berechnungsergebnissen der nationalen koordinierungsstelle emissionsberichterstattung stromerzeugung erneuerbar und konventionell um 6,3 prozent zurück united nations environment programme unfccc, 2015. paris agreement'. paris: united nations framework convention on climate change fakten und trends current challenges of germany's energy transition project and competing strategies of challengers and incumbents: the case of direct marketing of electricity from renewable energy sources we are grateful to all reviewers for helpful comments and suggestions that greatly improved earlier versions of the paper. a special thanks goes to all the experts on the uk and german energy sector that kindly provided us with their knowledge. we also thank adam lederer for language corrections. additional valuable input was provided by members of the coalexit research group, which we greatly appreciate. all potentially remaining errors are ours. the tef conceptualises industry environments and is based on evolutionary economics, neo-institutional theory, and economic sociology. it accommodates interactions between incumbent firms of a specific industry and a broader set of environments, including the economic and socio-political environments. the framework is based on schumpeter's idea to include social institutions relevant to economic behaviour in economic analyses (schumpeter 1942) . it recognises institutional change and includes strategic behaviour and power of actors. "embeddedness" means that the economy is embedded and not independent of social, political, and cultural dynamics.the tef framework builds on both selection theories and adaptation theories. it aims to integrate the two streams, not to choose either/or. the focus is on co-evolution, embeddedness, and bi-directional interactions (of industries and their economic, political, cultural, and social environments), which can only be analysed by combining the different theories and approaches. as geels (2014) states himself in the paper explaining the framework:' lewin and volberda (1999) suggest that selection theories and adaptation theories represent two levels of analyses, with the former emphasizing selection pressures that populations face from their environments, and the latter emphasizing (differences in) firmlevel strategies, capabilities and perceptions. […] "single-theme explanations for the adaptation-selection phenomenon have reached their limit. progress in the field requires combining and recombining multiple lenses instead of increasing fragmentation. we should consider the joint outcomes of managerial adaptation and environmental selection"' (geels, 2014, 262) .the tef framework is appropriate for our question and the coal industry, as it focusses on incumbent firms and the necessary pressure by policy makers and civil society to generate change (environmental selection). firms-in-industries are defined as "large, politically powerful, and scale-intensive with many sunk investments" (geels, 2014, 261) . further, the framework enables the analysis and comparison of coal phase-outs -non-linear processes involving a wide variety of actors -as it includes both lock-in and path dependence hindering reorientation of firms-in-industries and entire industriesas well as the reorientation of them, that means the change in the directionality of innovation. it includes insights from neo-institutional theory about the influences from the institutional environment, where "organizations compete for social fitness rather than economic efficiency" (geels, 2014, 264) . important is that key: cord-012518-ncrdwtdg authors: nan title: abstractband dog 2020 date: 2020-08-24 journal: ophthalmologe doi: 10.1007/s00347-020-01197-0 sha: doc_id: 12518 cord_uid: ncrdwtdg nan glaukom s65 kornea / konjunktiva s92 linse s100 pathologie / anatomie s102 plastische chirurgie, lider, orbita s106 refraktive chirurgie s112 retina / rpe / aderhaut / glaskörper s151 sehbahn / gehirn / neuro-ophthalmologie s155 strabologie / kinderophthalmologie s159 trauma s159 tumoren, hinterabschnitt s162 tumoren, vorderabschnitt und sonstige s166 uvea, iris, pupille, kammerwinkel s171 verkehrsophthalmologie s171 versorgungsforschung, gesundheitsökonomie und -politik s177 die abstracts sind zunächst nach subspezialitäten sortiert und dann alphabetisch nach erstautoren. liegend. beidseits gingen von der papille nach nasal ausgeprägte atrophien und pigmentepithelverdichtungen im sinne von knochenkörperchen aus. in der fundusautofluoreszenz (faf) zeigte sich in diesen bereichen eine ausgeprägt reduzierte faf, umgeben von einem saum erhöhter faf. die sd-oct der makula ergab eine regelrechte foveale senke und struktur, die oct-analyse der papille eine normale retinale nervenfaserschicht. in der kinetischen perimetrie (octopus 900, fa. haag-streit diagnostics, 90° bereich) stellte sich beidseits ein keilförmiges skotom der marke iii4 nach temporal superior dar, korrespondierend zu der lage und ausdehnung der betroffenen netzhautareale. im multifokalen elektroretinogramm fanden sich normwertige amplituden. im photopischen ganzfeld-elektroretinogramm (erg) waren die a-und b-welle leicht reduziert. flicker und ops waren unauffällig. die skotopischen erg antworten waren vor allem in der b-welle mittelgradig reduziert. schlussfolgerung: das alagille-syndrom kann zu pigmentretinopathien mit gesichtsfelddefekten führen, die einer retinitis pigmentosa ähneln. es handelt sich um den ersten beschriebenen fall eines sektoriellen retinalen befundes, der sich in bemerkenswerter weise spiegelsymmetrisch darstellt. the first examination no correction of ocular magnification was applied. in the second examination the previous focus value (of imaging 1) was applied as refraction data in the heyex-software to incorporate ocular magnification. the third examination was performed including anterior corneal curvature (as examined with zeiss iol master 700) and objective refraction, the standard parameters in the heyex-software for ocular magnification correction. segmentation of prnfl, bmo and mrw was conducted with heyex-software. intraclass correlation coefficient (icc) were computed. results: twenty eyes of 12 participants were included in this ongoing study. the mean value of se was -0. 6 conclusions: our preliminary data with small number of eyes showed that measurement of bmo area should incorporate biometric parameters for ocular magnification correction or at least the focus measurement as se data. in contract, prnfl and mrw measurements showed high agreement between the three measurements (no correction, using focus as se data for ocular magnification correction or the standard method). sessed by patient-reported visual functioning together with relevant clinical parameters and visual field (vf) changes over a longer time period. methods: 43 patients with glaucomatous optic nerve damage were enrolled in this prospective longitudinal observational study. assessment of qol was obtained by patient-reported visual functioning using the rasch-calibrated person estimate glaucoma activity limitation 9 (gal-9) questionnaire at baseline and after eight years together with best-corrected visual acuity (bcva), vf data, and many other parameters. results: bcva of the better eye changed from logmar 0,16 ± 0,22 to 0,21 ± 0,14 whereas there was change from 0,27 ± 0,25 to 1,39 ± 1,1 on the worse eye. values of gal-9 changed from -2,39 ± 2,14 to -1,38 ± 2,78, according to a gql sum score change from 79,17 ± 19,63 to 69,22 ± 27,95 . mean deviation (md) of the better eye changed from average -3,99 ± 4,55 to -4,83 ± 5,09 (p = 0,59) while the worse eye changed significantly from -8,86 ± 5,86 to -12,05 ± 8,07 (p = 0,02) . the change in pe-gal9 showed a highly significant correlation with the md at follow-up, especially with the worse eye (r = 0,43). the impact of the md at follow up on qol could also be well predicted in a regression model. conclusion: qol decreases significantly over time in glaucoma patients. especially the changes of the visual field of the worse eye have a great impact on reported functioning. careful treatment also of the eye with the worse glaucomatous damage is mandatory. long-term iop lowering and relationship to early iop response after a single bimatoprost implant administration in a phase 1/2 study klabe k. 1*, 2 , chen m. 3 , wang k. 3 , liu j. 3 , rivas m. 4 aims: this analysis assessed early iop response in a subset of patients in which a single administration of bim implant managed iop for up to 24 months without use of rescue topical medication. methods: in this prospective, paired-eye phase 1/2 study, bim implant was administered in the study eye; the fellow eye received once-daily topical bimatoprost 0.03 %. subgroup analysis evaluated early iop in patients without rescue/implant retreatment in the study eye who remained in the study through month 24 (long-term iop responders) vs all other patients. early iop response was defined as iop through 12 weeks (average iop from day 8 through week 12) . associations between early iop lowering and long-term iop responder status were explored using logistic regression analysis. results: of 75 enrolled patients, the 19 (25 %) long-term iop responders demonstrated early iop lowering after bim implant administration, with mean (sem) iop through 12 weeks of 15.8 (0.3) mmhg. patients with rescue/retreatment or early exit (all other pts) had a mean (sem) iop through 12 weeks of 18.0 (0.5) mmhg (p < 0.001 vs long-term iop responders). baseline mean (sem) iops for long-term iop responders and all other patients were 23.4 (0.3) and 25. 8 (0.5) (or 5.886 , >20 % vs ≤20 %). conclusions: early response to treatment can potentially be used to predict later response and guide clinical decision-making. the present phase 1/2 study results indicate an association between iop at early timepoints after bim implant administration and long-term iop lowering without additional treatment. cmv und rv au betrifft vorwiegend jüngere und männliche patienten, während vzv und hsv vau hauptsächlich in älteren und weiblichen patienten zu finden ist (p < 0.0001). n = 52 patienten (19 %) zeigten eine glaukomatöse schädigung, wovon n = 27 (10 % der gesamtkohorte) einen glaukomchirurgischen eingriff bei progression benötigten. es zeigte sich eine signifikante drucksenkung von präoperativ 28,18 ± 9,32 mmhg auf 14,72 ± 6,67 mmhg 2 jahre postoperativ (p = 0,004). in n = 10 patienten (37 %, n = 9 migs + n = 1 te) wurde ein zweiter operativer eingriff notwendig. schlussfolgerung: verschiedene virusentitäten der uveitis anterior stellen unterschiedliche spezifische risiken für die entwicklung eines glaukoms sowie notwendige folgeoperationen dar. migs kann als erstbehandlung in individuell ausgewählten fällen eingesetzt werden. das passende verfahren sollte von einem erfahrenen spezialisten sorgfältig ausgewählt werden. eine filtrierende glaukomoperation kann in vau patienten zur augeninnendrucksenkung über einen längeren zeitraum empfohlen werden. intraocular pressure 5 years after repeated anti-vegf treatment for age-related macular degeneration 38 the paediatric glaucoma diagnosing ability of macular segmentation by optical coherence tomography compared to peripapillary retinal nerve fibre layer thickness lever m. 1*, 2 , halfwassen c. 1, 2 , unterlauft j. d. 3 , bechrakis n. e. 1, 2 , manthey a. 1, 2 , böhm m. 1, 2 1 universitätsklinikum essen, essen, germany; 2 achim wessing institute for ophthalmological diagnosic (awio), essen, germany; 3 universitäts-augenklinik, leipzig, germany purpose: the decrease of peripapillary retinal nerve fiber layer (prnfl) and macular layers thickness measured by spectral domain-optical coherence tomography (sd-oct) is well documented in pediatric glaucoma. however, the diagnostic impact of these measurements in children has rarely been investigated. the aim of this study was to compare these measurements in pediatric glaucoma patients and healthy children to evaluate their respective glaucoma diagnosing ability. methods: retrospective observational study including 72 children aged 5-17 years (glaucoma: 19 [26.4 %] , healthy: 53 [73.6 %]) examined with sd-oct (spectralis®, heidelberg engineering). the thickness of prnfl sectors and of etdrs subfields after automated macular segmentation were compared between diseased and healthy control children. the correlation of those measurements with the presence of glaucoma was evaluated using logistic regression. the glaucoma discriminative capacity of single or combined prnfl and macular areas were calculated using area under the receiver-operating curves (auc). results: prnfl thickness is reduced in glaucoma patients (e. g. total-average-thickness: p < 0.0001). the thickness of the three inner macular layers is reduced in glaucoma patients (e. g., ganglion cell layer [gcl] , outer-inferior subfield: p = 0.0005). prnfl and macular segments' thickness correlate highly with the presence of glaucoma. correspondingly, analyzing these parameters revealed a high auc for the identification of glaucoma (prnfl: 0.83; gcl: 0.82). finally, the strongest glaucoma discriminating ability was observed for the combination of selected subfields of all macular segments (auc 0.96). the measurement of prnfl thickness and of selected macular segments showed a high glaucoma discriminative ability. the strongest diagnostic performance, however, was found combining data from all macular segments. further studies are needed to study more precisely how prnfl and macular thickness measurements using sd-oct could improve the diagnostic possibilities in pediatric glaucoma. pahlitzsch t. 1 , pahlitzsch m. 2 methodik: insgesamt wurden fünfzehn patienten (alter: 69,9 ± 7,1 jahre) mit moderatem bis fortgeschrittenem primären offenwinkelglaukom (powg) in die studie eingeschlossen. präoperativ, ein tag und drei monate postoperativ einer katarakt-operation wurden folgende untersuchungen durchgeführt: visus, gesichtsfeldprüfung (weiß-weiß-perimetrie octopus 900), augendruckmessung (goldmann applanationstonometrie), bestimmung der perfusionssituation mit der oct-angiographie (cirrus hd-oct 5000), der zentralen kornealen dicke (pentacam) und endothelzelldichte (nidek cem-530) sowie des flarewerts (kowa fm 700). ergebnisse: postoperativ verbesserte sich der bestkorrigierte dezimalvisus von 0,57 ± 0,16 auf 0,75 ± 0,11. es zeigte sich eine augeninnendruckreduktion von 19,7 ± 2,8 auf 14,0 ± 2,9 mmhg. eine abnahme der kornealen endothelzellzahl von 2502,4 ± 220,2 zellen/mm 2 auf 2304,3 ± 442,4 as to the optical coherence tomography results, the rate of the ganglion cell complex thinning in patients with pxf was 45.4 % higher than in patients without this syndrome. conclusions: according to the results of our study, the ganglion cell complex thinning is almost one and a half times higher in patients with openangle glaucoma and pseudoexfoliation syndrome in comparison with the ganglion cell complex thinning in patients with oag. methods: all patients who were admitted in january 2020 to our department for anti-vegf treatment and were on anti-vegf therapy (ranibizumab, aflibercept) for 5 years-from january 2015 or before, were included in our study. the retrospective study included 70 patients with namd (80 eyes). all the patients with glaucoma, ocular hypertension, myopic or diabetic maculopathy, eye trauma, uveitis were excluded from our study. the diabetes with or without diabetic retinopathy was also an exclusion criterion. only the patients with namd without any other ophthalmic pathology were involved. sustained iop was defined as a rise in iop above baseline ≥ 6 mmhg and/ or > 21 mmhg on two or more consecutive visits. untreated eyes were used for control group. results: the average age of our patients was 82,2 years (ranging from 68 to 96). the majority were women (76,1 %). except for clinical picture of namd other ophthalmological findings were normal. all patients were treated after "treat and extend" regiment receiving intravitreal ranubizimab (lucentis) or aflibercept (eylea) injections. bilateral manifestation was observed in 10 patients (20 eyes). in all eyes a decrease in iop from baseline was observed after 5 years of therapy with a mean reduction of 0.8 mmhg in treated eyes compared with an average increase of 0.5 mmhg in fellow untreated eyes. the difference was statistically significant. only in 2,5 % (2 of 80 eyes) iop elevation of 6 and 7 mmhg was observed. none of the patients in treated group experienced iop > 21 mmhg. in control group the elevation of 6 or more mmhg or > than 21 mmhg was not observed. conclusions: our study revealed that repeated intravitreal anti-vegf injections during a period of 5 years or more for namd in otherwise healthy eyes and subjects are not associated with iop elevation. we found a significant difference in iop between treated and untreated eyes with reduction in treated and elevation in untreated eyes. further studies are necessary to confirm our results. purpose: micro-invasive glaucoma surgery (migs) has become an important treatment approach for primary open angle glaucoma, while the long-term efficiency of these procedures remains yet to be substantiated. recently, we published a protocol for the assessment of aqueous humor outflow in vitro using an in rostock developed dedicated oculopressor (rop), which allows analysis of migs efficiency. in the present study the performance of an in house developed valve-controlled microstent was evaluated with regard to drainage capacity and effect on intraocular pressure (iop) using this protocol in rabbits in vivo. methods: six months after migs draining in the subconjunctival tenon space, aqueous humor outflow was assessed utilizing the rop protocol in anaesthetized new zealand white rabbits. a 23g cannula, connected to a scans (spectralis oct ii, heidelberg, germany) in 3 layers: superficial vascular plexus (svp), intermediate vascular plexus (ivp) , and deep capillary plexus (dcp). the local ethics committee approved the study. results: mean macula vd was significantly reduced in svp (28.91 ± 3.71 vs 32.33 ± 3.04), ivp (19.77 ± 3.54 vs 23.08 ± 2.49), and dcp (20.79 ± 4.29 vs 25.24 ± 2.98) of oht compared to healthy controls (p < 0.0001). additionally, mean macula vd of svp (26.28 ± 5.23), ivp (19.23 ± 3.75), and dcp (21.05 ± 4.60) were significantly reduced in pre-oag patients compared to controls (p < 0.0001). no significant differences were observed between oht and pre-oag patients in ivp, and dcp (p > 0.05), yet svp differed significantly (p = 0.018). conclusion: as a significant reduced macula vd was seen in all 3 retinal vascular layers of glaucoma suspects compared to healthy controls, the data of the present study might indicate microvascular changes in the macula region even early in glaucoma pathogenesis. vorstellung eines standardisierten schemas zur glaukomverlaufskontrolle in der augenärztlichen praxis rakitin m. fluid reservoir with buffered saline solution, was injected into the anterior chamber to adjust baseline iop to 20.0 ± 1.8 mmhg. for iop-measurements a second 23g cannula, connected to a pressure transducer, was inserted into the anterior chamber. the rop (weight = 60 g) was positioned on the central cornea and held in place for 4 min. iop value dynamics were continuously monitored and compared with the contralateral control eye which had not undergone surgical intervention (n = 3, each). results: immediately after rop placement iop peak values peaked of 44.0 ± 0.9 mmhg for control and 47.6 ± 1.4 mmhg for treated eyes were measured, respectively. iop of control and migs eyes was lowered during the four minutes of oculopression to similar values, 38.2 ± 0.6 mmhg and 38.2 ± 1.1 mmhg, respectively. however, iop decrease was significantly steeper in migs eyes compared to the untreated eyes. following rop removal iop levels were 4.1 ± 3.2 mmhg below adjusted baseline iop. the rop protocol was successfully utilized to evaluate aqueous humor outflow following migs in a standardized manner. the faster iop decrease in treated eyes in comparison to control eyes evidenced in vivo functionality of our proprietary, valve-controlled microstent and its facilitation of aqueous humor outflow six months after implantation. following this proof-of-principle in vivo study in a rabbit model, further investigations will include use of the rop protocol with glaucoma patients, before and after migs, to assess the contribution of the implanted drainage device to aqueous humor outflow. in vitro decrease of extracellular matrix deposition by suppression of the tgf-β pathway by a macrolide antibiotic in ocular fibroblasts sterenczak k. a. 1* , fuellen g. 2 purpose: a major complication after fistulating glaucoma surgeries are fibrotic processes causing postoperative decrease in the liquid drain. accordingly, the prevention of fibrotic processes represents a key aspect in the development of advanced therapeutic surgical concepts. kitasamycin represents an antibiotic molecule belonging to the group of macrolides and can act as a potential candidate to modulate fibrotic processes. herein we evaluated the antifibrotic potential of kitasamycin in a tgf-β mediated fibrotic human fibroblast in vitro model system. methods: primary ocular fibroblasts from human tenon capsule (htf) were isolated, cultivated and kitasamycin was tested for its highest possible non-toxic dose covering a concentration range from 1 to 100 µm. differentiation of fibroblasts towards myofibroblasts was performed by incubation with 10 ng/ml tgf-β1 for 48 h. fibroblasts were cultured on coverslips and fixed for immunocytochemical analysis. immunofluorescence was performed using antibodies against ecm-components (α-smooth muscle actin (sma), fibronectin). dapi staining was used for additional nucleic labeling. protein detection of ecm-components (α-sma, β-actin, β-tubulin, collagen i and vi, fibronectin, vimentin) was performed by western blot. results: in htfs exposed to different kitasaymcin concentrations no increase in α-sma and ecm-component expression was detected. tgf-β1 strongly induced fibrotic marker expression. the addition of kitasamycin inhibited the induced fibrotic marker expression in a concentration dependent manner (up to a highest concentration of 100 µm). conclusions: our study demonstrated a dose-dependent expression inhibition of fibrotic marker in tgf-β stimulated htfs by kitasamycin. these findings demonstrate that kitasamycin impairs the transformation of fibroblasts towards myofibroblasts and the expression of proteins involved in scarring processes. therefore, kitasamycin is a potential agent for the specific prevention of postoperative scarring processes and fibrosis following glaucoma filtration surgery. abstracts pensierte neovaskularisationsglaukom (nvg) bei gematchen patienten mit zentralvenenverschluss (zvv) und proliferativer diabetischer retinopathie (pdr) zu vergleichen. methodik: 83 patienten wurden mit 23-g-ppv, panretinaler full-scatter-endolaser, transskleraler zyklophotokoagulation (810-nm-diodenlaser, 2 w, 2 s, 20 herde) und intravitrealer bevacizumab-eingabe behandelt. phake augen wurden kombiniert mit katarakt operation versorgt. 12 zvv patienten konnten mit 12 pdr patienten auf iod gematcht werden mit coarsened exact matching (cem). analysiert wurden bestkorrigierter visus (logmar), augeninnendruck (iod, mmhg), medi-score (anzahl der glaukom medikamente) nach 1, 3, 6 und 12 monaten (m) , visuelle analoge schmerzskala (vaps, 0-10), erneute eingriffe und die erfolgsrate (iod ≤21 mmhg oder iod-reduktion ≥ 30 %, keine erblindung). ergebnisse: 12 zvv patienten konnten mit 12 pdr patienten mit dem präoperativen iod gematched werden (baseline iod: 49, 8 ± 8, 5 mmhg, p = 1, 0) . zvv patienten (80,9 ± 8,2 j) waren älter als pdr patienten (67,1 ± 11,3 j) (p = 0,002). männer waren in beiden gruppen häufiger betroffen (zvv 58 %, pdr 75 %). der visus zeigte nur in der pdr-gruppe einen signifikanten unterschied zwischen baseline (zvv 2,1 ± 0,4; pdr 1,8 ± 0,8) und 12 m follow up (zvv 2,0 ± 0,7; pdr 1,0 ± 0,7) (p < 0,002). der gematchte präoperative iod sank in beiden gruppen nach 12 m signifikant (zvv 13,4 ± 5,6 mmhg; pdr 13,2 ± 5,6 mmhg). es zeigte sich kein unterschied diesbezüglich zwischen den beiden gruppen (p = 0,230). der medi-score sank zwischen baseline (zvv 4, 3 ± 2, 4; pdr 4, 8 ± 2, 5) und 12 m stärker in der pdr gruppe (zvv 2,2 ± 1,6; pdr 1,0 ± 0,5) (p = 0,05). alle patienten waren postoperativ schmerzfrei. 10 zvv und 10 pdr augen (83,3 %) benötigten keine weiteren stationären behandlungen. die erfolgsrate betrug 83 und 75 % in der zvv beziehungsweise pdr-gruppe (p = 0,674). zusammenfassung: die kombinierte operationsstrategie senkte in beiden gruppen den iod und kontrollierte die schmerzen. in der pdr gruppe verbesserte sich der visus und sanken die drucksenkenden medikamente. 75 % der patienten erfüllten das erfolgskriterium nach einem jahr. kgd/iopstim: 37, 7 ± 5, 2/24, 7 ± 4, . streubreite der ezm: 3,2 ± 1,8/2,9 ± 1,3, (t-test: p = 0,36) intraclass korrelationskoeffizient (ikk) der ezm: 0,88/0,83. anova der ezm: p = 0,48/0,08. mrvp kgd minus mrvp iopstim: 13,0 ± 5,6. verhältnis mrvp kgd/mrvp iopstim: 1,56 ± 0,31. schlussfolgerungen: wie aus dem hohen ikk und dem anova-ergebnis zu schließen ist, werden mit dem iopstim und dem kgd gut reproduzierbare einzelwerte des rvp erhalten, deren streubreiten sich nur gering unterscheiden. der iopstim erscheint somit geeignet für den klinischen einsatz. das neue iopstim verfahren wurde erst einmal bei freiwilligen erprobt. weil bei diesen erwartungsgemäß eine spontane pulsation der zv (svp) vorliegt, musste unter valsalva-bedingungen untersucht werden, um die svp aufzuheben. diese bedingung und die unterschiedliche kraftrichtung bei kgd und iopstim kommen als ursache für den unterschied des mrvp in frage. strzalkowska a. fragestellung: wir benutzten exact matching, um einen ausgewogenen vergleich zwischen der ab-interno-trabekulektomie (ait) mit dem trabektom und der trabekulektomie (trab) mit mitomycin c (mmc) zu erstellen. methodik: 96 patienten, bei denen eine trab durchgeführt wurde, wurden mit 5485 ait patienten verglichen. die patienten wurden anhand des augeninnendrucks (iod), der anzahl der glaukommedikamente (medikamente) und der glaukomarten unter verwendung von exact matching und nach dem alter durch nearest-neighbor-matching verglichen. die ergebnisse wurden nach 1, 3, 6, 12, 18 und 24 monaten analysiert. ergebnis: 165 ait konnten mit 165 trab gematcht werden. der mittlere präoperative iod betrug 22,3 ± 5,6 mmhg und die grundlinienzahl der glaukommedikamente betrug in beiden gruppen 2,7 ± 1,1. nach 24 monaten sank der iod in ait auf 15,8 ± 5,2 mmhg und in trab auf 12,4 ± 4,7 mmhg. der iod war bei allen weiteren follow-ups niedriger als der ausgangswert (p < 0,01) und in trab niedriger als der in ait (p < 0,01). die anzahl der glaukomtropfen sank bei ait auf 2,1 ± 1,3 und bei trab auf 0,2 ± 0,8. im vergleich zum ausgangswert verwendeten die patienten bei allen follow-ups weniger medikamente (p < 0,01) und bei trab weniger als bei ait (p < 0,01). sekundäre chirurgische eingriffe (revisionen oder weitere glaukom-operationen) hatten den größten einfluss auf das überleben und wurden bei 15 ait-und 59 trab-patienten notwendig. schlussfolgerung: ait und trab führten zu einer verringerung des augeninnendrucks und der medikamente, die bei trab stärker ausgeprägt war, jedoch auf kosten von viermal so vielen sekundären interventionen. subgruppenanalyse nach kombinierter operationsstrategie für das dekompensierte neovaskularisationsglaukom -eine retrospektive coarsened exact matching fallserie strzalkowski p. 1* , strzalkowska a. 1 , loewen n. 1 uluk y. 1* , baulig c. 1 in der gruppe ts cya zeigte sich eine iod-senkung von präoperativ im median 19,5 mmhg (q25 10 mmhg/q75 23 mmhg) auf im median 15 mmhg (q25 11 mmhg/q75 17,5 mmhg) (p = 0,000) nach 12 monaten. die drucksenkenden wirkstoffe konnten im median von 2 (q25 0/q75 3) auf im median 0 (q25 0/q75 2) (p = 0,004) reduziert werden. ein complete success wurde bei 39 % und ein qualified success bei 58 % der patienten erreicht. die durchschnittliche prozentuale drucksenkung lag in beiden gruppen (ts nativ und ts cya) bei 22 % nach 12 monaten. der unterschied zwischen den beiden gruppen war nicht signifikant (p = 1000). schlussfolgerung: beide gruppen zeigten nach ts erwartungsgemäß eine signifikante senkung des iod, sowie der drucksenkenden wirkstoffe. die anwendung von topischem cya in der postoperativen therapie der ts zeigt keinen vorteil bezogen auf iod oder drucksenkende wirkstoffe. 6-75,9 %) in der trabekulektomie gruppe und 58,5 % (95 %-ki: 47,6-69,4 %) in der xen gruppe. es gab keinen signifikanten unterschied zwischen den beiden gruppen hinsichtlich des primären endpunktes ‚kompletter chirurgischer erfolg' nach einem jahr (or = 0,66 [95 %-ci: 0,32-1,34]; p = 0,26). mittels multivariater logistischer regressionsanalyse konnte kein einfluss auf den kompletten chirurgischen erfolg nach einem jahr für folgende faktoren gefunden werden: geschlecht, alter, präoperativer iod oder die anzahl topischer medikamentenklassen präoperativ. nach einem jahr war die iod reduktion (sekundärer endpunkt) in der trabekulektomie gruppe (10,5 ± 9,2 mmhg) signifikant höher verglichen mit der xen-gruppe (7,2 ± 8,2 mmhg) (p = 0,003). schlussfolgerung: xen-implantation und trabekulektomie zeigen ähnliche erfolgsraten nach einem jahr bei patienten mit refraktärem offenwinkelglaukom, und sind daher chirurgische optionen für die klinische routine. die trabekulektomie zeigte allerdings eine höhere drucksenkung im vergleich zum xen-stent. eine randomisierte klinische studie fehlt, um einen möglichen unterschied in den langzeitergebnissen zwischen den beiden gruppen zu zeigen. aim: glaucoma is a chronic disease that frequently requires long-term treatment with topical ocular hypotensive eyedrops. dry eye symptoms frequently coexists with glaucoma and may be initiated or exacerbated by topical glaucoma medications. thealoz duo® is a novel artificial tear preparation containing two active ingredients: trehalose, a natural alpha-linked disaccharide with high water retention capabilities and sodium hyaluronate, an anionic glycosaminoglycan polysaccharide found in various connective tissue which has lubricant and water-retaining properties. the aim of this study was to evaluate the efficacy of trehalose/hyaluronate tear substitute (thealoz duo®) in recovering the tear film changes in glaucoma patients with mild to moderate dry eye symptoms. methodology and results: the group of glaucoma patients (n = 15, 8 males and 7 females) under topical medical treatment and age-matched controls (n = 15, 7 males and 8 females) were reviewed. the mean age of all patients was (respectively, mean±sd) 56.3 ± 15.9 years, 15 males and 15 females. age, gender, number of glaucoma medications used, the duration of glaucoma treatment and the presence of dry eye symptoms were recorded. after initial evaluation, patients were instructed to administer thealoz duo® with the regimen of one drop/eye/three times daily. patients were observed in 2 visits: day 0 (baseline) and after one month of treatment (endpoint). tear film quality was measured using tear break-up time (tbut) test. significant changes at endpoint as compared to baseline were found in both groups (p < 0,05). tbut test results improved in both, glaucoma (7,33 ± 3,90 vs 8,08 ± 3,88 s) and non-glaucoma (8,36 ± 3,66 vs 8,80 ± 3,47 s) patients. the improvement in tear film quality (measured by tbut) was shown after application of trehalose/hyaluronate tear substitute for one month in both, glaucoma and control group patients with mild to moderate dry eye symptoms. however, randomized studies are required to truly ascertain the magnitude of their clinical value. vidinova c. n. 1* , pravoslava g. 2 1 military medical academy, sofia, bulgaria; 2 eye clinic zrenie, sofia, bulgaria our ability to help patients often depends on technology. oct is essentially used in the diagnosis and treatment of glaucoma. understanding the types of artifacts commonly seen is vital in the clinical practice. purpose: the purpose of our study is to determine the frequency and distribution of oct and oct a errors and imaging artifacts in patients evaluated for glaucoma. to provide examples for the most common mistakes. methods: in our prospective study 82 patients with primary open-angle glaucoma were enrolled. they all underwent a complete ophthalmological examination including va, perimetry and oct-both sd-oct (rtvue, examined patients were divided into 2 groups: the main group included 124 workers (248 eyes) who were in direct contact with the petrochemical products; the control group included 243 workers (486 eyes) who were not in direct contact with the petrochemical products. results: the mean age of workers in the main and control groups was 51.1 ± 0.7 and 49.2 ± 0.6 years, respectively (56.7 % male). the mean work experience of workers in the main and control groups was 22.5 ± 0.9 years and 19.2 ± 0.7 years (p > 0.05). the eye pathology was revealed in 189 (51.5 %) workers of modern petrochemical industry. the prevalence of eye pathology in the main and control groups was 80.6 ± 3.5 % and 36.6 %±3.1, the difference was statistically significant (р < 0,001). the prevalence of different eye pathologies in the both groups were 49.2 ± 4.5 % and 21 ± 2.6 % for the dry eye syndrome, 20.2 ± 3.6 % and 2.5 ± 1.0 %-conjunctival melanosis, 19.4 ± 3.5 % and 4.9 ± 1.4 %-blepharoconjunctivitis, 14.5 ± 3.2 % and 6.2 ± 1.5 %-pingvecula, 14.5 ± 3.2 % and 4.1 ± 1.3 %-conjunctival nevus, 6.2 ± 2.2 % and 1.2 ± 0.7 %-pterygium, 0.8 ± 0.8 % and 0.4 ± 0.4 %-glaucoma. the mean iop in the main and control groups was 16.1 ± 0.2 mmhg and 16.5 ± 0.1 mmhg, the difference was not statistically significant. conclusions: despite the decrease of the harmful factors' influence on the eye due to the use of modern petrochemical industry technologies in azerbaijan they still could cause occupational diseases of the eye. the prevalence of eye pathology in the workers of modern petrochemical industry with direct contact with the petrochemical products is higher than in workers without direct contact. ashikova p. 1* , oganesyan o. 1 , khandjan a. 1 , getadaryan v. 1 , grdikanyan a. 1 , oganesyan c. 2 1 the helmholtz moscow institute of eye diseases, moscow, russian federation; 2 the charles university, prague, czech republic introduction: a pterygium is a pinkish, triangular fibrovascular tissue growth on the cornea, which occurs more often from the medial side. in different countries the frequency (incidence?) of the disease varies from 0.7 to 31 %. over 100 surgical methods have been offered for the pterygium treatment. however, the rate of recurrence is still relevant and can reach up to 70 %. to decrease the reoccurrence of the pterygium, conjunctival and limbal autografts, amniotic membrane transplantation, beta-irradiation, antimetabolites are used. after any pterygium surgery, there is no bowman layer in intervention area. the bowman layer transplantation can restore not only normal anatomy of cornea, but also intercellular interactions (relationships?) as well. this may reduce the frequency of the reoccurrence of the pterygium. purpose: to describe the feasibility of bl transplantation during pterygium surgery and to study short-term results. methods: two female patients 35 and 62 years old with pterygium (stage ii) were operated. decimal uncorrected visual acuity was 1.0. patients were examined before the surgery, after 1 day, 1 and 3 months after surgery by biomicroscopy, keratotomography, and optical coherence tomography (oct). briefly, the surgery technique was following. after the pterygium excision, a simple conjunctival closure was performed. phototherapeutic keratectomy (ptk) recipient cornea and the stromal surface of bl graft were performed. the bl graft (3.5 and 4.0 mm in diameter) was placed in the postptk bed. a contact lens was placed on the cornea. postoperatively corticosteroids and antibiotics were prescribed 3 times per day for 3 weeks. results: the surgeries and 3 months follow-up were uneventful. the cornea area of intervention are clear, the grafts are in place, and the edges of grafts are fully adapted. the data of visual acuity and central keratometry stay unchanged. according to oct the grafts thickness are 68 and 73 microns 3 months after surgery. clinical efficacy of cypass microstent with 18 months follow-up wörn m. 1* , hohberger b. 1 , ennen m. 1 , lämmer r. 1 1 univ.-augenklinik, erlangen, germany propose: minimal invasive glaucoma surgery (migs) offers a good intraocular pressure (iop) lowering effect and high safety profile. as ab interno approach, the stents enable glaucoma surgery without conjunctival dissection. cypass microstent (available 04/2017-08/2018) was the only migs option, draining the aqueous humor suprachoroidally. it was the aim of the present study to investigate efficacy of cypass microstent as stand-alone and combined procedure with cataract surgery with a 18 months follow-up. methods: eighty-four eyes from 84 open-angle glaucoma (oag; 45 female, 39 male) patients received stand-alone cypass microstent implantation (n = 50) or combination with cataract surgery (n = 34). patients were grouped into cohort 1 with preoperative iop ≥ 21 mmhg (n = 44) and cohort 2 with preoperative iop < 21 mmhg (n = 40). efficacy outcome included change of mean iop and number of glaucoma medication through 18 months follow-up. results: preoperative iop was 28.25 ± 7.68 mmhg (cohort 1) and 16.37 ± 3.21 mmhg (cohort 2). mean iop was significantly decreased after 6 months (14.52 ± 4.91 mmhg; -48.60 %), 12 months (16.47 ± 3.65 mmhg; -41.70 %), and 18 months (18.80 ± 8.08 mmhg; -33.45 %) compared to preoperative iop for cohort 1 (p < 0.001). yet, no significant differences were observed for mean iop after 6 months (15.88 ± 5.49 mmhg, -2.99 %), 12 months (15.48 ± 3.65 mmhg, -5.44 %), and 18 months (15.53 ± 5.67 mmhg, -5.13 %) compared to preoperative iop in cohort 2 (p > 0.05). glaucoma medication was significantly reduced in both cohorts after 6 months (cohort 1: p < 0.001, cohort 2: p = 0.002) and 12 months (cohort 1: p < 0.001, cohort 2: p = 0.007) compared to baseline. at the end of the follow-up number of glaucoma medication was significantly decreased in cohort 1 (2.38 ± 1.12 vs 1.82 ± 1.31, p < 0.001) and slightly in cohort 2 (2.27 ± 1.30 vs 1.72 ± 1.31, p = 0.043) compared to preoperative. surgery procedure of stand-alone implantation or combination with phacoemulsification did not influence outcome of iop or glaucoma medication (p > 0.05). conclusion: iop reduction after cypass microstent implantation was larger in oag eyes with higher preoperative iop than with preoperative iop< 21 mmhg. number of locally administered medication was significantly reduced in both cohorts. aghayeva f. a. 1* , ibrahimova s. n. 1 , kasimov e. m. 1 1 national centre of ophthalmology named after academician zarifa aliyeva, baku, azerbaijan introduction: the petrochemical industry occupies a leading position among the sectors of the azerbaijan economy. thus, health care of the oil industry employees that are exposed to the complex of chemical, physical and psycho-emotional harmful factors is becoming more socially significant. in the 1980s academician zarifa aliyeva conducted research on occupational eye diseases in workers of petrochemical industry. taking into account the use of new modern industry technologies continuation of such type of studies are of great interest. the aim: of this study was to study the prevalence of eye pathology and intraocular pressure (iop) in workers of modern petrochemical industry of azerbaijan. methods: analysis of eye condition and iop was performed in 367 workers (734 eyes) of the oil refinery factory named after heydar aliyev. all first results of a prospective multicentre phase i/iia clinical trial on application of purified allogenic abcb5+ limbal stem cells for treatment of severe limbal stem cell deficiency auffarth g. 1* , meller d. 2 , cursiefen c. 3 , wasielica-poslednik j. 4 , chodosh j. 5 1 universitäts-augenklinik heidelberg, heidelberg, germany; 2 universitäts-augenklinik jena, jena, germany; 3 universitäts-augenklinik köln, cologne, germany; 4 universitäts-augenklinik mainz, mainz, germany; 5 department of ophthalmology, harvard university, boston, united states background: limbal stem cell deficiency (lscd) is characterized by a loss or deficiency of limbal stem cells (lscs), which causes a loss of the regeneration potential of the cornea, a loss of the barrier function between conjunctiva and cornea and severely impaired vision up to blindness. methods: in this study purified lscs from cadaveric donors were prepared by using the lsc marker abcb5. the aim of this study is to test this lscbased atmp in a first-in-human multicenter phase i/iia clinical trial to evaluate the safety and efficacy of ascending doses of allogeneic abcb5 + lscs for the treatment of lscd. the lscs are expected to permanently engraft in the limbal stem cell niche, so that the limbal barrier is restored and a transparent cornea is regenerated. the study is planned with 16 lscd patients in 4 ascending dose groups of 4 patients each. the inclusion criteria involve patients with secondary lscd with vessel penetration of at least 2 quadrants with central cornea involvement. cells are topically applied on the entire corneal and limbal area following surgical dissection of conjunctival pannus tissue from the corneal surface. to date, 2 patients have been recently treated with the imp, but as the primary efficacy endpoint is measured after 1 year it is not yet possible to present the primary efficacy endpoints. in preclinical studies, transplantation of abcb5+ lscs in mice with induced lscd led to normal and transparent human corneas without corneal neovascularization. the inclusion criteria involve patients with secondary lscd with vessel penetration of at least 2 quadrants with central cornea involvement. cells are topically applied on the entire corneal and limbal area following surgical dissection of conjunctival pannus tissue. to date, 2 patients have been recently treated with the imp, but as the primary efficacy endpoint is measured after 1 year it is not yet possible to present the primary efficacy endpoints. in preclinical studies, transplantation of abcb5+ lscs in mice with induced lscd led to normal and transparent human corneas without corneal neovascularization. discussion: abcb5+ lscs are a promising new treatment method that could possibly be a permanent cure to patients with lscd, but before any statistically significant statement about efficacy is possible, more patients need to be treated and the already treated patients need to be observed for a longer period. purpose: to assess possible parameters that may affect endothelial cell density (ecd) decrease after descemet membrane endothelial keratoplasty (dmek) by comparing eyes in the low versus high quartile of ecd decrease over a follow-up period of four years. methods: for 351 eyes (275 patients) who underwent dmek for fuchs endothelial corneal dystrophy (fecd), donor ecd decrease as compared to preoperative donor ecd was evaluated up to four years after surgery. eyes with a postoperative ecd decrease in the lower quartile at all available follow-up moments were assigned to group 1 (n = 51), and those in the upper quartile to group 2 (n = 42). multinominal regression was used to assess which covariates were related to the different patterns of ecd decrease (i. e. high versus low ecd decrease). from the retina to the cornea and to increase the in-plane resolution. furthermore, a piezo actuator is implemented to control the focal plane within the cornea. measurements on various corneal layers were performed ex vivo on pig and lamb eyes as well as in vivo on human eyes. results: in general, imaging could be performed at all investigated wavelengths and species. exemplifying the found imaging patterns, the cell borders and nuclei of the human superficial epithelium are shown, which have a stronger reflectance using the blue or green compared to near-infrared light. at deeper layers, almost all cellular structures (nerves, keratocyte nuclei) are comparable, but the image quality decreases with shorter wavelengths due to scattering (not shown here). interestingly, although the endothelium is the deepest layer, the cellular structure could only be resolved by the blue or green light and not by the near-infrared light. conclusions: most differences in image quality can be attributed to depthand wavelength-dependent scattering. however, there are also layers that vary in spectral reflectance of certain cellular structures (e. g. endothelium). this demonstrates the importance of choosing the right wavelength for certain target structures. in order to answer the question of the optimal wavelength, various aspects have to be considered: (1) corneal target structures (being a compromise between low scatting and high resolution), (2) light hazard and (3) glaring the patient. the optimal solution would be a multimodal instrument using different wavelengths. fallbericht: interstitielle keratitis im rahmen einer symptomtrias borgardts k. the filatov institute of eye diseases and tissue therapy nams of ukraine, odessa, ukraine; 2 lviv regional clinical hospital, lviv, ukraine the aim: was to determine the cytological features in conjunctival epithelium in patients with subclinical and manifest hypothyroidism and dry eye materials and methods: the impression cytology of 29 hypothyroid patients (18 with subclinical, and 11-with manifest) and dry eye was performed. all patients underwent ophthalmological investigation: biomicroscopy with fluorescein test, determination of the ocular surface disease index (osdi), tear film stability (test norn) and tear production (schirmer test i), the lipcof test. cytological material was taken using standart method of impression cytology by contact compression of the bulbar conjunctiva with millipore filter (millicell cm). the material was fixed in a mixture of alcohol and ether 1:1 with followed staining with hematotoxillin and eosin. were analyzed the following signs: state of a layer of epithelial cells (ec), metaplasia, degree of keratinization, type of ec change nuclei, goblet cells density, presence of the inflammatory cells. in the subclinical hypothyroidism a layer of ec was detected without significant structural changes. a distribution of ec was evenly. the ec were almost the same size without disturbances in intracellular contacts. the cells nuclei were round or oval. the ratio of nucleus to the cytoplasm was 1:2. changes in ec nucleus due to slight karyopicnosis were rarely observed. single cells were seen in a state of keratinization. no evidence of epithelial cells metaplasia and inflammatory cells was observed. the goblet cells density was slightly reduced. cytological features of the manifest hypothyroidism are more pronounced structural changes in the epithelial layer. in all cases, it was not possible to obtain a single layer of epithelial cells. in the epithelial layer focal epithelial hyperplasia, keratinization of ec, disturbance of intracellular contacts were observed. the nuclei of ec had various shapes, from round to rod-shaped. karyopicnosis and snow-like dissociation of ec nuclei were observed frequently. the goblet cells were in a state of degeneration, their density was significantly reduced. conclusion: сytological examination of the bulbar konjunctiva of patients with hypothyroidism and dry eye revealed degenerative changes in epithelial and goblet cells of various degrees, which depended on the stage of hypothyroidism. the severity of degenerative changes were more pronounced in patients with manifest hypothyroidism. el halabi m. 1* , seitz b. 1 , quintin a. 1 , suffo s. 1 , flockerzi f. 2 . further studies showed that stimulation of its activity is involved in this process through promoting both keratocyte transdifferentiation into myofibroblasts and extracellular matrix remodeling. based on these findings, the purpose of this study was to investigate whether there is an interplay between trpv1 and another trp subtype channel such as trp ankyrin 1 (trpa1) cold receptor, which is closely related to trpv1. methods: an established hck cell line was used as a cell model for corneal keratocytes. single cell fluorescence calcium imaging was used to measure the intracellular calcium concentration ([ca 2+ ] i ). specifically, the fluorescent dye fura-2/am was used to measure a fluorescence ratio (f340/f380), which is proportional to [ca 2+ ] i . results: the trpa1 agonist icilin (10 µm) increased the fluorescence ratio f340/f380 from 0.1010 ± 0.0004 to 0.2129 ± 0.0130 (n = 13; p < 0.0001). this increase could be suppressed by the trpa1 antagonist hc-030031 (10 µm) to 0.1051 ± 0.0008 (n = 51; p < 0.0001). interestingly, icilin was also able to suppress the cap-induced increase of f340/f380 from 0.1279 ± 0.0028 (n = 10) to 0.0838 ± 0.0118 (n = 30; p < 0.0398) (both 10 µm). conclusion: for the first time, our studies show a functional expression of the cold receptor trpa1 and its interplay with trpv1 in hck. consequently, the use of cold receptor agonists may be an interesting option to modulate stromal scarring through suppressing injury induced intrinsic trpv1 activity. abstracts bilität der befunde über den beobachtungszeitraum von derzeit 6 monaten. die beschriebene kohorte wird längsschnittlich weiterverfolgt. schayan, f. 1* , flockerzi e. 1 , hamon l. 1 , langenbucher a. 2 (b) , dünnste pachymetrie (c) und die bestkorrigierte sehschärfe (d) jeweils in den ausprägungsstadien 0 bis 4. alle werte wurden prä-und 6 monate postoperativ verglichen. ergebnisse: sechs monate postoperativ zeigte sich eine verbesserung sowohl des mittleren sc-visus (logmar) von 0,8 ± 0,3 auf 0,5 ± 0,3 (p < 0,001) als auch des mittleren cc-visus (logmar) von 0,4 ± 0,2 auf 0,3 ± 0,2 (p = 0,09). hinsichtlich der refraktion zeigte sich eine reduktion der sphäre von -3,0 ± 4,3 dioptrien (d) auf -0,7 ± 3,4 d (p < 0,001) und des zylinders von -4,7 ± 2,0 d auf -2,1 ± 3,3 d (p = 0,002 gibt es einen zusammenhang zwischen geburtsgewicht und hornhautaberrationen im erwachsenenalter? ergebnisse der bevölkerungsbasierten gutenberg-gesundheitsstudie (ghs) fieß a. 1* , urschitz m. 2 , nagler m. 3 , nickels s. 1 , münzel t. 4 , wild p. 3 , beutel m. 5 , lackner k. j. 6 , pfeiffer n. 1 astigmatismus (z(2,-2); z(2,2)), koma (z(3,-1); z(3,1)), trifoil (z(3,-3); z(3,3)), sphärische aberration (z(4,0)) und aberrationen höherer ordnung (hoa) und niederer ordnung (loa). ergebnisse: insgesamt wurden 5628 teilnehmer in die vorliegende analyse einbezogen (3004 frauen; alter: 56,0 +/-10,3 jahren). in der multivariablen analyse war ein niedriges gg mit höherer sphärischen aberration (b = -0,006 µm/500 g, 95 %-ki: [-0,008; -0,003]; p < 0,001) und höheren hoa (b = -0,007 [-0,010; -0,003] µm/500 g; p < 0,001) assoziiert. horizontales koma (z(3,1)) zeigte einen möglicherweise schwachen zusammenhang (b = 0,002 [-0,000; 0,003] µm/500 g; p = 0,051) wohingegen die anderen aberrationen keinen statistischen zusammenhang aufwiesen (astigmatismus (z(2,-2) p = 0,24; z(2,2) p = 0,80), koma (z(3,-1) p = 0,29), trifoil (z(3,-3) p = 0,06; z(3,3) p = 0,63). schlussfolgerung: unsere ergebnisse zeigen einen zusammenhang zwischen geburtsgewicht und sphärischer aberration bei erwachsenen im alter von 40 bis 80 jahren. dies deutet darauf hin, dass ein niedriges geburtsgewicht möglicherweise einen einfluss auf eine veränderte entwicklung der hornhautform haben könnte, was auf die optische bildqualität auswirkung haben könnte. duces involuntary eye movements and avoids compression artifacts, have proven to increase the image quality and 2d/3d reconstruction possibilities in all planes. in this study, we examine the clinical usability of the new system and present findings in patients with specific corneal alterations. methods: five patients with brittle corneal syndrome (bcs), mucopolysaccharidosis, small moldering myeloma (smm), schnyder corneal dystrophy and keratitis of unknown origin were examined with the hrtii-rcm 2.0 modality. subsequent, dedicated image alignment procedures and stack quality evaluation were conducted. these results were compared with conventional digital slit lamp examinations as well as anterior segment oct. results: the overall investigation time was less than 10 min for each patient. the recorded data was adequate for further analysis in all cases, almost independent of patient compliance. particular findings worth mentioning were observed in all patients. for example in the patient with bcs, an increased level of stromalhaze, alterations such as the elongation of keratocyte nuclei and clustering of cells at the anterior stroma, and dark bands in the posterior stroma were observed. in the patient with smm, confocal microscopy visualized diffuse localized deposits throughout the epithelium and stroma. the highly reflective deposits were intracellular as well as extracellular and were present as deep as anterior to descemet's membrane. this was in line with the slit lamp examination, which showed white dot-like opacities throughout the entire depth of the cornea. the rcm 2.0 corneal imaging concept enables reliable recordings of expanded in vivo human corneal image stacks, even in patients with limited compliance. this allows optimal imaging plane selection according to individual clinical questions and offers new insights into cellular processes in vivo. the rcm 2.0 enhances the slit lamp imaging concept to a cellular level. the influence of tonebp on lymphatic and electrolytic homeostasis of the cornea hadrian k. 1*, 2 , bock f. 1, 2 , cursiefen c. 1, 2 , eming s. a. 2, 3 , hos d. 1 objectives: lymphangiogenesis plays an important role in the regulation of various inflammatory processes in the cornea. furthermore, a positive function of lymphatic vessels in the regulation of corneal edema has recently been demonstrated. in addition to their function as mediators of (lymph)angiogenesis, macrophages have been shown to play a crucial role in the regulation of the salt/water balance in the skin. the mechanism underlying these processes involves the activation of tonicity-responsive enhancer binding protein (tonebp). tonebp is an osmosensitive transcription factor and can be induced in macrophages by proinflammatory stimuli. thus, aim of this work was to characterize the role of macrophagederived tonebp in the regulation of corneal inflammation and edema. methods: the role of tonebp in lymphatic and electrolyte homeostasis was investigated in two different corneal injury models in mice. for this purpose, six to eight weeks old wildtype c57bl/6n mice and mice with a tamoxifen-inducible tonebp knockout (ubcc-cre tonebp fl/fl) underwent intrastromal suture placement or incision of the central cornea to induce corneal inflammation and/or edema. tonebp expression was evaluated using immunofluorescence as well as qrt-pcr. furthermore, the co-expression pattern of tonebp/f4/80 (macrophages) and tonebp/ cd45 (leucocytes) in both models was evaluated. in addition, expression of vegf-c, a main regulator of lymphangiogenesis, was evaluated using qrt-pcr. results: tonebp is expressed in both corneal epithelium and stroma. the expression pattern of tonebp differs in the naïve cornea compared to injured corneas: whereas the majority of tonebp positive cells in the naïve corneal stroma are fibroblasts, the majority of tonebp cells in injured corneas are f4/80 as well as cd45 positive. although the overall expression schulz a. 2 , nickels s. 1 , wild p. 2 , schmidtmann i. 3 , münzel t. 4 , beutel m. 5 , lackner k. j. 6 , pfeiffer n cursiefen c. 3 , pfeiffer n. 4 , , viestenz a. 6 , geerling g. 7 the treatment of acute hydrops in keratoconus: first results from our centre and a preview of a germany-wide registry study händel a. 1* , siebelmann s. 1 , hos d. 1 , matthaei m. 1 , cursiefen c. 1 , bachmann b. 1 1 zentrum für augenheilkunde -universitätsklinikum köln, köln, germany purpose: the treatment of acute keratoconus varies considerably from conservative to surgical approaches. thus, aim of this study was to 1) obtain national data on treatment modalities of other centres and 2) compare two surgical treatment options for acute corneal hydrops in keratoconus (mini-descemet membrane endothelial keratoplasty (mini-dmek) or predescemetal sutures). methods: a germany-wide registry study on acute keratoconus was initiated. in addition, a retrospective analysis of thirteen patients who presented in our clinic with a corneal hydrops treated by mini-dmek (n = 5, group 1) or predescemetal sutures (n = 7, group 2) was conducted. results: ten centres agreed to participate in the registry study. in our retrospective analysis, both groups showed reductions in corneal thickness and increased visual acuity after surgical treatment of acute hydrops. in group 1 (age 32 years (± 7 years)) best corrected visual acuity increased from logmar 1.88 (± 0.5) before mini-dmek to 1.02 (± 0.6) 30 days after mini-dmek (p = 0.037). corneal thickness significantly decreased from 1230 µm (± 322 µm) preoperatively to 500 µm (± 50 µm) 30 days after mini-dmek (p = 0.011). group 2 (age 37 years (± 10 years)) showed a preoperative corneal thickness of 1420 µm (± 297 µm) and a visual acuity of logmar 1.67 (± 0.9). after surgical treatment using predescemetal suturing, there was a reduction in corneal thickness to 524 µm (± 186 µm) (p = 0.011) and visual acuity increased to logmar 0.96 (± 0.5) (p = 0.102) respectively after 30 days. there was no relapse of the hydrops and the corneas remained dehydrated to physiological levels. conclusions: both techniques, mini-dmek and predescemetal sutures, are effective treatments for acute hydrops in keratoconus. mini-dmek appears to be beneficial in large dm tears. as the treatment of acute keratoconus is different in many centres in germany, the initiated germanywide registry study on acute keratoconus will combine the results of many centres and lead to larger case numbers. clinical сase early therapeutic deep anterior lamellar keratoplasty following of pseudomonas corneal ulcers with descemetocele associated with contact lenses wearing. in the future we need to perform further antibody validation for the remaining markers to confirm these findings. purpose: the purpose of the study was to analyze mistakes made during surgical treatment for pterygium based on medical records of patients treated at the corneal pathology department of the filatov institute. materials and methods: one hundred and three patients (105 eyes) who had been followed up after surgery for ptergygium stage 3 or 4 during the recent two years were involved in this study. postoperative complications were observed in 27 eyes (25.7 %) of patients after primary pterygium excision. results: some patients seeking care at our institute had undergone 4-5 previous surgeries for pterygium in one eye. of the surgical complications, there were frequent recurrences with multiple early repeat surgeries (4 to 5 surgeries within a year) with the development of extensive vascular fibrous tissue and decreased vision (10 eyes), corneal ulceration and perforation in bilateral pterygium surgery in patients with concomitant autoimmune disorders (4 cases), and corneal ulceration and perforation due to deep pterygium excision (5 cases). conclusion: first, pterygium should be excised with minimal trauma to the limbus and cornea. special attention should be given to autoimmune disorders, in which a corneal trauma may result in keratomalacia. second, current medicamentous and surgical techniques should be used to prevent pterygium recurrence. third, one should not perform bilateral pterygium surgery or repeat pterygium excision early (≤ 6 months) after primary pterygium surgery. finally, any patient after pterygium excision should be followed by the ophthalmologist to promptly prevent or arrest the recurrence of pterygium. in situ donor keratometry in deceased patients as a new screening technique for eye banking? quintin a. objective: to evaluate the dynamics of biomechanical properties (corneal hysteresis (ch) and corneal resistance factor (crf)), topographical corneal thickness changes and corneal densitometry (corneal optical density (cod) in first-time soft contact lenses (scl) users (3 months) and in longterm scl wearers (over 3 years). methods: 20 patients (39 eyes) with low and moderate myopia took part in our trial. mean age of the participants was 31 ± 5 years. all patients were divided in 2 groups: i group-first-time scl users (20 eyes), ii grouplong-term scl users (19 eyes). keratoconic patients were excluded from the study. the participants of both groups underwent corneal pachymetry mapping, corneal densitometry (pentacam, oculus) and corneal biomechanical properties evaluation-ch and crf (ora, reichert technologies). in i group patients were examined before the first time and after 3 months of scl wear. in the group ii the same examinations were held during long-term (over 3 years) scl wear and after ≥1 week of scl removal. the i group showed reduction in central cornea thickness (by 4 mkm) and insignificant decrease of peripheral corneal pachymetry (by 4,35 mkm, p > 0,05) during 3 months of scl wear. cod reduced by 4 %. ch and crf were significantly increased compared to baseline by 0,1 and 0,6 mmhg respectively (p < 0,05). the ii group showed reduction in central cornea thickness (by 19,5 mkm) and in peripheral corneal thickness (by 22,5 mkm) during scl wear. cod increased by 14 %. ch and crf were significantly decreased by 0,5 and 0,35 mmhg respectively compared to the results after ≥1 week of scl removal (p < 0,05). conclusions: long-term scl wear can show morphological corneal changes. we observed the same dynamics of corneal pachymetry in both first-time and long-term groups. increase of ch and crf as well as cod reduction in the i group can show clinically insignificant water content changes in different corneal layers during first months of scl wear. significant ch and crf decrease with cod increase in long-term scl users can point at structural and dystrophic corneal changes. femtosekundenlaser assistierte transplantation der bowman-lamelle: laser assistierte donorpräparation und empfängerkonditionierung parlak m. abstracts the cornea (-1.1 d, -1.0 d, -0.9 d; and -0.1 d, -0.5 d, -0.5 d respectively; p > 0.05) did differ between groups. conclusions: after penetrating keratoplasty (pkp) with both sutures in place, a smaller graft diameter seems to result in a flatter curvature at the anterior surface of the cornea, but does not affect the astigmatism. this information may be indicative for iol power calculation in relation to graft diameter in a triple pkp procedure, depending of the individual size of the cornea. combined "muraine sutures" and intracameral air tamponade for the management of corneal hydrops in acute keratoconus razafimino s. 1* , daas l. 1 , quintin a. 1 , seitz b. 1 1 universitätsklinikum des saarlandes, homburg/saar, germany purpose: typically corneal transplantation in case of acute keratoconus with corneal hydrops has to be postponed for 3 to 6 months until the edema has resolved and a stable scar has formed. the purpose of this study was to analyze the short-term clinical outcomes in patients undergoing combined multiple predescemetal interrupted corneal sutures (so-called "muraine sutures") with intracameral air tamponade in acute corneal hydrops. methods: this retrospective uncontrolled case series enrolled 8 patients with keratoconus who presented with a corneal hydrops of recent onset. all patients underwent combined intracameral air tamponade and 3 to 6 predescemetal 10-0-nylon sutures placed orthogonally to the axis of the rupture of descemet's membrane. a prophylactic 6 o' clock iridotomy was preoperatively performed to avoid acute air-bloc intraocular pressure (iop) elevation. main outcome measures included pre-and postoperative median pachymetry in the center of the hydrops measured by anterior segment optical coherence tomography casia 2 (tomey corp., nagoya, japan) and best-corrected visual acuity (bcva in logmar). the median corneal pachymetry decreased significantly (p = 0.02) in all our patients. the median preoperative corneal pachymetry was 1605 µm (range 805-2423 µm), which significantly reduced to 1039 µm (range 573-1395 µm) at day 1 and to 672 µm (range 398-871 µm) 6 weeks after surgery. bcva improved from 1.5 preoperative to 0.95 log-mar (p = 0.46) 6 weeks after surgery. bcva on day 1 was finger counting (1.9 logmar) due to air fill of the anterior chamber. no acute elevation of the iop occurred. conclusions: our results show a rapid reduction of the corneal thickness and non-significant improvement of bcva after combined intracameral air injection and predescemetal compression sutures. "muraine sutures" seem to be an effective and safe procedure to reduce corneal swelling in acute corneal hydrops, thus allowing quicker readaptation of contact lenses and earlier corneal transplantation if necessary. die kontaktlinsenanpassung beim keratokonus -retrospektive untersuchung des verlaufes der linsenanpassung bei 200 patienten richter k. wir dokumentierten un-measured in situ 5 times repeatedly < 24 h after death using the portable retinomax k-plus 3 (bon, tokyo, japan). the means of the obtained keratometric readings were compared to the routinely measured 581 donor corneas using a mann-whitney u test. the respective values of the whole globes, from which sclerocorneal discs were removed for organ culture in the eye bank, were also compared to those obtained after measuring the same sclerocorneal discs in medium i after 6 ± 4 days using a wilcoxon signed-rank test. the mean standard deviation of the five in situ measurements was 1.7 d and 1.0 d for the dioptric power (p) at the steep and flat meridian of the cornea, and 1.0 d for keratometric astigmatism. p at the steep meridian of in situ corneas (44.7 d) remained unchanged after preserving sclerocorneal discs in medium i (43.8 d; p = 0.11). however, p at the flat meridian of in situ measured corneas (41.1 d) increased (p < 0.01) to 42.6 d, whereas keratometric astigmatism (3.6 d) decreased (p < 0.01) to 1.2 d after preservation in medium i. the comparison of the in situ values with the 581 routinely measured different donor corneas in medium ii showed no difference in p at the steep meridian (44.6 d; p = 0.34), but again a greater p at the flat meridian (43.1 d; p < 0.01) and a smaller keratometric astigmatism (1.5 d; p < 0.01). measuring deceased patients' eyes in situ with the portable retinomax k-plus 3 could be, in the absence of an anterior segment optical coherence tomograph (as-oct), an alternative and moderately reproducible screening technique in the eye bank. in comparison to the as-oct, the portable retinomax k-plus 3 estimates a similar power at the steep meridian of the cornea but seems to underestimate the power at the flat meridian and to overestimate the keratometric astigmatism. impact of graft diameter on the relation between sterile donor tomography in the eye bank and graft tomography after penetrating keratoplasty quintin a. background and purpose: sterile donor tomography in the eye bank can be used to avoid refractive surprises after corneal transplantation. the purpose of this study was to assess the impact of graft diameter on the relation between preoperative donor tomography and postoperative graft tomography after penetrating keratoplasty (pkp). patients and methods: this retrospective study enrolled 117 eye bank corneal tissues that underwent elective pkp with application of a double-running suture. donor and recipient trephination were performed using the 193-nm excimer laser (schwind amaris 1050rs). diameters were 7.5 mm (13 %), 8.0 mm (73 %) and 8.5 mm (14 %), with a graft oversize of 0.1 mm. preoperative measurements, taken through the cell culture flask using the anterior segment optical coherence tomograph casia 2 (tomey corp., nagoya, japan), were repeated postoperatively after 5 ± 4 months with all sutures in place. differences between post-and preoperative values (∆) were compared in function of the graft diameter using a mann-whitney u test. results: the ∆ keratometric power (p) at the steep meridian of the anterior surface of the cornea in the 7.5 mm grafts (-2.9 d) was significantly smaller than that in the 8.0 mm grafts (+0.6 d; p < 0.01) and than that in the 8.5 mm grafts (+1.4 d; p < 0.01). no statistically significant differences occurred for this parameter between the 8.0 mm and the 8.5 mm grafts (p = 0.23). at the flat meridian of the anterior surface of the cornea, ∆ p in the 8.5 mm grafts (-1.0 d) was significantly greater than that in the 8.0 mm grafts (-3.1 d; p = 0.03) and than that in the 7.5 mm grafts (-6.7 d; p < 0.01). no statistically significant differences occurred for this parameter between the 7.5 mm and the 8.0 mm grafts (p = 0.05). neither ∆ astigmatism (+3. purpose: to evaluate ocular symptoms in european non-hospitalized patients with severe acute respiratory syndrome-related coronavirus 2 (sars-cov-2) and to investigate associations with the demographic data as well as general physical symptoms. methods: in this prospective, observational study, 108 non-hospitalized patients with polymerase chain reaction (pcr) confirmed sars-cov-2 infection not requiring intensive care were asked using a standardized questionnaire regarding disease-associated ocular symptoms, demographic data, and general physical and nasal symptoms. total ocular symptom score (toss) was evaluated during and retrospectively prior to coronavirus disease 19 infection. associations between toss and demographic data as well as general and nasal symptoms were evaluated. results: 75 out of 108 non-hospitalized patients with covid-19 infection (69 %) suffered from ocular symptoms mostly including burning sensations, epiphora, and redness, compatible with conjunctivitis. these symptoms occurred in most cases in the first three days of covid-19 but were rather mild. toss was significantly higher during than prior to the covid-19 infection (p < 0.001). there were no significant associations between toss and demographics, general physical and nasal symptoms. conclusions: ocular involvement in european non-hospitalized patients with covid-19 seems to be highly underestimated and needs further attention. overall, these ocular symptoms including burning sensations, epiphora and redness seem to be mild. abstracts schlussfolgerung: die bildqualität der µoct scheint ausreichend, um durch quantitative auswertungen diagnostisch relevante aussagen zur sbp treffen zu können. aufgrund der variierenden nervendichte innerhalb der hornhaut ist die aufnahmefläche des µoct von derzeit 1 × 1 mm vorteilhaft, sodass sich zusammen mit der kontaktlosen anwendung des µoct ein klinisch erfolgversprechendes anwendungsfeld ergibt. epithelimplantationszyste der kornea schölles k. the "knights of the blind"-eye banking and lions, a successful cooperation since 1952 scholtz s. 1*, 2 , auffarth g. 3 , hellwinkel o. 4 , kampik d. 5 , maier p. 6 , seitz b. 7 , wegner t. 8 , krogmann f. 9 , rosenbaum k. 10 der prä-zu postoperative ecl in gruppe 1 betrug 36 % (n = 12) nach 6 monaten und 37 % nach 12 monaten (n = 5). in gruppe 2 lag der ecl bei 44 % (n = 11) nach 6 monaten, 36 % (n = 9) nach 12 monaten, 33 % (n = 9) nach 2 jahren und 27 % (n = 3) nach 3 jahren. die postoperative zentrale hornhautdicke nach 1, 2 und 3 jahren war in gruppe 1 im mittel (± sd) 751 ± 507 µm, 590 ± 174 µm und 552 ± 73 µm beziehungsweise in gruppe 2621 ± 227 µm, 642 ± 231 µm und 509 ± 43 µm (p = 0923, p = 0309 beziehungsweise p = 0,221). schlussfolgerung: unsere daten legen nahe, dass die dmek die sehschärfe bei patienten mit endotheldekompensation nach den hier untersuchten glaukomoperationen, gdd-implantation und te, deutlich verbessert. diese augen zeigen jedoch ein erhöhtes risiko zum transplantatversagen mit notwendigkeit zur re-dmek. allerdings besteht hierbei kein signifikanter unterschied zwischen den beiden untersuchten glaukomoperationen. sinicin e. 1* , bartram m. 1 purpose: to compare the efficacy of epi-off customized corneal crosslinking (cxl) with and without supplementary oxygen. methods: in this prospective study, 40 eyes of 40 patients with documented progressive primary keratoconus were treated either with epi-off customized cxl under a supplementary oxygen environment (o2 > 90 %, n = 20) or with customized cxl under atmospheric oxygen conditions (o2 = 21 %, n = 20) and followed for 1 year. customized irradiation patterns had an irradiance of 15 mw/cm 2 and radiant exposure levels ranging from 5.4j/cm 2 up to 10j/cm 2 centered on the maximal posterior elevation. analyzed parameters were placido topography, scheimpflug tomography, endothelial cell count, bscva and anterior segment oct. results: kmax showed significant changes after 1 year for both groups with a significantly higher regression in the supplementary oxygen subgroup (-3.5 ± 1.9d vs. -1.3 ± 1.3d). six out of 19 eyes (32 %) in the supplementary oxygen group showed a flattering of >5d, whereas only 2 out of 19 (11 %) did in the atmospheric oxygen group (p < 0.05). improvement in bscva and reduction in corneal coma were also significantly higher in the supplementary oxygen group, however, sterile infiltrates occurred more frequently, and corneal haze appeared denser. the regularization index (ri) was significantly better in the customized cxl group with supplementary oxygen. endothelial cell count maintained stable in both groups. conclusions: supplementary oxygen enhances the efficacy of epi-off customized cxl with stronger qualitative and quantitative flattening in kmax and corneal regularization. substantial flattening rates of more than 5d can be achieved in about 30 % of the treated cases. inselspital bern, bern, switzerland; 2 iroc, zürich, switzerland purpose: to measure the oxygen consumption and concentration during corneal crosslinking (cxl) in different depths and compare different protocols with and without supplementary oxygen. methods: in de-epithelialized porcine eyes, a femtosecond-laser generated channel was used to place a fiber-probe in corneal depths of 100, 200 and 300 microns to measure the local oxygen concentration. after a 10 min imbibition of 0.1 % riboflavin the corneas were irradiated at 3, 9, 18 and 30 mw/cm2 while the oxygen concentration was continuously measured. to assess the benefit of supplementary oxygen, all experiments were performed under atmospheric (21 % o2) as well as under hyperoxygenic (>95 % o2) conditions. results: the equilibrium oxygen concentration under atmospheric oxygen conditions at 3 mw/cm2 was 5 % in 100 microns decreasing to 3 % in 200 microns and 0 % at 300 microns. with 9, 18 and 30 mw/cm2 no oxygen was available in 100 microns or deeper. using a hyperoxygenic environment the oxygen concentration was 44 % using 3 mw/cm2 in 100 microns decreasing to 39 % in 200 microns and 33 % in 300 microns. at 9 mw/cm2 the concentrations were 5 %, 3 % and 1 % in 100, 200 and 300 microns, respectively. using 18 and 30 mw/cm2 all oxygen was depleted during cxl and no equilibrium was established, however, the time until all oxygen was consumed was longer in the 18 mw/cm2 than in the 30 mw/cm2. conclusion: supplementary environmental oxygen increases the stromal oxygen-availability during cxl. in particular at higher irradiances with increased oxygen consumption, supplementary oxygen is beneficial and eliminates the bottleneck of oxygen and, therefore, enhances more crosslinks. steindor f. a. 1* , borgardts k. 1 the benefits of using the amniotic dry membrane in corneal ulcers stanila d. m. 1*, 2, 3 , 2, 3 , stanila a. epithelial defects of the cornea heal quickly and without incidents. when these defects are not healed in time, defined in the literature in two weeks, they become known as persistent epithelial defects (ped) and sometimes become corneal persistent ulcers. the purpose of this paper is to present using omnigen refrigerated dry amniotic membrane and omnilens in corneal ulcers. we applied omnigen amniotic membrane and omnilens contact lens in 15 eyes with corneal ulcers, 8 eyes with neurotrophic keratopathy and 2 were perforated, 2 eyes with steven-johnson syndrome, 3 eyes with mooren ulcer and 2 were perforated, 1 eye with corneal lattice dystrophy and perforation, 1 eye with corneal transplant. we applied the amniotic membrane after rehydration in 10 cases without suture and 5 cases with suture. at the same time we applied the omnilenz contact lens on ocular surface until membrane was absorbed. omnigen is a transportable biological matrix that can be used as a regenerative therapy for corneal ulcers or other diseases of the ocular surface. the application of the membrane has been made according to the existing lesion. the use of the membrane was like graft with epithelial position up in 3 cases and patch with epithelial position down in 12 cases. the results were good, obtaining the anatomical reconstruction of the ocular surface. in 3 cases we repeated the membrane application. we must speak about unmet needs in our country regarding the treatment of corneal ulcers with or without perforation. because we have not a tissue bank we cannot perform corneal transplant and we resort in urgency to amniotransplant for the tectonic purpose. conclusions: omnigen's amniotic membrane together with the omnilenz therapeutic contact lens can be a solution for the treatment of corneal ulcers with perforated or no perforated cornea. korneale aberrationen höherer ordnung nach superfizieller keratektomie bei phscb walckling m. purpose: implementation of a systematic analytical approach for detection of antigen-presenting cells (apc) in human corneal tissue using facs. methods: to implement facs analysis of apc in the human cornea, we set up a novel protocol for cell harvesting from human corneal tissue. the corneas used were stored in organ culture, they were unsuitable for transplantation and consent for scientific use was available. the harvested cells were analysed by facs; special attention was paid to the detection of inflammatory and regulatory macrophage populations. among the live cells, cd14+/cd11b+/cd68+ cells were defined as macrophages. m1 macrophages were defined as cells expressing cd282+/cd86+/ hla-dr+/cd284+ in addition to the markers mentioned above; while m2 macrophages were defined by their additional expression of cd206+/ cd163+. subsequently, cell populations were assessed with respect to their response to different stimuli (lps/il-10/il-4 with il-13) as well as dynamics of apc in organ culture. results: facs allowed the identification as well as quantification of innate immune cells in processed human corneal samples. the use of lps as an innate stimulus led to a down-regulation of cd206 (p > 0.05), a regulatory maker in apc. co-expression of cd282+ and cd284+ is down-regulated by regulatory interleukins like il-10 (p < 0.001) and il4+il-13 (p < 0.05). in addition, hla-dr, as an inflammatory maker, was also down regulated by il-10 (p < 0.001). a cell loss over time during organ culture of the corneas was observed in both m1 and m2 macrophages (n = 15, p = 0.04). in the present study we report on the implementation of a facs protocol for processing and phenotyping human corneal cells. we demonstrate that inflammatory and regulatory macrophage subtypes are present in the cornea and that these cells show a functional response to different interleukins. we also demonstrate for the first time the dynamics of apc cell loss in organ culture. zwingelberg s. questionnaire. moreover, diabetes duration, blood sugar levels, and type of therapy for diabetes were assessed. results: palpebral demodicosis was twice as common in patients with t2 dm duration more than 10 years as in those with t2 dm duration less than 10 years (p = 0.002). our meibography study found changes in meibomian glands in 90 % of diabetics with palpebral demodicosis, with the mean meibograde score of 5.0 ± 0.9 points, which indicated a predominance of moderate mgd. abstracts skop. dabei wurde das 3d-system auf das vorhandene mikroskop angepasst, so dass die mikroskop-optik unverändert blieb. in beiden gruppen wurde entweder eine femto-sekunden-laser-assistierte kataraktoperation (flacs) oder eine traditionelle phakoemulsifikation durchgeführt. es wurden retrospektiv komplikationen erfasst und analysiert. ergebnisse: der anteil von flacs betrug in der 3d gruppe 17,1 % und in der 2d gruppe 15,1 %. eine kapselruptur trat bei 10 augen auf (3d: n = 4 (0,4 %), vordere vitrektomie: n = 3; 2d: n = 6 fällen (0,6 %), vordere vitrektomie: n = 5). ein kurzfristiger irisprolaps trat bei 3 augen auf (3d: n = 2, 2d: n = 1). in zwei augen kam es zu einer zonulolyse (3d: n = 1, 2d: n = 1). insgesamt zeigte sich kein statistisch signifikanter unterschied zwischen den beiden gruppen (p > 0,5). schlussfolgerung: in einer großen serie von 2000 augen zeigte sich hinsichtlich des sicherheitsprofils bei katarakt-operationen kein signifikanter unterschied zwischen der 3d und der 2d chirurgie. die 3d-chirurgie ist daher für die katarakt-op ohne zusätzliches risiko einsetzbar. protection of the cornea during cataract phaco (group 1) and 40 patients with pes who operated without protection (group 2). the average age is 71.8 ± 2.6 years. visual acuity before surgery 0.02 ± 0.15. in group 1, after capsulorexis was performed using micro-forceps,a soft contact lens (scl) was inserted into the anterior chamber (ac), and then a viscoprotector was introduced into the ac. the viscoprotector was located between the cornea and the lens and in the ac behind the lens. scl was removed after surgery at the iol implantation stage. results and conclusions: on the 1st day after surgery in 12 (30 %) patients of the 1st group and in 24 patients (60 %) of the 2nd group, diffuse edema of the entire area of the cornea with involvement of the stroma and epithelium was determined, edema of the upper corneal sector with folds of the descemet membrane in 8 patients (20 %) of the 1st group and 15 patients (37.5 %) of the second group. visual acuity in group 1 on the 1st day after phaco was also statistically significantly higher than before surgery and in group 2, averaging 0.52 ± 0.02. on the 7th day-0.67 ± 0.05, on the 30th day 0.71 ± 0.04, and this difference was significantly higher than in group 2 (p < 0,05). visual acuity in the 2nd group of patients on the 1st day after phaco was 0.34 ± 0.06, on the 7th day-0.46 ± 0.02 and on the 30th day-0.62 ± 0.03. our results indicate that the use of scl during phaco helps to improve the postoperative state of the cornea (to reduce the presence of stromal edema), which reduces the risk of intraoperative damage to the corneal endothelium, accelerates the restoration of corneal transparency, and thereby ensures higher indicators of visual acuity after surgery. ergebnis: die vorläufige auswertung in dieser laufenden studie ergab eine wunddehnung von 0,18 mm ± 0,1 mm (von 2,20 ± 0,03 mm vor implantation auf 2,38 ± 0,09 mm nach der implantation) in den mit dem autono-me-injektor behandelten augen und eine wunddehnung von 0,24 mm ± 0,07 mm (von 2,08 ± 0,08 mm vor implantation auf 2,31 ± 0,05 mm nach der implantation) in den mit dem isert-injektor behandelten augen. die licht-und rasterelektronenmikroskopie der injektorspitzen zeigte folgen learning yamane-10 tips for a better start -videobeitrag shajari m. comparing the effective lens position and refractive outcome of a novel rhexis-fixated lens to established lens designs shajari m. benefits and new features of modern international internet database "iolcon" for updated and optimized iol constants scholtz s. pre-and three months postoperatively. lens constant optimization was performed. results: seventy eyes of 56 subjects were included. elp for rhexis fixated iol was shortest (4.29 ± 0.24 mm), followed by c-loop haptic (4.41 ± 0.42 mm) and plate-haptic (4.51 ± 0.26 mm) iol. difference in elp was significant between rhexis fixated iol and both plate-haptic (p = 0.001) and c-loop haptic iol (p = 0.000). acd adjustment based on lens design showed a significant effect on refraction and iol power predictions for all formulas and lenses (p < 0.05). for the rhexis fixated iol the differences in refraction ranged from +0.04d for the hill-rbf to +0.096d for haigis. the other two lenses showed mean differences in refraction between -0.046d for hill-rbf. the difference in iol fixation and its resulting position in the capsular bag have a significant effect on the effective lens position and consequently a significant effect on the prediction of postoperative refraction. usov v. y. 1* , kolomiychuk s. g. 1 , tarik a. t. 1 1 the problem: age-related cataracts, especially when combined with the development of inflammatory diseases of the cornea-important medical and social problem. imbalance in the prooxidant-antioxidant eye tissues system is the main reason of the progression of pathological changes in the lens and in the cornea during inflammatory and degenerative diseases of the eye. the aim-to study the relationship between the indicators of the prooxidant-antioxidant system and pathological changes in the lens during the inflammatory process in the cornea. methodology: chinchilla breed rabbits with and without modeling bacterial keratitis were modeling a light cataract. the severity of pathological changes in the lens was determined by ophthalmobiomicroscopy. the activity of glutathione peroxidase and catalase, the content of malondialdehyde (mda) and diene conjugates (dc) were measured in the lens, chamber moisture and tear fluid of rabbits. the total antioxidant activity, the content of mda and dc were measured in the tear fluid of patients with keratitis with and without age-related cataract. results: a negative correlation between the indices of lipid peroxidation (mda and dc) and the antioxidant state rabbit eye tissues with cataract with concomitant keratitis was found. the relationship between the lens state and biochemical parameters was evaluated, with the spearman correlation coefficient being: negative for glutathione peroxidase (r = -0.82,p < 0.01) and catalase (r = -0.69, p < 0.05), positive for mda (r = 0.76, p < 0.05) and dc (r = 0.58, p > 0.05). significant abnormalities in the prooxidant-antioxidant eye tissues system were found to be depended significantly on the severity of pathological changes in the lens of rabbits with light cataracts, especially during the inflammatory process in the cornea. maximal changes in the prooxidant-antioxidant system in the tear fluid were observed in patients with age-related immature cortical, nuclear and posterior capsular cataracts and visual acuity less than 0.3 with keratitis. particularly pronounced corneal disorders were observed in patients with cortical and nuclear mature cataracts with visual acuity less than 0.1. the presence of correlation between the indices of lipid peroxidation products and antioxidant activity indicates the important role of these metabolic disorders in the formation of structural and functional changes in the lens of experimental animals and patients during the inflammatory process in the cornea. freiburger ophthalmopathologie im wandel der zeit: 1945-1989 glegola m. purpose: to assess a potential relationship between the location of the maximal extent of parapapillary gamma zone and the location of the foveola. the population-based beijing eye study 2011 consisted of 3468 subjects (mean age: 64.6 ± 9.8 years) who underwent a detailed ophthalmological examination including biometry and fundus photography. using fundus photographs we measured the angle between the disc-fovea line and the horizontal, and the location and width of parapapillary gamma zone. the present study consisted of 315 individuals with a mean age of 60.0 ± 8.1. years (range: 50-83) and an axial length of 24.1 ± 1.5 mm (range: 21.48,28.68). the angle between the disc-fovea line and the horizontal increased significantly (i. e., the fovea was located more inferiorly) with increasing width of parapapillary gamma in the inferior sector, and vice versa (disc-fovea angle = 0.004 × maximal gamma zone width + 11.3; standardized regression coefficient beta: 0.22; p = 0.002). this relationship was independent of axial length (p = 0.24). conclusions: an inferior location of gamma zone is spatially correlated with a relatively inferior location of the foveola and vice versa. this spatial relationship between location of gamma zone and fovea location supports the notion of bruch's membrane pushing backward during axial elongation and of a bruch's membrane opening shift parallel to a change in the fovea location. excessive collagen formation) is considered to be crucial for the congenital glaucoma development as well as for the elaboration of pathogenetically based treatment of this disease. peripapillary border tissues of the optic nerve head: associations with gamma zone and delta zone and potential biomechanical importance universität zu köln, cologne, germany; 2 univ.-augenklinik, erlangen, germany purpose: to assess the relationship between the peripapillary border tissue (pbt) of the choroid (pbtc) (jacoby) and of the peripapillary scleral flange (pbts) (elschnig) with the presence and length of parapapillary gamma zone and delta zone. methods: the histomorphometric investigation included histologic sections of enucleated eyes of caucasian patients. using light microscopy, the pbt dimensions were measured. the study included 85 eyes (age: 62.0 ± 14.1 years) (range: 37-87 years). mean axial length was 26.7 ± 3.5 mm) (range: 21.0-37.0 mm). the length of pbtc was strongly and positively associated with the length parapapillary gamma zone (p < 0.001; standardized regression coefficient beta: 0.92) and delta zone (p < 0.001, beta: 0.86). in contrast, the length of the pbts was strongly and negatively associated with gamma zone length (p = 0.009; beta: -0.32) and delta zone length (p < 0.001; beta:-0.54). the length of the peripapillary border tissues are strongly associated with the width of the parapapillary zones gamma and delta. since the whole inner ocular shell (choroid, bruch's membrane, retina) are connected to the outer shell (i. e., sclera) only through the scleral spur anteriorly and through the pbtc posteriorly, the findings may have importance for the physiology and biomechanics of the eye. optic disc fovea distance and axial length: highly myopic eyes versus non-highly myopic eyes purpose: to assess a relationship between the optic disc center-fovea distance and axial length in eyes divided into highly myopic eyes (axial length ≥ 26.5 mm and non-highly myopic eyes. methods: the population-based beijing eye study 2011 consisted of 3468 subjects (mean age: 64.6 ± 9.8 years) who underwent a detailed ophthalmological examination including biometry and fundus photography. the optic disc center-fovea distance was measured on the fundus photographs. the present study included fundus images of 111 individuals with a mean refractive error of -9.3 ± 3.8 diopters (range:-20.8, +1.75) and an axial length of 26.8 ± 1.9 mm (range: 22.55, 30.88) results: the disc-fovea distance increased significantly with longer axial length, with a relatively flat slope in the non-highly myopic eyes (disc-fovea-distance = 24.3 × axial length (mm)+514) and a steeper slope in the highly myopic group (disc-fovea-distance = 58.7 × axial length (mm)-460). the increase in the disc-fovea distance was strongly correlated with an increase in the width of parapapillary gamma zone, while the distance between the peripheral gamma zone border and the fovea was not significantly associated with axial length. the disc-fovea distance increases slightly with longer axial length in non-highly myopic eyes, and increases more steeply in highly myopic eyes, with a cut-off value of 26.5 mm axial length for the definition of high myopia. the results confirm the notion of bruch's membrane opening (bmo) shifting to the temporal side in non-highly myopic eyes, leading to the development of parapapillary gamma zone and thus an inthe problem of high risk of blindness and disability in a given population by reason of glaucoma dictated the relevance of this research. infringement of eye hydrodynamics plays the main role in glaucoma pathogenesis. purpose of this work was to establish patterns of development and structure of the human eye drainage system in embryogenesis and in postnatal ontogenesis. methodology: the human iris-corneal angles were studied in 40 embryos from 10 to 70 mm of parietal-coccygeal length (pcl) and on the anatomical preparations of 46 heads. the obtained histological sections of iris-corneal angles were stained with hematoxylin and eosin. the iris-corneal angle in embryos 21 mm pcl was represented by the accumulation of mesenchymal cells. it began to be identified in embryos 32 mm pcl when the border between the cornea and sclera started to be determined. in embryos 41 mm pcl the iris and ciliary body were shifting posteriorly and the spaces of schlemm's canal (sc) appeared above the trabecula. on histological sections the transition zone cornea-sclera was located in front of sc. background: jones tube surgery-creating a bypass between the caruncular conjunctiva and the nasal cavity using a glass tube drawing tears-is the gold standard for managing epiphora secondary to upper lacrimal outflow obstructions. however, tube extrusion occurred in up to 50 % of the cases. recently, the stoploss jones tube (sljt) with an internal silicone flange was reported to reduce the risk for tube extrusion. besides, tube insertion requires a bony ostium, which might preexist due to previous failed dacryocystorhinostomy (dcr). however, in patients without previous dcr, an external transcutaneous approach is needed for correct tube placement. purpose: to describe a novel transcaruncular laser-assisted sljt procedure without any skin incision for the treatment of lacrimal canalicular obstructions. methods: under general anesthesia, sharp scissors were used for a 3-mm caruncular incision and gently advancing dissection in inferomedial direction towards the nasal bone. a laser fiber optic with 300 µm-diameter connected to an 810 µm-wavelength diode laser was inserted into the track and positioned under visual control using nasal endoscopy, so that the aiming beam appeared at the anterior margin of the middle turbinate. subsequently, laser energy created a bony ostium with a 3-mm diameter, and a specially designed dilator was applied to enlarge the track. after measuring the distance between the caruncle and the nasal mucosa using a sizer, a sljt of the required length was passed down the guidewire until the silicone flange opened within the nasal cavity. the guidewire was removed, and a suture was passed around the neck of the tube and secured to the caruncular conjunctiva. results: twelve consecutive patients (12 eyes) with absolute canalicular dacryostenosis were enrolled. eleven eyes were surgically successful. in one case the inserted sljt was too long and had to be replaced by a 2-mm shorter one. in 3 cases, conjunctival scarring, conjunctival granuloma and tube-associated irritation of the ocular surface required conjunctival revision. there were no cases of tube extrusion or sink-in. conclusions: this novel technique is a promising strategy for the treatment of lacrimal canalicular obstructions. advantages include the lack of skin incisions and visible scars, the less risk of bleeding due to the vaporization method, and the reduced risk of tube migration or extrusion due to the stoploss jones tube with endonasal silicone flange. purpose: currently therapeutic management of patients with graves' orbitopathy (go) relies on clinical assessments and mri scans. however, monitoring of inflammation remains difficult since external inflammatory signs like injection and chemosis do not necessarily represent the orbital disease activity.therefore, we aimed to evaluate the diagnostic value of fdg-pet/mri to assess the inflammation of go patients. methods: patients with new onset of go who were examined in our eu-gogo tertiary referral center were enrolled in this trial. all patients underwent ophthalmological and orthoptic examinations to evaluate the activity and severity of go, as well as an 18f-fdg-pet/mri (siemens biograph mmrt). a subset of pet parameters including maximum standardized uptake value (suvmax), metabolic target volume (mtv), and total lesion glycolysis (tlg) were obtained separately per-eye and per-extraocular eye muscle (eom). eom thickness was measured on the coregistered mri. subsequently, differences in pet parameters among nospecs classes were compared and statistically analysed, as well as tested for correlation with clinical findings. results: fourteen go patients were enrolled and analyzed. three showed mild, six moderate-to-severe and four sight-threatening go. patients with severe go showed statistically significant higher values for tlg and mtv on the early static images, whereas suvmax did not differ among groups. pet parameters did not show statistically significant differences on the late acquisition images. pet parameters obtained both from early as well as late static images showed no correlation with eom thickness. pet parameters obtained from individual eoms were not correlated with its motility. discussion: tlg and mtv derived from pea of early static images appear to be good discriminators for severe vs. mild to moderate go. our results suggest that in go early static acquisition is superior to late static acquisition. as expected pet parameters of individual eye muscles were not correlated with associated eye motility, since fibrosis is responsible and not inflammation is mainly responsible for motility disorders. in conclusion, 18f-fdg-pet/mri appears to be a promising modality for the assessment of go severity with the potential to address limitations of the current diagnostic standard procedures. therefore, its diagnostic value should be subject of further studies on larger collectives. purpose: evaluation of the ocular and adnexa pathological signs and the results of their treatment in patients who previously underwent radiation therapy for eyelid skin cancer. material and methods: the study included 9 patients (3 men, 6 women, mean age 56.3 ± 2.7 years) who previously underwent radiation therapy for eyelid skin cancer. follow up period from 4 months to 3 years. all patients suffered from lid cosmetic defect, had different grades of cicatricial lagophthalmos and corneal lesions. lagophthalmos value ranged from 2 to 14 mm (m±m, mm: 7.1 ± 0.37), it was mild-in 1, moderate-in 3, severe-in 5 cases. cornea was involved in all cases (keratopathy-1, keratoconjunctivitis-2, neurotrophic keratitis-4, corneal ulcer-2 cases). dry eye diagnosed in all cases. radiation induced cataract was detected in 5 cases. bcva varied from 20/200 to 20/20 in 7 patients. results: all patients underwent medical therapy and one or more surgical steps. surgical correction of the eyelids position was performed in all cases. the type of surgical intervention depended on the grade of lagophthalmos: fixing of edges of a cartilage of a lower eyelid to the periosteum or canthoplasty, z-plasty, transposed flaps. in the presence of severe complications of the cornea, the following surgical procedures were performed: transplantation of the amniotic membrane-3, autoconjunctival flap-5 eyes. as a result of the combined treatment, the state of the eye anterior segment has improved in all cases. cataract extraction with iol implantation was performed after stabilization of the cornea. conclusions: radiation therapy for skin cancer of the eyelids can lead not only to a cosmetic defect, but also to severe dry eye and serious complications of the cornea and adnexa. the optimal combination of medical and surgical treatment can achieve a satisfactory position of the eyelids and stabilization of the ocular surface, which significantly improves the quality of life of patients. eine ptosis als wegweiser zur systemerkrankung neumann c. postoperativer astigmatismus nach photorefraktiver keratektomie bei augen mit rein sphärischer myopie ezzeldin m. 1* , frings a. 2, 3 , linke s. j. 4, 5 , steinberg j. 5, 6 objective: to determine the incidence of tearing in patients after radioiodine therapy (i 131 ), as well as to conduct a correlation analysis of tearing and some clinical and laboratory parameters. material and methods: a questionnaire survey of 235 patients was conducted (average age 55 ± 12 years, median 57 years) who underwent single (210 cases) or repeated (25 cases, up to 6 times) i 131 therapy in a dose of 1.5 to 6 gbq (average 3,6 ± 0.6 gbq) for thyroid cancer 2 to 61 months ago (average 31 ± 17 months, median 32 months). the severity of tearing was determined in points according to the munk scale. a statistical analysis was performed, including a spearman correlation analysis. the tearing was absent in 123 respondents (58.6 %), the severity of tearing was 1 point according to the munk scale in 39 cases (18.6 %), 2 points in 19 cases (9.0 %), 3 points in 14 cases (6.7 %), 4 points in 15 cases (7.1 %). a significant correlation was revealed between the severity of tearing and the frequency of i 131 therapy (p < 0.001, r = 0.5). the complaints of significant tearing after a single therapy with radioactive iodine (4 points) were determined in patients for periods of 5 to 46 months. the incidence of significant tearing (4 points) in patients after a single i 131 therapy increased with age (p < 0.001, r = 0.4), as well as with an increase in the effective radiation dose (p < 0.001, r = 0.5). conclusion: the significant tearing probably caused by a secondary obliteration of the nasolacrimal duct occurs in 7.1 % of cases. the probability of this complication development is dose-dependent and is related to the eines hochviskösen viskoelastikums im rahmen der icl-implantation beziehungsweise des icl-austausches entstehen. bei urrets-zavalia-syndrom nach icl-implantation stellen zwei gegeneinander versetzte aniridie-ringe im kapselsack zur dauerhaften reduktion der blendempfindlichkeit eine praktikable therapieoption dar. hamon l. 1* , flockerzi e. 1 (16) preloaded iols (20.0 d-21.0 d) per treatment group were delivered into the anterior chamber of human cadaver eyes through a 2.0 mm (vivinex) or 2.2 mm (autonome, ultrasert, and itec) incision size. corneal incision morphology was evaluated using optical coherence tomography (oct) and incision sizes were measured using asico incision gauges before and after iol delivery. differences in mean incision enlargement between delivery devices were evaluated using a paired t-test. results: autonome (0.29 ± 0.03 mm) and ultrasert (0.29 ± 0.03 mm) had the smallest average incision enlargement compared with itec (0.31 ± 0.03 mm) and vivinex (0.36 ± 0.06 mm). vivinex had the largest corneal incision enlargement and was significantly larger than autonome (p = 0.001), ultrasert (p = 0.001) and itec (p = 0.002). representative oct images of pre-and post-implantation incisions (cross sectional images of cornea) showed more incision gaping, corneal stromal damage and distortion for delivery systems with the largest incision enlargement: vivinex and itec. the new autonome preloaded delivery system protects the corneal incision during iol implantation and causes smaller incision enlargement and less corneal stromal damage compared to itec and vivinex. further clinical studies are necessary to confirm the effect of incision enlargement on wound healing and post-operative corneal morphology. lwowski c. m purpose: to assess the morphological and functional outcome of intravitreal aflibercept following treat and extend regime compared to fixed regime for treatment of eyes with neovascular age-related macular degeneration. this retrospective study included 128 eyes with primary onset neovascular age-related macular degeneration followed for 12 months. all eyes were treated with 0.5 mg/0.05 ml aflibercept. all eyes received three aflibercept injections monthly as upload phase. then, we compared two groups of eyes. for group 1, 57 eyes were treated following treat and extend regime. for group 2, 71 eyes were treated following fixed regime (fixed 8-weekly interval). main outcome measures included: best corrected visual acuity (bcva), central macular thickness (cmt) and number of injections. results: bcva (logmar) in group 1 vs group 2 was (0.61 ± 0.3 vs 0.72 ± 0.3, p = 0.09) before treatment and (0.48 ± 0.3 vs 0.51 ± 0.3, p = 0.6) after one year of treatment. the visual improvement was (0.10 ± 0.1 vs 0.14 ± 0.1, p = 0.1). cmt in group 1 vs group 2 was (360 ± 106 µm vs 393 ± 108 µm, p = 0.09) before treatment and (284 ± 58 µm vs 290 ± 67 µm, p = 0.5) after one year of treatment. the decrease in cmt was (76 ± 102 µm vs 102 ± 110 µm, p = 0.1). number of injections/eye in group 1 vs group 2 was (8.8 ± 1.4 vs 7.0 ± 0.0, p = 0.004). in group 2, six patients (11 %) were reverted back to monthly treatment as the treatment interval was extended over 8 weeks. conclusions: eyes treated with aflibercept following treat and extend regime had no significant differences regarding bcva and central macular thickness compared to eyes treated with fixed regime. however, eyes treated following treat and extend regime received a significantly higher number of injections during the first year of treatment. ergebnis: bei 11 augen erfolgte der revisionseingriff nach primärer linsenchirurgie (iol-gruppe: 10 multifokal, 1 monofokal), bei 11 augen war eine hornhautchirurgische operation vorausgegangen (hh-gruppe: 3 femto-lasik, 6 transprk, 2 lasek). die refraktion betrug präoperativ in der iol-gruppe +0,4 ± 1,5 dpt (-2,5 bis +2,25 dpt) im sphärischen äquivalent (sphä), in der hh-gruppe -0,54 ±0,75 dpt (-2,0 bis +0,5 dpt). der unkorrigierte visus (ucva) lag präoperativ bei 0,53 ± 0,23 (iol-gruppe: 0,38 ±0,21 (0,1 bis 0,6), hh-gruppe: 0,69 ±0,16 (0,4 bis 0,9). in 3 augen der hh-gruppe wurde eine wellenfrontgeführte ablation durchgeführt. 20 von 22 augen lagen postoperativ im zielbereich von ± 0,5 dpt. die ucva betrug 3 monate postoperativ im mittel 0,92 ±0,16 (0,6 bis 1,1) (iol-gruppe: 0,82 ±0,16 (0,6 bis 1,0), hh-gruppe: 0,98 ±0,06 (0,9-1,1). in der aberrometrie zeigte sich in der iol-gruppe eine zunahme, in der hh-gruppe eine dezente abnahme der aberrationen höherer ordnung (hoa, bei 6 mm pupille) (iol-gruppe: rms +0,244 µm, coma +0,025 µm, trefoil +0,208 µm, sphab -0,119 µm; hh-gruppe: rms -0,083 µm, coma -0,028 µm, trefoil -0,086 µm, sphab +0,028 µm). in keinem fall kam es zu postoperativen komplikationen, und es waren bei subjektiver patientenzufriedenheit keine weiteren revisionen notwendig. schlussfolgerung: excimer-laser-gestützte revisionseingriffe stellen bei residueller ametropie nach primärer linsen-oder hornhautoperation, speziell im refraktiv-chirurgischen bereich, eine sichere und effektive therapieoption dar, um dem patientenwunsch zu entsprechen. rayamajhi a. 1 objective: the mechanism of the two-photon absorption by photoreceptors and their visual pigment chromophore isomerization lays the ground for a better understanding of how infrared light (ir) triggers color perception in an unaided eye. the association between retinal disease and expected changes in ir-light sensitivity has yet to be investigated, though. this study aimed to measure and compare scotopic eye sensitivity of healthy and diabetic-retinopathy patients using an ir-light microperimeter. method: this research was carried out at the ophthalmology department of the heidelberg university hospital. among 69 included eyes, 28 were healthy, and 41 were diabetic. all participants underwent a comprehensive eye exam, including visual acuity (va) and contrast sensitivity tests, optical coherence tomography and slit-lamp examination. the ir threshold was measured following 30-min dark-adaptation with a goldman ii size stimulus and the method of adjustment. to this end, we used the ir-light microperimeter with integrated pulsed laser light (1045 nm) for sensitivity assessment and scanning laser ophthalmoscopy for fundus imaging. the mean age of the diabetic patients (61.2 ± 12.7 years) and the control group (56.2 ± 15.7 years) was not statistically significant (p = 0.15). the mean logmar va of the diabetic patients (0.12 ±0.17) was worse than in the healthy group (-0.04 ±0.08), which was significantly different (p < 0.001). furthermore, the contrast sensitivity of the diabetic patients was lower than that of the healthy group, especially at 6 and 18 cycles/degree. a statistically significant (p = 0.04) difference was found in the mean retinal thickness between the diabetic patients (300.0 ± 50.0 µm) and the healthy group (277.1 ± 19.5 µm). the mean retinal sensitivity to ir light in the diabetic patients (11.6 ± 2.1 db) was significantly (p < 0.001) lower than in the healthy group (15.5 ± 1.3 db). kharkiv national medical university, kharkiv, ukraine background: nowadays phacoemulsification is a standard in the cataract surgery. however, it is impossible to exclude the negative effect of low-frequency ultrasound, cavitation and thermal energy during surgery. changes in the posterior segment of the eye after phacoemulsification require clarification. aim: was to analyze the changes of the macular morphology and posterior vitreous among patients with unchanged posterior segment of the eye after the phacoemulsification of the cataract with and without posterior capsulorhexis. methods: in this prospective study 68 patients with senile cataract and unchanged posterior segment of the eye were included. all patients underwent uncomplicated phacoemulsification. posterior capsulorhexis was not performed to 42 patients of the first group whereas it was applied to 26 patients of the second group because of the opacification of the posterior capsule of the lens. oct was performed to all patients preoperatively and postoperatively on the 1st day, the 1st week, and the 1st, the 3rd, and the 6th months. results: there were no significant differences in the macular thickness among patients of the first and the second group before the surgery. significant difference in the macular thickness was detected on the 1st day (p = 0.03), the 1st week (p = 0.02), and the 1st month (p = 0.03). but this difference in both groups became insignificant by the 3rd and 6th month of observation. during the 1st week after the surgery one patient (3.8 %) of the 2nd group manifested cystoid macular edema that was resolved by the 3rd month of the observation. there were no changes of the posterior vitreous among patients of the 1st group. partial detachment of the posterior hyaloid membrane was observed in 1 patient (3.8 %) of the 2nd group one month after the operation. three months later partial detachment of the posterior hyaloid membrane was diagnosed in 2 more patients (7.7 %) of the 2nd group. during 6 months of observation partial detachment of the posterior hyaloid membrane did not lead to a significant change in retinal thickness among all 3 patients. summary: potentially, posterior capsulorexis during uncomplicated phacoemulsification leads to a more pronounced effect on the vitreous body and macular zone. however, the morphological changes in the posterior segment of the eye in this case are either reversible or do not lead to gross structural changes that could affect visual function within six months of observation. berlin m. edema-grade-dme study busch c. purpose: to evaluate the proportion of functional and anatomical responders and non-responders to the first dexamethasone (dex) implant in naïve and non-naïve diabetic macular edema (dme); to assess different response patterns; and to propose a grading classification for dme treatment response. methods: retrospective, multicenter, observational study. naïve and nonnaïve dme patients treated with dex, with visual acuity (va) ≥ 0.2 log-mar (≤ 0.8 decimal) and central subfield thickness (cst) of ≥ 300 µm measured on spectral domain optical coherence tomography (sd-oct). va and cst were recorded at baseline, month 2 and month 4 after first dex treatment. functional and anatomical responses were graded, and different response patterns were analyzed. results: we included 417 eyes from 388 patients, of which 42 % (175 eyes) were treatment-naïve. mean baseline va was 0.63 ± 0.30 logmar and mean cst was 534.5 ± 144.0 µm. the proportion of very good functional responders (va gain ≥ 10 letters) after 2 and 4 months was 56 and 57 % for naïve eyes, and 33 and 28 % for non-naïve eyes (naïve vs. non-naïve eyes: p < 0.001). proportion of functional non-responders (va gain < 5 letters) after 2 and 4 months were 18 and 16 % for naïve eyes, and 49 and 53 % for non-naïve eyes (naïve vs. non-naïve eyes: p < 0.001). anatomical non-responders (cst reduction < 10 % or cst increase) at 2 months were rare in both groups (naïve: 4 %, non-naïve: 12 %, p = 0.003). naïve eyes usually demonstrated an early and stable functional and anatomical improvement, while in non-naïve eyes a persistent non-response pattern was the 223 visusrelevante prognostische parameter bei fibrovaskulärer umwandlung der cnv unter anti-vegf-therapie bei neovaskulärer amd book m. methods: endothelial cell growth medium (ecgm-mv) and dmem were supplemented with 5 % fetal bovine serum, endothelial cell growth supplement, 90 µg/ml heparin and 100 nm cortisol, and confluent ibrec were exposed to both cell culture media or mixes thereof for 1-4 d. the cell index (ci) of confluent cells cultivated gold electrodes was continuously determined to evaluate permeability, cell adhesion or cell viability for which the activities of nad(p)h-dependent oxidoreductases were also measured. we assessed expression and/or subcellular localization of proteins specific for (microvascular) ec and those involved in regulation of paracellular flow and transport. results: the ci dramatically dropped to 10 % within 6 h after exposure of the cells to dmem, slowly recovering to normal values over the following 4 days. expression of tight junction (tj) protein claudin-5 specific for microvascular ec was significantly lower even after 1d and barely detectable on day 4. in contrast, expression of tj-protein claudin-1 was higher after 1d, but significantly declined during prolonged cultivation of the cells in dmem. adherens junction protein vecadherin was only slightly affected, but expression of caveolin-involved in transcellular transport-was also significantly lower. in accordance, plasma membrane localization of claudin-1 or claudin-5 was reduced and that of vecadherin more diffuse after exposure to dmem for 3d. the cells then still expressed ec-specific von willebrand factor although to a lesser extent. interestingly, the nad(p)hdependent oxidoreductases were more active. all changes were observed with ibrec cultivated in mixes of ecgm-mv and dmem containing at least 50 % dmem. conclusion: cultivating ibrec in dmem results in a rapid loss of the phenotype and properties typical for microvascular ec. these changes include barrier dysfunction and loss of specific marker proteins. therefore, observations made with (retinal) ec cultivated in dmem have to be evaluated with caution. falahat p. 1* , wintergerst m. w. m. 1 , holz f. g. 1 , schaefer c. 2 einfluss der wwc-proteine auf die gefäßbildung der netzhaut der maus egbring c. non-adhärenz und non-persistenz in der ivom-therapie -eine systematische literaturrecherche ehlken c. 1* , ziemssen f. 2 , eter n. 3 , lanzl i. 4 , kaymak h. 5 , lommatzsch a. 6 ex-vivo retinal dystrophy models via blue light induced neurodegeneration fietz a. 1* , hurst j. 1 , leinwetter m. 1 , schnichels s. 1 1 universitäts-augenklinik, tübingen, germany background: the hallmark of many retinal diseases like age-related macular degeneration (amd) or retinitis pigmentosa (rp) are neurodegenerative processes in the retina, leading to damaged photoreceptor cells and ultimately blindness. there is strong evidence that photochemical oxidative stress plays a role in amd and rp pathogenesis as well as in several other eye diseases. blue light triggers the production of reactive oxygen species (ros) and might therefore be a promising non-chemical-based inducer of oxidative stress. the aim of this project was to establish a ros based degeneration model on ex vivo porcine cells and retinae. methods: primary porcine retinal cells and organ cultures were exposed to blue light in varying intensities, treatment numbers and periods of time. ros production was measured and the apoptotic state of the retinal cells and cultures was determined. furthermore, the degree of degeneration was analyzed via immunhistology, western blot and qrt-pcr. cell and disease specific, as well as cell death and cellular stress markers were evaluated. results: primary rpe and müller cells were exposed to blue light for 1 h and the ros-level was determined. both exhibited significant ros increase after 6 and 24 h of cultivation (1.3-2-fold). furthermore, müller, rpe and dissociated retinal cells were exposed for 2 h and the rosamount was measured after 6 and 24 h. the strongest ros-increase was detected after 6 h (2-10-fold), proving that blue light irradiation causes oxidative stress in primary retinal cells. to investigate the effect of blue light on retinal explants, specific markers like parp-1, hsp70, p53, caspase 3, and opsin were analyzed. in blue-light-irradiated retinal explants, a significant increase of parp-1 and other stress markers was demonstrated, whereas opsin expression was decreased (2-fold). conclusion: we successfully established an oxidative stress based disease model with blue-light-induced neurodegeneration. this model can be used to test novel therapeutic approaches for amd-or rp-treatment exvivo. due to a similar morphology of the pig eye to the human eye, this setting is close to the human condition. the features of vitrectomy and vitreous chamber tamponade in the surgical treatment of severe forms of proliferative diabetic retinopathy golovin a. germany, the netherlands and switzerland. here, we will focus on the dme subgroup. the pacific study is a non-interventional, open label, multicentric study aiming to collect real world evidence regarding the use of ranibizumab. 5.014 patients were recruited in 186 sites across germany (ger), the netherlands (nl), and switzerland (ch). we present data of at least 12 months dme treatment with ranibizumab (299 naive and 411 pretreated patients-full analysis set) in a real-life setting, with a focus on treatment outcome, supply and therapy schemes applied. the percentage of female patients were 40.7 % (ger), 60.0 % (nl) and 31.8 % (ch). the entire population presented with a mean age of 66.4 (ger), 68.1 (nl) and 61.0 (ch) years. in ger most patients were already pretreated (p-t). mean bcva at baseline was 62.8 ± 15.8 (ger), 68.3 ± 9.4 (ch) for treatment-naïve (t-n, no patients nl) and 65.9 ± 16.1 (ger) 65.5 ± 12.7 (nl), 78.0 ± 5.4 (ch) letters for p-t patients. after 24 months ranibizumab treatment, 17.5 % more t-n than p-t patients achieved a bcva gain ≥ 15 letters. within 12 months t-n patients received an average of 6.2 ± 2.9 (ger), 6.3 ± 6.5 (ch) ranibizumab injections which was comparable to p-t patients ( markova k. 2 , rößler b. 3 , iwersen m. 3 , michel u. 3 , beeke e. 4 , gamael a. 5 , scheffler m. 6 , ter the lesion. number and length of regenerating rgc axons in the distal optic nerve stumps were analyzed one month after the lesion. results: the number of surviving rgcs in retinas treated simultaneously with gdnf and cntf, gdnf only, or cntf only was significantly higher than in retinas that received injections of control nscs at all post-lesion time points. importantly, the simultaneous administration of both neuroprotective factors rescued rgcs more effectively from injury-induced cell death than the separate administration of either gdnf or cntf. haritoglou c. 1* , conclusions: data indicate that combinatorial neuroprotective approaches represent a promising strategy to effectively rescue rgcs from lesion-induced cell death even when the treatment is started at a time point when a significant fraction of rgcs is already lost. jiang j. 1* 1 universitätsaugenklinik freiburg, freiburg, germany background: retinopathy of prematurity (rop) is a major cause of blindness in children. hyperoxic insults to the premature retina after birth, followed by a hypoxic phase subsequently, lead to neovascularisation. without timely treatment, retinal detachment ensues. diode laser photocoagulation is the current gold standard treatment but may not be widely available in developing and middle-income countries, where a surge in the incidence of rop has been observed. therefore, trials are ongoing to find time-saving and less invasive alternatives. objectives: this literature review aims to discuss potential treatment options explored recently (from 2010 onwards) and to examine how they relate to the pathophysiology of rop. embase and the cochrane database of systematic reviews (cdsr). key words such as "retinopathy of prematurity", "anti-vegf", "igf-1" and "beta blockers" were searched using medical subject headings (mesh), free text terms and synonyms. trials published in english and within the last ten years (2010-2019) were evaluated. results: thirty-five trials were relevant to this literature review and generated findings for the use of anti-vegf, igf-1, beta blockers, oxygen saturation targets and postnatal nutrition. the largest body of evidence relates to anti-vegf agents, but their side effect profile and optimal dosages are areas to be addressed before widespread clinical use can be advocated. other promising interventions include beta blockers and fish oil supplementations, which should be further evaluated in large-scale randomised trials. discussion: despite the advantages associated with anti-vegf therapy, more high-quality studies are required and determination of the safety of this class of drugs is paramount. up to date guidelines for ophthalmologists in the treatment of rop are also needed. there is much interest in managing a range of co-morbidities associated with prematurity by means of a single intervention, which has proven to be an ambitious but challenging task. purpose: to address the need for a reduced treatment burden for the management of neovascular age-related macular degeneration (namd), the port delivery system with ranibizumab (pds) was designed as a refillable, indwelling implant providing diffusion-mediated continuous intravitreal delivery of ranibizumab (rbz). the ladder trial (nct02510794) is evaluating the efficacy and safety of the pds with 3 rbz formulations compared with monthly intravitreal rbz 0.5 mg injections for the treatment of namd. the primary study endpoint, defined as time to first required implant refill assessed when the last enrolled patient completed the month 9 visit, has been reached. the phase 3 archway (nct03677934) is an ongoing randomized clinical trial directly comparing pds 100 mg/ml with fixed 24-week refills with intravitreal rbz 0.5 mg monthly. results: the ladder primary analysis population was 220 patients: 59, 62, and 58 patients in the pds 100, 40, and 10 mg/ml arms, respectively, and 41 patients in the monthly intravitreal rbz arm. the median time to first refill was 15.0, 13.0, and 8.7 months with 79.8 %, 71.3 %, and 63.5 % patients not meeting implant refill criteria until ≥ 6 months for the pds 100, 40, and 10 mg/ml arms, respectively. at month 9, mean change in bcva from baseline was +4.3, -0.5, -3.2, and +3.3 letters for pds 100, 40, and 10 mg/ml, and monthly intravitreal rbz arms, respectively. at the time of primary analysis, patients in the pds 100, 40, and 10 mg/ml arms had received a mean total of 2.4, 2.6, and 3.7 rbz treatments during a mean time on study of 16.4, 17.0, and 16.9 months, respectively, compared to a mean total of 16.8 rbz treatments received by patients in the monthly intravitreal rbz arm during 16.4 month mean time on study. the optimized pds implant insertion surgery and refill procedures were well tolerated. case examples from the ladder study and mechanism of action of continuous delivery will be presented at the meeting. conclusions: with 80 % of patients not needing an implant refill until ≥ 6 months in the pds 100 mg/ml arm and comparable efficacy outcomes between pds 100 mg/ml and monthly intravitreal rbz treatment, ladder results support the archway design of pds 100 mg/ml with fixed 24-week refills. pds has the potential to reduce high intravitreal treatment burden and improve patient outcomes in clinical practice. hintergrund: beschreibung der klinischen variabilität in augen mit retinitis pigmentosa assoziiert mit pathogenen eys genmutationen. methoden: neun patienten (mittleres alter: 46,2, 29-70 jahre bei erstuntersuchung, 4 frauen) mit zwei pathogenen eys genmutation bestätigt durch molekulargenetische diagnostik wurden klinisch untersucht mit s128 abstracts these patients. faricimab is a first-in-class bispecific monoclonal antibody directed against vegf-a and ang-2 and designed for intravitreal use. we aimed to assess efficacy and durability of faricimab in patients with dme and namd (boulevard and stairway). methods: boulevard (nct02699450) analysed treatment-naïve dme patients receiving intravitreal 1.5 or 6.0 mg faricimab or 0.3 mg ranibizumab (rbz), and previously anti-vegf-treated patients 6.0 mg faricimab or 0.3 mg q4 w for 20 weeks. stairway (nct03038880) analysed patients with namd treated intravitreal with 6.0 mg faricimab, q16 w flex or q12 w after initiation, or with 0.5 mg ranibizumab (rbz) q4 w. we here present the results of both phase 2 studies regarding central subfield thickness (cst), diabetic retinopathy severity score, size of choroidal neovascularisation (cnv) lesions and safety signals. results: cst improved from baseline at week 24 in all dme patients (adj. mean change, microns: 1.5 mg faricimab, -217.1; 6.0 mg faricimab, -225.8; 0.3 mg rbz, -204.7). 28 %, 39 % and 12 % of dme patients experienced a ≥ 2-step improvement in diabetic retinopathy severity score with 1.5 mg faricimab, 6.0 mg faricimab or 0.3 mg rbz, respectively. more patients treated with 6.0 mg faricimab maintained disease stability with stable cst during the off-treatment period at both 8 and 12 weeks after the last dose compared to rbz. 65 % (36/55) of faricimab-treated namd patients had no disease activity at week 24. q16 w flex and q12 w faricimab-treated namd patients showed similar cst and cnv lesion size reductions vs q4 w rbz. in both studies no new or unexpected safety findings were identified. conclusions: maintenance of disease stability was improved with faricimab in dme patients, and extended faricimab dosing (q16 w flex and q12 w) revealed similar outcomes compared to q4 w rbz in namd patients. these results support further investigation of the efficacy, safety and durability of response, addressing an important unmet need for treatment options for these patients. karzinomassoziierte retinopathie -zwei fallberichte kilic a. purpose: multicentre trials have demonstrated the safety and efficacy of the fac implant in the management of dme over a 3 year period. there are few real-world studies with sufficiently large populations of patients with dme reporting 3 year outcomes. the current study reports the safety and effectiveness findings from the iriss study methods: iriss is a post-authorisation observational open label, registry safety study of the fac implant in dme, conducted in the united kingdom, germany and portugal. the study was designed to collect the data from 550 patients that had been treated with the fac implantation according to the european label. the present analysis focused on only those patients (n = 295 patients/n = 343 eyes) completing 3 years of monitoring post-treatment with the fac implant. safety was assessed in terms of the occurrence of intra-ocular pressure (iop) events and their management. effectiveness was determined from changes best-recorded visual acuity (va), particularly on the percentage of patients maintaining or achieving 6/12 (20/40 or 70 etdrs letters) vision. results: mean age of the patients was 66.3 ± 10.8 (mean±standard deviation [sd]) years and the duration of dme was 4.95 years. the majority (85.7 %) of eyes treated with the fac implant were pseudophakic. mean iop was 15.6 ± 3.6 mmhg at baseline and remained below 21 mmhg throughout 36 months of follow up, with a small increase from baseline at month 36 (+1.2 mmhg, p = 0.033). topical iop-lowering drops were required in 39.9 % of eyes to control elevation in pressure and only 3.2 % (n = 11/343) of eyes requiring iop-lowering surgery. mean va improved/ was maintained in 71 % of eyes at year 3 and 32 % achieved 6/12 vision compared to 18.6 % at baseline. eyes with a starting va of 6/12 had no significant change in va over 3 years. conclusions: these real-life outcomes confirm the long-term safety and effectiveness of the fac implant in the treatment of dme after three years of treatment and in a cohort of patients larger than the eu registration trial population. the results from clinical practice show changes in iop and visual acuity similar to those reported in the pivotal fame trials. these data also show that when patients with good starting va (≥70 letters) were treated with the fac implant, vision was stabilised for the full three years of therapy. khoramnia r. 1* , patel s. 2 , khanani a. m. 3 , sahni j. 4 , sadikhov s. 4 , basu k. 5 , grzeschik s. 5 , szczesny p. 4 purpose: the importance of vegf-a inhibition in the treatment of neurovascular age-related macular degeneration (namd) and diabetic macular edema (dme) has been well demonstrated. still, real-world response rates are not sufficiently satisfying and treatment burdens are high due to high injection frequencies (q4 w). additional simultaneous inhibition of angiopoietin-2 (ang-2) might improve treatment efficacy and durability in berlin a. 1 , radun v. 1 , reichel c. 1 augenklinik der lmu münchen, munich, germany; 2 universität ulm, ulm, germany introduction: foveal alterations in asymptomatic patients can be very challenging in order to facilitate a diagnosis. methods: a female 21-year-old patient was referred to us by a general ophthalmologist to clarify a foveal lesion, that had been observed in a routine fundus examination of the right eye. in addition to her medical history and a standard clinical examination we performed the following examinations: fundus imaging, widefield imaging, heidelberger autofluorescence, oct imaging, fluorescein and icg angiography. the patient presented without any medical or ophthalmological history. the vision was 20/20. the clinical examination of the anterior segment of the eye showed no pathological findings. the posterior segment of the right eye showed a circular central lesion of the fovea. in fundus imaging and widefield imaging this lesion could be documented. heidelberger autofluorescence showed no foveal enhancement. in oct imaging the retinal layers were normal, however, we observed uncommon dilated choroidal vessels. in fluorescein and icg angiography the dilatation and central anastomosis of choroidal vessels were clearly documented. discussion: this is one of the rare examples of an asymptomatic patient who presented with an alterated fovea, which is most likely due to choroidal anastomosis and dilatation. bewertung der eignung des serum vegf spiegels als prognostischer faktor bei einem makulaödem bedingt durch einen venenastverschluss/zentralvenenverschluss unter therapie mit lucentis® (ranibizumab) lang m. purpose: neuronal ceroid lipofuscinosis (ncl) is a clinically and genetically heterogeneous group of neurodegenerative lysosomal storage disorders of mainly childhood. characteristic clinical symptoms of this fatal disorder include cognitive decline, mental deterioration, motor impairment, seizures, vision loss as a result of retinal degeneration, and premature death. enzyme replacement strategies represent promising treatment options for ncl forms caused by dysfunctional lysosomal enzymes. cln10 disease is caused by mutations in the gene encoding the lysosomal enzyme cathepsin d (ctsd). here, we analyzed the efficacy of two enzyme replacement strategies to attenuate retinal degeneration in a ctsd knockout (ko) mouse. methods: functional murine ctsd was administered to the ctsd ko retina prior to the onset of retinal degeneration by intravitreal injections of either a ctsd-overexpressing neural stem cell (nsc) line or ctsd encoding adeno-associated virus (aav) vectors. the impact of the treatments on accumulation of storage material, reactive astrogliosis and microgliosis, dysregulation of various lysosomal proteins and the loss of different retinal cell types was analyzed at the end-stage of the disease using immunohistochemistry and western blot analyses. results: both treatment strategies resulted in a reduction of storage material and attenuation of reactive microgliosis. treatments also normalized the dysregulated expression of various lysosomal proteins. however, significant attenuation of retinal degeneration was only observed in animals treated with ctsd-encoding aav vectors but not in animals that received injections of ctsd-overexpressing nscs. ctsd levels were significantly higher in aav-treated than in nsc-treated ctsd ko retinas. conclusions: a sustained administration of functional ctsd, either through a cell-or an aav-based approach, resulted in attenuation of the retinal pathology in an animal model of cln10 disease, the most severe ncl form. however, delivery of therapeutically relevant amounts of the lysosomal enzyme to the ctsd ko retina was only achieved through the gene therapy approach, as indicated by a significant rescue of retinal cell types. bioadhere -maßgeschneiderte bioadhäsiva für epiretinale netzhautstimulatoren okuläre beteiligung bei einer systemischen attr-amyloidose mit einer p.v50m mutation lubbad a. verlauf: im labor fielen ein kaliumwert von 2,5 mmol/l, sowie 600.000 leukozyten mit deutlicher linksverschiebung auf. bei v. a. leukämie erfolgte eine stationäre aufnahme in der hämatoonkologie. mittels zytound molekulargenetischer analyse wurde die translokation t(9;22) (q34;q11) mit nachweis des bcr-abl transkriptes ermittelt und die diagnose einer cml gestellt. daraufhin wurde die zytoreduktive therapie (mit hydroxycarbamid gefolgt von einem tyrosinkinaseinhibitor) eingeleitet. unter der therapie normalisierten sich die blutwerte und funduskopischen veränderungen und der visus stieg beidseits auf 1,2. in der fag zeigten sich die tortuositas und blutungen rückläufig. in der sd-oct kam es beidseits zum rückgang des mös unter der cml-therapie. diskussion: bei einem zvv erfolgt die diagnostik meist ohne notfallindikation über den hausarzt. bei bilateralem auftreten und jungen patienten (< 50 jahre) muss eine ursachenabklärung durchgeführt werden, da häufig eine systemische erkrankung assoziiert ist. die sofortige blutuntersuchung sowie die hämatoonkologische therapie sind bei diesen patienten essentiell. ein effekt der systemischen therapie auf das mö sollte für bis zu 6 wochen nach symptombeginn abgewartet werden, bevor eine anti-vegf therapie begonnen wird. so könnte in manchen fällen eine anti-vegf therapie eingespart werden. besteht das mö ungeachtet der purpose: our increasingly aging society leads to a growing incidence of neurodegenerative diseases. as the pathological mechanisms are inadequately known, the establishment of defined therapies is impeded. additive gene therapeutic approaches for the increased expression of a protective factor are considered as promising treatment modality. our approach relies on the genetic modification of retinal pigment epithelial (rpe) cells. we have already established their transfection to stably overexpress the genes coding for pigment epithelium-derived factor (pedf) and brainderived neurotrophic factor (bdnf), respectively. here, we describe the analysis of the neuroprotective function of the transfected rpe cells using a co-culture model. methods: arpe-19 cells were transfected with the genes coding for pedf and bdnf using the non-viral sleeping beauty transposon system. pedf and bdnf gene expression were analyzed via quantitative real-time pcr. pedf and bdnf secretion were evaluated by immunoblotting and quantified by elisa. transfected and non-transfected arpe-19 cells were cocultured with sh-sy5y cells, a human neuroblastoma cell line exhibiting neurite outgrowth in the presence of neurotrophic factors. prior to the co-culture, sh-sy5y cells were oxidatively stressed with 150 µm h 2 o 2 for 24 h to mimic damaged cells (oxsh-sy5y). after 48 and 96 h, the neurites were stained with an anti-neuron-specific β-iii tubulin antibody and visualized by confocal microscopy. neurite outgrowth was analyzed using the software simple neurite tracer. the transfection of arpe-19 cells resulted in a significant increase in pedf and bdnf gene expression as well as protein secretion. first results indicated that bdnf-transfected arpe-19 (trarpe-19) cells significantly stimulated neurite outgrowth (p < 0.01). the neurite length in trarpe-19/oxsh-sy5y co-cultures was 1.81-fold extended compared to oxsh-sy5y cells alone (64.1 ± 2.5 µm vs. 35.5 ± 1.2 µm), whereas nontransfected arpe-19 cells showed no significant effect on oxsh-sy5y cells (41.9 ± 1.3 µm). however, for pedf, no significant neurite elongation was observed until now. with the establishment of the sh-sy5y co-culture model, we were able to analyze the neuroprotective function of a cell-based gene therapy. the next steps include the co-cultivation of sh-sy5y cells with transfected primary pigment epithelial cells and the translation of the method to an ex-vivo retinal organ co-culture model. traumatische zyklodialyse -von zyklopexie ab interno bis zur dmek martin c. oct (0, 4, 8, 12, 16, 24, 32, 40, 48 wochen) auch die mikroperimetrie (maia mikroperimetrie, 0, 4, 12, 24, 48 wochen) . verglichen wurde der verlauf der fixationsstabillität (p1) und die zunahme und abnahme der retinalen empfindlichkeit an einzelnen punkten der mikroperimetrie. ergebnisse: nach 48 wochen zeigt sich ein anstieg der sehschärfe im mittel von 55, 7 ± 15, 9 auf 63, 9 ± 16, 8 buchstaben etdrs (p = 0,005) und eine abnahme der crt von 436,3 ± 205,8 µm auf 273,2 ± 152,4 µm (p < 0,001). p1 verbesserte sich von 63,5 ± 28,2 % auf 76, 8 ± 26,8 % (p = 0,019) . während zu beginn der therapie deutlich mehr punkte eine verbesserung der retinalen empfindlichkeit als eine verschlechterung zeigen (nach 4 wochen: 19, 1 ± 8, 1 vs. 8, 4 ± 6, 3 (p < 0, 001), gesamtsumme db: 90, 4 ± 68, 9 vs. 32, 7 ± 30, 9 (p < 0, 001) ; nach 8 wochen: 16,2 ± 7,0 vs. 9,8 ± 5,1 (p = 0,005), gesamtsumme db: 60,4 ± 48,0 vs. 29,4 ± 18,2 (p = 0,027), sind bei der letzten visite verbesserte und reduzierte punkte gleich häufig (12, 5 ± 7, 0 vs. 12, 5 ± 5, 5 (p = 0, 67), gesamtsumme db: 55, 8 ± 30, 5, 48, 2 ± 33, 0 (p = 0, 48) . diskussion: unsere ergebnisse zeigen, dass eine eylea-therapie mit fixem schema zu einer kurz-und langfristig signifikanten verbesserung der fixationsstabilität sowie der sensitivität einzelner bereiche der mikroperimetrie führt. es kann also mit einer konsequenten therapie eine langfristige verbesserung des sehens erzielt werden. in all eyes a complete ophthalmic examination, swept source oct and ss-oct a was performed. we measured central retinal thickness (crt), central choroidal thickness (cct) and the area of the fovea avascular zone in superficial (sfaz) and deep retina (dfaz) vessels layer. results: 38 eyes were included into this retrospective analysis. 24 patients were included into the "observation only group". during the 15 months observation period traction spontaneously released in 5/24 eyes (20 %), and the traction release was statistically significant (p = 0.01). in this group improvement of visual acuity was noted (from 0.5 to 0.7 snellen). in mulmüller p. l. 1*,2 , treis t. 3 , pfau m. 2, 4 , esposti s. d. 1 , alsaedi a. 1 , maloca p. 1, 5 , balaskas k. 1 , webster a. 1 , egan c. 1 mikroperimetrie bei amd patienten während des ersten jahres fixer therapie mit aflibercept (eylea) muto e. s. 1* , dulz s. 1 , spitzer m. 1 , wagenfeld l. drils at the boundary between the ganglion cell-inner plexiform complex and inner nuclear layer improved in 79/143 eyes (55 %, p = 0.002) one month after dex treatment. drils between the inner nuclear layer and outer plexiform layer improved slightly (p = 0,453). the occurrence of drils at baseline correlate with worse bcva and crt at baseline and one month after dex implantation. furthermore, an improvement of drils could be shown after one month. therefore, dril may serve as a predictive and robust biomarker of visual outcome in uveitic cme treated by dex injection. pauleikhoff l. 1* , vössing c. 2 , song f. 2 , usman m. 2 , joachimsen l. 1 , reiff c. m. 1, 3 nelis p. 1*,2 , schmidt c. 3 , lehmann f. 3 , ertmer c. 3 , heßler m. 3 oct-based automated vitreous inflammation score: a promising biomarker in dexamethasone implant treated uveitis patients pohlmann d. 1* , terheyden j. h. 2 , berger m. 3 , ometto g. 4 , montesano g. 4 , neuber f. 1 , langner m. 2 , wintergerst m. w. m. 2 , aslam t. 5 , liu x. 6 , holz f. g. 2 , keane p. a. 7 , crabb d. p. 4 , denniston a. 6 purpose: to objectively detect and evaluate vitreous inflammation before and after dexamethasone implant in patients with uveitis by using a recently developed optical coherence tomography (oct)-based algorithm. methods: in this multicenter, retrospective, cross-sectional study, 302 eyes of 223 uveitis patients were included. clinical and oct data (spectralis; heidelberg engineering inc) of all patients were collected. the inflammation score was obtained using an available automated oct-based algorithm. data of pre and post dexamethasone implantation were compared using a random effects model. results: patients with cystoid macular edema in uveitis anterior (n = 18 eyes), intermediate (n = 106 eyes), posterior (n = 153 eyes), and panuveitis (n = 25 eyes) were treated with a dexamethasone implant. the mean of age was 61 years ± 15 years and a range of 22 to 88 years. we registered 277 follow-ups visits at 1 month (up to 2 months after injection) and 265 followup visits at 3 months (>2 months to 4 months). the mean inflammation score at baseline was 0.135, and changed significantly to 0.077 (p < 0.0001) 1-2 months and to 0.079 (p < 0.0001) 3 months after dexamethasone injection. correlations with clinical ratings of intraocular inflammation are currently evaluated. conclusion: automated oct-based objective quantification of vitreous inflammation captures the expected decrease in vitreous inflammation following intravitreal dexamethasone implant. thus, the automated octbased quantification of vitreous inflammation may be a promising alternative and a potentially relevant biomarker compared to current subjective clinical estimates of vitreous inflammation in uveitis. dunkeladaptation und skotopische mikroperimetrie bei patienten mit sorsby fundusdystrophie raming k. 1*, 2 , hess k. 1, 2 , pfau m. 1, 3 , birtel j. 1, 2 , müller p. l. 4 , gliem m. 5 , issa p. c. 6 , holz f. g. 1, 2 vesselness-basiertes ki-verfahren zur automatischen 3d-segmentierung der gefäße in der oct-angiographie (octa) rothaus k. 1* , kuhlmann j. 2 , faatz h. 1 , jiang x. 2 , lommatzsch a. 1, 3 longitudinale struktur-funktions-analyse bei intermediärer altersabhängiger makuladegeneration saßmannshausen m. 1*, 2 , zhou j. 1, 3 , pfau m. 1, 2, 4 , thiele s. 1, 2 , fleckenstein m. 5 , holz f. g. 1, 2 , a smart phone app for individual risk assessment for progression of diabetic retinopathy scholtz s. 1* , aspelund t. 2 , einarsson s. 3 , gudmundsdottir a. 4 , jonsdottir s. 3 , steinarsson a. t. 3 , stefansson e schultheiss m. 1* , wenzel d. a. 2 , kromer r. 1 , poli s. 3 vorstellung der revision-studie zur therapie des nicht arteritischen zentralarterienverschlusses schultheiss m. 1* , poli s. 2 in-vitro-bewertung von hydrogelen auf alginat-und hyaluronsäurebasis als glaskörperersatz schulz a. 1*, 2 , rickmann a. 1, 2 , wahl s. 1, 2 , germann a. 3 , stanzel b. 1, 2, 3 the pachychoroid disease spectrum-and the need for a uniform classification system purpose: introduced in 2013, the term pachychoroid has recently generated worldwide interest explaining disorders associated with a thick choroid. the spectrum of pachychoroid disorders includes pachychoroid pigment epitheliopathy (ppe), central serous chorioretinopathy (csc), pachychoroid neovasculopathy (pnv; defined by cnv), and aneurysmal type 1 cnv (at1). in contrast to these new findings, cnv complicating csc has already been described in a multitude of publications; and, moreover, polypoidal choroidal vasculopathy (pcv) has long been known to present with a thick choroid. currently, it is unclear how ophthalmologists implement the old (csc with cnv, pcv) and new terminology (pnv, at1). the online database pubmed.com was searched for the terms "pachychoroid", "ppe", "csc", "pnv", "pcv", "at1" and "cnv". articles published between august 1st 2013 until october 29th 2019 were included. results: in total, 122 articles containing the term "pachychoroid" were identified. using the keyword "ppe", 30 articles were found. in the 994 articles on "csc", only 58 (5.8 %) also contained the keyword "pachychoroid", while 116 (11.7 %) also contained the term "cnv". in contrast, 37 articles were found on "pnv". only one article referencing "csc with cnv" and "pnv" as an overlapping disease was found (0.8 %). reporting the outcomes of anti-vegf in neovascular non-aneurysmatic pachychoroid disease, 7 articles on "csc with cnv", and 6 articles on "pnv" were found. in the 669 articles on "pcv", only 37 (5.5 %) also contained the keyword "pachychoroid". in contrast, "at1" was referred to in 3 articles. of these, all 3 (100 %) also referenced "pcv". conclusion: there is a significant amount of overlapping studies reporting similar findings in pachychoroid disease using different names for the disorders involved. most importantly, this involves the seemingly interchangeable use of the terms "csc with cnv" and "pnv". to unify the terminology and to spur validating research on the idea that pachychoroid diseases of the macula represent a continuum, we recommend a new classification system as follows: rod function rescue after gene therapy with voretigene neparvovec: time window for intervention? stingl k. background: voretigene neparvovec is a gene therapeutic agent for treatment of retinal degenerations due to bi-allelic mutations in rpe65. rpe65 is part of the phototransduction cycle in the retinal pigment epithelium. the rescue of rods is the short-term therapy effect of voretigene neparvovec. in the long term it should stop the natural course of retinal degeneration leading to blindness. we analysed the prediction value of patient's age for the short-term rod functional rescue by voretigene neparvovec using three readouts of dark adapted retinal function. methods: six eyes of four patients (age 16, 21, 24 and 33 years) have been treated with voretigene neparvovec and followed-up for at least 1 month. the dark adapted threshold was assessed by the full-field stimulation threshold (fst) with chromatic stimuli. the local rod function was assessed by dark adapted chromatic perimetry (dacp) using a new protocol applying 36 test points (cyan and red) in the central 30° visual field. additionally, the local rod function was also evaluated objectively by scotopic chromatic pupil campimetry (cpc), a novel test of the local rod function based on pupil contraction analysis following local scotopic stimuli inside of the 30° central visual field. all tests have been performed at baseline and one month after the treatment. results: all three methods indicated no measurable rod function before and showed clear improvement in the two younger subjects, and small or no improvements in the older subjects. fst with blue light improved by up to 40 db one month after the treatment, the red-blue thresholds difference improved to normal values (25 db) in the youngest subject. corresponding to the treated area, dacp showed an improvement of the retinal sensitivity of up to 40 db. scotopic cpc in that area improved to up to 30 % of normal values indicating functional rescue of 30 % of rod population in the same area. in the oldest subject, readouts improved only minimally after the therapy. all three readouts at one month after the treatment correlated highly with age: r 2 = 0,84 for fst blue threshold; r 2 = 0,85 for the improvement of dacp hill of vision volume and r 2 = 0,6 for the maximal pupil constriction amplitude in the scotopic cpc. conclusions: voretigene neparvovec can restore local rod function, measurable already one month after the treatment. the presented data indicate that the age may be a valid prediction factor for the short term outcome of rod rescue. medical university of plovdiv, plovdiv, bulgaria introduction: age-related macular degeneration (amd) is the leading cause of visual impairment in individuals over the age of 55 years worldwide. wet (exudative, neovascular) amd represents only about 10 % of all amd patients, but it is responsible for 90 % of blindness secondary to amd. choroidal neovascularization (cnv) is the hallmark of neovascular amd. according to the localization of cnv by optical coherence tomography (oct) and oct-angiography (oct-a) it may be classified as cnv 1 (under retinal pigment epithelium-rpe), cnv 2 (above rpe), cnv 3-retinal angiomatous proliferation (rap), and cnv 4 (mixed, cnv 1+cnv 2 subtype). objectives: different subtypes of cnv related to amd were established in randomly selected patient population using oct-a. aims: to investigate the frequency distribution and morphological types of the different cnv secondary to wet amd using oct-a. method: a total of 43 eyes (43 consecutive patients, mean age 72 ± 6 years (range 59-87), 53.5 % females) with wet amd were enrolled in a prospective study. all patients underwent a complete ophthalmologic examination, including best-corrected visual acuity (bcva) using snellen charts, slit-lamp biomicroscopy, tonometry, fluorescein angiography (fa), and oct-a (cirrus hd-oct, angioplex, carl zeiss meditec, dublin, ca). written informed consent was obtained from all patients. results: all cnvs were confirmed by fa and oct-a. in 36 eyes (83.7 %) there was late leakage from an undetermined source on fa and a pathologic vascular complex under rpe on oct-a correlating with cnv 1. in 2 eyes (4.7 %) there was leakage with a classic pattern on fa and a neovascular membrane into the avascular part of retina on oct-a suspected for cnv 2. in 5 eyes (11.6 %) we found cnv 4 (mixed cnv). cnv 3 (rap) was not established in the examined group of patients. three different morphological subtypes of cnv were visualized-"medusa-like" in 18 eyes (41.8 %), "seafan-like" in 15 eyes (35.0 %), and cnv with indistinct form in the rest 10 eyes (23.2 %). the results in this study confirmed the higher incidence of cnv 1 against the other cnv subtypes which correlates with the cited frequency in the literature. in addition, "medusa-like" and "seafan-like" cnv present an active subtype, while cnv with an indistinct form-inactive subtype. oct-a is a reliable and appropriate technique for easily visualizing and localizing different subtypes of cnv in patients with wet amd. oct-a characteristics of neovascular membranes in wet amdpossible correlation to the prognosis vidinova c. n. 1* , dafina a. 1 a rare case of serous retinal detachment at the posterior pole after high dose intravenous steroids due to renal-graft rejection wiecha c. background: non-adherence (na) continues to be a major challenge in the real-life treatment with anti-vascular endothelial growth factor. however, there is still a gap of sufficient evidence related to the reasons for na in patients treated with intravitreal injection therapy (ivt). furthermore, there is no reliable measure, which can be used in clinical routine to detect potential barriers in time. the aim of this investigation was to develop a questionnaire (adherence barriers questionnaire-ivt: abq-ivt), based on an earlier version of the abq. the existing abq was discussed in an expert panel and revised according to specifications of ivt. initially, the abq-ivt consisted of 24 items formulated as statements (4-point-likert-scale ranging from "strongly agree" to "strongly disagree"). the abq-ivt was applied in a cross-sectional survey of german patients with neoavascular age-related macular degeneration (namd) and diabetic macular edema (dme). evaluation of the questionnaire included an assessment of internal consistency as well as factor analysis. the occurrence of potential barriers in the patient sample investigated was evaluated using descriptive statistics. results: 234 of 253 patients (92 %) were able to complete the abq-ivt. on the basis of the answers given and the analysis of their independence, a possibility was used to condense the questionnaire-without reducing its informative value. the final abq-ivt has been reduced within the reliability analysis to 17-items and demonstrated a good internal consistency (cronbach's alpha = 0.78). factor analysis showed no evidence for subscales of the questionnaire. nearly half of the patients (49 %) reported being affected by at least three different barriers. on average, a patient was affected by 3.1 barriers. most reported barriers were "challenge due to time commitment of physician visits" (45 % of the patients) followed by "depression" (29 %), "travel and opportunity costs" (27 %), and "burden for family members" (25 %). the prevalence of specific barriers differs by patient characteristics. the items "cost of treatment" and "too old for therapy to be worthwhile" were reported more frequently in older patients compared to younger. the abq-ivt is a practical and reliable instrument for identifying patient-specific barriers of adhering to ivt treatment. in practice, it might be useful to assess, whether individual patients are at higher risk of na due to specific adherence barriers. optical coherence tomography angiography in patients with optic nerve head drusen and anterior ischemic optic neuropathystructural and functional analysis problem: to evaluate the quantitative characteristics of the radial peripapillary capillary (rpc) network in the eyes with anterior ischemic optic neuropathy (aion) in association with optic nerve head drusen (onhd), and age matched healthy eyes using optical coherence tomography angiography (oct-a). to determine correlations between rpc density and structural and functional damage of the retinal nerve fiber layer (rnfl). methodology: 16 eyes (mean 32.3 ± 5.6 years) with aion caused by onhd (exposed drusen) were enrolled, and also 10 eyes from the same patients with onhd (buried drusen), but without aion. the control group contained 30 eyes (age matched mean 31.8 ± 2.7 years). foto fundus, fundus autofluorescence and goldmann visual field testing were done in all patients. also peripapillary rnfl and oct a images were acquired using the optovue oct (angiovue; optovue). standard 3.4 mm diameter circular scans were used to record disc parameters and average rnfl in 8 sectors, and 4.5 × 4.5 mm scans of the optic disc were used to record oct-a rpc density (%) in 8 sectors. correlations between main outcome parameters (sectoral rpc density and rnfl thickness, disc area size and visual field index) were determined and compared to the control group. results: both corresponding rpc and rnfl sectoral parameters were significantly lower in eyes affected by aion than in buried drusen group and control group (p < 0.05). there was no significant difference between these parameters in buried drusen group compared to control group. sectoral rpc density was strongly correlated with corresponding sectoral rnfl (p < 0.001) and also visual field index (p < 0.01). no significant correlation was found between disc area size and rpc and rnfl damage. conclusions: this study shows significant peripapillary microvascular damage in eyes with onhd and aion correlating with the same sectorial rnfl reduction and visual field defects. the same patients' eyes with onhd deeply buried without aion and rnfl defects had the same finding of rpc as the age matched control group. our data suggest that disc area size is not an indicative risk factor for aion associated with onhd. oct-a could be a useful method in the analysis of capillary density in patients with onhd. introduction: childhood optic nerve glioma (ong) is typically a slowgrowing tumor that can lead to blindness. the first six years of life constitute the greatest risk of developing the disease. treatment of ong is currently controversial, and involves simple observation, radiation, chemotherapy, and surgical excision. objectives: chemotherapy has emerged as the preferred treatment modality for childhood ong. however, no study has previously analyzed long-term sequelae of chemotherapeutic treatment in children aged 0-1 years-old compared to older children, which is the objective of this study. aims: we aim to investigate whether the use of chemotherapy alone in the treatment of childhood optic nerve glioma correlates with different overall survival (os) patterns in infants as compared to its use in treating older children. methods: a retrospective, epidemiological analysis of children with optic nerve glioma was conducted on patient data extracted from the surveillance, epidemiology, and end result (seer) registry. patients were divided into two groups: group 1 included patients 0-1 years old while group 2 included patients 2-15 years old. survival analysis was performed in patients treated with chemotherapy only. results: 580 ong cases were identified, of which 21 % were in group 1. 457 patients were white. female:male ratio was 1:1. introduction: carotid cavernous fistula (ccf) is an abnormal communication between the cavernous sinus and the carotid arterial system. ccfs are classified into 4 subtypes based on their communication. these patients may present with signs as conjunctival chemosis, proptosis,exophthalmos, diplopia, ophthalmoplegia, orbital pain etc. case report: a 70-year-old male patient, presented diplopia and proptosis. his history revealed that he had previously been treated for one month with unspecified drops, after which he was admitted to a local hospital for a month. during that admission, he was given retrobulbar and subconjunctival triamcinolone. the situation with eye improved and he was discharged. an orbital ct scan was performed while in hospital. a few days later, the situation worsened, he developed diplopia, a reddish eye with protrusion. on presentation to our clinical centre, he had bcva 1.0, was hypermetropic. iop re was 14 mmhg and le 35 mmhg. slit-lamp and fundus examination of the right eye showed normal findings, while on the left eye, showed episcleral congestion with corkscrew blood vessels, reddish eye, slightly oedema cornea. fundus showed normal onh with dilated retinal blood vessels. both angles were wide open on gonioscopy, re schaffer 3, and le schaffer 1. the right motility was normal and the left was limited in elevation and abduction (hesse lancaster). the optic nerve head and maculae were both normal on oct. a b scan ultrasound showed a vertical hypoechogenic space just behind the optic nerve in the left orbit. the patient was given timolol 0.5 % 2 ×, brimonidine 2 ×, acetazolamide tablets and tobramycin/dexamethasone drop. he then underwent ct angiography, as mri was not possible because of a metallic foreign body sustained during the war. ct angiography showed dilated blood vessels crossing over the optic nerve, without much clear detail. the patient was referred to a neurosurgeon and underwent digital subtraction angiography which showed a carotid-cavernous fistula requiring neurosurgical treatment. brimonidine and tobramycin/dexamethasone drops with acetazolamide tablets were continued while he awaits surgery. the eye is quiet, the proptosis and diplopia have resolved. conclusion: many patients with ccf may initially present to an ophthalmologist, who should be able to make a presumptive diagnosis in most cases. an ophthalmologist should properly refer the patient to a neurosurgeon. lhon-therapie mit idebenon -wann beginnen, wie lange therapieren? eine langzeitbeobachtung über 14 jahre schuart c. o augenklinik, universitätsklinikum dresden, dresden, germany aim: uveitis is associated with visual impairment and blindness. in western countries idiopathic inflammation is the most common cause. nonbiologic treatment for autoimmune/idiopathic uveitis is not based on published strong evidence. this study was to evaluate high-dose intravenous methylprednisolone (ivmp) treatment in patients with juvenile autoimmune/idiopathic uveitis. method: a retrospective chart review was conducted in two tertiary referral centres to investigate treatment response to ivmp in children and adolescents with autoimmune uveitis between 2003 and 2016. disease activity, outcomes, and additional treatments were documented at 0, 3 and 6 months after ivmp. purpose: to evaluate the effect of prism adaptation test (pat) on the angle of squint as well as eye muscle surgery dosage in decompensated microesotropia (dekmet) and decompensated esophoria (dekeph). methods: in this single-centre retrospective study we reviewed the medical records of all patients with the diagnosis of dekmet or dekeph, aged at least 12 years, who were treated by strabismus surgery for the first time between 2003 and 2019. the maximum angle of squint (aos), pat results before surgery, surgical dosing and aos one day postoperatively were considered. pat included wearing a prism based on the largest angle for over 60 min. results: 82 patients (mean age 28 ± 13years) were included in the dekmet group, 100 patients (mean age 37 ± 17years) in the dekeph group. for dekmet, before surgery aos was 28.8 ± 10.6 pdpt for far (f), 30.9 ± 11.8 pdpt for near fixation (n). during pat (30.17 ± 10.47 pdpt), the aos increased significantly by 4.1 ± 5.7 to 32.5 pdpt (f) and by 3.7 ± 6.1 to 34.4 pdpt (n). postoperatively, aos was reduced to 5.3 ± 4.8 pdpt (f) and 5.8 ± 5.7 pdpt (n) . for dekeph, before surgery aos was 25.5 ± 8.8 pdpt (f) and 23.5 ± 9.8 pdpt (n). during pat (25,1 ± 8.6 pdpt), the aos increased significantly by 2.7 ± 4.3 to 28.2 ± 8.6 pdpt (f) and by 4.9 ± 4.5 to 28.3 ± 9.5 pdpt (n). postoperatively, aos was reduced to 3.3 ± 3.5 pdpt (f), and 2.5 ± 4.3 pdpt (n). before pat there was a significant distance-near difference (dnd) in both groups which differed significantly between both groups: mean dnd before pat: dekmet -2.2 ± 6.4pdpt (aos(f) lower than (n)), dekeph +2.0 ± 5.9pdpt (aos(f) higher than (n)). after pat there was still a significant dnd in the dekmet group, but no longer in the dekeph group. considering only eyes with ametropia up to 6 dpt spherical equivalent, the mean dosage applied for combined medial and lateral rectus muscle surgery (referring to the mean of aos (f) and (n) after pat) was significantly different between the groups (dekmet 3.18 ± 0.45 pdpt/mm, de-keph 2.93 ± 0.42 pdpt/mm). the mean dose-effect was not significantly different (dekmet 2.70 ±0.56 pdpt/mm, dekeph 2.61 ±0.42 pdpt/mm). the aos before pat in patients with dekmet showed significant dnd with greater angle of squint at near fixation, in the dekeph group the angle of squint was significantly greater at far fixation. after pat, the difference persisted in dekmet with the near and far angle in-s158 abstracts 353 ,3 ± 2,5 und die normalgeborenen 9,4 ± 3,2 jahre alt. results: dvd improved significantly (p < 0.05) in the two groups of the study. in group i, the mean vertical deviation improved from 18.21 ± 4.73 prism diopter (pd) preoperatively to 7.82 ± 5.61 pd 9 months after surgery (p < 0.05), with a mean correction of vertical deviation of 10.21 ± 3.52 pd and a mean correction of asymmetry of 2.1 ± 1.6 pd. four patients needed inferior rectus tucking for residual or recurrent manifest dvd. in group ii, the mean vertical deviation improved from 17.97 ± 6.89 pd preoperatively to 6.97 ± 5.46 pd 9 months after surgery (p < 0.05), with a mean correction of vertical deviation of 11.34 ± 2.71 pd and a mean correction of asymmetry of 2.5 ± 1.3 pd. five patients needed inferior rectus retucking for residual manifest dvd. conclusion: inferior rectus tucking is as effective as superior rectus recession with posterior fixation sutures for the primary treatment of dvd without inferior oblique overaction. inferior rectus tucking can also be used effectively for the treatment of residual and recurrent dvd; further studies are recommended in this field. do extraocular muscle pulley bands or does retrobulbar fat keep the eye muscles in place, and thereby acts like a pulley? simonsz h. j. 1* extraocular muscle pulley bands were described by tenon in 1805 as "faisceaux tendineux", acting as "poulies de renvoi". sappey described the band's smooth muscle fibers in 1888. in tenon's, sappey's, miller's and demer's pulley concepts, this connective-tissue band between muscle and bony orbital rim limits vertical shift of a horizontal rectus muscle belly in up-and downgaze caused by the muscle's tendency to follow the shortest path from insertion to origin. it thereby redirects muscle force like a pulley. however, the band's attachment to the muscle moves 20 mm sagittally when looking from 50° left to 50° right, limiting its ability to stabilize the muscle vertically. the band should be elastic to stabilize the muscle vertically while permitting large horizontal eye movements. we measured it after orbital exenteration: it was slack but, once extended, very stiff. in 1984, our research group in amsterdam compared horizontal-rectusmuscle positions in up-gaze with those in down-gaze using ct and found no vertical shift. in primary gaze the muscle path was curved outwards, indicative of retrobulbar pressure resulting from rectus muscles pulling the eye into the orbit and contained by muscles and connective tissue sheets enveloping retrobulbar fat including the intermuscular membrane. the eye ball is held in place with the retrobulbar pressure. later, we repeated the ct study in a crouzon patient whose bony orbital rim was displaced 2 cm posteriorly. the connective tissue band could not attach to bone. nevertheless, no vertical shift of horizontal rectus muscles occurred when the patient looked up or down. the muscles were kept in place by the retrobulbar fat and its enveloping connective tissue sheets including the intermuscular membrane. this mechanism was simulated in a soft-tissue finite-element model of the orbit, the muscles, the fat and the eye ball moving with 6 degrees of freedom. although retrobulbar fat was assigned a low elasticity as found in vivo, it not only kept the eye ball in place, but also horizontal rectus muscle bellies in up-and down-gaze and vertical rectus muscle bellies in left-and right-gaze. the force keeping the muscle in place is delivered either by fat, connective tissue and orbital wall, or by the eye. in the former case, overall muscle force is redirected and pulley action occurs. if both force components contribute equally, listings' law is implemented naturally. "faisceaux tendineux" are likely to be check ligaments. visualisierung von intraokularen fremdkörpern im vorderen augenabschnitt mittels infraroter transillumination (pilotstudie) kogan the incidence of ocular melanoma in germany alfaar a. 1*, 2 , rehak m. 1 , saad a. 3 introduction: ocular melanoma is the most common ocular malignancy in adults. its reported age-adjusted incidence in usa is 5.1 per million. many european countries have reported incidence and showed an increase from 2 per million in south europe to more than 8 per million in northern countries. our aim is to assess the incidence of uveal melanoma in germany between 2009 and 2013. methods: data were collected from the german centre for cancer registry data. we have used the icd-o-3 topography codes c69.0 to c69.9 and histology codes 8720/3 to 8780/3 for mining and reporting the data. the tumors were limited to those with malignant behavior. we have compared the incidence between northern and southern states. german states of schleswig-holstein, hamburg, lower saxony, bremen, north rhine-westphalia, berlin, brandenburg, mecklenburg-west pomerania, and saxony-anhalt were grouped as northern while the states of hessen, rhineland-palatinate, baden-württemberg, bavaria, saarland, saxony, and thuringia were grouped as southern states. results: our study included 2966 patients diagnosed with melanoma including 2645 patients (89.18 %) with ocular melanoma and 232 with an orbital disease (7.82 %). the diagnosis was histologically confirmed in 73.5 % of the patients, and 3.5 % were registered from death certificates only. the die rnfl war in allen quadranten außer dem temporalen signifikant geringer bei frühgeborenen im vergleich zu normalgeborenen. folgende parameter der papille waren signifikant größer bei frühgeborenen im vergleich zu normalgeborenen: die exkavationsfläche (0,47 ± 0,48 vs. 0,29 ± 0,37 mm 2 , p = 0,036), das exkavationsvolumen (0,10 ± 0,15 vs. 0,04 ± 0,06 mm 3 , p = 0,012), der flächenquotient cdr (0,25 ± 0,25 vs. 0,14 ± 0,15, p = 0,009), die horizontale cdr (0,49 ± 0,31 vs. 0,38 ± 0,27, p = 0,041) und die vertikale cdr (0,44 ± 0,29 vs. 0,33 ± 0,24 mm, p = 0,034). die randsaumfläche war signifikant dünner bei den frühgeborenen (1,40 ± 0,62 vs. 1,73 ± 0,56 mm, p = 0,005). bei den frühgeborenen zeigte sich eine signifikant positive korrelation des geburtsgewichts und des gestationsalters mit dem superioren quadranten und der gesamtdicke der rnfl. eine negative korrelation fand sich mit der exkavationsfläche, dem exkavationsvolumen und der horizontalen cdr. frühgeborenenretinopathie hatte keinen einfluss, die neurologischen erkrankungen waren hingegen der stärkste prädiktive faktor für eine dünnere rnfl in allen quadranten außer dem temporalen. schlussfolgerung: aufgrund der dünneren rnfl haben die papillen bei frühgeborenen eine dünnere randsaumfläche und eine größere exkavation. insbesondere wird die rnfl durch die neurologischen begleiterkrankungen negativ beeinflusst. diese faktoren sollen berücksichtigt werden, bevor bei einem frühgeborenen kind die diagnose eines glaukoms gestellt wird. results: according to bett closed globe eye injury was observed in 72 cases (97.3 %), incl. eye contusion in 70 cases and lamellar corneal injury in 2 cases. in 3 cases (4.0 %) there was open globe injury (with corneal perforating injury and with lens and iris prolapse in one case). left eye was injuried in 64.5 %, right-35.5 %. the character of the injury and its incidence was the following: corneal erosion-5 cases (6,6 %), corneal edema-1 (1.3 %), retinal edema-10 (13.3 %), eye hypertension-19 (25.0 %), hyphema, accompanied by vitreous hemorrhage-29 cases (38.6 %), vitreous opacity-3 (4.0 %), subretinal hemorrhage-20 (26.6 %). in 6 cases macular hole was observed, in 3 cases-retinal detachment. iris changes was in 19 cases (incl. aniridia in 1 case). in 16 cases (21.3 %) va was light perception; in 19 cases (25.3 %)-0,01-0,06; in 20 cases (26.6 %)-0,1-0,5 and in 20 cases (26.6 %)-0,6-1,0. in 24 cases (32.0 %) surgery was necessary and was performed. conclusion: bottles of sparkling wine can cause severe ocular trauma due to the high-impact energy. so, there is evident the necessity of educational work and protective measures among population for the prophylaxis of such eye trauma. the real rate of eye injury due to cork of sparkling wine supposed to be much higher. the bottle must be properly handled (although it is not the guarantee for trauma avoidance). it is advisable to avoid the bottle to undue heat and agitation and to direct cork away from the face when opening the bottle. all bottles of sparkling wine must have warning labels. the consumer can reduce the risk of eye trauma by keeping bottles in a cool place and not to shake it before use. introduction: retinoblastoma (rb) is the most common intraocular pediatric malignancy. many studies have analyzed its epidemiological trends and have shown varying results over the years. objective: the purpose of our study is to analyze the most recent epidemiological trends of retinoblastoma. aims: to analyze the epidemiological trends of retinoblastoma during the years 2009 to 2016 methodology: a retrospective, population-based analysis was conducted on children younger than 5 years of age. data were extracted from the surveillance, epidemiology, and end results registry from 2009 to 2016. incidence data from 2001-2008 were accessed for comparative purposes relative to results from the timeframe of our study. all incidence (ir) data were estimated as cases per million. incidence rate ratios (irr) were calculated in comparison to the entity with the highest value to determine statistical significance. results: 571 cases were identified. 52 % were females. the overall ir was 12.6. incidence rates in male and female subgroups were not statistically different (11.9 and 13.4 per million, respectively [p = 0.17]). ir was highest in black patients (13.9), but was not significantly different from other races; irr were: non-hispanic white (0.88, p = 0.35), asian/pacific islander (0.88, p = 0.53), and hispanic (0.9, p = 0.44). ir was significantly highest in the 1st year of life (29.5) and irr for presentation at the 2nd, 3rd, 4th, and 5th year of lives were: 0.48 (p < 0.001), 0.44 (p < 0.001), 0.17 (p < 0.001), and 0.06 (p < 0.001), respectively. 69 % of patients presented with unilateral (ul) disease. ul disease incidence was significantly higher than that of bilateral (bl) disease (8.7 and 3.9, respectively p < 0.001 epidemiological analysis and comparison of sporadic and inherited retinoblastoma, 2000-2016 mikhael s. 1* , tadrosse a. 2 , yassa a. 2 , mikhael m. 2, 3 , eloy j. a. 2,4,5 1 lewis (8.8) was significantly higher than that of bl (3.9) [irr 2.29; p < 0.001]. no change was noted in ul (apc -0.2; p = 0.6) or bl (apc 0.5; p = 0.6) ir from 2000 to 2016. stratification by sex showed no difference in ir within either ul or bl. in the ul and bl, there was no statistical significance in ir between non-hispanic white, black, pacific islander or hispanic groups. ir of ul cases were significantly higher than those of bl cases across all races. in the ul, 32 % of cases were diagnosed during the 1st year of life, overall age-adjusted incidence of ocular melanoma was 5 per million. generally, the incidence of ocular melanoma was higher in males than females consistently across all age groups except the group 45-49 years old. the crude incidence in males reached a peak in the age group 70-74 while the peak appeared between 80-84 in females. about 56 % of the patients were diagnosed at the age of 60 years or older. the choroid was the most common site for intra-ocular melanomas (86.65 %, n = 2292) followed by the ciliary body (12.85 %, n = 340). on the other hand, the conjunctiva was the most common site for orbital tumors (90.95 % of orbital melanomas, n = 211) followed by orbital adnexa (7.76 %, n = 18). northern states showed higher age-adjusted per/year incidence rates (6.2 per million) in comparison to southern states (3.9 per million). conclusion: the incidence of ocular melanoma followed the european rates and patterns. further studies are required for studying the trends, and the risk factors. kiefer t. 1* , schlüter s. 1 , bornfeld n. 1 in 20 % der fälle konnte der befund mittels einer lokalen therapie (brachytherapie, laser-und oder kryokoagulation) konsolidiert werden. in 35 % der fälle konnte der bulbus mittels iac, protonentherapie oder systemischer chemotherapie erhalten werden. 6 augen (30 %) mussten im mittel nach 15 monaten (0,5-47 monate) enukleiert werden. hiervon waren jedoch nur in 2 fällen die durch ivc behandelten tumore der grund der enukleation. diskussion: in unserem patientenkollektiv zeigte sich ein geringfügiges ansprechen der soliden tumore oder peripheren rezidive auf ivc. in vielen fällen konnte allerdings im weiteren verlauf nach ivc eine konsolidierende therapie mit bulbuserhalt durchgeführt werden. über die limitierten therapiemöglichkeiten der ivc bei soliden tumoren und die potentiell toxischen nebenwirkungen sind die betroffenen familien aufzuklären. analysis of the epidemiological trends of retinoblastoma during the years 2009 to 2016 mikhael s. 1* , tadrosse a. 2 , yassa a. 2 , mikhael m. 2, 3 , eloy j. a. 2,4,5 1 lewis objectives: the expediency of surgical removal of uveal melanoma (um) after its irradiation by various methods, including gamma knife radiosurgery (gkrs), remains debatable. unresolved questions are the indications and timing for um resection. aims: we analyzed the results of eye-conserving treatment of um after gkrs to determine the indications for um resection, as well as the optimal timing of surgery. methods: 68 patients (29 male, 39 female) between 28 and 79 years of age were treated using gkrs with a dose of 60-70 gy. 37 patients (54.4 %) with large um (t3n0m0 in 96.7 % of cases) and widespread secondary retinal detachment (srd) with macula involvement underwent tumor resection after gkrs (transscleral resection-7, endoresection-30). the remaining 31 patients (45.6 %) with medium-sized um (t2n0m0 in 71 % of cases) and local srd without macula involvement mainly were treated without surgery. patients of both groups received local adjuvant therapy (lat) -angiogenesis inhibitors and triamcinolone. the timing of operations after gkrs ranged from 3 days to 6 months. last years resection was performed only if there was no effect from lat for 3-6 months. follow-up period ranged from 6 to 86 months. results: in the group with um resection, enucleation was performed in one patient (2.7 %) due to tumor recurrence 35 months after surgery. 6 in which ir (14.3) was significantly higher than that in the 2nd, 4th and 5th years of life (p = 0.04, <0.001, and <0.001, respectively). in bl, 69 % of cases were diagnosed during the 1st year of life; ir (13.4) was significantly higher than that during the 2nd, 3rd, 4th, and 5th years of life (p < 0.001). the ir of ul cases were significantly higher than that of bl during all other age groups except during the 1st year of life. mean survival was not statistically different during 2000-2011: 114.5 months (ul) and 116.0 (bl). survival was also not significantly different between both groups based on race, age and sex. regression analysis showed that laterality was not a predictor for survival (hr: 1.497; p = 0.479). the incidence of sporadic cases of retinoblastoma is greater than that of hereditary ones, but similar during the 1st year of life. incidence is not affected by either sex or race. incidence during the 1st year of life is higher than that during the 2nd, 4th, and 5th years of life in sporadic cases and higher than all other age groups in inherited ones. sporadic and inherited cases of rb share comparable survival patterns. einfluss von papillendosis und bestrahlter optikuslänge bei protonentherapie choroidaler melanome riechardt a. i. 1* , stroux a. 2 mikhael s. 1* , tadrosse a. 2 , yassa a. 2 , mikhael m. 2, 3 , eloy j. a. 2,4,5 1 lewis introduction: uveal melanoma is the most common intraocular malignancy in adults. objectives: the purpose of this study is to analyze epidemiological trends in incidence and survival of uveal melanoma specifically arising from the ciliary body. aims: to identify epidemiological and survival trends of ciliary melanoma. methods: a retrospective, population-based analysis was conducted using the surveillance, epidemiology, and end results registry from 1973-2008. all incidence (ir) data were estimated in cases/million/year. incidence rate ratios (irr) were calculated in comparison to the entity with the highest value to determine statistical significance. treatment data were available only post year 1998. results: 743 cases of ciliary melanoma (cm) were identified. overall ir of cm was 0.92. 98 % of cases were white. ir for the white subgroup (1.08) was significantly higher than that for the black group (0.82) [irr: 0.038, p < 0.001]. ir was also significantly higher in males (1.04) than in females (0.82) [irr 0.79, p < 0.00]. patients aged 61-80 years had an ir of 3.22, which is statistically higher than that of 0-20, 21-40, and 41-60 years intervals [irr = 0.022 (p < 0.001), 0.09 (p < 0.001), and 0.039 (p < 0.001), respectively], but not the 81-100 years interval (p = 0.22). average ir during the years 1973-1990 (1.2) was significantly higher than that for the years 1991-2008 (0.68) [irr = 0.57; p < 0.001]. annual percent change (-3.19 ) over the time frame of the study displays a significant decrease in incidence (p < 0.001). 10-year overall survival (os) in 1973-83 (63 %) was significantly higher than that in 1996-2006 (54.1 %) [p = 0.048]. 30.6 % of cm cases received radiotherapy treatment and 65.8 % underwent surgery. 10-yr os in patients with localized malignancy who received only radiotherapy (60.5 %) was similar to that of those who were treated with surgery alone (57.8 %) [p = 0.508]. conclusion: the incidence of ciliary melanoma is highest in the white, male, and elderly population. although overall incidence is decreasing, overall survival patterns are declining, which requires further studies to pinpoint the underlying cause. radiotherapy does not exhibit survival advantage over surgery in the management of localized disease, suggesting that long-term sequelae of radiotherapy can be avoided with tumor excision, while maintaining similar survival rates. okkultes basalzellkarzinom innerhalb einer seborrhoischen keratose des augenlids: ein klinisch-pathologischer fallbericht nüßle s. beutel m. 4 , schmidtmann i. 5 augenzentrum am st franziskus hospital hautklinik -experimentelle dermatologie und immunologie der haut abteilung für nephrologie, immunologie und osteologie am st. franziskus hospital schmidtmann i. 10 , beutel m. 11 die konversion eines retinalen venenverschlusses (rvo) in ein neovaskuläres glaukom (nvg) ist eine visusbedrohende komplikation. unser ziel war es, die umwandlungsrate von rvo in nvg innerhalb einer klinik für augenheilkunde/kath. krankenhaus hagen, hagen, deutschland abstracts anzahl über zwei jahre erreichen. dabei konnten für 1/3 der patienten die injektionsintervalle verlängert werden. wir halten unser frühes te-schema für geeignet im anschluss kam ein 4-punkte-evaluationsbogen unter verwendung von "visual analogue scales" (bereich: 0-100) zum einsatz. sämtliche nachfolgenden ergebnisse sind als median/interquartilsabstand (iqr) dargestellt. ergebnisse: im rahmen dieser pilotstudie wurden 5 versuchspersonen im alter von 21/9 j. untersucht. die sehschärfe der untersuchten augen lag bei 1,7/0,8. schlussfolgerung: für deutlich überschwellige optotypen lagen sowohl die validität, als auch die retest-reliabilität aller eingabemethoden hoch. die untersuchungsdauer war bei sprachlicher rückmeldung mehr als 1 untersuchungen mit höherer fallzahl sind nötig lauermann p. 1* , gebest j. 1 , pfeiffer s. 2 , feltgen n. 1 , hoerauf h. 1 ausschlusskriterien waren faktoren, von denen bekannt war, dass sie den iod beeinflussen, wie z. b. die kataraktoperation während der nachsorge, die verlängerte anwendung von steroiden, die kryotherapie und die silikonöl-endotamponade. als primärer endpunkt wurde die relative veränderung des iod (operiertes auge im vergleich zum nachbarauge) [6] [7] [8] [9] [10] [11] [12] monate nach der operation definiert. sekundäre endpunkte waren die relative änderung des iod nach 3-6 und 12-24 monaten. mögliche beeinflussende kofaktoren wurden mittels ancova analysiert. ergebnisse: der primäre endpunkt zeigte keine signifikante iod-reduktion des operierten auges im vergleich zum nachbarauge (p = 0,089, n = 84). allerdings war der iod des operierten auges allein nach 6-12 und 12-24 monaten nach der operation signifikant reduziert (-0,75 ± 2,80 und -1,22 ± 3,29 mmhg, p = 0,008 bzw. 0,007). der augeninnendruck des nachbarauges war nach 12-24 monaten ebenfalls signifikant reduziert (-0,75 ± 2,73 mmhg, p = 0,008). in der subgruppenanalyse war die größe der vitrektomie ein signifikant beeinflussender kofaktor, der zu einem niedrigeren iod nach 20g im vergleich zur 23g-vitrektomie führte (p = 0,04). zusammenfassung: die pars plana vitrektomie bewirkte keine signifikante langfristige iod-reduktion im vergleich zum kontralateralen auge. wir beobachteten jedoch ein iod-senkungspotenzial bei der 20g-vitrektomie. analyse der choriokapillären flussdichte bei patienten mit systemischem lupus erythematodes -eine optische kohärenztomographie-angiographie-studie leclaire m. d. 1* , mihailovic n. 1 , brücher v. c. 1 , eter n. 1 1 universitätsaugenklinik münster, münster, deutschland hintergrund/ziel: kollagenosen wie der systemische lupus erythematodes (sle) können einfluss auf die retinale durchblutung haben. immunofluoreszenz-und elektronenmikroskopisch konnten einlagerungen von immunkomplexen in der choroidea von sle-patienten demonstriert werden. ferner zeigten sich veränderungen der choriokapillaris bei sle-patienten in der konventionellen kohärenztomographie. ziel der vorliegenden studie war die quantitative analyse der retinalen flussdichte (fd) gemessen mittels optischer kohärenztomographie angiographie (oct-a) bei patienten mit sle unter hydroxychloroquin (hcq)-therapie unter besonderer berücksichtigung der choriokapillaris. methoden: es erfolgte eine messung der fd im oberflächlichen und tiefen kapillären plexus sowie der choriokapillaris im makulären 3 × 3mm 2 oct-angiogramm (rt vue xravanti, optovue inc., fremont, kalifornien, usa) bei patienten mit sle (n = 19) und bei gesunden, alters-und geschlechtskorrelierten probanden (n = 19). nur patienten ohne hinweis für eine hcq-induzierte retinopathie wurden eingeschlossen. die sle-patienten wurden in eine hochrisiko-(hcq-therapiedauer >5 jahre) und eine niedrigrisikogruppe (hcq-therapiedauer < 5 jahre) eingeteilt. korrelationskoeffizienten in hinblick auf die fd und die kumulative dosis hcq wurden berechnet. ergebnisse: das mittlere alter der patienten lag bei 40,1 ± 11,5 jahren, das alter der gesunden kontrollen bei 38,2 ± 12,6 jahren (p = 0,63). in beiden inhaltet die gruppe der w12-responder alle patienten, die innerhalb von 12 wochen auf die standardtherapie reagiert haben, d. h. dass erstmals kein makulaödem im oct entweder zu visite 3, 4 oder 5 nachweisbar war und bis visite 5 auch kein rezidiv dokumentiert war. insgesamt zählen 17 patienten (51,5 %) zu den w12-respondern und 13 patienten (39,4 %) zu den w12-non-respondern und 3 patienten (9,1 %) sind vor visite 5 aus der studie ausgeschieden. bezüglich des primären endpunktes sind somit n = 30 patienten auswertbar. betrachtet man die baselinewerte der einzelnen studienpatienten, so zeigt sich eine deutliche streuungsbreite. der mittelwert liegt bei 226 pg/ml. insgesamt zeigt die gruppe der overall-responder deutlich niedrigere serum-vegf werte als die gruppe der non-responder. was auch, berücksichtigt man die pathophysiologie, zu erwarten war. schlussfolgerung: primäres studienziel war die frage, ob das erneute auftreten eines makulaödems innerhalb von 3 monaten bzw. eine erhöhung eines vorbestehenden makulaödems durch die bestimmung des serum vegf-wertes vorausgesagt werden könnte. hierbei konnten keine signifikanten unterschiede zwischen den respondern und non-respondern in der relativen vegf veränderung festgestellt werden. oct veränderungen der neurosensorischen netzhaut bei zerebraler mikroangiopathie langner s. m. 1* , terheyden j. h. 1 , geerling c. f. 1 , kindler c. 2, 3 , keil v. c. 4 , turski c. 1 , turski g. n. 1 , wintergerst m. w. m. 1 , holz f. g. 1 , gabor p. 2, 3 , finger r. p. 1 1 universitäts-augenklinik bonn, bonn, deutschland; 2 klinik für neurologie, universitätsklinikum bonn, bonn, deutschland; 3 deutsches zentrum für neurodegenerative erkrankungen (dzne), bonn, deutschland; 4 klinik für radiologie, universitätsklinikum bonn, bonn, deutschland fragestellung: zerebrale mikroangiopathien (zma) sind ein risikofaktor für neurodegenerative erkrankungen. bei letzteren wurden bereits retinale veränderungen in der optischen kohärenztomographie (oct) gezeigt, für die zma gibt es hierzu bislang jedoch keine daten. daher haben wir strukturelle retinale veränderungen bei probanden mit zma untersucht. methodik: 62 probanden wurden rekrutiert. ausschlusskriterien umfassten ophthalmologische vorerkrankungen und höhere myopie (< -6 dpt). bei allen probanden wurden eine cmrt, eine klinisch-ophthalmologische untersuchung, ein montreal cognitive assessment (moca) test, und ein oct-volumen-scan der makula sowie ein bmo-scan der papille (spectralis, heidelberg engineering) durchgeführt. die einzelnen scans wurden von der software des geräteherstellers vorsegmentiert und manuell korrigiert. probanden mit zma wurden statistisch mit gleichaltrigen kontrollen verglichen. die auswertung erfolgte mit der software spss 26 (chicago, il, usa). ergebnis: 36 probanden (21 weiblich, 15 männlich; mittleres alter 63 ± 10 jahre) wurden in die auswertung eingeschlossen. der vergleich von neun probanden mit zma und neun altersangepassten kontrollen (mittleres alter jeweils 57 jahre) ergab einen signifikanten unterschied des ganglienzellschichtvolumens (gcl, p = 0,008). in einer regressionsanalyse der 27 eingeschlossenen probanden mit zma waren die volumina der äußeren nukleären (onl, p = 0,034) und äußeren plexiformen schicht (opl, p = 0,035), das gesamte retinale volumen (p = 0,049), sowie anteile der retinalen nervenfaserschichtdicken (rnfl: papillomakuläres bündel, p = 0,007, temporal superiore, p = 0,03, temporal inferior, p = 0,011 und nasale anteile, p = 0,045) signifikant mit der läsionslast in der mrt assoziiert. schlussfolgerung: unsere studie zeigt erstmals sowohl veränderungen der neurosensorischen netzhaut in der oct bei zma gegenüber altersgleichen gesunden als auch veränderungen bei erkrankten, die mit der läsionslast in der mrt korrelieren. insbesondere die parameter opl, onl, gcl und rnfl sollten in prospektiven folgestudien weiter hinsichtlich ihrer eignung zur diagnose oder verlaufsbeurteilung bei der zma untersucht werden. neurovascular dysfunction in diabetes mellitus: the relationship between different stages of diabetic retinopathy and diabetic optic neuropathy karliychuk m. 1* , bezditko p. 2 , pinchuk s. 3 1 bukovinian state medical university, chernivtsi, ukraine; 2 kharkiv national medical university, kharkiv, ukraine; 3 eye microsurgery center ‚vash zir' , chernivtsi, ukrainediabetes mellitus (dm) causes neurodegenerative changes, and retinal neurodegeneration can be found in early stages of dm, even before the development of clinically detectable microvascular damage. the objective was to analyze the relationship between different stages of diabetic retinopathy (dr) and different types and stages of diabetic optical neuropathy (don). methods: a total of 575 patients (1150 eyes) aged 55,9 ± 7.8 years with type 2 dm were analyzed. in addition to routine eye examination, optical coherent tomography of the retina and optic nerve was performed. subclinical stage of chronic axial don was found in 50.2 % of eyes (231 eyes); initial stage-in 20.7 % of eyes (95 eyes); advanced stage-in 19.8 % (91 eyes); dystrophic stage-in 9.3 % of eyes (43 eyes). diabetic anterior ischemic neuropathy was found in 24 eyes (2.1 %), diabetic papillopathyin 6 eyes (0.05 %) of patients. results: frequency of dr in initial stage of axial don was 2.8 times, in advanced stage-6.2 times, in dystrophic stage-7.2 times, in anterior ischemic don and in diabetic papillopathy-6 times higher than in subclinical stage of don (p < 0,05). in absence of don dr was not detected. the difference in frequency of different stages of dr, depending on type and stage of don was noticed. the incidence of nonproliferative form of dr in initial stage of don was found to be 2.8 times, in advanced stage-3.3 times, in dystrophic stage-1.7 times, in anterior ischemic don-3.3 times, in diabetic papillopathy-3.6 times more often than in subclinical stage (p < 0.001). preproliferative and proliferative stages of dr were found only in advanced and dystrophic stages of axial don, and in anterior ischemic don and diabetic papillopathy, most often-in dystrophic stage of axial don. so, there was a direct correlation between the stage of dr and the type and stage of don (r = 0.72, p < 0.001). the frequency of preproliferative dr in advanced stage of axial don was 1.8 times, in anterior ischemic don-1.9 times, in diabetic papillopathy-2.4 times less than in dystrophic stage of axial don. the frequency of proliferative dr in advanced stage of axial don was 2 times, with ischemic don and diabetic papillopathy-2.2 times less than in dystrophic stage of axial don. so, in dm there was a direct correlation between the frequency and form of dr and the type and stage of don that confirms the close relationship between neuronal and vascular dysfunction in dm. vergleich subjektiver visusangaben mit elektrophysiologischen verfahren zur visusevaluation kitsche m. sakkadenmessung bei phenylketonurie -sinnvoll oder nicht? hopf s. 1* , nowak c. 2 , hennermann j. b. 3 , schmidtmann i. 4 , pfeiffer n. 1 mehmed b. 1* , fronius m. 1 , pohl t. 1 , schramm c. 2 , spieth b. 2 inferior rectus tucking versus combined superior rectus recession with posterior fixation suture (faden) for the treatment of dissociated vertical deviation without inferior oblique overaction milisic s. tumor recurrence was observed in 2 patients (6.4 %) in the group without um resection. 2 other patients (6.4 %) died from metastatic disease 12 and 15 months after gkrs. the degree of vision loss was more pronounced in the group with um resection. the highest rate of subretinal fluid resorption and regression of even large um was observed in patients with initially local srd on the background of regular lat without resection. the expediency of um resection in the absence of metastases should be determined 3-6 months after gkrs on the background of regular lat. complete regression of large um without resection is possible. the indication for um resection is not the size of the tumor only (t3n0m0), but a combination of several characteristics: lack of regression of the um with preservation of permanent secondary retinal detachment involving the macula after regular lat or the development of ng. the incidence and survival of adult orbital tumors in germany alfaar a. 1*, 2 , rehak m. 1 , hassan w. 3 , mehanna m. 3 , saad a. 4 , jansen l. 5 der ophthalmologe · suppl 2 · 2020 s165 conclusions: socioeconomic factors play an undeniable role in the disease management process for patients diagnosed with sebaceous adenocarcinoma of the eyelid. our results show that married patients tend to enjoy higher survival rates than unmarried ones. marriage or social union may translate into better compliance to treatments and a greater capability to endure the challenges brought forth by the disease. higher income levels, likewise, have been shown to correlate with better survival patterns. socioeconomic considerations should be a crucial aspect of the treatment process in caring for patients with eyelid sebaceous adenocarcinoma. wolf j. 1* , schlecht a. 1 lgl and acc patients, respectively. incidence was significantly higher for lgl than acc (0.33 and 0.06, respectively [p < 0.001]). acc incidence was highest in the black (bl) and lowest in the american indian (ai) subgroups (0.13 and 0.01, respectively [p = 0.001]); incidence for the non-hispanic white (w) and pacific islander (pi) groups were 0.05 and 0.11, respectively. the difference between racial groups with the highest and lowest incidence for lgl was not significant, 0.33 (w) and 0.27 (pi), respectively (p = 0.944); the ai and bl groups showed an incidence of 0.37 and 0.32, respectively. average incidence of lgl based on geographical location was highest in alaska (4.00); the second highest incidence was in california (0.68). alaska showed an lgl incidence spike of 24 cases/million in 2011. acc, contrarily, did not show varying regional incidence. 5-year overall survival was statistically higher for lgl than acc (86.2 and 66.8 %, respectively [p = 0.013]). sex, race, and disease stage did not impact survival for either lgl or acc patients. conclusions: females represent most lgl and acc cases. lgl incidence is significantly higher than that of acc. while acc incidence varies by race, lgl's is influenced by geography. alaska witnessed an lgl incidence spike in 2011, which accounts for its overall higher average incidence compared to other regions studied. overall survival in lgl is higher than that in acc.lgl survival considerably improved compared to that documented in previous studies, while acc survival remains similar to recently reported values. introduction: sebaceous adenocarcinoma of the eyelid is a slow-growing tumor. although many studies have analyzed its epidemiology and pathogenesis, no study has investigated the impact of socioeconomic disparities on its prognosis. objectives: to provide the first analysis on the effect of socioeconomic disparities in the management of sebaceous adenocarcinoma of the eyelid. aims: to provide insight on the impact of marital status and income level on survival rates of sebaceous adenocarcinoma of the eyelid using a population-based study. methods: a retrospective, epidemiological analysis of patients with sebaceous adenocarcinoma of the eyelid was conducted on patient data extracted from the surveillance, epidemiology, and end result (seer) registry from 1975 to 2016. survival analysis was performed using the kaplan-meier method. we performed data analysis on 1102 cases of sebaceous adenocarcinoma of the eyelid. 42.8 % were married, 8.7 % single, and 48.5 % separated/divorced/widowed (sdw). median survival in months were: 137 (married), 108 (single), and 83 (sdw). 10-year overall survival patterns based on marital status were: 56.5 % (married), 47.3 % (single), and 32.9 % (sdw). married patients showed significantly higher survival than sdw patients (p < 0.001). univariate cox regression analysis showed that the married group had a significantly lower hazard ratio than the widowed group (0.47; 95 % ci: 0.385-0.576 %; p < 0.001). 10-year survival in patients with median annual income between $ 50,000-$ 75,000 was significantly higher than that of patients with $ 20,000-$ 50,000 income (37.8 and 31.5 %, respectively [p = 0.048]). in der spa-gruppe war s100a9 gegenüber der jiau-gruppe erhöht (medium, lps, fsl-1, pha; p < 0,05). die t-zell-proliferation unterschied sich nicht signifikant zwischen den gruppen. schlussfolgerungen: die pbmc der jia-und spa-assoziierten uveitispatienten unterschieden sich hinsichtlich der zytokinmuster nach den unterschiedlichen tlr-stimulationen. die jiau-gruppe war durch erhöhte arginase-1-, aber niedrige il-6-und tnf-a-konzentration gekennzeichnet, während die spa-gruppe durch eine höhere s100a9-freisetzung charakterisiert war. anti-citrullinated protein/peptide antibodies in convalescence stage may be a marker of autoimmune uveitis panchenko m. 1* , shevchenko n. 2 single studies showed the existence of netosis in cytokine-induced ocular inflammation in a mouse model and in patients with behcet's disease. (barliya t et al., 2017; perazzio sf et al., 2017; safi r et al., 2018) . according to research data, the serum levels of myeloperoxidase (mpo)-dna complexes (net remnants) in patients with rheumatoid arthritis correlates with the level of anti-citrullinated protein/peptide antibodies. (wang w et al., 2018) . the aim of the work was to study serum levels of anti-citrullinated protein/ peptide antibodies in patients with uveitis. methodology: 39 patients (48 eyes) with idiopathic posterior, intermediate and panuveitis were examined and treated. 16 men and 23 women in the age group from 5 to 68 years were included in the study. the duration of the disease ranged from 3 months to 12 years. the first attack of uveitis was diagnosed in 15 patients, the chronic uveitis was in 24 patients. all the patients underwent standard ophthalmic examinations, including ultrasound biomicroscopy and optical coherence tomography. serum levels of anti-citrullinated protein/peptide antibodies were defined with the help of the enzyme immunoassay using a standard reagent kit. the control group included the serum of 25 healthy donors. results: high serum levels of anti-citrullinated protein/peptide antibodies were identified in 4 patients (26.7 %) in the active stage of first attack of uveitis and in 10 patients (41.7 %; p > 0.05) with chronic uveitis. anti-citrullinated protein/peptide antibodies were detected in the blood serum of 6.6 % of patients in the stage of convalescence of uveitis first attack and in 37.5 % of patients (p < 0.05) with chronic uveitis. conclusions: serum anti-citrullinated protein/peptide antibodies in patients with uveitis in the stage of convalescence may be a marker of the autoimmune process. the problem: macular edema (me) during anterior uveitis (au) reduces visual function, leads to dystrophic changes. currently, its pathogenesis has been little studied. objective: to study the frequency of me and the expression features of the icam-1 and cd-95 marker in patients with uncomplicated and complicated au. methodology: 104 patients with au were examined. 23 persons had the primary process (5-90 days) and 81-had chronic recurrent au (215-9490 days). a control group-27 healthy volunteers. the absolute (cell/µl) and relative (%) level of expression on the venous blood lymphocytes of the icam-1intercellular adhesion molecules marker, which is considered a functional inflammation biomarker, and the apoptosis marker cd-95 using monoclonal antibodies by the histoimmunocytochemical method were determined. results: monocular au proceeded without complications in 61.6 %, and binocular in 43.2 % (χ 2 = 4.8;p = 0.027). me (diffuse or cystic), which during the au in only one eye was found in 15 % cases, during au in both eyes-in 28.4 % (χ 2 = 3.6;p = 0.05) cases. the absolute level of icam-1 me = 458; q l-u (356-517) cells/µl with uncomplicated au, and in patients with me-me = 617; (580-817) cells/µl, which is 34.7 % higher (χ 2 = 7.9; p = 0.004). in the control group, the expression of icam-1 me = 113.3; q l-u (87-168) cells/µl. so during au, icam-1 is 4-5.5 times higher (p < 0.05). the relative index of icam-1 during au is 26 ± 1.2 %, during au with me is higher by 15.4 % (p = 0.03)-equal to 29.9 ± 1.8 %. in the control group, the relative amount of icam-1 is 8.5 ± 0.3 %, which is lower than during au in 3.1 and with complication of au-3.5 times (p < 0.05). absolute level of apoptosis marker cd-95 for au was me = 375; q l-u (303-477) cells/ µl, and for au with me-me = 426; q l-u (363-585) cells/µl (p = 0.4), which does not differ and 3.3 times higher (p < 0.05) than in the control group-me = 120; q l-u (87.6-226.6) cells/µl. the relative level of cd-95 during au is 24.3 ± 1.1 % and during au with me is below 21.5 ± 1.9 % (p = 0.07). in the control group, cd-95 is 23.1 ± 0.8 %. daniel m. application of the pattern electroretinogram in evaluation of the risk of myopia progression grudzińska e. introduction: myopia is an important social problem due to the significant increase in its incidence. myopia, especially of high degree, is associated with the risk of vision-threatening complications such as myopic macular degeneration, retinal detachment, cataract or glaucoma. the current state of knowledge does not allow to determine the early indicators of complications in people without degenerative changes at the eye fundus. the aim of the study was to assess whether the perg may be an independent, objective risk indicator for myopia progression. material and methods: 32 eyes of 17 patients aged 29.4 ± 4.8 years with myopia of medium degree were qualified to the study. the spherical equivalent of the refractive error was -4.54 ± 0.8d (range -3d--6d). the control group consisted of healthy individuals aged 20-40 years with refractive error ±1d. the following examinations were performed in the study and control group: an interview (in subjects with myopia including known risk factors of myopia onset and progression), assessment of the visual acuity, intraocular pressure, refractive error, assessment of anterior and posterior segment of the eye in a slit lamp, measurement of the axial length, structure and thickness of the macula and optic nerve disc in oct and perg. results: in the group with myopia, the spherical equivalent of refractive error was significantly lower than in the control group. the values of p50 and n95 wave amplitudes did not differ significantly between the groups. however, significant differences were observed in the p50 peak time. it was significantly longer in myopia than in the control group. the analysis of myopia progression risk factors showed a statistically significant positive correlation between the number of hours of physical activity per week and the amplitude of p50 wave. there was also a statistically significant positive correlation between the peak time of p50 wave and the amount of time spent on near work. conclusions: it is very possible that the perg may be an early prognostic marker in identifying those patients with myopia who will develop ophthalmic complications in the future. the assessment of p50 peak time, which correlates with the best known risk factor of myopia progression which is near work, seems particularly valuable. on the other hand, the increase of p50 wave amplitude may indicate a protective role of physical activity in myopia progression. analyse des stellenwertes von "elearning" in der augenheilkunde und evaluierung einer "elearning-app" grabowski e. published continously up to now. in 1852 he founded famous private eye clinic in berlin, where he treated many eye patients and educated many prominent ophthalmologists. at the age of 29 he became associate professor of ophthalmology, the first with such a title in germany. conclusion: albrecht von graefe was founder of modern ophthalmology and separated it from surgery. graefe's contacts, correspondency and meetings at ophthalmological congresses with his teachers, assistants, colleagues also contributed to international co-operation and internationalization in ophthalmology. although albrecht von graefe died before 150 years, he still provokes great admiration and respect in the world of ophthalmology. digitale weitwinkelfotografie bei der früherkennung der diabetischen retinopathie kaya s. ophthalmology service, makarska, croatiaobjective: a short review of the work of a german ophthalmologist albrecht von graefe is given in this presentation, on the occasion of the 150th anniversary of his death. he is regarded as the greatest ophthalmologist of the 19th century. modern and scientific ophthalmology owes its beginning to him. methods: extensive literature research is made and contacts with institutions for history of medicine as well as medico-historians in the field of ophthalmology.results: his contributions to ophthalmology were multiple. von graefe was the first to introduce iridectomy in acute glaucoma treatment, initiated visual field testing and developed the first tonometer. he made the first classification of glaucoma. von graefe was the ophthalmologist who created a special knife for cataract surgery. he was also the first to use helmholtz' ophthalmoscope. he founded the first ophthalmological society in the world and the second ophthalmology journal which has been lahme l. 1* , marchiori e. 2 , mihailovic n. 1 , nelis p. 3 , oberhuber a. 2 der ophthalmologe · suppl 2 · 2020 s181 of postoperative surgical interventions was observed within one year. the outcome was considered to be stable when no further surgical interventions were needed for 6 months before the last follow-up. the mean diameter of the corneal grafts was 8.7 ± 1.6 mm. the mean corneal thickness after one year of follow-up was 602 ± 110 µm. the bcva increased from 2.4 ± 6.9 to 1.7 ± 7.2, the mean intraocular pressure was 15.2 ± 7.2 mmhg. three patients (23 %) needed no further surgical interventions. four patients (31 %) needed amniotic membrane transplantation, three patients (23 %) needed cyclophotocoagulation for glaucoma, two patients (15 %) needed repeated ppv, one patient needed a repeat pole-to-pole-surgery because of corneal graft failure with panophthalmitis, and two patients were subject to enucleation due to recurrent endophthalmitis. 11 of 13 patients were considered stable during the one year of follow-up. conclusion: pole-to-pole-surgery seems to be a viable option to attempt the salvage of the eye globe suffering simultaneously from severely affected corneal and retinal pathologies, thus preserving an ambulatory vision. ist das u-net zur objektivierung manueller markierungen in phasenkontrastmikroskopbildern geeignet? maßnahmen der hochschulambulanz einer augenklinik während der covid-19-pandemie schmid a. purpose: drug delivery to treat ocular diseases still is a challenge in ophthalmology. one way to achieve drug delivery currently investigated topical administration of drug-loaded polymeric nanoparticles (nps) that are able to penetrate ocular barriers. the purpose of this study is optimal preparation of nps made from pseudo-proteins and evaluation of their ability to penetrate ocular tissues. methods: biodegradable nps of various types were prepared by nanoprecipitation of a pseudo-protein composed of l-leucine, 1,6-hexanediol and sebacic acid (8l6). arginine-based cationic polyester amides 8r6 and comb-like polyester amides containing lateral peg-2000 chains along with 8l6 anchoring fragments in the backbones were used to construct positively charged and pegylated nps. the nps were loaded with fluorescein diacetate (fda) or rhodamine 6g (rh6g) as fluorescent probes. suspensions of the nps were given to cultivated microglial cells and retinal pigment epithelial (rpe) cells as well as topically on eyes of c57bl/6 mice. penetration of nps into the eyes was checked by fluorescence analysis. results: nps were prepared and their physicochemical properties were characterised. cultured microglial cells and rpe cells took up the nps. after topical administration penetration of nps into the cornea of the eyes could be clearly shown. small amounts of fluorescent dyes were also found in the lens, the retina and the sclera depending on the type of nps. the results show that the new nps penetrate ocular tissues after topical administration and are internalized by the cells. this raises confidence that the nps may be useful carriers of therapeutic agents for ocular delivery. key: cord-005147-mvoq9vln authors: nan title: autorenregister date: 2017-02-23 journal: med genet doi: 10.1007/s11825-017-0126-6 sha: doc_id: 5147 cord_uid: mvoq9vln nan complex mechanisms of dosage compensation regulate the mammalian x chromosome due to the presence of one copy in males (xy) and two in females (xx). x inactivation silences one x chromosome in females in early development, leading to specific epigenetic and structural changes. the inactive x chromosome becomes condensed and forms a bipartite structure within the nucleus, as we have shown by chromatin conformation analyses. specific long non-coding rnas are implicated in the formation of this unique structure. the inactive x chromosome is preferentially located near the lamina or the nucleolus. genes that escape x inactivation tend to be located at the periphery of the condensed inactive x chromosome. such genes are more highly expressed in females, and thus associated with sex-specific differences manifested even in early development. we have found that significant sex bias in gene expression are associated with escape from x inactivation in human tissues from normal males and females, and in tissues from individuals with sex chromosome aneuploidy, including turner or klinefelter individuals. institute for genomic medicine, columbia university medical center, new york, usa a central challenge in human disease genetics is the identification of pathogenic mutations. one key approach to distinguishing benign and pathogenic mutations is to use population genetic data to identify regions of the human genome under purifying selection. here i describe how the residual variation intolerance scoring framework has been applied to identifying pathogenic mutations in and outside protein encoding regions of the genome. next i report how these are related approaches are being used to identify pathogenic mutations in large-scale scale studies in epilepsy and other neurodevelopmental diseases. finally, i discuss how the identification of genetic causes of disease can inform treatment choices. scents indicate things, make promises, attract attention and stimulate imagination, feed anxieties and hopes: they are the salt in the atmospheric soup. we regard seeing and hearing as more important sensory functions, because they contribute more to conscious, cognitive processes of perception -but at moments of the greatest enjoyment we close our eyes and taste the scent, smell the taste. before the spirit and beauty of a person can fascinate us, our nose must become infatuated. the olfactory system in the nose acts as a window, monitoring environmental chemical information and convert chemical stimuli in electrical nerve impulses which are conducted along the olfactory sensory neuron to their glomerular target in the brain. olfactory receptors (ors) activation shows the distinguished (camp-based) transduction pathway for odorant perception. in 1991 buck and axel discovered the olfactory gene family, the largest gene family in the human genome, and postulated an exclusive expression in the olfactory epithelium. however, recent whole genome sequencing data from our and other labs show that ors have been found in every tissue of human body which was analyzed by next generation sequencing. the importance of such ectopic expression of ors is raised since the physiological function of some of ors was characterized. when identifying additional expression profiles and functions of or in non-olfactory tissue, there are limitations posed by the deorphanization of ors concerning the activated ligands and by the small number of antibodies available. in contrast to the olfactory sensory neurons which are believed to express all 350 functional or genes (only one or type per cell), cells in non-olfactory tissues tend to express more than one individual or gene per cell. in addition, some of the signaling pathways in non-olfactory tissues seem to involve completely different components in comparison to the olfactory neurons. what is the functional role of these ectopically expressed olfactory receptors? evidences rapidly accumulate that ors participate in important cellular processes outside its primary sensorial organ where they function in odor detection and discrimination. in our lab the functional expression of the first was demonstrated in spermatozoa (2004) . in the meantime we could show the existence and function of ors in the cardiovascular system (heart, blood cells), the gastrointestinal system (small intestine, liver, pancreas), the genito-urinary system (kidney, testis, spermatozoa, prostate), the respiratory system (lung, smooth muscle cells), the skin (keratinocytes, melanocytes) and sensory organs (retina). interestingly we found a broad spectrum of important functions like cell-cell communication and recognition, tissue injury, repair and regeneration, cancer growth, progression and metastasis, nutrient sensing and muscle contraction. nevertheless the functional importance of ectopic ors is still not sufficiently understood. studies seeking to determine the function of ectopic ors are still in its infancy and require further intensive exploration. however, the potential of ors to serve a target for a wide range of clinical approaches is indeed given. this hold promises that the knowledge gained by future investigations would lead to deepen our understanding of or function in health and disease and may provide the basis for the development of applications in diagnosis and therapies in near future. enzyme replacement therapies have been developed over the last 25 years for several of the lysosomal storage disorders (lsd's). the success of enzyme replacement therapy for gaucher disease paved the way for the development of similar treatments for the mucopolysaccharidoses, fabry and pompe disease and lately also for neuronopathic lysosomal storage disorders by intrathecal or intracerebral injections. in addition, small molecule approaches have been developed including substrate reduction therapies and chaperones, which can be used orally. while in gaucher disease enzyme as well as substrate reduction therapy results in reversibility of disease manifestations, with decreases in hepatosplenomegaly, normalization of blood counts and prevention of skeletal disease, this is unfortunately not the case for all patients affected with other lysosomal storage disorders. an important concept is the "window of opportunity for treatment" which is different for these disorders. for example, in fabry disease, early fibrosis fects are well defined, neither the specific mechanisms underlying neurological abnormalities nor the role of decreased cholesterol versus sterol precursor accumulation in disease pathogenesis have been clearly delineated. to identify cellular phenotypes and causative signaling pathways, we derived induced pluripotent stem cells (ipscs) from slos and lath subjects to model these diseases in vitro. slos subjects were known carriers of the most common dhcr7 mutations, including the intronic splice acceptor mutation c.964-1g>c and the missense mutation p.t93m. while all ipscs demonstrated the expected biochemical defects due to dhcr7 or sc5d mutations, cellular assays uncovered a defect in neural stem cell maintenance resulting in accelerated neuronal formation in slos ipscs. further molecular and biochemical analyses demonstrated inhibition of cholesterol-wnt interactions and loss of wnt/β-catenin activity mediated cellular phenotypes. however, this cellular phenotype was exclusive to slos, as lath ipscs did not exhibit a neural progenitor defect or inhibition of wnt/β-catenin activity. while this work demonstrates the utility of ipscs for modeling rare diseases and identifies signaling deficits potentially underlying slos phenotypes, questions remain regarding cellular and functional consequences, the specificity of lipid-wnt interactions, and the role of other disrupted signaling pathways in mediating developmental and functional deficits in these diseases. unpublished work using a variety of approaches will be discussed comparing the specific effects of cholesterol synthesis mutations on cell fate, functional activity, and lipid modulated signaling pathways to more precisely define the consequences of cholesterol synthesis defects and identify potential targets for patient therapy. induced pluripotent stem cell (ipsc) technology has become one of the major approaches for disease modeling since its first report in 2006. the ability to reprogram cells from somatic into embryonic stem cell-like state and to differentiate them into desired cell types in the culture dish has allowed scientists to carry out the study of several diseases in cells such as neurons which, in the past, could not be isolated from living subjects. williams syndrome (ws), a genetic neurodevelopmental disorder where 25-28 genes are hemizygously deleted, is among those. despite cardiovascular abnormalities, its unique neurological phenotypes i. e. hypersociability is of our interest. for several decades, research on different neurological aspects of ws has been conducted in a variety of models such as patient-derived cell lines (lymphoblastoid cells and fibroblasts), post mortem tissue, and mouse models. however, the lack of physiologically relevant cell types such as neural progenitor cells (npcs) and neurons has left a critical gap in our knowledge the disease's cellular and molecular phenotypes. to fill this gap, we took the advantage of the reprogramming technology to capture the genomes of ws subjects in ipscs, which could be then differentiated into npcs and neurons, enabling evaluation of whether the captured genome with hemizygous deletion of those genes leads to relevant neuronal cellular phenotypes. dental pulp cells-derived ipscs of classical ws, rare ws and typical developing (td) subjects were neurally induced via dual-smad inhibition in order to generate npcs and neurons. we discovered that classical ws npcs exhibited increased apoptosis, and, therefore, doubling time, compared to td neurons. this could possibly contribute to the reduction in cortical surface area in classical ws individuals as assessed by magnetic resonance imaging. surprisingly, we found that rare ws npcs behaved similarly to td npcs rather than to classical ws npcs in terms of apoptosis. we confirmed that frizzled9, which is deleted in the classical ws but not in our rare ws genome, is responsible for such phenotype via gain-and loss-of-function assays. moreover, classical ws neurons in general showed increased frequency of activity-dependent calcium transient compared to td neurons. finally, classical ws neurons acid alpha-oxidation, and (4.) glyoxylate detoxification. with respect to peroxisomal fatty acid oxidation peroxisomes catalyze the chain-shortening of certain fatty acids including very-long-chain fatty acids, but requires the active help of mitochondria to catalyze the degradation of acetyl-coa and the reoxidation of nadh as produced in peroxisomes. furthermore, with respect to ether phospholipid biosynthesis peroxisomes heavily rely on the endoplasmic reticulum to complete formation of ether phospholipids whereas fatty acid alpha-oxidation also requires the functional interplay between peroxisomes and mitochondria and the same is true for glyoxylate detoxification. recent evidence holds that the interaction between peroxisomes and the different subcellular organelles, including mitochondria and endoplasmic reticulum, is mediated by specific tethering protein complexes which bring organelles physically together thereby allowing metabolism to proceed smoothly. the importance of peroxisomes in metabolism is stressed by the existence of a large group of single peroxisomal enzyme deficiencies of which x-linked adrenoleukodystrophy is best known. our current state of knowledge with respect to the role of peroxisomes in metabolism and the peroxisomal enzyme deficiencies will be presented at the meeting. huntington's disease: rna-sequencing, small rna-sequencing, chip-sequencing and gwas data. department of neurology, boston university school of medicine, boston, ma 02118, usa huntington's disease (hd) is a dominantly transmitted neurodegenerative disease of midlife onset. recently several different unbiased genome wide studies in hd have been performed. these analyses point to a variety of pathological pathways that are associated with important features of the disease, including age at onset, cag repeat size, and the extent of neuropathological involvement. genome wide association studies (gwas) have identified several regions of the genome that contain genes that are associated with the age at onset for hd. the strongest of these is located at 15q13.3 for rs146353869 for which a very rare allele (maf = 1.1%) is associated with an approximate 6-year younger age at onset for carriers of the minor allele. the same locus contains an independent effect for rs2140734 where a more common minor allele (maf = 30.2%) is associated with a 1. 4 year older age at onset for carriers of this allele. these single nucleotide polymorphisms are in the region of fan1, mtmr10 and several other genes; some of which are not expressed in brain and are not likely candidates for hd modification. eqtl analysis has not resolved which gene may be implicated. other gwas implicated loci include rs1037699 at 8q22.3 and rs144287831 at 3p22.2. we have sought to combine the information derived from multiple platforms to gain additional insight into the pathways that may be implicated in hd pathogenesis. in this strategy, we have performed mrna-sequencing, small rna-sequencing and chip-sequencing using the h3k4me3 mark for active transcription and the repressive mark h3k9me3 in human hd brain samples with gwas genotyping. while the striatum is most involved in hd, the extent of neurodegeneration in post-mortem tissue precludes meaningful comparison between disease and control samples, and consequently we studied prefrontal cortex (ba9). several common pathways were seen across these three platforms. mrna-sequencing and mirna-sequencing data identified altered transcriptional profiles implicating developmental pathways involving the hox genes and related homeo-box domain genes (e. g. pitx1, pou4f2, etc.) . notably, micrornas located in hox gene clusters were among those most increased and levels of these correlate with pathological involvement in the striatum. these genes, associated with early embryonic development, are commonly silent in normal adult brain, and were among the most differentially expressed genes in hd brain. these prominent statistical effects are driven by the near total absence of expression in normal brain. pathways implicated in mrna-seq and chip-seq studies, included immune function and regulation of gene expression. these associations were very strong, indicating a large immune reactive response in the hd in the heart is related to unresponsive disease and unfortunately fibrosis may occur already in an early stage, sometimes even without prior hypertrophy. whether earlier intervention will be beneficial is largely unknown. and then: what is early? many unresolved questions exist at this stage, including the following: -what is the natural history and "point of no return" for the different lsd's? -what is the natural history and "point of no return" for subgroups of patients within one lsd's? -what are the long term complications: treatments change the phenotype rather than cure the disease -what is the influence of antibody generation on clinical effectiveness? -how do we manage the extreme costs of these products, especially in light of the many unsolved issues with respect to effectiveness? surprisingly, so far healthcare professionals, governments and industry have failed to systematically address these issues, resulting in insufficient knowledge for potentially lifesaving treatments. early conditional access, followed by a strict, transparent, independent, collaborative evaluation in addition to fair pricing should be explored. after the recent explosion in sequencing throughput, variant interpretation has quickly become the bottleneck in our effort to usher in the era of genomic medicine. while homozygosity for apparently pathogenic variants in the context of disease states is a well-established phenomenon, homozygosity can uncover many medically relevant aspects of the human variome that are difficult to study otherwise. for example, seemingly benign variants may prove pathogenic in the homozygous state. this includes variants with benign prediction using in silico tools as well as variants in dominant genes with no phenotype in carriers because they represent bona fide recessive inheritance. variants that are associated with one phenotype in compound heterozygous states may express themselves quite differently phenotypically when homozygous. furthermore, previously reported pathogenic variants can be challenged when their presence in homozygosity is associated with no abnormal phenotype, thus improving the specificity of the annotation of the morbid genome. homozygosity for lof variants is a special scenario that allows us to study naturally occurring human "knockouts", a powerful tool to study the physiological context of genes in humans. finally, homozygosity in the context of autozygosity provides a robust mapping tool that can greatly aid in the identification of relevant variants, especially those that exert their pathogenic effect in ways that defy detection by our usual algorithms. by expanding the spectrum of phenotypes that are studied, one can unlock the full potential of homozygosity to understand the medical relevance of the human variome in it its full range from embryonic lethal to essentially benign. helmholtz zentrum münchen gmbh, institut für experimentelle genetik, ingolstädter landstr. 1, 85764 neuherberg, germany the inheritance of epigenetic information in mammals across generations has been controversial. some reports provided initial evidence that a paternal high fat diet may propagate obesity and glucose intolerance in offspring, but potential confounders such as molecular factors present in seminal fluid, paternal-induced alterations in maternal care or transmission of microbiomes were not ruled out in these studies. we have shown in mice that a parental high fat diet (hfd) renders offspring derived via in vitro fertilization (f1) more susceptible to develop excessive overweight and type 2 diabetes (t2d) in a gender and parent-of-origin specific mode. female, but not male, offspring from obese parents became significantly more obese during a hfd challenge than female offspring from lean parents. body weight trajectories and distribution patterns of individual body weights in female offspring from one obese and one lean parent demonstrate that paternal and maternal germline propagate obesity in a roughly equitable and additive fashion, but likely different mode of action. in contrast, a more deteriorated state of hfd-induced insulin resistance was observed in both f1 genders, albeit predominantly inherited via the maternal germline. towards the identification of epigenetic information in sperm and oocyte from hfd and low fat diet fed parents, we are currently analyzing their transcriptome and methylome signatures. the status of this analysis will be presented. we report for the first time epigenetic inheritance of an acquired metabolic disorder via mammalian oocytes and sperms excluding confounding factors. such an epigenetic mode of inheritance may contribute to the observed pandemic increase in obesity and t2d prevalence rates, especially in an environment where nutrition is abundant. brain which may be a major influence contributing to neurodegeneration. in many instances enrichment of h3k4me3 at transcription start sites was not accompanied by a corresponding increase in expression. the apparent inconsistency suggests that common regulatory mechanisms in the hd brain are disrupted and this may contribute to a complex interplay of factors contributing to the neurodegenerative process. often findings in human hd brain samples conflicted with those reported in hd transgenic mouse models, suggesting that one may wish to be cautious in interpreting the significance of either type of study in isolation. the causal pathogesis of huntington disease, new therapeutic approaches r. laufer senior vp discovery and product development global r&d, teva r&d product development mgmt. 12 hatrufa st, netanya, israel huntington disease (hd) is an autosomal dominant neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, cognitive decline and death 15-20 years after motor onset. hd is uniquely caused by a polyglutamine encoding cag expansion in the huntingtin gene (htt), which allows for identification of pre-manifest mutation carriers as much as decades before onset and should facilitate development of disease modifying therapies. yet over 20 years after identification of the hd mutation, available therapies offer only symptomatic relief and are fraught with side effects. development of safe small molecule therapies for hd has been hindered by difficulties identifying and validating tractable drug targets within the disorder's complex pathogenesis. teva pharmaceuticals is developing potential novel treatments based on a mechanistic understanding of disease pathways common to neurodegenerative diseases. the progress of these studies will be reviewed. rare complete gene knockouts in adult humans p. sulem statistics department, decode genetics, sturlugata 8, 105 reykjavik, iceland loss-of-function mutations cause many mendelian diseases. here have create a catalog of autosomal genes that are completely knocked out in humans by rare loss-of-function mutations. we sequenced the whole genomes of over 29,000 icelanders and imputed the sequence variants identified in this set into a total of 151 chip-genotyped and phased icelanders. of the genotyped icelanders, around 10% are homozygotes or compound heterozygotes for loss-of-function mutations with a minor allele frequency (maf) below 2% in close to 2000 genes (complete knockouts). genes that are highly expressed in the brain are less often completely knocked out than other genes. homozygous loss-of-function offspring of two heterozygous parents occurred less frequently than expected (deficit of 136 per 10,000 transmissions for variants with maf <2%, 95% confidence interval (ci) = 10-261). we are currently systematically phenotyping such human complete knock out. this phenotyping lasts 4 hours and attempts to cover most of the observable diversity in a non-invasive and cost efficient manner. i will demonstrate how using systematic phenotyping can advance the knowledge on individual gene knockout. we use results from in-house transcriptomics, existing animal models and complementary approaches to assess the observation in human. we will also discuss the scrutiny in other population in order to detect such complete knock-out. we will exemplify the impact of founder population and consanguinity in such an odissey. early onset and severe obesity can be inherited via loss of function mutations within the melanocortin pathway of hypothalamic body weight regulation. the most prominent player in this signalling pathway is the fat cell hormone leptin. leptin gene mutations were the first to be linked to monogenic early onset obesity. after binding of leptin to leptin receptors in the arcuate nucleus of the hypothalamus the neuropeptide msh is processed from the precursor pomc and acts as a ligand at the mc4 receptor. mutations in the leptin receptor gene, the pomc gene and the mc4 receptor gene were subsequently diagnosed in further patients with extreme early onset obesity. while leptin mutation patients can be treated with recombinant leptin -as shown already in the late 1990s -all other monogenic obesity forms are leptin resistants, and additional leptin failed to decrease body weight. only recently pomc gene deficient patients were successfully treated with the msh-analogue setmelanotide (kühnen et al. 2016) . common severe obesity is defined by the lack of disease causing monogenic defects. a plethora of gwas identified a large number of snps in common obesity associated with the individual bmi but only to a low amount of not more then 25%. however, almost all these common obese patients are characterized by high leptin levels suggesting sufficient generation of leptin in the increased fat tissue and a state of leptin resistance. several new data concerning the contribution of epigenetic and genetic variants in the pomc gene locus argue for a role of the melanocortin pathway also in common obesity and imply, therefore, a potentially new treatment option also in common obesity based on msh-analogues. genome-wide association studies have highlighted the role of genetic associations with susceptibility to common inflammatory diseases, highlighting potential new insights into disease pathogenesis and opportunities for therapy. however understanding the functional basis of these associations and delivering translational utility remains a significant challenge to the field. non-coding regulatory genetic variants are most commonly implicated in such studies. recent work highlights how such variants are also major drivers of diversity in the immune response transcriptome. this talk will discuss approaches we are taking to try and establish functional links between immune phenotype-associated regulatory genomic and epigenomic variation, and specific modulated genes and pathways. i will describe insights from the application of expression quantitative trait (eqtl) mapping to define genomic modulators of the global transcriptomic response in different primary immune cell populations and to specific innate immune stimuli in health and disease. this work highlights the extent of local and distant context-specific eqtl, enabling resolution of immunoregulatory variants and the identification of specific modulated genes involving disease associated loci. examples will be described showing how mapping trans-regulatory loci can be a powerful approach for discovery and dissection of gene networks informative for disease. i will also show how we have applied analysis of the genetics of gene expression in patients with sepsis admitted to intensive care, revealing new insights into disease pathogenesis. further progress in this area will require characterisation of associated variants in the context-specific disease relevant epigenomic landscape in which they may act, requiring careful consideration of relevant immune cell types and environmental modulators to study, to-abstracts aktuellen stellungnahme der deutschen forschungsgemeinschaft 1 sollen humane genomsequenzierungen die möglichkeit der rückmeldung von analyseergebnissen enthalten. als orientierung für einen verantwortungsvollen umgang mit dieser frage wird auf die projektgruppe eurat 2 verwiesen. dennoch bleibt das problem der einordnung mitteilungswürdiger ergebnisse aus forscher-und probandensicht und der bereitstellung der für aufklärungs-und rückmeldungsalgorithmen erforderlichen ressourcen. darüber hinaus ist bis heute nicht geklärt, welche kommunikativen prozesse eine ausreichende basis für ein informiertes einverständnis darstellen. diese und weitere fragen möchten wir mit frau prof. dr. med. dr. phil. eva winkler (nationales centrum für tumorerkrankungen, heidelberg), herrn dr. phil. martin langanke (theologische fakultät, universität greifswald) und herrn pd dr. phil. peter burgard (universitätskinderklinik heidelberg) kontrovers diskutieren. die organisatoren werden in die fragestellung einführen, anschließend sind impulsreferate (ca. 10 min) der referenten vorgesehen, bevor eine debatte mit dem publikum angeregt wird. fangerau, f. söhner (düsseldorf) in den 1960er jahren erlebte die humangenetik wie eine reihe anderer medizinischer disziplinen auch eine erhebliche institutionelle ausweitung. in den 1960er und 1970er jahren wurde beispielsweise an den universitäten der brd das gros der humangenetischen lehrstühle, in den ausgehenden 1970er und 1980er jahren auch in der ddr, eingerichtet. 1969 ging vom symposium "genetik und gesellschaft" im rahmen des marburger "forum philippinum" die initiative aus, in der ganzen bundesrepublik genetische beratungsstellen zu gründen und damit die genetische forschung (wieder) medizinisch nutzbar zu machen. in dieser phase der etablierung der humangenetik auf akademischer und praktischer ebene setzt das geplante zeitzeugenprojekt ein. es will die entwicklung der humangenetik in ihrem selbstverständnis als quer-und als längsschnittfach (pfadenhauer 2003:66) im deutschsprachigen raum ab den 1970er jahren mit hilfe von expertengesprächen dokumentieren und analysieren. im forschungsprojekt sollen zwei komplexe von fragestellungen bearbeitet werden: ein wissenschaftshistorischer, in dem die entwicklung und anwendung von diagnostischen und therapeutischen techniken im mittelpunkt steht, und ein sozialhistorischer, in dem es um die etablierung und den ausbau der institutionen der humangenetik sowie um den verlauf der das fach betreffenden gesellschaftlichen debatten geht. neben der gründung von instituten und der fachgesellschaft sowie der normierung der ausbildung für ärztliche und naturwissenschaftlich ausgebildete humangenetiker sollen die funktion der historischen reflexion und bearbeitung der facheigenen nationalsozialistischen vergangenheit in den 1980er jahren für die etablierung des fachs und der schwierige institutionelle trennungsprozess von der anthropologie mit ihren wirkungen auf das selbst-und fremdbild der humangenetik analysiert werden (weingart, kroll, bayertz 1992) . zusätzlich zur entwicklung in der brd sollen dabei auch die entwicklung des fachs in der ddr und mögliche deutsch-deutsche kooperationen zur sprache kommen (in jena befand sich z. b. in den 70er jahren das zentrale referenz-institut für genetische beratung in der ddr (vogel 1999:416) . das projekt kann sich auf zahlreiche arbeiten zur geschichte der deutschen humangenetik stützen (vgl. kröner 1997 , 1998 cottebrune 2006 cottebrune , 2008 weingart 1988; bennike 1992 dysmorphism carrying a pathogenic variant in the ebf3 gene detected by whole-exome sequencing. five missense, two nonsense, one 9-bp duplication, and one splice-site variant in ebf3 were found; the mutation occurred de novo in eight individuals, and the missense variant c. 625c>t [p.(arg209trp) ] was inherited by two affected siblings from their healthy mother who is a mosaic. ebf3 belongs to the early b-cell factor family (also known as olf, coe, or o/e) and encodes a transcription factor involved in neuronal differentiation and maturation. structural assessment predicts perturbing effects of the five amino acid substitutions on dna-binding of ebf3. transient expression of ebf3 mutant proteins in hek 293t cells revealed mislocalization of all but one mutant in the cytoplasm in addition to nuclear localization. by transactivation assays, all ebf3 mutants showed significantly reduced or no ability to activate transcription of the reporter gene under control of the cdkn1a promotor that corresponds well with loose association of ebf3 mutants with chromatin as demonstrated by in situ subcellular fractionation experiments. finally, rna-seq and chip-seq experiments demonstrate that ebf3 acts as a transcriptional regulator at cis-regulatory sequences and ebf3 mutant had reduced function due to partial disruption of the dna-binding domain. these findings demonstrate that ebf3-mediated dysregulation of gene expression has profound effects on neuronal development in humans and add ebf3 to the growing list of genes in which mutations cause syndromic forms of intellectual disability. step, we performed wes in further unelucidated uhs cases and identified homozygous nonsense mutations in tgm3 (transglutaminase 3) and in tchh (trichohyalin), respectively. elucidation of the molecular outcomes of the disease causing mutations by cell culture experiments of padi3 and tgm3 and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild type proteins. by immunofluorescence analysis, we could demonstrate a diffuse homogenous cytoplasmic distribution of the wt padi3, whereas in the mutants the proteins were observed to form large aggregates throughout the cytoplasm. by use of human anti-citrullinated protein autoantibodies, we could show a strong labelling in the wt whereas the staining of the mutants was barely above background. in order to demonstrate the importance of padi3 in hair shaft formation, we generated padi3 knockout mice. electron microscopy observations revealed morphological alterations in hair coat of padi3 knockout mice. for tgm3, we performed a transglutaminase activity assay. the analysis results revealed that the wt had a significantly higher transglutaminase activity in comparison to the truncated protein. here, we report for the first time the identification of uhs causative mutations located in the three genes padi3, tgm3 and tchh. the two enzymes responsible for posttranslational protein modifications, and their target structural protein, are all involved in hair shaft formation through their sequential interactions. these findings provide valuable information regarding the pathophysiology of uhs and contribute to a better understanding of this protein interaction cascade. this could be of further value for cosmetics and pharmaceutics industries paving the way for development of novel products. deadenylases are best known for degrading the poly(a) tail during mrna decay. the deadenylase family has expanded throughout evolution and, in mammals, consists of 12 mg 2+ -dependent 3' end ribonucleases with mostly unknown substrate specificity. pontocerebellar hypoplasia type 7 (pch7) is a unique recessive syndrome characterized by neurodegeneration with ambiguous genitalia (mim%614969). we studied 12 human families with pch7, uncovering biallelic, loss of function mutations in toe1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed mid-and hind-brain degeneration, modeling pch-like structural defects in vivo. surprisingly, we found toe1 associated with incompletely processed small nuclear (sn)rnas of the spliceosome, which is responsible for pre-mrna splicing. these pre-snrnas contained 3' genome-encoded tails often followed by post-transcriptionally added adenosines. human cells with reduced levels of toe1 accumulated 3' end-extended pre-snr-nas, and immuno-isolated toe1 complex was sufficient for 3' end maturation of snrnas. our findings reveal the cause of a neurodegenerative syndrome linked to snrna maturation and uncover a key factor involved in processing of snrna 3' ends. the kidney maintains acid-base homeostasis and electrolyte balance through highly specialized cells. in the distal nephron acid secretion is mediated by type a intercalated cells (a-ics) , which contain v-type at-pase-rich vesicles that fuse with the apical plasma membrane on demand. intracellular bicarbonate generated by luminal h+ secretion is removed by the basolateral anion-exchanger ae1. dysfunction of type a intercalated cells results in distal renal tubular acidosis (drta) and human mutations in v-atpase subunits and ae1 are causative for this condition. for the ae1 r607h mutation a dominant-negative trafficking mechanism was proposed to explain ae1-associated dominant drta based on studies in mdck monolayers. to test this hypothesis in vivo and to test potential rescue strategies correcting this mistargeting defect, we have generated a r607h knock-in mouse strain, which corresponds to the most common dominant drta mutation in human ae1, r589h. heterozygous and homozygous r607h knock-in mice displayed incomplete drta characterized by compensatory upregulation of the na+/hco3-cotransporter, nbcn1. as expected for the r607h mutation, red blood cell ae1-mediated anion-exchange activity and surface polypeptide expression were unchanged. surprisingly, basolateral targeting of the mutant ae1 in a-ics was preserved in contrast to previous studies in mdck cells. instead, we found ae1 expression in a-ics strongly reduced in a r607h dosage-dependent manner. additional cell culture studies in two widely used immortalized renal cell lines verified that targeting and half-life time of mutant ae1 protein was indeed preserved. surprisingly, atpase expression was reduced and its plasma membrane targeting upon acid challenge compromised. ultrastructural analysis revealed a loss of apical vesicles in a-ics, while we observed lysosomal inclusions and multilamellar bodies. accumulation of p62-and ubiquitin-positive material in a-ics of knock-in mice suggest a defect in the degradative pathway, which may ultimately lead to loss of a-ics. highlighting the expression of ae1 specifically in a-ics, type b intercalated cells were unaffected. we propose that reduced basolateral anion-exchange activity in a-ics inhibits trafficking and regulation of v-type atpase, compromising luminal h+ secretion and possibly also lysosomal acidification. our findings illustrate the considerable, context-dependent complexity of ae1-related kidney disease. b. vona 1 , d. liedtke 1 , k. rak 2, 3 , r. katana 4 , l. jürgens 3 , pr. senthilan 5 , i. nanda 1 , c. neuner 1 , mah. hofrichter 1 , l. schnapp 1 , j. schröder 1 , u. zechner 6 , s. herms 7, 8, 9 , p. hoffmann 10 , t. müller 11 , m. dittrich 1, 11 , o. bartsch 6 , pm. krawitz 12 , e. klopocki 1 , w. shehata-dieler 2 , mc. göpfert 4 , t. haaf 1 although many genes have already been identified as causing non-syndromic hearing loss (nshl), diagnostic rates of approximately 50% among hearing impaired patients suggest that many more genes are remaining to be identified. nshl is the most common sensory deficit that has a prevalence between one and two per 1000 newborns. furthermore, it demonstrates classic genetic heterogeneity with as many as 1% of coding genes in the genome anticipated to be involved in non-syndromic forms of deafness. autosomal recessive (75-80%) and autosomal dominant (15-20%) forms dominate inheritance patterns of deafness; however, in a small fraction of cases, x-linked deafness (1-4%) can be observed. whole exome sequencing of a german family with diagnostically unresolved nshl revealed a novel missense variant predicted as pathogenic in the gene ferm and pdz domains containing protein 4 (frmpd4) on chromosome xp22.2. this gene, also known as preso1, was first described as a regulator of dendritic spine morphogenesis. previous screening of pathogenic cnvs in array based comparative genomic hybridization among families with heterogeneous x-linked intellectual disability (xlid) showed duplication of xp22.2 including part of frmpd4 which implicated the gene in xlid. interestingly, a segregating truncating and a de novo missense mutation in frmpd4 have associated this gene with xlid, a phenotype not observed in our family. mouse expression studies localize frmpd4 to spiral ganglion neuron peripheral dendrites of the developing cochlea. in addition, we analyzed frm-pd4 knockdown and loss-of-function zebrafish mutants for innervation and structural defects in the otic vesicle and lateral line neuromasts. posterior lateral line neuromasts are observed with reduced axonal outgrowth that is also likely reduced in the lateral line nerve. abnormal innervation is also apparent in the otic vesicle. fluorescent neuromast labeling marked a significant reduction of overall otic vesicle and lateral line neuromasts in mutants versus wild type zebrafish. scanning electron microscopy revealed a pronounced absence of kinocilia in posterior lateral line neuromasts of frmpd4 -/zebrafish. furthermore, adult frmpd4 mutants show significantly delayed acoustically evoked behavioural responses compared to wild type fish indicating hearing impairment. investigation of transgenic drosophila insertion mutants detected a mild auditory phenotype i. e. a reduction in mechanical amplification gain and associated reduction in antennal fluctuation power. our results associate frmpd4 with x-linked hearing loss and suggest mutations in this gene are correlated with pleiotropic effects abstracts has also been demonstrated to be an important pathobiochemical feature in rtt. to test whether common deficits in mtor signaling could be responsible for the molecular pathogenesis underlying both syndromes, we generated and studied a novel cdkl5 knockout (cdkl5 -/y) mouse model and performed in vitro experiments in human cells. in cdkl5 -/y knockout mice loss of cdkl5 is accompanied by reduced phosphorylation levels of critical components of the mtor signaling cascade. these findings point at a regulatory role of cdkl5/cdkl5 on mtor activity and function. to gain further insights into the possible mechanism through which cdkl5/pi3k interaction could regulate mtor signaling, we used hek-t cells as cellular model. following knock-down of cdkl5, the amount of pi3k protein was significantly reduced compared to controls. to evaluate the contribution of our findings to pathogenesis, we performed rescue experiments in cdkl5 knock-down hek-t cells using wild-type and patient-specific mutant cdkl5 constructs. further experiments are ongoing to clarify the molecular mechanism by which cdkl5 regulates pi3k protein level in the cells. inferring expressed genes by whole-genome sequencing of plasma dna medical university graz, graz, austria, 2 university of technology, graz, austria the analysis of cell-free dna (cfdna) in plasma represents a rapidly advancing field in medicine. cfdna consists predominantly of nucleosome-protected dna shed into the bloodstream by cells undergoing apoptosis. we performed whole-genome sequencing of plasma dna and identified two discrete regions at transcription start sites (tsss) where nucleosome occupancy results in different read depth coverage patterns for expressed and silent genes. by employing machine learning for gene classification, we found that the plasma dna read depth patterns from healthy donors reflected the expression signature of hematopoietic cells. in patients with cancer having metastatic disease, we were able to classify expressed cancer driver genes in regions with somatic copy number gains with high accuracy. we were able to determine the expressed isoform of genes with several tsss, as confirmed by rna-seq analysis of the matching primary tumor. our analyses provide functional information about cells releasing their dna into the circulation. institute of human genetics, fau-erlangen-nürnberg, erlangen, germany, 2 institute of medical genetics, university of zurich, schlieren, switzerland rna-splicing is an important mechanism for eukaryotic gene expression and regulation. defective splicing significantly contributes to monogenic disease in humans. indeed, the mutational space for variants affecting splicing is larger than for coding variants. several computational methods have been developed to predict a variant's effect on splicing but lack predictive value outside the canonical splice sites and do not predict aberrant transcripts. thus the plethora of dna variants generated by recent advances in "next-generation" based sequencing (ngs) can be scored for a possible splicing effect, but a laborious wet-lab based confirmation and characterization is still required. rna-seq is widely used for quantification of gene expression and can be used to detect splicing events, but is limited for this use by the variable and often low read coverage of individual congenital anomalies of the kidneys and urinary tract (cakut) are the most common cause of chronic kidney disease in children. as cakut is a genetically heterogeneous disorder and most cases are genetically unexplained, we aimed to identify new cakut causing genes. using whole-exome sequencing and trio-based de novo analysis, we identified a novel heterozygous de novo frameshift variant in the leukemia inhibitory factor receptor (lifr) gene causing instability of the mrna in a patient presenting with bilateral cakut and requiring kidney transplantation at one year of age. lifr encodes a transmembrane receptor utilized by il-6 family cytokines, mainly by the leukemia inhibitory factor (lif). mutational analysis of 121 further patients with severe cakut yielded two rare heterozygous lifr missense variants predicted to be pathogenic in three unrelated patients. lifr mutants showed decreased half-life and cell membrane localization resulting in reduced lif-stimulated stat3 phosphorylation. lifr showed high expression in human fetal kidney and the human ureter, and was also expressed in the developing murine urogenital system. lifr knockout mice displayed urinary tract malformations including hydronephrosis, hydroureter, ureter ectopia, and, consistently, reduced ureteral lumen and muscular hypertrophy, similar to the phenotypes observed in patients carrying lifr variants. additionally, a form of cryptorchidism was detected in all lifr -/mice and the patient carrying the lifr frameshift mutation. altogether, we demonstrate heterozygous novel or rare lifr mutations in 3.3% of cakut patients, and provide evidence that lifr deficiency and deactivating lifr mutations cause highly similar anomalies of the urogenital tract in mice and humans. loss of cdkl5 associated with deficient mammalian target of rapamycin (mtor) signaling in mice and human cells we and other groups have shown that mutations in the x-linked cyclin-dependent kinase-like 5 (cdkl5) gene cause a severe neurodevelopmental disorder with clinical features including intellectual disability, early-onset intractable seizures and autism, that are closely related to those present in rett syndrome (rtt) patients. rtt is caused by mutations in the x-linked mecp2 gene. cdkl5 is a serine/threonine kinase and to date knowledge about its functional roles is scarce. we searched for cdkl5 interacting proteins by yeast-two hybrid screens. one of the candidates identified in these screens is a subunit of the phosphatidylinositol-4,5-biphosphate 3-kinase (pi3k). the results obtained in yeast could be confirmed in vitro in mammalian cells and in mouse brain by immunoprecipitation experiments and by co-localization studies. pi3k phosphorylates membrane lipids which act as docking sites to recruit targets upstream of mtor and thereby regulate among major cellular processes synaptic plasticity, which is the cellular basis for learning and memory. alteration of mtor signaling gene tubes for stabilizing rna immediately after drawing the samples. the subsequent rt-pcr analysis showed that 9 of the 11 variants located at potential splicing sites indeed affect splicing. thus 8 of these variants could be classified as deleterious (iarc class 5), while one chek2 variant could not be unequivocally classified as the rt-pcr analysis identified only 20% of the mutant transcript indicating continued usage of the constitutive splice acceptor site. this led to the classification as a probably hypomorphic allele. the variants in cdh1 and mlh1 did not affect splicing and were classified as benign (iarc class 1). none of the 5 rare synonymous and nonsynonymous exonic variants showed any effect on splicing. in conclusion, this analysis allowed the disambiguation of 10 out 11 vus at potential splice sites into a definite category (either iarc class 5 or 1). this work highlights the importance of computational splicing prediction and validation using rt-pcr of peripheral blood rna to assess the pathogenicity of vus. this in turn, allows more accurate genetic counseling and clinical management of affected families. gliomas present the major group of neoplasia in the central nervous system. they typically show invasive growth and high recurrence rate and are currently not curable. idh mutations are detected in nearly 70% of low grade gliomas and are considered to play a key role in low grade glioma development. while it is known that idh1/2 mutation leads to high-levels of 2-hydroxyglutarate (2-hg) that functions as an oncometabolite, little is known about the influence of idh1/2 mutations on energy metabolism and metabolic reprogramming in the tumor cells. since patient derived idh mutant cells do not grow in cell culture, previous studies from our group and others used transduced cell lines that overexpress idh1. in order to develop in vitro models with reduced side effects, we used crispr/ cas to introduce the idh1r132h mutation in a patient derived glioblastoma cell line. the edited cells expressed idh1r132h in western blot and expression levels of idh1 were comparable to the expression in wild type cells. the mutation was stable in long time culture experiments, without signs of senescence. moreover we found elevated 2-hg levels, proving that the idh1r132h neoenzymatic function is present in our cell lines. thus, we were able to edit and culture genomic idh1r132h mutated glioma cells for the functional analysis of the idh1r132h mutation for the first time without the effects of overexpression models. edited idh1r132h cell lines showed extended doubling times compared to wildtype cells. measurement of krebs cycle metabolites using mass spectrometry revealed elevated glutamate levels. we found enhanced atp-levels that could be a consequence of decreased atp consumption. additionally, the cells showed reduced viability compared to wildtype cells when cultivated in glycolysis inhibiting media, pointing out the enhanced dependency on glycolysis in idh1r132h cells. these results indicate changes in tumor cell metabolism and energy household induced by the idh1r132h mutation. since we and others could show that idh1r132h can alter nad+ and nadph levels, we tested if the idh1r132h mutated cells are more susceptible to selective inhibition of nad/p regenerating enzymes. esirna-silencing of nampt specifically decreased cell viability in idh1r132h but not wildtype cells with a concomitant increase of dead cells. in conclusion, we developed genes. thus, a standardized ngs based approach to characterize potential splice variants is lacking. hence we investigated the utility of hybridization based gene-panel enrichment and ngs of cdna. based on results of computational simulation we selected twenty rna-samples of patients with a known pathogenic splice-site variant in an inherited cancer predisposition gene. these variants were previously characterized by rt-pcr in our lab or in the literature. after rrna depletion and dna digestion we performed first and second strand cdna synthesis followed by "tagmentation"-based library preparation, targeted enrichment using the trusight cancer panel and sequencing on an illumina miseq platform. a computational pipeline was established to enable automated detection of aberrant splicing events by implementing different alignment and splice-junction detection algorithms together with filtering against control data sets. we also considered variant calling for the detection of allelic imbalance and gene-level expression analysis in this data. breast and ovarian cancer (bc/oc) predisposition has been associated with a number of high-and low-penetrance susceptibility genes. advances in sequencing technology has made multigene testing a practical option when searching for genetic variants associated with risk for bc/oc. variants of uncertain significance (vus), though, represent a major problem. we now studied 581 patients fulfilling criteria for brca1 and 2 testing using the next generation sequencing based trusight sequencing cancer panel on a miseq platform (illumina). data was analyzed after remapping with bwa to hg19 (grch37) using seqnext software (jsi) for variants in 14 known high and moderate penetrance susceptibility genes (brca1/2, atm, chek2, palb2, rad51c, rad51d, nbn, cdh1, tp53, mlh1, msh2, msh6, pms2) . besides 106 deleterious mutations we also identified 89 vus. of these, 11 variants (1 each in brca1, brca2, palb2, rad51c, rad51d, cdh1, and mlh1, and 2 in chek2 and mlh1, respectively) affect possible splicing sites. in addition, 5 synonymous and nonsynonymous variants outside the splicing sites (1 in brca1, brca2 and cdh1, respectively, 2 in rad51d) were not reported in exome variant server or exome aggregation consortium (exac) databases, so far. no families were available to study familial segregation. for all these variants a potential effect on splicing efficiency was predicted by three different computational algorithms (bdgp: splice site prediction by neural network, netgene2 server and the human splice finder (hsf 3.0) algorithm). we took advantage that these genes are ubiquitously expressed to investigate possible effects of these variants on mrna splicing using easily accessible peripheral blood. as mrna is notoriously unstable, we used pax-abstracts was evaluated for both the individual markers and their combinations derived from multiple algorithms. pronounced demethylation of all 3 markers was observed at baseline among cases compared to controls. risk of developing lc increased with decreasing dna methylation levels, with adjusted ors (95% ci) of 15.86 (4.18-60.17), 8.12 (2.69-4.48) , and 10.55 (3.44-32.31 ), respectively, for participants in the lowest quartile of ahrr, 6p21.33, and f2rl3 compared to participants in the highest 2 quartiles of each site among controls. the individual 3 markers exhibited similar accuracy in predicting lc incidence, with aucs ranging from 0.79 to 0.81. combination of the 3 markers did not improve the predictive performance (auc = 0.80). the individual markers or their combination outperformed self-reported smoking exposure particularly in light smokers. no variation in risk prediction was identified with respect to age, follow-up time, and histological subtypes. ahrr, 6p21.33, and f2rl3 methylation in blood dna are predictive of lc development, which might be useful for identification of risk groups for further specific lc screening, such as ct examination. over the past decades the search for disease causing variants has been focusing exclusively on the coding genome. this highly selective approach has been extremely successful however, recent data have revealed the importance of the non-coding genome in fundamental processes such as gene regulation, 3d chromatin folding, and pinpointed its role in disease. in this study, we systematically investigate the cis-regulatory landscape of pitx1, a homeodomain transcription factor that is exclusively expressed in the hindlimb. mutations and non-coding structural variations at the pitx1 locus have been shown to associate with a variety of congenital limb defects including club feet, polydactyly, and arm-to-leg transformation (liebenberg syndrome). we performed in vivo enhancer reporter essays in transgenic mice and identified several limb enhancer elements at the pitx1 locus; surprisingly they all showed both forelimb and hindlimb activity, although pitx1 is never expressed in the forelimb. capture hi-c experiments revealed a hindlimb-specific chromatin-organization at the pitx1 locus, which enables its promoter to contact several enhancers bearing a pan-limb activity only in the hindlimb. this tissue-specific chromatin folding plays a determinant role to refine the unspecific limb regulatory landscape toward a highly controlled and hindlimb delimited transcriptional output. to gain a better understanding of the pathology of pitx1 associated limb defects, we used crispr/cas9 to generate a set of deletions and inversions in the pitx1 cis-regulatory landscape in mice. genetic perturbations of the regulated 3d chromatin conformation lead to an ectopic forelimb expression of pitx1, resulting in an arm-to-leg transformation in mice and in human patients respectively. our data further highlight the role of non-coding mutations affecting chromatin folding in congenital disease and give new insights into the regulation of pitx1 during development and the pathomechanism of associated limb defects. hoxb13, a member of the embryonic homeobox transcription regulators, has been identified as the first susceptibility gene specific for prostate cancer (prca). the founder missense mutation g84e, which likely originated from finland, can be found in most populations of european ancestry. we determined the frequency of hoxb13 g84e for the german population, assessed in a cohort of 379 unrelated cases, each with positive family history of prca, 367 sporadic prca cases and in 1015 controls. additional 646 affected relatives from prca families were included to explore association with aggressive disease in subgroups with high gleason score (>7), advanced tumor stage, or psa at diagnosis >20 ng/ml. carriers of g84e were rare in controls (0.4%) and showed increased frequencies in both sporadic (1.6%) and familial prca cases (3.2%). estimated risks were or = 4.2 (p = 0.026) and or = 8.3 (p = 0.0003), respectively. the risk effect size increased with the number of affected individuals per pedigree: or = 12.6 (p < 0.0001) for 3 or more, and or = 14.4 (p < 0.0001) for 4 or more affected men. the strongest association with clinical features was observed between g84e and advanced tumor stage (or = 9.2; p < 0.0001). in conclusion, the observed frequency of hoxb13 g84e mutation carriers in our study cohort was intermediate as compared to the common prevalence in scandinavia and the rare occurrence in mixed european populations from the us. the risk estimates of hoxb13 g84e and the stronger effect sizes in families with increasing number of affected relatives were in line with a high penetrant germline predisposition. the association between g84e status and tumor stage may be of greater interest for clinical practice, but needs further validation. the absolute penetrance of the hoxb13 g84e mutation should be investigated in further studies in order to elucidate its suitability as a genetic predictor for prca. smoking-associated dna methylation markers predict lung cancer incidence homozygous smn1 loss causes spinal muscular atrophy (sma), the most common lethal genetic childhood motor neuron disease. smn1 encodes smn, a ubiquitously expressed housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. sma individuals harbor low smn expression from one to six smn2 copy genes, which is insufficient to functionally compensate for smn1 loss. however, rarely individuals with homozygous absence of smn1 and only three to four smn2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). previously, we identified plastin 3 (pls3) overexpression as an sma protective modifier in humans and showed that smn deficit impairs endocytosis, which is rescued by pls3 overexpression. here, we identify reduction of the neuronal calcium sensor neurocalcin delta (ncald) as a protective sma modifier in five asymptomatic smn1-deleted individuals carrying only four smn2 copies. we demonstrate that ncald is a ca 2+ -dependent negative regulator of endocytosis, as ncald knockdown improves endocytosis in sma models and ameliorates pharmacologically induced endocytosis defects in zebrafish. importantly, ncald knockdown effectively ameliorates sma-associated pathological defects across species, including worm, zebrafish and mouse. in conclusion, our study identifies a previously unknown protective sma modifier in humans, demonstrates modifier impact in three different sma animal models and suggests a potential combinatorial therapeutic strategy to efficiently treat sma. since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in sma and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies. mutations affecting coding or regulatory regions of smc2 cause dysregulation of condensins resulting in a phenotype reminiscent of cohesinopathies cornelia de lange syndrome (cdls) is a dominantly inherited malformation syndrome caused by mutations in genes encoding subunits (smc1a, smc3, rad21) or regulators (nipbl, hdac8) of the cohesin complex. this dna-bound complex regulates several chromatin-related processes such as chromosome segregation, dna-damage repair, transcription and chromatin structure. the project presented initially started with two children and their mother who showed clinical features reminiscent of cdls. while various sequencing approaches failed to identify the disease-causing mutation, a 60 kb spanning deletion co-segregating with the phenotype was identified by array-cgh. besides the last exons of cylc2, encoding a sperm head protein, no other genes were affected. subsequent in-silico analyses predicted the existence of a ~3 kb tissue-specific regulatory element within this region, located approximately 1 mb distant from the next protein-coding gene smc2, which encodes a subunit of the cohesin-related condensin complex. significant reduction of smc2 expression was verified in patient's fibroblasts by qpcr analysis. accordingly, a strong dysregulation of smc2 was observed in hek293 and sh-sy5y cells deficient for the putative 3 kb regulatory element, which was deleted by crispr/cas9 genome editing. reporter gene assays further highlighted the functional relevance of the identified regulatory element in regulating the smc2 gene promoter. interestingly, we could prove on protein as well as on mrna level that alterations in smc2 expression are correlated with the dysregulation of other condensin subunits such as smc4 in patient's samples as well as in cris-pr/cas9-generated cells. in a large exome sequencing project we have identified a smc2 frameshift mutation in an additional family with two patients who show clinical features overlapping with those seen in our initial family. quantitative pcr analyses in fibroblasts of both subjects also showed significant reduction of smc2 and smc4 expression, which is consistent with our findings in the first family. to further investigate whether alterations in condensin gene expression are specific for the dysregulation of smc2, we have decreased smc2 levels in different cell types by sirna. quantitative protein as well as mrna analyses revealed reduced smc4/smc4 expression. our data show for the first time the coordinated expression of different condensin subunits and its relevance for human disease. abstracts human pedigree. cardiac valves initially form through a process called endothelial-to-mesenchymal transition (emt) then subsequently elongate and mature during early juvenile life. expression analysis throughout embryonic and postnatal stages of adamts19-/-mice revealed an expression in all cardiac valves after valve formation. high resolution, digital echocardiography showed that mice without adamts19 expression develop dysfunctional aortic valves early in life, reminiscent of the human phenotype. notably, the expression of adamts19 in the valve was restricted to valvular interstitial cells and not observed in endothelial cells. functional analysis using proteomic approaches suggest that the presence of ad-amts19 is necessary to maintain extracellular matrix remodelling during valve development and its maturation. not only do the lof mice fully recapitulate the human phenotype, they also highlight adamts19 as a novel marker for valvular interstitial cells to specifically target initial post-emt processes as well as serve as an important model to understand an ageing valve phenotype in humans. exome sequencing of 55 bipolar disorder patients with rapid cycling implicates novel candidate genes in disease development bipolar disorder (bd) is a severe neuropsychiatric disorder characterized by recurrent episodes of mania and depression. bd has a lifetime prevalence of about 1% and a high heritability of about 70%. although recent genome-wide association studies identified the first susceptibility genes contributing to disease development, the cumulative impact of common alleles with small effect may only explain around 38% of the phenotypic variance (lee et al. 2011) . in consequence, rare variants of high penetrance have been suggested to additionally contribute to bd susceptibility. in the present study we focused on bd patients with rapid cycling (rc). rc is a course specifier of bd defined as having at least four recurrent episodes of acute illness within one year. since rc showed strong evidence for familiarity, we hypothesized that bd patients with rc might represent a more defined etiological subgroup and that rare variants of high penetrance might contribute to the development of rc in bd patients. we selected 55 unrelated bd patients with rc of german origin and performed exome sequencing using the illumina hiseq2500 platform. for data analysis, the varbank pipeline of the cologne center for genomics was used. we filtered for rare (minor allele frequency <0.1%), heterozygous and non-synonymous variants that were predicted to be possibly damaging or disease causing by at least 4 of 5 applied prediction tools. after these filtering steps, we identified a total of 110 different genes which harbored rare functional variants in at least three independent patients. gene set analysis for these genes using consensuspathdb revealed 165 decker 7 , g. nuernberg 8, 9 , d. hassel 2 , g. a. rappold 1, 10 mutations in the homeobox gene shox cause shox deficiency, the most frequent monogenic cause of short stature. the clinical severity of shox deficiency varies widely, ranging from short stature without dysmorphic signs to mesomelic skeletal dysplasia (léri-weill dyschondrosteosis, lwd). in rare cases, individuals with shox deficiency are asymptomatic. to elucidate the factors that modify disease severity/penetrance, we studied a three-generation family with five affected individuals with lwd using whole genome linkage analysis and whole exome sequencing. the variant p.phe508cys of the retinoic acid catabolizing enzyme cyp26c1 co-segregated with the shox variant p.val161ala in the five affected individuals, while the shox mutant alone was present in three asymptomatic individuals. two further independent lwd cases with shox deficiency and damaging cyp26c1 variants were identified. the identified damaging variants in cyp26c1 affected its catabolic activity, leading to an increased level of retinoic acid. we also provide evidence that high levels of retinoic acid significantly decrease shox expression in human primary chondrocytes and zebrafish embryos. individual morpholino knock-down of either gene shortens the pectoral fins, whereas depletion of both genes leads to a more severe phenotype. together our findings demonstrate that shox and cyp26c1 act in a common molecular pathway controlling limb growth and describe cyp26c1 as the first genetic modifier for shox deficiency. heart valve dysfunction in men and mice is caused by loss of function mutations in adamts19, a novel marker for valvular interstitial cells on a global perspective defects of the cardiac valves are one of the most common heart abnormalities in humans, with a substantial number of them requiring surgical intervention at least once in their life. several mechanisms have been proposed ranging from acquired to developmental causes, but thus far the majority can not be explained on the molecular level. here we report on the identification of a unique human family affected by multiple dysfunctional cardiac valves early in life. genetic screening revealed a homozygous deletion of the first eight exons in ad-amts19, a novel candidate gene for valvular heart defects. to investigate its role in heart valve development, we designed a transgenic mouse model that reconstitutes the loss of function (lof) in adamts19 found in the statistically analyzing de novo mutations identified in >5,000 id patients highlighted ppm1d as a candidate id gene. ppm1d is a type 2c phosphatase that functions as a negative regulator of cell stress response pathways by mediating a feedback loop of p38-p53 signaling, thereby contributing to growth inhibition and suppression of stress induced apoptosis. we identified 14 patients with mild-moderate id and a de novo truncating ppm1d mutation. deep-phenotyping of the patients revealed in addition to id overlap for behavioural problems (adhd and anxiety disorder), hypotonia, broad based gait, facial dysmorphisms and periods of fever and vomiting. ppm1d is shown to be expressed during fetal (brain) development and in the adult brain. all mutations were located in the last, or penultimate exon, suggestive of escaping nonsense-mediated mrna decay. both ppm1d expression analysis and cdna sequencing in patient ebv-lcls support the presence of a stable, but truncated transcript, consistent with this hypothesis. exposure of patient's cells to ionizing radiation resulted in normal p53 activation suggesting that p53 signaling is not affected by the truncated protein. however, a cell growth disadvantage was observed. thus, we show that de novo truncating ppm1d mutations in the last and penultimate exon cause syndromic id which provides novel insights in the role of cell cycle checkpoint genes in neurodevelopmental disorders. de novo truncating variants in asxl2 are associated with a unique and recognizable clinical phenotype harvard stem cell institute, department of stem cell and regenerative enriched pathways (q < 0.05) including actin cytoskeleton and calcium ion binding. subsequently we applied the residual variation intolerance score (rvis) and identified 41 genes which were ranked among the 25% most intolerant genes in the genome. these genes included the previously reported genome-wide significant bd risk genes syne1 and mll2. in addition, we identified novel, promising candidate genes which have not previously been implicated in bd development such as ryanodine receptor 3 (ryr3, affected in six patients) and huntingtin (htt, 4 patients). both genes are ranked among the 0.5% most intolerant genes of the genome. ryr3 encodes a brain expressed intracellular cation channel that mediates the rapid release of ca2+ from the endoplasmic reticulum, thus making it a highly plausible candidate gene for contributing to rc. abnormal expansion of a trinucleotide repeat in the htt gene causes huntington disease which is a neurodegenerative disease characterized by motor, cognitive and psychiatric symptoms. the seven most promising genes are currently being followed up by resequencing in larger cohorts of 2500 independent bd cases (including 250 patients with rc) and 2500 controls of european ancestry using the single molecule molecular inversion probes (smmips) technology. de novo truncating mutations in the last and penultimate exon of ppm1d cause a novel intellectual disability syndrome abstracts with gα. signaling properties of g protein complexes carrying mutant gβ1 subunits were further analyzed by their ability to couple to dopamine d1r receptors by real-time bioluminescence resonance energy transfer (bret) assays. these studies revealed altered functionality of the missense mutations r52g, g64v, a92t, p94s, p96l, a106t, and d118g but not for l30f, h91r, and k337q. in conclusion, we demonstrate a pathogenic role of de novo and autosomal dominant mutations in gnb1 as a cause of gdd and provide functional evidence for a loss-of-function mechanism underlying the disease. comprehensive phenotyping and trio-exome analysis of 50 children with neurodevelopmental disease whole exome sequencing (wes) has been proven as a powerful analytical tool to dissect the genetic basis of human hereditary disorders. here, we report on a prospective deep phenotyping and trio-wes study of 50 children affected by previously undiagnosed and diverse complex neuropediatric disorders. all children underwent a standardised and comprehensive clinical work-up in a single centre that included detailed clinical evaluations by pediatricians and clinical geneticists, extensive laboratory and metabolic analyses, analyses of cerebrospinal fluid, mri of the brain and eeg, followed by trio-wes analysis. this systematic approach allowed to identify a pathogenic mutation in a known disease gene in altogether 21 children (42%) and discovered a convincing candidate disease gene in additional 22 children (44%). taken together, this translates into a successful genetic diagnosis of up to 86% in this cohort. in 3 children with mutations in a known disease gene (3/21 = 14.3%) the molecular diagnosis substantially influenced the clinical management and drug treatment. we further document an expansion of the phenotype in known disease entities in 4 individuals. the extraordinary high gene discovery rate in our cohort emphasizes the potential of trio-wes even in a clinically inhomogeneous group of individuals with likely genetic disease. however, this requires a multidisciplinary approach including deep and sometimes reverse phenotyping, research-based interpretation of trio-wes identified genetic alterations, extensive review of the literature, use of several mutation prediction and protein-modelling tools, as well as openness and exchange of data with national and international researchers and clinicians working on similar diseases. exome sequencing of pooled dna samples for large-scale screening in individuals with sporadic intellectual disability b. popp, a. ekici, s. uebe, c. thiel, j. hoyer, a. wiesener, a. reis, c. zweier institute of human genetics, fau-erlangen-nürnberg, erlangen, germany high throughput sequencing has enabled identification of many novel disease genes and empowered diagnostic testing for heterogeneous disorders, especially for intellectual disability (id) where more than 1000 genes have been implicated. due to this extreme heterogeneity gene panels are ineffective, and expensive exome or genome sequencing is necessary. furthermore, many affected individuals have to be sequenced to confirm candidate genes and to refine the phenotypic spectrum. we now explored if pooling strategies could satisfy the need for a genome-wide, simple, cheap and fast screening technology. the asxl genes (asxl1, asxl2 and asxl3) participate in body patterning during embryogenesis and encode for proteins that are involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. germline de novo truncating variants in asxl1 and asxl3 have been respectively implicated in causing bohring-opitz and bainbridge-ropers syndromes, resulting in overlapping features of severe intellectual disability and dysmorphic features. to our knowledge, asxl2 has not yet been associated with a human mendelian disorder. in this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. all six had de novo truncating variants in asxl2. a careful review en abled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia and developmental disabilities. although overlapping features with bohring-opitz syndrome and bainbridge-ropers syndromes exist, features that distinguish the asxl2-associated condition from asxl1-and asxl3-related disorders are macrocephaly, absence of growth retardation and more variability in the degree of intellectual disabilities. we were also able to demonstrate with mrna studies that these variants are likely to exert a dominant negative effect, since both alleles are expressed in blood, with the mutated asxl2 transcripts escaping nonsense mediated decay. in conclusion, de novo truncating variants in asxl2 underlie a new neurodevelopmental syndrome, with a clinically recognizable phenotype. this work expands the germline disorders that are linked to the asxl genes. functional characterization of novel gnb1 mutations as a rare cause of global developmental delay over the past years, prioritization strategies that combined the molecular predictors of sequence variants from exomes and genomes of patients with rare mendelian disorders with computer-readable phenotype information became a highly effective method for detecting disease-causing mutations. the drawback of phenotype-based prioritization, however, is that they require a deep and comprehensive feature description to gain good performance. but in routine diagnostics, the naming of phenotypic features varies among clinicians, and sometimes a comprehensive phenotypic overview is not possible because of missing terminology. these gaps can be reduced by including a new layer of phenotypic information using facial recognition technology to detect dysmorphic features from two-dimensional photographs. automated image analysis is in principle able to identify any deviation from the norm and to quantify it objectively. we therefore developed an approach that combines facial dysmorphology novel analysis (fdna) technology with standard phenotypic and genomic features to identify pathogenic mutations in exome data. we have started collecting data from a diverse spectrum of patients with molecularly confirmed diagnoses in a multi-center study, and we present the current results. at the time of abstract submission more than 300 patients from over 10 contributing institutions were evaluated and used for simulation of a training set of exomes. automated facial recognition yields the correct diagnosis amongst the first ten suggested syndromes in more than two thirds of the cases and shows a high correlation with syndrome predictions that were based on expert annotated features. hereby, we could also confirm the diagnosis in cases with only subtle facial features. consequently, we used classical machine learning approaches to integrate scores based on the image analysis, phenotypic description and exome se-after initial evaluation of available computational methods by virtual pooling of exome data or simulated reads using different pooling fractions, we decided to exome sequence 96 individuals with sporadic id in 8 pools of 12 samples each. this was suggested to be the optimal combination with a 90% detection rate. dna was mixed in equimolar concentrations and submitted for exome sequencing. read data was aligned to the human reference, and variants were called using a ploidy of 24. resulting variant calls in 893 known id genes (sysid database) were then filtered for loss-of-function (lof) variants and for missense variants that were either previously reported as pathogenic or computationally predicted to be deleterious. furthermore, we screened 523 id candidate genes and 1694 haploinsufficiency intolerant genes for lof variants. subsequently, sanger sequencing was used to determine the individual carrying each variant in the respective pool and to test segregation in the parents. this approach resulted in the identification of 15 pathogenic variants (assumed or confirmed de novo) in known id genes (ahdc1, ankrd11, atp6v1b2, cask, chd8, kcnq2, kmt2a, kras, med13l, rit1, setd5, tcf4, wac, zbtb18), two pathogenic variants inherited from a symptomatic or healthy parent, respectively, (zmynd11, ifih1), and a homozygous variant in the recessive trappc11 gene. this included 13 loss-of-function and 5 missense variants. additionally, we identified 4 de novo variants in candidate genes. in our id cohort this resulted in a high mutation detection rate of 23%. thus, detection of rare variants from exome sequenced dna pools (pool-seq) is feasible and has a high detection rate similar other screening approaches. compared to affected-only exome sequencing this method can reduce costs by more than 90% with only marginal increase in sanger-sequencing costs and significantly speed up wet lab work with an acceptable increase in computational complexity. in contrast to targeted sequencing methods like molecular inversion probes or hybridization-based panels, our method has the advantage of allowing flexible re-analysis of the same data for new genes. in conclusion, we established exome pool-seq as a method for large-scale, cost-efficient and flexible sequencing in highly heterogeneous but well characterized disorders like id. three years of experience with targeted next-generation sequencing of developmental delay next-generation sequencing (ngs) has opened up new possibilities especially in the search for disease-causing mutations in disorders with common clinical features but a heterogeneous genetic background. the identification of the underlying genetic defect provides a clear diagnosis for patients more and more influencing their management and occasionally even their therapy, and it is the prerequisite for prenatal or preimplantation decisions in the affected family. ngs panels are used widely in clinical settings to identify genetic causes of various monogenic disease groups, such as intellectual disability (hu et al. 2015) , neurodevelopmental and neuromuscular disorders, among others. however, many new challenges have been introduced both at the technical level and at the bioinformatic level, with consequences including new requirements for interpretation of results, and for genetic counseling. we report on our experience with a targeted ngs panel comprising over 1200 brain related genes (mpimg-1-test) in the routine clinical diagnostics of patients with syndromic and non-sydromic forms of developmental delay as well as patients with neuromuscular disorders. 202 patients (age 1-71; mean 16) with syndromic (s) or non-syndromic (ns) developmental delay or with neuromuscular symptoms (nm), seen at the genetic counselling unit of our institute, were analyzed with targeted exon enrichment and ngs. chromosomal re-arrangements and copy number variations were excluded in all 202 patients previously by conven-it was recently shown that that clonal hematopoiesis can be driven by somatic point mutations. these acquired mutations occur with normal aging in up to 10% of older (>65y) individuals (1) (2) (3) and few reports in younger individuals. here we present a targeted re-sequencing assay that combines high throughput with ultra-high sensitivity based on single-molecule molecular inversion probes (smmip) (1) . we have now analyzed dna from 2000 healthy blood donors from 5 different age groups (20-29; 30-39; 40-49; 50-59 and 60-69y) , with no previous diagnosis of cancer, for somatic mutation in 100 loci. those loci included 50 known drivers of clonal hematopoiesis (2) (3) (4) and 50 novel or candidate loci. the improved assay allows low-frequency variant detection with variant levels down to <0.1%. this improved sensitivity allowed the identification of somatic mutations in a limited set of loci in >15% of old individuals, but also report those mutations in individuals of the youngest age group. most prevalent mutations include known hotspot mutations in dnmt3a and asxl1. here we show that somatic drivers of clonal hematopoiesis are more prevalent and occur in younger individuals than previously reported. these somatic events are age-related. however, the high prevalence and their occurrence in relatively young individuals implicates their origin as a common biological process involved in normal aging. a. m. nissen, j. graf, c. rapp, m. locher, a. laner, a. benet-pagès, e. holinski-feder medizinisch genetisches zentrum -mgz, munich, germany gene dosage abnormalities account for a significant proportion of pathogenic mutations in rare genetic disease related genes. in times of next generation sequencing (ngs), a single analysis approach to detect snvs and cnvs from the same data source would be of great benefit for routine diagnostics. however, cnv detection from exon-capture ngs data has no standard methods or quality measures so far. current bioinformatics tools depend solely on read depth which is systematically biased. we developed a novel approach based on: 1. utilization of five independent detection tools to increase sensitivity, 2. different reference sets for different kits and normalization against samples from the same sequencing run to improve robustness against workflow conditions, 3. definition of special quality thresholds for single exon events to minimize false negatives, 4. identification of reliable regions by assessment of capture efficiency using a reference set of cnv negative patients to minimize false positives. a cnv quence of the patients and could predict the pathogenic mutation among the top 10 positions in a prioritized exome in more than 90% of the monogenic cases in our cohort. hence, our results show that computer-assisted facial recognition is not only a promising technology that could be applied in the routine diagnostic workflow, but also a technology that allows diagnosis in cases with non-typical clinical presentation and boosts the diagnostic yield in exome studies. the added value of rapid exome sequencing in critical clinical situations for critical clinical situations, turnaround times (tats) of exome sequencing need to be fast in order to have an impact on clinical decision making. we therefore set out to develop a fast exome sequencing approach (max. 14 days). urgent exomes are preferably sequenced as trios to enable de novo analysis and assist data interpretation. dna library preparation is performed using the sureselect qxt protocol (v5, agilent), and sequencing is done on a nextseq500 (illumina) with a high coverage (200-300x). automated file handling allows rapid bwa mapping, gatk variant calling and annotation. a total of one-hundred samples have been sequenced until now using this rapid procedure: 28 trio's, 1x mother and child, 2x parents plus 2 children, and 6 single cases. six trios with known aberrations were used for experimental setup. of the remaining 31 families, in 14 families (possible) pathogenic snvs were identified, of which some still need further follow up, whereas 17 families remained negative after inspection of snvs and small indels. for cnv analysis, a trio based reference-free cnv approach is still under development. preliminary data show that all control cnvs (53kb-6mb) are detected correctly, and retrospective cnv analyses of the other samples identified three possibly de novo cnvs that need further follow up. shorter tats days were already beneficial for some patients, i. e. an adult male suffering from myelofibrosis and autoinflammatory symptoms. a sting-like phenotype (= stimulator of ifn genes) was suspected, with a possible involvement of the jak/stat pathway. urgent exome sequencing was performed and results were available within 9 days. interestingly, both a somatic variant in mpl (= trombopoetine receptor > myelofibrose) and a heterozygous variant in acp5 (trap, known immune dysregulation disorder) were identified, both fitting to the patients phenotype. based on these results the medication of the patient was changed, resulting in a substantial improvement of the patients constitution. in conclusion, we have implemented a rapid exome sequencing workflow for urgent cases. the rapid identification of pathogenic variants already had implications on patient treatment, underlying the added value of a fast genetic diagnosis. ement insertion, however, was spliced into the mrna of nabp1, leading to a frameshift mutation and a premature stop codon, potentially altering or abolishing gene function. in summary, we have shown that transposon insertions, both common variants as well as rare or de novo variants, can be detected in wes data. such insertions in coding or regulatory regions of disease-relevant genes might therefore explain some of the cases in which no pathogenic coding mutation can be identified by wes. the influence of human genetic variation on epstein-barr virus sequence diversity: a genome-to-genome approach c. hammer 1, 2 , a. loetscher 3, 4 , em. zdobnov 3, 4 genome-wide association studies (gwas) have identified common genetic polymorphisms that associate with clinical manifestation and immune response parameters of various infections. we here present an alternative approach, using variation in the virus sequence as phenotype, which is specific by nature and unique to genomic research in infectious diseases, for genome-to-genome (g2g) association studies. building on the unprecedented possibility to combine large-scale human and viral genomic data, we explored interactions between human genetic variation and viral sequence diversity in individuals infected with epstein-barr virus (ebv). the major goal is the identification of key genetic players in the evolutionary 'arms race' between pathogen and host. ebv is the pathogenic agent of infectious mononucleosis and is associated with a broad spectrum of lymphoid and epithelial malignancies, including lymphomas and nasopharyngeal carcinomas. there is also evidence for a role of ebv in the pathoetiology of multiple sclerosis. its genome is approximately 190 kbp long and encodes around 80 proteins, not all of which have been definitely identified or characterized. it is known that high loads of ebv are present in patients with advanced human immunodeficiency virus (hiv)-induced immunodeficiency. we therefore selected 780 immunosuppressed patients included in the swiss hiv cohort study (shcs) with low cd4+ t cell counts, and quantified ebv copy number in peripheral blood mononuclear cells (pbmcs). 290 cell samples contained more than 2,000 viral copies in total and were subjected to target isolation and subsequent enrichment using the sureselect method by agilent biotechnologies, followed by illumina whole-genome sequencing. after data processing and quality control, variable amino acids were called as binary variables, resulting in >200 variable positions per individual in average. the same patients also underwent genome-wide genotyping to obtain host genetic variation, followed by imputation based on the haplotype reference consortium reference panel. the association analyses are currently ongoing, and we will present the results at the conference. we use logistic regression to test for association between host single nucleotide polymorphisms (snps) and binary ebv amino acid variants. bonferroni correction is applied for multiple testing correction on the sides of both host and pathogen. stratification is taken into consideration by including principal components (pcs) for the host, and phylogenetic pcs for the virus. this project will offer a global description of the adaptive forces acting on ebv during natural infection. we have shown before for hiv that a virus genome associates much more strongly with human genetic variants than clinical endpoints. the analysis of all signals resulting from the interaction between human and viral genomes has the potential to identify novel host defense mechanisms, which could serve as future diagnostic and therapeutic targets. is called in a reliable region if at least two out of five tools are concordant for the respective cnv. the pipeline shows a sensitivity of 80% and a precision of 95%. within routine gene panel diagnostics we analyzed a total of 1088 patients indicated to have rare mendelian diseases for snv and cnvs. in 32 patients a cnv was detected in genes associated with the respective individual phenotype. interestingly, in several cases the cnv completed the patients report as it was detected in genes with a recessive mode of inheritance where previously only a heterozygous pathogenic snv was found. overall, with the additional analysis of cnvs we increased the diagnostic yield from 15% (class 4, 5 single nucleotide events) to 18%. however, there are still issues in the detection of cnvs from ngs data for routine diagnostics. cnv pipelines are very prone to errors caused by enrichment inconsistencies compared to snv detection tools. the assessment of sensitivity and specificity is difficult due to the lack of datasets to validate cnv detection pipelines. originally, the analysis of cnvs was performed mainly in patients with mental retardation disorders, resulting in a paucity of cnv data linked to other mendelian diseases. moreover, the identification of the actual size and thus the assessment of pathogenicity of a cnv is difficult, because targeted ngs gene panels do not cover all genes. in conclusion, ngs data is a suitable data source for the simultaneous detection of snvs and cnvs for clinical diagnosis; however, with the current tools it is only applicable in accurately validated regions. identification of transposon insertions in whole-exome sequencing data s. lukassen, n. übelmesser, ab. ekici, u. hüffmeier, ct. thiel, c. zweier, a. winterpacht humangenetisches institut, erlangen, germany 45% of the human genome consists of transposable element derived sequences, the most abundant of which are l1 and alu elements, followed by endogenous retroviruses. several hundred of these elements remain active, leading to insertion frequencies of up to one in 20 live births for alu elements and posing a threat to genome integrity. while most studies on transposons employ whole-genome sequencing (wgs) or target-enrichment based sequencing approaches, the most commonly used form of diagnostic high-throughput sequencing is currently whole-exome sequencing (wes). we were therefore interested in investigating transposon insertions in wes data as a possible source of disease causing mutations. we developed a software to call non-reference transposon insertions from single-end wes datasets by split-read mapping and analyzed 385 exomes this way. on average, 188 non-reference insertions were identified in each exome, with an average of 1.7 sites per patient identified in < = 0.5% of other patients. of these rare variants, 91% were deemed plausible by visual inspection. automated confidence calls of the software were concordant with visual inspection in 87% of cases. in 5% of cases a plausible insertion was awarded a lower score by the algorithm and in another 5% not called at all. in 1% of cases the automated call appeared to be falsely positive, in another 1% at the wrong position within the same 100bp window. laboratory validation of 11 convincing insertions revealed a 73% true positive rate, leading to an estimated specificity of 67%. when performing calls for reference l1 insertions on 10 exomes, 65% (49% -70%) of known elements whose flanking regions were covered by at least two reads were correctly identified, leading to a sensitivity of 65%. we thus estimate the average number of non-reference transposon insertions in our wes dataset to be 194 (153-235). 67% and 22.2% of sites identified were associated with alu and l1 elements, respectively, with the majority of calls stemming from evolutionary young transposons still assumed to be active. 10.3% of sites were located within the cds, 3.4% in the utrs of genes, 0.5% spanned an intron/ exon border, 48.5% were intronic and 37.3% of insertions were found in intergenic regions. we then chose 8 insertions within intronic (7) or utr (1) regions for further analysis. seven were not detected in the mrna. one intronic alu el-abstracts ws6-002 novel insights into male-pattern baldness pathobiology via integration of differential hair follicle mirna and mrna expression profiles with gwas data male pattern baldness (mbp) is a highly heritable condition and the most common form of hair loss in men. the phenotype is characterized by a distinct pattern of androgen-dependent progressive hair loss from the scalp that is restricted to hair follicles (hf) in the frontal and vertex scalp area. the molecular mechanisms that underlie this characteristic pattern and the differences in androgen-sensitivity between hf subpopulations in the frontal/vertex and the occipital scalp remain however elusive. to gain novel insights into the underlying biology and contributing genes and pathways, we systematically investigated for a differential expression (de) of mirna-and mrna-genes in hf samples from the frontal and occipital scalp area of 24 healthy male donors. array-based genome-wide mirna and mrna profiling revealed expression of 823 mirnas and 21,247 mrnas in human hf, of which 144 mirnas (17%) and 3,230 mr-nas (15%) showed a de between hf subpopulations. the strongest de mirnas included mir-4674, mir-6075 and mir-3185. among the strongest de mrnas were the wnt-signaling inhibitor dkk1, the protein kinase pak1 and the retinoid acid receptor rora. a subsequent pathway-based analysis in mirpathdb revealed that de mirnas targeted numerous interesting pathways. among them the wnt-and mtor signaling pathway which have been implicated in the control of hair follicle cycling, a mechanism that is disturbed in mpb affected hf and other plausible candidate pathways such as estrogen, thyroid hormone signaling or epidermal growth factor binding which have not yet been implicated mpb pathobiology. to yield further evidence for an involvement of de mirnas and mr-nas in the developement of mpb, we subsequently integrated our expression data with association data from a large gwas meta-analysis on mpb (n = 22,518). of the de mirnas and mrnas, only 1 mirna (mir-193b) and 49 mrnas were located within 1 mb of one of 63 genome-wide significant mpb risk loci. notably, the analysis revealed a co-localization of de mirna, de mrna, and nominally significant association signals (p < 10 -5 ) at 9 other genomic loci, pointing towards a role of these genomic regions in mpb pathogenesis. among them a locus on chromosome 3q22.2 that comprises the genes encoding the ephrin-type-b receptor 1 (ephb1) and the prostaglandin transporter slco2a1. interestingly, ephrins have been shown to be regulated by androgens and to play a role in hf formation, proliferation and hair cycling. and expression of prostaglandin d2, which is transported by slco2a1, has been found to be upregulated in balding scalp where it inhibits hair growth. in summary, our systematic analysis of differential mirna and mrna expression and the subsequent integration with genetic association data identified 9 novel potential risk loci for mpb and numerous candidate genes and pathways that are likely to play a role in mpb pathogenesis and emphasizes the importance of data integration of large-scale omic-analyses. palaeontological genomic analyses have shown that interbreeding between anatomically modern humans and neandertals occurred in europe and asia 40.000-50000 years ago. approximately 1.5-2% of the modern european and asian genome consists of introgressed dna from neandertals. some of these introgressed regions have been suggested to contribute to several traits and phenotypes including major depression and other mood disorders. in order to further assess the role of neandertal ancestry in cognition and the contribution of genetic risk for psychiatric disorders, we performed genome-wide analyses of neandertal alleles in publicly available psychiatric genomics consortium (pgc) gwas summary statistics with samples sizes ranging from about 6000 to 293723 individuals for the following phenotypes: educational attainment, attention deficit hyperactivity disorder (adhd), anorexia nervosa, anxiety disorders, autism spectrum disorder, bipolar disorder, major depressive disorder and schizophrenia. we estimated the proportion of heritability explained by snps in neandertal introgressed regions using stratified ld score regression (ldsc) and two sets of previously inferred neandertal introgressed regions. in a secondary analysis, we investigated whether specific functional annotations such as 3'utr, promoter regions or histone marks within neandertal regions were significantly associated with selected phenotypes. we identified a modest enrichment of heritability in neandertal introgressed regions in anorexia nervosa, autism spectrum disorder, bipolar disorder and major depressive disorder, although none of the results were statistically significant. several functional annotations, such as h3k4me1 histone marks within neandertal introgressed regions, appeared significantly enriched for snps contributing to the heritability of anorexia nervosa and autism spectrum disorder. in bipolar disorder, dnasei digital genomic footprinting regions, h3k9ac histone marks and super enhancer regions within neandertal regions appeared particularly enriched for heritability. on the other hand, both sets of neandertal regions were slightly depleted of snps contributing to the heritability of schizophrenia. for example, one set of neandertal regions that contained 33% of all analysed snps only contributed to 29% of the variance of risk (standard error: 0.04; p-value: 3.86 × 10 -3 ). in comparison to the rest of the genome, neandertal introgressed regions also contributed less to the heritability of educational attainment, adhd and anxiety disorders, although these findings were not statistically significant. to our knowledge this is the first study to systematically investigate the extent to which snps attributable to neandertal introgressed regions contribute to the heritability of several psychiatric/cognitive phenotypes. we are currently increasing our power to detect snp heritability in neandertal regions by applying the ldsc method to larger pgc datasets. harbor genes involved in the complement system, high density lipoprotein metabolism or extracellular matrix homeostasis. these pathways are known for their pleiotropic role in other conditions, such as cardiovascular disease, auto-immune diseases and cancer. here we aimed to investigate the extend of overlap between the genetic risk of various complex diseases and traits and the genetic risk for amd. methods: first, we catalogued 2,331 previously published, genome-wide significant variations associated with 82 complex diseases or traits. next, we computed a genetic score by calculating the (weighted) sum of risk increasing alleles for each disease or trait. consequently, a higher genetic score indicates that an individual has more risk/trait increasing alleles of a given disease or trait. for each score, we computed the association with late stage amd using a dataset provided by the international amd genomics consortium (iamdgc) including 16,144late stage amd cases and 17,832 controls. we also assessed the association of each variation individually with late stage amd risk in order to identify novel disease loci with strong evidence for pleiotropy. results: nineteen genetic scores of complex diseases and traits were significantly associated with amd risk (fdr < 0.01). most notably, all genetic scores related to autoimmunity were elevated in amd patients (p < 5.85×10-09 ), while scores related to cardiovascular disease were reduced in amd patients compared to controls (p < 3.10×10 -05 ). we also found that the genetic scores of melanoma and related malignancies were higher in amd patients (p < 8.43×10 -05 ). in addition, 32 out of 2,331 variants, which were used to compute the genetic scores, were significantly associated with amd (fdr < 0.01), implicating 25 novel, pleiotropic loci in amd risk. conclusion: our findings demonstrate a substantial overlap between the genetic risk of complex diseases/traits and the genetic risk for amd and provide evidence for 25 novel, pleiotropic loci associated with amd. while our findings highlight common disease pathways that may facilitate to develop multi-use drug targets, they also challenge the notion that gene/genome manipulation could be applied in general terms to eradicate risk for a defined complex disease. worldwide genetic association study of exfoliation syndrome and glaucoma identifies common genetic variants at five new susceptibility loci exfoliation syndrome (pex), a complex systemic disorder of the extracellular matrix, is the commonest cause of secondary glaucoma in aging population and thus a major cause of blindness globally, affecting 60-70 million subjects worldwide. inside a large, international collaboration project a genome-wide association study (gwas) was carried out on 9,035 pex cases and 17,008 controls, recruited from 24 countries across six inhabited continents, with replication in a further independent 4,585 cases and 92,829 controls from 15 countries. significant association was observed at seven loci, of which two confirmed the already known associated loci at the genetic markers mapping to loxl1-and cana1a-gene, five are new (p < 5×10 -8 ). the five new loci map to chromosomes 13q12 (rs7329408 near flt1-pomp-slc46a3, p = 9.41 × 10 -16 ), 11q23.3 (rs11827818 near tmem136-arhgef12, p = 1.21 × 10 -10 ), 6p21 (rs3130283 at agpat1, p = 2.12 × 10 -9 ), 3p24 (rs12490863 at rbms3, p = 3.9 × 10 -8 ) and 5q23 (rs10072088 near sema6a, p = 3.64 × 10 -8 ). to determine the pathophysiological role of the three most significantly associated loci (13q12, 11q23.3, 6p21), we investigated the expression and localization of the six related genes (flt1, pomp, slc46a3, tmem136, arhgef12 and agmale-pattern baldness (mpb) is characterized by a progressive hair loss from the frontal and vertex scalp that affects ~80% of men at the age of 80 years. epidemiological studies have shown positive associations between mpb and coronary heart disease (chd) and related phenotypes such as blood pressure (bp), diabetes (dm) or elevated blood lipid levels. the results however vary with regard to the associated pattern of hair loss (frontal or vertex) and the assessed endpoint measures for chd. and so far no study has investigated for a shared genetic determinant between the traits. using data from the heinz nixdorf recall study (n = 1,675 males) and a large meta-analysis on mpb (n = 22,518), we aimed at a systematic investigation of the association between mpb and chd on (i) an epidemiological and (ii) a genetic level. , men with vertex balding showed a higher bmi (β = 1.4 kg/m2), elevated fasting triglyceride (β = 8.0 mg/dl) and lower hdl-c levels (β = -2.7 mg/dl). to assess the genetic overlap between mpb and chd, we created a risk score (rs) from 63 mpb lead snps (p < 5×10 -8 ) and tested for association with chd and related traits phenotypes. no significant associations were observed. however, an age-stratified analysis revealed a 4% per allele risk increase for chd (hr = 1.04, 95%ci:0.97;1.17) and a decrease in fasting triglyceride levels (β = -0.5). we next used ld score regression analysis in to test for genome-wide genetic correlation between mpb and chd. the analysis revealed no significant correlations with cardiometabolic (n = 3), lipid (n = 4) or metabolic traits (n = 103). finally, to investigate for a genetic overlap at single loci, we compared the mpb risk loci with reported gwas signals for chd. the analysis identified seven overlapping associations between mpb and bp (n = 3); qt-interval length; atrial fibrillation; sudden cardiac arrest; and dm. for the majority of loci, the direction of effect differed between mpb and chd, opposing previous epidemiological findings. positive associations were identified between mpb and diastolic bp (fgf5, 4q21.21) and sudden cardiac arrest (atf1, 12q13.12). interestingly, fgf5 is known to stimulate cell growth and proliferation in multiple cell types, including cardiac myocytes and hair follicle (hf) cells, and atf1 is a hf expressed regulator of cell growth and differentiation that has been shown to prevent foam cell formation, which suggests that fgf5and atf1-signaling contribute to both traits. thus, our data support an association between mpb and chd related phenotypes and suggest that mpb deserves further evaluation as an additional risk factor for chd. pleiotropic effect of genetic variants associated with complex diseases and traits in age-related macular degeneration purpose: age-related macular degeneration (amd) is the leading cause of vision loss in western societies and is caused by both environmental and genetic risk factors. with regard to the latter, several associated risk loci abstracts otyping, results of which will be presented at the conference. of note, for nonsyndromic cleft lip with/without cleft palate (nscl/p), the most frequent form of orofacial clefting, 20 risk loci have been detected by gwas so far, with some of them reaching (nearly) genome-wide or significant p-values in samples much smaller than 550 cases. in the imputed nscpo dataset none of the presently known nscl/p risk loci showed a p-value < 10 -5 . our data so far confirm previous molecular and epidemiological findings, that nscpo is genetically distinct from nscl/ p. furthermore, the results indicate that common variants alone might not contribute to the same extent to nscpo as compared to nscl/ p. the correlation between defects at specific imprinted loci and distinct imprinting disorder (id) was accepted for a long time. however, it is now put into question because of a growing number of patients with multilocus imprinting disturbances (mlid), i. e. the aberrant methylation at more than one imprinted locus. in particular, mlid is present in individuals with silver-russell syndrome (srs) and beckwith-wiedemann syndrome (bws), and it has meanwhile turned out that patients with opposite phenotypes can share common epimutation patterns. on the other hand, mlid always occurs as mosaicism and varies in different tissues of the same individual. interestingly, the majority of mlid carriers show only one specific id phenotype, though loci of other ids are affected in addition to the one specific for the phenotype. we become aware of a growing number of patients with unexpected and even contradictory molecular findings in respect to the clinical diagnosis for referral. amongst others, we detected the srs specific icr1 hypomethylation in 11p15 in two of our patients referred as bws. in the first case, the icr1 hypomethylation was detected only in lymphocytes but was not present in buccal swab dna. the patient only had a slight asymmetry, but showed normal growth and did not exhibit any other feature compatible with bws, nor with srs. the reason for the lack of clinical features is unclear, but is comparable to the observation in monozygotic, but clinically discordant srs and bws twins. here the unaffected twin often carries the epimutation only in lymphocytes whereas the affected one shows the alteration in additional tissues. a reason might be sharing of hematopoietic stem. it can be postulated that the patient presented here is born after an (undetected) twin pregnancy with early loss of the affected twin. in the second case, the initial diagnosis of bws was made due to asymmetry, though overgrowth or other features were not present. further clinical ascertainment did not confirm this diagnosis, but growth of the patient was in the lower percentiles, in concordance with the icr1 hypomethylation. these cases as well as further cases in our cohort confirm that there is an urgent need to provide detailed clinical data upon requesting molecular diagnostics for imprinting disorders. in fact, the growing number of patients with unexpected results complicates the interpretation and illustrates the broad phenotypic range, but also provides further insights in the etiology of ids and setting of imprinting marks pat1) by qrt-pcr, immunohistochemical-and western-blot analysis in genotyped ocular tissues of pex and control patients. all six genes displayed moderate mrna expression in all ocular tissues analysed, with highest levels in iris, ciliary body, and retina. however, only pomp showed a trend towards reduced expression in the presence of the rs7329408 risk allele, in both pex and control patients. in general, both mrna and protein expression of pomp and tmem136 were significantly reduced up to 45% (p < 0.005) in anterior segment tissues in pex eyes compared to controls. no differences in mrna and protein expression were detected for the remaining genes analysed. immunofluorescence analysis showed that pomp, a proteasome maturation protein, is ubiquitously expressed in most ocular cell types and that tmem136, a transmembrane protein of unknown function, is primarily localized to endothelial cells of blood vessels and aqueous outflow structures. additionally, protein staining intensities for pomp and tmem136 were markedly reduced in anterior segment tissues of pex eyes compared to controls and co-localized to abnormal accumulation of pex material on ocular surfaces and in blood vessel walls. thus, at least two of the newly identified loci provide new biological insights into the pathology of pex syndrome/glaucoma and highlight a role for impaired proteasome function as well as vascular and trabecular endothelial dysfunction in the disease pathogenesis. nonsyndromic cleft palate only -evidence for a limited contribution of common variants in contrast to nonsyndromic cleft lip ± palate cleft palate only (cpo) is a common congenital malformation which might occur as part of a syndrome or in an isolated form, i. e., nonsyndromic cpo (nscpo). nscpo has a prevalence of 1:2500 and is considered multifactorial with genetic as well as environmental factors contributing to the disorder. in a recent study we identified the first genome-wide significant locus for nscpo which has been independently confirmed in another study. in order to discover more nscpo risk loci we performed a genome-wide imputation study with gwas data from 550 case-parent trios with european, asian and african ancestry which was retrieved from db-gap upon approved data access. notably, this gwas dataset had not yet been imputed, and we hypothesized that we can increase power to identify novel genetic associations by increasing the marker density and follow-up of suggestive findings by independent replication. genome-wide genotypes were imputed using impute2 based on 1000 genomes haplotypes, and snps were selected based on info-score > 0.4 and minor allele frequency > 1%. the imputation did not reveal any genome-wide significant snp, however, 83 snps at 26 loci showed p-values < 10 -5 . loci with more than two variants below this threshold (n = 19) were to be replicated using the massarray system (agena bioscience). three independent samples were used: two case/control replication cohorts from central europe (92 cases, 335 controls) and yemen (37 cases, 231 controls), and one european case-parent trio replication cohort (eurocran study; 223 trios). in a first round we genotyped 18 snps at eleven loci. one variant, rs6809420 at chr. 3q13, showed p < 0.1 in the replication cohort and after combining replication and gwas data, resulted in a decrease of p-value from 1.14×10 -03 to 3.22×10 -04 . this indicates that this locus, which includes candidate genes such as igs11, a known cell-adhesion molecule with yet unknown function in craniofacial development, might harbour a common risk variant with low effect size. we are currently performing a second round of gen-we report biallelic mutations in cad, encoding an enzyme of de novo pyrimidine biosynthesis, in four patients with developmental disability, epileptic encephalopathy, anaemia, and anisopoikilocytosis. two children died after a neurodegenerative disease course. treatment of two surviving children with oral uridine led to immediate cessation of seizures in both. a four-year-old girl, who was previously in minimal conscious state, started to communicate and walk with assistance after nine weeks of treatment. a three-year-old girl likewise showed developmental progress. blood smears normalised and anaemia resolved. our findings support the efficacy of uridine supplementation rendering cad deficiency a treatable neurometabolic disorder. delineation of the grin2a phenotypic spectrum alterations of the n-methyl-d-aspartate (nmda) receptor subunit glu-n2a, encoded by the gene grin2a, have been associated with a spectrum of neurodevelopmental, speech and epilepsy disorders. we identified 48 previously unreported patients with heterozygous pathogenic variants in grin2a, including 30 novel variants. after re-evaluation of all published grin2a cases, 104 previously reported patients met the acmg criteria for being pathogenic or likely pathogenic. thus, we are able to collectively review genotypes and phenotypes of 152 individuals with grin2a-related disorders. we show that the known phenotypic spectrum is expanded and ranges from near-normal development to severe and unspecific encephalopathy, comprising any disorder of speech development. furthermore, some patients do not display seizures. in contrast to previous reports, gri-n2a missense variants cluster within the functionally most relevant domains. we are the first to describe genotype-phenotype correlations in grin2a-related disorders, where carriers of pathogenic missense variants tend to have more severe neurodevelopmental phenotypes compared to carriers of truncating variants. the most severe end of the phenotypic spectrum was found to include novel features, such as infantile spasms and arthrogryposis and was associated with pathogenic variants in the pore-forming domain of grin2a. the eponymic name galloway-mowat syndrome (gamos; omim 251300) has been coined for the association of early-onset nephrotic glomerulopathy, microcephaly with variable brain anomalies, and facultative diaphragmatic hernia. it is supposed to be inherited as an autosomal recessive trait and clinical as well as genetic heterogeneity has been suggested. in 2014, wdr73 mutations were identified as a cause of gamos, but only a few cases have been reported to date. over the last 15 years, we have collected dna samples and clinical data from 56 unrelated families with one or more children affected by gamos or a gamos-like syndrome (glomerulopathy plus variable anomalies of brain morphology or function as inclusion criteria), including 15 consanguineous families. in this cohort, we performed whole exome sequencing followed by targeted analysis by sanger and ngs multigene panel resequencing. in a total of 25 families of this cohort (46%) the probable underlying genetic defect could be identified. in affected individuals from two consanguineous families, homozygous mutations of wdr73 could be found (vodopiutz et al., 2015) . thus, this gene accounted for only 4% of cases of our cohort. the affected child of another family had a novel homozygous mutation in arhgdia. this gene has previously been described in three families to cause early-onset steroid-resistant nephrotic syndrome (gupta et al., 2013; gee et al., 2013) , but there is some evidence that non-specific brain anomalies may also be part of the arhgdia-associated phenotype. fourteen and three index patients from unrelated families had mutations in one autosomal (osgep) and one x-linked gene (lage3), respectively, both encoding for components of the keops protein complex that has been implicated in transcription, telomere maintenance and chromosome segregation. no human phenotype has previously been assigned to mutations in this complex. notably, eight unrelated families with an identical mutation originated from the east asian population where the carrier frequency for this allele is 0.0008. in one consanguineous family with multiple affected children the disease segregated with a homozygous mutation in the sgpl1 gene encoding for sphingosine-1-phosphate lyase. in four families, the kidney phenotype could be attributed to mutations in genes for non-syndromic nephrosis (nphs1, plce1, one novel gene), while the brain phenotype was apparently independent. in conclusion, the molecular genetic findings in this cohort confirmed that gamos is exceedingly heterogeneous, and still in almost half of the patients with a gamos-like phenotype the genetic cause remained unclear. on the basis of our findings we are now able to define new biologic mechanisms that are critically involved in both, brain development and integrity of the glomerular filtration barrier. genotype phenotype correlations are emerging. finally, we demonstrate that gamos can also be inherited as an x-linked trait. abstracts taminase 1 gene (tgm1) is mutated in the majority of patients (around 30%), and its gene product, tgase1, is therefore primarily targeted in our approach for protein substitution. patients with arci have an impaired skin barrier function, most of them are born with a collodion membrane and suffer subsequently from varying degrees of hyperkeratosis, erythema, transepidermal water loss and infections. the disease can be life threatening neonatally but lacks a causative therapy and is still only treated symptomatically. therefore, our aim is to develop a personalised, causative therapy where the defective protein is substituted topically via a nanocarrier. therapeutic, human tgase1 was synthesized in hek 293 cells and assessed by western blot and flow cytometry analysis. enzyme activity was measured by in vitro assay. tgase1 was then coupled to a polyglycerol-based nanogel (dpg-ng) containing the thermoresponsive linker poly(n-isopropyl)acrylamide (pnipam), stabilising the enzyme as well as adding a thermal protein release trigger at 35°c, which is favorable for cutaneous applications. immunocytochemical stainings for tgase1 on monolayered basal keratinocytes that lack tgm1 expression confirmed the successful uptake of extrinsic tgase1 into the cells. further analysis over time showed that the enzyme was no longer detectable after 48 h and consequently led us to define a treatment schedule for the following experiments. 3d full thickness skin models were used as in vitro system to determine barrier function and enzyme activity after treatment with varying concentrations of the dpg-ng/tgase1 complex. three different sets of skin models were used for these experiments: normal models mimicking the healthy skin with an intact barrier function, models where tgm1 was knocked down, and models made with arci patient cells with tgm1 mutations. franz cell tests on treated skin models lacking intrinsic tgase1 confirmed the impaired barrier activity in disease models, demonstrated an improved barrier function after repeated treatments with dpg-ng/ tgase1 and showed restored tgase1 activity using an in situ assay. furthermore, first toxicity tests using mtt revealed high biocompatibility of dpg-ng/tgase1 after treatment of 2d and 3d cell cultures. these findings are successful steps for an advanced topical drug delivery system and are a promising approach for causative therapeutic intervention in arci. after further optimization concerning protein dosage and thorough toxicity tests, we will adapt this system also for the use with other proteins involved in arci. pigmentation disorders (pds) comprise a large group of rare and heterogeneous disorders that are mainly characterized by various coloration abnormalities affecting single parts of the body or the complete integument. the large group of pds includes the autosomal dominant inherited hyperpigmentation disorder dowling-degos disease (ddd). ddd is genetically heterogeneous, and to date causal mutations in three genes, namely krt5, pofut1 and poglut1 have been identified. after exclusion of mutations in these genes, we performed exome-and sanger-sequencing in six unrelated ddd-patients/families and identified six heterozygous truncating mutations in psenen encoding the presenilin enhancer protein 2. on closer examination of the histological sections, we came upon a novel feature that distinguished these individuals from previous ddd-cases by the presence of follicular hyperkeratosis. to assess the functional significance of psenen mutations in ddd pathogenesis, we performed mammalian cell culture based studies and knockdown experiments of psenen homolog psenen in zebrafish larvae (zfl). knockdown of psenen in zfl resulted in a phenotype with scattered pigmentation, which mimicked human ddd. in vivo-monitoring of pigment cells in the developing zfl suggested that disturbances in melanocyte migration and differentiation underlie ddd pathogenesis. interestingly, six of the psenen mutation carriers presented with co-morbid acne inversa (ai), an inflammatory hair follicle disorder. all individuals had a history of nicotine abuse and/or obesity, which are known trigger-factors for ai. although psenen mutations have been identified in a small subset (<1%) of familial ai previously and the co-manifestation of ddd and ai has been reported for decades, our study is the first to demonstrate experimentally that mutations in psenen indeed can cause co-manifestation of ddd and ai, most likely triggered by predisposing factors for ai. thus, the present report describes a clinically and histopathologically novel ddd subphenotype in psenen mutation carriers, which is associated with an increased susceptibility to ai. protein substitution therapy for autosomal recessive congenital ichthyosis (arci) overall burkitt lymphoma showed a low genomic complexity with a low number of snvs and svs. however, the integration of cnas, snvs and svs allowed us to identify recurrently affected genes, which are involved predominately in the pi(3) kinase pathway, tonic bcr signaling, and cell cycle regulation, chromatin composition and germinal center development. burkitt lymphoma (bl) is the most common mature aggressive b-cell lymphoma in childhood. the genetic hallmark of bl is a chromosomal translocation involving the myc oncogene and one of the immunoglobulin loci leading to myc deregulation. three epidemiologic variants of bl are differentiated: endemic (ebl), which occurs predominantly in equatorial africa and is associated with ebv-infection, sporadic (sbl), which occurs in westernized countries and immunodeficiency-associated. in addition, burkitt leukemia (b-al) is differentiated from bl in cases with more than 25% of the bone marrow cells being lymphoma cells. another rare bl-variant is myc-positive precursor b-cell acute lymphoblastic leukemia coexpressing tdt and myc (tdt+bl). finally, we recently described a myc-negative variant which shows a typical alteration on chromosome 11 (mnbll). the aim of the present study was to examine the epigenetic landscape of these bl variants. to this end, we analyzed the dna methylation of 116 bl (60 sbl, 29 ebl, 10 b-al, 15 mnbll, 2 tdt+bl) using the humanmethylation450 beadchip and contrasted the findings to 24 diffuse-large b-cell lymphoma (dlbcl) and 30 follicular lymphoma (fl). the majority of lymphoma were recruited in the framework of the icgc mmml-seq and mmml projects. the ebl were obtained from the nci ghana burkitt project. as controls, we used public available dna methylation data from 93 b-cell burkitt lymphoma (bl), including its leukemic variant burkitt leukemia (b-al), is the most common type of pediatric b-cell lymphoma accounting for 30-40% of new cases. its biological hallmark is the ig-myc translocation involving myc and mostly the immunoglobulin heavy (igh) locus or more rarely one of the immunoglobulin (ig) light chain loci. at the cytogenetic level the ig-myc translocation is the sole abnormality in around 40% of cases. overall, bl is characterized by a low genomic complexity. the aim of the present study was to analyze the genomic and transcriptomic landscape of pediatric/adolescent burkitt lymphoma by sequencing according to the guidelines of the international cancer genome consortium. a total of 39 samples of bl/ b-al from pediatric/adolescent patients entered this sequencing study. all patients were treated in population-based prospective clinical trials. inclusion criteria were besides availability of suitable materials, consent to participate in the study and appropriate diagnosis: age at diagnosis (≤ 19 years), the presence of ig-myc rearrangement detected by fish and/or whole genome sequence (wgs), absence of rearrangements of bcl2 or bcl6 genes. we performed wgs of tumor and matched control as well as transcriptome sequencing of the tumor cells according to the standards of the icgc (www.icgc.org). the pathognomonic ig-myc translocation was detected in 37of 39 of the cases using wgs, but was observed in all cases by fish. an igh-myc juxtaposition was detected in 34 patients and its variants igk-myc and igl-myc in 1 and 4 cases, respectively. we identified two different expression patterns of myc transcripts which were associated with the translocation breakpoint location. on the one hand the canonical myc transcript and on the other hand an alternative transcript with a transcription start site before the second exon. the latter produces an mrna which contains 486 nucleotides not included in the canonical transcript but nevertheless it encodes the identical protein. the integration of single nucleotide variants (snv) and copy number aberrations (cna) identified a total of 128 recurrently (≥2 samples) mutated genes. myc, id3, tp53, ccnd3, smarca4, arid1a, fbxo11, ddx3x were mutated in ≥20% of samples. in 33/39 (85%) cases, the id3/ abstracts ed. the annotation with the chromatin segmentation data of cd8 + t-cells from the blueprint project revealed enrichment of changes in methylation in distinct genomic regulatory elements in t-lgl. these differentially methylated functional regions were enriched for a set of transcription factor binding sites, known to be relevant in other lymphoid neoplasms. by bioinformatic analysis of methylation data and integration with gene expression data we identified hypermethylated and hypomethylated genes (e. g. bcl11b, themis, zeb2, hivep3) which point to candidate pathways potentially deregulated in the pathogenesis of t-lgl. conclusion: our study identified dna methylation changes in a set of candidate genes involved in various signaling pathways, which could potentially be used for diagnosis, prognosis and may become targets for novel treatment options. burkitt lymphoma (bl) is a mature aggressive b-cell lymphoma genetically characterized by a chromosomal translocation leading to ig-myc juxtaposition. treatment of bl is usually very successful particularly in children, with a cure rate of over 90% even among patients with advanced stage disease. however, the prognosis of the remaining patients experiencing disease progression and/or relapse is still very poor. bl has an overall low genomic complexity, thus secondary chromosomal changes in addition to the ig-myc translocation are rare. however, genomic complexity has been associated with aggressive disease and poor prognosis in various lymphomas including bl. because little is currently known about the underlying genetics of disease progression in bl we aimed at characterizing the molecular changes and characteristics that might lead to the relapse of bl. sequential tumor biopsies from initial diagnosis (id) and follow-up were available from a total of 8 patients (4-15 years at id), which were divided into two groups: five patients experienced a relapse from their initial bl diagnosed 58-210 days after id (group 1). in contrast, three patients developed twice a bl, i. e. presented with bl as secondary neoplasms diagnosed 3-5 years after id (group 2). dna extracted from archival formalin-fixed, paraffin-embedded (ffpe) tissue was used to analyze genome-wide copy number alterations (cna) using the oncoscan® platform (affymetrix) and mutational landscape by whole exome sequencing (wes). analysis of the cna in the 5 paired bl samples (group 1) revealed an increase in genomic complexity in 4/5 pairs as in id a mean of 8 cna was detected in contrast to 13.4 cna in relapse samples (p = 0.113). of note is that in all pairs, the relapse shared almost all cna which were present in id. wes analysis of group 1 showed similar results in all analyzed pairs. in total, 46.6% of mutations (median number of mutations = 106) were shared in id and relapse. nevertheless, a considerable amount of mutations were unique in id and relapse with a median of 18 (11.8%) and 51 (41.7%) mutations, respectively. on the other hand, mutations detected in samples populations of various differential stages. furthermore, we investigated whole-genome bisulfite sequencing (wgbs) data of 12 sbl and 6 b-al in comparison to 4 germinal center b-cell populations from healthy donors to decipher differentially methylated regions (dmr). these are defined as 10 or more cpgs differentially methylated between two groups. unsupervised dna methylation analysis of bl, fl and dlbcl revealed that all bl variants cluster apart from the non-bl cases. thus, supporting on epigenetic level that all analyzed bl samples are bl variants. multigroup comparison (σ/σ max = 0.4, q < 1e -13 ) separated the bl variants roughly in 3 groups: ebl, ebv-positive sbl and all other bl variants. furthermore, this analysis revealed ebl to harbor a massive hypermethylation in comparison to all other bl variants. comparison of the dna methylation using the humanmethylation450 beadchip data of sbl and b-al revealed 199 cpgs to be differentially methylated (σ/σ max = 0.4, q < 0.005). in contrast, using the wgbs data of the same samples a total of 4712 dmrs could be identified which were mostly located in enhancer and polycomb target regions. in conclusion, we show that all analyzed bl variants share a similar dna methylation profile. interestingly, dmrs between sbl and b-al were mainly located in enhancer and polycomb regions. in contrast, ebl showed a massive hypermethylation in comparison to the other bl variants. thus, the differences identified by dna methylation analysis can improve the understanding of the biological and clinical differences of the bl variants. dürig 1, 8 introduction: t-cell large granular lymphocytic leukemia (t-lgl) is a mature t-cell leukemia which often arises in the context of autoimmune disease. genetic changes like recurrent chromosomal aberrations are rare. recent studies identified somatic stat3 and tnfaip3 mutations in t-lgl cells. however, the molecular events driving leukemogenesis remain largely unknown. objectives: the goal of our study was to characterize the epigenetic basis of t-lgl to better understand leukemogenesis and potentially identify druggable pathways or diagnostic biomarkers for t-lgl. p. johansson 1,2 , l. klein-hitpass 2 , g. castellano 3 , k. kentouche 4 , f. nicolau 5 , i. oschlies 6 , e. carrillo-de santa pau 5 , m. przekopowitz 2 , a. queiros 3 , m. seifert 2 , a. valencia 5 , ij. martin-subero 3 , em. murga penas 7 , o. ammerpohl 7 , u. dührsen 1 , r. küppers 2 , j. we analyzed the dna methylome of facs sorted tumor cells of 11 t-lgl cases in comparison to benign αβ t-cell subsets. the infinium human methylation 450 bead chip was used for analysis. we annotated our data with the publicly available chromatin segmentation data of cd8 + t-cells from the ihec/blueprint project. the expression levels of selected genes were tested by reverse transcription real-time pcr. results supervised analysis of t-lgl compared to benign cd8 + memory cells resulted in 1,083 cpg loci significantly (q < 0.001) differentially methylatkrawitz 5, 6 , a. knaus 5, 6 , m. jäger 5, 6 , r. flöttmann 5 , t. eggermann 7 , b. hoechsmann 8 , h. schrezenmeier 8 paroxysmal nocturnal hemoglobinuria (pnh) is an acquired disorder of the blood-forming system. typically, affected hematopoietic stem cells (hscs) in pnh harbor a single somatic loss-of-function mutation in the x-linked piga gene. previously, a pnh patient with a different molecular etiology has been described and herein we report three more cases of this new subgroup: a predisposing germline mutation in pigt, which is an autosomal gene of the glycosylphosphatidylinositol (gpi)-anchor synthesis pathway, is followed by a second somatic hit. by means of deep sequencing and array-cgh, we observed acquired deletions of 8 mb to 18 mb on chromosome 20q in pnh cells that include pigt as well as a region that is commonly deleted in myeloproliferative neoplasms and myelodysplastic syndromes and that is known to be differentially methylated. this results in a complete loss of expression of certain genes at this locus which is also thought to contribute to the clonal expansion. the deficiency of gpi-anchored proteins on pnh cells results in a lack of the complement regulatory proteins cd59 and daf/cd55 on the cell surface and leaves them more vulnerable to the c5b-9 membrane attack complex. in contrast to classical pnh without any fully synthesized gpi-anchors, pigt mutations impair the transamidase that links the substrate to the anchor and thus result in an accumulation of unbound gpi molecules. this difference in the pathophysiology can also be visualized in flow cytometric analysis of peripheral blood: while cd55 and cd59 surface levels are reduced in all pnh cells, the atypical pnh cells due to a transamidase deficiency can be discriminated by a specific antibody, t5 mab, that binds free gpi anchors. besides the classical pnh symptoms of anemia, thrombosis, and hemolysis, patients with pigt mutations also manifest with additional autoinflammatory symptoms, such as urticaria, fever, arthralgia and meningitis, and it is hypothesized that the free gpi-anchor that accumulates in affected cells is causally related to autoinflammation. based on these findings, we propose the new entity of atypical pnh. background: the prevalence of metabolic disorders, in particular obesity has dramatically increased worldwide. genetic variants explain only a minor part of this obesity epidemics induced by physical inactivity and over nutrition. epidemiological studies in humans and animal models of di-from patients with secondary neoplasm (group 2) were mostly unique to id (= 109, 43.9%) whereas only 26.1% of all mutations were shared in id and secondary neoplasm samples (= 49). furthermore there were no shared cna in the corresponding samples identified by oncoscan® analysis. to sum up, the oncoscan® and wes analysis, of the paired bl group (1) provide strong evidence for a linear clonal evolution, meaning relapses may directly evolve from the previous lymphoma clone rather than a common precursor. in contrast, results obtained for patients with secondary neoplasm (group 2) showed no indication for linear but rather for divergent evolution. thus, analysis of recurrent mutations shared in id and second neoplasm samples can provide important information about disease progression and are therefore subject of ongoing analysis. y. murakami 1 , t. hirata 1 , s. murata 1 , t. kinoshita 1 , m. kawamoto 2 , s. murase 2 , h. yoshimura 2 , n. kohara 2 , n. inoue 3 , m. osato 4 , j. nishimura 4 , y. ueda 4 , y. kanakura 4 , p. m. in runx1 mutated aml the number of runx1 mutations, loss of the wild-type allele and the number and kind of additional mutations impact on prognosis a. stengel, w. kern, m. meggendorfer, k. perglerovà, t. haferlach, c. haferlach mll munich leukemia laboratory, munich, germany, 2 mll2, praha, czech republic aml with mutated runx1 show a distinct pattern of cytogenetic and molecular genetic abnormalities and an adverse prognosis. we analyzed the impact of multiple runx1 mutations and runx1 wild-type (wt) loss on associated genetic alterations and survival. for this, 467 aml cases with runx1 mutations (mut) were split in (1) runx1 wt loss (n = 53), (2) >1 runx1mut (n = 94) and 1 runx1mut (n = 323). 163 cases were selected for mutation analyses of 28 genes. in cases with 1 runx1mut, +8 was frequently found, whereas in wt loss +13 was the most abundant trisomy (+8: 66% in 1 runx1mut vs. 31% in wt loss, p = 0.022; +13: 15% vs. 62%, p < 0.001). cases with >1 runx1mut showed an intermediate distribution (+8: 44%, +13: 50%). missense mutations were the most abundant mutation type in wt loss cases (53% vs. 31%, p = 0.006), whereas in 1 runx1mut, frameshift mutations were found more frequently (45% vs. 28%, p = 0.016). in cases with >1 runx1mut, both were observed at similar frequencies (missense: 36%, frameshift: 38%). mutation analyses of 163 selected cases revealed 411 additional molecular mutations. 95% of cases showed at least one runx1-accompying mutation (range: 0-6). the median of accompanying mutations was n = 2 in the total cohort and in cases with 1 runx-1mut and >1 runx1mut, whereas it was n = 3 in runx1 wt loss. srsf2 (39%), asxl1 (36%), dnmt3a (19%), idh2 (17%), sf3b1 (17%), tet2 (17%) and bcor (16%) were revealed as most frequently mutated genes. cases with runx1 wt loss showed a higher frequency of asxl1mut compared to the other cases (50% vs. 29%, p = 0.009), while u2af1mut were absent from this group (0% vs. 10%, p = 0.019). median overall survival (os) in the total cohort was 14 months. wt loss (os: 5 months) and >1 runx1mut (14 months) showed an adverse impact on prognosis compared to 1 runx1mut (22 months; p = 0.002 and p = 0.048, respectively). mutations in asxl1 and kras and the presence of ≥2 additional mutations also negatively impacted os (10 vs. 18 months, p = 0.028; 1 vs. 15 months, p < 0.001; 12 vs. 20 months, p = 0.017). in univariate cox regression analysis runx1 wt loss (hr = 1.6; p = 0.024), ≥2 additional mutations (hr = 1.9; p = 0.019), asxl1mut (hr = 1.6; p = 0.030) and kr-asmut (hr = 4.4; p = 0.001) had an adverse impact on os. multivariate cox regression analysis revealed an independent adverse effect on os for runx1 wt loss (hr = 1.6; p = 0.039) and krasmut (hr = 4.2; p = 0.001). for 216/467 cases we received samples during course of the disease. in none of these cases, an evidence for a runx1 germline mutation was found by analyzing the mutation loads, thus all runx1 mutations are somatically acquired. taken together, we found strong differences between the subgroups in regard of cytogenetic and molecular genetic aberrations as well as regarding prognosis. thus, not only the presence and number of runx1 mutations but also the conservation of an intact runx1 allele as well as the number and kind of additional mutations is biologically and clinically relevant. abstracts different chromatin states, where methylation is inversely correlated with active histone marks. using the hardy-weinberg law, we estimate that there are 692 dmrs with a maf>0.05. we hypothesized that cis-acting dna polymorphisms could be responsible for the inter-individual variation of the dmrs methylation levels. we genotyped 2.5 million snps in the five donors and found that 82/157 (52%) dmrs have methylation levels highly correlated (>0.9) with the genotype of at least one nearby snp (± 6 kb window). this correlation was verified in 6/7 dmrs by targeted bisulfite sequencing in monocytes from 4 individuals used for wgbs and from 2 additional individuals. to validate our results in a larger population and possibly find correlating snps outside the ± 6 kb window for the remaining dmrs, we performed genome-wide association studies (gwas) using snp genotypes and illumina 450k cpg methylation data from blood samples of 1131 individuals from the heinz nixdorf recall study. these methylation arrays encompass only 51 cpgs contained in 30 of our dmrs, showing that they fail to identify a great number of potentially important regions. we certified that for these 51 cpgs, monocyte and whole blood dmrs methylation levels were correlated, and performed a gwas with ~500,000 snp for each of the 51 cpgs. for 48/51 cpgs, the correlation peak was near the cpg position. for each gwas, the snp with lowest p-value (in most cases p < 1e -200 ) was designated as lead-snp. snps in high linkage disequilibrium (r 2 > 0.8) to the lead-snps were located within the corresponding dmr or 16 bp to ~116 kb from it. many regions are bound by ctcf and other transcription factors. it is likely that snps affect the binding of these factors and thus the methylation state of the region. we conclude that these inter-individual differences in dna methylation are mainly driven by genetic factors. the dystonia 6 (dyt6) protein thap1 recruits the histone deacetylase hdac3 to mediate gene repression sektion für funktionelle genetik am institut für humangenetik, universität zu lübeck, lübeck, germany, 2 institut für neurogenetik, universität zu lübeck, lübeck, germany dystonia describes a heterogeneous group of neurological movement disorders characterized by contractions in various muscles resulting in abnormal postures, involuntary twisting and repetitive movements. dystonia 6 (dyt6), a primary torsions dystonia that first has an impact on cranio-cervical muscles causing problems with speaking and eating, is caused by mutations in the thap1 gene (thanatos-associated domain-containing apoptosis-associated protein 1). thap1 belongs to the family of thap proteins that are characterized by the presence of an evolutionarily conserved specific dna-binding thap zinc finger motif at their n-terminus. in humans 12 thap family members are known, designated thap0 to thap11. interestingly, most of the dyt6-causing mutations affect this thap domain. while we have previously described thap1-mediated repression of specific target genes, the molecular mechanisms how thap1 regulates promoter activity are rather unknown. it is known, that other members of the thap family such as thap7 and thap11 interact with the histone deacetylase hdac3 to mediate transcriptional repression. we have performed yeast-two-hybrid and gst pulldown assays to identify a specific interaction of thap1 with hdac3. by the use of truncated thap1 fragments we were able to narrow down hdac3 binding to the n-terminal thap-domain. for further functional characterization we have decreased hdac3 levels by sirna treatment or chemical inhibition and used taqman analyses to quantify the effect on thap1-target genes expression. thus, a significant increase of thap1-target genes expression was detected in those cells treated with hdac3 sirna. to further investigate whether the observed increase in gene expression is due to alterations of histone acetylation within the promoter regions we performed chromatin immunoprecipitation (chip) assays followed by qpcr using antibodies specific for different acetylated n-terminal residues of histone 3 as markers for transcriptional active promoters. by this, we detected an increased acetylation within the promoter regions of thap1 target genes that are dysregulated in cells treated with decreased hdac3 levels. et-induced obesity indicate that epigenetic changes associated with adverse parental and/or intrauterine factors may contribute to the missing heritability of metabolic disorders. possible adverse paternal effects are likely transmitted by the sperm to the next generation. to prove this hypothesis, we have systematically analyzed the effects of paternal obesity on the sperm epigenome and its implications for the next generation. results: to study the possible transmission of paternal bmi effects to the next generation, methylation levels of eight paternally expressed imprinted genes (peg1, peg3, peg4, peg5, peg9, peg10, nespas and igf2), two maternally expressed imprinted genes (meg3 and h19), and the obesity related gene hif3a were quantified by bisulphite pyrosequencing in sperm of 109 donors (undergoing ivf/icsi) and 121 fetal cord blood (fcb) of resulting offspring (conceived by ivf/icsi with the same sperm samples). hif3a showed a significant positive correlation between sperm methylation and paternal bmi. this effect on the sperm epigenome was replicated in an independent cohort of 188 sperm samples. for hif3a, paternal bmi also showed a significant positive correlation with fcb methylation. on the other hand, peg5/nnat exhibited a significant negative correlation between paternal bmi and fcb methylation. in contrast to pyrosequencing, deep bisulphite sequencing (dbs) allows one to study dna methylation at the single molecule level and enables us to distinguish between maternal and paternal alleles in fcb samples with an informative snp. epimutations which are defined as alleles showing >50% aberrantly (de)methylated cpg sites can also be identified with dbs. upon performing dbs on sperm samples, we observed a higher epimutation rate in the high bmi (28-40) group when compared to the low bmi (19-24) group across the four studied genes (peg1, hif3a, h19 and nespas). we are presently analyzing dbs data in selected cord blood samples with an informative snp to separately quantify methylation at the paternal and maternal alleles. it is important to decipher the methylation of the paternal allele when studying whether sperm methylation alterations are transmitted to the offspring. conclusions: our results suggest that male obesity is associated with modification of the sperm dna methylome, which may affect the epigenome (in fetal cord blood) of the next generation. allele-specific dna methylation occurs at functionally different regions: 1) at imprinting control elements, 2) on the silent x chromosome in females and 3) across the genome and probably dependent on the dna sequence in cis. the latter is termed haplotype-dependent allele-specific methylation and may contribute to inter-individual phenotypic variation. in a previous study on monocyte to macrophage differentiation, we showed that dna methylation differences between individuals were greater than between the two cell types. to study the genetic basis of these inter-individual differences in dna methylation, we analysed the methylome obtained by whole genome bisulfite sequencing (wgbs) of monocytes from five unrelated donors. for identifying differentially methylated regions (dmrs), we created two synthetic methylomes: one with the highest methylation values of each cpg in the five samples and one with the lowest methylation values. defining a dmr as a region of at least 4 cpgs with a methylation level difference of at least 0.8, we identified 157 dmrs, which cover 1165 cpgs and fall into cer, respectively. we show that ns-associated rit1 mutants intensified signal flux through the mek-erk pathway upon growth factor stimulation. by using heterologous expression systems, we identified the p21-activated kinase 1 (pak1) as novel effector of rit1. we found that rit1 interacts with the rho gtpases cdc42 and rac1, both of which are crucial upstream regulators of pak1. disease-causing rit1 mutations enhance protein-protein interactions and uncouple complex formation from growth factors. expression of both wild-type rit1 and its mutant forms resulted in dissolution of stress fibers and paxillin-containing focal adhesions from the cell center and increased cell movement. we conclude that rit1 is a potent regulator of actin dynamics, and dysregulated rac1/cdc42-pak1 signaling controlling cell adhesion and migration may be one aspect of the molecular basis of ns. medical systems biology, tu dresden, germany, 2 institute for clinical genetics, tu dresden, germany, 3 cancer science institute of singapore, national university of singapore, singapore, 4 institute of molecular biology, mainz, germany telomeres are short repetitive ttaggg sequences that cap the ends of chromosomes. these stretches of dna are covered by proteins and rnas which together protect the putative double strand break from dna repair mechanisms and facilitate replication. however, telomeres shorten with every cell division due to the end replication problem. the ribonucleoprotein telomerase counteracts this process by de novo elongation of telomeric repeats but its expression is mostly confined to the germ line and stem cells. even in the latter its activity is usually not sufficient to completely prevent telomere shortening. all cancer cells are also faced with this challenge and while the majority of cancer cells rely on telomerase, approximately 15% of cancers ensure sufficient telomere length via the recombination-based alternative lengthening of telomeres (alt) mechanism. to better understand telomere biology we aimed to identify novel telomeric factors by systematically screening for telomere-binding proteins in cell lines from 16 different vertebrates. here, we identified and characterized zbtb48, a zinc finger protein, as a novel direct telomere-binding protein across the vertebrate lineage. zbtb48 is directly binding to telomeric dna in vitro and it is localizing to telomeres in vivo via one specific zinc finger domain in both telomerase-and alt-positive cancer cells. interestingly, zbtb48 knock-out cells have longer telomeres, suggesting that zbtb48 limits telomere elongation. in addition, the combination of chipseq, rnaseq and proteome analysis revealed a transcription factor activity for a small, but specific set of target genes of zbtb48, linking its telomeric functions to mitochondrial metabolism. in conclusion, zbtb48 is a novel direct telomere binding protein with transcription factor activity that acts as negative regulator of telomere length. our data show for the first time a functional interaction of the 'dystonia 6 protein' thap1 with the histone deacetylase hdac3 and therefore give new insights into the molecular mechanisms of thap1-mediated gene repression. interestingly, previous functional studies as well as structure analyses revealed that only a subset of the dyt6-causing mutations affecting the n-terminal thap domain alter thap1-binding to dna. in ongoing studies we want to investigate the consequences of dyt6-causing mutations on thap1-hdac3 complex formation and its relevance in the molecular pathology of dystonia. reproductive homeobox (rhox) genes are clustered on the x chromosome and share a unique 60 amino acid helix-turn-helix dna binding homeodomain. they were identified in several species as having important roles in reproductive tissues, notably in the testis. the human rhox cluster is composed of three genes: rhoxf1 and two copies of rhoxf2 (rhoxf2a, rhoxf2b) which are referred to as rhoxf2/2b. rhox proteins are expressed exclusively by germ cells in human testis and aberrant rhox methylation is associated with several sperm parameters. because little is known about the molecular mechanism of rhox function in humans, the aim of the study was to identify target genes of human rhox proteins and to investigate the impact of rhox mutations on protein function. using gene expression profiling, we identified genes regulated by members of the human rhox gene cluster. some genes were uniquely regulated by rhoxf1 or rhoxf2/2b, while others were regulated by both of these transcription factors. several of these regulated genes encode proteins involved in processes relevant to spermatogenesis, e. g. stress protection and cell survival. one of the target genes of rhoxf2/2b is rhoxf1, suggesting cross-regulation to enhance transcriptional responses. the potential role of rhox in human infertility was addressed by sequencing rhox in a group of 250 patients with severe oligozoospermia. this revealed two mutations in rhoxf1 (c.515g>a and c.522c>t) and four in rhoxf2/2b (-73c>g, c.202g>a, c.411c>t and c.679g>a), of which only one (c.202g>a) was found in a control group of men with normal sperm concentration. functional analysis demonstrated that c.202g>a and c.679g>a significantly impaired the ability of rhoxf2/2b to regulate downstream genes. molecular modelling suggested that these mutations alter rhoxf2/f2b protein conformation. by combining clinical data with in vitro functional analysis, we demonstrate how the x-linked rhox gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility. colorectal cancer (crc). here, we proposed the possible molecular mechanisms responsible for crc initiation, progression and invasion using a network biology approach. materials and methods: in order to investigate the underlying crc pathogenesis, the dataset gse21510 consisting of normal tissues, stage i, stage ii, stage iii and stage iv of crc were obtained from gene expression omnibus (geo) and further examined. the differentially expressed genes (degs) were subjected to protein-protein interaction databases and a ppi network was constructed for each crc stage. topological analysis of resulted ppi networks revealed functional hub genes and involved in crc development. furthermore, the overlap genes between four studied crc stages were determined and deeply evaluated to identify deregulat ed biological networks during crc development. a standard real-time pcr was performed to validate the in silico findings utilizing sw620 and ncm460 cell lines. results: the most important hub genes (cdk1 for stage i, ubc for stage ii, esr1 for stage iii and atxn1 for stage iv) and sub-networks were identified in crc stages. moreover, several novel biomarkers were also introduced for each crc stage. gene ontology (go) and signaling pathway enrichment uncovered the important roles of wnt, mapk and jak-stat signaling pathways in regulation of crc pathogenesis. functional annotation of overlap genes revealed that cell cycle regulating genes are the most highly regulated genes during crc initiation, progression and invasion. in vitro analyses confirmed deregulation of atxn1 and cdk1, two hub genes of stage iv, in metastatic colon sw620 cells compared to normal colon ncm460 cell line. our study provides a new insight into the distinct molecular mechanisms underlying the pathogenesis of crc. the functional hub genes, sub-networks, prioritizes key pathways and novel crc biomarkers were also provided that can be useful in therapeutic programs. targeted next-generation sequencing approaches as well as next-generation whole exome sequencing are becoming more widespread in routine molecular diagnostics for patients with ataxia. however, since ngs at present is not suitable to detect (trinucleotide) repeat expansions, a pre-ngs testing for common polyglutamine expansion scas seems mandatory. but also sca subtypes caused by expansions in non-coding regions of genes like sca8, sca10, sca12, and sca36 as well as other ataxias known to be associated with repeat expansions like the fragile x-associated tremor ataxia syndrome (fxtas) should be taken into account before applying ngs-based diagnostics. in order to find an optimal diagnostic strategy in future more information about the frequency and phenotypic characteristics of rare repeat expansion disorders associated with ataxia would be helpful. we therefore analyzed a cohort of 441 patients with symptoms of cerebellar ataxia, dysarthria and other unspecific symptoms who were referred to our center for sca diagnostics and showed alleles in the normal range for the most common sca subtypes sca1-3, sca6, sca7, and sca17. these patients were screened for expansions in sca8, sca10, sca12, sca36 and fxtas as well as for the pathogenic hexanucleotide repeat in the c9orf72 gene. no expanded repeats for sca10, sca12 or sca36 were found in the analyzed patients. five patients with ataxia of unknown etiology showed sca8 cta/ctg combined alleles (83-129) that are discussed to be potentially pathogenic. one 51-year-old male patient with unclear dementia syndromes was diagnosed with a large ggggcc repeat expansion in c9orf72. and the analysis of the fmr1 gene identified one patient with a permutation (>50 cgg repeats) and seven patients poster *** = für den posterpreis nominiert preventive genetic counseling in neurogenetic disorders needs a better collaborative approach between genetic and neurology clinics -a report of four siblings with unverricht-lundborg disease: genetic counseling is the process of helping people to understand and adapt the medical, psychological and familial implications of genetic contributions to disease. for parents with a previous child or other family member with a known genetic syndrome expands options for preimplantation or prenatal diagnosis for the current or the future pregnancies. however, timely referral by health providers to genetic counselor and for discussing with couples regarding possible options is important. additionally, other factors such as personal decision making especially due to high price of some genetic services and uncertain results cause considerably delays to genetic testing. there are more than 200 various types of inherited neurological disorders in which alterations in genes lead to an inherited condition such as huntington disease, inherited forms of alzheimer disease, ataxia, muscular dystrophies and epilepsies. the knowledge of the causative gene mutations in the affected individual is critical in the possible prenatal diagnosis in other members of the pedigree. therefore a multidisciplinary care team, including neurologist and genetic counselor for the conditions diagnosed as inherited neurological disorders is critical in prenatal setting and consideration of an effective management. here, our report of four siblings affected by a rare form of inherited epilepsy (unverricht-lundborg disease) with an autosomal recessive pattern highlights the importance of the needs for a better collaborative approach in the neurogenetic setting. in fact, the birth of four successive siblings affected by similar neurogenetics disorders in a specific family is showing the need for more attention to this important issue, especially in terms of intersectoral collaboration. poorebrahim 6 hort: 6/10 differentially expressed transcription units). these differences in gene expression we detected did not correlate with dna methylation changes at the corresponding transcription regulatory sites. from our results we conclude that altered expression of imprinted genes indeed plays a role in tumorigenesis of germinal center derived b-cell lymphomas. however, the altered transcriptional regulation of these genes seems not to rely on the usual epigenetic mechanisms known from constitutional imprinting disorders. mf. abazari 1 , h. bokharaie 2 , m. asghari 3 , v. poortahmasebi 4 , h. askari 5 , m. investigating the expression of genes associated with autism spectrum disorders to identify sex related differences s. berkel, a. eltokhi, g. rappold institute of human genetics, heidelberg university hospital, heidelberg, germany neurodevelopmental disorders such as autism, attention deficit and hyperactivity syndrome as well as language problems and learning difficulties have a higher prevalence in male individuals compared to females. autism is characterized by impairments in social interaction, communication deficits and restricted and repetitive behaviors. boys are more frequently affected than girls; the ratio of affected boys compared to girls is 4:1 for autism and 11:1 for asperger syndrome. in this study we aim to elucidate the reason for this gender difference by following up two hypotheses: (1) risk genes for autism spectrum disorders (asd) might be expressed at different levels in males and females and (2) asd risk genes might interact with sexually dimorphic pathways. first, we investigated the expression of genes associated with autism spectrum disorders, including the shank gene family, in the brain of male and female mice to identify sex-dependent differences. the rna expression levels were analyzed in five different brain regions (cortex, hippocampus, striatum, cerebellum, thalamus) at different developmental stages (e15, e17, p1, p7, p12 and adult) in male and female mice. we identified a sex dimorphic expression of shank1 and shank3, but not of shank2. due to the fact that early brain development is strongly influenced by sex hormones (estrogen, testosterone), we further investigated the influence of these hormones on shank expression in human neuroblastoma cells (sh-sy5y) and primary mouse hippocampal neurons. a better understanding of the sex differences in the brain might help to explain the vulnerability for neuropsychiatric disorders like autism and paves the way to discover putative risk or protective factors for these disorders. imprinting defects in temple syndrome are caused by a failure in imprint establishment and/or maintenance j. beygo, c. mertel, g. gillessen-kaesbach, b. horsthemke, k. buiting institut für humangenetik, universitätsklinikum essen, universität duisburg-essen, essen, germany, 2 institut für humangenetik, universität zu lübeck, lübeck, germany temple syndrome (ts14) is a rare imprinting disorder characterised by low birth weight and height, muscular hypotonia and feeding difficulties in the infant period, early puberty and short stature with small hands and feet and often truncal obesity. in a subset of patients with ts14, the disease is caused by an imprinting defect (id) affecting the paternal allele of the imprinted region 14q32. the id results in aberrant methylation of the three known differentially methylated regions (dmrs), the germline-derived primary dlk1/meg3 intergenic (ig-)dmr (meg3/dlk1:ig-dmr), the postfertilization-derived, secondary dmr at the meg3 promoter (meg3:tss-dmr), and the postfertilization-derived, secondary intragenic meg8-dmr (meg8:int2-dmr). the meg3/dlk1:ig-dmr and the meg3:tss-dmr are methylated on the paternal chromosome and hypomethylated in patients with ts14 and an imprinting defect. the meg8:int2-dmr is unmethylated on the paternal chromosome and hypermethylated in these patients. both the meg3/dlk1:ig-dmr and the with alleles in the grey zone (41 to 54 cgg repeats), thus suggesting that individuals with fmr1 repeat expansions in the gray zone may also present with neurological signs. bernhart 3, 4, 5 , h. kretzmer 3, 4 , r. wagener 1 , c. mmml 6 some genes are subject to the mechanism of imprinting, i. e. their expression depends on parental origin. they primarily function in the control of proliferation, fetal development and cellular differentiation. constitutional imprinting disorders are in part also associated with an increased tumor risk. loss of imprinting has been also described as somatic event in tumorigenesis. while this phenomenon has been broadly analyzed in solid tumors, data on alterations of imprinting in lymphatic neoplasms are largely missing. we analyzed the rna expression of 321 transcription units/regions known or supposed to be subject to imprinting in two cohorts of normal b-cells and germinal center derived b-cell lymphomas. the first cohort (mmml) contains 686 samples: 56 burkitt lymphomas (bl), 600 non-burkitt lymphomas (non-bl, including various subtypes like follicular and diffuse large b-cell lymphoma) and 30 normal germinal center b-cell samples (gcbc, as controls). the second cohort (icgc mmml-seq) comprised 201 samples with 20 bl, 176 non-bl and 5 gcbc samples. gene expression was analyzed with affymetrix u133a genechips in the mmml cohort and by rna sequencing in the icgc mmml-seq cohort. results of the transcriptional analyses in the icgc mmml-seq cohort were compared to the dna methylation available from a subset of the analyzed samples (kretzmer et al., nat genet, 2015) . of the 321 transcription units 114 sites, corresponding to 64 transcription units, were present on the applied array used for the analysis of the mmml cohort. a two group comparison revealed 53 significantly differentially expressed sites corresponding to 31 transcription units between bl and non-bl including the plagl1 and peg10 genes. in total, 19 and 16 sites corresponding to 16 and 10 transcription units are differentially expressed between bl versus gcbc and non-bl versus gcbc, respectively. comparison of gene expression in the icgc cohort revealed 70 differentially expressed sites corresponding to 68 transcription units between bl and non-bl (overlap with mmml cohort: 24/31 differentially expressed transcription units), including again peg10 and plagl1, 37 differentially expressed sites corresponding to 37 transcription units between bl and gcbc (overlap with mmml cohort: 7/16 differentially expressed transcription units) and 44 differentially expressed sites corresponding to 39 transcription units between non-bl and gcbc (overlap with mmml co-abstracts maintenance dna methylation of l1 promoters, spoc1 could function in targeting g9a to l1 sequences. in conclusion our data implicate the epigenetic reader spoc1 in the suppression of line elements during germ cell development. s. bens 1 , j. kolarova 1 , m. kreuz 2 , sh. characterization of the expression of the imprinted kcnk9-gene in specific brain regions and the phenotypic analysis of kcnk9knockout mice a kcnk9/kcnk9 is a maternally expressed imprinted gene whose mutations are responsible for the maternally inherited birk-barel mental retardation dysmorphism syndrome. it encodes a member of the superfamily of k+channels with two pore-forming domains and is involved in the modulation of the resting membrane potential and excitability of neuronal cells. so far, only homozygous kcnk9 knockout mice with inactivation of both parental alleles were phenotypically characterized. these mice displayed cognitive deficits as well as a reduction of k+ leak current by 50%. in the light of maternal-specific imprinted expression of kcnk9/kcnk9 and the maternal inheritance of the birk-barel mental retardation dysmorphism, a thorough phenotypic analysis of heterozygous kcnk9 knockout mice with inactivation of only the maternally inherited allele is also warranted. as first aim of our study, we characterized the parental allele-specific expression of kcnk9 in various regions of the mouse brain. quantification of allele-specific expression by pyrosequencing (quasep) method was performed for different brain areas from several developmental stages of (c57bl/6xcast/ei) f1 hybrid mice. exclusive expression from the maternal kcnk9 allele was detected in the dentate gyrus, hippocampus, mesencephalon, medulla oblongata, thalamus and pons. biallelic expression with, however, a strong bias towards the maternal kcnk9 allele (94-99% of the transcripts) was observed in the olfactory bulbs, cortex, cerebellum, striatum and olfactory tubercles. as the second aim of our study, the phenotypes of wildtype, heterozygous kcnk9 knockout mice with maternal inherited knockout allele (kcnk9komat) and homozygous kcnk9 knockout mice (kcnk9kohom) were comparatively examined in a behavioral test battery. due to the already known cognitive defects of kcnk9kohom animals and especially the phenotype of the patients with birk-barel syndrome, it was assumed that kcnk9komat and kcnk9kohom animals show deficits in some of the tests. the spontaneous alternation in the y-maze test was significantly reduced by approximately 10-20% in kcnk9komat and kcnk9kohom mice compared to wildtype mice indicating a clearly impaired working memory. in addition, kcnk9komat and kcnk9kohom mice displayed a reduced prepulse inhibition of startle response compared to wildtype mice indicating an impairment of sensomotoric gating, a process to filter out irrelevant information. acoustic startle response as a measure of anxiety levels was also significantly decreased, but only in kcnk9kohom mice. our findings shall further elucidate the role of kcnk9/kcnk9 in brain physiology and pathophysiology and open new avenues for treatment of cognitive dysfunctions in birk-barel syndrome. meg3:tss-dmr act as imprinting control centres, although the meg3/ dlk1:ig-dmr functions as an upstream regulator of the meg3-dmr. so far, the function and regulation of the meg8-dmr is unknown. the hypomethylation of the paternal allele in ts14-id patients at the meg3/ dlk1:ig-dmr and the meg3:tss-dmr point to a failure in the establishment of the methylation imprint or to maintain the methylation imprint after fertilization. in this case, the incorrectly imprinted chromosome 14 would be inherited from either the paternal grandfather or grandmother. to prove this assumption we are investigating the grandparental origin of the affected chromosome 14 in our cohort of ten ts14-id families by studying the parent-of-origin specific methylation of the three dmrs in combination with informative single nucleotide variants (snps). at the moment we have identified three families informative for the meg3/ dlk1:ig-dmr, two families for the meg3:tss-dmr and two families for the meg8:int2-dmr. so far we have obtained results in two families for the meg3:tss-dmr. we found that in one case the allele harbouring the id was inherited from the paternal grandmother, but in the second case from the paternal grandfather, indicating that the id occurred after erasure of the parental methylation imprints. a complete lack of methylation observed in the majority of ts14-id patients is therefore likely due to a problem in establishing methylation on the paternal chromosome, whereas in rare cases with methylation mosaicism, the id is probably due to a problem to maintain the paternal imprint after fertilization. bosch, s. lukassen, j. kaindl, j. schwarz, c. nelkenbrecher, a. herrmann, a. reichel, a. ekici, t. gramberg, t. stamminger, a. winterpacht humangenetisches institut, erlangen, germany, 2 virologisches institut, erlangen, germany spoc1/phf13 is a gene located on human chromosome region 1p36.31 and mouse chromosome 4qe2. the protein was first described in patients with epithelial ovarian cancer, where its expression correlated with tumour progression and reduced survival time. spoc1 is a reader of the epigenetic mark h3k4me2/3, dynamically associates with chromatin during mitosis and plays a role in chromosome condensation. spoc1 deficient mice show a pronounced hypoplasia of the testis with a progressive loss of germ cells. although loss of spoc1 leads to a significantly reduced chromatin condensation of the sex chromosomes in meiosis, the protein is not expressed in spermatocytes but in the undifferentiated precursor cells, the spermatogonial stem cells (sscs). here, we present chip-seq data of mouse testis tissue demonstrating that spoc1 strongly binds to evolutionary young l1 elements in undifferentiated spermatogonia. we show that in hek cells overexpression of spoc1 leads to repression of transposition activity of line-elements strongly indicating a role of spoc1 in l1 element suppression. the cell has developed several lines of defence against retrotransposition to maintain genomic integrity, including dna methylation. these defence mechanisms are most elaborate in spermatogonial stem cells since transposition events in these cells would have a dramatic impact on the next generation. therefore, the repression of retrotransposition is of fundamental importance for germ cell development and ultimately the quality of the gametes. moreover, we present medip results showing that the methylation levels of l1 sequences decrease upon spoc1-knockout and demonstrate that the histone methyltransferase g9a is strongly upregulated in preleptotene meiocytes of spoc1 -/mice. g9a is expressed from spermatogonia until early meiosis where it regulates h3k9 di-methylation and has been shown to be involved in the repression of l1 element in mouse spermatogonia. we are able to demonstrate that h3k9me2 levels are unaltered in spoc1 -/mice, suggesting a potential functional link between g9a and spoc1 that does not affect the catalytic activity of g9a. since g9a can regulate de novo and medizinische genetik 1 · 2017 121 lated to investigate ciliogenesis. data resulting from rnaseq experiments are analyzed by established informatics tools (tophat, cufflinks and derivatives thereof). we will show results from our work in progress and we hope to convince people to intensify rna analyses even in routine labs to uncover hidden mechanism and/or mutations impacting mrna splicing and thereby causing human disease. telomeres are located at the ends of chromosomes and have an essential role in the maintenance of genome stability. after each cell division, a small part of this specialized sequence is lost. when telomeres reach a critically reduced length, the cell either dies through apoptosis or enters a state of permanent cell cycle arrest. it has been demonstrated that telomere biology is directly linked to basic biological phenomena such as aging, tumorigenesis and maintenance of dna integrity. it is known that oxidative stress accelerates telomere shortening in cells, resulting in premature cell senescence. shorter leukocyte telomeres have been observed in type ii diabetes or degenerative disease like dementia and alzheimer disease as well in chromosomal instability syndrome, such as fanconi anemia (fa) and nijmegen breakage syndrome (nbs). any link between telomere length and inflammation has not yet been extensively studied in autoimmune diseases. accelerated length shortening might be related to autoimmune disease predisposition. yet the reasons for this shortening are likely manifold, including the individual genetic background, oxidative stress and chronic inflammation. in order to shed light on these relationships, we investigate genomic dna extracted from blood of patients diagnosed with multiple sclerosis and from patients with huntington disease. the samples were divided into age groups. stepone q-pcr was applied to detect the relative telomere length as a function of age. initially identified differences in telomere lengths still have to be confirmed in larger cohorts. background: intrauterine exposure to gestational diabetes mellitus (gdm) confers a lifelong increased risk for metabolic and other complex disorders to the offspring. gdm-induced epigenetic modifications modulating gene regulation and persisting into later life are generally assumed to mediate these increased disease risks. to identify candidate genes for fetal programming, we compared genome-wide methylation patterns of fetal cord bloods (fcbs) from gdm and control pregnancies. methods and results: using illumina's 450k methylation arrays and following correction for multiple testing, 65 cpg sites (52 of which are associated with genes) displayed significant methylation differences between gdm and control samples. three of four candidate genes, atp5a1, prkch, and slc17a4, from our methylation screen and one, hif3a, from the literature were validated by bisulfite pyrosequencing. the gdm effect on fcb methylation was more pronounced in women with insulin-dependent gdm who had a more severe metabolic phenotype than women with dietetically treated gdm. however, the effect remained significant after adjustment for the maternal bmi and gestational week in a multivariate regression model. e. g. dekomien 1, 2 , b. bellenberg 2, 3 , n. trampe 2, 4 , r. schneider 2, 4 , c. prehn 2, 4 , c. krogias 2, 4 , r. kropatsch 1, 2 , m. regensburger 5, 6 , c. lukas 2, 3 , r. gold 2, 4 , 4 1 human genetics, bochum, germany, 2 ruhr-university bochum, germany, 3 radiology, st. josef-hospital, bochum, germany, 4 neurology, st. josef-hospital, bochum, germany, 5 molecular neurology, erlangen, germany, 6 university of erlangen, germany a pair of monozygotic 22-year-old twins suffering from hereditary spastic paraplegia 11 (spg11) is described. patients underwent thorough clinical examination and magnetic resonance imaging (mri) and mr-spectroscopy (mrs) at 3 tesla. genetic testing was performed by sanger sequencing and alternative splicing by rna analysis. clinically the patients presented a similar spectrum of symptoms with a higher level of disability in one of the patients. mri studies including morphometry and regional microstructural analysis by diffusion tensor imaging (dti) of the corpus callosum (cc) revealed marked thinning and corresponding increases of axial diffusivity (ad), radial diffusivity (rd) and apparent diffusion coefficient (adc) and reduction of the fractional anisotropy (fa) as compared to healthy controls in all cc sections, particularly in the anterior callosal body. there was marked supratentorial white matter reduction and to a lesser extent grey matter reduction in both patients. involvement of the cortico-spinal tracts was reflected by fa and rd alterations and cervical cord atrophy. the more strongly affected patient showed a higher degree of callosal microstructural damage and cervical cord atrophy. genetic testing of the spg11 gene revealed two mutations in compound heterozygous state, a known frameshift mutation as well as a novel synonymous exonic splice site mutation. this study shows similar but distinct clinical and imaging findings in monozygotic twins suffering from spg11, suggesting individual downstream genetic effects. targeted next generation sequencing techniques tremendously improved our ability to identify sequence variants. however fixing disease causing mutation still lack behind because of several reasons: inappropriate gene specific data bank, insufficient prediction tools, incomplete analysis and others. in addition identified sequence variants are a mixture of severe disease causing mutations and a myriad of variants of unknown pathogenicity. in addition an unknown number of silent mutations, neutral polymorphism and sequence variants deeply buried in introns might severely influence splicing of the premature rna molecule. by solely analysis of the dna sequence this impact onto the integrity of the mrna is completely ignored. in order to catalog the mrna isoforms derived from genes of our interest we started to set up rnaseq technologies in our routine lab. to reduce the amount of data, to improve the power of analyses and to identify rare isoforms of transcripts we use targeted rnaseq to characterize the mrna molecules originating from those genes we are interested in (e. g.: hereditary breast cancer core genes (10 genes), hereditary colon cancer (23 genes), primary ciliary dyskinesia (pcd)(40 genes). genes involved in pcd offer the invaluable advantage that the respiratory epithelium where these genes are normally expressed can be sampled from the inferior turbinate of the nose by brush biopsy either from healthy probands or from patients suffering from pcd. in addition to direct preparation of rna from these cilia, cilia carrying cells or tissue can be cultured and manipu-abstracts long-range pcr and direct sequence analysis. the comparative analysis of parental haplotypes with the sequences flanking the deletion breakpoints in the patients revealed the absence of any de novo mutations in breakpoint-flanking regions of 19 prs2-mediated and 6 prs1-mediated type-1 nf1 deletions. we conclude that although nahr is a mutational mechanism causing large nf1 deletions, there is no evidence for a local mutagenic effect of these recombination events. hence it is unlikely that nahr underlying type-1 nf1 deletions involves error-prone translesion polymerases that would increase the de novo mutation rate in breakpoint flanking regions. furthermore, the detailed haplotype analysis of prs2, a highly active nahr hotspot mediating the majority of large nf1 deletions, revealed that non-allelic homologous gene conversion (nahgc) between nf1-repa and nf1-repc, which results from non-crossover resolution of recombination intermediates, is the major driving force responsible for the haplotype diversity in this region. remarkably, the haplotype diversity patterns observed for nf1-repa and nf1-repc were markedly different indicating that during nahgc, nf1-repa is disporportionately more often the donor sequence used to repair mismachtes in heteroduplex regions than nf1-repc. we also noticed a correlation between haplotype diversity and the number of prdm9 a-allele binding sites suggesting that haplotype diversity and hence the nahgc rate within prs2 in nf-repa is regulated by prdm9. heidelberg center for personalized oncology dkfz-hipo dkfz, heidelberg, germany dna methylation aberrations at differentially methylated region of imprinted genes interfere with the naturally parental-specific mono-allelic expression. that leads to a bi-allelic or absent expression of the imprinted gene, a cause of imprinting disorders (ids). we aimed at analyzing the genome wide dna methylation pattern of two patients with ids, namely transient neonatal diabetes mellitus (tndm) and multi locus imprinting disturbance (mlid), and their respective parents. the dna methylation profiles of these individuals were obtained by whole genome bisulfite sequencing (wgbs) on b cells sorted by magnetic cell isolation. the sequencing libraries were prepared as described in kretzmer et al. [1] and sequenced on an illumina hiseq 2000 machine. wgbs data were processed with the methylctools toolkit. briefly, bisulphite-treated sequences were aligned with bwa-mem using a three-letter approach, and the methylation ratios were quantified for ~26.9 million out of 28.2 million cpg sites (coverage>5) genome-wide. quality control was performed to assess the quality of the dna methylation profiles and genetic fingerprinting was performed on the wgbs data confirming sample origin and family relationship. the wgbs data was further compared to already existing genome-wide human snp array 6.0 (snp array) and humanmethylation 450k bead array (450k) data, resulting in a good accordance with pearson's correlation coefficients >0.97. we detected an overall dna methylation level around 75% in all samples. already known dna methylation alterations, e. g. hypomethylation in plagl1 were validated by wgbs. by searching for differentially methylated regions (dmrs), defined as regions composed of at least five consecutive cpg loci showing a methylation difference between patient and corresponding parents above 30%, we identified 442 dmrs in the mlid our study supports an association between maternal gdm and the epigenetic status of the exposed offspring. consistent with a multifactorial disease model, the observed fcb methylation changes are of small effect size but affect multiple genes/loci. the identified genes are primary candidates for transmitting gdm effects to the next generation. they also may provide useful biomarkers for the diagnosis and prognosis of adverse prenatal exposures and assessing the success of interventions during pregnancy. the nuclease hsnm1b/apollo has a dual function in both dna-repair and maintenance of telomeres. as to the repair of dna interstrand crosslinks (icl), hsnm1b/apollo is linked to the fanconi anemia (fa) pathway and cells depleted for hsnm1b/apollo (sirna) resemble those from patients with fa. we have identified a single nucleotide polymorphism, rs6674384, which is associated with quantitative differences in hsnm1b/apollo expression (mrna). we analyze whether the differential expression relates to the degree of cellular sensitivity to the dna interstrand crosslinks inducing mutagen mitomycin c (mmc) and ionising radiation (ir), which induces, among other lesions, dna double strand breaks. all experiments are realized using lymphoblastoid cells derived from generally healthy donors. results of rt-pcr analysis of hsnm1b/apollo expression and of the cell viability assays will be presented and discussed in the context of the potential usefulness of considering rs6674384 in predicting individual sensitivity to mutagens relevant in anti-cancer treatment. p-basepi-014 nahr events causing type-1 nf1 microdeletions are not associated with an increased mutation rate in breakpoint-flanking regions m. hillmer 1,2 , a. summerer 1,2 , v. f. mautner 3, 4 , l. messiaen 5, 6 , h. 2 1 institute of human genetics, ulm, germany, 2 university of ulm, ulm, germany, 3 department of neurology, hamburg, germany, 4 university hospital hamburg eppendorf, hamburg, germany, 5 department of genetics, birmingham, usa, 6 university of alabama at birmingham, birmingham, usa large deletions of the nf1 gene and its flanking regions are the most frequent recurrent mutations in patients with neurofibromatosis type 1 (nf1). different types of large nf1 deletions have been identified which are distinguishable in terms of their size and breakpoint position. most frequent are type-1 nf1 deletions spanning 1.4-mb and characterized by breakpoints located within the low-copy-repeats nf1-repa and nf1-repc which exhibit 97.5% sequence homology within 51-kb. type-1 nf1 deletions are caused by non-allelic homologous recombination (nahr). two nahr hotspots have been identified termed prs1 and prs2 which encompass 5-kb and 4-kb, respectively. approximately 80% of all type-1 nf1 deletion breakpoints cluster within the prs1 and prs2 nahr hotspots. in this study, we analysed whether the nahr events causing type-1 nf1 deletions would be associated with an increased de novo mutation rate of sequences located in breakpoint-flanking regions. to do so, we sequenced the deletion breakpoint-flanking regions in the patients and compared these sequences with the homologous regions amplified from dna of the patients' parents who are not affected by nf1. however, in the germline of these parents, the deletions were mediated by nahr and then transmitted to their offspring. the parental haplotypes within the prs1 or prs2 regions of nf1-repa and nf1-repc were analysed by in the alpl gene and inherited as an autosomal dominant trait can cause milder forms. so far detailed knowledge of the milder forms is lacking. 39 patients with a mutation in the alpl gene were interviewed in a standardized questionaire concerning the different disease manifestations: teeth, bone fractures, pain of bones and muscles and quality of life. subgroups were formed with regard to the localization of the mutations in the three protein domains. patients with mutations clustering in the catalytic site of the molecule showed the most severe odontohypophosphatasia: one individual had premature primary tooth loss, 31% of patients showed adult tooth loss, 77% suffered from dental caries. the majority had the first manifestation before the age of 18. persons suffering from mutations in the two other domains reported a relatively high quality of live with low pain of muscles and bones. unexpectedly in all groups there was no significant difference in the portion of patients with bone fracture. conclusion: the clinical signs of dominant hpp are mostly unspecific. especially dental problems like severe adult teeth loss, an early manifestation of dental caries or enlarged pulp chambers can be a sign of odontohypophosphatasia and a dominant inherited mild hpp. mutations in the catalytic site of the alpl molecule are associated with a more severe odontohypophosphatasia. screening of non-neoplastic lymphatic tissues from children for the igh-myc fusion using a highly sensitive 4-color fish-assay burkitt lymphoma is a mature b-cell lymphoma which on the genetic level is characterized by the burkitt translocation t(8;14)(q24;q32) juxtaposing the igh locus in 14q32 next to the myc locus in 8q24. in a minor part of burkitt lymphomas, immunoglobulin light chain variants of the translocation result in overexpression of myc. despite being pathognomonic for burkitt lymphoma, the ig-myc juxtaposition alone is not sufficient on its own for a malignant transformation of the cell. other igh rearrangements like the igh-bcl2 fusion, typical for follicular lymphoma, were detected in a significant number of healthy individuals. for the igh-myc translocation, only scarce data in healthy individuals exist. this is most likely due to scattering of the breakpoints which are far more difficult to target by pcr than the igh-bcl2 translocation. therefore, we aimed at investigating if myc-translocation positive cells can also be detected in normal b-cell maturation. considering the epidemiology of burkitt lymphoma being the most common b-cell lymphoma in children, we focused on samples from young individuals. on the one hand, we analyzed non-neoplastic tissue specimen of bone marrow (n = 14) (age range 3-18, median age 8.5 years) and lymph nodes (n = 19)(age range 2-18 years, median age 12 years). on the other hand, considering the typical clinical presentation of burkitt lymphoma, we included non-neoplastic tissue specimen containing peyer patches (n = 25)(age range 48hours-20years, median age 17years). the specimen were analyzed using a four-color fluorescence in situ hybridisation (fish) assay with probes flanking the breakpoints on chromosomes 8 and 14. in this setting, a positive result comprised the break on both chromosomes (seen as signal split for each locus) and fusion of the involved genes (leading to two different fusion signals). the assay was first validated on controls of cells with a normal male karyotype from healthy individuals as well as on five burkitt cell lines and each five ffpe embedded t(8;14) negative and positive tissues as negative and positive controls. the assay was then applied for the screening of a igh-myc fusion in the above mentioned paraffin-embedded tissues. successful hybridizations of overall 9, 15 and 17 ffpe sections from bone marrow, lymph nodes and peyer patches respectively could be obtained. trio and 1776 dmrs in the tndm trio. in the mlid trio 238/442 dmrs showed increased and 204 dmrs decreased dna methylation in the patient's sample. of 442 dmrs, 325 are located in regions potentially associated with transcriptional regulation. further analysis revealed that 34/442 dmrs are associated with imprinted genes. in the tndm trio, we detected 1705/1776 dmrs to show hypermethylation in the patient compared to her parents and 71/1776 dmrs with lower dna methylation. in these trio 1166/1776 dmrs are associated with regions potentially correlated to transcriptional regulation and 13 dmrs with imprinted genes summarized, our results show that wgbs is a well suited and valid method for analyzing dna methylation. while the overall dna methylation levels does not differ between the analyzed patients and parents, a detailed analysis of smaller regions revealed the existence of 442 respectively 1776 differentially methylated regions between the analyzed mlid and tndm patient and their parents. supported by bmbf through fkz: 01gm0886 und 01gm1114 and 01gm1513 p-basepi-016 characteristic mutational profile in children of individuals exposed to ionizing radiation p. krawitz, h. manuel, a. knaus, g. hildebrandt, m. jäger, m. schubach, m. rodriguez de los santos, t. pantel, d. beule, s. mundlos, k. sperling institute for medical genetics and human genetics charite, berlin, germany the dna damaging effects of ionizing radiation are deliberately used in cancer therapy as well as feared in accidents related to nuclear technology. despite its influence on the exposed organism, irradiation was believed to have no major effect on succeeding generations, as irreparable dna damages were thought to result in cell death. recently, however, genome-wide mutation screenings in offsprings of male mice that were irradiated with high dosages showed an accumulation for certain de novo events. we therefore focused on these mutational classes in a small cohort of human individuals that were conceived while or after their fathers were exposed to high frequency radiation. in the whole genome sequences of 22 such offsprings we could confirm de novo rates for single nucleotide variants in the order of 10 -8 per base as previously reported. interestingly, however, we found de novo translocations of paternal origin as well as increased numbers of clustered de novo mutations that resemble the results from animal studies. this characteristic mutation profile might thus be used as an indicator of irradiation exposure in one of the individual's parents. from the upstream regular rb1 promoter. to test this hypothesis, we generated a genetic model carrying modifications in the rb1 promoter and in cpg85 using crispr/cas9 technology. data on the establishment of the model and first results will be presented. the gid/ctlh protein complex with its seven core protein members is conserved in all eukaryotic cells. in saccharomyces cerevisiae it functions as an ubiquitin ligase complex and regulates the metabolic switch from gluconeogenesis to glycolysis (1) . recently, we could show that the vertebrate gid/ctlh complex also functions as an ubiquitin ligase, however substrates and exact function remain unknown (2) . a growing number of components of the ubiquitin protein system (ups) are described to be regulators of ciliogenesis (3). defects in such genes are considered to cause ciliopathies, genetic disorders with typical phenotypic variations in patients and model organisms (4) . first data supports our hypothesis that the ctlh complex plays a major role in ciliogenesis, e. g. the ctlh subunit rmnd5a localizes to the basal body which is a modified centriole of primary cilia in nih-3t3 cells and rmnd5 knock down in xenopus laevis leads to defects in cilia formation of epidermal multiciliated cells. n. reich, m. sandbothe, r. buurman, b. schlegelberger, t. illig, b . skawran department of human genetics, hannover, germany background and aims: hepatocellular carcinoma (hcc) is characterized by genetic and epigenetic changes that lead to a deregulation of important tumor suppressors and oncogenes in a multistep process. one of these epigenetic changes is the elevated expression of histone-deacetylases (hdacs) which contribute to a transcriptional repression of certain genomic regions by remodeling the chromatin structure. thereby, not only the expression of tumor-relevant genes is affected, but also the expression of micrornas (mirnas). selected mirnas have been shown to play important roles in carcinogenesis. we aimed to identify mirnas deregulated by histone deacetylation and to understand their functional consequences in hcc tumorigenesis. methods: histone acetylation was induced by the global hdac inhibitor trichostatin a (tsa) in four hcc cell lines (hle, hlf, huh7, hepg2) and two immortalized liver cell lines (thle-2 and thle-3) in order to identify differentially expressed mirnas and messenger rnas (mrnas) by global expression profiling. findings were validated by transfection of microrna mimics and sirna-mediated knockdown in hcc cell lines, quantitative pcr, western blotting and luciferase reporter assays. results: after hdac-inhibition, hsa-mir-129-5p was significantly upregulated. the mir-129-5p holds tumor suppressive potential and its expression is reduced in different types of tumors. one predicted target gene of mir-129-5p is the hepatoma-derived growth factor (hdgf). this mitogenic growth factor is highly expressed in a variety of cancers, for example in hcc, and its expression correlates with a poor prognosis, irrespective of the tumor type. hdgf is a multifunctional protein that is involved in several signaling pathways, contributing to proliferation and metastasis of cancer cells, induction of angiogenesis and inhibition of apoptosis. incubation of hcc cells with tsa or transfection with mir-129-5p reduced expression of hdgf. luciferase assays indicate a direct regulation of hdgf by mir-129-5p. moreover, expression of the death receptor fas, which is a potential downstream target of hdgf, is also regulated by the mir-129-5p. a translocation t(8;14)(q24;q32) was not detectable in any of the investigated tissues. with the established assay we were able to provide a highly sensitive tool for the detection of the translocation t(8;14)(q24;q32). however, we did not detect normal b-cells carrying this translocation. this does not exclude that such cells exist. alternatively, the growth advantage conferred by myc may promote the acquisition of secondary genetic changes. this may result in a rapid tumorigenesis, that if occurring these cells only present as full blown burkitt lymphoma. myotonic dystrophy: links to the nuclear envelope p. meinke, s. hintze, s. limmer, b. schoser friedrich-baur-institute, munich, germany myotonic dystrophies (dm) are slowly progressing multisystemic diseases with a predominant muscular dystrophy -making dm the most frequent muscular dystrophy in adulthood. dm is caused by heterozygous dna-repeat expansions in the dmpk gene (dm1) or the cnbp gene (dm2). the repeat-containing rna accumulates in ribonuclear foci and splicing factors are sequestered to these foci, resulting in abnormal regulation of alternative splicing. dm patients show overlapping phenotype presentations with progeroid laminopathies, which are caused by mutations in nuclear envelope proteins. in search for molecular signatures of this overlap, we found an enrichment of nucleoplasmic reticuli in dm1 and dm2 patient myoblasts. additional, we found an alternative splicing of the lmna gene -both effects that are associated with progeroid laminopathies. this implies possible shared pathomechanism between dm and progeroid laminopathies. retinoblastoma is a tumor of the retina occurring in young children up to the age of five. it is caused by biallelic inactivation of the tumor suppressor gene rb1. we have shown that human rb1 is an imprinted gene and as such characterized by differential dna methylation of a cpg island (cpg85) in rb1 intron 2. cpg85 is not methylated on the paternal allele and acts as a promoter for the alternative rb1 transcript, rb1-e2b. it is argued that expression of rb1-e2b is causative of the observed skewing of regular rb1 expression in favor of the maternal allele. a true gametic differentially methylated region (gdmrs) is established in only one of the parental germ lines. it is supposed to be stable during early embryonic development and to be passed on to all daughter cells. we could show that cpg85 is free of methylation in human sperm. publicly available methylome data on oocytes revealed that cpg85 is fully methylated in human oocytes. these data are in agreement with cpg85 being a maternal methylated gdmr. we showed that the level of cpg85 methylation is 60 percent in blood, as expected. however, in eight tissues of three individuals we observed a gain of methylation at cpg85 ranging from 60 to 65 percent in liver and skin, and increasing to 70 to 85 percent in the other tissues (heart, kidney, muscle, brain, lung and spleen). interestingly, the degree of methylation was lower in fetal tissue than in adult tissue, as determined for brain and muscle. we also observed gain of methylation at cpg85 in two human embryonic stem cell lines and induced pluripotent stem cells. this is consistent with the finding of complete methylation at cpg85 in eight different retinoblastoma cell lines. we therefore conclude that cpg85 is an unstable dmr. in oocytes, dna methylation of gdmrs is established by transcriptional read-through from an upstream promoter. therefore, we hypothesize that gain of dna methylation at cpg85 is caused by run-through transcription erozygous state (nomenclature according to hgvs; reference sequence nm_000051.3). in silico-analysis by alamut (version 2.8.1) predict the loss of the donor splice site of intron 27 of the atm gene. cdna-analysis was performed and revealed the loss of exon 27 of the atm by a complex activation of two kryptic splice sites. a premature stop codon was generated giving rise to a truncated protein that leads to a pathogenic variant. the results of the genetic analysis are discussed in the context of the clinical findings. identification of the underlying genetic causes of gastric cancer will give a better view of the mechanisms that contribute to the pathophysiology of the disease. gemeinschaftspraxis für humangenetik und genetische labore, hamburg, germany, 2 zentrum für diabetologie bergedorf, schwerpunktpraxis, hamburg, germany about 2-5% of all pregnant women develop gestational diabetes mellitus (gdm) during their pregnancies and diabetes complicating pregnancy is associated with adverse maternal and perinatal outcomes, notably, risk of fetal macrosomia and neonatal hypoglycemia and development of diabetes after pregnancy. gdm is considered to result from interaction between genetic and environmental risk factors. the case of a 35-year old female german patient with a novel mutation in the pax4 gene (rare mody gene type 9) as a cause of gestational diabetes mellitus is presented. we describe clinical, biochemical and genetic features of the patient, who developed gdm and gave birth to her child by cesarean section. mody genes type 1-11 were analyzed. sequencing the pax4 gene revealed a novel mutation in exon 8, pax4,c.778delc, p.(leu260cysfs*24); reference sequence nm_006193.2), a deletion of a cytosine leading to a truncated, non-functional protein. to date, no small deletion has been detected in the pax4 gene. identification of the underlying genetic causes of gdm will give a better view of the mechanisms that contribute to the pathophysiology of the disease. furthermore, early identification may improve options to prevent gdm and complications for the mother and her child. the results of the genetic analysis are discussed in the context of the clinical findings. the modulation of dna methylation is highly flexible and plays an important role during cell differentiation. furthermore, the dna methylome alters considerably during aging. age related changes in the dna methylation of regulatory genes are assumed to have a major impact on carcinogenesis (teschendorff, 2010) . moreover, it was demonstrated that the chronological age of a human donor can be predicted with high accuracy by analyzing the dna methylation of a specific minor set of cpg loci which are aberrantly methylated during aging (horvath, 2013) . hence, we intended to investigate the effect of epimutations identified in different lymphoma entities in comparison with the influence of epigenetic changes in sequential b cell differentiation stages on the epigenetic age. the altered expression of the tumor suppressor mir-129-5p due to chromatin remodeling may play a fundamental role in hepatocarcinogenesis. we expect that histone deacetylation and putative target genes of epigenetically deregulated mir-129-5p can be targeted by new therapeutic agents. the microrna-449 family inhibits tgf-β-mediated liver cancer cell migration by targeting sox4 introduction: modulation of microrna expression is considered for treatment of hepatocellular carcinoma (hcc). therefore, we characterized the epigenetically regulated microrna-449 family (mir-449a, mir-449b, mir-449c) with regards to its functional effects and target genes in hcc. methods: after transfection of mir-449a, mir-449b, and/or mir-449c, tumor-relevant functional effects were analyzed using in vitro assays and a xenograft mouse model. binding specificities, target genes, and regulated pathways of each microrna were identified by microarray analyses. target genes were validated by luciferase reporter assays and expression analyses in vitro. furthermore, target gene expression was analyzed in 61 primary human hccs compared to normal liver tissue. results: tumor suppressive effects, binding specificities, target genes, and regulated pathways of mir-449a and mir-449b differed from those of mir-449c. transfection of mir-449a, mir-449b, and/or mir-449c inhibited cell proliferation and migration, induced apoptosis, and reduced tumor growth to different extents. importantly, mir-449a, mir-449b, and, to a lesser degree, mir-449c directly targeted sox4, which codes for a transcription factor involved in epithelial-mesenchymal transition and hcc metastasis, and thereby inhibited tgf-β-mediated cell migration. conclusions: this study provides detailed insights into the regulatory network of the epigenetically regulated microrna-449 family and, for the first time, describes distinct tumor suppressive effects and target specificities of mir-449a, mir-449b, and mir-449c. our results indicate that particularly mir-449a and mir-449b may be considered for mirna replacement therapy to prevent hcc progression and metastasis. novel mutation in the atm gene and activaton of two kryptic splice sites in an 52 year old female patient with gastric cancer gemeinschaftspraxis für humangenetik und genetische labore, hamburg, germany, 2 schwerpunktpraxis, hämatologie, onkologie und palliativmedizin, hamburg, germany, 3 israelitisches krankenhaus, chirurgische klinik, hamburg, germany gastric cancer is a global public health concern, ranking as the third leading cause of cancer mortality. familial aggregation of gastric cancer is common in about 10% of the cases, and about half of these can be attributed to hereditary germline mutations. however, for most gastric cancer cases, whether genetic events contribute to cancer susceptibility remains unknown. here we present a case report of a patient with gastric cancer, a family history of breast cancer and a novel mutation leading to complex cryptic splicing in the atm gene. ngs panel sequencing and cnv/mlpa analysis of 18 genes associated with gastric and breast cancer were performed. sequencing revealed a novel mutation in intron 27 of the atm gene, atm,c.4109 + 1g>a in an het-abstracts tions remained unidentified since positive deletion-junction pcr products could not be amplified. to identify the breakpoints of the 15 deletions, we performed custom-designed microarray cgh analysis with a high resolution of probes located within and flanking the nahr hotspots prs1 and prs2. the array analysis suggested that 11 of the 15 deletions exhibit breakpoints within prs2, even although previously performed breakpoint-spanning pcrs with primers designed according to the reference sequence of the human genome have been negative in these 11 cases. since prs2 exhibits high sequence diversity resulting from frequent nonallelic homologous recombination events without crossover, we surmised that haplotype diversity is responsible for the failure of the breakpoint-spanning pcrs performed with primers designed according to the reference sequence. therefore we characterized the haplotype diversity of prs2 in 30 human individuals and designed deletion-junction pcr primers that facilitate the amplification of rare prs2 haplotypes. so far, we have identified the breakpoints of four of the 11 type-1 nf1 deletions predicted to have been mediated by prs2 according to the array results. we are confident to identify further breakpoints by extending these analyses using primers suitable to amplify rare prs2 haplotypes. our findings indicate that the characterisation of nahr hotspots in terms of haplotype diversity is a premise to identify the breakpoints of nahr-mediated microdeletions by means of deletion-junction pcrs. p-basepi-028 *** array-based dna methylome analyses of primary lymphomas of the central nervous system ulm university, ulm, germany, 2 university of cologne, cologne, germany, 3 christian-albrechts-university kiel, kiel, germany, 4 university hospital muenster, muenster, germany primary lymphomas of the central nervous system (pcnsl) are defined as diffuse large b-cell lymphomas (dlbcl) that are confined to the central nervous system (cns). although pcnsl cannot be distinguished from dlbcl by their morphology as well as their histology, they differ in prognostic outcome. the aim of the present study was to compare the epigenomic landscape of pcnsl and dlbcl. to this end, we analyzed the dna methylation of a total of 26 pcnsl (cryopreserved or formalin fixed and paraffin embedded (ffpe)) using the infinium humanmethylation450 beadchip array (illumina) and contrasted these findings to 79 dlbcl (kretzmer et al., 2015) . as controls, we used publicly available dna methylation data from a total of 50 normal brain samples derived from different regions of the cns (gilbert et al., 2105; jaffe et al., 2016; kurscheid et al., 2015; mur et al., 2013; wockner et al., 2014) . after normalization of the data we performed thorough filtering and removed the random snps, all loci located on gonosomes, as well as those loci with a detection p-value >0.01 in at least one of the samples, leading to 457,951 loci entering subsequent analyses. when comparing the dna methylation profiles of pcnsl versus dlbcl we identified 8279 differentially methylated loci (σ/σ max = 0.4; q < 1e-4). in order to remove those loci which represent a "brain signature", we compared dlbcl versus brain (σ/ σ max = 0.4; q < 0.05) based on the list of the previously identified 8279 loci. after removing this "brain signature", we ended up with 2231 loci that are differentially methylated between pcnsl and dlbcl. in a next step we wanted to make sure that the differences in methylation at these 2231 loci are not due to differences in starting material (cryopreserved versus ffpe) which is known to influence the outcome of the beadchip analysis. therefore, we compared the dna methylation profiles of five cryopreserved versus ffpe samples (derived from the same tissue samples) and identified 318 differentially methylated loci (σ/σ max = 0.4, q < 0.05). only five loci of both lists overlapped, which were subsequently removed from further analysis so that we ended up with a final list of 2226 loci which are differentially methylated between pcnsl and dlbcl. in order to analyze the biological implications of the differentially methylated loci we evaluated an enrichment of known functional groups (ku-additionally, our aim was to analyze whether entity-specific differences in the resetting of the epigenetic clock are generated during lymphomagenesis or derive from modified dna methylation in the germinal center b cells of origin. to address these issues, we performed dna methylation profiling (hum-anmethylation450 beadchip) of 72 burkitt lymphoma samples (age 2-76 yrs), 119 diffuse large b cell lymphoma samples (age 3-93 yrs) and 103 follicular lymphoma samples (age 22-80 yrs) from the icgc mmml-seq and mmml-consortium (kretzmer et al., 2015) and the hematopathology section kiel as well as 145 peripheral blood samples of healthy individuals (0-63 yrs) available from the same project and current publications of our group (kolarova et al., 2015; friemel et al., 2014) . in addition, we received 61 b cell subpopulation samples (0-66 yrs) covering different stages of b cell differentiation that were measured in the same way (kulis et al., 2015; lee et al., 2012) . the epigenetic age of the samples was predicted using the "online age calculator" accessible at https://dnamage.genetics.ucla.edu and compared with the corresponding chronological age of the donors. in fact, the epigenetic age of peripheral blood samples of healthy donors was in high accordance with their chronological age (pearson's r 0.968, p-value <0.001) while the correlation between epigenetic and chronological age of sequential b cell differentiation stages was slightly lower (pearson's r 0.893, p-value<0.001). in contrast, the predicted epigenetic age of the burkitt lymphoma samples was significantly higher than the corresponding chronological age. this deviation may be interpreted as "epigenetic pre-aging". nevertheless, the epigenetic age of diffuse large b cell lymphomas and follicular lymphomas tended to be less affected. in conclusion, we found significant epigenetic pre-aging in burkitt lymphoma samples that seems to be induced during lymphomagenesis and does not derive from altered dna methylation patterns in the germinal center b cells of origin. moreover, no significant shift of the epigenetic age was observed for the other lymphoma entities, healthy blood samples and b cells of sequential differentiation stages. identification of type-1 nf1 deletion breakpoints mediated by rare prs2 haplotypes a. summerer 1, 2 , m. hillmer 1,2 , v. f. mautner 3, 4 , l. messiaen 5, 6 , h. 2 1 institute of human genetics, ulm, germany, 2 university of ulm, ulm, germany, 3 department of neurology, hamburg, germany, 4 university hospital hamburg eppendorf, hamburg, germany, 5 department of genetics, birmingham, usa, 6 university of alabama at birmingham, birmingham, usa neurofibromatosis type 1 (nf1) is a hereditary cancer syndrome with an incidence of 1 in 3000. in 5% of all nf1 patients, large deletions encompassing the nf1 gene and its flanking regions are causing the disease. the majority of all large nf1 deletions are of type-1; they encompass 1.4-mb and are mediated by nonallelic homologous recombination (nahr) with crossover. the breakpoints of type-1 deletions are located within the lowcopy repeats nf1-repa and nf1-repc which exhibit high sequence homology to one another. previous studies suggested that type-1 deletion breakpoints cluster within the paralogous recombination sites prs1 and prs2 spanning 5-kb and 4-kb, respectively. in our present study, we investigated 218 patients with type-1 nf1 deletions using long-range pcrs to detect breakpoints located within prs1 or prs2. according to these analyses, 157 (72%) of the breakpoints are located within prs2 and 34 (15.6%) in prs1. however, 27 (12.4%) of the type-1 deletions were not positive for these deletion-junction pcrs. we surmised that some of these deletions may have breakpoints within the 14-kb region located between prs1 and prs2. this 14-kb region also exhibits high sequence homologoy between the nf1-reps which is a prerequisite for nahr. indeed, 12 of the 27 type-1 nf1 deletions exhibited breakpoints within this 14-kb region as determined by the analysis of seven overlapping deletion-junction pcrs. however, the breakpoints of 15 dele-esophageal adenocarcinoma (ea) represents one of the most rapidly increasing cancer types in high-income countries. barrett's esophagus (be) is a premalignant precursor of ea and has an estimated prevalence of 5-6% in the population. however, only 0.1 to 0.3% of be patients develop ea. within an international consortium, we carried out a gwas meta-analysis in 6167 be patients, 4112 ae patients and 17.159 controls (gharahkhani et al., lancet oncology, 2016) . in a comprised be/ae analysis, we identified 14 genome-wide significant risk loci, of which seven were previously unreported. the strongest associated new risk variant was identified for rs17451754 (p = 4.8×10 -10 ), which maps within intron 21 of the cftr gene. cftr encodes a protein that functions as a chloride channel and that is mutated in patients with cystic fibrosis (cf). mutations in cf lead to abnormal viscous secretions with altered chemical composition, resulting in dysfunction of the respiratory system and the gastrointestinal tract. the most common cf mutation is δf508, a deletion of three nucleotides in cftr that results in the loss of a single codon for phenylalanine on protein position 508. interestingly, cf patients show a highly increased incidence of gastroesophageal reflux, which represents the major risk factor for be and ae. in view of the phenotypic overlap for gastroesophageal reflux and cystic fibrosis, and for gastroesophageal reflux and both be and ae, combined with the association of cftr risk variants in patients with be and ae, it seems plausible that a common pathophysiological mechanism is triggered by cftr. in order to test this hypothesis, we analyzed the association of δf508 in a european case-control cohort with be and ae patients. for this, we performed a genotyping assay of δf508 in 1037 be patients, 1609 ae patients and 941 controls. we could not observe a significant association (p = 0.57). this might be (i) due to insufficient sample power or (ii) due to the fact, that not δf508 but other genetic variants at the locus might explain the underlying functional mechanism of the association. fine mapping of all genetic variation at the cftr locus and exten-lis et al., 2015). remarkably, cpg loci that are differentially methylated during normal b-cell maturation were significantly depleted. in turn we saw an enrichment of loci located in heterochromatin. in summary, we detected more than 2000 loci that are differentially methylated between pcnsl and dlbcl, which do not play a functional role in normal b-cell differentiation. replication study of gwas-identified genetic modifiers of age at huntington's disease onset although there is a strong correlation between cag repeat length and age at onset (ao) of motor symptoms, individual huntington disease (hd) patients may differ dramatically in onset age and disease manifestations despite similar cag repeat lengths. since the modifier variations described so far only account for a small fraction of the heritable contribution to the ao, the identification of loci and genes using genome-wide methods appears highly promising. against the background of incomplete understanding of the hd disease pathophysiology, the hypothesis-free approach of gwas offers an ideal starting point for the search of modifier genes. recently, a combined analysis of all gwa data to hd modifiers identified different loci with genome-wide significant signals for association to residual age at motor onset [gem-h. consortium]. interestingly, none of the most significant association signals and none of the trending snps in the european gwa analysis corresponded to any previously suggested candidate modifier genes. in order to be able to better assess these data, we tried to replicate the top ten associated gwas variants in a comprehensive cohort of 505 german hd patients. we only found modest association with one of the top ranked snps (rs72810940), all remaining variations showed no correlation with the ao. this inconsistency highlights once again the difficulties of modifier searching in hd or any other monogenic disorder, which faces the same challenges as the genetic characterization of complex disorders. with an incidence of 0.2-0.3/100.000 malignant tumors of the thymus are a rather rare type of cancer. here we report the case of a man of german descent, who presented with a thymoma at age 55. in the pathological report the thymus tumor was described as an extremely unusual thymoma with partial loss of keratin and massive proliferation of myoid cells. it was subsumed to a primary thymic, partially epithelial neoplasia, resembling an uncommon b2/b3-thymoma. after the patient's death his widow looked for genetic advice concerning the risk of disease for her children. detailed personal and familial history brought up surprising information: thymoma was one of four cancers in our patient. he developed adenocarcinoma of the colon at age 35, squamous cell cancer of the nose/upper lip at 54 years and in addition current cancer staging revealed a papillary renal cell carcinoma. according to family history his father and his uncle developed colon cancer with 58 and 66 years, the son of this uncle was diagnosed with colon cancer at age 47. this cousin of the propositus was referred to genetic counseling, because of msi-high-status and loss of mlh1 and psm2 in immunohistochemistry. he was found to have a deleterious mutation in exon 14 of mlh1gene (c.2644c>a, p.tyr548stop) resulting in a premature termination of mlh1-protein. our patient has never been tested for hnpcc. however a post mortem performed immunhistochemical examination of thymic cancer cells revealed an almost complete loss of mlh1 nuclear expression suggesting the presence of a mlh1 germline mutation and indicated hnpcc. considering the loss of mlh1 in tumor cells it is more than likely that the development of thymoma was the consequence of deficient dna mismatch-repair. there have been reports of rare tumors in hnpcc families in the last years (i. e. clear cell renal carcinoma and uterine sarcoma). pande et al. reported one case of thymoma in their registration of cancer occurrences in 368 mutations carries from 176 hnpcc families [1] . our case emphasizes the importance of detailed family history and contributes to the discussion of widening the inclusion criteria for genetic counseling and testing for hnpcc. to this day the revised criteria of bethesda are used to identify families at risk. we propose that the established criteria have to be revised and rare tumors should be included. unknown partner genes in leukemias with rare translocations can be identified using targeted rna sequencing c. haferlach, n. nadarajah, m. meggendorfer, n. dicht, a. stengel, w. kern, t. haferlach mll, munic, germany in hematological malignancies fusion genes play an important role and function as therapeutic targets, impressively shown for e. g. bcr-abl1 and etv6-pdgfrb. thus, the identification of fusion genes is the basis for precision medicine, selecting treatment based on genotype and providing markers for disease monitoring. the aim of this study was to test the value of targeted rna sequencing in a routine diagnostic work up. 51 cases were selected harboring rearrangements of kmt2a (n = 10), runx1 (n = 19), etv6 (n = 11), pdgfrb (n = 6), npm1 (n = 2), rara (n = 2) and jak2 (n = 1) identified by chromosome banding (cba) and fish analyses. in none of the cases the partner gene could be identified using standard methods. targeted rna sequencing was performed using the trusight rna fusion panel (illumina, san diego, ca) consisting of 7690 probes covering 507 genes known to be involved in gene fusions. this assay allows the capture of all targeted transcripts. sequencing was performed on nextseq (illumina, san diego, ca). analysis was performed with the rna-seq alignment app (basespace sequence hub) using star for alignment and manta for gene fusion calling with default parameters (illumina). sive functional analysis are needed to find the causal variant that explains how the cftr locus interferes with the pathomechanism of be and ae. a recent functional study indicated cftr as a tumor suppressor gene in murine and human intestinal cancer, providing further evidence for cftr as a true disease gene for be and ae. background: it has long been established that mutations in brca2 predispose for pancreatic adenocarcinoma with brca2 germline mutations identified in 6-10% of familial pancreatic cancer cases. consequently, screening for pancreatic cancer has been recommended for mutation carriers with an affected first-degree relative since early detection has been shown to significantly improve 5-year survival from 4-7% to 24%. for brca1 mutations, however, relevance in pancreatic tumorigenesis is still being discussed with several studies questioning an elevated risk of pancreatic cancer in families with brca1 mutations while others are suggesting that brca1 may also play an important role in predisposing to pancreatic cancer. clinical screening for pancreatic cancer commonly remains unavailable to brca1 mutation carriers and it has even been questioned whether brca1 should be analyzed in familial pancreatic cancer at all. clinical report: here we report on a 59 year old woman with metastatic pancreatic cancer whose sister had died of pancreatic cancer at 50 years of age. in this family we identified a pathogenic brca1-germline mutation (brca1: nm_007294.3:c.1292dupt,p.(leu431phefs*5)) by next-generation sequencing using a 94-gene panel. the index patients' tumor was available for genetic analysis and showed loss-of heterozygosity for brca1. this strongly suggests the brca1 mutation to be causative of the pancreatic cancer development in this patient. when the family was first introduced to genetic counselling there was no evidence of breast-or ovarian cancer in any relatives. only after identification of the mutation did the index person reach out to distant family members and it was thereby revealed that a distant branch of the family had independently been counselled for hereditary breast and ovarian cancer. in this part of the family, however, there had not been any cases of pancreatic cancer. subsequent predictive testing was offered to healthy family members and 3 further mutation carriers could be identified. two women were referred to breast cancer screening. additionally, the mutation was identified in a relative with recurrent metastatic breast cancer at the age of 38 years. for her and the index patient parp-inhibition therapy thus became a possible further treatment option. conclusions: in conclusion we propose next-generation sequencing approaches including the analysis of brca1 to be used in familial pancreatic cancer. we also argue that brca1 mutation carriers with pancreatic cancer cases in their family should be offered the same screening program as brca2 mutation carriers. within the framework of a study this could allow for more precise risk stratification in the future. contralateral dcis is unknown. only the male breast carcinoma was herceptin receptor positive, all other breast carcinomas of the chek2 mutation carriers were her2 negative. among our chek2 positive families we noticed the association with chek2 mutation and female breast cancer. we observed a contralateral breast cancer, male breast cancer and other tumors in our families as well. the majority of the observed breast cancers was estrogen and progesterone receptor positive and herceptin negative. while benign uterine smooth muscle tumors are among the most frequent human symptomatic tumors, their malignant or borderline lesions are only rare findings. both lesions can show somatic copy number alterations, but their patterns differ, thus constituting helpful diagnostic tools. aimed at an advanced classification of the lesions we have performed molecular inversion probe array analyses of these tumors. besides complex patterns of genomic alterations seen in nearly all cases, two of the lesions presented with copy number neutral uniparental disomies i. e. normal copy numbers with an apparent monoallelic origin. in one case, an upd of part of the long arm of chromosome 22 was detected in a uterine leiomyosarcoma. the tumor showed genetic heterogeneity with gains and losses. in addition, the 11.45 mb segment located at 22q12.1-q13.1 was clearly of monoallelic origin throughout all cells investigated. all other genetic alterations were restricted only to part of the cells of the sample thus reflecting the presence of tumor cells as well as normal bystander cells which in general characterizes mutations that had arisen during tumor development. in contrast, the upd that was detected in all examined cells clearly suggests its germline occurrence. the second tumor was a leiomyoma-variant of the type with bizarre nuclei. again, besides gains and losses an apparent germline upd was found that covered a 3.71 mb segment on chromosomal segment 8q11.21. upd for even the whole arm of chromosome 22 repeatedly has been reported not to coincide with phenotypic manifestations. nevertheless, the question arises whether or not the observed upds might be related to a familiar predisposition for uterine muscle tumors. of note, as a result of genome-wide association studies snps on 22q recurrently have been found to be significantly associated with fibroid development. triple negativity is an independent predictor of germline mutations in breast cancer predisposing genes breast cancer is the most common cancer in women. 12-15% of all tumors are triple-negative breast cancers (tnbc) lacking expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. so far, tnbc have been mainly associated with mutations in brca1, although recent studies also found mutations in other breast cancer susceptibility genes. a brca1/2-centered perspective thus may ignore the significance of other predisposing genes, whose relevance appears obvious as dna damage repair by homologous recombination is a complex process involving many proteins. in 32/51 cases with rearrangements involving kmt2a (n = 10), runx1 (n = 8), etv6 (n = 6), pdfgrb (n = 4), rara (n = 2), npm1 (n = 1) or jak2 (n = 1) the partner genes were identified. these were in kmt2a rearranged cases: mllt10 (n = 2), mllt1 (n = 2), itpr2, flnc, asxl2, dcp1b, maml1 and arhgef12. in runx1 translocated cases partner genes were plag1 (n = 2), prdm16, mecom, zfpm2, man1a2, n6amt2, and kiaa1549l. prdm1, mecom and zfpm2 have previously been described in the literature as runx1 partner genes but were not suspected in our cases as partner genes due to complex cytogenetic rearrangements. the other identified partner genes have not been described so far. interestingly, prdm1, mecom, zfpm2 and the newly identified plag1 are all members of the c2h2type zinc finger gene family. partner genes identified in etv6 rearranged cases were: abl1, ccdc126, clptm1l, erg, foxo1 and cflar-as1. wdr48, zbtb11, nfia and mprip were identified as partner genes of pdgfrb and rpp30 in an npm1-translocated aml. in an all patient a jak2-ppfibp1 fusion was identified leading to classification as a bcr-abl1-like all. in an apl patient showing an ins(17;11) (q12;q14q23) a zbtb16-rara fusion was identified and thus resistance to all-trans retinoic acid, arsenic trioxide, and anthracyclines can be predicted. further in a case with t(17;19)(q21;q13) an irf2bp1-rara fusion was detected. conclusions: targeted rna sequencing was able to characterize rare gene fusions and provided the basis for the design of rt-pcr based assays for monitoring mrd. targetable genetic aberrations were identified, which were not detected by cba enabling more individualized treatment. targeted rna sequencing may be a valuable tool in routine diagnostics for patients with rearrangements unresolved by standard techniques. female carriers of a pathogenic mutation in the chek2 gene are reported to have a life time risk of about 20-45% to develop breast cancer. there is evidence for increased risks for contralateral breast cancer, male breast carcinoma and other types of tumors. in addition to well-known mutation chek2:c.1100del, other pathologic mutations are being identified in the gene due to the inclusion of the gene in most breast cancer gene panels for dna testing. between 2012-2016 the center for hereditary breast and ovarian cancer regensburg cares for 10 families with pathogenic or probably pathogenic mutations in the chek2 gene affecting nine female patients and one male patient (6 × c.1100del, 1 × deletion of exon 10, and 3 × variants considered as likely pathogenic: c.1408g> c, c.1561c> t, c.1169a> c). the mean age of diagnosis of breast cancer (both sexes) was 45.1 years (range 25-61 years). the patient with the deletion of exon 10 was first diagnosed at 25 years of age and developed a contralateral breast carcinoma (dcis) at 35 years of age. the male patient was diagnosed with breast cancer at 58 years of age and at 59 years with a renal carcinoma. one patient was diagnosed with a papillary thyroid carcinoma at age 26 years and developed breast cancer with 35 years. in 8 out of the 10 families, breast carcinoma diagnosed with 51.3 years on average, was reported in the family history. in addition, there were additional malignancies such as prostatic carcinoma, thyroid carcinoma, colorectal cancer, gastric carcinoma, leukemia, cervical carcinoma and malignant melanoma. none of the affected family members was tested for the respective chek2 mutation. the tumors with an initial diagnosis at 25 years and 35 years were estrogen-receptor-negative and progesterone-receptor-negative. the other 8 of the 11 breast cancers were positive for the estrogen receptor, 7 of the 11 tumors were positive for the progesterone receptor. the receptor status of the abstracts it has been shown that in 3d culture hescs can be differentiated into neural retina containing organoids. we established this differentiation schedule and started comparative differentiation of wildtype h1 hescs and the rb1 null derivative (g4, rb1 mt/mt ) into neural retina. during the first weeks of differentiation into neural retina organoids generated from the rb1 mt/mt hescs have a smaller diameter and thinner retina layer compared to wildtype organoids. however, during the time-course the mutant organoids began to catch up. thus, at later stages no difference in size and thickness could be observed anymore. comparative immunostainings of cryosections at d19 show no difference in expression of the markers pax6 and sox2 between the wildtype and mutant hescs. further comparative immunostainings for markers specific for neural retina like e. g. rx and vsx2 at d19 and d33 are ongoing and will be presented. exome sequencing identified potential causative candidate genes for unexplained cowden syndrome purpose: cowden syndrome (cs) is a cancer predisposition syndrome characterized by the occurrence of breast cancer, epithelial thyroid cancer, endometrial carcinoma and various other findings such as mucocutaneous lesions and macrocephaly. cs belongs to the pten hamartoma tumor syndrome (phts) primarily associated with germline mutations in pten. in recent years, germline mutations in additional genes (sdhb, sdhc, sdhd, pik3ca, akt1, sec23b) have been described in few patients; however, to date, in 20-75% of patients meeting clinical criteria for cs the underlying cause remains unclear. methods: to uncover predisposing causative genes, the exomes of 11 clinically well characterized, mutation negative patients with suspected cs were sequenced (illumina hiseq) using leukocyte dna. assuming a monogenic disease model, the called variants were filtered for rare (minor allele frequency ≤1% for homozygous/compound heterozygous variants and ≤0.01% for heterozygous variants according to dbsnp, evs, and exac), truncating (nonsense, frameshift, highly conserved splice sites), and missense germline variants (predicted to be pathogenic by at least 2/3 in-silico tools). for data analysis and variant filtering the gatk software and the cartagenia bench lab ngs software were applied. all candidate genes were included in a pathway analysis (ingenuity). in a first preliminary analysis, we focused on known cancer genes and genes interacting with pten. results: after stringent filtering steps, comparison with large datasets from population-based controls, and detailed manual inspection to exclude artifacts, 75 genes were affected by presumed biallelic variants (16 homozygous and 59 putative compound-heterozygous), one of these is a known cancer gene (cbfa2t3); in 17 genes biallelic variants were found in 2-6 patients. heterozygous variants were found in 23 genes in 2-6 patients, but none of these are known cancer genes. in 132 genes, heterozygous truncating mutations occurred in only one patient, 4 of these are cancer genes (msh6, wrn, kdm5a, pml) . the phenotype of the patient with a msh6 frameshift deletion fulfilled key features of cs (early-onset metachronous papillary thyroid cancer, breast cancer, endometrial and colorectal cancer), however, the tumor spectrum is partly compatible with lynch syndrome/ hnpcc. examination of the colorectal cancer demonstrated microsatellite instability and a loss of msh6 protein expression. the pathway analysis of the remaining candidate genes identified several interacting partners of pten (grhl3, ehhadh, cstf3). conclusions: preliminary data indicate that exome sequencing might identify potentially relevant causative genes for cs, some of which are recurrently mutated. the present work-up consists of the inclusion of further non-cancer genes, validation of variants by sanger sequencing, testing of to determine the prevalence of mutations we performed panel-based germline mutation testing of 10 high and low-moderate penetrance breast cancer susceptibility genes (brca1, brca2, atm, cdh1, chek2, nbn, palb2, rad51c, rad51d and tp53) in 229 consecutive individuals affected with tnbc unselected for age at diagnosis or breast and ovarian cancer family history. age at diagnosis ranged from 23 to 80 years with an average of 50.2 and a median of 48 years. in 60 women (26.2%) we detected a pathogenic mutation, with a higher frequency (31.3%) in the group manifesting cancer before 60 years. deleterious brca1 mutations occurred in 14.8% of tnbc patients, predominantly frameshifting (24/34, 70.6%). the most frequent, both among brca1 mutations and in total, were the founder mutations c.5266dupc and c.2411_2412delag. deleterious brca2 mutations occurred in 5.7% of patients, all but one (c.1813du-pa) being unique. while no mutations were found in cdh1 and tp53, 15 mutations (25%) were detected in one of the six other predisposition genes (palb2, chek2, atm, nbn, rad51c, rad51d). no individual presented more than one mutation. almost half of all deleterious mutations (42.5%) were detected in very young women aged 35 years or less. the median age at diagnosis was significantly younger for brca1 (40 years) and brca2 (41.5 years) carriers compared to patients without a mutation (p = 2.746e-05; mann-whitney) or compared to non-brca1/2 mutation carriers (p = 0.022). in contrast, patients with non-brca1/2 mutations were not significantly younger than mutation negative women (p = 0.5288). interestingly, family history had an independent influence on age at diagnosis. taken as a whole, women with family history had a median age at diagnosis 6 years earlier than those without (p = 0.00057). this difference was lost in mutation carriers while it remained in cases without mutation. in summary, our data confirm and expand previous studies of a high frequency of germline mutations in genes associated with ineffective repair of dna damage by homologous recombination in women with tnbcs. many of these women would go untested with current restrictive criteria. in order that each patient receives therapies tailored to her genetic status, gene panel based mutation testing should be offered to all women diagnosed with tnbc, irrespective of age at diagnosis or family history. p-cancg-037 *** neural retina differentiation of hescs as an in vitro model for retinoblastoma d. kanber, m. hiber, d. lohmann, l. steenpass institute of human genetics, university hospital essen, university duisburg-essen, essen, germany retinoblastoma is the most common eye tumor of early childhood. inactivation of both alleles of the retinoblastoma gene (rb1) results in the development of retinoblastoma. our aim is to establish a human cell-based model for retinoblastoma. using the crispr/cas9 system we have generated human embryonic stem cells (hescs) carrying a mutation either on one or both rb1 alleles. all the detected mutations are located in exon 3 of the rb1 gene and close to the splice donor site of this exon. analyses on dna, rna and protein level were performed for three mutant and one double-mutant clone. the following genotypes were identified by deep sequencing (nm_000321.2(rb1_v001)): clone c2, c.364_380del, heterozygous; clones c7 and g3, c.372_378del, heterozygous; clone g4, c.[372_378del; c.367_368dup] (complex mutation on one allele), homozygous (loss of heterozygosity). the mutations of all four clones result in a premature stop codon in exon 4. on rna level we detected expression of mutant rb1 transcripts reflecting the genotype in all clones and an additional mutant rb1 transcript with skipping of exon 3 in three clones. as the heterozygous clones also showed expression of the wildtype rb1 transcript, rb1 protein (prb) could be detected for these clones (c2, c7, g3) by western blot analysis. however, the double-mutant clone g4 showed no expression of prb. so far, we have characterized 3 heterozygous and one homozygous clone. another three double-mutant clones are under investigation. the recurrent germline missense mutation g84e in the hoxb13 gene has been demonstrated to predispose to hereditary prostate cancer (prca), despite the underlying pathogenic mechanism is not yet understood. molecular examination of a first set of g84e positive tumors sought for somatic characteristics, and suggested that oncogenic ets gene fusions may appear at unusually low frequencies as compared to the general prevalence of ets fusions in prca (22% vs approx. 50%). hypothesizing that hoxb13 could predispose to ets fusion negative prca, we have analyzed 942 cases from three european ancestry populations (finland, germany and us) for the coincidence of hoxb13 g84e and the most common ets fusion, tmprss2:erg, in corresponding tumor samples. while the prevalence of tmprss2:erg fusions was similar among the three study groups (range: 56.5-60.7%), the frequency of g84e genotypes differed markedly between us (1.5%), german (3.6%) and finnish samples (8.3%). despite the expected frequency gradient among study populations, all subsamples showed a strong enrichment of g84e mutation carriers among tmprss2:erg fusion negative cases as compared to fusion positive cases (center adjusted or = 4.96; 95%ci = 2.30-11.9; p = 0.0001). consistent with the previous study, the crude frequency of the tmprss2:erg fusion in hoxb13 g84e carriers was 23.5% (range 16.7% -28.6%). examination of disease characteristics highlighted age at diagnosis to be associated with tmprss2:erg negative status (per year or = 1.04, p = 0,00007) and by trend, also with the presence of the g84e germline variant (per year or = 0.97, p = 0.14). within the subtype of tmprss2:erg fusion negative carcinoma carriers of g84e were diagnosed 3.5 years earlier as compared to non-carriers (61.6 ± 1.4 years versus 65.1 ± 0.4 years, p = 0.017). in conclusion, this study demonstrated a significant tumor subtype specific association for hoxb13 g84e mutation carriers having a higher frequency of tmprss2:erg fusion negative prca. meta-analyses from case control comparisons suggested that subtype specific risk of hoxb13 g84e for tmprss2:erg negative prca could be as high as or = 19.0, as compared to or = 9.9, when prca is regarded as one entity regardless of fusion status. finally, although tmprss2:erg negative prca is usually known to be associated with later ages of diagnoses, hoxb13 mutations may indicate a subgroup of earlier onset cases within the fusion negative entity. relatives to determine the phase of assumed biallelic variants and segregation with the phenotype where applicable. with 1 to 2 cases in 1 million inhabitants per year adrenocortical cancer (acc) is a rare disease. due to often late diagnosis and limited treatment options prognosis for patients are poor with a 5 year overall survival rate of 7 to 35%. though knowledge about molecular genetic events in acc increased over the last few years no reliable molecular prognostic factors, no effective targeted cancer therapy and no personalized treatment approach has emerged to date. that's why we intend to establish a reliable method to define a molecular signature of accs that could be used for a prognostic classification of adrenocortical cancers, for planning an individualized therapeutic approach and for the identification of known or potential targetable molecular events in the single patient. in a retrospective study dna from acc and matched blood samples is sequenced to detect somatic single nucleotide variants (snv), small insertions and deletions (indel) and copy number alterations (cnv). sequencing data are then compared to clinical data e. g. tumor stage, resection status, ki67-index and time of progression free and overall survival to define molecular prognostic factors. target enrichment of 160 genes that are known to be associated with different entities of cancer is performed with the human comprehensive cancer panel (qiagen) and sequenced on a nextseq500 (illumina). data are analysed with gensearchngs (phenosystems). znrf3, a gene that was also described to be involved in the development and the progression of acc a few years ago, is sequenced separately with sanger and analysed with gensearch (phenosystems). to date tumor samples and matched blood samples from 43 patients were analysed. one or more tumors comprise one or more snvs or small indels in 48 of 160 genes of the panel and in znrf3. snvs and small indels are most often found in tp53, ctnnb1 and znrf3 with frequencies of 28%, 26% and 19% respectively. in 37 of 160 genes cnvs -duplications and deletions -occur. cdk4 is duplicated in over 50% of the cohort. mdm2 gains are found in over 40%. one can also find three types of cn patterns: a quiet type with low number of copy alterations, a noisy one with high number of chromosomal breakages and a chromosomal one with high frequency of alterations of chromosomal arms. while no correlation between snvs and small indels and clinical outcome could be found so far, cn patterns of the accs seem to correlate with progression free survival and overall survival. patients with a noisy cn pattern have a shorter progression free and overall survival than patients with chromosomal and quiet type. though tendencies in the correlation of molecular markers and prognosis for patients suffering acc can be recognized, further samples need to be analysed to confirm the results. it is planned to sequence another 60 tumor samples and matched blood samples for this retrospective study and to validate the results in a prospective study with another 100 patients. expression of mir-371a-3p and mir-367-3p was analysed in serum samples by quantitative pcr. the cohort of 27 gcnis patients consisted of 11 patients with a solitary testicle, who had undergone orchiectomy for contralateral tgct, and 16 patients with two testicles, one of which with gcnis, but no concurrent tgct. twenty men with non-malignant testicular disease served as controls. additionally, in situ hybridisation (ish) with a probe against mir-371a-3p was performed on four testicular biopsy specimens known to harbour gcnis. sequential step sections of the corresponding tissue blocks were analysed immunohistochemically, using oct4 antibody to visualise gcnis. the median expression value of mir-371a-3p in gcnis-patients was 5.2 (interquartile range [iqr] = 35.8) which is significantly higher than the median expression of 0.0 (iqr = 0.0) in controls. both of the two gcnis subgroups had significantly higher mir-371a-3p levels than controls, with a median expression of 18.2 (iqr = 37.3) and 2.7 (iqr = 32.7), respectively. regarding mir-367-3p expression, there were no significant differences between gcnis and controls. using a relative quantity of 5 as a cut-off value, the mir-371a-3p was able to detect 51.2% (95% confidence interval [95% ci] = 31.9-71.3%) of gcnis, while only 5% (95% ci = 0.1-24.9%) of the controls were positive. in the subgroup with previous tgct 63.6% (95% ci = 30.8-89.1%) of gcnis could be detected and in the subgroup without previous tumour the rate was 43.8% (95% ci = 19.8-70.1%). the detection rates for all gcnis and for both subgroups were significantly higher than for the controls. ish staining demonstrated the expression of mir-371a-3p in gcnis cells in two of the four cases. in conclusion, this study indicates a new and minimal-invasive way of diagnosing gcnis by measuring serum levels of mir371a-3p. this approach is endorsed by the demonstration of mir371a-3p in gcnis cells by ish staining. however, the sensitivity is still low and thus, the method certainly needs refinement possibly by applying a panel of additional mi-crornas. nonetheless, measuring serum levels of mir371a-3p may constitute a valuable aid in clinical assessment of men afflicted with high-risk factors of tgct. p-cancg-043 *** the mir-371a-3p is a highly specific and sensitive serum-based marker for the diagnosis and follow-up of testicular germ cell tumours testicular germ cell tumours (tgct) are a paradigm of curable malignancies. clinical management largely relies on measuring the serum biomarkers. inopportunely, the markers beta-hcg, afp and ldh are only elevated in about 60% of patients. therefore, micrornas of the clusters mir-371-3 and mir-302/367 were proposed as novel serum-based markers. we evaluated four of the candidate mirnas (mir-371a-3p, mir-372-3p, mir-373-3p and mir367-3p) with regard to their usefulness as tgct markers. overall, serum samples from 166 tgct-patients and from 106 controls were analysed using quantitative pcr. the first 50 consecutive patients and 20 controls were analysed for all four mirnas. after roc-analysis only the marker with the greatest discriminative power was studied further. the decline of mirna expression after orchiectomy was quantified in 134 cases and in 27 metastasized cases the marker was analysed repeatedly during the course of chemotherapy. additionally 10 cases with relapsing disease were studied. the mir-371a-3p featured the highest discriminative power (area under the curve: 0.94; 95% confidence interval [95% ci]: 0.874-0.982). in the entire cohort, patients could be distinguished from controls with a sensitivity of 88.7% (95% ci: 82.5-93.3%) and a specificity of 93.4% (95% ci: neuroendocrine tumor of the adrenal gland: an unusual manifestation of tsc c. müller-hofstede, j. horvath, b. dworniczak, p. wieacker institute of human genetics, university of münster, germany we report on a young woman asking for the recurrence risk of the neuroendocrine tumor of her mother deceased at the age of 38. her mother clinically presented because of therapy-resistent hypertonia, dyspnoe, progressive edema in the legs and face and a caput medusae. mri scan revealed a tumor (11×12 cm) in the right adrenal gland with lymph node metastases compressing the v. cava inferior and synchronous metastases in lung and liver. laboratory examinations showed highly elevated levels of cortisol and adrenocorticotropin (acth). cerebral mri was normal suggesting an ectopic acth secretion by a non-pituitary tumor. histologically, an undifferentiated, largely necrotic tumor was described so that the neuroendocrine nature of the tumor could not be proven. she died within three weeks after diagnosis. on suspicion of multiple endocrine neoplasia type 1 we initially performed a sequence analysis of men1 on tumor dna by next generation sequencing without detection of a pathogenic mutation. thereupon the molecular genetic panel analysis (nf1, ret, sdhb-d,tmem127, tsc1, tsc2, vhl) uncovered the heterozygous mutation c.3379c>t (p.arg1127trp) in the gene tsc2. this mutation is already described as pathogenic (hu et al. 2014 ). in the tumor dna the allele frequency of the normal allele mounted up to 10%, whereas the allele frequency of the mutant allele came to 90% pointing to a loss of heterozygosity (loh). the mutation was confirmed by sanger sequencing. taken all together, we assumed, that the mutation in the gene tsc2 represents a germline mutation. mutations in the suppressor genes tsc1 and tsc2 cause tuberous sclerosis, an autosomal-dominant disorder, resulting in hamartomatous tumors in the heart, brain, kidneys, skin and other organs. once in a while it is discussed whether neuroendocrine tumors (nets) represent a characteristic of tsc. there are some case reports describing nets in the context of tsc, but mainly in connection with nets of the pancreas (e. g. insulinoma) or the pituitary. to the best of our knowledge there exists only one case report of a bronchial carcinoid as a result of a germline mutation in tsc1 (dworakowska et al. 2009 ) and no description of net of the adrenal gland due to a mutation in tsc1 or tsc2. ngs provides the opportunity of wide-spread testing, even post-mortem, in order to get clarification for the descendants. although in our case we could not distinguish if the mutation detected represents a germline mutation or a somatic mutation, we were able to offer a predictive testing to the daughter and other family members. we report on a rare case of net of the adrenal gland because of mutation in the gene tsc2. this case illustrates that in the differential diagnosis of nets, tsc genes should also be considered. germ cell neoplasia in situ (gcnis) is the precursor lesion of testicular germ cell tumours (tgct). if detected clinically, this lesion may herald a pending tgct. unfortunately, the only way of diagnosing gcnis is by testicular biopsy and subsequent immunohistochemical examination. therefore, non-invasive methods of diagnosis are required. mirnas of the mir-371-3 and mir-302/367 cluster had been suggested as serum biomarkers of full-blown tgcts. we aimed to explore the utility of these mirnas for the detection of the pre-invasive stage of tgcts and we looked to the expression of two mirnas in serum samples of 27 gcnis patients. psmc3ip located on chromosome 17q21 is a putative tumor suppressor gene that encodes for the nuclear psmc3 interacting protein. the protein functions as coactivator of steroid hormone mediated gene expression and is important for rad51 and dmc1-mediated homologous recombination during dna repair of double-strand breaks. recently germline variants in psmc3ip, also known as gt198, tbip, and hop2, have been identified with low frequency in early onset familial breast and ovarian cancer (hboc) patients and in a patient with apparently sporadic early onset breast cancer. somatic variants in psmc3ip are frequently observed in breast, ovarian, and fallopian tube cancers. in this study, we analyzed a cohort of 166 brca1/2 mutation-negative hboc (n = 158) or early onset sporadic breast cancer patients (n = 8) for variants in psmc3ip. we identified seven different heterozygous variants in 8 out of 166 index patients: c.-115g>a (rs191843707); c.-70t>a (rs752276800); c.-37a>t (rs199620968); c.-24c>g (rs200359709); c.519g>a p.(trp173*); c.537 + 51g>c (rs375509656); c.*24g>a. these variants were not listed or at very low frequency (<1%) in the exac database. carriers of psmc3ip germline variants were mostly (6/8) affected by early onset breast cancer (median age of onset 36 years). for three out of seven different variants (c.-115g>a, c.519g>a, and c*24a>g), a possible impact on psmc3ip expression or function was observed. the stop mutation c.519g>a p.(trp173*) was found in two sisters, which were both diagnosed with unilateral breast cancer at age 33. the premature stop codon is located within the dna-binding domain of psmc3ip and is predicted to induce nonsense-mediated mrna decay (nmd). remarkably, c.-115g>a was already described in familial breast and ovarian cancer, and was found once in this study in a female that developed unilateral breast cancer at the age of 33 years. the variant c.-115g>a (rs191843707) was shown to induce a slightly, albeit significant decrease of reporter gene expression. the c.24*g>a variant was identified in a woman diagnosed with unilateral breast cancer at the age of 36 years. luciferase reporter assays indicated an impaired effect of c.24*g>a on microrna binding. germline variants in psmc3ip are present in breast and ovarian cancer families. whether mutated psmc3ip is a new risk factor for early onset breast/ovarian cancer in families with hboc and/or apparently sporadic early onset breast cancer remains to be shown. 86.9-97.3%) with this marker. in patients without metastases the mir-371a-3p expression declined significantly after surgery. in metastasized cases the levels dropped sharply after chemotherapy. all of the 10 relapses had elevated mir-levels, and expression decreased upon chemotherapy. mir-371a-3p has significantly higher sensitivity than each one of the classical tgct markers and than a combined panel of beta-hcg, afp and ldh (87.8% vs 50.4%). in non-metastasised seminoma the mir-371a-3p expression depended significantly on tumour size. mir-371a-3p is highly sensitive and specific for tgct. it correlates with the stage of disease and with treatment effects and it therefore fulfils the prerequisites of a valuable serum-based biomarker. the significant association with tumour bulk in localised disease provides evidence for the tgct being the primary source of mir expression. the sensitivity of mir-371a-3p surpasses that of classical tgct markers by far, and thus it may become the new gold standard for serum diagnostics of tgct in the coming years. co-occurrence of radioulnar synostosis and amegakaryocytic thrombocytopenia (rusat) was initially described as an inherited thrombocytopenia syndrome that is caused by a mutation in hoxa11. in three simplex patients, de novo missense mutations in mecom were reported as an alternative origin of the disease (rusat2). mecom, identified as a common ecotropic viral integration site 1 (evi1) in murine myeloid leukemia, is known as a key transcriptional regulator in hematopoiesis and sporadic myeloid leukemia. we report here on a novel mecom mutation cys766gly (uniprot q03112-1) identified by whole exome sequencing in a family with rusat, hearing impairment, hand dysmorphisms, and patellar hypoplasia in four patients spanning three generations. notably, two of four affected individuals in our family developed a myeloid malignancy. the novel mecom missense mutation cys766gly affects a heavily conserved cysteine residue in c 2 h 2 -zinc finger motif 9 in the c-terminal zinc finger domain of mecom. this residue is crucial for the tetrahedral coordination of a zinc ion stabilizing the zinc finger conformation and thus, is essential for dna binding of the c-terminal zinc finger domain. our findings reconfirm the causality of mecom mutations and indicate that mecom mutations also need to be considered in familial rusat patients. in addition, we report for the first time that mecom germline mutations targeting the c-terminal zinc finger domain are associated with an increased risk for myeloid malignancies. this extends the rusat2-associated phenotype and proposes that mecom germline mutations can cause a genetic predisposition to myeloid malignancy. (z., b. and s., d. contributed equally to this work) many genes that harbor rare mutations which entail a medium or high risk for breast cancer (bc) belong to dna double strand repair and have been identified by linkage analysis or by sequencing of candidate genes in bc families. in addition, a considerable number of common but low risk germline variants have been found in genome wide association studies. however, these predisposing factors yet only explain a fraction of bc cases. with the intention to identify low frequency variants conferring an intermediate bc risk, we performed an association study using a candidate gene approach and testing dna repair capacity with the micronucleus test (mnt) as an additional second phenotype. rs3810813 in the slx4/fancp gene showed an association with bc that was pronounced in younger cases and was confirmed in a verification cohort (combined analysis of 1,448 cases, 2593 controls: or = 2.19 (1.45-3.30) , p = 0.00012 for cases ≤40 years). genotyping additional snps and imputation revealed a specific european haplotype of ca. 150kb length that spans slx4 and adjacent genes. it is tagged by 6 the observed mutual dependence of the two phenotypes allowed a considerably improved interpretation of the results: (i) the unknown causal variant on the haplotype can be assumed to be present mostly in cases, indicating a rare variant with a rather strong effect; (ii) using this information on the two phenotypes in the association between the mnt results and bc improved considerably the identification of the specific risk among cases (<60 years) who carried the haplotype. roc curves for bc depending on mnt results revealed that the stratification on carriers of the haplotype increased the auc from 0.65 (p = 0.0007) to 0.94 (p = 0.0001). both associations can be best explained by a risk variant carried by a fraction of the haplotypes that is enriched in early onset bc cases. slx4 is the only gene in the tagged region which can be functionally related to both associated phenotypes, while for the other genes no connection to bc or dna repair is reported. inherited dna repair mutations: are they modifiers of brca1 and brca2 penetrance and age at onset of hereditary breast and ovarian cancer? background: inherited mutations in brca1 and brca2 are the most common causes of hereditary breast and ovarian cancer (hboc). the risk of developing breast cancer by age 70 in women carrying a brca1 mutation is 57-65% and 45-55% in brca2 carriers. however, mutations in brca1 and brca2 only explain about 25% of all hboc cases. the lifetime risk varies between families and even within affected individuals of the same family. the cause of this variability is unknown but it is hypothesized that additional mutations or rare variants in genes that are possibly interacting with brca1/2 in different dna-repair pathways contribute to this phenomenon. methods: we obtained samples of 181 patients positive for brca1 or brca2 mutations and an age-of-onset (aoo) of breast cancer below 35 or above 60 years of age from the german consortium for hereditary breast and ovarian cancer. panel sequencing was done to screen germline dna for mutations in 311 genes involved in different dna-repair pathways. variants were classified into five classes according to a modified version of plon et al (2008) . only truncating mutations and known pathogenic missense mutations were considered pathogenic or likely pathogenic. results: the patient group with an early aoo (93 women) had developed breast cancer at a mean age of 26.4 years (± 2.06) and the control group (88 women) had developed breast cancer at a mean age of 69.5 years (± 7.05). a total of 4,293 variants were detected in all patients and 54 of these (1.3%; 95% ci, 0.96%-1.64%) were presumed to be deleterious. mutations were found in 45 genes other than brca1 and brca2. mutations were mainly found in single-strand break repair (ssbr 29%), double-strand break repair (dsbr 26%) and checkpoint factors (13%). the rest were found in genes with other functions such as brca1/2 interactors, centrosome formation, and signal transduction. the putative mutations were found in 26 women of the control group (29.5%; 95%ci, 20.3%-40.2%) compared to 36 women of the patient group (38.7%; 95% ci, 28.8%-49.4%). the incidence of germline mutations in dna-repair genes did not differ according to the age of onset (p = 0.2). prevalence of additional germline mutations in dsbr in patients (13%) was not significantly different from prevalence of dsbr mutations in controls 10.2% (p = 0.6) conclusions: the preliminary results failed to show a difference in mutation load between the two cohorts of brca1/2 carriers sorted by age of onset. larger studies are needed and may provide further insight into the role of mutation load in hboc age of onset of brca1/2 carriers. objective: glioblastoma stem-like cells (gscs) carry stem cell features and therefore seem to be responsible for tumor initiating, maintaining, recurrence and chemo-and/or radiotherapy resisting. the current knowledge on genetic and transcriptomic characteristics of these cells especially in comparison to glioblastoma tissue is still limited. the aim of this study is to compare the genetic and genomic profile of glioblastoma tissue and gscs. thereby, differences in involved genes and affected pathways on dna level as well as on gene expression level are identified. material and methods: peripheral blood and tumor tissue were obtained from patients with glioblastoma. tumor tissue derived explant cell culture and serum-free culture were established. based on multi-parameter magnetic-activated cell sorting (macs) technique, cd15 and cd133 labeled cell subpopulations of gscs could be isolated. the tumor tissue, serum-free culture, and the isolated cell subpopulations as well as blood were analyzed by snp array and gene expression (excluding blood) in a paired design. for preliminary characterization of gscs in the serum-free culture we confirmed the stem cell features of gscs by the expression of nestin, sox2, and cd133 (applying immunofluorescence staining). results: our results of snp array analyses showed genetic aberrations in all analyzed cellular entities (tumor tissue and cell subpopulations, e. g. gain of chromosome 7, loss of 10q23.31, loss of 10q11.1->q26.3, and complete loss of chromosome 10). furthermore, distinct genetic differences between the cell subpopulations and tumor tissue were observed (e. g. loss of chromosome 4 and segmental uniparental disomy of 9p24.3->p21.3, only in cancer stem-like cell subpopulations). in addition, we detected many possibly candidate cancer genes and pathways which may have an influence on tumorigenesis. gene expression analyses revealed strongest differences between fresh tumor tissue and serum-free culture based cells, where more than a third of investigated genes were affected. when contrasting fresh tumor tissue with stem cell marker positive serum-free cultured cells, 1,106 genes were upregulated in the stem cell marker positive cells, whereas 1,533 genes where upregulated in fresh tumor tissue. within these genes, strongest enriched pathways in stem cell marker positive cells included positive regulation of cell cycle and cancer-related pathways, whereas in fresh tumor tissue predominantly immune-related pathways were found, e. g. myeloid leukocyte activation, inflammatory response and phagocytosis. conclusion: differences between gscs and tumor tissue using snp array analyses and gene expression were detected. our results may help to get more information about the molecular pathomechanisms of glioblastoma. it still needs more investigations on the field of genetic and genomic analyses between gscs and glioblastoma tissue to identify novel potential targets for therapy development. background: fanconi anemia (fa) is a rare inherited chromosomal instability syndrome associated with bone marrow failure as well as myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). one-third of fa individuals exhibit bone marrow cytogenetic clones, notably gains of 1q and 3q and/or loss of 7/7q, and 15% to 30% of fa patients developed mds/aml. in recent years, the application of high throughput technologies has revealed recurrent somatic mutations in genes implicated in myeloid malignancies. as additional genetic maladies facilitating mds/aml development in fa is lacking, we aimed to elucidate whether these mutations would be present in fa patients with mds/aml. methods: using illumina trusighttm myeloid sequencing panel (san diego, ca), we performed next-generation sequencing (ngs) on dna extracted from bone marrow specimens from 17 fa mds/aml patients registered in the european working group of childhood mds. the sequencing panel targeted 54 genes frequently mutated in hematologic neoplasms. results:. ten of the 16 (62.5%) evaluable patients had 18 lesions (1 to 3 mutations per patient; 14 missense, 1 nonsense, 2 insertion and 1 duplication) in 13 genes. the presence of a somatic mutation did not appear to correlate with complex karyotype or −7/7q. all affected genes occurred in isolation with exception of runx1 and kras. while 13 of the mutations were pathogenic, 5 were variance of unknown significance. mutations in genes involved in epigenetics (dna methylation, chromatin maintenance and cohesin complex; idh2, tet2, dnmt3a, idh1, ezh2, rad21 and asxl1) and mutations in transcription factor genes (runx1, ikzf1 and etv6) represented the most frequently affected genes. this was followed by mutations of genes encoding signaling molecules including the ras pathway (kras and ptpn11). altered runx1 was the most common lesion and occurred in individuals with aml, raeb or raeb-t. one patient with refractory anemia with ring sideroblasts (rars) had a mutation in the spliceosomal gene, sf3b1. conclusions: while the most common mutations encountered in sporadic cases of mds were in genes involved in rna splicing and epigenetics, these two broad categories of genes appeared to have less influence in our fa patients. most mutations were nonrecurring suggesting that there is no specific mutation pattern of these genes in fa-related mds/aml. however, runx1 mutations and also mutations involved in genes of the ras pathway appear to play a pathogenic role in fa mds/aml development. taken together, the data suggests that mutations in genes that cause clonal hematopoiesis in the population at large do not contribute significantly to fa hematopoietic clonal disease; however, particularly acquisition of runx1 and ras pathway alterations promote malignant myeloid disease progression. more extensive studies analyzing more patients are necessary to further define the secondary hits leading to fa myeloid disease. chromatin remodeling is a complex process shaping the nucleosome landscape, thereby regulating the accessibility of transcription factors to regulatory regions of target genes and ultimately managing gene expression. the swi/snf (switch/sucrose nonfermentable) complex remodels the nucleosome landscape in an atp-dependent manner and is divided into the two major subclasses brahma-associated factor (baf) and polybromo brahma-associated factor (pbaf) complex. somatic mutations in subunits of the swi/snf complex have been associated with different cancers, while germline mutations have been associated with autism spectrum disorder and the neurodevelopmental disorders coffin-siris (css) and nicolaides-baraitser syndromes (ncbrs). css is characterized by intellectual disability (id), coarsening of the face and hypoplasia or absence of the fifth finger-and/or toenails. so far, variants in five of the swi/snf subunit-encoding genes arid1b, smarca4, smarcb1, arid1a and smarce1 as well as variants in the transcription factor-encoding gene sox11 have been identified in css-affected individuals. arid2 is a member of the pbaf subcomplex, which until recently had not been linked to any neurodevelopmental phenotypes. in 2015, mutations in the arid2 gene were associated with intellectual disability. in this study, we report on two individuals with private de novo arid2 frameshift mutations. both individuals present with css including id, coarsening of facial features, other recognizable facial dysmorphisms and hypoplasia of the fifth toenails. hence, this study identifies mutations in the arid2 gene as a novel and rare cause for css and enlarges the list of css-associated genes. the ubiquitin pathway is an enzymatic cascade including activating e1, conjugating e2, and ligating e3 enzymes, which governs protein degradation and sorting. it is crucial for many physiological processes. compromised function of members of the ubiquitin pathway leads to a wide range of human diseases, such as cancer, neurodegenerative diseases, and neurodevelopmental disorders. mutations in the thyroid hormone receptor interactor 12 (trip12) gene (omim 604506), which encodes an e3 ligase in the ubiquitin pathway, have been associated with autism spectrum disorder (asd). in addition to autistic features, trip12 mutation carriers showed intellectual disability (id). more recently, trip12 was postulated as a novel candidate gene for intellectual disability in a meta-analysis of published id cohorts. however, detailed clinical information characterizing the phenotype of these individuals was not provided. in this study, we present seven novel individuals with private trip12 mutations including two splice site mutations, one nonsense mutation, three missense mutations, and one translocation case with a breakpoint in intron 1 of the trip12 gene and clinically review four previously published cases. the trip12 mutation-positive individuals presented with mild to moderate id (10/11) or learning disability [intelligence quotient (iq) 76 in one individual], asd (8/11) and some of them with unspecific craniofacial dysmorphism and other anomalies. in this study, we provide detailed clinical information of eleven trip12 mutation-positive individuals and thereby due to its heterogeneous etiology, primordial growth retardation is often a challenge for geneticists and clinicians in respect of diagnosis, therapy and prognosis. thus, pinpointing its genetic origin is required for a personalized treatment and prognosis. one syndrome mainly characterized by intrauterine and postnatal growth is silver-russell syndrome (srs), a clinically and molecularly heterogeneous disorder with a considerable overlap with other syndromes. in only 60% of patients with the characteristic srs phenotype the diagnosis can be confirmed molecularly, but 40% of cases remain without molecular diagnosis. in fact, in clinically less well characterized patients referred for diagnostic testing, the detection rate is less than 20%. however, systematic investigations on the contribution of mutations in genes which may be considered in the differential diagnoses of srs are still missing. we examined 60 patients referred for molecular testing of srs but without molecular alterations associated with srs by ngs. a targeted ngs approach comprising 26 genes implicated in the differential diagnoses of srs or suggested as srs candidate genes was performed. in 5 patients fulfilling the criteria of srs accordingly to our recently developed clinical scoring system, disease-causing variants were found. these patients carried mutations in genes associated with bloom syndrome, mulibrey nanism, kbg syndrome, short syndromes or ig-f1r-associated short stature, respectively. indeed, some of the differential diagnoses detected in our cohort have a major impact on clinical management, including cancer screening because of a high risk for tumor development. furthermore, we did not identify any pathogenic mutation in one of proposed srs candidate genes (e. g. mest, grb10, copg2), thus raising the question whether these genes are indeed involved in the etiology of srs. we show that a (targeted) ngs approach is an important tool to identify the genetic cause in patients with unexplained growth retardation. furthermore, our data show (positive) clinical scoring in srs should not impede the consideration of differential diagnoses and other molecular causes. submicroscopic deletions of chromosome band 2p25.3 have been reported in more than 20 patients. common clinical features include intellectual disability/developmental delay, central obesity and behavioural difficulties. myt1l became the main candidate gene for id and obesity since it is deleted or disrupted in all published patients. however, reports of deletions affecting only this gene and even more so of deleterious myt1l sequence variants are very rare. to our knowledge, until now only two patients with de novo myt1l point mutations have been reported. in the present study, we analysed a cohort of individuals with intellectual disability of unknown aetiology and their unaffected parents by whole exome sequencing. we identified de novo myt1l sequence variants in two out of 311 patients. patient 1 carried a nonsense mutation (c.1531g>t, nm_015025.2; gly511*) whereas patient 2 carried a direct splice site mutation (c.2769-2a>g). according to prediction algorithms, both detected myt1l variants are deleterious (patient 1: sift score 0, cadd score 42; patient 2: cadd score 24.6). in addition, patient 2 carried a de novo splice site variant in setd1b. however, this variant is predicted to be benign (cadd score 2.5) as well as a known snv (rs749218728, maf 0.0000323). a comprehensive clinical characterisation of the two patients yielded only mild or moderate intellectual disability, behavioural problems and muscular hypotonia as common clinical signs. surprisingly, obesity was only present in patient 2. postnatal tall stature and transient microcephaly were present in one patient each. this clinical picture is compared to the published phenotypes of patients with myt1l point mutations, patients with microdeletions of only myt1l and patients with larger 2p25.3 deletions. with the reduced penetrance regarding obesity, the clinical picture of patients with myt1l mutations is becoming more and more unspecific. the retina and anterior neural fold homeobox gene (rax) controls the embryonic eye development and is involved in human autosomal-recessive microphthalmia. so far only a few compound heterozygous mutations in rax have been described in microphthalmia patients. we report a first case of microphthalmia caused by a novel homozygous mutation in rax. the 8-month-old patient was born to consanguineous parents and presented with extreme microphthalmia, panhypopituitarism and developmental delay. mri of the brain showed bilateral agenesis of the anterior visual pathway and tractus opticus. ocular ultrasound confirmed bilateral anophthalmia. additionally, dysgenesis of the corpus callosum and an abnormal pituitary gland have been detected. the first child of these parents, who died shortly after birth, had also been diagnosed with bilateral anophthalmia. using panel diagnosis of the disease associated genome, we identified the homozygous pathogenic variant c.112del, (p. 138fs) in rax. we performed a segregation analysis and confirmed that both parents are heterozygous for this variant. so far developmental delay and panhypo-expand the clinical spectrum of the trip12 gene in non-syndromic intellectual disability with or without asd. background: whole exome sequencing (wes) using next generation sequencing has proven to be a powerful tool in determining the underlying genetic cause of rare disorders. here, we show, that clinical follow-up and diagnostic re-evaluation can be crucial for uncovering further disease-causing mutations. clinical report and genetic findings: we report follow-up data of a previously published consanguineous family with two children, a boy and a girl, suffering from severe encephalopathy, hypotonia, microcephaly and retinal dystrophy. wes had shown a homozygous intronic splice variant in pgap1 (c.1090-2a>g;p.?) causative for the symptoms. both parents were heterozygous carrier for the pgap1 variant (granzow, paramasivam et al, mol cell probes 2015) . in the next pregnancy, the unborn child presented hydrops fetalis, omphalocele, short tubular bones and cystic kidneys. chorionic villus sampling showed the fetus to be homozygous for the pgap1 variant. however, neither of these symptoms fit with a pgap1-associated disorder. additional wes of fetal dna and re-evaluation in the family showed a homozygous nonsense variant in ift140 (c.g3577t;p.e1193*) consistent with a diagnosis of mainzer-saldino syndrome (mss) which is characterised by the association of renal disease, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia, as a second diagnosis in the fetus. again, both parents were shown to be a heterozygous carrier for the ift140 variant. yet, as omphalocele was not accounted for by any of the identified conditions, a third genetic cause cannot entirely be excluded. alternatively, omphalocele may be a rare manifestation of mss, or be the result of a combination of both disorders. the couple opted for induced abortion. discussion: it is estimated that an individual carries multiple heterozygous variants for autosomal recessive disorders in his or her genome. especially in consanguineous families, this results in an elevated risk for children with more than one disorder. in recent publications of clinical exomes, double diagnoses have been reported in 0 to 12% of investigated subjects. thus, the possibility of more than one causative gene should be carefully explored when working with wes and re-evaluation in case of additional clinical symptoms within a family should be considered. also, follow-up of families with rare genetic disorders may lead the clinical geneticist beyond the assumed single cause to multiple single gene disorders in the same family. conclusion: using wes, we have identified two independent single gene disorders in a consanguineous family demonstrating that clinical follow-up and diagnostic re-evaluation can be crucial for uncovering multiple disease-causing mutations in one family. we present a case study using molecular cytogenetic approaches on a 3 year old boy presenting with microcephaly-brachycephaly, macroglossia and absent speech development. the boy is the first child of healthy, consanguineous parents of pakistani origin. following an uncomplicated pregnancy, the hypotrophic newborn was delivered at 37 weeks weighing 2610 g. in the third month of life, the baby had viral meningitis. regular pediatric follow-up revealed psychomotor delay with hypotonia. creatine kinase, lactate and fibroblast growth factor 21 measured in serum were high. subsequent investigations at the age of 16 months included brain mri, electroencephalogram and muscle biopsy that gave hints of a mitochondriopathy or potential neuropathy of axonal type. due to the suspicion of a complex mitochondriopathy, whole exome sequencing was performed using a sureselect human all exon kit (agilent, 50mb v5) on a hiseq 2500 (illumina). the analysis revealed a heterozygous microdeletion of 121 kb on chromosome 9q34.3 which was classified as an unclear variant (uv), ehmt1-gen was not affected. re-examination of proband's dna using array cgh detected a larger 180 kb heterozygous deletion in the 9q34.3 region (arr[hg19] 9q34. 3(140,395,510-140,575,736) x1), encompassing exon 1 of ehmt1 (euchromatin histone methyltransferase 1; transcript nm_ 024757.4). haploinsufficiency of this gene results in kleefstra syndrome (omim 610253), a multisystem disorder due to either microdeletions in 9q34.3 encompassing ehmt1 or intragenic point mutations. mlpa analysis (ehmt1 mpla-kit p340) of parental dna further indicated a de novo origin of the deletion in our proband. a similar deletion has been described previously in a case presenting with clinical features of kleefstra syndrome [2] strengthening the importance to include the 5' part of ehmt1 in sequencing as well as cnv screening. in summary, our study clearly shows that array cgh is a valuable complementary approach to ngs especially for poorly covered regions in ngs i. e. exon 1 of many transcripts. we report on a three-generation family with variable manifestations including delayed and incomplete tooth eruption, early tooth loss due to short dental roots, acroosteolysis, osteoporosis, tendon ruptures, joint hypermobility, muscle weekness, glaucoma, neurological features, and psoriasis. after detection of elevated cd169/siglec1-expression on monocytes and an upregulation of interferon-stimulated gene transcripts, singleton-merten syndrome was diagnosed. the novel heterozygous mutation c.992c>g (p.thr331arg) in ifih1 was found in three affected family members. singleton-merten syndrome is a very rare autosomal dominant interferonopathy, so far described in not more than four families. until now, only two different gain-of-function mutations in ifih1 have been detected. mutations in ifih1 are also associated with aicardi-goutière syndrome and recently features of both conditions were found in the same family. our findings expand the mutational spectrum of singleton-merten syndrome and demonstrate the high intrafamiliar variability associated with mutations in ifih1. pituitarism have not been described in association with rax mutations. therefore we conducted array comparative genomic hybridization and karyotyping in the index patient. both tests gave normal results. prenatal diagnosis by chorionic villus sampling in the next pregnancy excluded a homozygous carrier status for this rax mutation. mutations in the phf6 gene are associated with borjeson-forssman-lehmann syndrome (bfls), an x-linked intellectual disability disorder affecting mainly males. female carriers usually show no or mild clinical signs. however, recent studies described females with de novo phf6 gene defects (mutations, deletions) and severe phenotypes resembling coffin-siris syndrome [1, 2] . here, we report on a girl with a maternally inherited phf6 mutation and a phenotype resembling those described previously in affected females. the mother had learning difficulties and mild dysmorphological features (hypertelorism, prominent forehead). when seen at age 12 months, the proposita showed muscular hypotonia, was unable to sit and had limited head control (developmental delay 6 months). dysmorphic features included scaphocephaly, hypertelorism, a small flat nose with anteverted nares, low set, prominent ears, a high, narrow palate, absent labia minora and linear skin pigmentation on the thighs. ophthalmologic investigation identified strabism convergens of the left eye, hyperopia and an excavated papilla with a pale optical nerve. ultrasound showed patent foramen ovale, tricuspid insufficiency and a unilateral incomplete duplication of the renal pelvis. karyotyping performed elsewhere was normal (46,xx). results of a genome-wide snp array analysis (affymetrix cytoscan hd) were also normal. using a targeted ngs approach for syndromic and non-syndromic developmental delay encompassing over 1200 brain related genes (mpimg-1-test), we identified a heterozygous lof-mutation c.88c>t (p.gln30*) in the phf6 gene (encoding phd finger protein 6). in addition, a human androgen receptor (humara) assay using blood dna showed a highly skewed x-inactivation (91:9). segregation analysis indicated a maternal origin of the variant. the mother also had skewed x-inactivation in blood. her husband and other daughter tested normal for the c.88c>t variant. here, we describe the first female patient with a maternally inherited phf6 mutation and a severe phenotype. the mild phenotype in the mother might be due to different patterns of x-inactivation or to (undetected) mosaicism. this report represents the 12th case of a severely affected female patient with a phf6 gene defect. findings support the assumption [1] that the female phenotypes of bfls might be more common than previously estimated. toms typical for ada2 deficiency such as fever, livedo racemosa, abdominal colics, arthralgias, and raynaud's phenomenon were observed months later. cecr1 sequencing (nm_001282225.1) revealed two previously described pathogenic missense mutations: c.140g>c, p.(gly47ala) and c.506g>a, p.(arg169gln). compound heterozygosity was confirmed by parental analysis. to the best of our knowledge, this combination of mutations has not been described until now. the p.(arg169gln) is considered as founder mutation in the dutch population, but first phenotype-genotype analyses did not allow further prediction of clinical outcomes. ada2 deficiency should be considered in patients with childhood stroke despite the absence of systemic inflammation and cerebral vasculitis. congenital hyperinsulinism (chi) has been described as heterogeneous entity caused by at least 9 different genes. in 1991, tein et al. first described a defective activity of l-3-hydroxyacyl-coa dehydrogenase in a 16-year old patient with hypoketotic hypoglycemic encephalopathy. biochemical markers of l-3-hydroxyacyl-coa dehydrogenase deficiency (schad) are high 3-oh-glutarate excretion in urine and c4-oh-carnitine in plasma. the clinical presentation is very heterogeneous with regard to age of onset, severity of symptoms as well as response to medical treatment and leucine-sensitivity. in some patients even a near total pancreatectomy was performed. schad dependent hyperinsulinism (hhf4) is a rare autosomal recessive disorder that is caused by mutations in the gene hadh. here we describe 4 patients from 3 unrelated families out of a cohort of 136 chi patients mainly from central europe. patients 1 and 2 are siblings from unrelated parents. the older brother manifested with hypoglycemic convulsions at the age of 5 weeks. a subtotal pancreatectomy was performed in an outside academic hospital. in further course he developed epilepsy and has been treated with diazoxide and anticonvulsants. the 2nd child was born with hypoketotic hypoglycemia and chi was diagnosed in first days of life. diazoxide treatment stabilized blood glucose and both children were referred to our pediatric endocrinology at the age of 8 years and 10 months, respectively. mutational analysis revealed the homozygous variant c.547-3c>g within the region of the splice acceptor site in intron 4 of the hadh gene in both affected children. this change is neither registered in exac nor described in the mutation databases hgmd or in the literature and was predicted to disrupt proper splicing. we then completed mutational analysis in unidentified patients of our chi cohort with diazoxide responsiveness and known or suspected consanguinity. patient 3 was born to consanguineous parents and chi manifested in the girl at neonatal age with hypoketotic hypoglycemia. she was successfully treated with diaxozide. later, she developed convulsions and statomotoric developmental delay. the homozygous splice mutation c.636 + 471g>t in intron 5 of hadh was detected in the child. the parents were identified as heterozygous carriers. in patient 4, a girl born to consanguineous turkish parents, chi manifested at the age of 6 months with hypoglycemic seizures. she responded well to diazoxide treatment. a homozygous missense mutation (c.406a>g; p.lys136glu) in exon 3 of hadh was detected in the patient and her parents were heterozygous carriers. hadh mutations in case 3 and 4 have been previously described in probands of turkish descent and appear to be founder mutations in the turkish population. in conclusion, we recommend hadh mutation analysis to be considered in chi children with unknown cause and known consanguineous pedigrees or originating from populations with higher prevalence of consanguinity. homozygous or compound heterozygous mutations in cecr1 (cat eye syndrome chromosome region, candidate 1) have recently been identified to causing deficiency of adenosine deaminase 2 (ada2; dada2) with childhood polyarteritis nodosa (pan) (omim # 615688). this inflammatory vasculitis affects the skin, and inner organs (predominantly kidneys and gastrointestinal tract) and also shows a high risk of ischemic stroke, brain hemorrhage as well as peripheral neuropathy. using whole-exome sequencing it was also found that the six adult patients (aged 20-48) described by sneddon in 1965 (sneddon syndrome, omim # 182410) likewise carried compound heterozygous cecr1 mutations. sneddon syndrome is characterized by a combination of dermatologic features (livedo racemosa) and ischemic brain infarctions. recently, clinical and genetic data of more than sixty ada2 patients have been reviewed and underlined the wide clinical variability in age at onset, clinical findings, outcome of neurological involvement, and additional hematological symptoms. typically, stroke has been reported to follow systemic inflammatory disease and predominantly affects posterior and central brain areas. here we describe one of the rare patients in whom acute mesencephalic stroke preceded systemic inflammation and presented as initial clinical symptom. symptriploidy is a recurrent finding in prenatal diagnostics. in a small number of individuals, correction of triploidy has been suggested based on the finding of (mosaic) genome-wide uniparental disomy (upd). we here investigated uncultured und cultured amniotic cells (ac) and placental tissue from a fetus, in which ultrasound examination in the 17 + 0th week of gestation revealed growth retardation, left diaphragmatic hernia with parts of stomach and bowel localized in the chest, dextrocardia, short nasal bone and single umbilical artery. these findings were confirmed at the 18 + 1th week when the pregnancy was terminated. the pregnancy was conceived spontaneously by a 28-year-old mother and a 31-year-old father, both healthy and with uneventful family history. the parents were non-consanguineous and carry a normal karyotype. microsatellite analyses of uncultured ac obtained at initial presentation showed for chromosomes 13, 18 and 21 a pattern suggesting triploidy with only biallelic presentation. while y-chromosomal sequences were lacking the x-chromosome showed, unexpectedly a rather disomic pattern. metaphase yield on cultured ac was low but showed a mosaic karyotype 48,xxx-,+10[20]/47,xxx[17], which was confirmed for several chromosomes by interphase fish. remarkably, a triploid clone was cytogenetically not detected. thus, we performed further analyses using microsatellite markers, oncoscan technology and fish. these studies unraveled in uncultured ac a pattern suggestive of triploidy with the supernumerary chromosomal complement derived from a maternal isodisomy with the notable exception of the x chromosome. in cultured ac and placental tissue for all chromosomes, except x and 10, a diploid pattern was observed with alleles from both parents identical to those in the uncultured ac. trisomy x was confirmed in both tissues with the supernumerary chromosome x being of paternal isodisomic origin. the trisomy 10 was seen only in cultured ac, and likely represents a pseudomosaic which nevertheless could not be proven due to insufficient yield of mitoses from the parallel cultures. finally, retrospective interphase fish on remnant uncultured ac showed two diploid clones, one disomic (approximately 40% of nuclei) and one trisomic (60%) for the x chromosome. the most likely explanation for the findings is a mosaicism for one diploid clone with genome-wide maternal isodisomy and a second diploid but bi-parental cell line with paternal trisomy x. given the identity of the (maternal) alleles in both clones our findings suggest that originally a triploid clone due to a maternal division error/inclusion of a polar body ii existed which underwent (erroneous) triploidy rescue resulting in one diploid biparental clone and one haploid clone of maternal origin that underwent haploid rescue resulting in genome-wide maternal isodisomy. the biparental clone with trisomy x either resulted from a sperm with two x-chromosomes or an erroneous x-duplication during trisomy rescue. since the introduction of non-invasive prenatal diagnosis (nipd, e. g. har-mony® test) in 2013, this test is frequently demanded and routinely applied in prenatal centers and medical practices. mostly, nipd is intended to detect autosomal trisomies (13, 18, 21), but also offers the possibility to analyze sex chromosomes. therefore, also sex chromosome aneuploidies (sca) (e. g. monosomy x (turner syndrome), triple x, xxy (klinefelter syndrome), xyy) are incidentally found. so far, in our prenatal center sca were detected in 7 pregnancies by har-mony® test, consisting of three pregnancies with monosomy x (turner syndrome) and two pregnancies with klinefelter syndrome (xxy). triple x and xyy were detected one time each. of the three cases with suspected monosomy x, the diagnosis of turner syndrome could only be confirmed in one case. this fetus also had a hydrops at week 10 + 0. for the other two fetuses, the chromosomal analysis of amniotic fluid revealed normal female karyotypes (46,xx). in both cases with suspected klinefelter syndrome, this diagnosis could be disproved by amniocentesis (karyotype 46,xy). in the pregnancies with assumed triple x and xyy, the true fetal karyotype was not further determined yet. from our experience, the rate of false positive results concerning the sex chromosome aneuploidies is noticeably higher than reported in two studies of nicolaides 2014 and hooks 2015. this has to be strongly considered in the counselling of patients who wish to know the fetal sex by nipd. congenital myopathies and congenital muscular dystrophies: will genetic testing replace muscle biopsy in the near future? congenital myopathies and muscular dystrophies are a group of inherited neuromuscular diseases with early onset and broad genetic and histopathological overlap. the diagnostic approach has considerably changed with next generation sequencing methods available. here, we describe the diagnostic value of genetic and histological methods in a cohort of 117 index patients and hence the efficacy of diagnostic procedures. 78 of 117 patients had a muscle biopsy as a first-tier approach. in 54 of 78 patients muscle biopsy was informative, leading to a classification in subgroups of cm or cmd. however, in only a few of these cases biopsy led to a specific diagnosis (e. g. merosin deficiency). in 55 of 78 patients genetic testing (candidate gene sequencing or ngs) was performed additionally to muscle biopsy as a second-tier diagnostic step, while 39 patients of the whole cohort received genetic testing only. in almost two-thirds of these 94 patients genetic testing identified known pathogenic or most likely pathogenic variations. these findings illustrate that genetic testing is superior to muscle biopsy in accurately diagnosing cm or cmd. in conclusion, we suggest that invasive muscle biopsy should be replaced by genetic testing as first-tier diagnostic procedure in patients with clinical signs of cm or cmd. nmd inhibition increases the amount of gaa-rna in patient's lymphocytes as well as in the cells of his parents. the residual function of the resulting protein has to be investigated. discussion and conclusion: rna analysis in lymphocytes with and without nmd inhibition is a simple method for analysing splice defects in all monogenic disorders with expression of the disease causing gene in lymphocytes. a further advantage for the patient is the use of blood cells instead of fibroblasts, because a skin biopsy can be avoided and analysis times are reduced. the exact characterization of pathogenic variants is an important aspect of diagnosis, prediction of disease severity and genetic counselling. in vitro nmd inhibition in lymphocytes of affected patients allows the characterization of splice defects. in the future successful inhibition of nmd in vitro might help to identify patients, who may profit from a therapeutic intervention with nmd inhibitors. even expression of a partial protein with low or no activity reduces the risk for the patient to develop antibodies hampering enzyme/protein replacement therapy. p-cling-067 12q14 microdeletion syndrome: a family with short stature and silver-russel (srs)-like phenotype. introduction: the silver-russel syndrome (mim 180860), first described independently by silver and russell in 1953, is a condition with intrauterine growth retardation, postnatal growth failure and other characteristic features, including relative macrocephaly (defined as a head circumference at birth ≥1.5 sd score (sds) above birth weight and/or length sds), prominent forehead, body asymmetry and feeding difficulties as recently defined in an international consensus statement. patients and methods: we report here on 3 first degree relatives with a silver-russel syndrome phenotype who presented with prenatal-and postnatal growth retardation, feeding difficulties, a prominent forehead and a failure to thrive. additional features such as dysmorphic facial features, periodically increased sweating, and scoliosis were present in one of the family members only, whereas learning problems and cardiac arrhythmia were present in one other. none of the patients had relative macrocephaly. high resolution array-cgh was performed to screen for cncs and mlpa to confirm the array-cgh result. results: no hypo-methylation of the imprinting center on 11p15 nor uniparental disomy of chromosome 7 and 14 were found in the index-patients. high-resolution array-cgh identified a 12q14.3 microdeletion of 1.67 mb (arr[grch37 12q14.3(65, 863, 528 ,640)×1). the heterozygous loss was confirmed by mlpa in the index patient and the other two affected family members (i. e. her brother and mother). the deletion includes the genes hmga2, llph, tmbim4, irak3, helb, grip1, and the pseudogene rpsap52. conclusion: to the best of our knowledge this is the first report on familial presentation of a silver-russel syndrome due to a microdeletion in 12q14.3. none of the patients had relative macrocephaly. supporting the hypothesis by takenouchi et al. that the causative gene for relative macrocephaly resides centromeric to hmga2, the region centromeric of hmga2 is not included in the deletion in our family. spastic ataxia of charlevoix-saguenay (sacs) is an autosomal recessive neurodegenerative disorder and is caused by homozygous or compound heterozygous mutations in the sacs gene. first symptoms of sacs are walking difficulties due to unsteady gait. further typical clinical features include spasticity, ataxia, pyramidal tract signs, nystagmus and dysarthria. here, we report on a 16-year-old female patient who initially presented with disturbances in motor abilities including frequent falls and high arched foot. cranial mrt was normal while nerve conduction velocity was significantly reduced. the patient's parents did not show any clinical features. since no pmp22 duplication was detected we performed a gene panel including 64 genes that are associated with hereditary motor and sensory neuropathies (hmsn) and related disorders by using targeted next generation sequencing. we identified the two heterozygous stop mutations c.9305t>a (p.leu3102ter) and c.9305dupt (p.leu3102phefs*8), located at the same position in the sacs gene. sanger sequencing did not enable us to properly display that there is a transversion and a duplication of the same nucleotide at two different alleles. this exemplifies that, in contrast to sanger sequencing, ngs can illustrate both alleles separately. to conclude, this case was only resolvable by ngs which makes this method appropriate for the detection of compound heterozygous mutations, especially in the rare event when two mutations occur at the same position. background: the precise identification and characterization of genetic variants in monogenic diseases has a wide influence on diagnosis and therapy. about 10% of pathogenic variants are splicing variants. due to the complex mechanism of splicing regulation it is difficult to predict the effects of variants on mrna splicing. possible consequences are exon skipping, intron retention, generation of novel splice sites or the utilization of a cryptic splice site. common consequences are a frame-shift and the generation of premature termination codon. this leads to rna degradation via the nonsense mediated decay (nmd) pathway. in a patient with the clinical symptoms of non-classical infantile pompe disease and a confirmed acid alpha-glucosidase (gaa) deficiency, we detected two novel, exonic variants in the gaa gene. both base pair exchanges suggested either an amino acid exchange or a splice defect as consequences. however, conventional investigation of the leucocyte mrna of the patient and his parents was inconclusive. degradation of the respective mutated rna by nmd was suspected. we developed an approach in order to characterize novel splicing mutations in a simple and non-invasive manner. material and method: isolated blood lymphocytes from patient and his parents were cultured in standard leucocyte medium supplemented with different concentrations of the nmd inhibitors ocadaic acid, anisomycin, and wortmannin for 24 h. cells were harvested and rna was isolated. the reverse transcribed cdna was amplified in allele specific pcrs and qpcr assays. results: compared to the non-stimulated lymphocyte controls nonsense mediated rna decay was inhibited by anisomycin. the consequences of aberrant rna splicing were detectable: the maternal mutation results in exon skipping, the paternal mutation in intron retention. furthermore vascular ehlers-danlos syndrome (type iv) is considered to be an autosomal dominant disorder caused by heterozygous mutations in col3a1, which are missense or splice site variants in about 95% of cases. we here report on a three-year-old female of non-consanguineous parents born with bilateral clubfoot as well as dysmorphic facial features, joint laxity, and mild contractures of finger joints. developmental delay became evident. after trauma at 2 years of age she developed brain haemorrhage. mri diagnosis at this age revealed an additional frontal aneurism as well as frontoparietal polymicrogyria. we identified novel compound heterozygous col3a1 mutations: the nonsense mutation c.1282c>t (p.arg428*) and the c.2057delc (p.pro686leufs*105) frameshift mutation leading to a premature stop codon. further studies showed that the mutations were inherited from each parent who had no features for ehlers-danlos syndrome type iv. only two other families have been reported so far with recessive mutations of this gene and a severe vascular phenotype and polymicrogyria. biallelic mutations of col3a1 seem to be accompanied with a significantly worse outcome compared with heterozygous mutations and polymicrogyria is an additional phenotypic feature. here we describe five patients with epidermolytic epidermal nevi in different degrees of severity with the mosaic mutation c.466c>t (p.arg156cys) in krt10 gene. the same mutation has previously been described in patients with ei (bygum et al. 2013) . we analyzed dna from peripheral blood and/or skin biopsies from affected and unaffected skin with next generation sequencing (ngs) and sanger sequencing methods. using ngs we found this mutation in blood in mosaic states ranging from 6% to 23%. the mosaic could only be confirmed by sanger sequencing in the patient with the highest mosaic frequency of 23%. in four of our patients we investigated skin biopsies from affected and unaffected skin. it is noteworthy, that only one of four patients showed the mutation in heterozygous state of 50% in the affected skin, whereas the other patients presented a mosaic state also in the affected skin. to exclude a recurrent sequencing artefact at this position, we examined 100 control patients for this mosaic mutation using ngs. in none of these patients we found the same dna change. patients with epidermolytic epidermal nevi have a higher risk to have children with a full-blown ei phenotype. our results show the importance of ngs as the method of choice to explore the molecular genetic basis of epidermolytic epidermal nevi. strikingly, all our patients carry the same mosaic mutation c.466c>t in krt10. we suggest that this position is a hotspot for postzygotic mutations in krt10. non-syndromic hearing loss (nshl), with presently around 100 associated genes, is one of the most genetically heterogeneous disorders constituting nearly 70% of genetic deafness with a predominantly recessive inheritance pattern. thirty percent of hearing loss (hl) can be connected as a part of over 600 distinct syndromes. next generation sequencing (ngs) technologies have revolutionized pathogenic variant identification. different strategies enhance pathogenic variant detection supporting detailed hl investigation to overcome the many ambiguities associated with clinical heterogeneity. detection of the disease causing variant in correlation with the phenotype can be challenging in small families, in situations with ambiguous clinical histories and allelic heterogeneity. using a clinical and whole exome sequencing approach, we tested over 130 probands as part of a multicentre iranian and german genetics of hl study that included 29 probands primarily with sporadic or dominant hl in a parent-child or parent-sibling trio context. the majority of these probands were pre-screened for defects in gjb2 and strc. libraries were prepared using trusight one and nextera rapid capture exome enrichment and sequenced using the miseq and nextseq 500 desktop sequencers (illumina). analysis was performed using gensearchngs and an inhouse exome analysis pipeline. around 40% of cases were resolved from phenotype matching and segregation analysis. interestingly, the fraction of resolved cases was much higher in our iranian cohort (>50%) compared to our german cohort (>30%) which may be attributed in part to increased consanguinity in the iranian families. we observed likely disease causing variants in syndrome-associated genes including eya1 causing branchio-oto-renal syndrome, a phenotype that was retrospectively confirmed by acquisition of additional clinical information. with few exceptions, we observed a diverse collection of affected genes in probands from our german collected cohort. contrastingly, the iranian cohort revealed frequent mutations in myo15a and otof. furthermore, co-segregation of variants in myo6 and tecta, with expected dominant hl phenotype, was a hindrance overcome by extensive segregation testing. familial locus heterogeneity was also observed by mutations in cib2 and slc26a4 segregating in different branches of the same extended pedigree. success in the identification of disease causing variants in known hl genes is contingent upon analysis strategy, clinical information and opportunity for segregation testing. the ability to retrospectively connect an already apparent syndromic phenotype to a syndrome-associated gene without prior knowledge is a powerful application of comprehensive analysis that is not restricted to nshl genes. this work provides an improved understanding of population-specific genetic epidemiology of hereditary hl and highlights the challenges in defining genetic causes in a highly heterogeneous disorder such as hl. vere disproportionate microcephaly (-15,6 sd), corneal clouding, myopia, teeth abnormalities and dysmorphism. panel diagnostic by next generation sequencing for primary microcephaly, including all known genes for seckel syndrome, was unremarkable. microarray analysis (affymetrix® cy-toscan hd) revealed a heterozygous 65 kb deletion, spanning the plk4 gene. this deletion was confirmed by mlpa (multiplex ligation-dependent probe amplification) analysis. subsequent sequence analysis of the plk4 gene showed a variant of unknown significance on the second allele. in silico analysis of this variant indicated a significant decrease of the relative splice efficiency at the splice donor site. rt-pcr analysis confirmed altered splicing, resulting in a predominant loss of exon 11 of the transcript and predicting truncation of the plk4 protein. interestingly, a residual wild-type transcript was also detectable in patient rna, implying that this variant effects splicing only partially. by analysis of the parents, the splice variant and the large deletion were proven to be compound heterozygous. discussion: up to now, only a few patients with plk4 mutations have been described in the literature. the phenotype comprises primary microcephaly, primordial dwarfism and chorioretinopathy (mccrp2). to our knowledge, we describe the first case of a plk4 heterozygous whole gene deletion and at least partial biallelic inactivation of the gene, therefore expanding the genetic background of this disorder. furthermore, we give a detailed phenotypic description of a further individual with plk4 alterations. the girl does not show retinopathy so far. while generalised retinopathy was discussed to be one of the most prominent distinctive features between mccrp2 and primary microcephaly/seckel syndrome, we consider plk4 rather to be a further candidate gene pointing towards seckel syndrome. additional investigations on centriole function in patient-derived cells are in progress. pathogenic variants of mitochondrial dna cause a wide range of severe congenital disorders with maternal inheritance and a high transmission risk for female carriers. we report on eight families with an index case presenting with the common pathogenic variant m.8993t>g (p.leu156arg) in the mt-atp6 gene in virtually homoplasmic form. in five families the mutation was detectable in peripheral blood from the mother in heteroplasmic form. in three families with a sporadic case of leigh syndrome the mutation was not detectable in peripheral blood (or urinary or buccal cells) from the mother, possibly indicating a de novo event. furthermore, one family presented with a de novo nonsense mutation in the gene mt-atp8, which was present in peripheral blood of the index case in about 70% and was not detectable in the mother or the unaffected sister. two female carriers with a heteroplasmy level of 50% asked for prenatal testing. both pregnancies showed an apparently homoplasmic load of the mutation. mutations in lztfl1 (bbs17) may be associated with a severe renal phenotype huntington's disease (hd) is a rare autosomal dominant neurodegenerative disorder caused by expanded cag repeats as diagnosed via direct dna analysis. for asymptomatic individuals, predictive testing (pt) can facilitate life planning and diminish uncertainty, but it is also associated with substantial social and psychological challenges. we present a prospective case series of counselees seeking predictive hd testing at the huntington centre north-rhine westphalia (bochum, germany) between 2010 and 2012. the international protocol including several pre-test sessions was followed throughout. the aim of this study was to prospectively follow the decision-making process of individuals at risk in our centre and explore their experiences following the decision as well as the impacts of mutation test results by means of standardized questionnaires and a semi-standardized telephone interview one year after the initial counselling session. 72 individuals participated in at least one of the three phases of the survey, including 31 individuals for the telephone interview. in our cohort, almost all interviewees reported a balanced emotional state one year after initial counselling, regardless of the decision for or against the test. the most important motivations for a decision in favor of pt were the ability to plan private life and to eliminate uncertainty. the most important motivations against pt were the fear of an increasing risk for others (e. g. offspring) and the fear to obtain an unfavorable htt mutation result, followed by the considered, willful decision for "wanting to not know". furthermore, we identified evidence for gender-specific aspects in decision-making in line with and expanding our previous observations. this study represents one of the few comprehensive prospective evaluations regarding decision-making and coping strategies related to predictive testing for huntington's disease. we submit that gender-related aspects should be heeded in genetic counselling during the predictive testing and counselling processes. our findings could serve as a basis for more extended prospective evaluations with higher numbers of participants and longer follow-up intervals. institute of human genetics, heidelberg, germany, 2 department of conservative dentistry, heidelberg, germany, 3 cegat gmbh, center for genomics and transcriptomics, tübingen, germany background: plk4 (polo-like kinase 4) has been designated as "master regulator" of centriole assembly. complete loss of plk4 is lethal in mice, whereas biallelic plk4 mutations with some retained function have been described in a few patients with microcephaly, growth failure and retinopathy (mccrp2, omim #616171). this is a heterogeneous entity overlapping with mcph (primary microcephaly) and seckel syndrome. during the last years several new genes have been discovered associated with this spectrum. clinical report and genetic findings: we report on a 4 year old female patient with intellectual disability, primordial dwarfism (-6,9 sd), most se-abstracts linked to rare autosomal recessive diseases with poor prognosis. we then compared couples and filtered for variants present in genes overlapping in both partners. putative pathogenic variants were tested for co-segregation in affected fetuses where material was available and in unaffected siblings. out of eleven couples of mediterranean and arabian ancestry (c:8, nc:3) and two non-consanguineous couples of european ancestry, we found five cases (5/13, 38%, c:4, nc:1) with both parents being heterozygous carriers of rare potentially deleterious variants in one or more overlapping genes. in four of these couples the underlying genetic cause for pre-or early postnatal child death could be established, in two of the families the diagnosis was confirmed by homozygous detection of the parental variant in the available dna of the affected child. in a consanguineous couple with pathogenic variants for a severe autosomal recessive disorder identified in both parents, the molecular diagnosis for their child that had died at 5 months of age could not be established. out of 9 couples in whom no causative diagnosis could be achieved 4 consented to undergo further wes analysis. identified variants are now used for preimplantation and prenatal diagnostics in all four families in which a causative diagnosis was established. our data show that ngs based gene panel sequencing of selected genes involved in lethal autosomal recessive disorders is an effective tool for carrier screening in parents and for the identification of recessive gene defects in families that have experienced early child death and/or multiple miscarriages. k. komlósi 1 , s. diederich 1 , d. l. fend-guella 2 , u. zechner 2 , s. schweiger 2 , o. bartsch 2 1 recently an x-linked syndrome with maternally inherited or de novo mutations in taf1 was described with global developmental delay, intellectual disability (id), delayed speech, characteristic facial dysmorphology, generalized hypotonia and variable neurologic features (mrxs33, mim: #300966, xlr). there have been only three publications of 14 unrelated families, 11 with single-nucleotide changes and 3 with gene duplications including taf1 (kaya et al., 2012; o'rawe et al., 2015; hu et al., 2016) . we identified a german family in which two brothers (21 and 5 years) showed severe intellectual disability, absent speech and understanding, and hypotonia but different neurologic and behavioral phenotypes. besides severe id the older brother also had postnatal short stature (-3 sd), a severe lennox-gastaut epilepsy and a neurodegenerative course. the younger brother showed autistic behavior and lost his very limited skills at age 3 to 3.5 years. both showed mild dysmorphic features (prominent supraorbital ridges, sagging cheeks, long philtrum, long face, thin upper lip, and high-arched palate), oropharyngeal dysphagia and generalized hypotonia. a gluteal crease with a sacral caudal remnant described as a characteristic feature was not seen in our case, and hearing impairment, microcephaly, dystonic movements or tremor were not observed either. the family history was highly suggestive of x-linked inheritance with an affected maternal uncle, a maternal aunt with multiple miscarriages, and two aunts with learning disability. since a previous analysis of 107 known x-linked mental retardation genes had not revealed the cause in the older brother, we used a targeted ngs approach (mpimg1-test: >1200 brain related genes) for the analysis of the younger brother. following enrichment a 300 bp paired end sequencing was carried out on an illumina miseq system with >95% of target covered >20-fold (hu et al, 2014) . only taf1 fitted the x-linked model and the phenotype. the unreported hemizygous sequence variant c.2833g>a (p.asp945asn) in exon 18 of taf1 was deemed pathogenic. it affected a highly conserved residue in the central "duf3591" domain, where 4/11 previously described mutations had clustered. segregation analysis confirmed hemizygosity in the older brother and heterozygosity in the mother with completely skewed x-inactivation (100:0). gonadism and learning disabilities. because of the delayed onset of symptoms, the diagnosis is often established during late childhood. however, in some cases renal morphological changes detected by ultrasound may resemble those usually seen in autosomal recessive polycystic kidney disease (arpkd). however, histologically bbs-kidneys differ distinctly from other polycystic disorders by cystic orientation, localisation, extension, structure and size. 21 bbs genes have been identified to date. mutations in bbs2, bbs4, bbs6 and bbs10 have been found to cause an antenatal presentation of bbs that may in some aspects mimic meckel gruber syndrome (mks). there is increasing evidence that bbs, at least in some families, shows an oligogenic mode of inheritance with three mutations at two bbs loci. yet, only three patients in two families with bbs caused by mutations in lztfl1 (bbs17) have been reported. their diagnosis was established in childhood and all patients had mesoaxial polydactyly as a distinct manifestation. in contrast to previous lztfl1 cases, in our family the diagnosis of arpkd was suspected sonographically at 27 weeks gestation (wg). pregnancy was terminated at 30 wg. autopsy revealed postaxial polydactyly of both hands, enlarged spongy kidneys, hemivertebra t6 and some features of potter's sequence. histological examination of the kidneys showed multiple, not radially oriented thin walled cysts, internally lined by thickened pas-positive basement membranes and microcystic dilatation of collecting ducts. cystic changes were accentuated in the renal medulla. corticomedullar differentiation was mainly preserved. the tentative diagnosis was bbs. fetal dna was investigated using a next generation sequencing panel which included 17 known bbs causing genes. hereby a heterozygous nonsense variant (np_065080.1: p.glu260*) inherited from the mother and a heterozygous missense variant (p.glu92lys) inherited from the father of the lztfl1 gene were identified. furthermore a maternally inherited heterozygous missense variant of unknown clinical significance in bbs4 was detected (np_149017: p.pro443ala). our case shows for the first time that mutations in lztfl1 can lead to a severe prenatal presentation of bbs due to profound renal manifestations with a kidney histology that is not considerably milder but distinct from that observed in mks. it is not clear to which extent the bbs4 variant may act as a disease modifier. this may challenge genetic counselling and prenatal diagnosis in a further pregnancy. furthermore our case shows that mesoaxial polydactyly is not always present in bbs patients with lztfl1 mutations and further studies are necessary to establish the frequency of mesoaxial polydactyly and other genotype phenotype correlations for bbs patients with lztfl1 mutations. p-cling-075 *** targeted next-generation sequencing analysis in couples at increased risk for autosomal recessive disorders k. komlósi, s. diederich, d. l. fend-guella, j. winter, o. bartsch, u. zechner, s. schweiger genetic childhood disorders leading to prenatal, neonatal or early childhood death are genetically heterogeneous. many follow autosomal recessive or x-linked modes of inheritance and bear specific challenges for genetic counselling and prenatal diagnostics. parents are carriers but unaffected and diseases are typically very rare but with recurrence risks of 25% in the same family. often, affected children (or fetuses) die before a genetic diagnosis can be established, post-mortem analysis and phenotype descriptions are insufficient and dna material of affected fetuses or children is not available for later analysis. a genetic diagnosis showing biallelic mutations or mutations on the x-chromosome in male fetuses or children is, however, the requirement for targeted carrier testing in parents, risk calculations, and prenatal and preimplantation diagnostics in further pregnancies. we employed targeted next-generation sequencing (ngs) for carrier screening of autosomal recessive lethal disorders in 8 consanguineous (c) and 5 non-consanguineous (nc) couples with one or more affected children. we searched for heterozygous variants (non-synonymous coding or splice variants as well as cnvs) in parents' dnas in a set of 430 genes r. kropatsch 1, 2 , j. preine 3, 4 , jt. epplen 1,2 1 department of human genetics, 2 ruhr university, bochum, germany, 3 charcot-marie-tooth disease (cmt), also commonly called as hereditary motor sensory neuropathy, is the most common monogenetic disease of the peripheral nervous system with significant clinical and genetic heterogeneity. the main clinical manifestations of cmt include progressive distal muscle weakness and atrophy, impaired distal sensation, depressed tendon reflexes and high-arched feet. based upon electrophysiological and histopathological features cmt can be divided into predominantly demyelinating or axonal forms. an intermediate form also exists characterized by evidence of both, demyelinating and axonal, impairments. genetically cmt can be caused by mutations in over 40 genes, including the dnm2 gene encoding dynamin 2 protein, a large gtpase primarily involved in receptor-mediated endocytosis and membrane trafficking. only a small number of mutations in dnm2 causing cmt have been described so far. we report the case of a 48-year-old man presenting with backache, ataxic gait and distal muscle weakness of the lower limb considered to be a consequence of the pre-diagnosed disc prolapse 3 years ago. over the past months bilateral progressive weakness of ankle dorsiflexion, foot drop and tingling paresthesia in stocking distribution have occurred. neurological examination disclosed depressed tendon reflexes of the upper and lower limbs. neurophysiologic investigations revealed an axonal sensible polyneuropathy with normal distal motor latencies and nerve conduction velocities. the sural nerve biopsy indicated single unmyelinated or thinly myelinated axons, loss of myelinated nerve fibers, numerous clusters of regenerating fibers without onion bulb formations suggesting an intermediate form of cmt. by using next generation sequencing (ngs) and a multi-gene panel, consisting of 751 inherited neurological disease-associated genes, we identified a heterozygous missense mutation c.439g>a, p.asp147asn (rs370086632) in the dnm2 gene. this particular mutation is located in a highly conserved nucleotide region encoding the catalytic n-terminal gtpase domain. this evidence suggests a pathogenic phenotype caused by the described mutation, which is being underlined by the following facts: public 1000 genome database covering harmless variants of the human genome does not report it. additionally, the exac browser with exome sequencing data of >60,000 unrelated individuals, lists the mutation and shows a low allele frequency of 0.000008243, corresponding to one known heterozygous mutation carrier. other online prediction tools like the mutation taster, polyphen, sift and provean categorize it as pathogenic. in conclusion, the novel dnm2 mutation is responsible with high probability for the late-onset form of intermediate cmt of the investigated patient. heterozygous mutations in pcdh19 cause an x-linked female-limited form of an early infantile epilepsy (juberg-hellman syndrome). the phenotype of this syndrome is variable, ranging from benign focal epilepsy to severe, serial seizures, repeating up to more than 10 times a day for several consecutive days. the intellectual outcome of affected patients ranges from normal to severe intellectual disability. psychiatric disturbances are frequent and manifest as autism, schizophrenia or aggressive behavior. neurological features such as ataxia may also be present. women with triple x syndrome usually show a normal physical development. cognitive deficits in vivo functional modeling of taf1 has already provided evidence for an effect on a neuronal phenotype. the phenotype in patients can be reminiscent of rett syndrome, but with milder regression and normal movements lacking a specific stereotypic pattern (no hand wringing). while severe neurodegeneration has been described in duplications, the present probands clearly showed developmental regression associated with a missense mutation. the analysis of further family members is pending. our case adds to the phenotypic spectrum of x-linked syndromic mental retardation type 33. targeted enrichment sequencing was successfully applied to identify greenberg dysplasia as cause of fatal anomalies in one fetus of a dizygotic twin pregnancy. p. m. kroisel 1, 2 , b. csapo 2, 3 , m. häusler 2, 3 , l. michelitsch 4, 5 , s. verheyen 1, 2 , p. klaritsch 2, 3 , k. wagner 1, 2 1 institute of human genetics, 2 medical university of graz, graz, austria, 3 department of obstetrics and gynecology, 4 gynecologist and obstetrician, 5 weiz, austria in the first pregnancy of a 29 year old kosovarian woman and her 36 year old husband during the 16th week of gestation one of her dizygotic twins showed a severe skeletal dysplasia with all long bones extremely shortened and partially bended. the thorax was short and narrow. in addition a ventriculomegaly of the brain and an increased nuchal translucency was noticed. a very bad prognosis was expected and an achondrogenesis was suspected clinically. the other twin appeared to be normal. an amniocentesis was performed to potentially identify the genetic basis of the disorder. qf-pcr to rule out common trisomy's and cytogenetics revealed normal results and following a normal agilent 60k array cgh analysis as a next diagnostic step next generation sequencing by using the trusight one gene panel focusing on three genes including slc26a2, trip11 and col2a1 was performed. since no pathogenic mutation was found by this approach, a more extended bioinformatics study was initiated. by filtering out common variants in the more than 4800 genes of the panel in our own database or in the exac-and 1000 genome databases our search was extended to genes with rare homozygous or compound heterozygous variants. by this strategy it was possible to reduce the potentially causative gene mutations dramatically and among those remaining genes for known very severe skeletal phenotypes just in the lbr gene the homozygous missense mutation c.1639a>g, p.asn547asp was identified in our fetus. since this particular mutation is already known to be pathogenic leading to the lethal greenberg dysplasia (clayton et al., nucleus. 2010 ) the diagnosis could be achieved in the affected fetus of the pregnancy of our patient still before completion of the 23rd week of gestation. both parents were found to be heterozygous for this mutation in the lbr gene. recently it was shown that different mutations of the very same gene can also lead to less severe forms of bone dysplasia. the couple was informed about our results and possible consequences were discussed and offered. the couple however came to the decision not to draw any consequences. both fetuses especially the affected one were well documented sonographically including in a series of 3d images. in the 27th gestational week during a sonographic investigation the affected fetus did not show cardiac function and an oligohydramnios was found. since development of the second non affected fetus was still within the normal range, we hope the now single pregnancy will carry on normal until birth. from our finding we would propose that our chosen strategy is straightforward and can be applied in a wide range of pregnancies to identify various up to severe and fatal single gene disorders associated with sonographic anomalies within a few weeks which should provide substantial benefits for these families. after the working diagnosis ps had been established, molecular analyses regarding the recurrent akt1 mutation (p.glu17lys) were performed by sanger sequencing in available affected tissue specimen of all three individuals. this revealed a high level of mosaic state for the akt1 mutation c.49g>a, (p.glu17lys) in affected tissues from bone and in meningiomas. re-evaluation of the ngs data from blood (individual i) confirmed the absence of that mutation in all reads, and no mutation was detected by sanger sequencing in dna from blood in individuals ii and iii. thus, a somatic mosaicism leading to a mild proteus phenotype could be confirmed as the underlying genetic cause in all three affected individuals. in conclusion, mild forms of proteus syndrome caused by the recurrent akt1 mutation in patients with limited regional involvement may be particularly difficult to diagnose and might be underdiagnosed. distal gne-myopathy: rare differential diagnosis of polyneuropathy here, we report a case of a 37-year-old patient with presumed polyneuropathy and elevated creatine kinase levels (400-800 u/l). clinical features included atrophic and bilateral paresis of lower legs of the frontal and rear compartment without high arched foot, while sensibility was not affected. additionally, a myopathic emg in m. tibialis anterior and a slight axonal damage in the motor neurography was detected. due to this overlapping neuromyological phenotype we performed a gene panel including 155 genes associated with neuromuscular diseases using targeted next generation sequencing. gene panel analysis revealed the homozygous mutation c.829c>t (p.arg277trp) in the gne gene. this mutation is described in the literature as cause of a distal gne-myopathy and was also detected in an affected brother (ck 1900 u/l), having consanguineous parents. the current case emphasizes that a large gene panel analysis is recommended in case of an overlapping neuropathological and myopathological phenotype. c. landgraf, g. schmidt, s. morlot, b. schlegelberger, b. auber institute of human genetics, hannover medical school, hanover, germany ehlers-danlos syndrome (eds) is a heterogeneous group of connective tissue disorders. according to the 1997 villefranche classification, eds comprises six major types as well as some rare specific entities. one of these has been referred to as "eds, musculocontractural type" (mc-eds), "adducted thumb-clubfoot syndrome" or "eds, kosho type". first described in 1959 as an eds vi subtype, it recently was identified to be caused by biallelic changes in either the chst14 or dse genes, resulting in a loss of dermatan sulfate (ds) biosynthesis. characteristic symptoms are multiple congenital malformations such as contractures (club feet, adducted thumbs), visceral and ocular anomalies, generalized joint laxity, scoliosis, muscular hypotonia, fragile, hyperextensible and bruisable skin, as well as a typical craniofacial appearance. distinctive features include hypertelorism, down-slanting palpebral fissures, bluish sclerae, micro-corneae, short nose with hypoplastic columella and long philtrum, thin upper lip vermillion, small mouth, retrognathia, low-set and posteriorly-rotated ears. the psychomotor development is delayed. 31 (chst14) and 3 (dse) patients have been reported as yet. the patient, a 30-year-old woman using a wheelchair, had club feet, surgically corrected asd ii, muscular hypotonia, the characteristic face and hyperextensible skin with atrophic scars; particularly visible were those and learning disabilities are more common than in the general population and compared to siblings. their motor skills are likely to be somewhat impaired and coordination problems are frequent. in some patients psychological problems were described. furthermore, eeg abnormalities are occasionally observed, with clinical seizures present in up to 15% of patients. here we report on a 15-year-old girl with a 47,xxx karyotype and early infantile, intractable epileptic seizures, beginning at the age of 9 months. about three years later, she developed severe, serial seizures often related to febrile infectious diseases. in adolescence, the epileptic symptoms became less intense. she additionally showed autistic features, mental deficiencies, hypermobility of the joints and ataxia. array-cgh, fragile x-analysis as well as sanger sequencing and mlpa of the scn1a and mecp2 genes revealed no additional abnormalities, besides the xxx karyotype. in the pcdh19 gene the heterozygous missense mutation c.695a>g (p.asn232ser) was identified, whereby the mutated allele seemed to appear in a 2:1 ratio compared to the wild type allele. this mutation has been previously described as disease causing. furthermore, three in silico prediction programs (sift, polyphen-2, mutationtaster) classified the mutation as pathogenic. the patient's asymptomatic mother had a normal 46,xx karyotype and was not a carrier of the pcdh19 mutation. pcdh19-related epilepsy exhibits an unusual mode of inheritance in which only heterozygous females are affected and hemizygous males are asymptomatic carriers. random x-inactivation in the brain of females with pcdh19 mutations causes a cellular mosaicism, which likely accounts for the pathogenesis by altering the cell-cell-interactions ("cellular interference"). however, the precise mechanism is still unknown. hypothetically, the wide range of phenotypic expressions may be explained by partially skewed x-inactivation and thereby limitation of the cellular interference. hence, an unequal ratio of mutated to wild type cells should give a milder phenotype compared to the fifty-fifty situation. in contrast to this hypothesis, the phenotype in our patient was rather severe. nevertheless, we cannot exclude that the triple x status contributes additionally to the observed phenotypic expression. proteus syndrome (ps, omim 176920) is a highly variable disorder with asymmetric and disproportionate overgrowth of the body, connective tissue nevi, epidermal nevi, dysregulated adipose tissue, and vascular malformations, caused by a somatic activating akt1 mutation. we report on three unrelated individuals (two adults and one 6 year old boy) who showed similar clinical findings that not fulfilled the rigorous clinical criteria for ps (biesecker, 1999) . beside an asymmetric hyperostosis of the skull or facial bones, all three had an ocular dermoid. two individuals developed alveolar hyperostoses and intracranial calcifying meningiomas. only one individual showed skin changes. all three had normal feet and no vascular lesions. molecular analyses in individual i performed in blood revealed normal results for array karyotyping and no relevant variant in whole exome sequencing (trio approach). introduction: incontinentia pigmenti (ip) is a rare x-linked male lethal genodermatosis that affects the neuroectodermal tissue and is always associated with a bullous rash of the skin along blashko lines in female neonates. it is caused by mutations in ikbkg which encodes the regulatory subunit of the ikb kinase complex required for nf-kb activation. ikbkg has a pseudogene (ikpkgp) with identical exons 4 to 10. the most frequent ip mutation is a recurrent exon 4_10 deletion due to non-allelic homologous recombination with the pseudogene. here, we report a novel deletion of exons 4 and 5 in the ikbkg transcript recognized by rna analysis. patient: we investigated a 9 yr old girl with typical erythematous rash after birth which resolved within 2-3 m. apart from a small hyperpigmented area around the right mammilla there were no skin alterations. she had few conically shaped teeth, normal nail and hair structure, no neurological manifestation and normal intelligence. only clinical sign were repeated vitreous hemorrhage of the left eye from age 5 m. family history was negative. methods: analyses on genomic dna extracted from blood included testing for the common ikbkg exon 4_10 deletion by long range pcr and mlpa (p073-a1, mrc holland), x inactivation analysis in the androgen receptor gene as described, and massively parallel sequencing (mpseq) of the ikbkg gene (trusightone, nextseq, illumina®; data analysis with nextgene/geneticist assistant [softgenetics®] and seqnext [jsi®]; reference sequence grch37, hg19). rna was extracted from cultured blood lymphocytes, and the entire ikbkg transcript (nm_003639.4, 10 exons) was sanger sequenced on cdna level (the a of the start codon is in exon 2); the results were analyzed with sequencepilot (jsi). result: genomic dna analyses including mlpa of ikbkg and ikbkgp specific probes in our patient did not reveal a putative mutation. there was a completely skewed x-inactivation pattern. cdna sequencing of ikbkg demonstrated skipping of exon 4 and 5 (r.400_671del) which is predicted to cause a frame shift starting from codon p.gln134, a premature stop codon 28 amino acids downstream (p.gln134glyfs*29) and complete loss of protein function. the loss of exons 4 and 5 is most likely due to an intronic splice variant in intron 6; investigations regarding the origin of this deletion are ongoing. conclusion: the presence of the high homologous pseudogene makes sequence analysis of ikbkg challenging. we report a deletion of exons 4 and 5 in the ikbkg transcript that required rna analysis for its identification. due to the skewed x-inactivation and typical clinical picture causality of the detected deletion is certain. the exact genomic cause of this alteration remains to be clarified. also in the era of mpseq, rna analysis may be necessary for detection of deep intronic mutations or the study of genes with homologous pseudogenes, as shown here in the case of ikbkg. primary congenital glaucoma (pcg) and early onset glaucomas are one of the major causes of of blindness in children and young adults' worldwide. both autosomal recessive and dominant inheritance have been described resulting from bowel surgeries due to colon perforation. her older sister who died aged 19 following an acute abdomen had club feet and the typical facial appearance, while three healthy sisters seem to be unaffected. her parents are first cousins of turkish origin and do not show eds symptoms either. the two affected sisters had been diagnosed with a syndromic disorder that could represent a rare form of eds. however, neither a confirmation of the suspected diagnosis nor a classification had yet been achieved. due to the distinctive symptom complex and the presumed autosomal recessive inheritance pattern, we strongly suspected this to be a case of mc-eds. sequencing of the chst14 gene (reference sequence: lrg_600) revealed a formerly undescribed homozygous variant (c.644c>t; p.pro-215leu). the variant changes the highly conserved pro215 residue which is located in the critical 3'-phospho-5'-adenylyl sulfate binding site and can be classified as likely pathogenic (acmg standards and guidelines, richards et al., genetics in medicine 2015) . the parents are heterozygous carriers of this variant, respectively. this case represents a unique entity within the umbrella term eds and illustrates the importance of clinical assessment leading to a diagnosis confirmed by genetic analysis. the underlying genetic defect in patients with mitochondrial peo is either a primary mutation of the mitochondrial genome (single, large-scale mtdna deletion or mtdna point mutation) or recessively and dominantly-inherited mutations in nuclear genes involved in mtdna maintenance leading to clonally-expanded multiple mtdna deletions in muscle. the nuclear disease genes are largely implicated in the replication and stability of mtdna, and as such a pathogenic mutation leads to secondary instability of the mitochondrial genome. causal mtdna deletions can be found in a heteroplasmic (mixture of mutated and wild type mtdna) state. however, each tissue/cell has its own biochemical threshold of mutant mtdna load which needs to be exceeded before focal respiratory chain deficiency becomes evident. to investigate this, muscle biopsies of 17 patients with genetically-and clinically-characterized mitochondrial disease of nuclear origin (9 polg, 5 twnk, 2 rrm2b and 1 slc25a4 (ant1)) and 4 healthy controls were analysed using quadruple oxphos immunohistochemistry, quantifying the biochemical phenotype in individual muscle fibres of patient muscle biopsies. this technique is based on quadruple immunofluorescence to detect structural components of complexes i (ndufb8) and iv (coxi), as well as porin (a marker of mitochondrial mass) and laminin (a cell membrane marker to define the boundaries of muscle fibres). further studies on 7/17 patients (3 polg, 2 rrm2b, 1 twnk, 1 slc25a4 (ant1)) included the correlation of the biochemical deficiency with the mtdna abnormality in individual cells, following laser microcapture and determination of the size and level of clonally-expanded mtdna deletion within fibres by real-time pcr. our preliminary data from quadruple immunocytochemical studies show that the muscle biochemical phenotype is different in patients with multiple mtdna deletions compared to other mtdna mutations; work is continuing to determine the exact size and level of clonally-expanded mtdna deletion in individual muscle fibres and correlate this with the observed biochemical defects and disease thresholds. abstracts these families, and functional studies, together with phenotype descriptions in the literature, are essential for pathogenic grading. however, several difficulties remain, such as the huge size of the ttn gene (>100kb) impeding functional studies, the wide spectrum of phenotypes and variants, the still small patient cohort, and often unspecific immunohistochemical abnormalities in muscle biopsies. the clinical evaluation of ttn variants thus presents a great challenge to the field of human genetics diagnostics. compound heterozygous variants in the qars gene (omim 603727) have been identified in only four patients with autosomal recessive progressive microcephaly with seizures and cerebral and cerebellar atrophy (mscca), to date. these patients showed severe developmental delay, progressive primary microcephaly, intractable seizures, hypomyelination or delayed myelination, thin corpus callosum, and small cerebellar vermis on brain imaging. here we report on two unrelated girls with progressive primary microcephaly, epilepsy and brain anomalies. trio exome analysis in each of the families revealed two different combinations of compound heterozygous variants in qars. all four variants are highly conserved throughout vertebrates, not reported in any database, yet, and in silico analysis predicted the variants as possibly damaging or deleterious. the first patient was born to non-consanguineous german parents. at birth, she was too short (−2.8 sd) and mildly microcephalic (−2.3 sd). she developed intractable seizures within the first hour of life. her growth continued to be mildly retarded (−2.8 sd at age 9 years) but microcephaly was progressive (−6.5 sd at age 9 years). she did not achieve any of the motor or cognitive developmental milestones, she did not have eye contact. the only interaction with her surrounding was a diffuse reaction to being touched. cranial mri showed no myelination of the supratentorial region, corpus callosum agenesis, simplified gyral pattern of frontal lobes, enlarged cerebral ventricles, and normal brain stem and cerebellum. trio-exome sequencing revealed the compound heterozygous qars variants c.1132c>t, p.(arg-378cys) and c.1567c>t, p.(arg523*). segregation analysis by sanger sequencing confirmed the heterozygous variants in the parents and two non affected sibling of the index patient. the second patient was initially evaluated at 11 days of age when she exhibited myoclonic seizures, intrauterine growth retardation, microcephaly, and elevated lactic acid. at birth, she was microcephalic (hc 29 cm) and microcephaly was progressive (−5.4 sd at age 19 months). cranial mri suggested undersulcation. she has required a gastrostomy feeding tube. trio-exome sequencing revealed the compound heterozygous qars variants c.40g>a, p.(gly14ser) and c.1573c>t, p.(arg525trp). segregation was confirmed by sanger sequencing analysis. together with the four previously described patients we conclude that compound heterozygous variants in qars are associated with a primary and progressive microcephaly, early onset of intractable seizures and severe developmental delay. brain imaging in the neonate can show simplified gyral pattern as an early characteristic feature. overlapping phenotypes are seen in patients with epileptic encephalopathy, lissencephaly and primary microcephaly. application of ngs panels or exome technology will allow for early diagnosis and further collection of patients for better delineation of the phenotype. with involvement of several genes including cyp1b1, foxc1, pitx2, myoc and pax6. however, mutations in these genes explain only a small fraction of cases suggesting the presence of further candidate genes. to elucidate further genetic causes of these conditions we performed whole exome sequencing in a patient with pcg and retinal detachment and identified compound heterozygous variants in col1a1 (p.met264leu; p.ala-1083thr). targeted col1a1 screening of 26 additional patients detected three further heterozygous variants (p.arg253*, p.gly767ser and p.gly-154val) in three distinct subjects: two of them were diagnosed with early onset glaucoma and mild form of osteogenesis imperfecta (oi), one patient had a diagnosis of pcg at age 4 years. all five variants affected evolutionary, highly conserved amino acids indicating important functional restrictions. molecular modeling predicted that the heterozygous variants are dominant in effect and affect protein stability and thus the amount of available protein, while the compound heterozygous variants act as recessive alleles and impair binding affinity to two main col1a1 binding proteins: hsp47 and fibronectin. dominantly inherited mutations in col1a1 are known causes of connective tissues disorders such as oi. these disorders are also associated with different ocular abnormalities, although the common pathology for both features is seldom recognized. our findings expand the role of col1a1 mutations in different forms of early-onset glaucoma with and without signs of oi. thus, we suggest including col1a1 mutation screening in the genetic work-up of glaucoma cases and detailed ophthalmic examinations with fundus analysis in patients with oi. the gene ttn encodes the largest known protein, titin, which plays a key role in structural, mechanical, developmental and regulatory functions of cardiac and skeletal muscles. accordingly, titinopathies are characterized by great clinical and genetic heterogeneity. the clinical spectrum ranges from severe phenotypes with cardiac involvement to pure myopathies at the milder end, including autosomal recessive and dominant inheritance patterns (chauveau et al. 2014, hum mutat; 35:1046) . next generation sequencing analysis identifies a large number of variants of unknown clinical significance; the potential clinical relevance of these variants cannot be assessed with certainty without further studies. three case reports highlight the difficulties in human genetics diagnostics concerning ttn. the first case is of a 46 year-old woman with proximal muscle weakness, slightly elevated ck, scoliosis, and no family history. a heterozygous known pathogenic variant was identified in mex1, associated with autosomal recessive congenital core myopathy combined with primary heart disease. additionally, an unknown variant was detected. both variants could be clinically relevant with regard to the patient's phenotype, but this can be neither confirmed nor excluded at this time. the second case is of a 7 month-old finnish girl who presented with severe muscle hypotonia at birth and mental alertness with normal brain mri and eeg. congenital fiber-type disproportion was suspected. a homozygous frame-shift mutation in mex1 was identified which to our knowledge has not yet been described in the literature. this variant is likely of clinical relevance with regard to the patient's phenotype, but this can be neither confirmed nor excluded at this time. the third case is that of a 34 year-old woman with suspected myofibrillar myopathy. a known pathogenic homozygous frame-shift mutation in mex6 was detected which is associated with autosomal recessive congenital myopathy with central nuclei. segregation analysis revealed that the healthy parents are heterozygous carriers of this variant. the clinical diagnosis of a ttn-associated disease could therefore be confirmed. ttn variants need to be assessed in combination with detailed clinical and muscle biopsy data. segregation analysis is necessary but not sufficient for the clinical grading of variants. identification of a variant in several independent families, segregation of the variant with disease phenotype in gous pathogenic smad4 variant c.719dupt;p.(ala241fs) (ncbi reference sequence nm_005359.5). gastric as well as colonic cancer and polyposis was present in the paternal family history. conclusions: ts14 and other imprinting disorders are likely underdiagnosed, as the main clinical features (e. g. growth retardation, hypotonia) are distinct but unspecific. as exome sequencing becomes a more frequent diagnostic procedure, imprinting disorders caused by mutations in imprinting centers will presumably be diagnosed more often. methylation defects, however, will remain underdiagnosed, without a specific clinical differential diagnosis, which would guide to appropriate analysis of the methylation status. a bowel invagination in early childhood due to a single polyp can be a symptom of jps, especially in the context of a paternal history of polyposis and intestinal cancer; thus, family history should be carefully obtained. in the outpatient clinic, child psychiatrists as well as neurologists thoroughly work up phelan-mcdermid patients according to a standardized protocol by taking medical history, performing physical examination, and, if needed, organizing further supplementary examinations. in addition, a genetic analysis and hair/tissue sampling is performed. since its foundation, a steadily increasing number of so far 70 patients from all over germany has been seen and treated. the outpatient clinic aims at facilitating and accelerating the diagnosis of phelan-mcdermid syndrome, improving medical support for affected patients of all ages, and, last but not least, fostering a better understanding of the causes and pathomechanisms leading to the symptoms of the disease. t. m. neuhann, l. neuhann, c. rapp, a. laner, a. benet-pages, e. holinski-feder medizinisch genetisches zentrum, münchen, germany congenital eye malformations, such as the microphthalmia-anophthalmia-coloboma (mac) spectrum, congenital cataracts, anterior segment dysgenesis (asd), and congenital glaucoma, affect more than 1:8.000 newborns. the phenotypic spectrum of the aforementioned entities is highly variable and partially overlapping. eye malfomations are very heterogeneous; to date causative mutations have been described in more than 100 next-generation-sequencing (ngs) technology has revolutionized genomic research and has transformed clinical diagnostics. ngs offers enormous potential for providing accurate diagnoses to individuals with previously unresolved syndromes. in the pediatric endocrine clinic, clinicians are often faced with the task of making a diagnosis in children with syndromic short stature. as there may be considerable clinical overlap between short stature syndromes, deriving a clinical diagnosis may prove challenging. furthermore, even if a clinical differential diagnosis is established, often several genes would need to be tested before a molecular diagnosis is made. as access to genetic testing is limited in algeria, we conducted a pilot study on 10 algerian patients with syndromic short stature using a combination of two different ngs modalities, namely whole-exome-sequencing (wes) and mendeliome sequencing (trusight one sequencing panel). a molecular diagnosis could be established in 9/10 patients, making the diagnostic rate in this initial cohort 90%. as 7 patients had novel mutations we could expand the mutational spectra of several genes, namely cul7, npr2, sos1, vps13b, and znf81. we could thus substantiate the clinical utility of wes and the mendeliome in patients with a diverse array of syndromic short stature syndromes. chromosome 14 harbours an imprinted locus at 14q32. maternal uniparental disomy of chromosome 14, paternal deletions and paternal loss of methylation at the intergenic differentially methylated region (ig-dmr) and the somatic dmr within meg3 are associated with temple syndrome (ts14, mim 616222). the phenotype of ts14 consists of pre-and postnatal growth retardation, early feeding problems and muscular hypotonia, joint laxity, motor developmental delay, premature puberty, and truncal obesity. juvenile polyposis syndrome (jps, mim 174900) is characterized by predisposition to hamartomatous polyps in the gastrointestinal (gi) tract, specifically in the stomach, small intestine, colon, and rectum, including the risk for gastrointestinal cancer. pathogenic variants in the bmpr1a and smad4 gene are identified in about 40-50% of affected families. we report on a family with two female children. the index patient, an 8-year-old girl, was diagnosed to have ts14 due to hypomethylation at the somatic dmr within meg3 with clinical features reminiscent of prader-willi syndrome in early childhood and milder clinical signs at further age (i. e. mild global development delay, muscular hypotonia, suspected central obesity, no prominent facial dysmorphisms). snp array-cgh analysis was unsuspicious and no deletion of the imprinting center was observed. thus, ts14 is caused by a sporadic imprinting defect in our patient. her 10-year-old sister was diagnosed with smad4-associated jps after an episode of intestinal invagination due to a polyp, histologically diagnosed as peutz-jeghers polyp, in early infancy. sequencing identified a heterozy-abstracts coding vmat2 have only very recently been described as causal for brain dopamine-serotonin vesicular transport disease in two families with multiple affected children (rilstone et al., n engl j med 368, 2013, 543-550; jacobsen et al., j inherit metab dis 39, 2016, 305-308) . the index case presented here is a 7-year-old girl with severe mental retardation and a dystonic movement disorder. she is the tenth child of a consanguineous arabic couple and was initially referred to neuropaediatric examination at the age of four months due to recurrent oculogyric crises and muscular hypotonia. blood metabolic testing and cerebrospinal fluid (csf) analyses were inconclusive. notably, biogenic amines were within their normal ranges and the differential diagnosis of aromatic l-amino acid decarboxylase (aadc) deficiency could not be confirmed. conventional cytogenetics, subtelomeric screening, array-cgh and different ngs panel analyses did not identify a causative mutation. both parents and all eight living siblings are obviously unaffected. a brother with a known hypotonic movement disorder died at the age of three years due to prolonged seizures with hyperthermia and cerebral edema. by utilizing whole-exome sequencing, we identified a homozygous substitution in the slc18a2 gene of the index case causing an amino acid change (c.710c>a; p.pro237his) in a conserved transmembrane domain of vesicular monoamine transporter 2 (vmat2). homozygosity for this missense change could also be verified in a dna sample of her deceased brother. an obvious reduction in frequency of oculogyric crises was observed in our index case under therapy with pramipexole already within 4 weeks after start of treatment. furthermore the patient shows less dystonic movements under therapy. the case presented here highlights the importance of considering brain dopamine-serotonin vesicular transport disease as differential diagnosis for early-onset extrapyramidal movement disorders combined with mental retardation even if neurotransmitters in csf are normal. for a large number of individuals with intellectual disability (id), the molecular basis of the disorder is still unknown. however, whole exome sequencing (wes) is providing more and more insights into the genetic landscape of id. in the present study, we performed trio-based wes in 311 patients with unsolved id and additional clinical features, and identified homozygous cplx1 mutations in three patients with id from two unrelated families. all displayed marked developmental delay and migrating myoclonic epilepsy, and one showed a cerebellar cleft in addition. the encoded protein, complexin 1, is crucially involved in neuronal synaptic regulation, and homozygous cplx1 knockout mice have the earliest known onset of ataxia seen in a mouse model. recently, a homozygous truncating mutation in cplx1 was suggested to be causative for migrating epilepsy and structural brain abnormalities. id was not reported. the currently limited knowledge on cplx1 suggests that complete loss of complexin 1 function may lead to a complex but variable clinical phenotype, and our findings encourage further investigations of cplx1 in patients with id, developmental delay and myoclonic epilepsy to unravel the phenotypic spectrum of carriers of biallelic cplx1 mutations. genes. due their heterogeneity, diagnostic testing for congenital eye malformations was limited in the pre-ngs era. we performed exome analysis in 30 patients with congenital eye malformation (mac spectrum, asd, congenital cataract, congenital glaucoma). primarily, a gene panel comprising 112 genes associated with eye malformations was evaluated. additionally the exome data was evaluated in selected patients as a second step. the panel analysis revealed pathogenic sequence variants in 10 patients and 8 genes (mab21l2, bcor, nhs, prss56, cyp1b1, foxc1, pitx2, gcnt2). putatively causative sequence variants were identified additional patients. the diagnostic yield of the panel was highest in patients with non-syndromic microphthalmia/coloboma and congenital cataracts, and lowest in patients with syndromic mac spectrum (i. e. additional systemic features/malformations). ngs based panel testing is a strong diagnostic tool to determine the underlying causes of non-syndromic congenital eye malformations. due to the partially overlapping phenotypes and high heterogeneity it is more sensible to perform large gene panel analysis, as opposed to smaller single phenotype based panels. superactivity of phosphoribosyl¬pyrophosphate synthetase i (prpps) is a rare inborn error of purine metabolism that is characterized by increased levels of uric acid in blood and urine (omim 300661). the disorder is caused by gain-of-function mutations in the x-chromosomal gene prps1. in male patients, disease manifestation is in early childhood. additional clinical characteristics include intellectual disability, hypotonia, ataxia and hearing loss. heterozygous female mutation carriers have a later age of onset and a less severe clinical course. only seven families with prps1 gainof-function mutations have been reported to date. we report on a 7-year-old boy with congenital hyperuricemia, urolithiasis, developmental delay, short stature, hypospadias and facial dysmorphisms. his mother also had hyperuricemia that was diagnosed at age 17 years but was otherwise healthy. a novel prps1 missense mutation (c.573g>c, p. leu191phe) was detected in the proband and his mother. enzyme activity analyses confirmed superactivity of prpp synthetase. the family reported here broadens the clinical spectrum of prpps superactivity and indicates that this rare metabolic disorder is associated with a recognizable facial gestalt. homozygous and compound heterozygous mutations of the rnu4at-ac gene are associated with mopd1 and roifman syndrome. mopd1 is characterized by severe microcephaly with brain malformations including abnormal gyral pattern, corpus callosum agenesis or hypoplasia, vermis hypoplasia and intracranial cysts, psychomotor retardation, short stature, skeletal dysplasia, dry skin, sparse hair, flexion contractures, round face with beaked nose and protruding eyes, and premature death with a majority of the patients who die before the age of 28 months for unknown reasons. roifman syndrome was first described as a novel association of antibody deficiency, spondyloepiphyseal chondro-osseus dysplasia, retinal dystrophy, poor pre-and postnatal growth, cognitive delay and facial dysmorphism including long eyelashes, downslanting palpebral fissures, a long philtrum and a thin upper lip. all patients with roifman syndrome reported so far lack brain malformations. the rnu4atac gene encodes a small nuclear rna (snrna), which is essential for minor intron splicing. homozygous (g.51g>a, g.46g>a) and compound heterozygous mutations (g.51g>a;g.55g>a, g.51g>a;g124g>a and g.40c>t;g.124g>a) have been described in mopd1. all mutations involve the 5' or 3' stem loop of the u4atac snrna. in contrast, all cases with roifman syndrome investigated so far showed compound heterozygous rnu4atac mutations with one allele harboring a mutation in the mopd1 associated 5' stem loop and the other allele showing a mutation in the stem ii site of the u4atac snrna, which has not been involved in mopd1, so far. thus, the different pattern of the mutations observed in mopd1 and roifman syndrome may contribute to the distinct features of both syndromes. however, our patient shows, that features of mopd1, i. e., brain malformations, may also be present in patients who show roifman syndrome associated rnu4atac mutations. this indicates that both syndromes may represent overlapping features of the clinical spectrum of rnu4atac mutations. h. roth, h. stöhr, b. h. f. weber institute of human genetics, university of regensburg, germany introduction: inherited retinal degenerations comprise a genetically heterogeneous group of eye diseases with overlapping clinical presentations. up to now, more than 200 genes have been associated with different forms of retinal dystrophies (rd) such as retinitis pigmentosa (rp) or cone-rod-dystrophies (crd) with mutations in 84 and 34 causative genes, respectively. here, we present the results from three patients with remarkable findings and discuss their implications for risk prediction and genetic counseling. methods: targeted next-generation sequencing (ngs) technology based on agilent custom designed gene panels (sureselect) has been established in our diagnostics department to identify causative mutations in a large patient cohort with approximately 200 rd patients. high-throughput sequencing data are routinely analyzed with the clc biomedical workbench. classification of variants was based on bioinformatic analyses using alamut visual software, mutationtaster, sift and polyphen-2 prediction programs, allele frequencies, amino acid conservation and literature. results. ngs analysis revealed two patients with rp and one patient with crd, each of whom carry putative causative mutations in several rd genes. first, a male patient with a family history of crd, is a carrier of a nonsense mutation p.(arg1144ter) in rims1 and two likely pathogenic missense mutations in aipl1 (p.(tyr134phe)) and guca1a (p.(pro-50leu)), each in a heterozygous situation. mutations in all three genes can cause adcrd. in addition, the patient carried a hemizygous nonsense mutation p.(glu1017*) in the x-chromosomal rpgr gene. secondly, a female patient with simplex rp was found to be homozygous for a frameshift-causing deletion p.(ser527leufs*28) in the impg2 gene causing arrp. she also carried three heterozygous, likely pathogenic missense mutations in crx (p.(tyr142cys)) causing adrp, in the x-chromosomal rpgr (p.(ala365val)) and in ush2a (p.(ile1621val)) associated m. s. reuter 1 , a. riess 2 , u. moog 3 , t. a. briggs 4, 5 , k. e. chandler 4 background: disruptions of the foxp2 gene, encoding a forkhead transcription factor, are the first known monogenic cause of a speech and language disorder. so far, mainly chromosomal rearrangements such as translocations or larger deletions affecting foxp2 have been reported. intragenic deletions or convincingly pathogenic point mutations in foxp2 have up to date only been reported in three families. we thus aimed at a further characterization of the mutational and clinical spectrum. methods: chromosomal microarray testing, trio exome sequencing, multi gene panel sequencing and targeted sequencing of foxp2 were performed in individuals with variable developmental disorders, and speech and language deficits. results: we identified four different truncating mutations, two novel missense mutations within the forkhead domain and an intragenic deletion in foxp2 in fourteen individuals from eight unrelated families. mutations occurred de novo in four families and were inherited from an affected parent in the other four. all index patients presented with various manifestations of language and speech impairment. apart from two individuals with normal onset of speech, age of first words was between 4 and 7 years. articulation difficulties such as slurred speech, dyspraxia, stuttering or poor pronunciation were frequently noted. motor development was normal or only mildly delayed. mild cognitive impairment was reported for most individuals. conclusion: by identifying intragenic deletions or mutations in fourteen individuals from eight unrelated families with variable developmental delay/cognitive impairment and speech and language deficits, we considerably broaden the mutational and clinical spectrum associated with aberrations in foxp2. h. rieder, f. beleggia, d. wieczorek we report on a 4-year-old boy with microcephaly, arachnoidal cysts, pachygyria, microgyria, and severe intellectual disability. he also had short stature including shortening and deformation of the femora, brachydactyly, and short ribs with costochondral dysplasia. he showed facial dysmorphism with narrow palpebral fissures, a short nose with a depressed nasal bridge, and a broad mouth with full lips. clinical laboratory investigations demonstrated persistently slightly elevated liver enzymes. exome sequencing revealed compound heterozygous mutations of the rnu4at-ac gene, g.51g>a;g.16g>a, which has been described in an individual with roifman syndrome. we report on a seven-year-old girl, first child of non-consanguineous italian parents, with developmental delay, muscular hypotonia and distinctive craniofacial features (epicanthus inversus, ptosis, broad nasal bridge, mild retrognathia, low-set posteriorly rotated ears and malpositioned teeth in the mandible). because of the tentative diagnosis of blepharophimosis-ptosis-epicanthus inversus syndrome (bpes), conventional cytogenetic analysis, sanger sequencing and mlpa (multiplex ligation-dependent amplification) of foxl2 were initiated and showed unremarkable results. microarray-cgh revealed a 414 kb microduplication of genetic material on 15q11.2: arr[hg19] 15q11.2(22765628_23179948)x3 encompassing the genes tubgcp5, cyfip1, nipa1 and nipa2 of maternal origin. patients with 15q11.2 microduplication have been described to be affected by developmental delay, motor and/or expressive language delay, epilepsy, learning disabilities and/or behavioral problems. however, genotype phenotype correlation is complicated by incomplete penetrance. healthy and mildly affected carriers are reported in the literature. we speculate that the microduplication might contribute but does not fully explain the phenotype of our patient, in particular concerning the craniofacial features. subsequent trio whole-exome sequencing identified a de novo heterozygous mutation in setbp1 (c.3909t>a/ p.tyr1303*) leading to a premature stop codon and most probably resulting in a truncated and functionally impaired protein. mutations in the set binding protein 1 gene (setbp1) on 18q12.3 have been identified to cause schinzel-giedion syndrome (sgs, omim 269150), a rare autosomal dominant disorder characterized by postnatal growth failure, severe developmental delay, seizures, facial dysmorphism, genitourinary, skeletal, neurological, and cardiac defects. chromosomal deletions in 18q including setbp1 have been reported to cause a milder phenotype known as "autosomal dominant mental retardation-29" (mrd29, omim 616078). these observations suggest that the severe sgs phenotype might be the consequence of a gain-of-function or dominant-negative effect of the mutations and that setbp1 haploinsufficiency results in a different, milder phenotype. so far, the function of the set-bp1 protein is unknown. the presented case adds up to the yet small number of reported cases of mrd29 and thereby contributes to the clinical spectrum of setbp1 haploinsufficiency. this work was supported by "förderstiftung des uksh" (project number: 006_2016). the demand for genetic counseling had been constant, in germany, over many years. from 1996 to 2004 around 47.000 cases per year on the average and with minor fluctuations were reimbursed by the german sickness funds (public health insurance system; pabst and schmidtke, gendiagnostik in deutschland, bbaw, p. 195-203, 2007) . in connection with the "genbin2"-project, a new nationwide survey was initiated regarding with arrp. finally, in another female rp patient with no family history of rd, we detected a nonsense mutation p.(trp558ter) and a likely pathogenic splice site change (c.6078 + 3a>g) in the arrp gene eys, assuming compound heterozygosity. in this patient we also identified two heterozygous, likely pathogenic missense mutations in hmcn1 (p.(pro2226thr)) and cep290 (p.(arg2210cys)) underlying dominant and recessive forms of rd. in all three cases, specific mutation(s) could not be uniquely identified as causative. conclusion. results in the three rd cases emphasize that ngs can generate unexpected results that are difficult to interpret, particularly in the absence of segregation analysis and functional data on pathogenicity. the implications for genetic counselling and predictive testing will be discussed. smart qnipt study -detection of fetal trisomy 21 based on methylation-specific quantitative real-time pcr m. sachse, s. werler, j. bonnet, u. neder, h. sperling, s. busche, s. grömminger, w. hofmann lifecodexx ag, konstanz, germany objectives: current non-invasive prenatal testing (nipt) methods for the detection of fetal trisomy 21 (t21) are primarily based on next generation sequencing (ngs) strategies which are quite costly in clinical application and hence are limited to patients who can afford the testing. here, we describe the results of a blinded study with respect to the test accuracy of a newly developed nipt assay based on quantitative real-time pcr (qpcr) for prenatal testing of fetal trisomy 21 (qnipt). methods: in the study maternal plasma samples were collected from 1,044 pregnant women and blinded by an independent contract research organization. after extraction of cell-free dna using qiasymphony instrument and methylation-specific digestion of dna samples a multiplex qpcr was performed. the primary qpcr data were finally evaluated with our ce marked data analysis software. results from this analysis and from confirmatory ngs testing were compared with nipt results using ngs. the study results of successfully analysed maternal plasma samples (n = 966) demonstrated a positive percentage agreement (ppa; equates to sensitivity) of 100% (lower 1-sided 95% confidence interval of 91.8%; n = 35/35) and a negative percentage agreement (npa; equates to specificity; n = 931/931) of 100% compared to ngs-based results. the negative predictive value (npv) for the novel qnipt and confirmatory ngs testing was 100% (lower 1-sided 95% confidence interval of 99.68%). the average fetal fraction of the 966 examined blood samples was 8.1%. the qnipt assay provided reliable test results in 54 blood samples with a fetal fraction below 4% and as low as 2.4%. conclusion: our results suggest that the proprietary qnipt assay is a very reliable and robust method suitable for clinical routine in accordance with international medical associations. the assay represents a more cost-efficient solution over ngs testing and will also be able to provide results in the shortest possible time. while current nipt methods require a minimum fetal fraction of 4% in blood samples from singleton pregnancies, we could demonstrate in the study that our smart qnipt assay can be employed on blood samples with a fetal fraction of as low as 2.4%. in summary, the application of smart qnipt could have the potential to become a nipt solution on a global scale for pregnant women of all ages and risk groups. further studies which aim to include the determination of trisomy 13 and trisomy 18 are currently underway. with respect to the developmental delay of our index patient, chromosome analysis and array-cgh were performed. a microduplication in 3p26.2 (app. 50kb) of unknown significance and a microduplication in xq27.3 (app. 550kb), which comprises the fmr1-gene, were identified and shown to be of maternal origin (arr[hg19] 3p26.2 (2, 811, 861, 170)x3, xq27.3(146, 663, 212 ,089)x3). fmr1 is associated with fragile x syndrome, which is one of the most common causes for x-linked mental retardation. cgg-trinucleotide repeat expansions in the 5' untranslated region (>200 repeats) lead to aberrant hypermethylation of the fmr1-promotor and silencing of fmr1 expression. in contrast, premutations (55-200 repeats) lead to a higher expression of fmr1 and cause a clinical syndrome that is characterised by late progressive cerebellar ataxia (fxtas). in line with this gain-of-function mechanism, we hypothesize that the xq27.3 duplication, which could lead to an increased gene dosage of fmr1, causes a fra(x)-/fxtas-like syndrome and explain the clinical findings in our family. vengoechea et al. described a patient with a similar duplication, who was affected by developmental retardation, epilepsy and hyperactivity. they discussed the microduplication, which arose de novo in their patient, as the cause for the boy's symptoms (vengoechea j. et al., eur j hum genet., 2012 nov; 20(11):1197-200) . in conclusion, we assume a fmr1-duplication syndrome in our family with variable expressivity and a different impact on male and female patients. to further prove this hypothesis, we are planning to perform a segregation-analysis within the whole family. background: congenital myasthenic syndromes (cms) are a genetically heterogenous group of disorders leading to weakness of skeletal muscles -especially ocular, bulbar and limb muscles -with onset mostly at birth or in early childhood. the severity of cms can vary significantly ranging from death in early childhood due to respiratory insufficiency to only mild muscle weakness in adulthood. more than 25 genes that are highly expressed in the neuromuscular junctions are associated with cms. mutations in the chrne gene on chromosome 17p13.2 are responsible for about one half of genetically solved cms cases. they can cause different subtypes of cms with either autosomal dominant or autosomal recessive inheritance. results: here, we report a 3-year-old boy who was born with bilateral eyelid ptosis and congenital vertical talus of the right foot that needed surgical correction. the boy displayed muscular hypotonia with a myopathic facial expression and delayed motor development. ophthalmologic examination revealed external ophthalmoplegia. a next generation sequencing based gene panel for congenital myopathies detected the homozygous frameshift mutation c.750_769dup (p.leu257profs*50) in the chrne gene in the boy. gene dosage analysis did not show an exonic deletion in the chrne gene. sanger sequencing confirmed the mutation in a heterozygous state in the boy's father. however, his mother did not carry the mutation in the chrne gene. conclusions: these results suggest the rare event of a (partial) paternal uniparental isodisomy of chromosome 17 as cause of the homozygous c.750_769dup (p.leu257profs*50) in the chrne gene in the boy. further experiments are currently undertaken to confirm this hypothesis. the annual reimbursement frequencies of the relevant entries in the ebm fee schedule, 08572, 01792, 01837 and 11232, for which only specialists in human genetics and subspecialists in medical genetics can account, from 2009 until 2014. contrary to the findings in the earlier period the demand for genetic counseling has risen sharply: 41,243 (of a total of 54,360) cases in 2009; 45,525 (58,341) in 2010; 46,691 (59,724) in 2011; 51,316 (63,242) in 2012; 54,739 (69,732) in 2013; and 61,308 (74,780) in 2014. we speculate that the temporal correlation of the rise of genetic counseling demand with the enactment of the german act on testing (february 1, 2010) is not coincidental. further factors that might contribute to the increase in demand are the ensuing guidelines of the german commission on genetic testing and cme activities related to attaining a qualification for genetic counseling for specialties other than human genetics. in the course of these activities the awareness for the importance of genetic counseling delivered by specialists in human genetics and subspecialists in medical genetics may have risen. acknowledgements: the "genbin2"-project supported by the robert koch-institute through funding from the german federal ministry of health. we gratefully acknowledge the collaboration with dr. michael erhart, zentralinstitut der kassenärztlichen bundesvereinigung (zi-kbv), berlin, germany it is well known that duplications of down syndrome critical region (dscr) on chromosome 21q22 can cause down syndrome whereby the distinct phenotype is associated with the involved genes and the size of duplication. however, in literature are hardly any cases with mosaic duplications of dscr described. here we report on a 6 year old boy with some clinical features of down syndrome including distinctive craniofacial dysmorphism, simian crease and sandal gap as well as delayed motor and speech development. no other organ abnormalities are known. conventional chromosome analysis showed no numerical or structural aberration whereas interphase fish analysis revealed three signals for dscr in approx. 40% of lymphocytes and in approx. 80% of buccal mucosa cells. array-cgh analysis on dna from peripheral blood confirmed a 2,56 mb duplication of chromosome 21q22.13q22.2. the duplication involves among others the gene dyrk1a which is reported as a candidate gene for down syndrome. this case presents one of the smallest known duplications within dscr which causes even in a mosaic state a mild phenotype of down syndrome. 4-year-old boy was referred to our outpatient clinic due to global developmental delay mainly affecting his speech and his fine motor development. in addition, muscular hypotonia and an abnormal gait were reported by the referring paediatrician. his mother, his maternal grandmother, and numerous relatives are affected by gait ataxia. no causative mutation was detected in the maternal grandmother by means of a multi-gene panel for spinocerebellar ataxia encompassing 118 genes. genetic testing for friedreich ataxia was also without pathological findings. cer, but the patient mother's grandfather had a cancer of unknown origin and died at the age of 50 years. because of the suspicion of having a lynch syndrome an immunohistochemistry and microsatellite analysis have been performed on the tissue of the colorectal cancer and the hepatic metastasis. all four mmr proteins were properly expressed in immunohistochemistry in colorectal cancer. just the expression of mlh1 protein in the hepatic metastasis was focally weakened and inhomogeneous. the microsatellite markers bat25, bat26, d5s346 (apc), d2s123 and d17s250 (mfd) were all stable, a a146t-kras mutation was found. after performing a multi-gene panel (ngs, next-generation sequencing), a gross heterozygous deletion of exon 1 in msh6 gene has been found in the cnv analysis of the ngs data. this mutation was confirmed with a mlpa and quantitative real time pcr analyses. furthermore, rna expression of msh6 was reduced to 50% in blood lymphocytes in comparison to control samples pointing to a potential role of msh6 loss in the patient's tumor development. we are observing more and more patients with probably pathogenic and pathogenic mutations in one of the mmr genes with normal immunohistochemistry and microsatellite analysis. therefore, we propose that the criteria for performing a molecular genetic analysis of hnpcc/lynch syndrome should be revised. exome sequencing reveals gata1 mutation in a patient with partial delta-storage pool deficiency and mild thrombocytopenia objectives: we report about a 35-year-old male patient of russian background with severe and frequent epistaxis and hematoma since infancy. he presented with mild thrombocytopenia and increased mean platelet volume. von willebrand's disease and subhemophilia had been excluded. previously, he was diagnosed with immune thrombocytopenic purpura. he never underwent elective surgery. his parents were asymptomatic. however, his 4-year-old daughter also suffers from severe bleeding symptoms (multiple, light red hematoma in consequence of minimal trauma). methods: whole exome sequencing (wes) was carried out for the patient, his asymptomatic wife, his symptomatic daughter and her asymptomatic 8-year-old brother. platelet function was assessed by light transmission-, lumi-aggregometry and flow cytometry. lysates of gel-filtered platelets were analyzed for total granule p-selectin, cd63 and von willebrand factor (vwf) content by western blotting and for serotonin levels by elisa, respectively. results. platelet function and characterization of the patients granula suggested a delta-storage pool disease (spd). in most cases delta-spd occurs as part of a syndrome, e. g. combined with albinism, immunodeficiency or a thalassemic-like blood disorder. as the patient and his daughter did not show any conclusive phenotype, their dna was subjected to wes. exome sequencing revealed a not yet described gata1-mutation close to two zinc finger domains (znf1 and znf2) in a highly conserved region of the gata1 gene in the 4-year old daughter (c.886a>c, p.t296p, heterozygous) and her father (c.886a>c, p.t296p, hemizygous). this mutation was absent in 150 wildtype-controls but could also be demonstrated in the indexpatients' asymptomatic mother. only a few mutations are known to be located in this c-terminal region to date. mutations in gata1 may lead to different clinical presentations, depending on their location within gata1 (e. g. diamond-blakfan anemia (exon2), x-linked thrombocytopenia (znf1), transient myeloproliferative disorder (intron 1, exon 2, exon 3) and acute megakaryoblastic leukemia (intron 1, exon 2, exon 3) in case of down-syndrome). significantly increased hbf-levels (reference level: ≤0,8%) in the affected family members of 13,5% (4-year-old daughour proposita is a 42 years old woman, who was transferred to our genetic counselling department for the suspicion of m. osler (hereditary hemorrhagic teleangiectasia, hht). during routine check up an anemia was diagnosed. a tumor search was initiated and unexpectedly the ct of the abdomen showed a suspect coin lesion of about 2.5 cm in diameter localized in the basal part of the right lung. further investigations revealed a pulmonary arterio-venous malformation which was hemodynamically relevant and already led to chronic right heart overload. a coil embolization was performed. retrospectively, medical history of the patient included episodes of severe epistaxis in childhood and a neurosurgical intervention for intracerebral bleeding at the age of 16 years without permanent neurological deficits. during genetic counselling our proposita mentioned that her 8 years old daughter also suffered from anemia due to multiple polyps of the colon. after polypectomy her hemoglobin values normalized. although histologically the polyps appeared as juvenile ones a mutation search in the apc-gene was initiated by the gastroenterologists without identifying a pathogenic mutation. combining the two pieces of information, we offered a mutation search in the smad4 gene and a pathogenic mutation c.1081c>t (p.arg361cys) was found in both patients in heterozygosity. colonoscopy in the mother did not show juvenile polyps or gastrointestinal vascular malformations. vice versa, no cerebral or pulmonary arteriovenous malformations could be detected in the daughter. our family illustrates, that the same mutation within a family may phenotypically appear as different diseases. a careful taking of medical history and the knowledge of all relevant diagnostic findings (in this case e. g. the histology of the polyps) can enable the geneticist to offer a precise differential diagnosis leading to a well-directed molecular testing. to our opinion this is still relevant even in the era of ngs-based panels because the more precise the clinical diagnosis and the choice of the genes to analyse the less problems with unclassified variants will arise. the hereditary non-polyposis colon cancer (hnpcc, lynch syndrome) is caused by pathogenic germline mutations in mismatch repair genes (mlh1, msh2, msh6 and pms2) causing microsatellite instability (msi) and decreased or lost expression of the appropriate mismatch repair protein (mmr) in the immunohistochemistry (ihc) on tumor material. thus, ihc and msi testing help to identify the mmr gene, which most likely harbors a germline pathogenic variant. msi and ihc testing prior to germline analysis are specified in the s3 guidelines of hnpcc and if negative normally preclude further genetic analyses. here, we present a 51 year old patient with a synchronous colonic (ceacum) and renal cancer at the age of 50 years. at the time of the diagnosis hepatic metastases of the caecal adenocarcinoma have already been present. histologically, the colonic tumor was a poorly differentiated adenocarcinoma (pt3pn) with lymphangiosis and haemangiosis carcinomatosa. the renal cancer showed histology of a moderately differentiated clear cell renal carcinoma. in the family history the 50 year old sister and the parents are healthy. the twin sister of the patient's mother had a collateral breast cancer at the age of 46 years and died two years later. the mother's grandparents had no canmedizinische genetik 1 · 2017 155 p-cling-109 pitfalls in molecular genetic diagnostics a. tibelius, e. fey, k. hinderhofer institute of human genetics, heidelberg, germany probably every clinical laboratory geneticist may look back on at least one case in his career which has caused him quite a headache. this means those cases with completely unexpected and at a first glance implausible results which could be interpreted correctly only after intensive enquiry and additional testing. here, we report on three of such pitfall cases from our routine diagnostics. case 1: a 30-year-old woman, pregnant with monozygotic twins, was referred to prenatal cystic fibrosis diagnosis. she and her partner were carriers of mutations in the cftr gene (621 + 1g>t and g542x, respectively). prenatal molecular diagnosis demonstrated that both fetus had inherited only the paternal mutation. routinely, we performed maternal cell contamination analysis by comparing polymorphic microsatellite loci between the maternal and fetal dna. surprisingly, 12 of 16 tested microsatellite loci revealed a discrepancy between the maternal and fetal genotypes, meaning neither of both maternal alleles was present in fetal dnas. a potential confusion of samples was excluded. moreover, the presence of paternal mutation in fetal dnas indicated a correct genetic relationship between awaited children and the partner of pregnant woman. the only one plausible interpretation of the obtained result was a pregnancy by egg donation. afterwards, this suspicion was confirmed by the couple. case 2: we present a 38-year-old man with infertility resulting from azoospermia. conventional chromosomal analysis and an additional fish analysis using y probes indicated a 46,xx karyotype with no detectable sry. in parallel, a molecular azf (azoospermia factors) diagnostic was performed by a standard multiplex pcr. by this method the absence of sry region and a deletion of regions azfb and azfc, was identified, explaining the observed azoospermia. interestingly, the pcr showed that the azfa region was still present in patient chromosomes, contradicting cytogenetic and fish results. thus, a complementary fish analysis was performed in order to reveal a low-grade y-mosaicism and sry material was detected in 3% of the cells (a result under the threshold level). based on this observation, pcr conditions for the azf diagnostic were modified and a very weak sry-specific pcr product detected. case 3: a molecular diagnostics for frax (fragile x syndrome) was requested for a 4-year-old boy with a slight delay in speech development. his brother was already molecular-genetically diagnosed as having frax. the analysis by southern blot hybridisation in the patient revealed a smear of methylated fragments characteristic for an expanded allele in the full mutation range. surprisingly, two fragments of normal length, methylated and non-methylated, could also be detected in patient's dna. a subsequent aneuploidy mlpa confirmed a supernumerary x-chromosome in the patient consistent with a klinefelter syndrome. these results were verified by an independent cytogenetic analysis. the syndrome of congenital symmetric circumferential skin creases (cscsc1 and cscsc2) replaces the old term michelin-tire-baby syndrome (mim 156610) and is characterized by congenital circumferential skin folds, primarily of the limbs, facial dysmorphism, cleft palate and intellectual disability. mutations in the β-tubulin encoding gene tubb or in the microtubule end binding family member mapre2 are the underlying genetic cause. ter) and 2,8% (35-year-old indexpatient) suggested dyserythropoiesis, although thalassemic features of the blood count were lacking. conclusion: we describe a gata1 mutation as the cause of a delta-storage pool disorder. imbalanced x-chromosome inactivation might explain the different phenotypes of the gata1 mutation carriers and will be investigated through allele-quantification based on rna isolated from whole blood and from platelet rich plasma in case of the index patient and different family members. we saw three siblings (aged 62, 61, 54; two female, one male) with variable signs of retinal disease. all three developed night blindness till the 3rd decade, furthermore near-sightedness and deterioration of the visual acuity to a various degree at age 35-45. the clinical diagnosis was given as stargardt's disease, fundus flavimaculatus or unspecified retinal degeneration respectively. two more siblings (aged 59 and 57) and the mother (died age 62) were clinically unaffected. the father (died age 69) was reported to have had bad eyesight beginning in the 5th decade, his father similarly (no further information available). the three affected siblings have a total of five children (age 25-39), none of them showing clear signs of retinal disease up to now. considering the clinical diagnosis stargardt's disease we analysed the genes abca4, elovl4, prom1 and cngb3 by sanger sequencing. no pathogenic mutation could be detected. afterwards we performed next generation sequencing of several genes associated with retinal dystrophies and found a novel splice site mutation in the prph2 gene (mim #179605). the mutation was confirmed in all three affected siblings by sanger sequencing. considering that mutation pathogenic we could re-diagnose our patients with patterned macular dystrophy type 1 (mim #169150). that disease is inherited in an autosomal dominant fashion, corresponding to the pattern of inheritance evident in our family. the etiology of epileptic encephalopathies, characterized by severe, early-onset seizures accompanied by developmental delay or regression, is highly heterogeneous. in recent years, next-generation sequencing approaches have led to the discovery of numerous causative genes; however the spectrum of associated phenotypes still needs to be further explored for many of these genes. we performed multi-gene panel analysis in a little boy of german non-consanguineous parents who showed severe early-onset infantile epileptic encephalopathy and almost absent neurological development. in this patient we identified a novel heterozygous missense mutation in the gabrg2 gene which was absent in the parents. in silico analyses strongly suggested a pathogenic relevance of this sequence variation which resides within a highly conserved region. so far, gabrg2 mutations have mainly been associated with milder types of epilepsy such as febrile seizures and childhood absence epilepsy. therefore, our findings extend the phenotypic spectrum associated with mutations in this gene at the severe end. referring on a case report by al marzouqi et al. (2014) , who reported on a girl with bilateral amastia in the context of skewed x-inactivation, we want to underline the importance of mlpa analyses in the case of negative sequencing of the eda gene, as whole exon duplication can be the cause of hypohidrotic ectodermal dysplasia. to our knowledge, the report of al marzouqi et al. has been the only case about this genetic alteration in the literature so far. novel pogz mutation in a patient with intellectual disability, microcephaly, strabismus and sensorineural hearing loss we report on a 3-year-old male patient with severe intellectual disability, microcephaly, sensorineural hearing loss, ocular abnormalities (strabismus and hyperopia), congenital heart defect (atrial septum defect and pulmonary stenosis) and minor facial abnormalities (thin upper lip, frontal upsweep). next-generation sequencing analysis revealed a novel heterozygous de novo mutation in pogz: c.2703_2710del, p.(thr902serfs*39) . the clinical problems of this patient are in accordance with the findings in the previously reported pogz mutation carriers. reports of additional patients with pogz mutations will be needed to establish detailed phenotype-genotype correlations of this novel and probably underdiagnosed syndrome. novel clinical and molecular aspects in two patients with kleefstra syndrome d. wand, b. seebauer, k. heinimann, i. filges medical genetics, university hospital, basel, schweiz the phenotype of kleefstra syndrome is clinically variable but characterized by facial dysmorphism, intellectual disability, childhood hypotonia and variably associated other malformations. haploinsufficiency of ehmt1, caused by either heterozygous microdeletions in 9q34.3 or sequence mutations in ehmt1, has been identified to be the underlying causal mechanism. here we present two girls from two unrelated families with clinical signs of kleefstra syndrome. besides the main features such as facial dysmorphism, intellectual disability/developmental delay and childhood hypotonia, the 17 years old girl presented with additional accelerated growth whereas the other girl, 2 years old, showed failure to thrive. both children have no heart defect, renal or urogenital anomalies or severe respiratory infections. we identified two rare variants likely to be causal: a novel heterozygous splice site ehmt1 variant and a heterozygous microdeletion in chromosome 9q34.3, including exons 26 an 27 of the ehmt1-gene . these patients broaden the spectrum of kleefstra -associated ehtm1 causes, contribute to novel aspects of genotype-phenotype correlations and a better understanding of the clinical variability of the disorder. here, we present two girls from two non-consanguines families showing clinical aspects of the syndrome of congenital symmetric circumferential skin creases. additional features are present in both girls. patient 1 is the first child of healthy parents. she was born at 39 + 6 weeks of gestation with a weight of 2660 g, a length of 49 cm and a head circumferences of 34 cm. respiratory distress, a cleft palate, a heart defect (atrial septum defect), an anogenital malformation and facial dysmorphism were diagnosed after delivery, additionally to the skin creases phenotype. conventional karyotyping performed on blood lymphocyte cultures and cgh-array analysis showing normal results. patient 2 is the second child of unrelated healthy parents. her older sister is healthy as well. because of an intrauterine growth retardation an amniocentesis with chromosomal analysis was performed, showing a normal karyotype of 46,xx. patient 2 was born at 38 + 3 weeks of gestation by caesarean section. the birth weight was 2450 g, the birth length was 47 cm and the head circumferences was 34 cm and the apgar score was 1/3/5. due to respiratory distress and hypoxia a tracheotomy was initiated. she also presented with cleft palate, feeding difficulties, a heart defect (atrial and ventricular septum defect), asplenia and facial dysmorphism. the skin phenotype was remarkably similar to that of patient 1 with a prominent neck fold and skin creases mainly on the back part, but also at the limbs. in both children the skin folds gradually diminish over the time without any intervention like it was described for michelin-tire-baby syndrome/ cscsc1 and cscsc2 patients. for these reason, a disease-causing mutation in the genes mapre2 and tubb were excluded in both children. to identify a genetic cause, we performed a trio whole-exom sequencing, including the healthy parents and the affected child, in both families. a de novo stop mutation was detected in patient 1, while no promising results could be detected in patient 2. further studies, especially functional in vivo studies and analysis of further patients with a similar clinical presentation will answer the question if the above described phenotype is an expanded variant of the congenital symmetric circumferential skin creases or a unique new syndrome. clinical findings in a family with x-linked hypohidrotic ectodermal dysplasia due to a duplication of exon 2 in the eda gene -a case report we want to summarize the phenotypic spectrum in one affected three generation family with x-linked hypohidrotic ectodermal dysplasia. we report on one strongly affected male and 2 slightly affected female carriers, carrying a duplication of exon 2 in the eda gene. reason for genetic assessment was the request of a female for evaluation of a possible risk for occurrence of the familial disorder in a further planned pregnancy. the woman reported an inability to breastfeed and she and her daughter showed conical teeth, a dry skin and sparse hair. the affected male showed absent deciduous teeth, hypodontia of permanent teeth, missing regulation of the temperature due to a lack of sweat glands, bilateral nipple hypoplasia, a dry and wrinkled skin, missing eye lashes, eyebrows and scalp hair and sparse body hair. the fingernails were inconspicuous, whereas the nails of the toes, particularly the nails of the hallux were yellowish and thickened. the affected male had an operation due to a gallstone and cysts were found in one kidney. there was no increased predisposition to infections. the suspected diagnosis of x-linked hypohidrotic ectodermal dysplasia was confirmed by genetic analysis of the eda gene (sequencing and mlpa analysis). a duplication of exon 2 was detected in the affected male patient and was confirmed in the two mildly affected female relatives by realtime-pcr. terminator sequencing mix v1.1 on an abi3130xl genetic analyzer (applied biosystems) and detected an insertion of 77 bp between the exons 7 and 8. we located the insertion's sequence in intron 7 of the dmd gene and sequenced flanking sequences of gdna to find the underlying mutation causing the insertion. two hemizygous single nucleotide variants (snvs) surrounding the inserted fragment could be identified. the first variant (c.650-39575 a>c) is a common polymorphism (maf according to 1000 genomes project: 14.97%) at the position of an existing acceptor splice site. the second variant (c.650-39498a>g) is novel and creates a new cryptic donor splice site with high probability. these two cryptic splice sites create the formation of a 77 bp pseudoexon which produces a frameshift and a premature stop codon (p.asp217alafs*) in the dmd gene. in summary, we could genetically confirm the clinical diagnosis of a dystrophinopathy by two divs in dmd. although the insertion of the pseudoexon creates a premature stop codon the patient's clinical phenotype indicates a milder type of becker muscular dystrophy. this contradiction could be explained by the remaining existence of dmd wild type mrna most likely due to a not constantly active cryptic splice site. most interesting about the present case is the fact that a common snv facilitates the creation of a pseudoexon. this makes the region a potential hotspot for divs in the dmd gene which would be worthwhile further investigations. with more than 90% of all humans preferring to use their right hand, handedness is the most noticeable functional expression of cerebral lateralization in humans. however, the precise molecular mechanisms that regulate handedness and other related forms of cerebral lateralization remain largely elusive. therefore, the question which genetic, epigenetic and environmental factors contribute to human handedness is one of the central questions in research on lateral asymmetries. handedness is a complex, heritable trait, for which polygenic inheritance is assumed, meaning that a large number of genetic factors with a small additive effect contribute to the observed variance in hand preference. to date, genetic association studies have implicated only a few specific genes influencing handedness. particularly interesting is the association between the human androgen receptor (ar) gene and different aspects of handedness, since the interrelationships constitute a conceptual bridge between the theories that invoke testosterone as a factor in the development of cerebral asymmetries with theories proposing that the x chromosome contains a locus that influences the direction of hand preference. in an initial large association study in 1057 samples of healthy adults we already demonstrated that handedness in both sexes is associated with the ar cag-repeat length, with longer repeats being related to a higher incidence of non-right-handedness. in addition, we have performed a second association study in an independently collected healthy cohort of more than 1000 test persons with comprehensive data on the handedness phenotype. we were able to replicate the association with longer cag repeats being related to a higher incidence of non-right-handedness, especially in females. since longer cag-repeat blocks have been linked to less efficient ar function, these results implicate that differences in ar signaling in the developing brain might be one of the factors that determine individual differences in brain lateralization. dej. waschk, ac. tewes, p. wieacker, s. ledig institute of human genetics, münster, germany the mammalian female and male reproductive tracts develop from the paramesonephric ducts (müllerian ducts, md) and mesonephric ducts (wolffian ducts, wd), respectively. in the absence of testicular differentiation and anti-müllerian hormone, the wd regress and the md give rise to the fallopian tubes, uterus, cervix and the upper part of the vagina. disorders of normal md development in females can manifest as fusion anomalies of the uterus such as septate uterus, bicornuate uterus, unicornuate uterus and uterus didelphys, or more complex malformation patterns like mayer-rokitansky-küster-hauser syndrome (mrkh) or herlyn-werner-wunderlich syndrome. mrkh is characterized by congenital absence of the uterus and the upper two-thirds of the vagina in individuals with a normal female karyotype, most of whom show normal ovarian function. mrkh can further be associated with additional malformations e. g. of the kidneys and the skeletal system. herlyn-werner-wunderlich syndrome is characterized by uterus didelphys, obstructed hemivagina and ipsilateral renal agenesis. despite anomalies of the md occur quite frequently, the etiology of most cases with these disorders remains unknown. the homeodomain transcription factor emx2 (empty spiracles homeobox 2) was found to be critical for urogenital and central nervous system development. previous studies showed that in emx2 mutant mice, the kidneys, ureters, gonads and genital tracts were completely missing. in order to elucidate whether mutations in emx2 are causative for md anomalies in humans, we performed sequence analyses of emx2 (genbank nm_004098.3) in our study group of 142 female patients with clinically characterized disorders of the md including 62 patients with mrkh and 7 cases with herlyn-werner-wunderlich syndrome. we found the heterozygous intronic mutation c.592-17c>a twice in this cohort. this variant has been described earlier (rs41308651) and is listed in the exome aggregation consortium exac variant with a minor allele frequency of 0.23%. in silico analysis revealed no significant changes for the correct splicing of emx2. we therefore consider this variant to be a benign polymorphism. we conclude that mutations in emx2 are not causative for disorders of the md in our cohort. a. zaum, w. kreß, a. gehrig, s. rost institute of human genetics, würzburg, germany dystrophinopathies are x-linked muscle diseases caused by mutations in the dmd gene (omim: 300377). due to the huge size of this gene, the detection of mutations is sometimes challenging. despite multiplex ligation-dependent probe amplification (mlpa) and sequencing of all 79 exons, about 7% of patients do not show any mutations in coding regions and therefore remain without molecular diagnosis. we assume that the majority of these patients have deep intronic variations (div) which are not detectable by standard diagnostic techniques. the index patient analysed is a twelve-year-old boy who was by chance diagnosed with elevated ck levels (up to 15,000 iu/i) at eight weeks of age. today he is still able to walk without walking aids but needs assistance when climbing stairs. in 2008, a muscle biopsy revealed complete absence of dystrophin which established the diagnosis of dmd. for the molecular diagnosis, standard diagnostics ascertained no causative mutation. therefore we decided to search for deep intronic mutations. we isolated mrna from muscle tissue of the patient and amplified overlapping cdna fragments using rt-pcr. the fragments were analysed by gel electrophoresis for size differences compared to an unaffected control. the cdna product comprising exons 6-12 revealed an augmented fragment size compared to the control and the expected product size (about 1100 bp instead of the expected 1007 bp). we sequenced the altered cdna product using bigdye recent research in psoriasis has identified pustular psoriatic manifestations as either mendelian traits or as major genetic risk factors in contrast to the numerous associated snps in classical plaque psoriasis vulgaris. autosomal recessive mutations in il36rn have been identified in ~25-40% of patients with generalized pustular psoriasis (gpp), a rare, severe pustular variant of psoriasis. in addition, heterozygous missense variants in card14 and ap1s3 have been associated to pustular psoriasis as well and shown to be functionally relevant. in order discover how relevant those genes were in our large gpp cohort of 61 patients recruited all over germany, we screened them for coding variants in il36rn, card14 and ap1s3 by sanger sequencing and for quantitative aberrations by mlpa. we identified homozygous or compound heterozygous il36rn mutations in 15 of 61 gpp patients (25%) and single heterozygous mutations in 5 patients (8%). the most common mutations were c.338>c>t/ p.ser113leu and c.227c>t/ p.pro76leu, present on 49%/20% of mutated alleles, respectively. we also identified three so far unreported mutations: c.338c>a/ p.ser113x, c.295-300delacct-tc/ p.thr99_phe100del and c.130g>a/ p.val44met. according to molecular modeling, c.338c>a/ p.ser113x resulted in a shortened, de-stabilized protein analogous to c.280g>t/ p.glu94x. the other two mutations were also predicted to result in destabilized, likely disease-relevant, loss-of-function proteins. heterozygous ap1s3 mutations were detected in two gpp patients, both of whom carried additional homozygous or compound-heterozygous il-36rn mutations. 4 gpp patients were heterozygous carriers of rare missense variants in card14 (7%); of note, two of these patients carried additional mutations in il36rn. our genotype-phenotype correlation revealed a similar gender distribution in carriers of il36rn mutations and wildtype carriers, but a strong association between bi-allelic mutations in il36rn and early age of manifestation (p = 7.4x*10-04). as in other autosomal recessively inherited mutations, the frequency of parental consanguinity was significantly higher in patients with two il36rn mutations compared to non-carriers. overall, our genetic studies suggest a lower impact of variants in ap1s3 and card14 in pustular psoriasis than of those in il36rn. interestingly, the combination of il36rn mutations with either ap1s3 or card14 congenital prosopagnosia (cp), also known as developmental prosopagnosia or face blindness, describes the inability to recognize faces. cognitive functions such as intelligence as well as the sensory visual capabilities are usually not impaired but people with prosopagnosia are negatively affected in their social life because individuals with the disorder have difficulty in recognizing family members, close friends or colleagues. although the prevalence of cp is estimated at 2.5% and it appears to run in families, the contexts, which genetic, epigenetic and environmental factors contribute to this trait, are largely unknown. therefore we started to establish a large, well-characterized cp cohort for genetic studies. we hypothesize that rare highly penetrant non-synonymous genetic variants could explain some cases of cp. as part of a larger genetic study of patients with cp, we performed family based whole-exome sequencing and targeted re-analysing on four individuals from two families with multiple affected members. by obtaining samples from affected and unaffected members of the same family, we hope to effectively identify de novo and inherited variations. variations are considered on the basis of allele frequency, mutation type, literature and mutation prediction tools, thus generating a list of candidate variations/genes for each patient that is amenable to interpretation and further analyses in the extended cohort. through this approach, we hope to identify causal variations/genes in families and isolated patients with cp. erythrokeratodermia variabilis (ekv) is a rare, autosomal dominant genetic skin disorder with a highly heterogeneous phenotype. to date, three causative genes (gjb3, gjb4 and gja1) are described but further genetic heterogeneity is expected. card14 mutations are only described for psoriasis vulgaris, generalized pustular psoriasis and pityriasis rubra pilaris. for the first time, we present disease causing card14 mutations in patients with an "ekv-like" phenotype. it refers to one familial case with two affected individuals, with an autosomal dominant transmission from the mother to the daughter and one independent sporadic case. all patients present typical ekv symptoms. a rash of well-demarcated erythematous and scaly plaques interspersed with distinct islands of uninvolved skin or small reddish papules coalescing into large reticulated scaly plaques and palmoplantar keratoderma. to confirm the suspected diagnosis of ekv, we analyzed a custom designed multi-gene panel by next generation sequencing with 74 genes associated to hereditary skin diseases (agilent haloplex technology). the sequencing results did not reveal any mutation in the genes gjb3, gjb4 and gja1, but we found two pathogenic mutations in card14. in the familial case c.467t>c p.leu156pro (rs387907240, enst00000570421.5) was detected, while the sporadic case carries c.371t>c p.leu124pro. we hypothesize that different genetic and environmental factors are involved in the evolution of the phenotype in patients with card14 mutations. our cases show that classification of unusual skin phenotypes can be challenging without genetic testing. therefore, gene panel sequencing is a cost-efficient and time-saving solution for solving difficult cases with sometimes unexpected genetic background. bipolar disorder (bd) is a complex psychiatric disorder affecting more than 1% of the world's population. the highly heritable disease is characterized by recurrent episodes of manic and depressive symptoms. as the cumulative impact of common alleles with small effect may only explain around 38% of the phenotypic variance for bd, rare variants of high penetrance have been suggested to contribute to bd susceptibility. in the present study we investigated 226 individuals of 68 large multiply affected families of european origin using whole exome sequencing (wes). in each family, two to five affected individuals with bd or recurrent major depression were selected for sequencing. wes was performed on the illumina hiseq2500 platform and the varbank pipeline of the cologne center for genomics was used for data analysis. all identified variants shared within each family were filtered for a minor allele frequency <0.1% and potentially damaging effects predicted by at least four of five different bioinformatics tools. we identified a total of 1214 rare, segregating and functional variants implicating 1122 different genes, of which 903 were brain expressed. subsequently, we applied the residual variation intolerance scores (rvis, petrovski et al., 2013) and identified 294 genes that were ranked among the 20% most "intolerant" genes in the genome. gene enrichment analysis of these genes showed a significant enrichment for a total of 18 pathways (p < 0.001) including neuron projection, axon development and cell adhesion. for follow up analyses, we prioritized genes that were either found in at least two unrelated families in the present study or that were previously reported in next generation sequencing or gwas studies of bd. in addition, we enclosed the genes that were predominantly driving the significant pathways in the above mentioned gene enrichment analysis. the different approaches of prioritization yielded 42 candidate genes that are currently being followed up by resequencing in cohorts of about 2500 independent bd cases and 2500 controls of european ancestry. the candidate genes include cdh22 that encodes a calcium-dependent cell adhesion protein that may play an important role in the morphogenesis of neural cells during the development and maintenance of the brain. for resequencing we use the single molecule molecular inversion probes (smmips) technology that enables multiplex targeted resequencing in large cohorts. the smmips sequences were designed with an empirically variants in several patients indicated an oligogenic inheritance rather than a purely monogenic one. moreover, the oligogenic basis of this group of inflammatory diseases might currently be underestimated, as our study suggests that genetic risk factors other than il36rn mutations remain to be identified in the majority of gpp patients. exome sequencing of 46 multiply affected schizophrenia families provides new insights into the pathogenesis of the disorder schizophrenia (scz) is a multifactorial psychiatric disorder with a lifetime risk of ~ 1% and a heritability of about 60-80%. analysing multiply affected families using whole exome sequencing (wes) is a very promising approach to identify new scz risk factors. in these families, individuals are affected with scz over several generations. it is likely, that in multiply affected families genetic variations with particularly strong effect co-segregate with the disorder and contribute to the development of the psychiatric symptoms. to our knowledge, the present study is the largest study analysing multiply affected scz families using wes worldwide so far. we included 46 families with at least 3 affected members each. from each family, 3-5 individuals were exome sequenced on an illumina hiseq 2500 and analysed using the varbank pipeline of the cologne center for genomics (http://varbank.ccg.uni-koeln.de) and the clc bio biomedical genomics workbench. we included rare (allele frequency ≤ 0.1% in the exome aggregation consortium dataset) variants that were predicted to be pathogenic (combined annotation dependent depletion score ≥ 15; cadd.gs.washington.edu), confirmed by sanger sequencing and co-segregating with the disorder. in total we identified potentially pathogenic mutations in ~ 880 genes. a substantial proportion of these will not contribute to the pathogenesis of scz. in order to further tease out the most promising candidate genes we applied multiple strategies: (i) screening our mutations in independent patient and control cohorts through international cooperations (access to more than 3,000 scz patients), (ii) gene-based tests, (iii) pathway-and network-analyses, (iv) gene expression analyses and (v) sequencing of the candidate genes in 2,500 scz patients and 2,500 controls. analyses are ongoing and will be presented at the upcoming conference. abstracts and our evolution, we know very little about the different mutagenic processes in our germline. of particular interest are a handful of highly recurrent dnm associated with congenital disorders and/or rasopathies, that have been described as driver mutations expanding in the male germline. the mutation itself causes a change in the tyrosine kinase receptor/ras/ mapk pathway, which in turn confers the spermatogonial stem cell a proliferative advantage. selfish or driver mutations are quite common in cancer, but we still know very little about the selfish expansion in the male germline. the reason might be that mutations in the human germline are very rare, and it is rather difficulty to directly measure such rare events. most of our knowledge on germline mutagenesis comes from indirect sequence comparisons or whole genome sequencing of pedigree families, but it renders little information about individual mutagenic events. for this reason, we have adapted an ultrasensitive, next generation sequencing (uss) technology for the measurement of rare mutations to study the expansion of selfish genes in the male germline. as a proof-of-principle, we have sequenced at an extremely high coverage exon 10 and 15 of the fgfr3 gene in young and old sperm donors. we found an increased mutation frequency for the loci associated with achondroplasia and thanatophoric dysplasia ii in sperm of older donors. our results also show that we can distinguish ultra-rare mutations occurring at a frequency of one in hundred thousand wild type; thus, making this method ideal to discover potential driver dnm that might be expanding with paternal age. s. sivalingam 1, 2 , a. j. forstner 1, 2, 3 , s. herms 1, 2, 4 , a. maaser 1, 2 , c. s. reinbold 4, 5 , t. andlauer 6 , j. frank 7 , h. dukal 7 , d. schendel 7 , 2 , p. hoffmann 1, 2, 4 , t. kircher 8 , u. dannlowski 8, 9 , a. krug 8 , a. cichon 1, 2, 4 , s. witt 7 , m. rietschel 7 , m. m. nöthen 1, 2 introduction: affective disorders such as major depressive disorder (mdd) and bipolar disorder (bd) are genetically complex and heterogeneous disorders. both genetic and environmental risk factors contribute to the etiology of the diseases. however, the neurobiological correlates by which these risk factors influence the disease development are hardly understood. increasing evidence suggests that epigenetic modifications such as dna methylation have important implications on the development of psychiatric disorders including mdd and bd. several studies revealed that alterations in the dna methylation can modulate gene expression in response to the environment. to investigate this, genome-wide dna methylation analysis of 66 female individuals from three extreme groups (genetic-, environmental risk and healthy controls) was performed. methods: for the genome-wide dna methylation analysis we selected: (i) 22 individuals with genetic risk (at least one 1st degree relative with a life-time diagnosis of mdd or bd), (ii) 22 individuals with environmental risk (maltreatment in the childhood trauma questionaire) and (iii) 22 matched healthy controls. all individuals were of european origin. processing was done according to the manufacturer's protocol using the infinium methylationepic beadchip (illumina, san diego, ca, usa) covering more than 850.00 methylation sites at the life & brain center (bonn, germany). state-of-the-art data processing protocols, including correction for blood cell type heterogeneity, color correction, eliminating probes containing snps and cross reactive probes were used. after quality control trained design algorithm mipgen (boyle et al., 2014) and sequencing is currently performed on the illumina hiseq2500 platform. our preliminary results strongly suggest that rare and highly-penetrant variants in neuronal and cell adhesion genes contribute to bd etiology. the results of resequencing of a large case/control sample will provide further evidence for an involvement of particular pathways. the use of zebrafish as model system to quantitatively assess the impact of risk variants in non-coding regions in vivo s. l. mehrem 1, 2 , b. nagarajan 3 , n. ishorst 1, 2 , a. c. böhmer 1, 2 , e. mangold 2 , b. odermatt 3 , k. u. ludwig 1, 2 1 department of genomics, life & brain center, university of bonn, bonn, germany, 2 institute of human genetics, university of bonn, bonn, germany, 3 institute of anatomy, university of bonn, bonn, germany most human malformations occur early in embryonic development and are present immediately after birth. one common human birth defect is nonsyndromic cleft lip with or without cleft palate (nscl/p), affecting about 1 in 1,000 newborns. nscl/p has a multifactorial background with a strong genetic component. recent genome-wide association studies identified several loci as risk factors for nscl/ p. notably, most of them map to non-coding regions and are expected to have a functional impact through regulatory mechanisms. given the early developmental time point of facial development and the resulting lack of accessible human tissue, follow-up analyses of risk variants are challenging. we are hypothesizing here that we might be able to quantitatively detect differences in regulatory activity between wildtype and risk variants located in predicted enhancers by using the zebrafish as model organism. the advantages of using the zebrafish are (1) ex utero development, (2) transparency of the fish, (3) easy manipulation and (4) relatively short generation times. we applied a dual-luciferase assay plasmid system which is based on a sequential measurement of two luciferases (firefly and sea pansy luciferase) in fish lysates upon injection of a single plasmid. this plasmid, which contains a minimal promoter (minp) and the enhancer region of interest, is microinjected into zebrafish eggs of one-cell stage. after three days, fish are collected, lysed and luciferase activity is measured using a luminometer. for our proof-of-principle analysis we analyzed an nscl/p risk locus on chromosome 13q31 (ludwig et al. 2012) . through database research one enhancer was predicted that contained two strongly associated risk variants. in vivo fluorescence analysis using egfp in zebrafish embryos revealed this enhancer to be active in craniofacial development, but qualitative differences in activity were not observed by eye. upon cloning of the enhancer in the dual-luciferase system, our injection results in vivo indicate that the system is working in principle. however, a high standard deviation between single replicate measurements was observed, probably due to variability in transfection efficiency. we therefore are planning to adapt the protocol in order to screen for positively injected fish embryos. we are currently investigating the functionality of these screening constructs in zebrafish embryos. results will be presented at the meeting. once successful, our approach represents a practical method to quantify the activity of regulatory elements in real time in vivo. this will be of particular importance in the functional follow-up of genetic findings in non-coding regions for the majority of birth defects. previously genome-wide association methods in patients with classic bladder exstrophy (cbe) found association with isl1, a master control gene expressed in pericloacal mesenchyme. this study sought to further explore the genetics in a larger set of patients following-up on the most promising genomic regions previously reported. genotypes of 12 markers obtained from 268 cbe patients of australian, british, german ital-and normalization 780,467 cpg-sites were tested for genome-wide dna methylation by a linear regression model while accounting for biological as well technical covariates. results: the genome-wide dna methylation analysis of the three extreme groups revealed 39 cpg sites (p < 1×10 -04 ) in the subgroup analysis "environmental risk vs. controls" and 35 cpg sites (p < 1×10 -04 ) in the analysis "genetic risk vs. controls". in addition, we identified 48 cpg sites (p < 1×10 -04 ) in the comparative analysis of "genetic risk vs. environmental risk". none of these cpgs showed significant differential dna methylation after correction for multiple testing. however the hierarchical clustering of the differentially methylated sites provided some evidence for differentially methylated patterns between the subgroups. discussion: our genome-wide dna methylation analysis of the extreme groups provided some evidence for differentially methylated cpg sites which unfortunately did not withstand correction for multiple testing. this may reflect at least in part that the sample size of the present study was too small to detect differentially methylated cpgs at the genome-wide level. a. tafazzoli 1, 2 , t. vaitsiakhovich 3 , l. pethukova 4, 5 , 2 , s. redler 1, 6 , r. kruse 7 , b. blaumeiser 8 , m. böhm 9 , g. lutz 10 , h. wolff 11 , , am. christiano 5 , p. kokordelis 1 , mm. nöthen 1, 2 , rc. betz 1, 2 1 alopecia areata (aa) is a common hair loss disorder that occurs in both sexes and all age groups. aa is thought to be a tissue-specific autoimmune disease directed against the hair follicle. both, gene-based and genome-wide association studies have identified more than 10 susceptibility loci for aa; however, a large percentage of the overall heritable risk still awaits identification. to provide further insight into the immune related nature of aa, we and our us collaborators had each performed an immunochip-based analysis. we recently performed a meta-analysis, combining the data from both studies, and are now aiming to follow-up the best results in an additional german cohort by use of a sequenom assay to identify novel susceptibility loci. we conducted the meta-analysis by using data from the above mentioned two studies on illumina beadchip arrays including a total of 1,096 cases and 3,176 controls of central european origin. method of synthesis of regression slopes (msrs) was used for the analyses which are implemented in metainer software package. for follow-up step, we chose the most promising candidate snps. these will be examined with the sequenom massarray iplex platform in an independent aa sample comprising 1,459 cases and 970 controls. by use of the meta-analysis combining data from the us and our sample, we identified 49 novel loci with a suggestive p-value of pbecker-wu ≤ 10 -3 (phet ≥ 0.01). among them, nfkb is the most significant locus (pbecker-wu = 1.5 × 10 -07 ). in order to achieve genome-wide significance, we plan to follow-up the most promising snps in an independent german sample. we considered the 19 most significant loci (lower p-becke-wu value) for the replication step. the experiments are ongoing and results will be presented at the meeting. despite the recent identification of susceptibility loci for aa, our understanding of the genetics of aa is incomplete. identification of new loci may provide novel insights into biological pathways and a better elucidation of disease pathophysiology. 22q13.1 encompassing only one gene: kcnj4. the duplication segregates with the phenotype in the family. kcnj4 belongs to the same subfamily of potassium channels as the known disease gene for cooks syndrome kcnj2 and both share several biological functions. recent data show that gain of function mutations in another potassium channel kcnh1 cause zimmermann-laband syndrome, a congenital malformation syndrome also associated with hypoplasia or aplasia of nails and terminal phalanges. therefore we propose that duplications of kcnj4 may also cause tissue specific misregulation resulting in digit and nail defects. taken together we show in a three generation pedigree that cooks syndrome is associated with a duplication of kcnj4. our data further highlight the emerging role of potassium channels in congenital digit and limb anomalies. case report: deletion of the terminal short arm of chromosome 5 (chromosome 5p deletion syndrome) without 5q-duplication with a familial history of a large pericentric inversion of chromosome 5 b. gläser, n. hirt, e. botzenhart, j. fischer, m. leipoldt institut für humangenetik, universitätsklinikum freiburg, freiburg, germany we report on a male patient referred as a newborn with typical clinical features of chromosome 5p deletion syndrome. conventional karyotyping of lymphocyte cultures confirmed a deletion of the terminal short arm of chromosome 5 with breakpoint in 5p14. the size of the deletion (22mb) could be refined by microarray analysis and assigned to pos 5:16497-22278242. parental cytogenetic investigations showed a normal karyotype in the mother whereas the father was revealed to be carrier of a large pericentric inversion of one chromosome 5. odd crossing-over in the inverted segment of a pericentric inversion in a parent can lead to unbalanced offspring caused by gametes with a terminal deletion of the p-arm together with a duplication of the q-arm or gametes with a duplication of the p-arm together with a deletion of the q-arm. in order to find out, whether the 5p-deletion in the child is the result of an independent event or if it is related to the structural chromosomal aberration of the father a microsatellite analysis is going to be performed p-cytog-130 a small supernumerary marker chromosome of the pericentric region of chromosome 8 in a child with intellectual disability: case report and literature review b. hoffmann, g. gillessen-kaesbach, i. hüning institut für humangenetik, universität zu lübeck, lübeck, germany small supernumerary marker chromosomes (ssmc) are reported in 0.043% of newborn infants. we report on a girl, which was born preterm at 28 weeks of gestation via cesarean section due to pathological ctg. the pregnancy was complicated by gestational diabetes. she presented with muscular hypotonia, multiple hemangiomas, dysmorphic features and feeding problems. the body measurements were in normal range. the feeding problems made a tube feeding necessary until the age of four months. facial features consisted in epicanthus, high palate, broad nasal tip and broad nasal root. a brain mri showed periventricular leukomalacia and hypoplasia of corpus callosum. drug-resistant seizures with hypsarrhythmia started at the age of ten months. the affected girl was the only child of healthy non-consanguineous parents. the father also presented with a few small hemangiomas in the face. there was no history of intellectual diasability in the extended family. karyotyping showed ssmc in mosaicism. molecular characterization by array-cgh showed that the ssmc consists of pericentric chromosomal material derived from chromosome 8 (arr [ h g 1 9 ] 8 p 1 1 . 1 p 2 1 . 3 ( 2 2 , 8 1 6 , 5 2 7 -4 3 , 3 9 6 , 7 7 6 ) x 2~3 , 8 q 1 1 . 1q11.21(47,673,716-52,164,874) x2~3 dn (grch37/hg19). ian, spanish and swedish origin and 1,354 ethnically matched controls and from 92 cbe case-parent trios from north america were analysed. only marker rs6874700 at the isl1 locus showed association (p = 2.22 × 10 -08 ). a meta-analysis of rs6874700 of our previous and present study showed a p value of 9.2 × 10 -19 . developmental biology models were used to clarify the location of isl1 activity in the forming urinary tract. genetic lineage analysis of isl1-expressing cells by the lineage tracer mouse model showed isl1-expressing cells in the urinary tract of mouse embryos at e10.5 and distributed in the bladder at e15.5. expression of isl1 in zebrafish larvae staged 14 hpf to 24 hpf was detected in the developing pronephros region. our study supports isl1 as a major susceptibility gene for cbe and as a regulator of urinary tract development. to date, seven patients with interstitial deletions at chromosome 8q22.2-q22.3 have been described in the literature. all patients reported had moderate to severe intellectual disability and a characteristic facial phenotype including blepharophimosis, telecanthus, epicanthus, flat malar region, and a thin upper lip vermillion. six of the seven patients had epileptic seizures. by analyzing the deletion's overlaps, two distinct critical regions have been suggested for the facial phenotype as well as for intellectual disability and seizures. here we present another patient with a de novo 3.6 mb deletion in 8q22. 2-q22.3 . the patient shows moderate mental retardation. he has an abnormal eeg, however, only one episode of clinical seizures has been observed so far. the facial gestalt resembles the typical dysmorphic features of the patients with 8q22.2-q22.3 deletions reported previously. minor anomalies were short fingers and toes, and a single palmar crease. our report supports the assumption that deletions in 8q22.2-q22.3 cause a distinctive and clinical recognizable microdeletion syndrome with characteristic facial features and intellectual disability. since the patient's deletion overlaps with most of the critical region for the dysmorphic phenotype but only with parts of the intellectual disability critical region, the molecular data presented here further narrow down the critical region for the intellectual disability seen in patients with 8q22.2-q22.3 microdeletions. p-cytog-128 *** cooks syndrome is associated with a duplication of the potassium channel kcnj4 mundlos 1, 2, 4 , d. horn 1 , m. spielmann 1, 2, 4 1 institute for medical genetics and human genetics, charité universitätsmedizin berlin, berlin, germany, 2 max planck institute for molecular genetics, berlin, germany, 3 northern ireland regional genetics service, belfast city hospital, belfast, ireland, 4 berlin-brandenburg school for regenerative therapies -bsrt, berlin, germany cooks syndrome (mim106995) is a rare autosomal dominant disorder classically characterized by onychodystrophy, and anonychia, with absence or hypoplasia of the distal phalanges of the hands and feet. large duplications including kcnj2 were shown to be causative for cooks syndrome. recently mouse studies revealed that tissue specific misregulation of kcnj2, a potassium channel of the subfamily j, cause hypoplasia of nail beds and abnormal distal phalanges, thus resembling the cooks phenotype. here we report on a three generation pedigree with typical features of cooks syndrome that was negative for kcnj2 testing. we performed high resolution array-cgh and identified 80 kb duplication on chromosome p-cytog-132 chromosome 17q23.1-q23.2 deletion syndrome -an additional case with sensorineural hearing loss. s. leubner, c. hennig, a. junge mitteldeutscher praxisverbund humangenetik, dresden, germany the chromosome 17q23.1-q23.2 deletion syndrome (mim #613355) is a contiguous gene syndrome caused by a deletion encompassing the chromosome region 17q23.1-q23.2. initially, it has been described by ballif et al. (2010) . up to now, only a few cases with a microdeletion 17q23.1 q23.2 have been reported. most of them carry a microdeletion with recurrent breakpoints and similar size of about 2.2 mb. the common clinical features of cases with the chromosome 17q23.1-q23.2 deletion syndrome comprise mild-to-moderate developmental delay, microcephaly, postnatal growth retardation, heart defects, limb anomalies, and hearing loss. we present an additional male patient with a 2.2 mb deletion of chromosome 17q23.1-q23.2, detected by array cgh (cytochip isca 4×180k v1.0, illumina). our index patient shows typical symptoms of the chromosome 17q23.1-q23.2 deletion syndrome like developmental retardation (in particular speech delay), a head circumference at the 3rd centile, postnatal growth retardation with a body height at the 4th centile, heart anomalies (right-sided aortic arch, patent ductus arteriosus), and sensorineural hearing loss on both sides. our data improve the characterization of the typical phenotype caused by a chromosome 17q23.1-q23.2 deletion and reinforce the suspicion that this region might be associated with sensorineural hearing loss. partial, homozygous deletions of ahi1 gene causes joubert syndrome type 3 d. meier, a. behnecke, jwg. janssen, u. moog, k. hinderhofer institute of human genetics, university hospital heidelberg, heidelberg, germany we describe a patient who is second child of consanguineous healthy parents from turkey. he was born after 37 weeks of gestation with normal birth parameters (2850 g, 51 cm, ofc 32 cm). at the age of three months atypical eye movements became apparent. psychomotor development was delayed from the beginning, he presented with hypotonia and later developed ataxia. an abnormal breathing pattern was not noticed. an abnormal mri with hypoplasia of the cerebella vermis at the age of 21 months and the clinical signs described above led to a clinical diagnosis of joubert syndrome. at that time no diagnostic testing was available. he returned to the outpatient clinic of the institute of human genetics at the age of 22 years having developed retinopathy in the meantime. his height was below average (~3 cm, <p3) but still within his family's range. aside from the known irregular eye movements he showed nail hypoplasia of the thumbs, and small hands and feet. he was working at a sheltered workshop. the year before, the pediatric department of the university hospital heidelberg had initiated testing of 7 of the by then known genes causative for joubert with no mutation being found (nphp1, tmem216, cc2d2a, mks3, rp-grip1l, ofd-1 and mks4). the gene associated with joubert syndrome type 3 (omim #608629) was not included though. with the external mri not being available at that time we decided to first perform chromosome and array analyseis. the conventional chromosome analysis was without pathological findings. subsequent array analysis by affymetrix® cytoscan hd oligo/snp showed an 11 kb homozygous deletion in 6q23.3 within the ahi1 gene, the gene for joubert syndrome type 3. the deletion encompasses exon 10 to 13 which includes parts of the wd-repeats (tryptophan-aspartic acid (w-d) dipeptide) on the protein level. the homozygous deletion was confirmed by mlpa in the patient and both parents identified as heterozygous carriers. so far, alterations in ahi1 leading to joubert syndrome comprise homozygous point mutations or small heterozygous deletion/duplications in combination with a second pathogenic mutation. so we here present the first case of a large homozygous deletion in the ahi1 gene, being causative for joubert syndrome type 3. we conclude that in un-a review of the literature showed that the few patients with a ssmc of the pericentric regions of chromosome 8 described in literature show early feeding problems, developmental delay, delay in acquired language, difficulties in social skills, difficulties in attention and activity levels or autistic-like behavior. the facial dysmorphic features were not specific. multiple hemangiomas have been reported in some cases. west syndrome or seizures have not been reported so far. the phenotype of our patient is consistent with the literature data, however there are no patients with a ssmc showing the same breakpoints reported so far. especially due to the mosaicism and the rare number of patients described in the literature, no precise clinical prognosis for this patient was possible. the generation of patient-specific induced pluripotent stem cells (ipscs) by reprogramming of adult somatic cells (e. g. skin fibroblasts) represents a novel technology for studying disease mechanisms. for generation of ipscs several reprogramming methods, including the use of integrating vectors and non-integrating vectors, have been developed. since genetic abnormalities can arise during the reprogramming process, genetic quality assurance is indispensable. the aim of this analysis was to compare the genetic stability of ipscs generated using either a dna-integrating or a dna-non-integrating reprogramming technique in order to reveal possible method-specific differences. we studied ipscs derived from patients with parkinson's disease (pd), the second most common neurodegenerative disease worldwide. to monitor the genetic stability, we investigated copy number variants (cnvs) in addition to conventional chromosome analyses in ipscs of ten pd patients and six healthy control individuals. using high-resolution chromosomal microarray analysis (cma), we monitored the formation of relevant cnvs during reprogramming to pluripotency by comparing fibroblasts and ip-scs of the respective individual. to date, fibroblast cultures of all 16 selected probands as well as 47 retroviral reprogrammed ipsc clones and eight sendai-viral reprogrammed ipsc clones were analyzed. aneuploidies were detected in three fibroblast cultures (19%), in five retroviral ipsc clones (11%), and in none of the sendai ipsc clones. de novo cnvs were identified in 33 retroviral ipscs (70%) -19 clones showed one (40%), nine clones showed two (19%), four clones showed three (9%), and one clone showed four newly arisen cnvs (2%). in seven of eight sendai clones, de novo cnvs were detected (88%). two de novo cnvs were identified in three clones (38%), three cnvs in two clones (25%), four in one clone (13%), and even six de novo cnvs were detected in one clone (13%). additionally, a mosaic gain of the whole long arm of one chromosome 9 was identified in one sendai clone. only 14 of the retroviral reprogrammed clones (30%) as well as one sendai reprogrammed clone (13%) showed no newly arisen cnvs. the cnvs were between 106 kb and 6.4 mb in length in retroviral clones and between 106 kb and 926 kb in sendai clones. because almost all cnvs contained genes, including genes involved in neurodevelopment and transcriptional regulation, a biological relevance seems highly probable and changes in cell phenotype cannot be excluded. since ipscs and cells derived thereof -independent of reprogramming methods -often contain aberrations not detected by standard chromosomal analysis, our findings highlight the importance of high resolution cma procedures. in spite of differences in the analyzed number of clones, our results indicate more and larger cnvs in sendai clones than in retroviral reprogrammed clones. supported by forips, bavarian research network induced pluripotent stem cells. abstracts mother's lymphocytes showed a balanced form of the aberrant karyotype found in her son. the healthy mother possesses the insertion ins(12;4) (q24.33;q24q28), the simultaneous resection of the 12q2?3-12q24.3 material and generation of the additional ring chromosome with this material. during meiosis a normal chromosome 4, the derivative chromosome 12 and the ring chromosome were passed on to the son leading to the unbalanced karyotype as described above. further analysis with pan-centromeric probes and immunohistochemistry with an antibody against cenp-a might confirm the existence of a neocentromere on the ring chromosome which was meiotically transmitted from the mother to the son. the mtor (mechanistic target of rapamycin) kinase is the most important regulator of local dendritic and presynaptic protein translation in the brain. it has been shown to fundamentally influence ampa receptor activity by shifting glua1 to glua2 balance and to control the synthesis of several proteins involved in synaptic function and plasticity. both, loss and gain of mtor activity lead to significant disturbances of brain homeostasis and neuronal function resulting in intellectual disability (id), epilepsy, autism and behavioural alterations. in order to analyse mechanisms of homeostatic regulation of mtor-dependent synaptic function we are using a heterozygous tsc2 knockout (ko) mouse model for tuberous sclerosis (ts). tsc2 (together with tsc1) is part of a complex that inhibits the mtor kinase. mutations in either of the two genes result in increased mtor activity and upregulated downstream signalling and are causative for autosomal dominant ts. it had been shown previously that mtor hyperactivity leads to a set point shift and significantly influences neuronal homeostasis by altering cell excitability and e/i balance in heterozygous tsc2ko animals. according to these studies we hypothesize that this shift significantly leads to synaptic connectivity and plasticity as well as network alternations that result in progressive development of cognitive dysfunction, epilepsy, autism and behavioural alterations over time in ts. we have established a behaviour battery, which consists of a novel object recognition test (nort), a morris water maze test (mwm) and an evaluation of nest building and social interaction to study social behaviour and cognitive performance in heterozygous tsc2ko mice in different age groups. interestingly we found that, while two months old animals do not show any alterations, three to four months old animals develop significant disturbances in social behaviour in the social interaction test and in nest building. furthermore, we could show significant changes in cognitive performance of seven to eight months old heterozygous tsc2ko mice in the nort compared to three to four months old heterozygous tsc2ko animals. these data show that the ts phenotype builds up on shifts of the e/i balance that develop at a very early stage. pharmacological intervention with e/i balance is a promising therapeutic strategy to prevent brain damage caused by congenital hyperexcitability and homeostatic dysfunction. clear cases of a joubert phenotype an array-cgh is recommended besides sequencing to clarify the molecular basis of the phenotype. recently, a new syndrome of intellectual disability (id) with dysmorphisms due to deletions within the chromosomal band 3q26.32 harboring the tbl1xr1 gene was described. additionally, a specific point mutation in tbl1xr1 causes pierpont syndrome, a distinct mental retardation syndrome. we report four patients in which array-cgh analysis and real-time quantitative pcr of genomic dna revealed tbl1xr1 microduplication. adjacent genes were not affected. the microduplication was verified as de novo by real-time pcr of parental dna in one patient. the other three cases occurred in two generations of a second, unrelated family. we compare the remarkably similar clinical findings in tbl1xr1 microdeletion, point mutation and microduplication cases and expand the tbl1xr1-associated phenotypic spectrum. among others, intellectual disability, hearing loss and autism spectrum disorders are common features of tbl1xr1-associated diseases. our clinical observations add to the increasing evidence of tbl1xr1's role in physiological brain development and simultaneously demonstrate that opposing genetic disease mechanisms affecting tbl1xr1 can lead to similar id phenotypes. in conclusion, the observed phenotypic overlap indicates that this novel microduplication is a genomic sister-disorder to the 3q26.32 microdeletion syndrome. neocentromere formation has been reported in more than 100 small supernumerary marker chromosomes (ssmc). here we report the formation of a putative neocentromere within a supernumerary ring chromosome derived from chromosome 12 in a five year old patient with generalized developmental delay, microcephaly and hexadactyly. chromosomal analysis (gtg-banding, resolution 550 bands) of the boy showed an aberrant karyotype with a derivative chromosome 12 demonstrating additional chromosome 4 specific material inserted into the distal part of the long arm (ins(12;4)(q24.33;q24q28)), a simultaneous deletion of the chromosomal region 12q2?3-q24.33 on the same homolog and an additional supernumerary small marker chromosome, presumably a ring chromosome in all metaphases tested. in parallel, array-cgh was performed and revealed a gain of 22.6 mb in chromosomal region 4q24-q28.1 without a detectable imbalance of chromosome 12 material within the limits of the method (400k array). fish analysis with wcp probes and telomeric probes for chromosomes 4 and 12 confirmed the interstitial insertion of chromosome 4 material into the long arm of the derivative chromosome 12. the marker chromosome stained completely with a wcp12 probe but centromere probes for chromosomes 4 and 12 were both negative suggesting the formation of a neocentromere within region 12q2?3q24.33. in total, the complex rearrangement resulted in a partial trisomy 4q in the boy as a presumable explanation for the phenotype. the mother has experienced one abortion and the parents have two more children who show a normal development so far. both parents were karyotyped. the analysis of the father was normal (46,xy). konventional chromosomal analysis of the in flvcr1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to hsan. using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the flvcr1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans. autosomal-dominant familial hypercholesterolemia (fh, omim 143890) is one of the most common genetic diseases with an estimated prevalence of 1: 200 to 1: 500. three major genes have been associated with the disease: low density lipoprotein receptor (ldlr), apolipoprotein b (apob) and proprotein convertase subtilisin/kexin 9 (pcsk9). we have recently published data on the mutation spectrum in these three genes in about 200 fh patients from germany, however, this screen for mutations was negative in about 40% of the cases. stap1 (signal transducing adaptor family member 1) was recently suggested to be the fourth fh gene. we therefore screened the stap1 gene for disease causing variants in a group of 70 fh patients negative for mutations in the three established fh genes by direct dna sequencing (sanger). the newly identified variants will be presented including a phenotype-genotype correlation. phenotype: we present a 13-year-old girl with developmental delay, craniofacial anomalies, congenital eye anomalies (4.5 dpt), short stature and behavioral disorder with auto-aggressions. preliminary examinations (array-cgh analysis, karyotyping, diagnostic of metabolism, eeg, cmrt, sequencing of srcap-gene) revealed normal results. methods: in course of the midas-project, we sequenced the index patient and her healthy, non-consanguineous parents on a nextseq500 platform (illumina, san diego, ca, usa) to perform a trio analysis. for library preparation we used an enzymatic fragmentation approach. exome capture was performed with a sureselect human all exon kit v6 (agilent, santa clara, ca, usa), targeting coding exons and conserved splicing sites of over 20.000 genes. the library of the index patient was sequenced with a 285-fold mean coverage as 151-bp paired-end reads. 94% of the target region were covered 20-fold or higher. data analysis and variant evaluation was done using the clc genomic workbench 9.0 (qiagen, hilden, germany) and annotations from commercial as well as public databases. results: the trio analysis of whole-exome sequencing data from the index patient and her parents revealed 58 variants. we identified the disease-causing de novo adnp mutation c.2188c>t (p.arg730*, adnp), resulting in the introduction of a stop codon in exon 5/5 and truncation of the corresponding protein. both of the parents did not carry this mutation. adnp is part of the atp-dependent baf chromatin-remodeling p-monog-137 the challenge to insert costello syndrome causing hras mutations into human keratinocytes using the crispr/cas9 editing technology. l. brandenstein, k. kutsche, g. rosenberger university medical center hamburg-eppendorf, hamburg, germany germline missense mutations in the hras gene cause costello syndrome, a rare developmental disorder characterized by a typical facial gestalt, postnatal growth deficiency, intellectual disability, and predisposition to malignancies as well as skeletal, cardiac and dermatological abnormalities. the molecular pathophysiology caused by heterozygous hras gain of function mutations has been analysed in various tissues and cell types, however, up to date the molecular basis for cutaneous manifestations in costello syndrome is largely unknown. to address this question in an appropriate model system, permanent human keratinocyte (hacat) cells carrying costello syndrome-associated mutations in the endogenous hras gene should be generated by using the crispr/cas9 technology. double strand breaks induced by cas9 can be repaired in two ways: the error-prone non-homologous end-joining pathway for the generation of knockout models or the homology directed repair pathway, which allows precise editing. the latter enables the introduction of specific point mutations into a cell line by using a single stranded dna (ssdna) as repair template. however, we found that this is a very rare event and its efficiency depends on various factors including used cell line, selected guide rna, length and amount of ssdna and also cas9 variant. nonetheless, by using cas9 wildtype, we could insert the disease-associated c.35g>t (p.g12v) mutation into genomic hras both in hacat cells (8 positive clones out of 800) and hek cells (3 positive clones out of 15). in contrast, using the cas9 nickase protein variant that prevents off target effects, did not result in positive clones. taken together, in 10 months of working with crispr/ cas9 we gradually gained experience with many problems and pitfalls of this technology and, finally, now we are able to introduce point mutations in cell lines. next, we will use the mutant hacat cell line to gain deeper insight into the function of hras for epidermal homeostasis and its deregulation in costello syndrome. ccdc66 could also play a role in prenatal development of the mouse retina and brain. embryonic stages of interest comprise <e10 at the initial development of the eye, e13 after formation of nasal pits and prominent brain growth, when brain vesicles are initially visible and e18 where the eye and nasal brain develops. this study aims at characterizing ccdc66 rna and ccdc66 protein expression during key steps of prenatal development in the wt mouse and respective ccdc66 reporter gene expression in the ccdc66 -/model. methods: ccdc66 rna expression was analyzed by quantitative ccdc66 qrt-pcr and ccdc66 protein expression was determined by sds page and western blot in eye and whole brain homogenates of the mouse at selected embryonic stages (<e10, e13 and e18). in addition, 15 µm sagittal cryosections of mouse embryos were hematoxylin and eosin stained and analyzed for ccdc66 reporter gene expression by x-gal staining or antibody staining followed by light or fluorescence microscopy. results: ccdc66 rna and ccdc66 protein is detected in the wt mouse at different developmental time points in the eye and brain tissue. accordingly, ccdc66 -/reporter gene expression is evident at prenatal developmental stages in cryosections of whole embryos. conclusion: expression studies of ccdc66 gene and products in the eye and brain in wt and ccdc66 -/mice in different developmental embryonic stages are of crucial interest, and the role of ccdc66 gene and products in prenatal development has to be evaluated in detail in further studies. nn. hauer, b. popp, e. schoeller, ab. ekici, a. reis, ct. thiel institute of human genetics friedrich-alexander-universität erlangen-nürnberg, erlangen, germany the application of next generation sequencing technologies (ngs) has been extremely successful in the identification of the underlying causes of genetic disorders. whereas, most variants are located in coding regions, variants within splice-sites that might lead to alternative splicing affecting protein function or causing mrna decay are less well characterized. even for canonical splice-sites, prediction of the splicing effects for an identified variant is cumbersome. this becomes even more apparent as ngs based exome and genome sequencing tremendously increase the number of variants potentially affecting splice-sites. in our study to identify the underlying causes of idiopathic short stature by whole exome sequencing in more than 200 patients, we observed 44 variants in consensus splice-sites defined as up to 12 bases from the 5' and 5 bases from the 3' end of an exon. we used several computational methods (splice site finder, maxentscan, nnsplice) to calculate their potential effect on splicing. these results were often inconclusive and did not predict the definite effect on transcripts. therefore we performed rtpcr amplification of the respective regions, electrophoretic evaluation of fragments and sanger sequencing. in case of a suspected alternative splicing the evaluation of the distinct resulting isoform, though, was often hampered by the overlap of sequences. this was especially severe when multiple naturally occurring alternative transcripts were present. to overcome these issues, we established a deep sequencing approach (targeted rtpcr-seq) to identify all present isoforms in the affected patients compared to controls. for this method the 44 rtpcr fragments were normalized to generate an equimolar mixture. libraries for patients and controls, respectively, were generated by ligation of distinct index adaptors for both amplicon pools and subsequently sequenced on an illumina miseq platform. the resulting reads were aligned and visualized as sashimi plots. observed splice-junctions in patients were compared to the controls and public databases. we found concordant results between standard rtpcr evaluation and our rtpcr-seq approach for 40 variants (alternative splicing for 8 variants). for these confirmed variants the specific effect was more obviously ascertained by the ngs based approach. for 1 variant only rtpcr-seq was able to separate multiple co-occurring distinct isoforms, both in controls complex, which is involved in the regulation of gene expression. an influence on neuronal cell differentiation is postulated. mutations in the adnp gene were previously reported in 10 patients with autism and mild to severe intellectual disability. all reported adnp mutations are characterized by de novo mutations that are clustering in exon 5 and severely disrupting the protein-coding sequence, either by the introduction of a stop codon or a frameshift induction. by additionally considering the clinical symptoms, we were able to diagnose the helsmoortel-van der aa-syndrome (hvdas; omim id: 615873). discussion: the challenge of next-generation sequencing (ngs) approaches in diagnostics is no longer the technical feasibility or costs but the medical interpretation of the large sequencing data sets. besides a robust genetic analysis with focus on disease-associated genes, family anamnesis and detailed information of clinical symptoms in a standardized scheme are important for a reliable diagnosis. here, we present one of over 50 cases that will help to establish the multiple integration of data annotation software (midas), which will enable phenotype-genotype-correlations to enhance the validity of results of genetic analysis, minimize the risk of misinterpretation and enable faster diagnosis in routine patient care. peripheral neuropathies are a group of clinically and genetically heterogeneous disorders, mutations in a large number of genes have been described so far. among patients with charcot marie tooth disease (cmt)/hereditary motor and sensory neuropathies (hmsn), a common duplication of the pmp22 gene on chromosome 17p12 is by far the most frequent mutation; sequence variants in the genes gjb1, mpz and mfn2 account for a considerable proportion of patients but all other associated genetic variants are extremely rare. deletions of the pmp22 gene lead to hereditary neuropathy with liability to pressure palsies (hnpp) but transition between the different phenotypes may be rather fluid in some instances. next generation sequencing (ngs) has revolutionized cmt diagnostics as a large number of genes can be investigated at a time but it also leads to challenging questions. use of ngs has certainly improved the diagnostic yield in cmt diagnostics and it has repeatedly resulted in unexpected findings. broad molecular testing has already disclosed overlaps between clinical conditions and more surprising results are yet to come. here we report our recent experiences with the diagnostics of neuropathies. our results, different testing strategies, implications for clinical management and genetic counselling will be discussed and possible further steps suggested. (gerding et al., hum mol genet., 2011) . retinal ccdc66 protein expression in the wildtype (wt) mouse demonstrates highest levels shortly after birth. moreover, ccdc66 protein can also be detected early postnatally in mouse brain tissue. therefore, there is strong evidence that p-monog-145 *** individuals with hereditary trichilemmal cysts present a combination of an inherited high-risk plcd1 allele (including two sequence variants) and additionally acquired somatic plcd1 variants in the cysts. trichilemmal cysts are benign tumors derived from the outer root sheath of the hair follicle. they occur most often solitary or multiple on the scalp but can also be found on other body parts. the cysts can develop sporadically or in an autosomal dominant mode of inheritance with incomplete penetrance. in 2005, the hereditary cyst form was mapped to the tricy1 locus on chromosome 3 p24-p21.2 in a familial genome-wide linkage study. we analyzed 5 affected individuals from 4 different tunisian families by whole-exome sequencing. the bioinformatic evaluation revealed no rare, disease causing mutations but the sequence variant c.1379c>t, p.s460l (maf = 0.025, rs75495843, enst00000334661.4) in the phospholipase c delta 1 (plcd1) gene within the tricy1 locus. furthermore, all five individuals present a second variant c.903a>g, p.p301p (maf = 0.27, rs9857730) in the same gene. plcd1 is a member of the phospholipase c family. the enzyme is involved in calcium-dependent intracellular signal transduction and catalyzes the hydrolysis of phosphatidylinositol 4, 5-bisphosphate into the second messenger diacylglycerol and inositol triphosphate. homozygous knockout-mice have hair defects and show aberrant skin development with increased progression of skin tumors and intradermal hair-follicle derived cysts. in humans, plcd1 is highly expressed in the hair follicle but so far only nonsense mutations have been described causing hereditary leukonychia totalis without any skin or hair abnormalities. a segregation analysis of plcd1 in a tunisian family cohort and one german family (13 families, 64 individuals, 38 affected) showed that all affected individuals contain the same two sequence variants. based on these results, we propose that plcd1 is responsible for the cyst phenotype. cdna sequencing from three different cysts revealed additional acquired somatic sequence variants in plcd1. we found c.2234c>t p.s745l in two cysts and the two variants c.2129c>t, p.s710f and c.2132c>t p.s711f in the third one. all three somatic variants are not described in the exac database and the 1000 genomes project. allele-specific rt-pcrs were performed with cyst cdna and we could show that the somatic variants are on the same allele as the inherited variants. the acquired somatic cyst sequence variants lie within or respectively near the c2 domain of the plcd1 protein. the c2 domain is involved in the calcium-dependent binding to membrane-integrated phospholipids. depletion of the domain leads to decreased membrane association and protein activity. we assume that the allele with the variants c.1379c>t and c.903a>g is a risk factor for hereditary trichilemmal cysts and that additional acquired rare sequence variants are the genetic trigger for the development of the cysts. and patients. discordant results for 4 variants proposed a higher specificity of the rtpcr-seq. thus, we established a simple amplicon based deep sequencing approach for standard rtpcr fragments to ascertain the effects of specific splice site variants. this technique has proven to be highly scalable, fast and efficient to analyze splice-site variants. p-monog-144 eif2s3 mutations associated with severe x -linked intellectual disability syndrome mehmo mehmo (mim %300148), is a rare x-linked syndrome characterized by profound intellectual disability, epileptic seizures, hypogonadism, hypogenitalism, microcephaly, and obesity. in 1998 steinmüller and colleagues described a large family with mehmo syndrome with five affected males in two generations and assigned the disease locus to the short arm of chromosome x (xp11. 3-22.13 ). we took advantage of massively parallel sequencing in four families with mehmo syndrome, including the family reported by steinmüller et al. to identify the underlying genetic cause if this severe disorder. we here show mehmo syndrome is associated with mutations in the x chromosome gene eif2s3. in three families we identified a c-terminal frameshift mutation (p.ile465serfs) and in an unrelated boy who is less severely affected, we identified a novel maternally inherited missense mutation (p.ser108arg) in eif2s3. eif2s3 encodes the gamma subunit of the eukaryotic translation initiation factor 2 (eif2). eif2 is essential for eukaryotic translation initiation and regulation of the integrated stress response (isr). subsequent studies in patient fibroblasts (p.ile465serfs) showed increased isr activation due to the mutation and functional assays in yeast demonstrated that the p.ile465serfs mutation impairs eif2 gamma function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the affected males with ile465serfs mutation. our results suggest that more severe mutations in eif2s3 cause the full mehmo syndrome, while less deleterious mutations are associated with a milder form of the syndrome with only a subset of the symptoms. introduction: larger structural genomic duplications or deletions (copy number variations = cnvs) are routinely detected by array comparative genomic hybridization (acgh). while acgh has been established as a robust and effective approach for cnv screening, it remains expensive and is limited by resolution. in addition, commercial mlpa testing today allows the identification of exon deletions or duplications for a limited number of core genes. more recently, massive parallel sequencing of multi gene panels (mgps) has been introduced as a fast and cost-effective tool in routine genetic diagnostic testing to identify causal intragenic sequence alterations not only in core genes but also in those with small contribution to the respective phenotypes. the obtained mgps data may also be bioinformatically assessed to detect exon deletions or duplications within the analyzed genes panels and thus can be expected to further improve the diagnostic yield. methods: more than 100 patients were sequenced with phenotype specific gene panels on a miseq platform (illumina) and analyzed with our bioinformatic diagnostic workflow including a quantitative data assessment using our in house java based bioinformatic script to search for gene or exon deletions. detected deletions were confirmed by an independent method (e. g. mlpa, pcr amplification of the junction fragment or linkage analysis), if available. results: we here report details for six suspected deletions in seven patients detected by our in house bioinformatic workflow: heterozygous gene deletions of pafah1b1, spastin or arfgef2, respectively; two heterozygous cftr deletions of exon 2 and 3, one homozygous partial sftpb deletion and a homozygous ispd exon deletion. the complete gene deletions of pa-fah1b1 and spastin as well as the cftr deletion of exon 2 and 3 could be confirmed by mlpa. for the partial sftpb deletion both breakpoints could be precisely located within the readout, allowing determining correct deletion size and design of primers to amplify the junction fragment. the homozygous deletion of exon 6 in the ispd gene could be confirmed by pcr and linkage analysis. discussion: diagnostic multi gene panel sequencing after nextera enrichment allows sufficient homogeneity of the obtained patient and control data per target to quantitatively search for constitutional deletions covering two or more adjacent targets. combined data assessment considering the individual clinical data will not only further increase the diagnostic yield but can also be expected to further delineate the mutational spectrum for specific phenotypes by the simultaneous detection of clinically relevant sequence variants as well as cnvs. mgps sequence assessment may also allow to gain new insights into the genomic architecture and origin of target regions and haplotypes, involved in common structural variations. c. niemietz, v. sauer, l. fleischhauer, s. guttmann, s. reinartz groba, a. zibert, h. schmidt universitätsklinikum münster, klinik für transplantationsmedizin, münster, germany various types of somatic cells have been reprogrammed to induced pluripotent stem cells (ipsc) followed by differentiation into hepatocyte-like cells (hlc). recently, cells that shed from the renal epithelial system were shown to be a suitable and convenient source for ipsc generation. in the current study, urine-derived cells (ucs) were isolated from urine donations of patients having familial amyloid polyneuropathy (fap), a neurodegenerative disease caused by mutation of the transthyretin (ttr) gene and wilson disease (wd), a genetic disorder of atp7b causing copper accumulation, predominantly in liver and brain. patient-specific hlcs were differentiated in order to study disease-specific mechanisms and to investigate the efficacy of novel compounds. for isolation of renal epithelial cells, urine of fap and wd patients was processed. ucs were reprogramed into ipscs using plasmids resulting in transient expression of factors sox2, oct3/4, klf4, and c-myc. after characterization of ipsc that expressed high levels of pluripotency markers, like oct4 and nanog, a 3-step hepatocyte differentiation protocol was performed. ipscs were subjected to a treatment with growth factors (activin a, wnt3a, fgf2, hgf) for 14 days. the hepatic, patient-specific character of differentiated hlcs was assessed by functional analysis, gene expression profiling, genotype analysis, and immunostainings. therapeutic oligonucleotide efficacy targeting ttr was determined by immunocytochemistry, qrt-pcr and western blot analysis. ttr-stabilizing activity of tafamidis was investigated by means of thermal shift assay and western blot analysis. copper chelation by methanobactin was determined by atomic absorption spectroscopy. reprogramming of ucs resulted in stable ipsc lines with characteristic pluripotent marker expression. differentiated hlcs showed high similarity to human hepatocytes in terms of genetic profile and functional activity. small-interfering rnas (sirnas), antisense oligonucleotides (aso) (niemietz et al. 2016, plosone 11(9) :e0161455), and the ttr stabilizing compound tafamidis that are currently assessed in clinical studies were studied in hlcs derived from fap patients. a novel chelator was used to determine intracellular copper accumulation in hlcs derived from wd patients (lichtmannegger et al. 2016, jci 126 (7):2721-35). fap-specific hlcs revealed differently expression of key regulators of the protein quality control (pqc) system. our results demonstrate that ipsc derived from urine are excellently suited to study hereditary liver diseases. hlcs could be investigated in the patient-specific genetic background. the efficacy of novel compounds was assessed and individual responses were monitored. involved in gene regulation. by parent-patient trio whole-exome sequencing, we were able to characterize the unterlying genetic cause in the patient, who has undergone multiple diangostic test before without receiving a diagnosis. discussion: although for many congenital syndromic diseases the disease-associated genes are known it remains difficult for physicians to interpret the highly variable phenotypes as well as their variable nomenclature in order to request the appropriate molecular analysis. here we present one of more than 50 cases that will help to establish a database connecting specific phenotypes, using the human phenotype ontology terms, with the each corresponding disease-causing genes (midas). novel compound heterozygous nalcn variants in two brothers with muscular hypotonia and global development delay l. segebrecht 1 , c. weiß 2 , m. jäger 1,3 , t. zemojtel 1, 4, 5 , d. horn 1 , n. ehmke 1, 6 1 institute of medical and human genetics, charité -universitätsmedizin berlin, berlin, germany, 2 spz dpt. pediatric neurology, charité -universitätsmedizin berlin, berlin, germany, 3 berlin-brandenburg center for regenerative therapies -bcrt, charité -universitätsmedizin berlin, berlin, germany, 4 institute of bioorganic chemistry, polish academy of sciences, poznań, poland, 5 labor berlin -charité vivantes gmbh, berlin, germany, 6 berlin institute of health, berlin, germany we report on two brothers aged two and three years, with muscular hypotonia, global development delay, abnormal respiratory rhythm, mild facial dysmorphism, recurrent respiratory infections, and failure to thrive. sequencing of 3089 disease related genes identified compound heterozygosity for two novel mutations in nalcn: c.4281 c>a (p.phe1427leu) and c.4103 + 2 t>c. nalcn encodes a voltage-independent, non-selective cation channel, which is involved in regulation of neuronal excitability. the missense variant c.4281 c>a affects the highly conserved amino acid position phe1427 which is located in segment s6 of domain iv in the pore-forming unit of nalcn. the variant c.4103 + 2 t>c alters the donor splice site in intron 36 and is predicted to cause skipping of exon 36, resulting in loss of function of nalcn. biallelic mutations of nalcn are associated with infantile hypotonia, psychomotor retardation and characteristic facies (ihprf1), whereas heterozygous de novo mutations cause congenital contractures of the limbs and face, muscular hypotonia, and global developmental delay. the clinical features of our patients resemble mild ihprf1, caused by a biallelic missense mutation in segment s3 of domain iv. it has been suggested that variants in or close to s5 and s6 of the pore-forming domains lead to the above mentioned autosomal dominant condition whereas variants in other regions or loss of function mutations result in autosomal recessive inheritance. this is the first report of a mutation in a s6 segment, inherited in autosomal recessive manner. our findings indicate that phenotype-genotype correlations in nalcn are more complex than suggested so far. phenotype: in a young undiagnosed patient with developmental delay, intellectual disability and craniofacial dysmorphic anomalies, whole-exome sequencing (wes) identified a de novo insertion in the gene arid1b, known to cause the rare congenital coffin-siris syndrome and nicolaides-baraitser syndrome, respectively. methods: as part of the midas genotype-phenotype-correlation project, we sequenced the index patient and his healthy parents on a next-seq500 platform (illumina, san diego, ca, usa) and performed a trio analysis. for library preparation we used an enzymatic fragmentation approach. exome capture was performed using the sureselect human all exon kit v6 (agilent, santa clara, ca, usa) to target most of the over 20.000 genes. the libraries were sequenced to approximately 190-fold mean coverage as 151bp paired end reads. 92% of the target region was covered 20-fold or higher. data analysis and variant evaluation was performed using the clc genomic workbench 9.0 (qiagen, hilden, germany) and annotations from commercial as well as public databases (dbsnp, hgmd, clinvar, exac). results: we identified a de novo 1bp insertion in the arid1b gene, causing a frameshift mutation that leads to the truncated protein. arid1b is part of the atp-dependent chromatin remodeling baf-complex, which is abstracts tient affected by psoriasis carried the surrogate marker snp rs4406237-a for the psors1 risk variant hla-cw 0602 haplotype homozygously; and the same snp was found heterozygous in his prp-affected father. neither the pathogenic variant in card14, nor the risk variants for psoriasis described above were found in the healthy mother. whole exome sequencing revealed genetic variants, predicted to have serious consequences in further genes involved in the nf-κb as well as the notch pathway. these variants either segregate with prp or are present in the psoriasis affected individual only. the presence of an individual carrying the same card14 mutation as his prp-affected relatives but suffering from psoriasis instead strengthens the relation between prp and psoriasis, which has been repeatedly suggested in literature. we propose a balance between familial prp and psoriasis in the family investigated in this study and present genetic variants, which might influence this balance in addition to variants in card14. wilson's disease (wd) is an autosomal recessive disease resulting from copper (cu) excess due to mutations in the atp7b gene coding for a cu-transporting atpase. wd pathogenesis, however, can not only be explained by gene coding mutations since phenotypes exhibit strong variations despite the same exonic dna makeup in the gene. also in several patients with clinical wd symptoms no gene coding variants are detectable. our former studies revealed decreased liver atp7b mrna expression in some wd patients. this decrease was not only observed in patients with nonsense atp7b mutations leading to rapid mrna decay, but also in patients with missense mutations and also in some patients with suspected wd without atp7b mutations. patients with low atp7b expression presented with a more fulminant disease progression. however, we could not detect mutations in the atp7b promoter region (c.-900 to atg) in those patients. there are possibly other deregulating mechanisms responsible for decreased atp7b mrna expression. up to now, atp7b transcriptional regulation is only poorly characterized. it is known, that four metal responsive elements (mre a, c, d and e) are located within the atp7b promoter. gene regulation through mres is often metal-dependent. liver atp7b mrna expression revealed also to increase under cu addition in several species by an unknown mechanism. up to now, only one atp7b transcription factor (tf), the mrea binding ku protein, is known. the aim of our work was to further analyze the regulation of the atp7b gene, especially through mres. to screen for tf-mre interactions and to narrow down the binding site of tf, we performed electrophoretic mobility shift assays (emsa) by incubating nuclear extracts of the liver cell line hle with probes corresponding to atp7b mrec, d and e. to identify mre-binding tf matinspector analysis was performed. identified candidate tf were coexpressed with atp7b promoter-driven reporter gene to evaluate their impact on reporter gene expression. one in the reporter assay positively tested tf was validated by different emsa experiments. further it was overexpressed with and without addition of metal ions in hle to investigate the impact on endogenous atp7b expression. we showed that tf mtf1 is able to bind to mree within the atp7b promoter and significantly increases atp7b promoter driven reporter gene expression. mtf1 binding was primarily mediated by the first three bases of the mre consensus sequence. also for mrec and mred specific protein interaction could be shown and the protein binding site was narrowed down by emsa with protein identification still pending. fur-polyglutamine diseases is the formation of the so called neuronal intranuclear inclusion bodies (nii). as ataxin-3 is predominantly located in the cytoplasm, the formation of protein aggregates in the nucleus require a nucleocytoplasmic shuttling of ataxin-3. we already demonstrated in vivo using transgenic mouse models that the toxicity of expanded ataxin-3 depends on its intracellular localization: while nuclear ataxin-3 gave rise to a strong phenotype with a high number of protein aggregates, purely cytoplasmic ataxin 3, however, even with a highly expanded polyglutamine repeat (148 glutamines), was not able to induce a phenotype and even did not aggregate. we further identified and characterized intracellular transport signals (two nuclear export signals, nes, and one nuclear localization signal, nls) within the coding sequence of ataxin-3. therefore, it is evident that proteins involved in the nucleocytoplasmic transport machinery recognize these localization signals, control the intracellular localization of ataxin-3, thereby influence the toxicity and aggregation of ataxin-3 and, thus, the pathogenesis of sca3. we now screened a library of transport proteins in order to identify the transport protein which is critically involved in the nucleocytoplasmic shuttling of ataxin-3. we indeed identified a transport protein which modifies both the formation of aggregates and the intracellular localization of ataxin 3. while the overexpression of this protein moved ataxin-3 into the nucleus, its downregulation kept it out of the nucleus. we replicated this correlation in vivo in drosophila and observed in addition to this again a clear link between the intracellular localization of ataxin-3 and its toxicity i. e. its ability of induce neurodegeneration and a behavioral phenotype. likewise we even confirmed in a mouse model of sca3 the importance of the identified transport protein as its knockout largely prevented ataxin-3 from aggregating and alleviated behavioral and movement deficits. understanding the mechanisms behind the intracellular transport of ataxin-3 could give us clues into the pathogenic functions of expanded ataxin-3 and ways to mediate the progression of neuronal degeneration in sca3. evidence for genetic factors outside card14 influencing the phenotype of a family with familial pityriasis rubra pilaris and psoriasis familial pityriasis rubra pilaris (prp) is an erythematous inflammatory skin disease caused by heterozygous activating mutations in card14, a known activator of the nf-κb pathway. different genetic variants within card14 have been associated with psoriasis. the purpose of our study was to clinically and genetically investigate affected as well as unaffected members of a family with prp in order to determine the mutation responsible for this severe skin disease in the three affected family members. a father, three of his adult children as well as the mother of one child affected by prp were investigated clinically. in addition we extracted genomic dna from the blood of each individual and performed whole exome sequencing as well as direct sequencing of single genes. clinical investigation confirmed that the father and two of his children were affected by familial prp, with the skin showing the characteristic pattern of prp, early onset and chronic course. a third child was unaffected by prp, suffered however from psoriasis. the mother of one child affected by prp showed no sign of skin disease. genetic investigation revealed a heterozygous missense mutation in exon 4 of card14, c.[371c>t], p. present in all investigated individuals with prp or psoriasis. the same mutation has been described before as being pathogenic in a different family with prp. regarding genetic variants associated with psoriasis, we found the risk alleles of three coding variants in card14: rs2066964, c. short stature is a common condition of great concern to patients and their families. in most cases it is genetic in origin but the underlying cause often remains elusive due to clinical and genetic heterogeneity. in an unbiased approach we carefully phenotyped 565 patients and randomly selected 200 for whole exome sequencing. sequence variants were analyzed for pathogenicity and the affected genes characterized regarding their functional relevance for growth. all patients received extensive clinical and endocrinological examinations, careful clinical genetic phenotypic evaluation followed by targeted diagnostic assessment for suspected diagnoses. we identified a known disease-cause in only 14% of patients, the most common causes being cnvs found in 7%, followed by syndromic monogenic causes in 5% and turner syndrome in 2%. whole exome sequencing identified additional mutations in known short stature associated genes (27) in 17% of patients who manifested only part of the symptomatology precluding an early clinical diagnosis. here, heterozygous carriers of recessive skeletal dysplasia alleles (acan, npr2) were a surprisingly frequent cause of idiopathic short stature found in 3.5% of cases. we next selected known short stature genes with mutations for pathway analyses of the affected proteins and found that 54% are involved in the main functional categories cartilage formation, chromatin modification and ras-mapk signaling. in addition we identified 37 further strong candidate genes, of which seven had deleterious mutations in at least two families. interestingly, 48% of these candidate genes are involved in the 10 main functional categories already identified for the known short stature associated genes further supporting their pathogenicity. finally, in 16% of the 200 sequenced individuals our findings were of significant clinical relevance regarding preventive measures, symptomatic or even targeted treatment. besides evaluation for orthopedic or developmental issues especially screening for neoplasias (trim37, ptpn11, nf1), symptomatic treatment for chronic kidney disease (clcn5) and targeted treatment for severe hypertension (pde3a) were of clinical relevance for the affected individuals. these results demonstrated that deep phenotyping combined with targeted genetic testing and whole exome sequencing is able to increase the diagnostic yield in short stature up to 31% with concomitant improvement in treatment and prevention. rigorous variant analysis considering phenotypic data further led us to the identification of further 37 probable novel candidate genes. thermore, we found the endogenous atp7b mrna expression to be significantly increased in hle after cu treatment or cu treatment and concurrent mtf1 overexpression. sole mtf1 overexpression did not alter atp7b expression. we newly identified mtf1 to bind mree within the atp7b promoter. its in vivo role in the pathogenesis of wd needs to be further elucidated. modifying genes have been identified for lung function in cystic fibrosis [1] , disease severity [2] and several comorbidities [3] . within the european cf twin and sibling study, we focus on genes that modify the basic defect, assessed as defective chloride conductance in cftr-expressing epithelia, which we could describe by an association study on patient cohorts selected for informative endophenotypes among f508del-cftr homozygous patients [2, 4, 5] . as a first example, rs7901656 in fas modifies fas gene expression (p = 0.0009, data from 16 intestinal biopsies [6] ), cf disease phenotype (praw = 0.0039, comparing 13 concordant mildly affected f508del homozygous sib pairs and 12 concordant severely affected sib pairs; praw = 0.0047, comparing 20 unrelated f508del homozygous index cases without residual chloride secretion by nasal potential difference measurement (npd) to 13 patients with cftr-mediated chloride secretion; [2] ) and alters binding affinity for the transcription factors nf-kbp50, nf-kbp65 and hif1a [7] which govern the cellular response to infection and hypoxia. as a second example, the transcription factor ehf, derived as a positional candidate from a north american genome wide scan [1] , is associated with 2 npd-defined phenotypes (praw = 0.0082, comparing 17 index cases with high response to amiloride in npd to 13 index cases with low response to amiloride in npd; praw = 0.0268, comparing 14 index cases without chloride secretion in intestinal current measurement (icm) to 9 index cases with cftr-mediated residual chloride secretion in icm [5] ) and affects the transcriptome of cf patients' intestinal biopsies in favor of a better processing of f508del-cftr [5] which we could confirm in epithelial cell lines as sirna provided against ehf results in a downregulation of mgat2 and mgat4, both of which are key enzymes for the complex glycosylation of proteins such as cftr. these examples indicate that small, albeit carefully selected subpopulations facilitate the identification of genetic variants by an association study that can be validated in functional assays. furthermore, we suggest that while the selection of subsamples within a population with a rare disease such as cystic fibrosis results in a loss of power, findings obtained for more than one endophenotype are indicative for a true-positive finding of a modifying gene. finally, transcriptional regulation influences the cf basic defect. interference with these pathways may results in better f508del-cftr maturation, leading to better cftr function in patient's tissue and thereby promoting health in cystic fibrosis. funding by: deutsches zentrum für lungenforschung dzl; mukoviszidose institut ggmbh abstracts at presentation at age 21 years her head circumference was on the 75th centile, her height below the 3rd centile, and her weight on the 3rd centile. she showed a disproportional short stature and mild skeletal signs like clinodactyly of the 5ths fingers. minor facial dysmorphisms included an oval face, epicanthic folds, upslanting palpebral fissures and a bulbous nose. karyotype was normal and copy number variants were excluded by high-resolution cma. as no aetiological diagnosis could be made clinically we performed whole exome sequencing (wes) and detected a homozygous splice site mutation (c.1640 + 1 g>t) in pik3c2a (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha) (nm_002645). sequencing of rt-pcr products from cdna of patient's fibroblasts showed in-frame skipping of exon 5 and 6, equally affecting all known isoforms. phosphatidylinositol 3-kinases (pi3ks) are lipid kinases involved in a large set of biological processes, including membrane receptor signaling, cytoskeletal organization, and endocytic trafficking. pi3kc2a is ubiquitously expressed and has been proposed to play an important role in clathrin-mediated endocytosis and regulation of phosphatidylinositol 3-phosphate (ptdins3p) levels. furthermore pik3c2a has been implicated in the biology of the primary cilium. the patient's distinct phenotype resembles the previously described phenotype in pik3c2a hypomorphic mice with pre-and postnatal growth retardation and a broad spectrum of renal abnormalities. the complete knockout of mouse pik3c2a showed embryonic lethality. ongoing studies on the exact consequences of the splicing defect will determine if and how much residual wild-type transcript is retained and if this is a hypomorphic variant. this case is to our knowledge the first description of a pik3c2a human phenotype. adaption of the crispr/cas genome editing system as a platform for the mutation of nipal4 as a representative of arci-associated genes in hela cells n. ballin, m-a. rauschendorf, j. fischer institute of human genetics, freiburg, germany autosomal recessive congenital ichthyosis (arci) is a rare genetic disorder with known disease-causing mutations in 9 genes. functional implementations of identified mutations are in most cases still unknown, which is amongst others due to the limited amount of skin biopsies of arci-patients. thus, suitable cell culture models for the investigation of keratinocyte differentiation are highly needed. during the last years the prokaryotic clustered regularly interspaced short palindromic repeats (crispr)/crispr-associated (cas) system has been turned into a potent tool in the field of genome engineering. the cas9 endonuclease is directed by a short guide rna (grna) to its target sequence, where it generates a dna double strand break (dsb). as cellular repair mechanisms often fail in reconstituting the original sequence, insertion/ deletion (indel) mutations occur potentially leading to a complete gene knock-out (ko). consequently, the crispr/cas system offers a simple rna-programmable tool for in vitro mutation of arci-associated genes. hence, this system was applied in hela cells to target nipa like domain containing 4 (nipal4), the second most frequently mutated gene in arci patients. in this context, functional studies were used to validate nipal4 as suitable target in hela cells. successful application of the crispr/cas system lead to the generation of a new clonal hela cell line carrying a one basepair insertion in nipal4 exon 1 (c.106insg (ccds 47328.1) ). this mutation was further characterized and potentially results in a complete ko of nipal4. this system can now be used as a basis for targeting further arci-associated genes and for transferring the system into keratinocytes, which are the amyotrophic lateral sclerosis (als) is a late-onset progressive, neurodegenerative syndrome. most als cases are sporadic (≈90%). in familial forms, mutations in several different genes have been identified with a repeat expansion in c9orf72 and mutations in sod1 being the most prevalent. no non-genetic cause of als has been identified. since there are no overt clinical or pathoanatomical differences between sporadic or familial cases, de novo mutations have been suggests in disease pathogenesis and two previous studies provided some evidence for this hypothesis. we present data of 82 patient-parents trios from an international collaborative study. by whole exome sequencing we identified 69 non-synonymous de novo mutations in the patients, however all of them occurred in different genes. there was no concordance between the mutated genes found in our trio set and the two earlier smaller trio studies. in silico analyses suggest that none of the here identified mutations are part of any of the previously postulated molecular pathways. also, gene-gene-interaction analyses failed to find an enrichment of interacting genes. lastly, we demonstrate that the de novo mutations in als patients in this and the two earlier studies are located in genes prone for de novo mutations in general. our results thus indicate that, in contrast to previous reports, de novo mutations do not seem to be a major contributor for als. disproportioned short stature and multiple anomalies in a patient with a homozygous pik3c2a mutation -a new ciliopathy? we present a 21 year old female with disproportional growth retardation, eye and renal anomalies. she was born small for gestational age as the first child of healthy, consanguineous tunisian parents at 37 weeks after an uneventful pregnancy (2470 g, 47 cm). during her first year of life, there were severe feeding problems with regurgitation and she developed postnatal growth retardation. developmental milestones were normal. during the second year the girl developed strabismus in both eyes. later on, a cataracta polaris anterior, anomalies of the cornea, hyperopia and progressive retinal degeneration led to profound visual impairment. furthermore, a right kidney agenesis and a complex tubulopathy, as well as increased echogenicity of the single left kidney were diagnosed. r. brumm, m. schmuck, y. dinçer, s. schulz, s. wilson, i. rost, hg. klein, sh. eck center for human genetics and laboratory diagnostics dr. klein, dr. rost and colleagues, martinsried, germany next-generation sequencing may lead to a significant improvement in diagnostic yield for rare, heterogeneous disorders through the ability to simultaneously sequence all genes contributing to a certain indication at a cost and speed that is superior to traditional sequencing approaches. on the other hand, the practical implementation of ngs in a clinical diagnostic setting involves a variety of new challenges which need to be overcome. among these are the generation, analysis and storage of unprecedented amounts of data, strict control of sequencing performance, validation of results, interpretation of detected variants and reporting. especially the variant interpretation emerges as current bottleneck of the diagnostic workflow. here we present midas (multiple integration of data annotation software), a central software system for data integration in a diagnostic laboratory. the goal of midas is to construct a modular software system to integrate data from laboratory information management system (lims), data from the routine sanger sequencing workflow as well as ngs sequencing results and correlate the identified variants with the patients' phenotypical features to aid in variant interpretation and accelerate reporting. the phenotype is systematically recorded using the human phenotype ontology standard nomenclature. in particular, genotype-phenotype correlations identified in one patient are made available for all other cases, to aid the interpretation and build a comprehensive knowledge base. the midas software may thus serve as central information system all diagnostic patient data. midas is implemented in java using direct database access via jdbc, and javafx as graphical user interface. its architecture is designed modular including a dynamic module loader, a user management with ldab connection and basic search functionality. according to available modules the user management and search form are adjusted; granting access for module specific views. as an advantage of this architecture, other molecular diagnostic data, such as arraycgh or mlpa results can easily be integrated by implementing new modules. midas aims to aid molecular diagnostics by simplifying and accelerating data analysis and interpretation, improving patient care. midas is being developed as part of a prospective multicentric study including clinical, diagnostic and software development partners. a grant by the bavarian ministry of economy, media, energy and technology is used to fund this effort. institute of human genetics -medical faculty -rwth aachen university, aachen, germany, 2 izkf aachen -rwth aachen university, aachen, germany with the implementation of next generation sequencing (ngs) based assays as key tool in dna-sequencing, conventional sanger-sequencing has become a standard method to verify single nucleotide polymorphisms (snps) of interest. however, designing specific pcr-and sanger-sequencing primers has always been a rather time consuming task in terms of searching the right genomic sequence, considering known snps that could result in allele-specific amplification, and converting selected primer-sequences to upload them to web-based services. to circumvent this, we developed optimus primer, a python script, that automatically designs respective primers. the script uses the database of the ucsc genome browser to download positional information of genomic sequence (refseq gene: hg38) and common snps (snp147common: hg38) in the region of interest. the genomic sequence is downloaded via the ucsc primarily affected cell type in the skin of arci patients. furthermore, it is only a small step to expand the system from generating gene ko to gene editing allowing the introduction of patient-specific mutations in non-patient derived keratinocyte cells. hence, setting-up the crispr/cas system to target an arci-associated gene is an important starting point for future studies to investigate pathogenic effects of arci-causing mutations and to understand arci pathogenesis on a molecular level. in this context, the transfer of the established protocol into keratinocytes offers the possibility to generate 3d cell culture models of mutations in arci-associated genes and allows in vitro investigation of their implications on the differentiation process. this system would further represent an organotypic model of arci-disease with potential application in screening and identification of chemical compounds for the complementation of existing clinical therapies. background: telomeres cap and protect chromosome ends from degradation and fusion, and are therefore essential for maintaining chromosome stability and genomic integrity. they have a length of 5 to 15 kb (depending on age, sex and cell populations). due to the end-replication problem there is a continuous telomere loss of 50 to 150 bp/cell cycle and thus throughout lifetime. in laboratory practice different methods of telomere length measurement are used to identify patients with bone marrow failure syndromes (e. g. dyskeratosis congenita, aplastic anemia), hematological diseases, or other telomeropathies. each telomere length measurement method has its advantages and disadvantages regarding material required the complexity and feasibility of the method and other parameters. methods: in this study we compared and validated four different methods for telomere length measurement, i. e. southern blot analysis, quantitative pcr (qpcr), quantitative fluorescence in situ hybridization (t/ c-fish) and flow cytometry-fish (flowfish). whenever possible, edta and/or heparin blood samples were collected from a population of 154 healthy individuals of different age groups (newborn -81 years). depending on the method dna (southern blot and qpcr), metaphases (t/ c-fish) or rather vital cells (flowfish) were analyzed. results: comparison and validation of the telomere length measurement methods allowed us to calculate percentiles for all age groups. percentile curves could be used in diagnostic to identify patients with short telomeres. all methods showed acceptable accuracy, but equally imply the necessity of validation and appropriate controls in each experiment. here, flowfish was the most precise, accurate and reproducible method compared to the other methods. discussion: our study emphasizes the influence of expertise and experience that is required in order to produce robust and reliable telomere length analyses. here, we provide advice on how to choose the appropriate method in general and for individual cases to safely discriminate between natural variability and pathological telomere shortening in individual cases. in the next generation sequencing age, the strongest challenges have shifted from genotyping to handling the myriad of variants detected. whole exome sequencing yields tens of thousands of coding and non-coding variants, a number increased to millions if the whole genome is sequenced. to master this mountain of data, computer-based identification of potential disease mutations is absolutely indispensable. however, current computational strategies are usually generated by computer scientists without integrating human geneticists into software development. this often leads to tools which fulfil their main purpose but are not ideally suited to the needs of human geneticists. to assess the relevance of a suggested disease mutation, geneticists need detailed information about the effect on the protein and about the gene or protein itself. to close this gap, we have developed mutationdistiller, a web-based tool for user-driven variant prioritisation based on biological disease properties. its analysis includes the potential role of a mutated gene in pathogenesis as well as the estimated effect of a variant on gene/protein function. thus, mu-tationdistiller allows human geneticists to use every piece of information they consider relevant. unlike similar tools, its input goes beyond the human phenotype ontology and can include complete diagnoses, biological pathways, gene expression, and gene function. potentially harmful variants are identified by mutationtaster, which provides a deleteriousness score together with data on the actual effect of the variants. mutationdistiller is not restricted to non-synonymous variants but can also handle intragenic non-coding or synonymus variants. moreover, the program incorporates the known modes of inheritance of disease genes and the genotype of the queried variants (including compound heterozygosity). the output page provides all information on one site: the core component is a concise overview table of the most likely disease genes and variants. here, the most crucial information such as the variants' effect on the protein, frequencies in polymorphism databases and known diseases caused by mutations within this gene and their mode of inheritance are listed. more detailed gene and disease information and hyperlinks to external data are provided below, hence offering a comprehensive overview of the available knowledge. this allows geneticists to draw their own conclusions without any tedious collection of relevant information from the internet. a beta-version of the program is freely available at http://www.mutationtaster.org. m. jäger 1,2 , m. schubach 1 , t. zemojtel 1 , k. reinert 3 , d. m. church 4 , p. n. robinson 1, 5, 6 1 institute for medical and human genetics, charité, berlin, germany, 2 bcrt, berlin, germany, 3 institute for bioinformatics, department of mathematics and computer science, freie universität, berlin, germany, 4 10x genomics, pleasanton ca, united states, 5 department of mathematics and computer science, freie universität, berlin, germany, 6 the jackson laboratory, farmington ct, united states with the grch37 human genome release in 2009 the genome resource consortium extended the previous linear, "golden-path" paradigm of the human genome and introduced a more graph-like model in the sense of regions with alternate loci, representing common alterations in sequence and structure. in whole-genome sequencing (wgs) these stretches of sequence are largely but not entirely identical between the primary assembly and an its corresponding alternate locus can result in multiple variant calls against regions of the primary assembly. this results in characteristic and recognizable patterns of variant calls at positions that we term alignable scaffold-discrepant positions (asdps). we developed an algorithm (asdpex) that analyzes these patterns in the 178 structurally variable regions of the current grch38 genome assembly. a heuristical approach then infers whether the pattern of variant calls of a sample contains sequences from the primary assembly, an alternative locus, or their heterozygous combination at each of these 178 regions. api and common snps are annotated. in the following step primers are picked using primer3 (whitehead-institute). the target sequence is either the snp-containing exon in case of exons smaller than 300 bp, or, the genomic region flanking the snp of interest. the positional information of the snp is gathered using the mutalyzer api (lumc). the last two bases of each primer are checked for all snps in the 3' end (snp147: hg38). the script will be integrated into a website for free easy access and usability (www.optimus-primer.com). the only information needed is the refseq id, the exon, and the cdna position (eg: nm_000.249.3:c.464t>g). the script optimus primer will provide the user with primers in the widely used primer3 format. the users are also able to download the source from our website, and to run it on a local unix system as a command line tool. mutationtaster3: moving towards a comprehensive evaluation of disease causing mutations o. ebner, jm. schwarz, d. hombach, m. schuelke, d. seelow charitè universitätsmedizin, berlin, germany mutationtaster is a free and user-oriented application for comprehensible evaluation of non-synonymous and synonymous as well as non-coding dna sequence variants. as 1500+ citations show, the software has been strongly embraced by the clinical and research community. however, its capacities for assessing the functional consequences of non-coding variants are still limited. while mutationtaster is able to predict the variant's effect on major intragenic regulatory features such as splice sites or polyadenylation signals, many other potential effects of intronic or utr variants are not covered by the software yet. variants in the non-coding region of the genome are frequent and thought to cause a substantial part of yet unsolved mendelian diseases. these variants can occur either in extragenic regulatory regions (see abstract on regulationspotter) or in the untranslated regions, including introns, of a gene. unfortunately, their effects are much harder to predict than those of non-synonymous variants. therefore, only few disease mutations outside the coding sequence have hitherto been found and experimentally confirmed. to close this gap, we are advancing the current version of mutationtaster to improve the analysis of intragenic non-coding variants. we do so by implementing additional tests for variants in the 5' and 3'utr to determine their effect on au-rich elements, microrna binding sites and the influence of secondary structure on gene expression. moreover, we plan to shift from the analysis of exclusively monogenic to complex diseases with cancer being the first disease model to be integrated into mutationtaster3. already implemented enhancements of mutationtaster3 comprise the improved splice site analysis using maxentscan and the integration of mitochondrial polymorphisms from the human mitochondrial genome database. the addition of links to the integrative genomics viewer and the lof-metrics of the exac browser (exome aggregation consortium) streamline the usage of the mutationtaster3 results and enable researchers to get a detailed view of the relevant variants and associated genomic sequences. taken together, mutationtaster3 will offer much better capabilities to predict the disease-causing potential of intragenic variants than its predecessor. the software is freely available at http://www.mutationtaster.org neurocure exzellenzcluster, berlin, germany, 2 neuropädiatrie charité-universitätsmedizin, berlin, germany, 3 medizinische genetik charité-universitätsklinikum, berlin, germany, 4 berliner institut für gesundheitsforschung bih, berlin, germany ifications, or genome-wide interactions. all variants which have the potential to influence one or several candidate genes are presented in a graphical interface and ranked according to their predicted effect on the target gene(s). instead of giving scores, we present the potentially affected regulatory features in an intuitive graphical matrix. the software can freely be used at http://www.mutationtaster.org p-techno-179 genecascade2017 -a one-stop shop for finding disease mutations d. seelow 1, 2 in the last years, our genecascade software suite for the elucidation of rare diseases has seen some major extensions, mainly for the discovery of non-coding mutations. the website contains a number of tools focusing on the different steps in studying genetic disorders. all applications are web-based, have easy-to-use interfaces and are aimed directly at human geneticists, without any need to install software, use the command line or to try to explain clinical features to it specialists. we think that software should adapt to the user -not the other way around. and, unlike other web shops, its use is completely free. homozygositymapper finds disease-linked regions in consanguineous families. users can upload genotypes from snp chips as well as wes and even wgs data. we display likely disease loci in intuitive graphical interfaces. the results can also be used in our 'downstream' applications such as genedistiller, mutationtaster, or mutationdistiller to identify the actual disease mutation. genedistiller provides a user-driven way to find the best candidate gene for a genetic disorder. users can specify various aspects of the patients' phenotypes and the results are presented in a comprehensive way without the need to manually query other internet resources to collect further information relevant to asses the disease potential. mutationtaster evaluates the disease potential of non-synonymous as well as of non-coding and synonymous dna variants within genes. it does not only display a prediction but also detailed information on the variants' likely effect on mrna and protein. it can either analyse single variants as found by sanger sequencing or complete vcf files from wes or wgs projects. regulationspotter lists information about potentially regulatory dna variants outside of genes. variants are ranked according to their predicted effect on gene regulation. in addition to a score, we provide a user-friendly summary of the functional effects a variant may have. mutationdistiller combines genedistiller and mutationtaster in a single application for convenient variant and gene prioritisation. it offers human geneticists much more freedom to enter the phenotype than similar applications and provides deeper information about the variant and the gene. cnvinspector is aimed at the study of copy number variants. users can upload their patients' (or cohorts') cnvs and compare them with their own healthy controls or public data stored in our database. we include deci-pher to indicate known diseases caused by cnvs at the same position. epossum examines the effect of dna variants on transcription factor binding. as tf binding site prediction is notoriously unreliable, we also give an indication of the statistical relevance of a result. the genecascade website can be accessed at http://www.mutationtaster.org. we investigate 121 in-house wgs datasets and found that on average 51.8 ± 3.8 of the 178 regions correspond to an alternate locus rather than the primary assembly sequence. filtering these genomes with our algorithm identified around 7900 variant calls per genome that colocalized with asdps. our findings suggest the potential of fully incorporating the resources of graph-like genome assemblies into variant calling. our algorithm already uses the information contained in the 178 structurally variable regions of the grch38 genome assembly to avoid spurious variant calls in cases where samples contain an alternate locus rather than the corresponding segment of the primary assembly. the human phenotype ontology project provides three resources, the ontology of clinical features, disease-phenotype associations and algorithms that enable analysis of data that is described using hpo. this resource is being used for computational deep phenotyping and precision medicine. it enables clinical data integration for translational research. the hpo is being increasingly adopted in software, research projects and companies world-wide. we will discuss the progress and recent developments that the hpo project has made since 2008. this will include the expansion of hpo for common (complex) diseases, novel algorithms for phenotype-driven analysis of genomic variation, cross-species mapping of phenotype data, translation of hpo into several languages and also the addition of a more patient-friendly terminology for hpo terms. millions of patients worldwide suffer from a rare genetic disease. to date, the omim database lists more than 8000 diseases with proven or suspected mendelian basis, but the molecular cause is known for less than 60%. although next generation sequencing has drastically facilitated the discovery of disease mutations, a substantial fraction of whole exome sequencing (wes) projects fail to identify the causal variant. this may be due to the fact that disease-causing mutations are not always located within the coding sequence. whole genome sequencing (wgs) could solve this problem, but we are not yet readily prepared to handle the vast amount of variants which are generated by this technique, as intuitive and reliable software solutions for variant evaluation are missing. the currently existing approaches are not well-suited for a diagnostic setting, because they output a battery of different scores whose interpretation is left to the users. however, human geneticists are specialists for the assessment of symptoms of genetic diseases -not for the analysis of numerical scores indicating different likelihoods of the occurrence of regulatory elements. this is especially true when it comes to the hundreds of thousands of extragenic variants found by wgs, for which effect predictions always face a high level of uncertainty. we consider it crucial to provide geneticists with the information they need to determine the significance of a variant, not to flood them with scores whose meanings are difficult to grasp. to facilitate the interpretation of extragenic variants, we are developing regulationspotter. in contrast to other approaches such as genomiser or cadd, we integrate as much knowledge as possible in a user-friendly fashion to pinpoint the variants which are most likely to disturb the expression of candidate genes. regulationspotter includes different data on regulatory dna elements such as dna methylation, transcription factor binding sites, histone mod-the human exon 1 in the 5' end of the murine huntingtin gene. by using a novel object recognition test with a 24 h interval between sample and test phase we have found a profound deficiency of hippocampus dependent long-term memory in heterozygous transgenics. this phenotype was detected as early as 12 weeks of age and is complementary to deficits that we have identified in the hdhcag111 mouse model previously. motor deficits as well as intranuclear aggregates are described at much later stages in both of these models. we have shown previously that in hd patients, mediated through mtor signaling, translation of mrna carrying expanded cag repeats is elevated (krauss et al., 2013) . we have also seen that the biguanid metformin antagonizes mtor signaling in neurons in-vitro and in-vivo (kickstein et al., 2010) . we show here that metformin, by interfering with the mtor kinase and its opposing phosphatase, pp2a, regulates local protein synthesis in the brain and is able to suppress the production of disease making protein in early hd. furthermore metformin leads to a significant improvement of movement abilities in a c-elegans model for hd and to a rescue of early cognitive symptoms in the hdhcag150 animal model. these data suggest that metformin is a very promising candidate for early phase treatment of hd patients. allele-specific suppression of dominant-negative bestrophin 1 mutations a. milenkovic, lm. braun, f. grassmann, b. h. f. weber institute of human genetics, regensburg, germany purpose: retinal pigment epithelium (rpe) differentiated from human induced pluripotent stem cells (hipsc) demonstrated degradation and mislocalization of mutant bestrophin 1 (best1) protein in autosomal dominant best disease (bd). importantly, mutated alleles revealed a dominant-negative effect leading to an impairment of volume-regulated chloride transport, the basic function of the homo-pentameric best1 channel. here, our study aimed for a proof-of-concept to treat bd by selectively eliminating best1 mutant transcripts in patient-derived hipscs prior to rpe differentiation via the crispr/cas9 genome editing technology. methods: adult human dermal fibroblast were obtained from skin biopsies of bd patients and reprogrammed into hipscs. single guide rna sequences (sgrna) targeting 6 best1 mutations were selected by the "optimised crispr design tool" (zhang lab, mit 2015) . editing efficiency and specificity of designed sgrnas were tested in hek293 cells using an established fluorescence-based assay. after transfection of hipscs the percentage of indel formation of on-and off-targets was determined by a pcr-based crispr/cas cleavage detection kit. cas9-treated stem cell populations were analyzed for pluripotency and selected for full genome sequencing before differentiation to rpe cells. results: computational design of 6 disease-causing best1 variants (n11k, v86m, s108r, q238r, a243v, and i295del) offered at least one sgrna with predicted high quality per mutant allele. the guide sequences were cloned into the cas9-expressing plasmid px459 and co-transfected with pcag-egxxfp plasmids containing genomic fragments of ~500 bp of either the mutated or wildtype sequence to the corresponding sgrna. as targeted cas9 cleavage results in reconstitution of the egfp expression cassette by homology dependent repair, the efficiency and specificity of cas9 cleavage was evaluated on a plate reader by quantifying egfp fluorescence after 48 h of transfection. as a result, 5 out of the 6 sgrnas tested showed high allele-specificity and are now used for targeted genome editing in hipscs. conclusion: so far, there is no treatment for bd although the molecular pathology of best1 has recently been established. our proof-of-concept study aims to determine whether haploinsufficiency of normal best1 protein is sufficient to fully or partly restore cellular function in cells of primary bd pathology, namely the rpe. to this end, we will determine the degree of rescue (i. e. reconstitution of volume-regulated chloride conductance) by whole-cell patch-clamp analysis. if successful, our crispr/ cas9-driven approach will be useful to treat other diseases with dominant negative effects of the mutated allele. p-therap-184 *** metformin rescues early cognitive symptoms in the hdhcag150 mouse model and is therefore a promising candidate for treatment of hd patients. huntington's disease (hd) is an autosomal dominant neurodegenerative disorder that is caused by an unstable glutamine (cag) trinucleotide repeat expansion within exon 1 of the huntingtin gene and leads to cognitive decline and affects motor abilities. in the prodromal phase of the disease patients develop mood swings, personality changes and subtle cognitive impairment. close understanding of clinical signs and molecular mechanisms behind this early stage of hd is an important step for the development of a causal therapy. we have analysed a knock-in mouse model that carries 150 cag repeats and single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation age-related clonal hematopoiesis associated with adverse outcomes clonal hematopoiesis and bloodcancer risk inferred from blood dna sequence age-related mutations associated with clonal hematopoietic expansion and malignancies cancer spectrum in dna mismatch repair gene mutation carriers: results from a hospital based lynch syndrome registry update on kleefstra syndrome characterization of a novel transcript of the ehmt1 gene reveals important diagnostic implications for kleefstra syndrome national institute of child health and human development abstracts p-monog-147 novel mutation in hoxc13 expand the mutation spectrum of pure hair and nail ectodermal dysplasia abstracts in conclusion, targeted sequencing represents an effective, fast, cost-efficient and flexible method, since new candidate genes can be added easily to the panel, for the sequential analysis in chh patients. furthermore, panel sequencing alleviates the uncovering of oligogenic inheritance in genetic traits like chh abstracts p-monog-159 neurodegeneration in the olfactory bulb and olfactory impairment in the ccdc66 -/-mouse model for retinal degeneration s. schreiber 1 , e. petrasch-parwez one hallmark of sca3 and other mechanisms that govern ccdc66-deficient degeneration, a detailed evaluation is performed in order to reveal processes that contribute to retinal degeneration during early postnatal development and adulthood. methods: the functional role of ccdc66 deficiency is investigated by two independent approaches: 1) gene expression profiles at p10 and p28 are analyzed in retinal tissue of cccdc66-/-and wildtype mice (genechip mouse gene 2.0 st array, affymetrix) followed by quantitative real-time pcr (qrt-pcr) and 2) potential retinal interaction partners of ccdc66 protein are identified by yeast-two hybrid screening in the wildtype mouse and further analyzed by immunohistochemistry. results: using two screening methods (rna-expression profiles and protein interaction partners), our results indicate that 1) the ccdc66 deficient mouse model reveals early changes in retinal rna gene expression already at p10 and the highest number of genes differential expressed at p28. most expression differences were related to genes associated with the extracellular matrix. further genes are involved in retinal degeneration, angiogenesis, transcription factors and proteolysis. 2) the ccdc66 protein interacts with the proteins eps8 (epidermal growth factor receptor kinase substrate 8) and mpdz (multiple pdz-domain protein). both proteins are expressed in several retinal layers of the retina, confirmed by immunohistochemistry. moreover, mutations in the mpdz gene were already identified in patients with retinitis pigmentosa/leber congenital amaurosis. conclusion: expression profiles reveal expression changes at an early time point of retinal degeneration in the ccdc66-/-mouse model enabling further studies on the role of genes and processes involved in early retinal degeneration. in addition, the interaction partners of ccdc66, eps8 and mpdz, are the basis for further studies examining the pathways of retinal degeneration in the mammalian retina including man -and possibly contribute to future studies in man and human disease. ccdc66 null mutation causes retinal degeneration and dysfunction of medical and molecular genetics and skeletal dysplasia multidisciplinary unit hospital unit of genetics of neurodegenerative and metabolic diseases sel-002, ws7-005, p-compl-125 ws3-004 p-cling-051, p-cling-052 ws1-003, ws4-006, p-cling-056, p-cling-068, p-cling-075 ws7-001, p-cling-050, p-cling-074, p-monog-141 p-cancg-042 p-cling-060, p-cling-073, p-cling-086 p-cling-063, p-cling-065 ws7-005, ws8-004, p-cling-088 ws7-001, p-cling-110 p-cancg-044, p-cancg-046, p-cling-082 ws2-001 medizinische genetik 1 · ws3-002, p-cling-052 ws5-001, p-cling-055 ddd study 0. p-cling-095 ws3-006, ws6-001, p-cling-054, p-compl-120 ws4-006, p-cling-056, p-cling-075 distelmaier f. ws7-003 ws9-004, p-basepi-010, p-cancg-041 ws7-001, ws8-006, p-cling-050, p-cling-067, p-cling-074 ws5-001, p-cling-058, p-monog-162 ws2-002 ekici ab. ws5-005, p-monog-143 sel-001 p-cancg-048, p-techno-171 plen 2, ws4-002 golla a dfg-workshop göpfert mc sel-004, sel-004 haferlach c. ws8-005 haferlach t. ws8-005 ws3-006, ws6-001, p-compl-120, p-compl-121 p-cancg-032, p-cancg-039, p-cancg-046 gilissen c. ws3-003 ws4-003, p-basepi-006, p-cling-089, p-cling-099 p-cling-063, p-cling-065, p-cling-081, p-cling-095 ws7-001, p-cling-050 p-cling-051, p-cling-052, p-cling-054 ws3-006, p-compl-121 p-monog-155 medizinische genetik 1 · ws2-002 hüffmeier u. ws5-005 ws4-003, p-cling-099 seq consortium ws8-002 ws1-003, ws3-006, p-compl-121 p-cling-053, p-cling-059, p-cling-072, p-cling-109 ws2-005, p-cancg-040 ws1-003, ws3-006, ws9-002, p-compl-121 p-cancg-044, p-cancg-045 ws3-003 ws5-001, p-cling-055, p-cling-058, p-cling-069, p-cytog-128 ws6-003, ws6-006, p-compl-120, p-compl-124, p-compl-125 ws2-003, p-cling-095 p-cling-052, p-cling-080 p-cling-063, p-cling-065 p-cling-050, p-cling-060, p-cling-074 ws9-004, p-cancg-042 ws4-006, p-cling-075 ws2-002, ws2-003, p-cancg-036 khor cc. ws6-005 sel-001, ws8-001 ws4-003 medizinische genetik 1 · p-cling-053, p-cling-072, p-cling-095 ws3-006, p-compl-120, p-compl-121 ws2-005, p-cancg-040 ws6-006, p-cling-054 meggendorfer m. ws8-005 ws7-004, p-cancg-046 p-cling-051, p-cling-052, p-cling-080 p-cling-081, p-cling-090 ws2-005, p-cancg-040 ws2-002, ws4-005, p-monog-143 ws8-001 ws3-004 ws2-002, p-cling-095 pasutto f. ws6-005 nanda i. ws1-003 nöthen mm. ws3-006, ws6-001, ws6-002, ws6-003, ws6-006 ws3-003 ws3-003, ws3-006, ws7-005, ws8-004, p-cling-088, p-compl-120, p-compl-121 sel-004 medizinische genetik 1 · p-cancg-044 schnapp l. ws1-003 ws2-004, p-cancg-031, p-cancg-046, p-cling-092 ws4-006, p-basepi-008, p-cling-056 p-cling-066, p-cling-106 p-cancg-031, p-cling-092 salaverria i. ws8-002 ws3-006, p-compl-121 ws2-003, ws4-005, ws6-005, p-cancg-036, p-cling-085, p-cling-095, p-cytog-131, p-monog-143, p-monog-167 p-cancg-044, p-cancg-045, p-cling-067, p-cling-089 ws9-005 sel-002, ws7-005, p-compl-125 the international gamos consortium e ws2-002, ws4-005, ws5-005, p-cancg-036, p-monog-143, p-monog-167 sel-002, ws3-006 ws9-004, p-cytog-127 p-cancg-031, p-cling-092, p-cling-112 p-cancg-044, p-cancg-045, p-cling-067 stengel a. ws8-005 p-cling-085, p-cling-094 ws3-002, ws4-004, ws7-003, p-cling-051, p-cling-052, p-cling-054, p-cling-080, p-cling-093 p-cancg-047, p-cling-051 ws8-003 ws2-001, p-cling-111 ws7-005 witsch-baumgartner m. p-cling-084 p-cancg-039 p-cancg-044 wunderle m. ws2-003 zechner u. ws1-003, ws4-006, p-basepi-008, p-cling-056, p-cling-068, p-cling-075 ws6-004, p-cancg-046, p-cling-097 edu 2, p-cling-051, p-cling-052, p-cling-054, p-cling-080, p-cling-094, p-cling-096 p-cling-051, p-cling-052, p-cling-054 wiesener a. ws4-005, p-monog-167 ws4-005, ws6-005, p-cancg-036, p-cling-085, p-compl-119, p-monog-167 ws2-002 ws2-005, p-cancg-040 autorenverzeichnis zenker m. ws7-002, ws9-005, p-cling-061, p-cling-080 dialog mit shg, p-cling-074 ws4-005, ws5-005, p-cling-095, p-monog-151 ac. koller 1,2 , j. strohmaier 3 , ku. ludwig 1,2 , fc. degenhardt 4 , m. wulff 1,2 , d. breuer 1,2 , l. winkler 1,2 , f. neukirch 1,2 , a. maaser 1,2 , a. forstner 1,2 , s. sivalingam 1,2 , a. reif 5 , a. ramirez 6 , w. maier 6 , d. rujescu 7 , i. giegling 7 , h. thiele 8 , p. nürnberg 8 ,2 , a. fischer 9 congenital hypogonadotropic hypogonadism (chh) is a rare and clinically and genetically heterogeneous disorder. chh is characterized by incomplete or absent puberty caused by the lack or deficient number of hypothalamic gonadotropin-releasing hormone (gnrh) neurons, disturbed secretion or action of gnrh, or both. chh is often associated with anosmia and is then termed kallmann syndrome (ks), as well as with other phenotypes like unilateral kidney agenesis, skeletal abnormalities, midline malformations, and hearing loss. x-linked, autosomal-dominant, and autosomal-recessive, as well as di-and oligogenic inheritance has been described for chh. in the meantime a multitude of genes has been reported to be associated with chh. actually, in fewer than 40% of the chh cases the underlying genetic cause can be identified. we analyzed a total of 19 patients with chh (4 female/15 male) by using targeted sequencing of 16 chh-associated genes kal1, chd7, fgf8, fgfr1, fshb, gnrh1, gnrhr, hs6st1, kiss1, kiss1r, nsmf, prok2, prokr2, spry4, tac3 and tacr3 and identified pathogenic mutations in 8 chh patients of our cohort. mutations were detected in kal1 (3 patients), tacr3 (2 patients), prokr2 (1 patient), hs6st1 (1 patient) and gnrhr (1 patient). furthermore, we found in two patients with described pathogenic mutations (one patient with prokr2 mutation and the other with kal1 mutation, respectively) additional mutations in nsmf gene and tacr3 gene, respectively, suggesting digenic inheritance in these cases. pain is necessary to alert us to actual or potential tissue damage. specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (hsan). although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the flvcr1 (feline leukemia virus subgroup c receptor 1) gene, which encodes a broadly expressed heme exporter. different flvcr1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. mutations institute of human genetics, bonn, germany, 2 the rild institute, wonford, exeter, uk introduction: ectodermal dysplasias (eds) are a large group of heterogeneous genetic disorders characterized by abnormal development in ectoderm-derived tissues and organs including skin, hair, and nails. among the eds, pure hair and nail ectodermal dysplasia (phned) is a rare genodermatosis characterized by nail dystrophy and sparse or absent hair on the scalp. materials and methods: a family of iranian origin was enrolled in this study. two children from a consanguineous marriage are affected from phned. in addition, the father has alopecia areata (aa) but does not show any nail dysplasia. the mother is unaffected. the paternal and maternal grandfathers had nail dysplasia almost similar to the siblings but did not manifest any hair loss or aa. homozygosity mapping and genedistiller analysis were performed to identify candidate genes. sanger sequencing was used to localize the mutations. results: in total, we identified 10 homozygous regions with almost 700 candidate genes. among these genes were also krt85 and hoxc13, already known to be related to the phenotype of our patients. therefore, we focused our additional analyses on krt85 and hoxc13. sanger sequencing showed a so far unknown homozygous insertion of 28 bp in exon 2 of hoxc13. conclusion: we identified an unknown mutation for phned which expands the spectrum of mutations for phned. the hair loss of the father seems rather be due to a distinct type of hair loss, namely aa, which is quite common in the general population. however, the nail dysplasia from both grandfathers is unclear and cannot be examined anymore. it still remains unclear if the nail dysplasia in the grandfathers was due to the same mutation in hoxc13 or is based on a different mutation. generation of a cell culture model for epidermodysplasia verruciformis by knock-out of ev3, a novel gene involved in this genodermatosis e. imahorn 1 , m. aushev 2 , sj. de jong 3 , e. jouanguy 4, 5, 6 , jl. casanova 4, 5, 6 , ph. itin 1, 7 , j. reichelt 2, 8 epidermodysplasia verruciformis (ev) is a rare hereditary skin disease leading susceptibility to certain types of cutaneous human papilloma viruses, mainly β-hpv, and a high risk for development of cutaneous squamous cell carcinoma. homozygous or compound heterozygous loss of function mutations either in tmc6 or in tmc8 have been described in several ev patients, but more than 1/3 of affected families do not have mutations in one of them. a third gene (ev3) has recently been identified to be mutated in ev patients without tmc6 or tmc8 mutation. investigations on the function of this gene may elucidate pathomechanisms of ev. we aim to generate an ev3 deficient model cell line to study the effects of ev3 despite the scarcity of patient material. detection of deletions during diagnostic massive parallel ngs gene panel sequencing neuronal differentiation at a later time point. a novel mutation c.4a>g, pser2gly in des gene in a family with catecholamine polymorphic ventricular tachycardia s. komatsuzaki, p. villavicencio lorini, k. hoffmann institute for human genetics, university hospital halle, germany background: myofibrillar myopathy (omim 601419) is a heterogeneous neuromuscular disease characterized by progressive muscle weakness, partially with cardiomyopathy and/or arrhythmia. pathohistological examinations show desmin-positive protein aggregations. the mutations in des, cryab, bag3, myot, ldb3, flnc, fhl1, and dnajb6 have been identified in patients with myofibrillar myopathy. desmin is a intermediate filaments and composed of non-helical n-and terminal head domain and a central a-helical rod domain. till now more than 67 mutations myofibrillar myopathy were identified in patients with and a genotype-phenotype was reported: mutations in rod domain tend to cause neuromuscular symptoms, and mutations in head domain is associated with cardiac symptoms. the head-domain is a serine rich domain and phosphorylated by protein kinase c. almost all reported mutations in head-domain in des are at serine residues (e. g. s2i, s7f, s13f, s46f, s26y, s26t). these findings suggested that the serine residues in head-domain could play an important role in biological function of desmin. here we report a novel mutation c.4a>g, ps2g in des in a family with catecholamine polymorphic ventricular tachycardia. case: a 58 years old patient developed a syncope in a cold winter. the cardiological investigations revealed a diagnosis of catecholamine polymorphic ventricular tachycardia and the implantation of permanent pacemaker was indicated. his older brother, father and paternal uncle suffered from arrhythmia and all of them received a permanent pacemaker implantation at around 60 years of age. his cousin was died at age of 24 years due to sudden cardiac arrest. we performed a genetic analysis for ryanodin-rezeptor 2-gen and lamin a gene and no mutation in these gene was identified. next, we performed an exome sequencing. here a novel heterozygous mutation c.4a>g, ps2g in des was identified. this mutation was also identified in uncle of index patient. the c.4a>g, ps2g in des was not reported neither in 1000 human genome project, nor in exome aggregation consortium. a mutation at serine 2 (p.s2i) was previously reported in patients with myofibrillar myopathy with cardiac symptoms. based on these findings, it was strongly supposed that the p.s2g in des gene is a causative mutation for myofibrillar myopathy with arrhythmia in our patients. discussion: recent studies suggest that the mutations in des influence not only on muscle stability and myocardial force generation, but also impaired ubiquitin proteasome system, which could be caused by aggregated desmine. generally, phosphorylation of serin and threonine in head-domain of desmin is related to disassembly of filaments. therefore the p.ser-2gly mutation in des could cause altered phosphorylation of desmin. the functional abnormalities caused by pser2gly in des gene need to be studied. gene dosage manipulation of the chromatin organizer ctcf in the nervous system of drosophila melanogaster results in neurological and morphological phenotypes e. konrad, a. gregor, m. brech, c. zweier institute of human genetics, fau-erlangen-nürnberg, erlangen, germanythree-dimensional organization of eukaryotic genomes is crucial for temporal and spatial regulation of gene expression. architectural proteins, like the ccctc-binding factor ctcf are responsible for establishing and maintaining this organization. ctcf is involved in virtually all chromatin regulating processes including enhancer function, insulation, alterna-for this purpose we used the crispr/cas9 system to delete the ev3 coding sequence in an immortalized keratinocyte line. after isolation by facs, single cell clones have been expanded. we screened 41 clones for deletion of the whole gene as well as for the expression of ev3. three clones showed no detectable gdna sequence or expression of the ev3 gene. we found only one wildtype clone without deletion of ev3 or alterations near the cas9 cut sites. all clones have been characterized by a snp-array as well as sequencing of the knockout site and the most probable offsite targets. these ev3 deficient keratinocyte lines are the first cell culture model for ev. it will be a valuable tool to identify cellular pathomechanisms of the disease and allow insight into the control of β-hpv in the general population. hemizygous loss of function of single x-chromosomal genes is the most frequent cause for genetic intellectual disability (id). while a long list of gene mutations have so far been described to be responsible for the disease phenotype, little is known about the underlying neuronal mechanisms. reprogramming of somatic cells into stem cells (ipscs) followed by differentiation into neuronal precursors (npcs) is an important tool for translating research allowing an understanding of network dysfunction in id patients. opitz bbb/g syndrome (os) is characterized by a number of ventral midline defects combined with learning disability, developmental delay and intellectual disability. it is caused by mutations in the x-linked mid1 gene, which, as we have shown previously, regulates mtor dependent local protein synthesis. in a mouse model, loss of mid1 function leads to significant disturbance of axonal outgrowth. we have generated ips cells from several patients with os and from one mother that carries a loss of function mutation in the mid1 gene. by sorting cell clones after reprogramming we have established ips cell clones from this female carrier of a 4bp deletion in the mid1 gene (c.1801_1804delctcc) either expressing from the mutated x-chromosome or the non-mutated x-chromosome and have shown that indeed the generated ipsc-clones express either 0% of the mutated or 0% of the wildtype mid1. we determined this directly using an allele-specific mid1-rt-pcr and indirectly by comparing the methylation of humara-alleles. comparison of mutation expressing ipsc-clones with non-mutation expressing ipsc-clones showed that the mid1 mutation results in significantly smaller cells with reduced s6 phosphorylation supporting aberrations in the mtor/pp2a signaling cascade. when differentiating ipscs into neuronal precursor cells, significantly bigger embryoid bodies (ebs) can be detected in the mutation expressing clones, while the total eb number is higher for the non-mutated mid1 expressing clones. when further differentiated, ebs from all ipsc-clones form neuronal rosette structures expressing beta-iii-tubulin, with a lower rosette structure count in the mutated mid1 expressing cells. these data clearly hint a defect in neurogenesis in cells with hemizygous mid1 mutations. interestingly, while ipscs stably kept the x-incativation pattern of the original fibroblast, during differentiation x-inactivation was lost leading to biallelic expression from both x-chromosomes in the npcs pointing towards an as yet unknown reactivation mechanism of the inactive x-chromosome. we are currently analyzing x-inactivation throughout the differmutations in the aminoacyl-trna-synthetase genes sars and wars2 are associated with autosomal recessive intellectual disability l intellectual disability (id) is the common feature of a very heterogeneous group of disorders, which comprises a broad variety of syndromic and non-syndromic phenotypes. here we present mutations in two aminoacyl-trna synthetases that are associated with id in two independent iranian families. in the first family, we found a missense mutation (c.514g>a, p.d172n) in the cytoplasmic seryl-trna synthetase (sars) gene that affects the enzymatic core domain of the protein and impairs its enzymatic activity. this probably leads to reduced trnaser concentrations in the cytoplasm. in silico analyses predicted the mutant protein to be unstable. this prediction could be experimentally substantiated by results obtained through studies with ectopic mutant sars in transfected hek293t cells. in the second family, we identified a compound heterozygous genotype of the mitochondrial tryptophanyl-trna synthetase (wars2) gene, consisting of a nonsense mutation (c.325dela, p.ser109alafs*15), which very likely leads to nonsense-mediated mrna decay, in combination with a missense mutation (c.37t>g, p.w13g). the p.w13g mutation affects the mitochondrial localization signal of wars2, leading to mislocalization of the mutant protein. thus, when taking aimp1 into account, which we have recently implicated in the aetiology of id as well, there are now three genes with a role in trna-aminoacylation that are associated with this condition. hence we propose that the functional integrity of t-rnas in general is an important constituent in the development and maintenance of human cognitive functions. dravet syndrome is a rare autosomal dominant genetic disorder with early-onset epileptic encephalopathy is mainly caused by different de novo mutations of the scn1a gene encoding the type 1 subunit of voltage-gated sodium channel. whole exome sequencing(wes) enables scanning a large number of genes which not only can confirm the diagnosis but also helpful in understading for possible relationship between clinical manifestations and mutaion. the authors investigate wes in a 18 months term boy with hypotonia and convulsion of normal and relative parent. she had uncontrolled sizure without fever, and developmental delay from 6 months.in treatment protocol anti covulsants changes to valorate, clonazepam and stiripentol as well. a deleterious novel heterozygous splice site mutation in scn1a tive splicing, imprinting, v(d)j recombination, chromatin loop formation and defining topologically associated domains (tads). recently, we identified de novo mutations in ctcf in patients with a surprisingly mild phenotype of variable developmental delay or intellectual disability, mild short stature and microcephaly, and behavioural anomalies. apart from observing brain malformations and early lethality or learning deficits in two conditional knockout mouse models, little is known about the role of ctcf in neuronal development and preservation so far. therefore, we utilized the model organism drosophila melanogaster to further explore the role of ctcf in cns development and function. similar to observations in knockout mice, complete knockout or ubiquitous knockdown of ctcf is embryonic lethal in drosophila. we therefore utilized the uas/gal4 system to induce tissue specific knockdown or overexpression of ctcf in the fly nervous system. we first investigated development and morphology of the larval neuromuscular junctions (nmjs), an established model for synaptic development. while pan-neuronal overexpression of ctcf showed no morphological nmj alterations, pan-neuronal knockdown resulted in fewer nmj branches than in a specific control. additionally, we observed a reduced number of active zones in a hypomorphic mutant line compared to a wildtype control. using the negative gravitaxis assay to examine gross neurological function, we found a highly significant impairment of geotaxis behavior in flies with ctcf knockdown in neurons, motoneurons and muscle and in flies with overexpression of ctcf in glia cells, muscle and motoneurons. currently we are testing learning and memory behavior with the courtship conditioning paradigm. our findings of various neurological and morphological anomalies upon manipulation of ctcf dosage in the fly nervous system emphasize the role of ctcf in nervous system development and function and provide a basis to further study the molecular mechanisms underlying cognitive dysfunction caused by ctcf-deficiency. congenital or early-onset nystagmus (cn) is characterized by involuntary eye movements and shows enormous clinical and genetic heterogeneity. cn may be an ambiguous sign of many different diseases, including retinal dysfunction/degeneration, ocular/oculocutaneous albinism, and severe central nervous system disorders, such as pelizaeus-merzbacher or pelizaeus-merzbacher-like diseases (pmld). due to enormous heterogeneity found among the diseases leading to cn, whole exome sequencing (wes) and panel-based bioinformatics was considered as an approach to rapidly identify disease-associated genetic sequence variants. we have analyzed 9 families with 16 cn-affected patients. herein, we present three clinically different patients who were initially affected with cn, but developed further clinical symptoms of various severities. one of these patients showed features of retinal dysfunction, including night blindness and myopia, while two other patients developed severe phenotypes including mental retardation or pmld. wes identified four genetic variants in genes associated with cn. the first patient showed a hemizygous splice-donor variant (c.2576 + 1g>a) in the calcium channel voltage-dependent alpha-1f subunit (cacna1f) gene, the second patient carried a hemizygous variant (c.1403g>a, p.r468h) in the ferm domain-containing protein 7 (frmd7) gene, and the third patient showed two heterozygous variants (c. 291c>g, p.y97* and c.716t>c, p.v293a) in the gap junction protein gamma-2 (gjc2) gene. sanger sequencing confirmed the identified variants in the index patients and verified co-segregation in several family members. our results suggest a beneficial role of wes to identify the molecular causes of cn and to rapidly confirm an initially unclear clinical diagnosis. especially, patients with rare and severe disorders (e. g. pmld) will benefit from a wes analysis performed in the early stage of the disease. the ccdc66-deficient (ccdc66-/-) mouse model exhibits slow retinal degeneration similar to a human retinitis pigmentosa (rp) phenotype (gerding et al., hum mol genet., 2011) . in order to determine whether ccdc66 gene expression might also play a role outside the retina, this study aimed at characterizing ccdc66 protein expression during early postnatal development of the mouse brain. furthermore, morphological and behavioral impact of ccdc66 deficiency in the mouse brain was analyzed. methods: ccdc66 protein expression was determined by sds page and western blot in whole brain homogenates and in selected brain regions of interest (olfactory bulb, hippocampus, cortex, striatum, cerebellum, brain stem) during early postnatal development and in adult wildtype (wt) mice. in addition, cryosections of the ccdc66-/-olfactory epithelium and bulb (during postnatal development) and the rostral migratory stream (in adult) were analyzed for ccdc66 reporter gene expression by x-gal staining. selected brain regions were additionally analyzed by electron microscopy. in order to correlate anatomical with behavioral data, olfactory performance was studied in aged ccdc66-/-mice compared to ccdc66+/+ controls by an olfactory habituation/dishabituation test (yang and crawley, curr protoc neurosci., 2009) , where olfactory exploration-time during the presentation of neutral and social odors is examined. results: ccdc66 protein was detected throughout the early postnatal development of the wt mouse brain, decreasing after birth. amongst analyzed brain regions, highest expression of ccdc66 protein was detected in the olfactory bulb exhibiting similar ccdc66 levels to retinal expression. accordingly, ccdc66 reporter gene expression was demonstrated in the mature olfactory bulb glomeruli, the adjacent olfactory epithelium and along the rostral migratory stream in the ccdc66-/-mouse brain. interestingly, strong ccdc66 reporter gene expression in glomeruli of the ccdc66-/-olfactory bulb was correlated with signs of degeneration in the ccdc66-/-mouse, but not in controls. the degeneration was also reflected by olfactory impairment in ccdc66-/-mice, which spent significantly less time for sniffing at initial presentation of unknown, neutral odors and barely responded to social odors. conclusion: besides the retina, ccdc66 protein plays a crucial role in the olfactory system as shown by its expression there as well as by ccdc66 deficiency resulting in neurodegeneration and alteration of olfaction-related behavior in the ccdc66-/-mice. as impairment of the olfactory sense in multiple neurodegenerative disorders is a common finding, the ccdc66-/mouse model is not only restricted to study retinal degeneration but possibly also degeneration of the central nervous system. background: the ccdc66-deficient (ccdc66-/-) mouse model exhibits slow retinal degeneration similar to a human retinitis pigmentosa (rp) phenotype (gerding et al. 2011) . in order to gain insights into the molecular disruptions in cilia structure or function lead to a class of human disorders called ciliopathies. joubert syndrome is characterized by a wide spectrum of symptoms, including a variable degree of intellectual disability, ataxia, and ocular abnormalities. here we report a novel homozygous missense variant (c.223 g>a; p.g75r) in the arl13b gene, which we identified by whole exome sequencing of a trio from a consanguineous family with multiple affected individuals suffering from intellectual disability, ataxia, ocular defects, and epilepsy. the same variant was also identified in a second family. we saw a striking difference in the severity of ataxia between affected male and female individuals in both families. functional analysis demonstrated that dihydrotestosterone treatment of sh-sy5y cells induced a down regulation of arl13b expression. both arl13b and arl13b-p.g75r expression rescued the cilia length and shh defects displayed by arl13bhennin (null) cells, indicating that the mutation did not disrupt either arl13b function. in contrast, arl13b-p.g75r displayed a marked loss of arl3 guanine nucleotide-exchange factor activity, despite retention of its gtpase activities, highlighting the correlation between its loss of function as an arl3 guanine nucleotide-exchange factor and joubert syndrome. s. renner, a. busch, t. bierhals, j. butter, v. kolbe, g. rosenberger institute of human genetics, university medical center hamburg-eppendorf, hamburg, germany taad (thoracic aortic aneurysm and dissection) is a heterogeneous disease that often remains silent until a life-threatening complication occurs. it belongs to the connective tissue disorders and causes 1% of death in industrial countries. several disease genes have been already identified; however, about 50% of patients with taad-associated syndromes do not show a mutation in these genes. thus, further heterogeneity is obvious. since individual risk stratification and therapeutic options highly depend on the individually mutated gene, it is very important to identify more disease genes which are aimed to be found by whole exome sequencing (wes). many of the known disease genes encode for proteins that are important for the structure and stabilization of the extracellular matrix as well as for the contraction of vascular smooth muscle cells. one central pathway is the tgf-beta signaling which functions among other proteins via the tgf-beta receptor, its ligand and its downstream target smad2/3. our project plan includes (i) exome sequencing both in affected individuals within families as well as in sporadic patients, (ii) filtering of raw data and prioritization of sequence variants by using a bioinformatic in house pipeline, (iii) verification of novel putative disease genes in a cohort of 200 mutation-negative patients with taad spectrum disease and (iv) functional analyses to gain deeper insight into the pathobiology of taad. in a first round of wes analysis and variant prioritization, we identified a highly conspicuous sequence variant in three family members with taad. screening of the respective gene in a large cohort of mutation negative patients revealed another variant in two siblings with taad. structural and functional considerations strongly support deleterious effects for both identified putative pathogenic missense variants that affect a novel cell cycle-and/or apoptosis regulating protein. functional analyses did not show an involvement of this protein in tgf-beta signaling. in ongoing experiments, we focus on mutation-induced consequences on cell proliferation, cell cycle progression and apoptosis. indeed, we found inhibitory effects of the missense variants on proliferation by affecting the cell cycle key protein cdkn1a. we hypothesize that dysregulation of proliferation and/ or apoptosis of specific cells, e. g. smooth muscle cells, underlies taad.abstracts for screening. positively tested compounds were reanalyzed by whole-cell patch clamp recordings and cell volume measurements. results: the halide assay revealed reproducible halide permeability across wells and, as a control, reliably detected mdckii cells overexpressing wild type best1 by a decrease of yfp fluorescence to 70% following 60 seconds iodide stimulation. cells harboring mutant best1 showed 85% of default yfp fluorescence after the same time interval. conclusion: the current study established an assay appropriate for high and small-scale compound screening targeting best1 localization and function. this assay will be used to screen for compounds in mutant cells lines best1-t6p and best1-y227n for their ability to improve trafficking to the pm or correcting protein folding to enhance ion permeability. background: neuropeptide y-y2 receptor (y2 receptor), an auto-receptor of neuropeptide y (npy) and attractive guanine nucleotide (g) protein-coupled receptor target, has been implicated as a potential therapeutic target for many clinical conditions, including epileptic seizure, depression, pain, and alcoholism. in huntington's disease (hd) patients and animal models of hd, npy-expressing striatal interneurons are selective preserved and increased with advancing disease. however, the potential role of y2 receptor in hd pathology remains under-explored. aims: to investigate whether activation of y2 receptor using npy and selective y2r ligands could ameliorate behavioral deficits and neuropathology in r6/2 mouse model of hd. methods/techniques: npy and selective y2 receptor agonist npy13-36 were intranasally administered to r6/2 mice, five days in a week, beginning from 4 weeks of age until 12 weeks of age. in the second study, r6/2 mice received daily intraperitoneal administration of selective non-peptide y2 receptor antagonist (sf-31) to selectively block y2 receptor. results/outcome: intranasal application of npy showed significant increase in rotarod performance compared to the saline and sf-31 treated r6/2 mice (*p < 0.05 and **p < 0.01 at 8 and 12 weeks of age respectively, n = 12). however, treatment with npy13-36 showed a clear trend towards increased rotarod performance at 12 weeks of age compared to the saline and sf-31 treated r6/2 mice but the difference did not reach significance. also, treatment with npy and npy13-36 showed no significant effect on body weight loss in r6/2 mice, contrasting with previous data obtained with single intracerebroventricular (icv) injection of npy in r6/2 mice. furthermore, intranasal application npy or npy13-36 led to decrease in mutant huntingtin (htt) aggregation and mediated increase in dopamine-and camp regulated phosphoprotein (darpp-32) and brain derived neurotrophic factor (bdnf) levels. additionally, we found that npy and npy13-36 attenuate microglial activation, inducible nitric oxide synthase (inos) expression, and proinflammatory cytokines production in r6/2 mice compared to the saline and sf-31 treated r6/2 mice. conclusion: taken together, our findings suggest that targeting npy-y2 receptor might be a potential neuroprotective therapy for hd and other neurodegenerative diseases. best of both world's: a novel, rapid capture protocol that overcomes drawbacks associated with dna fragmentation in established methods j. seggewiß, c. ruckert, p. wieacker institute of human genetics, westfaelian wilhelms-university of muenster, muenster, germany rapid capture protocols are an attractive proposition for clinical sequencing labs, as they enable quicker sample-to-sequencing turnaround times. the fragmentation of input dna for the construction of pre-capture libraries is a bottleneck in established protocols. mechanical shearing is the gold standard, but is laborious using single-tube covaris instruments; and higher-throughput instrumentation is cost-prohibitive to many smaller labs. "tagmentation"-based methods (e. g. the nextera rapid capture system from lllumina, or agilent's sureselect qxt system) employ transposases for fast ond simple library construction. however, these protocols are associated with significant sequence bias, especially with low-quality ffpe samples and are extremely sensitive to dna input -thus requiring meticulous quantification of viscous, high-molecular weight dna. here we describe a newly-developed rapid capture protocol that combines the kapa hyperplus kit (kapa biosystems) with integrated, low-bios enzymatic fragmentation, and agilent's proven sureselect xt target enrichment technology. the streamlined method follows for the preparation of high-quality, sequencing-ready libraries in one working day. the novel enzymatic fragmentation reagent does not require careful quantification of input dna, yielding reproducible fragmentation profiles optimal for capture over the 5fold range-tested (50-250 ng) the single-tube kapa hyperplus protocol results in very efficient conversion of input dna to precapture library thereby decreasing duplication rates and increasing the complexity of the library going into the modified, 90min sureselect fast xt hybridization protocol. our protocol represents a significant improvement for fast routine diagnostics, where robust and reproducible pipelines ore needed to support timely treatment decisions. lmj. braun, a. milenkovic, bhf. weber institute of human genetics, regensburg, germany purpose: human bestrophin-1 (best1) is a chloride channel controlled by ca 2+ and cell volume and is localized at the basolateral membrane of the retinal pigment epithelium (rpe). so far, there is no therapy for the best1-associated diseases, of which the most common is best vitelliforme macular dystrophies (bvmd). in this study, we developed an assay applicable for high and small-scale compound screening targeting best1 localization and function. methods: to assess best1 channel function we developed a halide assay. briefly, mdckii cell lines were established, stably expressing wildtype best1 or bvmd-associated best1 mutants together with a yellow fluorescent protein (yfp)-based halide sensor. in polarized mdckii cells, wildtype best1 and best1-r218c localize regularly at the basolateral plasma membrane (pm) while best1-l224m and best1-y227n appear significantly reduced in quantity and grossly mislocalized to cytosolic compartments. cells were stimulated with extracellular addition of iodide known to pass the pm through anion cannels and, as a consequence, intracellularly quench yfp fluorescence. variations in yfp fluorescence levels as a marker for best1 function were recorded in 96 well plates by a plate reader setup. a small-scale 2,560 compound library, commercially available as spectrum collection (microsource discovery systems, gaylordsville, usa) was used key: cord-006860-a3b8hyyr authors: nan title: 40th annual meeting of the gth (gesellschaft für thromboseund hämostaseforschung) date: 1996 journal: ann hematol doi: 10.1007/bf00641048 sha: doc_id: 6860 cord_uid: a3b8hyyr nan the variable molecular weight (mw) of vwf is due to differences in the number of subunits comprising the protein. it is assumed that endothelial cells secrete large polymeric forms of vwf and that smaller species arise from proteolytic cleavage. vwf has two main properties: it stabilizes factor viii protecting it from inactivation by activated protein c or factor xa, and it mediates platelet adhesion to subendothelium of the damaged blood vessel wall. each vwf subunit contains binding sites for collagen and for platelet giycoproteins gp ib and gp iib/i~a. multiple interactions of the multivalent vwf lead to extremely strong binding of platelets to subendothelial surface, that is capable of resisting high wall shear rate in the circulating blood. only the largest multimers are hemostatically active. lack of the largest vwf multimers was observed in patients with yon wiuebrand disease type 2a. unusually large molecular forms of vwf were found in patients with thrombotic thrombocytopenlc purpura. proteolytic enzyme(s) may be involved in the physiologic regulation of the polymeric size of vwf and thus play an important role in the pathogenesis of vwf abnormalities in some patients with congenital or acquired disorders of hemostasis. we have purified (-10,000-fold) from human plasma a vwf degrading proteas¢ using affinity chromatography and gel filtration. the proteolytic activity was associated with a high mw protein (mr -300 kd). vwf was resistant against the protease in a physiologic buffer but became degraded at low salt concentration or in the presence of 1 m urea. proteolytic activity had a ph optimum at g-9 and was not inhibited by serine protease inhibitors or sulfl~ydryl reagents. inhibition by chelating agents was best reversed by barium ions, the observed properties of the vwf degrading enzyme differ from those of all hitherto described pretenses. analysis of cleaved vwf showed that the peptide bond 842tyr-g43met had been cleaved -the same bond that has been proposed to be cleaved in rive. the endothelium releases the vasodilator nitric oxide (no) and the vasoconstrictor endothelin (et)-i. no is formed from l-arginine via the activity of constitutive nitric oxide synthase (cnos or enos). an inducible form of nos (inos) is activated by cytokines. no activates guanylyl cyclase in vascular smooth muscle and platelets, leading to the formation of cgmp which induces relaxation or platelet inhibition, respectively. in vessels, no is responsible for endothelium-dependent relaxations; in vivo it exerts a vasodilator tone which can be enhanced by shear forces and receptor-operated agonists such as acetylcholine, bradykinin, thrombin, atp and adp. infusion of no-inhibitors in vivo leads to vasoconstriction and increases in blood pressure and oral administration to hypertension in the rat. within the endothelium, no inhibits et gene expression and release of the peptide via cgmp. hence, no-induced hypertension is associated with increased plasma et levels. et, a 21-amino acid peptide, has potent vasoconstrictor properties via eta-and in part etb-raceptors on vascular smooth muscle. in endothelial cells, et activates etb-receptors linked to no and prostacyclin formation. under basal conditions, little et is formed, but is increased by thrombin, angiotensin ii, arginine vasopressin, cytokines and ox-ldl. et antagonists have been developed and allow to study the effects of et in vivo. et and no most likely play an important role in disease states such as hypertension, atherosclerosis, coronary artery disease, heart failure, pulmonary hypertension and subarachnoid hemorrhage. clinical trials to further define their role in these disease states are now under way. in summary, the endothelium is an important regulator of vascular tone and structure in vitro and in vivo. in disease states, their interaction is imbalanced leading to enhanced vasoconstriction, thrombus formation and structural changes of the blood vessel wall. pharmacological tools aiming to inhibit those changes are now being developed. j.m. harlan, r.k. w/nn, s. sharar, and n. vedder universit3, of washington, seattle, washington ischemia-reperfusion injury has been implicated in the pathogenesis of a wide variety of efinical disorders. [n preclinical models, tissue damage .clearly occurs during ischemia, but, parado.,dcally, may be exacerbated during reperfusion. this reperfusion injury appears to involve activation of the intlammato~, cascade with generation of complement 'components, lipoxygenase products, and chemokines as proximal mediators and neutrophils as final effectors of vascular and tissue damage. we have examined the role of leukocyte adhesion in reperfusion injury in two models -the rabbit ear as a model of isolated organ injury and hemorrhagic shock and resuscitation in the rabbit and primate as a model of traumatic shock and multiple organ failure. data regarding the efficacy, timing, and safety of leukocyte adhesion blockade using selectin-or integrindirected reagents in these models w/ll be presented. the current status of anti-adhesion therapy in other pre-clinical models and early clinical trials will be re~4ewed. an amidolytic assay for the determination of activated protein c (apc)-resistant factor va (fva) has been developed. this assay measures the cofactor activity of fva in diluted plasma samples via the rate of thrombin formation. the apc response is calculated from two fv determinations: one performed in the presence (apc-fv) and one in the absence of recombinant apc. the apc-fv activity is expressed as percentage of the initial fv activity and indicates the sensitivity of fva to apc. normal ranges were established by analysing plasma samples of 100 healthy individuals and an apc-fv activity above 60% was found to be indicative for apc-resistance (apc-r). in a control group of 34 patients the apc-r assay gave abnormal results in 15 patients. dna analysis confirmed heterozygous fv r506q mutation in all 15 patients and confirmed the non-carrier status in all of the 19 patients yielding normal results. an aptt-based apc-r assay performed on the same group of patients showed abnormal results in two of the non-carrier patients. one of these patients was diagnosed as positive for lupus anticoagulant, whereas the reason for the wrong positive result in the second patient remains unclear. eleven patients were analyzed before start of oral anticoagulation and during oral anticoagulant treatment. comparison of the assay results demonstrate a correlation of 96% indicating that the assay is independent of the activities of vitamin k-dependent clotting factors. the apc-r amidolytic assays allows specific and sensitive detection of fva-resistant to apc. the assay is performable in plasma samples of all persons in whom the diagnosis of apc-r may be indicated. in patients treated with oral anticoagulants or showing other clotting abnormalities affecting the aptt the apc-r amidolytic assay is helpful to establish the diagnosis of apc-r. dept of pediatrics, university hospitals kiel and mtinster, germany resistance to activated protein c (apcr), in the majority of cases associated with the arg 506 gin point mutation in the factor v gene is present in more than 50 % of patients < 60 years of age with unexplained thrombophilia. to determine to what exteut this relativdy common gene mutation affects the risk of thromboembolie events in infants and children, its occurrence was investigated in a population of children with unexplained venous or arterial thromboernbotism: thrombosis of the central nervous system (cns, n=4), vena portae (n=4), deep vein thrombosis (n=4), vena caval occlusion (n=4), neonatal renal venous thrombosis (rvi'; n--3), neonatal stroke (n=14), stroke (n=3), arteria femoralis ocdusion (n=2). four ont of these 38 patients showed a positive history of unexplained familial thrombophilia. apcr was measured in an activated thromboplastin time (afit) according to dahlbtick. the results were expressed as apc-ratios: clotting time obtained using the apc/caci2 -solution divided by dotting time obtained with cac12. concerning the special properties of the childhood hemostatic system, infants and children with apcr < 2 were considered to be apc-resistent only when the results were confirmed in a 1: l 1 dilution with factor v deficient plasma (instrumentation laboratory munich, germany). plasma of 268 healthy children served as controls. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mul i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. consistent with the ap'it based method 10 out of 19 children with venous (v) thrombosis and eight out of 19 patients with arterial (a) vascular insults showed the common factor v mutation. additional coagulation defects (autithrombin, protein c type i, enhanced antiphospbolipid igg, enhanced lipoprotein (a)) were found in 30% (v) and 28% (a). furthermore, we diagnosed exogenlc reasons (septicemia, postpastal asphyxia, fetopathia diabetica, central line and steroid/asparaginase administration) in six out of 10 (v) and three out of 8 (a) children with thrombosis and apcr. all four patients with a positive family history of thrombophilia (mothers only !) showed the common factor v mutation arg 506 gin. in the control group the prevalence of apcr was 5.1%. the high incidence of additional exengenic factors in children with apcr confirm literature data of previously described inherited coagulation disorders during infancy and childhood: an acquired risk of thromboembolic disorders masks the coagulation deficiency in the majority of patients with an inherited prethrombotic state. furthermore, the incidence of 42 % apc resistant children with arterial insults in this study challenge the view that apcr is associated with venous but not with arterial thrombo-st8. 12 activated protein c resistance and plasminogen deficiency in a family with thrombophilia m, zttger 1 , f. demarmels biasiutti 1, ch. mannhalter 2, m. furlan 1 , b.~e 1 1httmatologisches zentrallabor der universit~t, inselspital, ch-3010 bern 2klinisches institut fur medizinische und ehemische labordiagnostik, universit~t wien, a-1090 wien several hereditary defects of the proteins regulating blood coagulation have been associated with familial thrombophilia. since the recent discovery of activated protein c (apc) resistance due to the factor v rf06q mutation as a highly prevalent hereditary risk factor for venous thromboembolism (tel, evidence is accumulating that familial thrombophilia may be due to a combination of genetic defects. thus, protein c-or protein s-deficient patients having suffered from te seem to be more likely to carry the factor v r506q mutation than expected from its allelic frequency in the population. we report a family (see figure) in which plasminogen deficiency (0.60 u/ml) had been found in the propositus having had twice postoperative deep vein thrombosis (dvt) at ages of 29 and 31 yrs, respectively, as well as in 5 family members (0.53-0.69 u/ml). out of these 5 plasminogen deficient individuals, only the propositus' daughter had suffered from recurrent dvt at age <20yrs. reinvestigation of this family in 1995 showed factor v r506q mutation in the propositus, his daughter, an asymptomatic sister and a brother with postoperative pulmonary embolism (pe). his father had had postoperative pe; he is deceased and could not be examined. ~, [] plasminogen deficiency ~ ,rll factor v rf06q mutation /~ propositus ~¢ bistory of dvt and/or pe e superficial phlebitis j2~,,~ not investigated "1" deceased even though this family is small for establishing unequivocal association of te with known defects, the two most severely affected individuals with recurrent te at ages <30yrs had combined plasminogen deficiency and apc resistance whereas those with isolated plasminogen deficiency were asymptomatic. these data support the concept of multigenie interactions leading to familial thrombophilia. resistance to activated protein c (apc resistance) is the most common dsk factor for venous thrombosis (vt). in most cases apc resistance is caused by a single point mutation at position arg 506 in the factor v gene (factor v leiden). while ample data in hetarozygous patients have been published, reports in homozygous patients are limited. we studied 29 patients (12 males [m] , 17 females it]) in whom a homozygous mutation had been verified by dna analysis. the median age at the time of the study was 38.8 years (y) (range 9-83 y). twenty-five patients had experienced vt (10 m, 15 f). four patients were discovered dunng family studies and were asymptomatic, three were children (between 9 and 13 y) and one patient was a 62 y old man. in males the first thrombosis occurred at a median age of 38 y (range 21-82 y), in females this was at a significantly younger median age of 26 y (range 17-49 y). twelve of the 15 symptomatic females had taken oral contraceptives (oc, estregen content 0.02-0.ling) for 6 to 150 months (median 71 m) pdor to thrombosis. in 2 women vt occurred during pregnancy, in 1 female it was precipitated by hormone replacement therapy. in contrast, in 8,<10 males the thrombosis happened spontaneously, in 2 males it followed surgery. the sites of thrombosis were dvt in 9 males and 14 females, dvt and pulmonary embolism (pe) in 4 females and 1 male, dvt and caval vein thrombosis in 1 female and superficial thrombophlebitis in 4 males and 1 female. eight females had at least one pregnancy, in total 11 children and 5 abortions. two had thrombotic events during pregnancy and 2 after delivery. all homozygous patients showed apc ratios between 1.12 and 1.68 (mean 1.39 + 0.19). conclusion: patients with homozygous fv leiden have similar clinical symptoms as patients with deficiencies of antithrombin-, protein c or protein s deficiency. however, in contrast to these defects a very high dsk dudng oral contraceptive medication leading to an ealier manifestation in females can be observed. several synthetic (efegatran, argatroban, inogatran and napsagatran) and recombinant (hirudin, peg-hirudin and hirulog) antithrombin agents are in different stages of nlinical development for cardiovascular and thrombotic indications. while the specificity of these agents for thrombin is a concern, little has been done to study the effects of these agents on other serine proteases involved in coagulation and fibrinolytic processes. fihrinolytic compromise by site directed thrombin inhibitors has been reported recently (thromb res 74(3): 193-205, 1994) . while these agents have been shown to inhibit plasmin and related enzymes, little or no informatienentheir effects onthe generation and functionality of apc is available. sinceapc plays an increasingly important role both as an antienagulant enzyme, by inhibiting factors v and viii, end a pro-fibrinelytic enzyme, by stimulating the release of t-pa fi'om endothelial sites, an inhibition of apc may result in both pro-coagulant state and fibrinolytic deficit. representative thrombin inhibitors (dup 714 -a prototype boronic acid peptide derivative, efega~an, argatroban, hirulog, hirudin and feg-hirudin) have been compared for their ability to inhibit apc (american red cross). these bionhemically defined studies in which the remaining activity of apc after incubation with a thrombin inhibitor was determined speclrophotometrically with a ehromogenic substrate (s-2288, pharmacia, franklin, oh) , demonstrated that dup 714 and efegatran inhibit apc in a coneentrafinn dependent manner (ic~0=0.75 and 8,4 gm resp~tively), hirnlog inhibits apc weakly (60 p2¢i produced only 25% inhibition), while argatroban and ~ have no anti-apc activities, while hirulog, hirudin and argatroban produced no direct enti-apc activities, it is conceivable that they may inhibit thrombomodnlin-bound thrombin and thus prevent activation of protein c, resulting in a functional apc deficit and failure to improve clinical outcomes despite higher dosage. while initially it was thought that sole targeting of thrombin will provide monospacifin anticoagulant agents devoid of some of the adverse effects observed with heparin, the recent clinical trials clearly suggest that thrombin is not the only determinant of thrombogenesis. furthermore, potent antithrombin agents such as hirudin, hirulog and peptides, indirectly inhibit the generation of apc, by compromising thrombomodulin-bound thrornhin and such agents as efegalran and dup 714 also produce direct apc inhibition. endogenous inhibition of formed ape by thrombin inhibitors may therefore compromise the feedback regulatory funetiens of apc and may lead to thrombotic amplification in fully enticoagulated patients. these studies warrant prcelinlcal assessment of thrombin inhibitors to evaluate their relative inhibitory effects on apc. poor anticoagulant response to activated protein c (apc resistance) causes a significant portion of deep vein thrombosis (dvt) whereas its association with coronary artery disease (cad) and myocardial infarction (md is still controversial. therefore, we investigated 346 recently hospitalised patients suffering from cad with or without previous mi. the cad was proven by coronary angiography. apc resistance was analysed by using the ap'l-i'-based assay coatest apc resistance (chromogenix). eleven patients showed an apc sensitivity index below 2.0 viewed as apc resistance. using pcr technology, the factor v mutation causing apc resistance 11691 g "-) a) has been shown in nine of these eleven patients. this represents 2.6 % (9/346), compared to 8,5 % found in healthy blood donors (8/94). one homozygous carrier (male, age 76) was identified (apc sensitivity index 1.4) who suffered from dvt at age 59. recent angiography demonstrated diffuse cad, no thrombotic events were reported in his family. in contrast, multiple thrombotic manifestations (dvt, mi, stroke) occurred in the relatives of four heterozygous patients. we conclude that the prevalence of apc resistance is rather low in patients with cad. nevertheless, the natural history of coronary manifestation of apc resistance seems to vary, probably depending on the presence and severity of cardiovascular risk factors. resitanee to activated protein c (apc resistance) is the most cormnon hereditary cause of thrombophilia and significantly linked to factor v leiden pcr based methods are used to identify the crucial point mutation in the factor v g(me. we designed primers in order to identify factor v leiden by allele-specific pcr amplification. amplification specificity for factor v was ensured by the 3'primer fv1001, located at the introng/intronl0 border of the g~ae. one sense and two antisense primers were used in ~vo separate primer mixes specific for factor v arts06 (wildtypo) or factor v otn506 (factor v leiden) yielding 235bp products each. in each pcr reaction a pair of primers amplifying a fragment of the human growth hormone gene was included, fimctioning as an internal positive amplification control (429bp pcrfragment). after an initial denaturation step 10/.tl samples (100rig genomic dna) were subjected to 10 two-temperature cycles fouowed by 20 threetemperature cycles. for visualisation 8p3 of the amplification product were run on a 2% agarose gel presmined with ethidittm bromide. the presence or absence of specific pcr amplification allowed defiu/te allele assignment without the need for any postamplifieation specificity step. the in~ernal positive control primers it~cate a sucessf-u/pcr amplification, allowing the assignment of homozygosity. in a prospective study 126 p~e.ients with tlaromboembolic events were analysed using this technique and enmpared with pcr -rflp according to bertina et al. the concordance between these techniques was 100 %. in 27 patients a heterozygous factor v ohas06 mutation was detected, whereas one pa6ent with recurrent thrombcembolism was homoz-ygous. no false-positive or false-negative results worn observed in the homozygous as well as hcterozygous samples. in addition in 15 samples identified to carry the point mutation by al/ele-specifin pcr anxplification automatic :~equencing confirmed the heterozygous or homozygous point mutation. due to its time-and cost-saving features allele-specific amplification should be considered for screening of factor v leiden. background: an initial intravenous course of tmfractionated heparin ~ljasted on the basis of the activated partial thromboplastin time is the currmt standard treatment for most patients with venous thrombosis. low-molecularweight hqmrin pre~a~tious can be administered subcutaneously, once or twice daily, without laboratory monitoring. we compared the relative effic.~y and safety of low-molecular weight heparin versus anfractionated heparin for the initial treatment of deep venous thrombosis. methods: english-language reports of randomized trials vtta'e identified th~ a medline search (1984 through 1995) and a complementary extensive manual search. reasons for exclusion from the analysis were no hepada dosage adjustments, the lack of um of obj~tive tests for deep venous thrombosis, dos~ranging studies that used higher doses of low-molecularweight heparin than are ctareatly in use, and the failure to provide blind endpoint ~sossmeat. we assessed the incidence of symptomatic recurrent vinous thromboembolic disease, the incidence of clinically ii~t bleeding and mortality. results: twelve of the 21 identified trials satisfied the predetermined criteria. the relative risk reductions for symptomatic thromboembolic complicatious, clinically ~t bleeding, and mortality varied firom 20.60% aad were all statistically significantly in favor of low-molecular-wedght hqtmrin. coadusions: low-mol~ular--weight hoparim administered subcutaneously in fixed doses adjusted for body weight aml without laboratory nmaitori~ =re more effective and safea" tlum adjn~_-dose standard h~. sauce low molec~dar weight hqmrim vary in o~apositiou =ad pharma~ogical im)fil~ the benefits of each ~ shodd ~tabllsbcd separately. unfraetionated hcparin (uh) and low molecular weight heparin (lmwh) are widely used for the prevention and treatment of thrombotic disorders. uh and lmwh induce platelet aggregation in vitro. rgd peptides compete with fibrinogm for the binding to the glycoprotein receptor (gp lib-ilia) of platelets and inhibit platelet aggregation. to inhibit the heparin-indueed platelet ~tion and prolong the half-life in blood of rgd peptides, we linked ac-rgdv-ssggs-ahx-yk eovalently to lmwh in a ratio 1:1. the peptide is composed of three regions: a. rgd-gives the specificity for the receptor gp lib-ilia; b..ssggs-ahx-is the spacer between carrier and ligand, which should facilitate the intnraetion between the conjugate and the gp lib-ills receptor; c. -yk arc functional antino acids for iodination (y) and for covalent attachment (k) to the cattier lmwh. the aggregation achieved with different concentrations of lmwh, lmwh-eonjugate and lmwh/rgd-peptide mixture in a ratio 1:1 was mea.~ared after 20 rain.; maximum aggregation after platelet activation with 10 pm adp was set equal to 100%. platelet aggregation in normal human plateletrich eitrated plasma (prp; 220 000/p.l) was induced by lmwh in a dose ~ndent manner. heparin can induce antbodies which interact with platelets and endothelial cells. this causes thrombocytopenia and thromboembolic complications. hitpatients do need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory prooven hit (hipa-test) with recombinant (r-)hirudin. as these patients had been preseleeted by their immunological response during heparin treatment and the treatment duration of the study was longer than in any other study using thlrudin, all patient samples were investigated for anti-r-hirudin antibodies himdin antibodies were screened by a sandwich elisa using r-hirudin fixed to the solid phase as antigen. all plasma samples were screened for anti-hirudin antibodies of the igg class, but solar only a suset of samples for lge anti-r-himdin antibodies. 38 of 82 patients (46.3%) developed anti-hirudin antibodies of the igg class. anti-hirudin antibodies were not detectable not before 6 days of r-hirudin administration solar no ige anti-hirudin antibodies were found. none of the patients devdoped thromboeytopenia or allergic symptoms. however, in a subset of patients the anti-hirudin antibodies enhanced the anticoagulatory effect of r-hirudin. in 5 patients the hirudin dosage had to be decreased by 2-3 fold to maintain a stable aptt level, in 3 patients, despite stable r-hirudin maintenance dose the aptt increased to values >100 see.. during the study 4 patients with anti-hirudin antibodies had to be reexposed to a second course of r-hirudin for parenteral anticoaguhtion none of these patients developed any allergic reaction. in conclusion we found a high proportion of anti-hirudin antibodies in hatpatients treated with r-hirudin for more than 7 days. these antibodies seem to have minor clinical relevance in regard to allergic reactions. however, one has to consider that these antibodies may influence the pharmacokinetics of rhimdin and thereby enhance its antieoagulatory potency. therefore, aptt must be monitored closely in patients receiying r-hirudin for more than 5 days a major concern in the use of hirndin, the most potent and specific thrombin inhibitor, is the risk of bleeding associated with the potential effect of this drug on hemostasis, particularly when the antithrombotic therapy is combined with invasivo procedures, fibrinolytic treatment, or patient's predisposition to abnormal bleeding. thus, availability of an antagonist to hirudin would be essential for instant neutralization of the antithrombotie action. however, thueh a hirudin antagonist is unknown in nature. to prepare an antagonist to hirudin, a mutant derivative of human prothrombin, in which active site aspartate at position 419 is replaced by an asparagine, has been designed, expressed in recombinant chinese hamster ovary cells, and purified to homogeneity. d419n-prothrombin was converted to the related molecules d419n-meizothrombin and d419n-thrombin by limited proteolysis by e. carinatue venom and o. scvutellatus venom, respectively. both d419n-thrombin and d419nmeizothrombin exhibited no thrombin activity and titration resulted no detection of the active site. however, binding to solid phase immobilized hirudin and fluorescence studies confirmed that the binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins, hi vitro examinations showed that d419n-thrombin and d419nmeizothrombin bind to immobilized hirudin, neutralize hirudin as well as in the purified system and in human blood plasma and re-activate the thrombin-hirudin complex. animal model studies confirmed that d419nthrombin and d419n-meizothromi.,in act as hirudin antagonist in blood cireulatlon without detectable effects on the coagulation system. while i.v. injections of hirudin in mice resulted in an increase in partial thromboplastin time, thrombin time and anti-thrombin potential, additional injections of d419n-thrombin and d419n-meizothrombin resulted in a normalization of these coagulation parameters. elevation of plasma homocysteine is a hereditary disorder of methionine metabolism associated with a high risk of arterial vascular disease. however, as yet relatively little attention has been directed towards the association between hyperhomocysteinemia and juvenile venous thromboembolism (vte). consequently the aim of our study was to evaluate the prevalence of hyperhomocysteinemia (hyper-hcys) and juvenile vte. patients: 85 patients (29 men, median age 42 ys; 56 women, median age 35 ys) who had at least one verified episode of vte before the age of 45 ys were investigated in regard to their total plasma hcys levels. none of the patients had renal or liver dysfunction or evidence of any autoimmune or neoplastic disease. methods: plasma total homocysteine levels were determined by hplc with fluorescence detection. hyperhomocysteinemia was defined as hcys levels exceeding the upper limit of the normal range obtained in our laboratory from 80 healthy control subjects (40 males, median age 25 ys, hcys 95% ci: 2.02-5.67 pmol/l; 40 females, median age 27.5 ys, hcys 95% ci: 2.99-5.40 ,gruel/l). resuits: 13 out of 85 patients had hyper-hcys, giving a prevalence of 15.3 %. of these 13 patients, 9 were male and 4 female, indicating that the relation between elevated plasma hcys levels and vte may not be as strong in woman as in men. discussion: according to previous reports, our study shows that there is a high prevalence of hyper-hcys in patients with juvenile vte. however, the mechanisms by which hyper-hcys can provoke vte and whether hcys is an exclusive risk factor or if it contributes to other existing predispositions, possibly working as a trigger factor is unknown yet. some authors suggest hcys-iaduced effect on factor v activation or inhibition of thrombomodalin-dependent protein c activation. in addition an influence on thrombocyte aggregation has been postulated. conclusion: measurement of hcys levels may be useful in the evaluation of patients with a history of juvenile venous thromboembolism and could be clinically important as hyper-hcys is easily corrected by vitamin supplementation. detailed determination of the pathogenesis of vte in patients with hyper-hcys should be the aim of further investigations. a deficiency of one of the coagulation inhibitors antithrombin (at), protein c (pc) or protein s (ps) and resistance to activated protein c (apc resistance) are established risk factors for venous thromboembolism (vte). in the majority of patients with apc resistance, the .tug 506 gin mutation (factor v leiden) is present. whereas deficiencies of one of the coagulation inhibitors are rare in the normal population, the allele frequency of factor v leiden is 2-7% in western europe. heterozygous individuals have a 3-7fold, homozygous an 80fold increased risk for vte the typical clinical features of all abnormalities are deep vein thrombosis, pulmonary embolism, superficial vein thrombosis and thrombosis at unusual sites, like mesenteric vein thrombosis or cerebral vein thrombosis. the thrombotic risk is low during childhood, but increases considerably after the 13th year of age. a retrospective study in adult patients out of families with a symptomatic deficiency of at, pc or ps revealed that around 30% of surgical interventions and traumas of the lower extremities were complicated by vte. therefore, these patients should receive thrombosis prophylaxis al~er surgery and trauma if their age is higher than 13 years. pregnancy is associated with a very high risk for vte in individuals with at deficiency and prophylaxis should be initiated already in the first trimester. after delivery, thrombosis prophylaxis is adviced for all females known to have an abnormality. oc increase the risk, especially in at deficient and in homozygous factor v leiden females and are therefore contraindicated in these individuals. oc do also increase the risk for vte in patients heterozygous for factor v leiden and females known to have this abnormality should be discouraged from taking oc or should at least be informed on their increased risk. university hospital-', jerusalem, israel, hospital bcan.iou ~. paris, france, increased frequency of thrombocmbolie events have been observed iu patients with b-thalassemia. our findings of shortened platelet survival and enhanced urinary excretion ofthmmboxanc a: metabolitcs (blood 77:1749 (blood 77: , 1991 suggested an increased platclet activation in tbese patients. we also fouud that isolated thalassemie rbc enhance prothronlbin activation, suggesting an increased membrane exposure of procoagulant phospholipids i.e, phasphatidylserine (am j. hematol. 44:33, 1993) . we now show that annoxin v, which has a high specificity and affinity for anionic phospholipids inhibits pmthrombm activation by factor xa, by binding to thalassemic rbc (ic~, = 0.32 nm). kerckhoff-klinik, bad nauheim ~, medizinische poliklinik bonn 2, institut for immunologie und transfusionsmedizin universit~ll greifswald a antibody-mediated intravascular platelet activation is believed to be the basis for both arterial and venous thrombosis in patients with hat. while the development of arterial thrombosis can explained sufficiently by intravascular platelet activation, it is a matter of discussion whether additional risk factors are involved in the pathogenesis of hat-related venous thrombosis. since resistance to activated protein c (apc) is the most common inherited risk factor for venous thrombosis described the frequency of apc resistance among a population of 68 hat patients has been studied. hat was diagnosed using the heparin-induced platelet aggregation assay and confirmed by the ~4c-serotoninrelease test. the diagnosis of apc resistance was established by two functional assays and genetic analysis. at time of diagnosis of hat, 38 patients showed venous thromboembolic complications. among these, 24 were found positive for apc-resistance. pulmonary embolism was diagnosed in 18 hat patients, 14 of them were apc resistance positive. none of the 18 hat patients who showed exclusively thrombocytopenia were apc resistance positive. early oral anticoagulation (oa) was initiated in 7 patients after the diagnosis of hat has been established. six of these patients developed serious thrombotic complications including skin necrosis. these results demonstrate that apc resistance is an additional and common risk factor for the development of hat-related venous thrombosis. early initiation of oa during an acute episode of hat dramatically increases the risk of thrombosis. therefore, oa in hat patients should be initiated only after platelet counts have been returned to baseline levels and effective parenteral anticoagulation is achieved. controlled trials for primary and secondary prevention of stroke g. de (3aetano, c. cerletti and v. bertel~ consorzio mario negri sud, santa maria imbaro, italy this presentation will review the antithrombotic treatments to prevent ischemic stroke that have been evaluated in controlled clinical trials. in two studies of aspirin therapy for pdmary prevention in male physicians there was no reduction in the incidence of stroke, while that of first myocardial infarction was significantly lowered. similar results were obtained in a prospective study in a large cohort of women taking aspirin daily. the incidence of vascular death was not modified by aspirin in any of these trials. this is possibly due .to an excess of strokes associated to aspirin treatment: indeed the four vascular events avoided in 1000 us physicians under aspirin prevention for five years would result from five myocardial infarction and one vascular death avoided and two additional strokes occurred. oral anticoagulant therapy decreases the relative risk of stroke in patients with non valvular atrial fibrillation. warfarin appears to be superior to aspirin, but the latter drug is a useful alternative when long-term anticoagulant therapy cannot be administered. a metanalysis of about 150 trials and over 100,000 patients with different vascular diseases treated with aspirin (at different doses) and/or other platelet inhibitors showed 25% overall reduction of vascular events including stroke. the optimal dose of aspirin for secondary stroke prevention could not be established. in patients with previous minor strokes or tia there was 22% reduction of vascular events and 23% of non fatal strokes. the avoidance of nine strokes of any cause among the 38 expected in 1000 patients at risk would result from the sum of 10 ischemic events avoided and a haemorrhagic one occurred in excess. ticlopidine was reported to reduce the risk of stroke in two large tdals (one in patients with major stroke), but there is no evidence that it is better or safer than aspirin. we compared the effect of the direct specific thrombin inhibitors, napsagatran (na) and rec. hirudin (rh) with unfractionated heparin (uh) on the further growth of preformed thrombi. as a model of thrombogenesls, an annular perfusion chamber exposing rabbit aortic subendothelium was perfused with native rabbit blood at an arterial wall shear rate (650/s). fibrin and platelet thrombi were allowed to form during a 10min perfusion period after which the test agents were given iv as a bolus and a continuous infusion (3 and 10pg/kg/min, n=6) and the perfusion continued for 20min. the control groups were perfused for 10 or 30 rain (n=6). fibrin deposited and platelet thrombi formed on subendothelium were evaluated by microscopic morphometry. the % surface coverage with fibrin was not reduced in the drug-treated groups since fibrin deposition was similar in the 10 and 30 min control groups (39+8% and 335:6%, respectively, mean:l:sem). platelet thrombus area (ta) in the control groups increased from 24+7pm2/pm after 10min to 97+32pm2/lim after 30rain perfusion. na at 1011g/kg/min reduced ta by 94% to values (6+_2ptm2/ ~m) lower than those of the 10min control group whereas rh at this dose reduced ta by 70% (30-j:141.tm2/i.tm). uh at both doses was ineffective. these findings show that in contrast to uh the direct thrombin inhibitors na and rh inhibit the growth of preexisting thrombi. these results could be explained by the higher potency of na and rh as compared to uh for inhibiting clotbound thrombin (gast et al., blood coagul fibrinol 1994, 5.' 879-87) and suggest that thrombus-bound thrombin is an important modulator of platelet thrombus growth and/or stability in this thrombosis model. platelet adhesion -the initial event of thrombosis -is believed to be completely prevented by intact endothelium. we challenged this theory by superfusing intact human umbilical vein endothelial monolayers with activated human platelet rich plasma utilizing the stagnation point flow adhesio-aggregometer (spaa). the spaa provides flow mediated contact of platelets with the superfused surface. heparinized (3.5 -4.0 u/ml) platelet rich plasma (prp) was obtained from healthy volunteers and activated by addition of adenosine diphosphate (adp 2"10 -6 m). platelet deposition was recorded on-line by video as well as by measuring scattered light. fixed samples were examined by phase contrast and electron microscopy, inhibition experiments were performed with either the tetrapeptide rgds, the non-peptide gpiib/llla-inhibitor ro-43-8857 or a monoclonal antibody directed against the gpilb/llla complex. stimulation with adp prompted platelets to adhere to intact endothelium single or as microaggregates of a diameter of up to 100 micrometer. adhesion was dependent upon convective transport resulting in platelet collision with the endothelial monolayer. infusion of rgds or ro-43-8857 into the flowing, adp-stimulated prp completely prevented platelet adhesion to the endothelium as well as subsequent aggregation. when the inhibitor inflow was stopped while adp stimulation persisted, adhesion and aggregation occurred immediately. re-establishing the inflow of the inhibitors -with still continued adp stimulation -led to disintegration of the adhering aggregates. when prp preincubated with the monoclonal antibody against gpllb/llla was superfused, platelet adhesion to the endothelium and aggregation were irreversibly blocked. our results suggest that convective transport and stimulation of platelets are prerequisites to overcome endothelial thromboresistance and that subsequent platelet adhesion to the endothelium is mediated via the platelet gpilb/llla receptor complex. prevent thrombus formation affer ptca i.p. 8tepanova t, g.v. bashkov 2, l.p.kapralova, 2 s.p. domogatsky ~ cardiology research center t and national haematology scientific cettter 2 russian academy of medical sciences, moscow, russia percutaneous transluminal coronary angioplasty (ptca) results in atheroselerotie plague rapture, vascular wall damage and thrombogenic collagen exposure. subendothelial collagen type i-lll is a very ~rong agonist of platelet-dependent thrombus formation in arteries. the anlithrombotic action of rabbit polyclonal antibodies to rat collagen type i-ill and their chemically synthetized conjugate with monoclonals to human recombinant two-chain/one-chain urokinase type plasminogen activator (rtcu-pa/rseu-pa), cross reacting with rat tcu-pa/scu-pa was studied both an in vifro and in vivo. anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like ~ructures induced by rat collagen immobilized with the polystiroi surface in a condition mimics the high shear rate in the large elastic-type arteries. the short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the platelet-dependent thrombus formation in the arterio-venous shunt in rats by 56_+4 % (p<0.001) as well as by the bispecific conjugate (44_+4%, p<0.001). the treatment of collagen-adsorbed conjugate by rtcu-pa did not increase the autithrombotic effect of bifunctional antibodies. the present date suggest, that the local administration of the anticollagen antibodies at the site of atherosclerotic plague rapture may tm the efficient tool for prophylaxis of platelet-dependent thrombus formation in arteries after ptca. increased levels of certain hemostatic factors have been shown to be related to an increased risk of cardiovascular events. hypercoagulability is suggested to predispose to arterial thrombosis and thereby to participate in atherogcncsis. we therefore assessed fibrinogen, prothrombin fragment 1+2 (fi+2) and yon willebrand factor (vwf) antigen in 80 consecutive patients (aged 59+5 years) with known coronary artery disease (cad) who all underwent coronary angiography. the extent of coronary artery disease was quantified according to modified criteria of the american heart association (total, proximal and distal "score"). furthermore the intima-media thickness (imt) was determined in the carotid and femoral arteries by standardized ultrasonographie measurement, vwf antigen was found to correlate positively with the total and proximal coronary score (r=029, p<0.05 and r=o.36, p< 0.01). while fi+2 showed no correlation with the coronary scores, it was significantly correlated with the imt values in the carotid arteries (r=0.27. p< 0.05). after differentiating tertiles of the parameters patients belonging to the upper tertile of fi+2 concentrations had significantly higher imt values of the carotid and femoral arteries (0.81_+0.11 mm vs. 0.72+_0.13 mm in the lower tertile, p<0.05:1.38_+0.44 mm vs. i. 15-+0.25 ram, p=0.05) whereas in patients belonging to the upper tertile of vwf antigen concentrations the proximal coronary artery score was significantly higher (!.71-+0.59 vs. 1.31+ 0.92 in the lower fertile, p<0.01). fro correlation of fibrinogen concentrations and extent of cad or imt values of the carotid and femoral arteries could be demonstrated. in conclusion procoagnlatory mechanisms as indicated by elevated concentrations of yon willehrand factor antigen and fi+2 may be contributing factors in atherogenesis. we have previously shown that pgei is a potent inhibitor of pdgf-ioducod proliferation of vascniar smooth muscle cells (vscm) and inhibits dna replication by a camp-related mechanism (grol~er et al, 1994) . the present study investigates of whether or not this aatimitogeni¢ activity of pget can be amplified by trapidil, a compound that has been shown recently to inhibit the incidence of restenosis of hmnan coronary arteries subsequenmt to ptca (maresta et al. 1994) , vsmc were prepared from coronary arteries of adult bovine hearts, passagod and kept under standard tissue culture conditions. cells of passage 4-9 wore incubated in serumfree medium for 24h in the presence of indomethacin (3 p.m). addition of pdgf-bb (10 ng/ml) under these conditions stimulated dna-replication as assessed from 'hthymidin lncm'poration, by 3.-4laid above control level. trapidil at 10 idvl caused a minor reduction of pdgf-induced mitogenesis whereas 10t) of the compound resulted in a marked reduction of dna replication by 69% (p < 0.05, n = 4). pgei at 0.3 nm diminished the incorporation rate by t 1% while the simultaneous administration of both pged and trapidil (100 idyll caused a significantly stronger response as seen from n reduction of ~h-thymidine incorporation rate by 82% (p < 0.05, n = 4). as a possible mechanism of action, trapidil might have inhibited phosphodiesterases. to establish this, we measured the camp-depcudont proteinkiaasc (pk) a activity in cell homogenates. trapfdil increased the basal fka-activity from 19% to 31% of the maximum response while the response to pget (10 am) amounted to 55%. coincubation of pgei with trapidil caused a 65% stimulation of pka activity, sugesting a small though detectable inhibition of vscm phosphodiesterases by trapidil at anttmitogenic concentrations. essentially similar results wore obtained when thrombin was used as the mitogenic agent. the data demonstrate a significant antimitogenic effect of trapidil at p.molar concentrations that are in the range of plasma levels after therapeutic administration of the compound in rive. at these concentratrations, pget induced inhibition of mitogenesis is markedly enhanced by trapidil. inc. i~enna, and ~cenlral itematnlogy laboroto~. , university hospital of bern pibrinogen (fg), yon willebrand factor antigen (vwf) and tissue-type plasminogeu activator antigen (l-pal have recently been shown in be independent risk factors for subsequent coronary events in patients with angina pectoris (nejm 1995; 332:635) although paul antigen has also been proposed as a risk factor, conclusive dam showing its predictive value is still lacking. furthermore, we have recently shown in a study investigating 200 survivors of myocardial infarction that not only are fg, t-pa and pai-i significantly increased in these patients when compared to a heahhy conlro[ group, but pci activity is also elevated (7hrornb. tfaemost. 1995;73:1137 abst.) , hi order to obtain cut.off points for the individual parameters, frequency histogram plotl; were transformed into straight line cumulative frequency (probit) plots (thromb i/aemost. 1995;74:718) . the cut-off valu~ for the four parameters were determined as follows: fg at 2.7 g/l, t-pa at 8.7 ng/ml, pal-i at 25 ng/ml and pc[ at 126% of a normal pooled plasma. utilising there cut.off points it was then possible to determine the accumulative discriminatow effectiveness of the parameters. when fg w;qs employed alone as the discriminatow factor, it was observed that 47% (941200) of the coronary heart dir, ease (chd) group eilher had the cul..off value or were below it aud 29% (29199) of the normal group were above the cut-off value, thus, resulting in 47% false ne$atives and 29% false positives. when a second additional risk factor, t-pa wa_~ introduced, the number of false negatives dropped to 21% [i.e. 79% (158/200) had two, risk factors elevated] and the number of false positives to 13% to investigate whether a third parameter could discriminate further, pai-i antigen was used to analyse the rcnudning false positives and negatives. an additional 4% could be detected, resuhing in 83% of the chd group having three risk factors elevated. similarly, the number of normal aubjecta with three parameters elevated dropped by 4% to 9% furthermore. when a fourth parameter was introduced, namely pci, it was round to discriminate a further 8% in the chd group, thereby increasing tile di~riminalion to 91%. the number of false positives dropped to 4%, additionally, determination of pci increased the discrimination of patienta having had multiple infarctions from 88°/= when thrce parameters were mcasured to 100%. from these results it can be concloded that determination of fibrinogen levels alone is not sumcicnt to separate patients from controls as t-pa adds significant discrimination. pai-i antigen which correlated strongly with t-pa did not significantly increase the discriminatory potential of both fg and i-pa. however, by employing pci as a fourth paramctcr, virtually complete separation between the chd and normal groups as well as rurthcr recoguitiou of' patients having had multiple infarctions could be obtained. to test the hypothesis that oral contraceptives (oc) enhance exercise-induced activation of blood coagulation we examined 11 women (27 + 3 (sd) years, bmi 20.4 + 2.0 kg/m 2, vozm.. 49 + 7 ml/kg/min) without oc between day 18 and 22 of the menstrual cycle and 10 women (24 + 2 (sd) years, bmi 20,6 ± 1,6 kg/m', vo2max 47 + 7 ml/kg/min) taking oc (150 mg desogestrel and 30 mg ethinylestradiol) between day 7 and 21 of drug intake. prothrombin fragment 1 +2 (ptf1 + 2) and fibrtnopeptide a (fpa) were measured before and after running for one hour on a treadmill at a speed corresponding to the anaerobic threshold. mean heart rate [191 ± 10 vs. 196 ± 12 min 1) and mean plasma lactate (3.3 ± 1.6 vs. 3.1 + 1.2 mmot/i) wera comparable during exercise between control and oc group, respectively. results for markers of thrombin and fibrin formation were: ptf1 +2 (nmol/i) fpa (ng/ml) control before 0,66 ± 0.19 1.0 + 0.2 after 0.73 + 0.23 2.2 + 1.2" oc before 0.82 + 0.31 1.0 + 0.2 after 1.11 + 0.48* + 5.8 -+ 6.0* + * p < 0.05 vs. baseline, + p < 0.05 between groups. we conclude that oral contraception with 150 mg desogestrel and 30 mg ethinylestradiol enhances exercise-induced thrombin and fibrin formation, our data suggest that exercise testing might be useful for evaluating the risk of thrombosis associated with different compositions of oc. a. haushofer +, wm. halbmayer +, j. radek +, m. dittel *, r. spiel *, h prachar *, j. mtczoch *, m. fischer + + zentraltaboratorium mit thrombose-und c~rinnungsambulanz -krankenhaus lai~: * 4. medizinische ab[eilung mtt ka~liolo$i¢ -krankenhaus lainz und ludwig bottzmann-lnstitut ftlr herzinfarktforsohung, wien fifty-one patients (age 61.6 ± 9.2a; 34 m / 171) implanted with 74 coronary stems 03 palm~-schatz, 27 gianturco-roubin, 14 micro stcnts) received a now antithrombotic treatment using a combination of ti¢lopidine (tic) 2 ×250 mg/d for 28 days and acetyl salicylic acid (asa) zoo mg/d for long-term treatment. patients (pat) only received 15000 tu standard hepartn as i.v. bolus immediately before stent implantation (day l ). side effects and changes in hematological (day i to 7, 14. 28 and 35 [= without t[cil liver and kidney parameters (day 7, 14, 28, 35) were monitored. thirty-eight pat (75%) came for the controls to our del~rtment and were additionally monitored by thromboelastograpy (teg) and bleeding time (bt) (day 2g and 35). the other pat were monitored externally, side effects were reported. thrombin geucration after stenting was monitored from day i to 7 by prothrombin fragment 1+2 (f 1+2) and thrombin-antithrombin-lll-comptex (tat). "k" of the "leg decreased (day 28 vs 35; p< 0.0l ). bt prolongation was negatively correlated with the bodysurf ace area (tic+asa: p< 0.05, asa: p< 0.0 l) and showed a reduction after withdrawal of tic (2 l0 sec, 180/ 300 so: [median, quartiles] vs. 120 sec, 881157 sec; p< 0.ix)01). f 1+2 and tat of day i (blood collection: 0, 2, 4, 6 h after intervention, f 1+2:0.84 nmol/i, 0.68/i.07 nmol/l: tat: 2.6 pg/1, 2.0/4.6 ilg/1) were lower compared to day 2 to 7 (f i +2: 1.31 nmol/l, ].08/i .62 nmol/l; tat: 4.8 pg/i, 3.2/7.2 ijg/1; p< 0.0001). tic scorns not to be a strong thrombin generation inhibitor. during stenting one pat (i.9%) sustained a non penetrating mci and one (1.9%) an ischaemic stroke. tic+asa were very effective, only with one pat (1.9%) stent thrombosis (acute) occurred. side effects: 8/15.7% gastrointestinal (one lead to hospitalization), 5/9.8% hematomas at the needle site in the groin (one surgical intervention), 5/9.8% leucopcnias (one agranulozytosis with hospitalization), 3/5.9% allergic skin reactions and 2/3.9% increased liver enzymes (got, gpt, "pgt, alkaline phosphatase; > 2 × of the j. ). with one pat with gastrointestinal disturbances and skin reactions tic had to be withdrawn and treatment was changed to oral anticoagulatlon + asa. one pat showed a combination of skin reactions, gastrointestinal distufl~aneas and on day 28 a heavy reaction of the liver enzymes ( j. after 5 weeks). a decrease of the white blood count (day 1:7.19 gh, 6.03/8.2 g/l, day 28:5.46 g/l, 4.63/6.47 g/l; p< 0.000 i) could be observed. the safety of the therapy with tic+asa should be elucidated and extensively discussed. the serpins c1 esterase inhibitor (cllnh), antithrombin iii (atiii), alantitrypsin (slat), and a2-antiplasmin (azap) are known inhibitors of coagulation factor xla (fxla). although initial studies suggested al at to be the main inhibitor of fxla, we recently demonstrated cllnh to be a predominant inhibitor of fxla in vitro in human plasma (wuillemin et el., blood 1995; 85:1517) . the present study was performed to investigate the plasma elimination kinetics of human fxla-fxla inhibitor complexes injected in rats. the amounts of complexes remaining in circulation were measured using elisas. the plasma tl/2 of clearance was 98 min for fxla-alat complexes, whereas it was 19, 1 8, and 1 5 min for fxla-cllnh, fxla-a2ap, and fxla-atiii complexes, respectively. thus, due to this different plasma tl/2, preferentially fxla-alat complexes may be detected in clinical samples. this was indeed shown in plasma samples from thirteen children with meningococcal septic shock (mss), a clinical syndrome which is complicated by activation of coagulation, fibrinolytic, and complement systems. fxla-fxla inhibitor complexes were assessed upon admittance to the intensive care unit. fxla-a 1at complexes were elevated in all patients, fxla-c11nh complexes in nine, fxla-atiii complexes in one patient, and no elevated fxla-a2ap complexes were found. we conclude from this study that, (1) although c1 inh is the predominant fxla inhibitor, fxla-alat complexes may be the best parameter to assess activation of fxi in clinical samples, (2) measuring fxla-fxla inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of fxla in rive, and (3) fxl is activated in patients with meningococcal septic shock. dudng the coagulation of plasma about 20 % of the (x2ap present is covalently crosslinked to fibrin by factor xiila (aoki und sakata 1980, thomb. res. 19: 149-155) . we investigated the binding of azap by factor xiila to soluble fibrin (desaabb-fibdno) whose polymerization was inhibited by an isolated fibrin ddomain named d=,,, (haverkate and tiemann 1977, thromb. res. 10: 803-812) . d==. is known to have an intact fibrin-polymerization site and is able to block the prolongation of the fibrin protofibrils at an early stage depending on its concentration. lateral association to fibrin fibers does not take place, since the inhibited protofibnls formed at the conditions used here do not reach a sufficient length (williams et el. 1981, biochem. j. 197: 661-668; hantgan et al. 1983, ann. n. y. acad sci. 408: 344-365) . material and method: soluble desaabb-fibrino was prepared by incubation of (lztl)-fibrinogen (0.48 mg/ml), d=1o (1.91 mg/ml; molar ratio d==o to fibrin 14:1) and 0.4 u/ml thmmbin for 45 min. then q2sl)-c~2ap (16 p.g/ml), faktor ×ill (2 ulml) and ca 2) (5 mmol/i) were added. the crosslinking reaction was stopped at different times of factor xiila-incubation by adding of urea/edtasolution. the suspension was analysed by ultracentrifugation on gradients containing saccharose, urea and sos. re~ultl: the elution profiles of the ultrecentifugation-gradients show the formation of cmsslinked fibrin oligomers of increasing size depending on the time of factor xiila-action. the crosslinked fibrin polymers contained about 80% of the fibrin initially added. although factor xiila acted well, crosslinking of azap in the fibrin oligomers could not be observed. conclusl0n: as we already demonstrated (kelach et el. 1994, ann, hematol. 70 (suppll) : a 60) the crosslinking of azap to fibnn clots depends on the structure of the fibdn network, especially on the degree of lateral association of the fibrin pmtofibdla. in desaabb-fibrino no lateral association of fibrin protofibnls takes place under the conditions chosen here. thus it is consistent with our theory that we did not observe any binding of aiap to the fibrin oligomers of desaabb-fibrino. human pci is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as tissue kallikrein and the sperm protease acrosin. it is synthesized in many organs including liver, pancreas, and testis. the physiological role of pci has not been defined yet. recently, we have cloned and sequenced the mouse pci gene (zechmeister-machhart etal., manuscript in prep.) . this enabled us to study pci gone expression in murino tissues using mouse pci edna and crna probes. by northern blot analysis, mouse pci tar.ha was exclusively found in the reproductive tract (testis, seminal vesicle, ovary), all other organs analyzed -including the liver were negative for pci mkna, indicating that in the mouse pci is not a plasma protein. to determine which cells of the reproductive tract synthesize pci, cellular localization was assessed by in situ hybridization of mouse testis and ovary sections. in testis, pci mrna was present in the spermstogonia layer and in leydig cells, while sertoli cells and peritubular myoid cells were negative. these results are consistent with the immunohistological localization of human pc1 (laurell et al,, 1992) . in the mouse ovary, stroma cells of the medulla and around the follicles were positive for pci mrna. no pci expression was detected in theca or granulosa cells. we also studied the regulation of mouse pci gone expression by steroid hormones in vivo. [n mature male mice castration caused an increase in pci mrna in seminal vesicles, which was reversible upon the administration of testosterone. in tissues of intact adult male and female mice, pci mrna levels decreased after injection of human chorionic gonedotropin (hcg), while in castrated male mice, hco had no effect on seminal vesicle pci mrna. progesterone and 17-b estradiol decreased ovarian pci mrna levels in immature female mice. these data suggest direct down-regulation of mouse pci by sex steroids. the different tissue specific pci-geoe expression in men and mice furthermore indicates a different biological role of this serpin in the two species. ctr. transgene technology, leuven "[' 1-tissue factor ('it) is a 47 kda glycoprotein mainly known a the primary cellular initiator of blood coagulation. whether tf expression may also play a role in development is unknown, but the lack of spontaneous viable mutations of the tf gene in rive leads to the speculation that its absence may not be compatible with normal embryonic development. to determine the significance of "if in ontogenesis, the pattern of tf expression in mouse development was examined and compared to the 'if distribution in human postlmplantation embryos and fetuses of corresponding gestational age. at early embryonic period of both murine (6.5 and 7.5 pc) and human (stage 5) development there is a strong tf expression in both ectodermal and entodermal cells. "if decoration was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express "if in the adult organism. surprisingly, during renal development and in adult organism tf expression differs between men and mice. in humans maturing stage glomerali were "if positive whereas in mice glomeruli were negative and instead epithelia of tubular segments were tf positive. in ncuroepithelial cells there was a striking 'if expression indicating a possible role of'if in neumlation. moreover, there was a robust tf expression in tissues such as skeletal muscle, and pancreas, which do not express in adult. in contrast to tf, its physiologic ligand factor vii was not expressed in early stages of human embryogenesis, but was detectable in fetal liver, the temporal and spatial pattern of tf expression during murine and human development support the hypothesis, that 'if serves as an important mo~hojzenic factor darinz embrvozenesis. to serve as an anticoagulant, protein c (pc) must be activated by a complex formed between the enzyme thrombin (t) and its cofactor thrombomodulin (tm). therefore, downregulation of endothelial cell surface expressed tm, for example, triggered by an inflammatory stimulus, could become a critical factor in effective pc activation. in order to develop a recombinant (r) pc mutant which can be activated independently of the tkm-complex, a peptide sequence including p1-7 in the activation peptide of pc was modified to be identical to the factor xa (fxa)-cleavage site in prothrombin. the mutant was expressed in hu 293 cells, purified and its anticoagulant properties characterized. using purified fxa the mutant showed activation rates between 0.02 and 0.13 nmlmin at pc concentrations between 97 and 770 nm, while the rpc wild type was insensitive for fxa activation. the activation reaction is calcium-dependent reaching maximal activation rates at a calcium concentration of 3 mm and was enhanced to 3.8-fold by addition of anionic phospholipids (pl). in contrast to the wild type pc the rpc mutant was insensitive for activation by the t/i-m complex. addition of the mutant to normal human plasma induces a prolongation of tissue-factor and p-it-based clotting assays. using normal human plasma as a source for fxa the the activation rates of the mutant were found 5-fold higher than in the purified system if tissue factor was used to generate fxa. in conclusion, our data demonstrate that the rpc mutant is effectively activated by fxa in a purified as well as in a plasma system. interestingly, the activation rates are enhanced in the presence of pl and normal human plasma. fudher studies should clarify the potential use of this mutant as a novel anticoagulant. thrombln plays a pivotal role in thrombotic events. the time course of thrombln concentration in blood or plasma after activation is of special interest to answer a variety of questions. with a chromogenic assay developed by hemker et el. [thromb. haemostas, 70, 617, 1993] it became possible to measure the generation of thrombin in activated plasma continuously. inhibitors of clotting enzymes which are to be developed as anticoagulants should be able to inhibit thrombin generation or to immediately block generated thrombin. we have used a test based on hemker's thrombin generation assay to elucidate which potency and specificity an inhibitor of factor xa needs to efficiently block thrombin generation in human plasma. thrombin generation after extrinsic (tissue factor) or intrinsic (ellagic acid) activation was followed using the chromogenic substrate h-~ala-gly-arg-pna (pentapharm ltd.). a series of synthetic low molecular weight inhibitors as well as naturally occurring inhibitors of factor xa with different potency were investigated. because of the inhibition of activated factor x the generation of thrombin in plasma is delayed and the amount of the generated thrombin is reduced. the concentrations which cause a 50 % inhibition of thrombin generation (icso) correlate with the k~ values of the inhibitors. low molecular weight inhibitors with k~ values of about 20 nmol/i inhibit the generation of thrombin after extrinsic activation with icso in micromolar range. after activation of the intrinsic pathway tenfold lower concentrations are effective. the strongest inhibitory activity after extrinsic as well as intrinsic activation is shown by recombinant tick anticoagulant peptide (r-tap) with ic~o of 0.049 pmol/i (axtdqsic) and 0.037 pmo/i (intdnsic). in the compadson of synthetic low molecular weight inhibitors of thrombin end factor xa which have similar k= values for the inhibition of the respective enzyme (lowest i<1 20 nmol/i), factor xa inhibitors are less effective tn the thrombin generation assay. in contrast, the highly potent xa inhibitor r-tap shows a stronger inhibition of thrombin generation than the tight binding thrombin inhibitor hirudin. background: resistance to degradation of coagulation factor v by activated protein c is associated with a point mutation in which adenine is substituted for guanine at nucleotide 1691 in the gene coding for factor v. to date this specifc mutation appears to be the most common inherited abnormality which predisposes patients to venous thrombosis. for this reason a reliable, fast and automatable system for the diagnosis of the described point mutation is required. the conventional methods used to identify the mutation are based on allele-specific restriction enzyme site analysis or direct sequencing. these methods have disadvantages for a large scale dna diagnosis, which include the need for electrophoresis or a high cost and time consumption. methods: an alternative strategy of dna diagnosis, the allele-specific oligonucleotide ligation assay, was adapted for the diagnosis of tile point mutation of factor v. following pcr amplification of the target dna, tile procedure was performed completely automatically on a robotic workstation with an integrated elisa reader using a 96-well microtiter plate. allelespecific restriction enzyme site analysis was performed to confirm the genotypes. results: in 10 patients with the mutation and in 20 individuals without the mutation the genotypes determined with the conventional allele-specific restriction enzyme site analysis were in 100% concordance with the elisabased oligonucleotide ligation assay. discussion: the pck-oligonucleotide ligation assay applied as automated detection system for the identification of the coagul;mon factor v point mutation allows tile rapid, reliable, and large scale analysis of patients at risk for thrombosis. resistance to the asticoa=m~lant activity of activated protein c (apc resistance) has emerged as the most con'anon inherited thrombophilic state. patients lreterozygous for factor v leiden are more likely to suffer from thromboembolie events than controls. this risk is even more pronounced in homozygotes. due to the low sensitivity and speeifity of most coagulation tests some investigators suggest to examine patients for the presence of factor v leiden mutation by pcr-based methods. re e~tly we presented an aptt-based functional test (acceleria inactivation test ait): 1:20 diluted plasma (50bi) is mixed with factor v deficient plasma (50~tl) and aptt reagent (501.d), incubated at 37°c and then coagulation is induced by caci2 and a.pc (50~d). using a standard curve, the clotting time (see) is transferred in per cent accelerin inactivation (%ai). using this test, the widely used apc-ratio as well as pcr-based factor v leiden detection (confirmed by direct sequencing) we prospectively studied 172 consecutive patients with thromboembolic events. patients without the factor v mntation eonsitently showed more flazm 50% al with the exception of one patient with severe factor deficiencies (including factor v) due to hepatic failure and heterozygous for factor v-leiden resulting in 62*/. ai, there was a complete concordance between the pcrbased method and dysaseelerinemia detected by ait. due to these result a specifity and sensitivity of ait above 95 % was calculated. furthermore, a clear discrimination could be obsoved beween 34 heterozygotes (20%<ai<50%) and 8 (incl. 5 samples obtained from jeaa) homozygotes (ai<20%). in contrast a.pc-ratio resulted in a great overlap between patients homozygous for the wildtype , hcterozgous or homozygous for factor v leiden. in addition we confirmed thc di.~eulties nsing the apc-rauo for patients trader anticoagulation, or with different types of other d~rombophilie defects such as lupus anticoagulant or protein s deficiency. due to the time and cost-saving featttres the/kit should be considered for the rapid detection of apc resistance without the use of pcr-based techniques. in summary we found, well in accordance with previous reports, that apcr due to the fv-leiden mutation is • highly prevalent defect in vte pts. pts with the fv-leiden mutation were eharacterised by a higher incidence of vte's without recognised prothrombotic risk factors and more frequent positive family history; no higher incidence of recurrent vte's or other prothrombotie coagulation defects was found; the age at initial manifestation of thrombophitia was comparable. acquired a recently identified mechanism for famdial thrombophilia is characterized by a poor anticoagulant response to actavated protein c (apc), it is now welt recoginzed to be due to a genetic defect in the factor v gene, which results in the syn-,thesis of an abnormal factor v molecule and to be a frequent risk factor for thromb.osis, however apc resistenee can also be found in certain acquired conditions. since pregnancy ~s known to be associated with a hypercongulable state resulting in a prevalance of thrombosis up to 1% and thromboembolic events, we determined the prevalence of apc resistance during the course of pregnancy at the city hoslfital ruesselsbeim. germany. blood samples from pregnant woman were drawn at mmester 1, 2, 3 and at term. besides the routine coagulatton screening, protein c and viii:c, the apc resistance were evaluated. a modified aptt method utilizing human plasma fracnonated apc (amencan red cross. bethesda, maryland, ~usa) was used to assay apc resistance. this method relies on the determination of two aptt's in the patient plasma, one in the absence of apc and one in the presence of a fixed amount of apc. clotting tune was determined after mcubation using the aptt reagent ( we investigated the sensitivity of various species towards activated, human protein c (apc) and found an impaired response in the order monkey < horse < pig < dog < rabbit. we assume that this effect is mainly caused by differences in the apc cleavage region of factor v, as recently described for a factor v mutation in bumat~s: factor v-leiden. this was corroborated by the finding that addition of human factor v similar to the factor v added with the animal plasma had only minor influence on test outcome. coagulation assays dependent on apc, such as for protein c and protein s determination, in the standard apc assay, the more factor v-leiden specific apc assay and in the pcat, a new screening assay for the whole protein c system, are based on the inactivation of factor va via apc. thus, in all these assays, 5% of rabbit plasma added to human plasma mimicked a apc response as observed for heterozygous carriers of factor v-leiden and 20% as for homozygous carriers. we exploited this factor v-leiden like behaviour to prepare plasma standards for calibration of apc dependent assays by mixing rabbit plasma with human plasma~ as reference for these standards the 0% value was assigned to the clotting time in the absence of apc, while the 100% value was assigned to the clotting time in presence of apc as obtained with a human, normal plasma pool. then these calibrated standards were used for establishing reference curves. on automated coagulation analyzers, the results obtained were automatically reported in '% of normal' or '% sensitivity'• this not only eases evaluation, more importantly, this procedure also considerably improves the comparability of results obtained with different reagents and instruments. in order to establish the methods for activated protein c resistance and the faktor v leiden-mutation we investigated 204 healthy individuals (128 women, 76 men, median age 37 ys) using the assay "contest ® apc-resistance" (chromogenix, sweden). the mean apc-ratio was 2.65, the cut-off level for low response to apc was calculated as 2.15, 46/128 women were oral-contraceptive users. they showed a slight decreased apc-ratio (mean 2.46). 24 of the 204 individuals (11,8%) had a poor response to apc (mean ratio 1.94) as a apc-resistance. for detecting the factor v gene mutation we a used non commercial own modified easy to handle pcr-based test. in 14 of the 204 individuals (6.9%) the factor v-leiden mutation could be found as heterozygotes. in this group, the apc-ratio was significant lower (mean 1.87) as in the group with apc-resistanee and without deteetiun of the factor v mutation (mean 2.04). nevertheless in one individual with beterozygote factor v-leiden, the apc-ratio was found in the normal range (2.24) , also in a few individuals with very iow apc-ratio the factor v mutation could not he detected. finally this data suggests a high prevalence of apc-resistance and factor v-leiden mutation in the population of the southwest of germany. resistance to activated protein c (apc) has been reported to interfere in clotting assays for protein c (pc) and protein s (ps) from various manufacturers, resulting in decreased values or even apparent deficiency (type ii') of pc. or is. in our laboratory, when using functional, avit-based assay kits for pc., is, and apc sensitivity from behring (marburg, germany) on an automatic turbidimctric coagulation analyzer (fibrintiraer a, behring), the interference in the is assay has been observed in a very pronounced manner soon after the introduction of apc sensitivity testing into the standard laboratory procedure for patients with thrumbophilia. yet we were not aware of an interferenca caused by apc resistance in the pc test. we therefore evaluated our results for pc eed ps with respnet to apc sensitivity status in a retrospective analysis. exclusion criteria were: oral anticoagulant therapy with vitamin k antagonists, impaired liver function, borderline result in the apc sensitivity test, known analytical intedercnc* other than apc resistance (e.g. elevated factor viii:c). resu/ts: apparant pc activity was slightly (12 %) lower and apparent ps activity was grossly (45 %) lower in ape resistent patients than in patients with normal apc sensitivity: there was no case with a presumed du~l defect of both at~ rnsistaac¢ and pc deficiency. in addition, none of the few patients diagnosed with pc deficiency before the introduction of the apc sensitivity assay turned out to ix: apc resistent. much more of a problem is the known sensitivity of the pc test to an elevated factor viii concentration which is quite a common finding among hospital patients but rarely known in an individual case. by aeccelerating the clotting process, elevated factor viii can cause apparent pc deficiency that can not be confirmed in a chromogunie pc assay. ,clearly, when combined with apc resistance, this problem is exspacted to be more severe. on the other hand, the comhination of both apc resistance and apparent is deficiency was found on a regular basis with many decreased and some bordeflinc values for is but only few in the lower normal range and virtually none in the upper non~al range. also, several #eats previously diagnosed with is deficiency turned out to be pc resistent, and the diagnosis had to be corrected, but dual defects cannot be ruled out. conclusion: for, the interpretation of pc and/or 1:"3 values from clotting assays the apc sensitivity status must be considered. when apc resistance and an apparent deficiency of pc or is is found, a type i deficiency may be confirmed or ruled out using antigen assays, but at present, there seems to be no reliable way other than genetic analysis to address the question of dual defects with presumed type ii deficiency, especially in the case of ps. therefore, there is a clear need for analytical improvements and the development of functional assays that are less sensitive to interference by apc resistance. the most common inherited risk factor for venous thrombosis is the resistance to activated protein c (apc). the main reason for this resistance is a point mutation in the gene of dotting factor v (f v). a nueleotide exchange leads to a different amino acid sequence in the protein and thus to the loss of one of the cleavage sides which is important for the inactivation of the procoagulatory form off v. the imbalance in the coagalation cascade leads to a strong risk for venous thrombosis, which is thought to be inoreaced by factor 7 to 10 for heterocygotes and about 80 for homocygote individuals. the prevalence in patients with a history of thrombosis is about 20%, among apcresistant patients as high as 80%. prevalence-off v ,leiden" is reported to be 2% of the normal population in leiden/netherlands and as high as 15% in a certain city in the south of sweden. due to the obviously different geographic distribution and the highly increased risk of venous thrombosis in carriers of the mutation investigated the distribution of this mutation in the normal population of different regions in germany. first attempts to assess the prevalence off v mutation by molecular methods (polymerase chain reaction followed by restriction fi'agment length polymorphism analysis) revealed that in the area of wiirzburg 7.7% of blood donors carry the mutation, which is significantly different (fischer t-test, level of significance 0.05%) from published data observed in freiburg (3.4%). homburg/saar and harmover revealed 5.8% and 8.5%, respectively, and were not significantly different from wfwzborg. further investigations of blood somples fi:om other regions should give an overview of the geographic distribution of this mutation in germany and might finally help to draw a map era regional risk of venous thrombosis. the regular apc resistance test is not applicable to plasma from orally anticoagulated (oac) or heparinized patients due to decreased levels of vitamin k-dependent clotting factors and to thrombin inhibition by antithrombin, respectively. the former limitation is overcome by prediluting samples 1-1-4 with fv-defident plasma. for this purpose a special quality of fv-deficient plasma has been prepared, which also allows analysis of heparinized plasmas. this fv-deficient plasma has now been evaluated with the coatest apc resistance assay. a complete ape ratio discrimination for the fv q506 mutation is obtained for plasmas from oac and heparin patients as well as from non-treated individuals. ratios above 2.2 are obtained in normal genotype plasmas, whereas heterozygotes for the mutation yield ratios in the range 1.6-2.0. the basal apt times for 15 oacplasmas with widely spread inr-values were reestablished into the normal range. .the stability of the reconstituted fv-deficient plasma was found to be' at least 3 hours at room tempetature, indicating its suitability for .routin.e u.~. f .u~ .e~mv~., the influence of preanalytical procedures is rmm~ single or double cenuifugation as well as freezing of samples do not alter the apc ratios to any significant extent. our results prove the usefulness of the modified coatest apc resistance kit method as a reliable, simple and rapid tool when screening for the presence of the fv q506 mutation. hereditary resistance against the anticoagulatory action of activated protein c (apc-resistance, apcr) which was first described by dahlb~ick et al. (pnas, 90:1004 , 1993 was identified as a possible new thrombophilic factor in a high percentage (17-60%) of young adults with thrombotic events. a single missense mutation (r506q) due to a g/a transition (g1691a) in exon 10 of the factor v gene (bertina et al., nature, 369:64, 1994) is tightly linked with apcr and is regarded as the causative molecular defect. identification of this mutation by pcr-based methods is easy to perform and avoids preanalytie and analytic errors in the eoagulometric assay for apcr. since the impact of this mutation in children with thromboembolic disease has not been determined to date, we initiated a multi-center prevalence study on two pediatric populations, with and without thromboembolie events. we compared 125 pediatric patients with thrombosis, divided into 3 different age groups (0 to <0,5 years; >0,5 to <10 years; >10 to <18 years) with a normal population of 159 children. the mutation g1691a was found with an unexpected high prevalenee of 12% in our normal controls. however, the prevalence was significantly higher in the age groups: 0 to< 0,5 years (25%) and >10 to <18 years (30%). in patients between >0,5 to <10 years the overall prevalence was similar to the control (13%). however in patients of this age with spontaneous thrombosis apcr was also a significant risk factor (29%). our results emphasize the impact of apcr for thrombogenesis in children. however, the significance is agedependent and does possibly reflect the different physiology of haemostasis in our three age groups. activated protein c (apc)-resistanec is a newly reeognised risk factor for thrombosis. in at least 90% of the cases it is caused by a single point mutation in the factor v gene (g->a at nucleotide 1691), which predicts replacement of arginin 506 with ghitamin. one of the apc cleavage sites in factor va is located c-terminal of arginin 506, and mutated factor va (factor v leiden) is resistant to apc-mediated inactivation. from epidemiologic studies it is known, that this abnormality can be found in about one third of patients with thrombosis. apc-resistance is a major basis for venous thromboembolism and is prevalent in about 5.10% of the general caucasian population. recurrent spontaneous abortion (rsa) affects 1-5% of couples and represents a major concern for reproductive medicine. in spite of extensive endocrine, genetic, serologic and anatomic evaluation some 30-40% of rsa women remain unexplained. a frequent morphologic finding in placentae of aborted pregnancies is an increase of fibrin deposition within the intervitlous space. because of these findings we studied the prevalence of apc-resistance in women with rsa (more than 3 miscarriages) of unknown origin. in 2 of 35 cases we found a pathologic apc-resistance, both patients had a history of recurrent thrombosis and were heterozygous for factor v leiden. the prevalance of apc-resistance is 5,7% and thus equals the prevalence in the general population. our data do not support the hypothesis that apc-resistanee is a risk factor for recurrent spontaneous abortion. h~matologisches zentrallabor der universit~t, inselspital, 3010 bern resistance to activated protein c (apc) due to the mutation 506 arg --~ (]in of factor v (factor v leiden mutation) is the most frequent hereditary thrombophilic defect known today, with a prevalence of 20 -50 % in patients with idiopathic venous thromboembolism and of about 3 -5 % in the general population. with an allele frequency of 2 % the expected number of homozygous individuals is about 4 in 10000. homozygous and heterozygons individuals differ considerably with respect to the relative risk of thrombosis (80 -fold versus 7 -fold) as well as to the age of the first thrombotic event (31 versus 44 years). deficiency of the vitamin k dependent protein s (p$), an important cofactor of apc, is another hereditary thrombophilia which is, however, much rarer than apc resistance with a prevalence of 5 to 8 % in patients with venous thromboembolism. factor v leiden mutation as well as ps deficiency are associated with impaired anticoagulatory activity of apc, which is most pronounced in case of the combination of the two defects. the combination of ps deficiency (with an assumed prevalence similar to that of pc deficiency) with heterozygous or homozygous apc resistance can be expected with a probability of 1: ~ 5000 or 1: ~ 500000, respectively. it is well known that ps levels decrease towards the low normal or even subnormal range during pregnancy. moreovar, there is increasing evidence that the sensitivity of plasma to the antieoagulatory effect of apc decreases during pregnancy resulting in an acquired apc resistance. these pregnancy associated effects art obviously much more relevant in case of preexisting ps deficiency or apc resistance and should contribute to the elevated thrombotic risk during pregnancy in a subject with either of the two defects, and even more so for a woman who suffers from both defects. we describe a young woman with a combination of homozygens apc resistance ( apc ratio 1.10, normal range: 2.02 -3.73), pronounced ps deficiency (free ps 0.ll u/i, total ps 0.30 u/i, normal range: 0.55 -1.20 u/1 and 0.65 -lag u/i, respectively) and, moreover, impaired fibrinolysis (no change of euglobulin -lysis time after 10 rain venous occlusion) who developed deep vein thrombosis after cesarean section in her first pregnancy. examination of her familiy showed heterozygous apc resistance in her asymptomatie father (apc -ratio 1.99) , combination of heterozygous apc resistance (apc -ratio 1.60) and ps deficiency (free ps 0.30 u/i, total ps 0.58 u/i) in her nsymptomatic mother and no defect in her sister. considering the fact that the mother was still thrombosis free at the age of 49 one may assume that the thrombosis risk in the proposita was mainly influenced by the homozygnsity for apc resistance. s. ehrenforth, m. adam, b. zwinge, i. scharrer university hospital, dept. of angiology, frankfurt a.m., germany introduction: apc resistance has been shown to be the most commonly inherited defect which constitutes a risk factor for venous thrombosis (vt). however, most of the present epidemiological studies concerning apc-r prevalence in thrombophilia were derived from results of tests conducted onplasmas collected under various conditions. this may influence the great differences reported on the prevalence of apc-r among these patients. for example, it has been shown that freezing of plasma specimens prior to analysis of apc-r causes a significant decrease in the assay results.the aim of our study was to evaluate the influence of eentrifugation conditions on the results obtained with the chromogenic apc-r assay. patients and methods: blood was collected from 31 patients (t9 women, 12 men; fv gent.type: 13 r/r 506, 14 r/q 506, 4 q/q 506) through veinpuncture into trisodmm ciwat (1:9). platelet-rieh and platelet-poor plasma was obtained by immediately centrifugation at 4"c for 10, 20, 30, 40, 50, 60 rain at 1000, 2000, 3000, 4000, 5000 and 6000 rpm. additional, pnp obtained from 14 healthy individuals (7 male, 7 female without hormonal trealraent) was prepared equally. apc-response was determined within one hour after centrifugation using the coatest apc resistance kit from chromogenix. results: for both, pnp and sin/gle plasma samples, we observed continuous higher af'c-ratios after increasing cenwifugation intensity. for example, an increase from 1000 to 6000 rpm resulted m an increased apcratio from 2.7 to 3.26 (20 min), from 2.88 to 3.326 (60 rain) respectively. even though less distinctive, similar results were observed concerning the duration ol eentrifugation: when the duration was increased from 10 to 60 minutes we observed a continuous increase in apc-ratio, for example from 2.74 to 3.12 when using 2000 rpm and from 3.09 to 3.36 when using 4000 rpm. the decrease of the ratio after low eentrifugation is the eonse-9nence of the shortening of affft in the presence of apc, without a signhcant influence of basal al:rl~ without apc. conclusion: our results demonstrate that centrifugation conditions are important to consider for the interpretation of apc-r results. supporting our observations, recent studies from sidelmann et al. have shown that an increase in plasma platelet concentration, low eentrifugation respectively, causes a signficant decrease in the apc-response. however, so far the mechanism responsible for the significant effect of both on apc-r assay results is unknown. although technically simple, the biochemical cemplexitiy inherent in the chromogenic apc-r assay necessitates a standardized plasma handling procedure to secure a reproducible determination of apc-il compapjson of different assays for determination of apc-resistance with the geno'fyping factor v (arg -> glu) g. siegert*, s. gehrisch*, e. runge**. r. naumann**, r. kn6fler*** *institute of clinical chemistry, **clinic of internal medicine, *** childrens hospital resistance to apc diagnosed on the basis of prolongated clotting time in the aptt assay" is now considered a major cause of thrombophilia. in the majority apc resistance is ~ted with a point mutation in factor v molecule (arg506glu), but both are not synonym. protongated baseline aptt is a limitation of the assay. following the determination is not possible in risk groups of patients (factor)ill deficiency, lupus anticoagulan0 and in patients under anticoagulant therapy. in these causes a dilution of plasma in factor v deficient plasma is recommended. the immunochrom assay is based on the inactivation of factor villa by apc. the aim of the study was to compare different functional apc response assays with the result of the dna analysis. apc response was tested in 100 healthy probands, 81 thro~ patients and 16 family members using the lmmtmochrem assay, the contest (chromogeaix) and the contest with 1+ 4 dilution of the plasma in native factor v deficient plasma (immune). the dna analysis was performed as described by bertina. one patiem was homozygoas for factor v mutstion~ a hetemzygous result was obtained in 4 members of the control group, in 28 patients and in 6 family members. in all cases with factor v mutation the ratio of the immunochrom assay was lower than the laboratory own value, independent on anticoagulant therapy. pathological ratios in this assay were also obtained in one member of a family" with high thrombotic incidence (dna arg/arg) and in 17 patients under anticoagulant therapy ( two of this patients are one cloned twins). in the contest a ai~ response was diagnosed in all cases with factor v mutation without anticoagulant therapy and in 40 % of heterozygous patients under anticoaglant therapy. results of the test using the dilution in factor v deficient plasma showed a good agreement vath the results of the dna analysis but the method is obviously only sensitive for the factor v mutation. the reason for pathogical ratios in the lrnmunechrem assay in wildtype patients is unclear. the majority of this patients is treated with anticoagulants, a comparison with the contest is not possible. interestingly in one patient under heparin and low ratio in the immunochrom assay' after reduction of hepann the ratio of the coatest was also low. it seems necessary to investigate in which distance to the thrombotic events the apc resistance should be tested. following pathological ratios in ftmctional apc assays must be discussed: high levels of factor viii and or v wiuebrand antigen (acute phase reactien), other mutations in factor v and viii. the factor v dilution assay should be replaced by the dna analysis. due to their differing compositions, the "sensitivities" of various aptf reagealts differ not only with respect to factor depletions, heparin and fibrin-fibrinogen degradation products, but also with regard to pathological inhibitors. for lupus anticoagulants this means that "lupus-sensitive" reagents can be delineated from "lupus-insensitive" reagents. with a "lupus-insensitive" ai~ reagent there is no or only slight prolongation of the aptt in the plasma under investigation, whereas with a "lupus-sensitive" reagent marked prolongation is observed. for the meaninof~l use of aptr reagents it is necessary to know the extent to which they are influenced by lupus anticoagulants. the following 14 apti' reagents were tested: • ptt-reageaz, p'rta, ptta liquid, ptt-la, pti'-lt (boehringer/stago) • pathromtin, pathromfin sl, necthroratin (behring) • platelin s, piatehn excel ls (organon tekinka) • actin-fs, actin-fsl (dade) • aptt silica lye, aptt silica liquid (instntmentation laboratory) the material for investigation consisted of 20 plasmas from patients with lupus anticoagulants. a confirmatory test (lupus anticoagulant test, immune) was positive for all of the patients. measurements were made using the sta coagulation analyser (boehringer/stago). it can be seen from the results that in some instances very different prolongations were obtained in identical plasmas by using differing aptt reagents. low susceptibility to lupus anticoagulants was shown by actirt fs (dade), ptt-reagenz (bcehrlnger) and neothromfin (behring). high susceptibility was shown by platetin excel ls (organon teknika), ptt-la and pti'-lt (boehringer/stago). lupus anticoaguhant screening with the aptt reaction is promising when two aptr reagents differing as greatly as possible in their lupus anticoagulant sensitivity are used. the resistance to the anticoagulant response of activated protein c (apc) is a major cause of venous thrombosis. apcresistance is due to a single mutation in factor v gene, which predicts replacement of arg 560 in the apc-cleavage site with gln (factor v leiden mutation). in contrast to other known genetic risk factors for thrombosis, this factor v 1691 g-a mutation has a high prevalence in the common population of western europe (average 4-5 %). we have determined the prevalence of the factor v 1691 g-a mutation in a population of 700 probands of north-eastern part of germany. the mutation was found in 7 %. (heterozygoty were found in 48 subjects 1 person was homozygous.) the results are compared with our studies of populations from argentine and poland. me analysed the factor v 1691 g-a mutation in 71 patients with thrombosis from germany and hungary. this mutation has been found in about 60 % of these patients. in contrast, the frequency of this mutation was strongly reduced in a group of 47 patients with thrombosis and pulmonary embolism of argentine (3 heterozygotes in 47 patients; 6 %). the results of these different populations will be described and discussed. past medical history: venous thromboembolic events (re) at 18, 19 and 2i years; intermittent oral anticoagulation (oac) without te's. diagnosis of autoimmune disorder with elevated antinuelear-antibody-fiters and positive lupus-anticoagulant test. no other relevant illnesses; family history uneventful. two weeks prior to the referral to us -acute febrile illness with nausea, diarrhea, abdominal pain; hospitalisation, treatment with iv antibiotics and anticoagulation with fraetionated heparin; development of extensive deep vein thrombosis (dvt) of the right leg; initiation of full-dose unfractionated heparin; decline of platelet count from 121 to a nadir of 21 g/l; referral to our department. on admission an extensive coagulation screen yielded the following results (n/normal, t/elevated, i/reduced, +/positive, -/negative): pt t, aptt t, tr n, factor ii, v, viii n, factor vii, ix, xi, xii /,, fibrinogan t, atiii n, protein c, s *, activated protein c sensitivity ratio 1.92 ($), fv-leidenmutation pcr -, fibrinolytic system n, tat t, ft÷2 t, lupus anticoagulant +, heparin induced platelet antibodies +; no diagnosis of a specific autoimmuna disorder could be made. an immunosuppressive therapy with corticosteroids and anticoagulation with recombinant hirudin were init'~at~; no p~ogr~zsion of the dvt oeeured and normalisation of the platelet count was observed. during follow-up under oac ) and low-dose corticosteroids, the patient was well, the pathologic coagulat;.on results, including lupusanticoagulant and activated protein c resistance, have returned to normal; no further te's have been observed. in summary we present a case of a complex coagulation disorder as part of an autoimmune process, resulting in a clinically manifest prothrombotie dysbalance including lupus anticoagulant, acquired resistance against activated protein c and heparin induced thrombocytopenia (type ii), entering complete remission under combined immunosuppressive and anticoagulant therapy. in the last 30 years, a vast number of simplified analytical procedures have been developed for the diagnosis of haemostatic disorders. today the detection method have evolved from the mechanical hooking method or ball coagulometry to optical systems, which additionally can utilise chromogenic substrates or immunological methods. in these systems the clotting time is derived from algorithms (e.g. threshold or maximum of the first or second integral). we studied 50 healthy subjects, aged 21 to 65 years and 118 patients, aged 9 to 93 years using a new aptt reagent (pathromtin $l). the results were compared with those obtained with a routinely used reagent (pathromtin). the reference range, factor-, heparin-and lupus anticoagulant sensitivity were determined. analysis was performed using the behring fibrintimer a (bfa) with optomechanical clot detection, the behring coagulation timer (bct) with op-"dcal clot detection by threshold and the dw test and dw confirm for lupus anticoagulant diagnostic. our results showed that the new pathromtin sl reagent met the demands for a higher factor and lupus anticoagulant sensitivity. it is highly suitable for monitoring heparin therapy and gave comparable results with the optical and the optomechanical analyser systems, hence reagent c~n be used for both systems. restenosis following percutaneous transluminal angioplasty (pta) continous to be a major clinical problem. neoinfimal hyperplasia, being the major undedying cause, can not be sufficiently avoided. vadous plasmatic coagulation and fibrinolytic factors, have been associated with artedal restenosis. anticardiolipin antibodies (act_) have been established as dsk factors for venous or arterial thrombosis. methods: in a cohort of 75 patients (53 men and 22 women, age 68±13 years) undergoing pta of a peripheral artery we prospectively evaluated whether acl could influence 6 months restenosis rate. patients were clinically examined before, 3 and 6 months after pta. noninvasive grading of artedal stenosis was done by duplex scanning of jet peak velocities. restenosis was arbitrarily defined as more than 50% occlusion of the lumen at the site of dilatation 6 months after successful intervention. laboratory investigation at the same time included acl and other known atherosklerosis risk markers, such as fibdnogen (fbg), yon willebrand factor (vwf), homocystein (hcy), c-reactive protein (crp). thrombin generation markers, such as thrombin-antithrombin iii complexes and prothrombin fragments 1+2, as well as thrombomodulin (fm) as an endothelial activation marker, were also measured. results: 31/75 (41.3%) patients were considered to have developed restenosis after 6 months. 9/75 (12%) patients were found to have positive igg-(19-35 gpl) and/or igm-acl (14-103 mpl) at all three measurements. 1/75 was negative before but seroconverted (igm) 3 months after pta. 2/10 (20%) acl-positive and 29165 (44.6%) acl-negative developed restenesis at 6 months (chi-square p-value=0.14). all above mentioned coagulation parameters did not differ between acl-positive and -negative patients, measured before or 6 months after pta. some of them are shown below (values before pta): fbg ( basilar artery stenosis is a rare event in young children. risk factors are head or neck trauma with consecutive dissection of the vertebral artery, cardiac diseases or hypercoagulability. elevated lipoprotein (a) (lp(a)) serum levels in adults can mediate atherosclerosis. in addition, lp(a) might interfere with fibrinolysis. here we report on a 3 year old boy , who presented with acute brain stem symptoms. history revealed neither trauma nor infectious disease. conventional and mr angiography showed stenosis of basilar artery without ischemic lesions. laboratory findings were normal in routine blood and csf tests. global coagulation parameters as well as procoagulant and anticoagulant factors were normal. cardiac and autoimmune disease could be ruled out. lp(a) serum levels were significantly elevated to 104 mg/dl (normal range <30 mg/dl). analysis of other family members revealed a hereditary hyperlipoproteinemia (a) which might explain family history of an increased incidence of myocardial infarction and cve in elderly family members. clinically the patient recovered completely from brain stem symptoms after heparinization and subsequent oral anticoagulation with phenprocoumon. however, radiological signs of basilar artery stenosis were progredient. in a recently developed specific test, an elevated anti-phosphatidylserin antibody titer was detected one year after primary diagnosis. in conclusion, this is the first report on a child with stenosis of the basilar artery and elevated levels of lp (a). it is unclear, whether apa contributed to the onset of basilar artery stenosis or developed secondary due to endothelial defects after thrombosis and anticoagulation. apa, however, might increase the risk of further thrombotic events in this patient. in 110 patients with thrombotic events respectively patients with systemic lupus erythematodes antioardiolipin antibodies (aca) aund lupus anticoagulant (la) were ~ea~ured. for aca detecting we use the assays from elias for igg-and ig~}-antibodies. we use as sensitive methods for detecting la in our laboratory the testkits from diagnostlca stago (staclot la with hexagonal array of phospholipids, ,ptt-la a very sensitive pttmethod and staclot p~p-a platelet neutralization procedure) and the ptt from organon teknik~ (platelin excel ls with two incubation times, 1 and 10 minutes). i"~e results of this tests were compared with three new or~e on german market: specktin apot (aktlvated plasma clotting time), specktin aptt (aptt wlth purified soy extract) and 8pecktin la (phospholipid preparation in concentrations between 10 and 500 ~g/ml); all wak chemie. traditional aptt reagents were developed for the sensitive detection of factor vib an ix as a cause of hemorhage. high sensitivity against lupus anticoagulants, which also prolong aptt, was not required for this purpose, with increasing recognition of the importance of antiphospholipid antibodies as a risk factor for thrombembolism, more sensitive reagents were designed, which now reliable detect this condition. using such reagents as a screening test in a general hospital makes it necessary to distinguish both conditions quickly. we here report an algorhythm, by which we use an inhibitor (lupus anticoagulant) sensitive (sta aptt, boehringer) and an inhibitor insensitive reagent (actin fs, dade) to distinguish anticoagulants and factor deficiencies as a cause of prolonged aptt. citrate plasma from 33 patients with various diseases showed an unexpectedly abnormal inhibitor sensitive aptt (>40s). 13 plasmas with factor deficiencies remained abnormal with the insensitive aptt reagent. a regular correction of their defect occured on mixing with normal plasma. by measurement of single coagulation factors five patients with contact factor xii deficiency were found. this condition is associated with thrombosis and very rarly with bleeding. three patients with factor xi deficiency and two patient with factor ix deficiency were also identified. antiplatelets, of any kind, permits a secondary prevention of myocard ischemic lesions. there is no general consensus regarding secondary prevention of cerebral ischemic lesions. aspirin remains the most common substance, ticlopidlne also brings about prevention, but with important secondary effects. european stroke prevention study i has demonstrated that the combination of antiplatelets, in particular aspirin/dipyridamole (persantln), is also very active. to collect more information, esps 2 was organized and 6602 patients receiving either a placebo,either 50 mg aspirin,either 400 mg sustained release form of dipyridamole (persantin (r)), or the combination aspirin/dipyrldamole, were recruited. it ended march 31st 1995 with the following conclusions: i-aspirin, 50 mg a day, brings about a significant secondary reduction of stroke (18.z %), after a two year follow-up. notwithstanding the low dose of aspirin, haemorrhages remain important. 2-dipyridamole, at 400 mg a day, brings about a significant reduction of stroke (i6.3~), similar to that of aspirin. one could thus substitute 50 mg aspirin by 400 mg dipyridamole. 3-the combination of 50 mg aspirin and 400 mg dipyridamole brings about a significantly greater reduction of stroke (37.0 ~). esps 2 revealed that a low dosage of aspirin is active, that dipyridamole alone is also active, but that the combination of both gives far better results. the study of the primary end-points,the study of the survival curves, the factorial statistical analysis and the pairwise comparison analysis, led to these conclusions. the conclusions drawn from esp£ 2 underline that the combination aspirin/dipyridamole is a privileged choice for cerebral ischemia, the state of activation of circulating platelets in acute cerebral ischemia is controversial. activation of platelets on single cell level can be assessed by determining the shape change or the expression of antigens such as p-selectin (cd62). shape change is an early and rapidly reversible event in platelet activation whereas p-selectin is irreversibly expressed on the platelet surface upon stimulation. methods: we investigated 20 untreated patients within one day after cerebral ischemia, 20 patients months after stroke treated with warfarin, and 21 age and sex matched control subjects without vascular risk factors. venous blood was collected into a fixation solution blocking the metabolic processes in platelets within milliseconds. we determined the fraction of resting discoid platelets by phase contrast microscopy. the expression of p-selectin was measured by flowcytometry. results: the fraction of platelets expressing p-selectin was higher in patients with acute cerebral ischemia (7.8_+4.7%) than in control subjects (1.9_+1.1%; p<0.001, u-test). patients with stroke (n=15, 7.8+4.5%) and patients with transient ischemic attack (tia; n=5, 7.6-+6.1%) had similar values. patients months after stroke still had higher values (4.3+9,3 %, p<0.05) than control subjects. the rate of discoid platelets was not different between patients with acute ischemia (n=17, 85.6-+8.8 %), patients months after stroke (n=19, 85.7-+5.1%) and control subjects (n=21, 86.7_+5.8 %). platelet count was not significantly different between groups. conclusion: the elevated proportion of platelets expressing pselectin indicates strong platelet activation in acute cerebral ischemia and in a majority of patients months after stroke. assessment of pselectin revealed a higher sensitivity for platelet activation after stroke or tia than analysing the reversible shape change. further studies have to clarify if monitoring of platelet activation by flowcytometry is helpful as a prognostic tool and to evaluate therapeutic strategies after stroke. vascular smooth muscle cell (smc) proliferation and migration into neointima are the hallmarks of atherogenesis. the complexity of these processes and their concerted action and interaction of molecules are yet to be fully elucidated, one crucial molecule seems to be the urokinase-type plasminogen activator receptor (upar) recently also assigned as cd87 antigen, upar serves a dual function: (1) it directs upa proteolytic activity to a special location on the cell surface and (2) induces cellular signals leading to various phenotypic changes. we have investigated the signal-transducing capacity of upar in human smcs and provide here a molecular explanation for uparrelated cellular events. activation of these cells with upa (even with inactivated catalytic center) results in the induction of tyrosine phosphorylation, suggesting modulation of upar-associated protein tyrosine kinases (ptks) upon ligand binding. we obtained patterns of tyrosine-phosphorylated proteins with molecular masses of ~ 55-65 and 35-40 kd. using antibodies against different types of ptks as well as immunoprecipitation-and immunoblotting techniques the ptks involved in the upar-signalling complex were identified to be members of the src-ptk family. the cotocalization of upar and ptks at the cell surface of smcs was further confirmed by confocal microscopy studies. we conclude that the upar-ptk complex is most likely involved in this signal transduction pathway that provides the coordinated action of extracellular proteolysis, adhesion, and cell activation, which is required for cell migration. this mechanism may be crucial for the progression of atherosclerotic plaques. activation markers of haemostasis have been found elevated in relation to diabetic vascular lesions. simultaneous pancreas-and kidney transplantation (pkt) in type i diabetes has been shown to improve diabetic complications and long term survival. we measured haemostatic vascular risk factors and activation markers in plasma of 18 patients after successful pki', 17 patients after pkt and rejection of the pancreas graft and 5 patients after pkt and rejection of the renal graft. blood samples were taken during routine ambulatory visits, patients were free of any ongoing acute disorder or transplant rejection and under continuous immunosuppressive medication. despite individually adjusted insulin therapy hba1 plasma levels increased after pancreas rejection (5,37 vs 7,i2, p<0.001). platelet counts and plasma levels of fibrinogen, f1+2 fragment, tat-, app-complex and-fibrin monomer were found significantly elevated as compared to diabetic controls but not significantly different with respect to complete or partial successful pkt. one major reason of the increased activation state of haemostasis may be cyclosporin treatment given to all patients, t-pa and pal i plasma levels were within the normal range and significantly correlated to plasma triglyceddes (r.0.049; p<0.005). d-dimer plasma levels were significantly lowered after pancreas rejection (772(375) vs 324(232) nglml; mean(sem) p<0.005), which might reflect impaired fibrin degradation related to increased glycosylation of fibrinolytic factors. in conclusion, despite the marked improvement of glucose and lipid metabolism, plasma markers of activation of coagulation and flbrinolysis are not decreased to normal after simultaneous pancreas and kidney transplantation. according to the investigations of fowler et al. and pepe et al. the probability of an ards occurring with one risk factor is 5-8%, and in the presence of several risk factors, 25%. goris et al. and johnson et al. determined the level of severity with the aid of a fixed scale: the injury severity score. all these investigations are however not to be interpreted as typical following coronary surgery. these investigations demonstrated that the kallikrein and factor xii systems are of great importance as intraoperative risk factors. here the factor xii system plays a major role with direct or indirect activation of the kauikrein-kinin system with the splitting products alpha-factor xiia and bfactor xha respectively. all ards scores take the pmn-elastase into account. if the pmn-elastase values (1000 pg/l) are constantly high postoperatively then lung complications are to be expected. patients developing an ards displayed significantly lower alpha2-macroglobulin values. patients who developed a highly significantly raised kallikrein-like acdvity (>60 u/i) after the beginning of bypass and showed constantly high values during ecc are difficult to keep under control due to the blood pressure behaviour. the platelet pal also shows a significant rise and intraoperatively runs analogous to platelet factor 4, only antiparailel, since it attacks the endothelium. we were able to show that pai-1 is suitable as an indirect marker for a possibly developing restenosis. 85% of the patients investigated with lowered pai-1 values in the postoperative phase did not develop a restenosis. however, with patients showing significantly rising pa[-1 values from the 1 st. to 3rd. postoperative day 50% of all the cases had a restenosis. a further risk factor in this respect are significantly raised fibrinogen levels which lay over 800% at the end of surgery. if these fibrinogen values do not fall from the 1st. postoperative day onwards a raised risk of thrombosis must be reckoned with in the absence of therapeutic intervention. the following parameters represented haemostaseological risk parameters with significant behaviour within the framework of this study: 1) regards the blood pressure behaviour, the kallikrein-like activity (>60 u/i); 2) with regards to the lung complications, aipha2-macroglobulin and pmn-elastase (>900 g/i); 3) and final/y as a possible marker for a developing restenosis pai-1 and fibrinogen (>800%). resulting from numerous clinical studies homocysteinemia is found to be an almost independent risk factor of atherosclerosis including thrombotic complications as well as of venous thromboembolism. experimental investigations on the underlying mechanisms suggest endothelial cell damage accompartied by the development of an atherogenic and thrombogenic potential, increased platelet reactivity, oxidative modification of ldl, and enhanced affinity of lp(a) for fibrin. to our knowledge no results are published on the influence of homoeysteine on leukoeytes although these cells are deeply involved in pathological events within the vasculature. therefore, as a first approach different functional parameters of human polymorphonuclear leukozytes (pmnl) were followed under incubation with 60, 300, and 600 i.tm (final concentration) dl-homocysteine (hc) in isolated fractions or whole blood, respectively: l) spontaneous mobility of pmnl, measured as migration distance into micropore filters in a modified boyden-chamber, is found to be significantly enhanced by the two smaller hc concentrations. 2) chemotaxis induced by 0.1 i.tm formylmethionylleueylphenylaianine (tmlp) shows no significant differences. 3)monitoring of chemiluminescence signals (autolumat lb953, berthold) is complicated as hc influences the luminol-mediated indicator reaction. adjusting appropriate conditions the following results are obtained: spontaneous chemiluminescence and that induced by zymosane, tmlp, and the ca2+-ionophore a23187 are entranced by the two higher hc concentrations. there are, however, differences between the blood donors as a minority does not respond to hc in repeated measurements. with phorbol 12-myristate 13 acetate the signal is diminished by hc in all cases and with all concentrations. 4) phagoeytosis induced by zymosane (microscopic evaluation) as well as by opsonized e. coil (cytoflowmetric evaluation) is significantly increased by the two higher hc concentrations. conclusion: the activation of human pmnl is enhanced with respect to the majority of investigated stimuli by hc in concentrations reached under pathophysiological condititions. the effect of pysical exercise on hemostatic parameters was studied in 12 patients (male, mean age: 55 [range 36-68] yrs) with angiographically documented coronary artery disease (cad) and in 11 controls (male, 53 [43-62] yrs) both participating in an 1 hour group exercise session for cardiac rehabilitation. in each group relevant arteriosclerotic lesions in carotid, abdominal and leg arteries were excluded by doppler ultrasound examinations. patients were all under 6-blocking agents and aspirin. plasma levels of prothrombin fragment 1+2 (ptfi+2) and fibrinopeptide a (fpa) reflecting formation of thrombin and fibrin, respectively, were measured at rest and immediately after 1 hour of exercise consisting of jogging, light gymnastics and ball games. training intensity in both groups was comparable as indicated by the mean heart rate during exercise corresponding in patients to 80+6% (mean-+sd) and in controls to 79-+5% of the maximal heart rate previously determined on a bicycle ergometer. baseline values for ptf1 +2 were significantly lower in oatients (0.67-+0.15 nmol/i; mean-+sd) than in controls (1.01-+0.21; p<0.001 i. after exercise we found an increase of ptf1 +2 in controls to 1.14-+0.24 nmol/i (p<0.001) while in patients ptfi+2 remained unchanged (0.67-+ 0.14 after). accordingly, exercise induced r se of fpa was more pronounced in controls (from 0.62-+0.24 to 1.60-+0.62ng/mt; p<0.001) than in patients (from 0.63-+0.26 to 1.20+0.46ng/ml; p<0.00t). we conclude that in terms of thrombin and fibrin generation exercise training does not exert detrimental effects on hemostasis in patients with cad. lower baseline values and lack of exercise induced increases of ptf1 +2 in patients with cad might be attributed to medication with aspirin and/or b-blocking agents. periodontitis marginalis (pm) is an inflammatory oral disease that is caused by gram-negative bacteria and that has a high incidence in the second half of the life. clinical signs of pm are gingival bleeding, periodontal pockets, alveolar bone destruction and loss of teeth. recent epidemiologlcal studies have provided some evidence for an association between pm and atherosclerosis. in the present paper we will summarise some of the results that we have obtained in studies on patients with pm as well as on patients with hypercholesterolaemia (hc) and atherosclerosis. pm was frequently found to be associated with hc (90 % in rapidly progressive pm) and increased reactivity of peripheral blood neutrophils and platelets (e.g. generation of oxygen radicals and paf-induced aggregation). patients with hc and atherosclerosis had a higher frequency of severe pm when compared with data on the community periodontal health. the severity of pm was higher in patients with plasma cholesterol levels _> 6.5 mm when compared to those with plasma cholesterol < 6.5 mm. in patients with coronary atherosclerosis the severity of pm was significantly correlated with plasma cholesterol level, systolic blood pressure and the number of diseased coronary arteries. these results provide further evidence for an association between pm, hc and atherosclerosis. it can be speculated that hc is not only a risk factor for atheroscterosis but also a risk factor for pm and acts by increasing the reactivity of neutrophils and platelets. on the other hand, pm as a mild chronic inflammation could promote the development of atherosclerosis due to effects of endotoxins on vessel wall, blood cells and haemostatic factors. it has been also speculated that phagocyting leukocytes in the inflamed periodontal tissues could contribute to oxidative modification of ldl. so far, there is no evidence that atherosclerosis may contribute to the pathogenesis of pro protein z (pz) is a vitamin k dependant plasma protein synthesized in the liver. it promotes the association of thrombin with phosphorlipid surfaces. recently it has been shown that a deficiency of pz may lead to a bleeding tendency. in patients undergoing chronic hemodialysis, disorders of hemostasis are common. to examine if plasma levels of pz are altered in patients with end stage renal disease we determined pz in plasma of patients at the beginning of hemodialysis treatment. the results were compared with a group of 50 healthy controls. the difference of pz levels in plasma of patients with end stage renal disease with the control group was not significant. control group was 2900 + 487 ng/ml and in patient group was 3133 + -1394 ng/ml. one patient with marked bleeding tendency after hemodialysis pz was 1387 ng/ml. we concluded that patients with bleeding disorders pz determination may be helpful. the normal range of actin fs was reinvestigated in a multicentric approach. a protocol was developed which requests from each center to assess the aptt with one common and one variable lot of actin fs in 50 samples of suspected normals. inclusion and exclusion criteria based upon the results of clotting assays, liver enzymes and clinical data were defined. results: a total of 1056 results was obtained. the majority of centers in this study used the electra 1000 or 1600 c (mla). results for the electra group (n = 6) showed a precision for the common lot of actin fs with a common lot of a three level control from 1.5 % (level 1) to 1.1% (level 3) with an excellent accuracy between the 6 centers. clotting times with the variable lots of actin fs were very similar. the results from normals, however, showed a somewhat higher dispersion using the common lot of actin fs. 4 of 6 centers had almost identical mean values (range 26.6 to 27.2 sue) whereas one reported shorter and one longer clotting times (25.2 and 29.7 sec). results with the variable lots gave almost identical results as the common one. a total of 556 results of all lots gave a normal range of 23.5 to 31.7 (5 -95 % percentiles) on electra. mean values on acl (n = 200) were 26.3, on bct, 27.65 sec, on amga coagulometric, 29.4 sec, on amga turbidimetric, 26.6 sec (n = 100 each). all centers used sarstedt monovettes with 3.2 sodium citrate. discussion: the results of this study demonstrate the lot to lot consistency of all lots of reagents included in this study since the common and variable lots showed very consistent results. interestingly in the large group of electra users the normal ranges showed some differences, though the controls in all centers were almost identical. this confirms the recommendation that a normal range as stated from the manufacturer should be used for orientation only and that each laboratory should assess its own range. direct acting anticoagulant agents such as hirudin (r-h), argatroban (arg), efegatran (efe) and peg-hirudin (ph), represent specific and potent inhibitors of thrombin. blood samples collected in r-h (10 ~g/ml), arg (50 ~tg/ml), efe (25 ~tg/ml) and ph (10 ~tg/ml) do not clot for extended periods (>24 hours), thus allowing for the collection of plasma for analytical purposes. unlike heparin, these agents do not require any plasma cofactor for their anticoagulant effect. in contrast to citrate, oxalate, edta and heparin, these antithrombin agents do not alter the electrolyte or protein composition of blood. thus, blood collected in these agents may provide a physiologically intact (native) sample for clinical laboratory profiling. we have used all of these agents to prepare whole blood and plasma samples for various diuical laboratory measuroments. plasma samples collected with these agents are obviously not suitable for global clotting tests (pt, aptt, thrombin time, fibrinogen); however, these agents are optimal anticoagulants for the collection of samples for the molecular markers of hemostatie activation, such as fibrinogen/fibrin related degradation products, prothrombin fragment, protease cleavage products, tfpi, tnf and other protein mediators. electrolytes, blood gases, enzymes and protein profiling can also be satisfactorily measured on blood samples collected with these agents. antithrombin anticoagelatad blood used fur hematologic analysis showed equivalent blood count and differential results as that obtained with edta blood. unlike other anticoagulants, these agents do not interfere in the cell staining process. washed blood cells can also be prepared using antithrombin aents supplemented buffers for morphologic and fuuctional studies. thrombin inhibitors such as hirudin have also been used for flow cytometry and image analysis of blood cells and tissue exudates. our observations suggest that these anticoagulants can be used as suitable anticoagulants for clinical laboratory blood sampling. these agents can also be used as a flush anticoagnlant fur most automated instruments as these exhibit superior anticoagulant properties to heparin. furthermore, the hematologic parameters obtained in antithrombin anticeagnlated blood may be physiologically more relevant than those determined on blood collected in edta, citrate or heparin. antithrombin ul determination is one of the most popular method for in vitro diagnostic of number of different disorders. human fhrombin a~nity purified on heparin-rnodified silica-based sorbents was used for level of antithrombine lu determination by abilgaard method in blood of 40 patients with pregnancy pathology, acute leukemia, thrombocytopenia and anemia. it was founded, that antithrombin level is decreased to 50-60% of normal values in case of pregnancy pathology, to <50% -in case of acute leukemia and thrombocytopenia, to s0% -in case of anemia. obtained results show the strong relationships between named disorders and patient antifhrombin iii level. therefore anfifhrombine iii estimation may be used as simple and quick method for preliminary diagnosis of above named disorders. bm coasys 110 is a complete automated analyzer system for coagulation tests. it is well suited for routine coagulation testing in random access in a medium throughput laboratory environment. analytical performance and practicability were tested by a common evaluation program in five hospital laboratories. within run and day to day cv's were below 5 % in different samples (controls, patients) . comparison in different therapeutic ranges confirms the declaration of the isi-value for calculating inr-values. normal values for coagulation tests with results in pdmary units were checked in 390 samples and confirmed. due to the optical measuring principle of the bm coasys 110 there was a little tendency to shorter times with the thrombin reagent. in conclusion the performance of coagulation tests with the bm coasys 110 was rated as well or better compared to existing systems in the laboratories with advantages due to short timed familiarizing and easy handling. flexibility and stability of the system permit optimal integration and innovation into the w0rkflow of the routine laboratory. the purified thrombin and antithrombin iii (at iii) have a great interest in the clinical diagnostic and treatment practice, so their isolation methods are very important. molecules of these proteins have some fragments replying for interaction with native glycosaminoglycan, heparin. this interaction is used for isolation and purification of thrombin and at ul from native materials, blood plasma or its fractional products. we have done comparative studying these proteins purification on heparin sorbenfs, which contain heparin immobilized on sificagel, modified by glycidooxipropyl, gamma-aminopropyl and tosyl chloride groups, or on cellulose: heparin-epoxy-silica (1), heparin-gammapropyl-silica (2), heparjn-tozylsilica (3), and heparin-cellulose (4). we founded that thrombin binds with all sorbents, while at iii doesn't binds with sorbents 2 and 3. there wasn't any difference between silica and cellulose sorbents in thrombin desorbfion by t m naci. at iii binds more stronger with heparin-ceuulose t[~,~n with silica sorbents but specific activity and purity degree were approximately the same on both kinds of sorbents. thrombin specific activity and purity degree were approximately twice higher on sorbents 2 and 3 in comparison with sorbents ! and 4 (2250-3000 nih units/mg versus 1260-t350 nih unlts/mg). therefore, sorbents 2 and 3 can be used for isolation and purification of thrombin and sorbents t and 4 can be used for isolation and puriiication of at ii1. we used these sorbents for large scale purification of named proteins. purified thrombin was used for production of diagnostic kits for anfithrombine iii, fibrinogen, fibrin/fibrinogen degradation products and thrombin time determination. after an aerobic or anaerobic physical exercise various alterations of the hemostatic system were detected. numerous investigations of the hemostatic system exist of running and of bicycle ergometer exercise but not of swimming. young volunteers (n=54; median age 25 years) were investigeted~ there was an aerobic exercise (achieved heart rate 130 --10/min, lactate < 4 mmol; n= 27) and an anaerobic exercise (achieved heart rate 150 ~ lo/min, lactate > 4 mmol/1; n= 27). in both groups there was a significant shortening of the ptt. under anaerobic conditions hematocrit and quick significantly increased. factor viii activity rose significantly in both groups. indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 only under anaerobic conditions (tat from 2,5 to 5,4 pg/1; f1+2 from 0,87 to 0,93 nmol/1). indicating activation of fibrinolysis t-pa activity increased significantly in the anaerobic group (from 0,1 to 0,4 iu/ml) but not in the other group. this findings indicate that there is e balance in the hemostatic system by activation of clotting as well as of fibrinolytic system in young volunteers during exercise by swimming dependend on the degree of exercise load. membranes as well as compact, porous disks are successfully used for fast analytical separations of biopolymers. as far as capacity, speed and performance of separation are concerned, the supports are as effective as other recently developed fast media for the separation of biopolymers °). so far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations. in this report, the use of a compact tube made of poly(glycidyl methacrylate) for fast preparative separations of proteins is shown as a possible solution of these problems. the units have yielded excellent results, regarding performance and speed of separation as well as capacity. the application of compact tubes made of poly(glycidyl methacrylate) for the preparative isolation of the coagulation factors viii and ix from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. in terms of yield and purity of the isolated proteins, this method was comparable to preparative column chromatography. the period of time required for separation was five times shorter than with corresponding column chromatographical methods. our measurements showed an excellent correlation of the two systems (r=0,99). the maximum amplitudes on the roteg were on average 2.2% higher than on the hteg, corresponding to a slightly lower reverse momentum of the measuring system in comparison to the hteg. we report first results out of the evaluation of sta compact (boehringer mannheim/diagnostica stack)). sta compact is designed for automated analyses of routine and special coagulation (chronometfic, photometric [405 nm] and turbidimetric [540 run]) tests. in addition, it does measure ,,derived" fihrinogen. tests as follows were evaluated: prothrombin time (pt), partial thromboplastin time (aptt), fibrinogen (clauss method), thrombin time, at iii (chromogen), hepato quick, as well as the factors ii, v, vii, x, and viii. results: within run cvs of the clotting tests were below 2% (calculated on the basis of seconds) in most cases, day to day cvs below 4% (not measured for factors, yet). at iii yielded within run cvs below 3% in the decision range. measuring ranges: at iii: 20-140 %; fibrinogen: 1.3-9.0 g/l (plasma -dilution 1/20), after rerun with other dilutions: from 0.3 g/1 (dilution: 1/5) to 18 g[l (dilution: 1/40). method comparisons, using sta as reference, yielded slopes close to 1.00 and negligible intercepts. throughput: with routine clotting tests about 100 tests/h, in a sample selective access mode. we conclude, that sta compact allows precise measurement of routine and special coagulation tests. it is also a reliable system for photometric tests and well suited for intermediate workloads as well as stat analyses. we did evaluate ptt lt, a new liquid, silica based ptt reagent. special attention was given to reference interval and heparin sensitivity. the new reagent is well suited for the measurement of intrinsic clotting factors and is reported to have high sensitivity for lupus anticoagulants (higher sensitivity than sta aptt [boehringer mannheim = bm]). it is stable for 4 days in the cooled compartment of the sta analyzer. methods: all experiments were made on sta. for comparison, we used three other ptt reagents (a lab. routine, silica based aptt, as well as sta aptt and sta ptt kaolin from bm). in addition, thrombin time (3 u/ml thrombin, sta thrombin reagent) and heparin (chromogenic xa test, rotachrom heparin) were measured. results: within mn imprecision (n=21) was below 0.7 % cv in the normal range and in two controls (mean values: 35 s, 76 s), and 1.4 % in a heparin plasma (mean: 81 s). between day imprecision (d=10) was below 3% in two controls ( mean values: 34 s and 58 s). the upper limit of the reference range is 40 s (97.5 th perc., median: 32 s; 90 patients with normal coagulation status [routine aptt, fib., pt], median age: 54 years); almost identical reference ranges were obtained with sta aptt and the routine ptt reagent, while sta ptt kaolin showed significantly lower values (97.5th perc.: 33 s, median 28 s). method comparison study: good agreement using plasmas from patients without heparin: (y= 0a + 0.98 x, n= 198, range of(x) from 25 to 79 s, r = 0.97; x = sta aptt). the median values from 54 patients under high dose heparin were: routine ptt: 81 s, sta aptt: 75 s, ptt -lt: 82 s0 sta ptt kaolin 54 s, thrombin time 37 s and heparin 0.5 iu/ml in conclusion, results of the new reagent compare well to our routine ptt and to sta aptt system reagent. it allows sensitive monitoring of high dose heparin therapy and is well suited for detecting abnormalities of the intrinsic clotting factor pathway. is a standard technique since many years. the interpretation of the thrombelastograms has been widely based on phenomenologic observations, while there is a lack of exact information concerning the coagulation mechanisms leading to the teg amplitude (a're~). the aa'ec is a measure for the mechanical stiffness of the clot and depends on: a) fibrin formation and adequate polymerisatiun of a 3-dlmensional network: measurements with nonrecalcified citrated blood activated with adp or epinephrine (both n=10) did not show any clot formation in the teg this relies on the need for a mechanical coupling between the teg pin and cup over a distance of 1 mm, which is accomplished by the fibrin network therefore, teg can only be performed under thrombin formation and thus under thrombin-activation of the platelets in the sample. factors, which inhibit platelet aggregation but don't limit thrombin-activation of platelets, cannot be monitored by teg. b) the attachment of the dot on the surface of the teg pin and cup. according to recent literature we suggest that the attachment of the clot in the teg relies exclusively on fibrinogen/fibrin adsorption to the surfaces of the pin and cup. interruption of this attachment can result in lower amplitudes or the so-called ,,stairway" phenomenon. we could show a complete interruption of the clot attachment by dipping the pin for one second in 30% albumine solution (n=10). c) the fibrinogen concentration (fg) and platelet count (pc) of the sample. in 50 volunteers we found only a poor correlation of the maximum amplitude (ma) with fg alone (r=0.40) or pc respectively (r=0.50), while there was a very good nonlinear correlation to the product of fg and pc. we suggest that the fibrin network forms the main structure of the clot while the thrombocytes enhance its stiffness in a concentration-dependent manner. this effect of the ptatelets can be completely reversed by gplibfllla antagonists. d) adequate coagulation activation: in nonactivated teg even small amounts of inhibitors can lead to a significant reduction of the ateg. conclusion: alterations in teg measurements can be judged more properly when the underlying mechanisms are understood. the consideration of the limitations of the method allows a more specific interpretation of the results. as a response on a customer request we did investigate the sample stability of blood samples for the aptt. the study was set up in a way that simulated the conditions of a large private laboratory in which the samples arrive several hours after blood collection. blood was drawn from 10 donors into 3.2 % sodium citrate and mixed well before it was divided into several aliquots which were kept at room temperature. the aliquots were centrifuged after ~ 0,5, 11, 23 and 29 h after venopuncture and the plasma was analyzed immediately with 3 different reagents on electra 1000. results: there was a clear difference in the change of the apttover time with these reagents. also f viii (determined with a chromogenic assay with complete and standardized activation) change considerably. 2 reagent a: ellagic acid, plant phospholipid, reagent b: sulfatide/kaolin, phopholipids, reagent c: ¢llagi¢ acid, plant and rabbit brain phospholipids the increase of aptt was apparently not a function of the decrease of fviii because the in vitro f viii sensitivity of reagent b. was inferior to reagent a though reagent b showed more prolongation of the aptt than reagent a. reagent c, however, showed only minor changes in the aptt. discussion: these data show that the sample stabifity of the aptt is reagent dependent and that it is not simply a function off viii sensitivity. other factors such as the buffer system but also the sensitivitiy towards other factors than f viii seem to contribute. a comparison of the technical principle of the roteg coagulation analyser and conventional thrombelastographic systems an. calatzis, p. fritzsche. al calatzis, +m. kling, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology yechnische universit~t m0nchen thrombelastography (teg) was introduced by hartert in 1948 as a method for continous registration of the coagulation process. in 1995 we presented the roteg coagulation analyser, using a newly developed technical method. in teg systems according to i/artert the sample (blood or plasma) is placed in a cup which is alternately rotated to the right and left by 4,75 °. a cylindrical pin, which is suspended freely on a torsion wire, is lowered into the blood. when coagulation starts, the clot begins to transfer the rotation of the cup to the pin against the reverse momentum of the torsion wire. the angle of the pin is electromagnetically detected, transformed to the teg amplitude and continously recorded. in the roteg the pin is attached to a short axis, which is guided by a ball beating. thus all possible movement is limited to rpotation (r_oteg). the cup is stationary, and the pin is rotated alternately by 5 ° to the right and left by a feather system. when a clot is formed, it attaches to the surfaces of the pin and cup and starts preventing their relative movements against the reverse momentum of the feather. here the reduction of the rotation of the pin, which is detected optically, is tranformed to the teg amplitude. as can be shown by theoretical analysis and by control measurements, the roteg provides the same measuring capabilities as conventional teg systems. the main advantage is the solid guiding of the measuring system, which makes the roteg easily transportable and less susceptible to shock or vibration during measurement. yhrombelastography (teg) is a standard monitoring procedure for evaluation of coagulation. usually only nonactivated native blood teg measurements (nateg) are performed, which leads to a) a long time interval until coagulation and fibrinolysis parameters are available b) very high susceptibility of the measurement to inhibitors like heparin, which disturbes the judgement of other components of coagulation, c) unspecific results. our aim was to develop a coagulation monitoring system based on teg providing fast and specific information on the different components of coagulation. methods: the following measurements are performed in paralel using disposable pins/cups (haemoscope): a) extrinsic activated teg (exteg): 3551al whole blood (wb) + 5~tl innovin (recombinant thromboplastin reagent, dade). b) intrinsic activated teg (integ): 3551al wb + 5~tl kaolin (suspension 5g/l, behring). c) aprotinin teg (apteg): exteg + 20 kie aprotinin (trasylol, bayer). d) heparinase teg (hepteg) as decribed in (1). results: exteg and integ provide information on the extrinsic/intrinsic system within 5-10 min and information on the platelet/fibrinogen status within 10-20 min. because of the addition of potent activators integ and exteg can be performed when inhibitors like heparin are present in the circulation. fibrinolysis effects can be seen on exteg and integ and by comparison of exteg and apteg (apteg: invitro-fibrinolysis inhibition by aprotinin). if fibrinolysis is detected by exteg or integ and aprotinin-susceptibility is verified by apteg, aprotinin therapy will be initiated. heparin effects are revealed by hepteg. discussion: by the comparison of parallel teg measurements which have been differently activated, specific and fast information on the different aspects of the clinical coagulation status is provided. the presented tests can be easily performed bedside and only a small specimen of whole blood is needed (0,4-1,8 ml). introduction: a severely prolonged aptt (333s; normal: 40~os) was observed during preoperative screening for a planned splenectomy in a 71-year-old man with an 8 year history of osteomyelofibrosis. fellewing neer-normal~atien (71s) of the ap'ci" after 10 rain preincubation in a kaolin based aptt assay, pk deficiency was suspected and studies were performed to further investigate the nature of the pk deficiency as well as the mechanism underlying the normalization of the prolonged aptt by increasing the preincubation time. methods: the apl-r assay was peal'armed using kaolin/inesithin. high molecular weight kininogen clotting activitiy (hk:c), fxii:c end pk:c were measured by an aptt based assay using neothromtin ® (behnng) and 1 rain (pk:c) or 4 min (hk:c, fxli:c) preincubation. pk amidolytic activity (pk:am) was assayed using cosset pk ~ (chromogenix) and pk antigen (pk:ag) by quantitative immunoblotting. fxll and hk proteolysis dunng activation of plasma by kaolin (10mg/ml at 37=c) or ds (12.5~tglml at 4=c) was demonstrated by immunotilotting assays of fxii and hk following sds-page. assay pk:c pk:am pk:a~i fxfi: the propositus had pk:c<5%, pk:am=5% and pi~ag<2.5% as compared to normal pooled human p(asma (nhp). his son and two daughters had pk:c-50% and normal aptt values, incubation of the propositus' plasma with ds did not result in fxii or hk cleavage within 180 rain, whereas jn nhp detectable f×ii and hk proteolysis occurred after 5 rain and complete proteolysis was observed after -120 rain. in contrast, kaolin activation of propositus' plasma led to slow activation of fxii after 10 rain, presumably by autoactivation, and to fxlla-induced hk proteolysis. near-normalization of the propositus' aptt by prolongation of the preincubation time paralleled fxii autoactivation as evidenced by immunobletting. we describe a propositus with severely prolonged aptt due to hereditary, crm negative pk deficiency suffering from omf. activation with a particulate suspension of kaolin led to slow fxii autoactivation and hk proteolysis, whereas ds in solution did not induce fxii or hk cleavage. fxii autoactivation seems to be responsible for the normalization of the prolonged aptt in pk deficiency after prolonged preincubation times. in our study we compared a conventional bag with silicone tubing (a) for blood donation with 2 new ones (]3 from biotrans and c from baxter) with a newly developed y-shaped adapter. this adapter is integrated into the tubing and therefore provides the advantage for drawing blood samples in a closed system. the 3 systems were identical in amount and content of anticoagulant, i. e. 63 ml of cpd per bag resulting in approximately 14% of the final whole blood volume. the purpose of the study was to determine whether the different tubings can influence the quality of plasma products conceming the blood coagulation system. in plasma samples we measured several factors of the procoagnlatory and fibrinolytic systems. intralndividual control eitrated (.135 m) blood samples were initially drawn from the contralateral cubital vein from the same male donor (34 in each group). in all bag samples we found small but significantly higher levels of the global test parameters ap'it and ti" compared to controls, indicating a higher amount of anticoagulant. pt, however, revealed no differences, thus suggesting that factor activities were not altered (statistics according to mann-whimey). increase of procoagulatory activity measured as tat complexes showed elevated levels in bags a and c whereas prothrombin fragments fl+2 decreased only in a. conceming the fibrinolytic system, plasminogen a~tivators and pai-1 values were diminished in all three systems 03 < a < c) compared to controls. d-dimers were lowest in a followed by slightly higher values in c, controls and b. fibrin monomers did not reveal any significant differences: a < c < controls < b. in summary, the quality, of the 3 different blood sampling devices was comparable to the intraindividual controls as to factor activities measured by global tests. the activation of the procoagulatory and fibrinotytic systems was slightly but in most cases significantly higher in the two new devices than in the conventional one. all values, however, obtained from the plasma samples did not exceed the normal range of healthy blood donors. therefore we concluded that the two new closed blood drawing systems are favorable for blood donating procedures. in 20 patients with acute myocardial infarction (ami) and thromholytie therapy (13 patients with rt-pa, 6 patients with streptokinase and one with heparln) with ck, myoglobin and ekg criterions the patients were divided in two groups (reperfusion/no fellow two hours after starting the thrombolytic therapy) . blood samples were taken before, 30 rain, i h, 2 h, 4 h, 8 h,12 h after lysis and than every day till day 10. because of the central role of factor xii in activation of coagulation, fibrinolysis, kallikreln-kinin-system and complement cascade we investlgate the role of factor xila initiated by ami and the relation of factor xiia to the thrombolytie agent and reoeclusion rate. for the investigatlens we take the kits from shield diagnostics (xiia), behring diagnostica (c~-inactivator, pl~nogen, ~-antip]~n~n, pap), chromogefiilx ab (prekallikrein) and di~nostica stage (vile). the results: there is an increase of factor xiia immediately after starting the fihrinolysis (max. 30 rain after starting); the increase 5/i independently of the thrombolytie agent. parallel to factor xiia raises factor viia without significant changes of c1-1naotor and prekalllkrein. that means: activation of xiia and fibrinolytic pathway leads to relatively mild c.hanges in kallikrein system, hut to significant activation of extrinsic system by vila-tissue factor. in some patients is an additional rise in the system xiia -viia, when the fibrinolytic system is already in the normal range. there will need further investigations to define the risk of reocclusion as a result of activation of faktor viia by faktor >li ia. autoimmune thrombocytopenic purpura (aitp) is a frequent complication of chronic lymphocytic leukemia (cll] which developes on different stages of the disease and needs special treatment measure. mechanism of autoimmune disorders in cll remains uncleared. we investigated immunologic phenotype of blood lymphoid cells in 22 patients suffering from cll with aitp. in these patients we did not observe disorders in expression of b-lineage markers as compared with cll patients without immune complications (13 patients). but in the 1st group of the patients the greater number of b-celts expressed markers of activation. according to ig heavy chain expression, the lymphocytes in most cases of cll complicated by aitp had more mature phenotype. in all patients with k phenofype of cll lymphocytes we found immune disorders. the development of aitp was accompanied by lowered level of t cells and changed dis'flibution of their immunoreguiatory subsets: diminished number of cdz~cells and increased one of cd~'÷lymphocytes. the results of our investigations undirectly proved that malignant b-cells in cll are involved in production of autoantibodies against blood cells. dysbalance in t-cell system with functional disturbances of immunoregulation are significant in development of autoimmune complications in cll a24 in women with severe fvii deficency (<10%) hypermenorrhagia may cause life threatening blood loss. therefore, hysterectomy at a young age is reported frequently in the literature. a 12 year old girl without history for a bleeding disorder was transfered with hypermenorrhagia. the initial laboratory data revealed an abnormal quick-test of 30% due to fvll of 9,0%, normal platelet count and hemoglobin level of 7,2 g/dl. antifibrinolytic therapy (tranexamic acid 4x15mg/kg bw/d) and lynestrenol substitution were started to reduce the hemorrrhage. despite treatment the daily blood loss increased to a maximum of 290ml. therefore, substitution therapy with recombinant fvila (rfvila) (novonordisk) was started at a dose of 15 ilg/kg bw q 6 h. subsequently blood loss decreased to 30ml/d, but even with an increasing dose of rfvlla up to 35 i~g/kg bwq 4h (fvil activity max. 7400% 10 min after injection) and additional hormonal support with a lh-fsh-anatgonist some hemorrhage remained. a short .course of methergin was stopped due to severe pain. ultrasound of the uterus revealed a hypertrophic endometrium causing the persistent bleeding. it decreased slowly over several weeks and hemorrhage stopped completely after 40 d. the total rfvlla dose administered was 118 rag. no side effects were observed. no transfusions of blood products were necessary. currently, menstrual cycle is suppressed by estriosuccinate. conclusion: due to close cooperation with a specialised gynecologist, hypermenorrhagia was controlled and in this woman with severe fvll deficiency hysterectomy was avoided. in three male members aged between 27 and 52 years of a family suffering from inherited bleeding disorders the diagnosis of protein z deficiency was established. plasma protein z evaluated by elisa (asserachrom protein z, diagnostica stago, france) ranged between 200 and 300 ng/ml. the patients mostly suffered from moderate bleeding complications like prolonged bleeding secondary to trauma or invasive measures and also spontaneous hematuria. previous laboratory investigations revealed variable platelet function deficiencies and transitory boderline decrease of von-willebrand factor. spontaneous bleedings were rarely recognized, however, they occured more frequently when analgetics were taken. bleeding complications showed good response to hemostyptic measures and antifibrinolytic therapy. the use of pcc containing a high level of protein z in these patients is restrained to severe bleeding disorders or major surgery. defibrotide is a mammalian polydeoxyribonucleotide derived anti-ischemic and antithrombotic drug (crinos s.p.a., v"flla guardia, italy). while the drug is known to produce polytherapeutic effects owing to its multicomponent nature, the exact mechanisms of its anti-ischemic effects remain unknown at this time. since defibrotide is found to be effective in ischemic disorders such as paod, vod related occlusive disorders and related rnicroangiopathic conditions, we studied the effect of this drug on the contraction of dog and pig arterial strip/rings obtained from various sites. in vitro supplementation ofdefibrotide to the organ bath containing control dog and pig arterial rings did not modulate the serotonin and thromboxane (generated) contraction, however, tissues obtained from dogs treated with 10 mg/kg defibrotide iv exhibited a profound desensitization to the agonist induced contractile process. the time course of these effects was found to be much larger than the plasma half-life of defibrotide. this presentation will provide additional data on the effect of defibrotide on the contraction of vascular smooth muscles as a possible explanation for the anti-ischemic effects of defibrotide. a. wehmeier, a. popescu, w. schneider klinik for h,~matologie, onkologie und klinische immunologie der heinrich-heine-universit&t d0sseldorf in chronic myeloid leukemia (cml), evolution of blast crisis is the limiting factor of survival. however, as in other chronic myeloproliferative disorders, bleeding and thrombotic complications are a major source of morbidity but their incidence has rarely been analysed in larger patient groups. we retrospectively evaluated 182 patients with cml during chronic phase (170 cases), accelerated disease (58 cases), and blast cdsis (72 cases), and determined the incidence of thrombohemorrhagic complications in relation to the stage of the disease. in chronic phase, 28 patients had bleeding complications (8.4%/patient year) and 15 patients thrombotic episodes (4%/patient year). the incidence of bleeding increased significantly in accelerated disease (18 patients, 51.2%/patient year) and blast crisis (37 patients, 347%/patient year), and many patients had repeated complications. contrary to our expectations, the incidence of thrombotic complications also increased to 10.2%/patient year in accelerated phase and 39.8% /patient year in blast crisis, tn chronic phase, 3 patients died because of bleeding events. in accelerated phase, 5 patients died due to bleeding and 1 patient due to thrombotic complications. in blast crisis, bleeding was associated with 21 deaths, and pulmonary embolism with 2 deaths. analysis of the cause of thrombohemorrhagic complications revealed that in chronic phase, bleeding was often associated with uncontrolled busutfan therapy, whereas in blast crisis, severe bleeding occurred mainly when platelet counts were low and peripheral blasts increased. however, there was no obvious explanation for thrombotic complications. we conclude that bleeding and thrombotic complications are a major source of morbidity and mortality also in cml, and that the incidence of such complications increase in advanced stages of the disease. klinik for innere medizin °, klinikum schwerin patients suffering from primary or secondary amyloidosis may occasionally acquire a coagulation disorder characterised by isolated factor x deficiency. we report on a 60-years-old man who presented with lower gastrointestinal bleeding and prolonged prothrombin time (quick 50 %). amyloidosis was suspected and proven using biopsy of the rectum and histological analysis. in addition, a monoclonal gammopathy of undetermined significance was diagnosed by immunofixation (light chain, type x). detailed investigation of the prolonged prothrombin time led to the discovery of a pronounced factor x deficiency (residual activity 4 %). inhibitors of coagulation factors could not be demonstrated. the treatment of the patient consisted of red blood cell transfusion and infusion of prothrombin complex concentrates. due to the extremely rapid clearance of infused factor x, no increase of its activity was observed. chemotherapy of the monoclonal gammopathy was initiated (melphalan/ prednisone). over the following six months the frequency of major bleeding episodes gradually decreased. however, subclinical occult bleeding continued. the factor x activity was repeatedly found between 10 and 12 %. we support the suggestion from literature data that clinically relevant bleeding episodes are likely to occur in patients with amyloidosis-associated factor x deficiency if the residual activity is below 10 %. sepsis and septic shock is a disease entity which is characterized by inflammatory reactions (sirs), coagulation abnormalities (dic), organ failure (mof) and severe hemodynamic alteration frequently leading to death in a shock. the aim of our studies was to investigate the efficacy of antithrombin iii (kybernin ®) on ~he outcome of septic shock in a pig endotoxemic model. pigs, in this model respond to lps with elevated tnflevels, decreased leukocytes and platelets counts, increased tat and fibrin monomer levels, hypotension and in increase of the pulmonary arterial pressure (pap), indicating impaired lung function. a total number of 13 male castrated juvenile domestic pigs (25 -30 kg) were anaesthetized, ventilated mechanically and infused with saimonella abortus equi lipopolysaccharide (s. equ-lps) over three hours (0.5 ~g/kg * h). a swan-ganz-catheter was inserted into the pulmonary artery to measure the pap. animals were allocated to two groups,, the treatment group (n = 7) received antithrombin iii (at iii) according to the following regimen: 250 u/kg (t = 60 -30, i. v. infusion), 125 u/kg (i. v. bolus, t = 0) and 250 u/kg (t = 180 -240 rain, i. v. infusion). the placebo group ( n = 6) received the appropriate amount of human serum albumin: 50 -25 -50 mg/kg (same schedule as with at iii). main objective was defined as the mortality rate at six hours a_~er s. equ-lps infusion. whereas in the placebo group 4 out of 6 animal died (mortality rate: 66 %) all at iii-treated pigs survived the observation period of 6 hours (p < 0.05, x2-test). the at iii group was shown to have a lower pap than the control group, especially the second peak of hypertension was abolished by at iii. it is therefore concluded that at iii should be a useful tool for the treatment of severe sepsis and septic shock. in a nationwide monthly survey all childrens hospitals in germany (esped) were asked to clinical and therapeutical informations about children suffering from pmi. during july 94 till june 95 299 children were registered. from these, 87 had either ecchymoses and/or necroses related to an increased mordibity and mortality (20%), whereas 212 showed no bleeding signs except for petechiae. of these children one died. the therapeutic interventions concerning hemostasis are listed according to the defined two risk ~oups. from the patients with ecchymoses or necroses, 13/87 received combination therapy (compared to 5/212 with petechiae or no bleeding sign) of at iii, heparin and/or plasma. only t child received protein c concentrate. the data show that children with low risk did in part receive higher doses of heparin and/or at iii concentrate than did high risk patients, whereas plasma therapy was adjusted to severity of eoagnlopathy. furthermore, the wide range of given therapeutics allows no information about the different medications. therefore, controlled studies with respect to the different therapeutic interventions in children with high risk pmi is desirable. a fully automated procedure for the reptilase time assay y. schmitt (1) and h.j. kolde (2) (1) institute for laboratory medicine, st~dtisches klinikum, darmstadt, frg, (2) dade diagnostics, unterschlei6heim the reptilase time assay is a relatively simple technique for the detection of fibrinogen degradation products and fibrinogen deficiency or abnormality. the procedure is performed with citrated plasma and batroxobin reagent, a snake venom enzyme from bothrops atrox. this enzyme cleaves fibrinogen by releasing fibdno peptide a only but not fibdno peptide b. in contrast to the physiological enzyme thrombin that is readily neutralized by antithrombin iii and hepadn batroxobin is not inactivated by physiological inhibitors. at present this assay is mainly performed manually or on mechanical instruments. we have adapted this assay to the electra 1000 fully automated coagulation analyzer (medical laboratory automation, pleasantville, n.y.) using the thrombin clotting time procedure in the instrument software with batroxobin reagent (dade diathe clot formation is registrated turbidimetrically and the dotting time is pdnted. the within run precision (n= 10) of this procedure was tested with two plasmas from the daily routine and was between 2.8 and 3.4 %. in 25 normal samples we found clotting times from 10.5 to 12.8 sec. in 30 samples with liver disease (confirmed by pseudochlinesterase < 2000 u/ml) or on thrombolysis therapy with streptokinase or urokinase the fully automated assay on the electra was compared to the semiautomatic method using a kc 10 coagulometer (amelung, lemgo, germany) based on a rolling metal ball pdnciple and magnetic endpoint detection. the two assays agreed very well with a correlation coefficient of r = 0,948 and a regression line according to passing and bablok of y = 1.0 x + 1.7. these data show that the reptilase time can be performed with good precision and with good correlation to the manual technique on mechanical instruments on the electra 1000. introduction: disseminated intravasal coagulation (dic), due to a massive activation of the coagulation system, is frequently observed in intensive care patients suffering from severe underlying diseases. laboratory diagnosis of dic is based on different coagulation tests, but unfortunately the routine haemostaseological parameters react with latency in the course of acute dic objective: in four cases from a cohort of 43 patients with severe sepsis and dic we analysed special haemostaseological parameters (tat, f1-t2, d-dimers, human-leucocyte-elastese (file), catepsin g and heparin cofactor ii (hc ii)) and correlated them with a mof-score in order to test their predictability on the prognosis of these patients. results: all patients were substituted with at iii concentrate. l,1 the investigated patients median time of treatment with at iii concentrate was 8 (6-9) days and median time of dic-duration was 6 (4-8) days. none of the presented patients died during observation period. all analysed parameters, except d-dimers, showed a sufficient correlation with the evaluated mof-score (tat: r= 0,78; f1-f2: r= 0,84; hle: r= 0,71; catepsin g: r=-0,75; hc ii: 1"=-0,88). the d-dimers did not correlate with the mof-score, which is probably due to the delayed reactive hyperfibrinolysis in the course of dic. furthermore, the decrease of the tat-complexes, f1-f2, hle and catepsin g levels were followed by an increase of at hi and hc ii activity. conclusion: in general the analysed activation markers and coagulation parameters are sufficiently to describe the ongoing process of the dic. the hyperfibrinolytic activity of dic is sufficiently represented by the d-dimer test, but is of defered reactivity in the course of dic. unfortunately these parameters are not established in the routine monitoring of dic on intensive care units and therefore further studies are needed to investigate the practicability and reliability in the daily routine monitoring. we have previously reported that notoginsenoside r1 (ng-r1) has an effect on counteracting lipopolysaccharide (lps) induced upregulation of plasminogen activator inhibitor-1 and tissue factor expression in cultured human umbilical vein endothelial ceils in vitro and in mice in vivo [fibrinolysis 1994;8:(suppl 1)119]. in this study we investigated the effect of ng-r1 on prevention of lps induced lethal toxicity in mice. because mice are relatively resistant to lps when applied as a single agent, we sensitized them by simultaneous treatment with d-galactosamlne. the 80% lethality induced by lps (1.5 mg/mouse) plus d-galactosamine (8 mg/mouse) in c3hs-ie mice was reduced to 16% by simultaneous administration of ng-r1 (1.5 mg/mouse) with lps/galactosamine (p<0.05 by x 2 test). ng-r1 also significantly delayed lps/galactosamine induced lethal toxicity from 12 hours to 30 hours with all animals surviving beyond 30 hours. because lethality induced by lps involves the synergistic effect of multiple effector molecules such as tumor necrosis factor (tnf)-ct, interleukin (il)-i, interferon 3' etc., we also investigated the effect of ng-r1 on lps induced tnf-ct production from leukocytes in cultured human whole blood cells (hwbcs) ex vivo. the production of tnf--ct induced by lps (1 ng/ml for 24 hours) in the supernatant of hwbcs was inhibited by 46% and 22% respectively, when the cells were incubated 1 ng/ml or 10 ng/ml lps together with 100 i~g/ml ng-r1, respectively (tnf-ct concentration, 1 ng/ml lps treated cells: 297+192 pg/ml, i ng/ml lps plus 100 l.tg/ml ng-ri treated cells: 162+137 pg/ml, p<0.01; 10 ng/ml lps treated cells: 3094_+487 pg/ml, 10 ng/ml lps plus 100 pg/ml ng-r1 treated cells 2423+713 pg/ml, /'=-0.02). the present results suggest that ng-r1 can prevent the onset of lps toxicity as well as the lps induction of cytokines. therefor ng-ri may be effective in preventing the effects of septic shock in gram-negative infections. to elucidate the mechanisms by which coagulation is initiated in septic patients in vivo, coagulation measurements were prospectively evaluated in patients with severe chemotherapyinduced neutropenia. this group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing prior to and during evolving sepsis or septic shock. 62 patients with febrile infectious events were accrued to the study. of these, 13 patients progressed to severe sepsis and an additional 13 patients to septic shock. at onset of fever, factor (f) vlla activity, f vii antigen and antithrombin iii (at iii) activity decreased from normal baseline revels and were significantly lower in the group of patients who progressed to septic shock compared to those that developed severe sepsis (medians: 0.3 versus 1.4 ng/ml, 21 versus 86 u/dl and 45 versus 95%; p < 0.001 ). the decrease of these variables in septic shock was accompanied by an increase in a marker of thrombin generation like prothrombin fragment 1 + 2 (medians: 3.6 versus 1.4 rim; p=o.05). these differences were sustained throughout the septic episode (p < 0.0001 ). f vlla and at ill levels of <0.8 ng/ml and <70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of 100 and 85%, and a specificity of 75 and 85%, respectively. in contrast, fxila-alpha antigen levels were not different between both groups at onset of fever and were only marginally higher further during the course of septic shock (p=o.001). thus, septic shock in neutropenia is associated with significant coagulation activation, presumably driven by the tissue factor pathway rather than the contact system. furthermore, in septicemia both f vlla and at iii measurements are sensitive markers of an unfavourable prognosis. hemostatic parameters in sepsis patients treated with anti-tnfct monoclonal antibodies c. salat 1, p. boekstegers 2, e. holler 1,3, b. reinhardt i, r. pihusch 1, k. werdan 2, m. kaul 4, t. beinert 2, e. hiller 1 med. klinik iii 1 und i 2, klinikum grosshadern der ludwig-maximilians-universitat monchen, h~imatologikum der gsf 3, knoll ag ludwigshafen 4 tumor necrosis factor et (tnfc~) is a central mediator in the pathogenesis of sepsis and septic shock. as administration of anti-tnfct monoclonal antibodies was able to protect animals from an otherwise lethal endotoxin challenge clinical studies were initiated in patients with sepis. tnfct exerts a procoagulant effect, e.g. by enhancing pai-i and activating thrombin as indicated by an increase in tat and pf 1/2 levels. therefore it may be involved in disseminated intravascular coagulation in sepsis. we determined tat, pf 1/2, d-dimers, tpa, upa, pai-i and vwf levels in 30 patients with sepsis or septic shock. 14 patients received the anti-tnfa monoclonal antibody mak 195f (knoll ag, ludwigshafen), whereas 16 patients served as controls. we found a significantly lower level ofupa in anti-tnfc~ treated patients. since the difference existed before onset of treatment it can not be attributed to tnfot antagonisation. all other parameters investigated did not differ significantly between the two groups throughout the study period. failure to detect modulation of hemostasis by anti-tnf~ might be explained by delayed initiation of treatment in clinical sepsis. in animal experiments it has been observed that the antibody prevented lethal endotoxin effects when given prophylactically or 30 minutes after endotoxin challenge, but not when it was administered 2.5 hours later. in addition, beneficial clinical and hemostatic effects of tnfet antagonisation might be observed only in subgroups of patients with hyperinflammatory sepsis. larger studies addressing this point are under way. protease receptors for thrombin and trypsin have been described for different cell lines. we investigated the ability of trypsin to activate human umbilical vein endothelial cells (huvec). cell activation was measured by the increase of intracellular free ca 2* (caff) with help of microscope fiuorometry (fura-2) and by the von willebrand factor release measured by a sandwich elisa. incubation of huvec with thrombin (1u/ml) or trypsin (10nm) showed a 2-10 fold increase of c~ff. a subsequent homologous stimulation after 80 s lead to a 2-5 fold lower concentration of ca~ 2÷ compared to the first stimulation. therefore cells have been desensitised by the first stimulation. inhibition of the proteolytical activity of trypsin by soybean trypsin inhibitor was followed by failure of trypsin inducing an increase of ca~ 2÷ concentration. in cross stimulation experiments with thrombin and trypsin, we could demonstrate, that cells first stimulated with thrombin showed a second maximal response by subsequent stimulation with trypsin. the same effect was measured with first stimulus trypsin and second stimulus thrombin. trypsin and thrombin induced a release of von willebrand factor (2-5 fold in comparison to unstimulated cells). we found a vwf release dependent on the concentration of trypsin similar to thrombin. an electrophoretic analysis of the released von willebrand factor showed a different multimeric composition of vwf between trypsin and thrombin stimulation. these results indicate, that there might be a protease receptor on huvec for trypsin being different from the thrombin receptor. clinical and laboratory findings of coagulopathy were investigated by an 1-year-survey to 320 children's hospitals. 291 meningococcal infections were evaluable. severe disease (characterized by need for mechanical ventilation, dialysis and/or catecholamines) was seen in 42 of these children; 29 of those survived and 13 died. clinical signs of severe coagulopathy were seen in 83 children: ecchymoses (n = 73) and skin necrosis (n = 36) were associated with increased mortality (16% and 20%, resp., compared to 4.5% overall mortality). five of 29 surviving children with skin necroses required surgical interventions (skin transplantation and/or amputations). petechiae were frequent (n = 156) and as isolated finding not related to severe disease or fatal outcome (6% mortaliy). platelet counts at admission were lower in non-survivors (10th-90th percentile: 30 -450.000/gl, median: 139.000/i.tl) than in survivors (10th-90th percentile: 140 -480.000/i.tl, median: 242.000/gl). at iii values showed no difference between survivors and non-survivors. protein c was available in few patients (n =14): in this subgroup, protein c was lowered in patients with limited disease (10th-90th percentile: 20 -105%, median: 48%) as well as severe disease (10th-90th percentile: 30 -75%, median: 60%). in conclusion, the findings "ecehymoses" and "skin necroses" were related to fatal outcome and therefore included in a prognostic score for severity of meningncoccal disease. the influence of irradiation on pai-i and vwf levels in human umbilical vein endothelial cell cultures k. fragiadaki, c. salat, r. pihusch, b. reinhardt, m penovici, e. hiller med. klinik iii, klinikum grosshadern der ludwig-maximilians-universitat monchen an elevation of pai-1 in bone marrow transplant recipients developing veno-occlusive disease (vod) of the liver has been described earlier. endothelial cell damage due to the preparative myeloablative radioehemotherapy is supposed to be an important step in the pathogenesis of the disease, which is characterized by an obstruction of small intrahepatic venules. in order to investigate a possible role of irradiation we studied the influence of several doses (0, 5, 15, 30 gy) on pai-1 and vwf levels in the supematant of human umbilical vein endothelial cell cultures (huvec). pai-1 antigen and vwf were determined by enzyme immunoassays. whereas pai-1 and vwf levels remained unchanged alter irradiation with 5 gy and in control cultures, a rise was observed one day after irradiation with 15 gy (mean day 0"-)day +1) in pai-1 (100,0% --)171,2 %) and vwf (100%--)159,7%) levels. the increase was more pronounced and reached levels of statistical significance after a dose of 30 cry (pai-1 100%--) 278,7% and vwf 100%--)168%). both pai-1 and vwf levels decreased on day 2 after irradiation with 15 and 30 gy. our results indicate that irradiation induces an increase of pal-1 and vwf in endothelial cells. nevertheless, this effect was observed only in doses above those ones used during conditioning when patients receive 3x4 gy. additional factors seem to be of significance. cytokines like tnfo~ enhance pai-1 and vwf in endothelial cell cultures and are known to be elevated in bmt-associated complications. it can be speculated that irradiation in concert with these factors may contribute to the development of veno-occlusive disease. disseminated intravascular coagulation is characterized by high consumption of coagulation factors, systemic elevation of fibrinolysis by tpa and concomitant elevation of pai-i secreted from inflamed endothelial cells. in an attempt to investigate the contribution of inflammatory cytokines, endothelial cells lines of microvascular origin were stimulated in vitro and pal-1 antigen was measured 2h, 4h and 24h after stimulation. in contrast to results published from experiments performed with macrovascular human umbilical vein cells (huve), our results obtained with 3 different microvascular endothelia isolated from skin, solid tumor tissue and bone marrow revealed that inflammatory cytokines reduced pal-1 antigen levels. in addition to tnf-a (25ng/ml) and lps (10pg/ml), we found that il-10 (100 u/ml) and gm-csf (100 u/rot) also reduced pai-i levels within the first 2h of incubation (from 120ng/ml to 80-110 ng/mll and the effect was even more pronounced after 4h and 24h (from 380 ng/ml to 250 ng/ml). il-1 (10 u/ml) and lps (10 pg/l) also reduced constitutive levels of pal-1 but the effect occured later than 4h after addition of the stimulator. the strongest synergistic effect was demonstrated with gm-csf plus il-1 resulting in pal-1 suppression of 50% after 2h and 30% after 24h. in contrast, g-csf (300 u/ml) induced the immediate (120 to 140 ng/ml after 2h and 380 to 420 ng/ml after 24h) upregulation of pal-1 antigen. stimulation of pat-1 levels was also observed with tgf-i~ (10 pg/ml), however not earlier than 18h of incubation. interestingly, both stimulatory cytokines, ie. g-csf and tgf-13, alone were able to counteract the decrease of pat-1 antigen by tnf-a but only a combination of g-csf plus tgf-g neutralized the effect by il-1. results indicate that inflammatory cytokines regulate pal-1 fibrinolysis in a synergistic and antagonistic fashion. we established the culture of human brain microvascular endothelial cells (hbmec) in order to investigate the pathophysiology of hu~man cerebral malada, which is still associated with a high mortality rate. it is widely accepted that among the reasons for the fatal outcome of cerebral malaria, the interaction of endothelial cells with cytokines and paras lites with subsequent changes in haemostaseological parameters is involved. the human microvascular endothelium may therefore play a deci §ive role in the pathophysiology of cerebral malaria. ery throcytes containing later stages of p. falciparum specifically bind to capillary ec in vivo (sequestration). tnf-cq il-1 and il-6 are considerably elevated in severe malaria. coagulation factors such as tissue factor and von willebrand factor are affected by malada suggesting the involvement of the hbmec in cerebral malada. so far, research on the involvement of the hbmec has been performed on ec cultured from human umblilical veins (huvec). the relevance of this model may be questioned on t, ,he grounds that the capillary endothelium probably plays a greater role than the endothelium of the large vessels. besides, some propertie.$ of the endothelioum seem to vary, upon the organ of origi/n. for the~ reasons, our laboratory has established the hbmec as a model to study the pathophysiology of human cerebral malaria. to demonstrate the relevance of this model in the context of malaria, hbmec were challenged with sera from different patients with severe p. falciparum malaria and with serum from a healthy donor. we can demonstrate that in cells challenged with malaria patient sera icam-1 and substance p were upregulated. on the other hand cells challenged with serum from a healthy donor expressed neither icam-1 nor substance p. these results strongly suggest the relevance of this model for vessel involvement in malaria. both, histamine and serotonin have been described as potent stimulators of yon willebrand factor (vwf) release from human umbilical vein endothelial cells (huvec). we performed experiments to differentiate the receptors for histamine and serotonin induced vwf release. absolutely unexpected we don't found any significant vwf release after the addition of serotonin to huvec or human artery endothelial cells (huaec) in concentrations from 0.1 ijm to 50 pm. in the case of histamine (0.1 pm -50 pm) we measured a vwf release 2-5 fold compared to unstimulated cells. this release was in the same order of magnitude as the release induced with 11u thrombin. to verify these results we measured the effect of histamine and serotonin on the intracellular ca 2÷ concentration (ca~ 2÷) in huvec and huaec. cells were labelled with fura-2 and the change in fluorescence after agonist addition was measured with a microscope fluorometer. using the same agonist concentrations as above we found an 5-10 fold increase of caj 2. with histamine or thrombin but no effect by addition of serotonin. this results indicate a similar activation of human endothelial cells by histamine and thrombin and that serotonin don't stimulate endothelial vwf release or increase of cay. activation and/or dysfunction of the endothelium can be triggered by cytokines (e.g. interleukin-2, tumor necrosis factor-alpha) or bacterial substances (e.g. endotoxins) and may contribute to shock and multi organ failure. pal-l and tm were assessed as parameters of activated endothelium following bsct in three to four days intervals from start of conditioning therapy through day +35. data were compared to the occurrence of sepsis, veno-occlusive disease (vod), capillary leakage syndrome (cls) and graftversus-host-disease (gvhd). patients with neither complication served as controls. no *days after stem cell tranplantation pai-1 and tm were increased in all patients with sepsis, cls~ vod and/or gvhd. pai-1 peaked at days 14 to 18 and the increase was highest in sepsis and lowest in cls. the increase in tm values was somewhat delayed (day +24) and was highest in vod and cls and lowest in gvhd. pai-1 and tm are sensitive markers of endothelial activation in sepsis, vod, cls, and/or gvhd, but they do not allow a differention between these complications. endothelin (et) is the most potent vasoconstrictor. it is known that et plasma concentration is correlated with a poor prognosis in patients with non ischemic cardiomyopathy (cm). the contribution of the heart to the production of et is still unknown. to investigate the pathogenetic mechanism in patients without coronary artery disease (cad), we examined 13 patients with hypertension ( . pulmonary capillary wedge pressure (pcwp) was measured in all patients. et and its precursor big-endothelin (bet) were determined at rest and after pharmacological stimulation with dipyridamole (0.5 mg/kg body weight), that increases coronary blood flow by factor 2 -4 on a non endothelial pathway. cardiac coronary et and bet concentrations were determined from the arterial blood samples, obtained from the aorta, and simultaneously from the coronary sinus (venous blood). blood samples were collected into ice chilled vacutainer tubes and stored after centrifugation at -70 *c. et and bet were analysed after extraction by a sepal< c 18 cartridge by radio immuno assay technique (immundiagnostik). it is concluded that et is increased with elevated filling pressures of the heart in patients with cm. it is not produced in considerable quantity by the heart neither at rest nor at increased blood flow. there4ore the lung has to be considered as the major organ for the production of et and bet in patients without cad. to characterize the incompatibility of blood with foreign surfaces valide in vitro methods especially in testing of platelet function are neceessary. it seems to be effective to use test systems which can also be helpful lateron in the clinic when foreign surfaces (e.g. venous catheters) are used and evaluated in so called phase-4-studies. we studied the influence of 21 reference polymers under standardized and controlled flow conditions on platelets in citrated blood specimen of healthy blood donors.the following tests were performed pre and post platelet-pol)aner contact: decrease of platelet count, platelet aggregation (wu-gmtemeyer index), analysis of platelet spreading capacity on standardized plastic surfaces by using a visual microscopic evaluation according to breddin and bfirck (1963) and an interactive computer-aided system (ibas, kontron gmbh, manchen, frg) by digitalizing the morphological picture of the platelet slides and area detection with a resolution of 512x512 pixels. results: platelet counts showed significant differences pre and post polymer contact, the wu-grotemeyer index demonstrated platelet activation only by blood contact with large volumes of polymeric material whereas both visual and computer-assisted evaluation of platelet spreading ability revealed a marked shift in the different classes of platelets: platelet activation results in a decrease of large structural elements and an increase of elements with spider threads. (pre contact (n=1000): 27:~-6 large forms of platelets, 700~-39 small forms and 275:l-41 spider forms; post contact (n=1000): 6-+-5 large forms, 510a:56 small forms and 484±58 platelets with spider threads). in some series there were significant differences between visual and computer-aided evaluation in the detection of small and spider forms. however, the relative increase of these nonspread spider forms could be stated with beth methods (wilcoxon test). we therefore conclude, that platelet morphometry with both methods is a sensitive and reliable ex vivo method to evaluate platelet interactions with artificial surfaces and can also be used lateron in phase-4-studies in patients. however, the ibas-system requires further maprovement in hard-and so,ware to reduce the high expenditure of this method. despite for the most part standardised methods such as hypothermia, cardioplegia the perioperative myocardial infartion rate is still high at approx. 6%. in cardiovascular surgery it is well known that various cardioplegic solutions are employed for myocardial protection during the ischemic phase. in order to evaluate the possible influence of these solutions we selected two of the most commonly used cardioplegic solutions for investigation in a randomised double-blind study: htk (group 1) and st. thomas (group 2). after randomisation each group consisted of 20 patients who had to undergo aortocoronary bypass surgery. aim of the investigation was to establish possible varying cellular changes during the reperfusion phase or in the early operative phase in order to be better able to apply reinforcing clinical measures. in the context of this study the classical enzyme-diagnostical methods ck,ck-mb and ldh as most useful, however not as convincing. still, we have in the meanwhile been able to show that the cardiac muscle troponin t proves a particularly sensitive parameter regards differentiated ischemic damage to the myocardium. ~his we were able to conflrm in extensive preliminary trials. cardiac troponin t was registered with a one-step lmmunoassay using two highly specific monoclonal antibodies directly via two different epitopes of cardiac troponin t. simultaneously the corresponding pre-and postoperative ecg was registered. further, within this context we investigated parameters that indicate cellular damage, such as platelet factor 4 (pf4), t-pa, interleukin-6 and pmn-elastase. in the reperfusion phase in group 2 there is a significant rise in tmponin t while in group 1 these values remain practically unchanged up to the 1st. postoperative day. of special importance is interleucin 6 since according to most recent studies the release of this substance leads to platelet activation via the arachidonic acid metabolism. this pathway must, further, be regarded within the context of free radical formation. on the 1st. postoperative day the 11 6 values in group 2 are significantly higher. the effects of membrane damage is also observed via pf4 and the pmn-elastase to be different in both groups. on the basis of this study we arrive at the conclusion that the htkcardioplegia is essentially less damaging than that of the st. thomas solution. (2) r. hetzer (2) (1) department of hematology and oncology, vimhow klinikum, humboldt university, berlin, germany (2) we investigated the influence of two different vad systems on these hemostatic changes. vads were implanted in 18 patients [11 bi-vad (berlin heart), 7 left vad (novacor n 100)] with end-stage heart disease who were awaiting heart transplantation. the following hemostatic parameters were measured during the first 51 days of bddging or until heart transplantation: thrombin-antithrembin iii (tat) complexes, prekallikrein, factor (f) xll, plasminogen, or2 -antiplasmin, and i?,thremboglobulin. results: during the first week of bridging, significantly higher tat levels were observed in novacor patients compared to berlin heart patients. prekallikrein activity levels were significantly lower in the berlin heart patients in the early bridging period. all other parameters were comparable in both groups throughout the entire observation period. differences in hemostatic parameters became apparent only in the early bridging period with more enhanced pmthrombin activation in the novacor group and more prominent contact activation in the berlin heart group. avoidance of the transmission of viral infections and saving in the use of blood products encouraged the use of apparatwe intraoperative autetransfusion techniques. patients and methods: arer randomization apparative intraoperative autotransfusion was performed in 5x7 patients during elective hip surgery using i-iaemonetics cell saver ill, haemonetics cell saver v, electromedics elmd, haemolite 3 and fresenius continuous autotransfnsion system (cats). at defined tmaes we detenmned a lab panel (clinical chemistry, lipids, proteolytic capacity, hemolysis, coagulation panel) at 9 determination points in the reservoir, the retransfused blood and in the patient. results: no significant differences concerning proteolytic capacity, prothrombin time, platelets, lipids, electrolytes. increased hemolysis (p<0.01) in the hcs iii group vs. the other groups (lo rain. after application of the retransfnsed blood). low heparin concentrations of retransfused blood in the hcs iii group( 0.32+-0.3 u/ml) vs. high concentrations in the cats group (0.47 +-0.3;p--0.01). parameters of thrombin generation were elevated in the hcs iii group vs. the other groups (p=0.02). conclusions: the use of 5 different apparative autotransfnsion systems dunng elective hip surgery results in dysturbances of hemocompatibility. the activation of the coagulation system during the collection and filtering is partly influenced by the elimination kinetics and the dose regime of heparin. however intraoperative autotransfusion must be roan~ged very carefully and possibly adverse effects of perioperadve heparin peak levels have to be considered. little information is available on the management of patients with factor viii deficiency who require cardiac surgery. we report the case of a 54 year old man with factor viii deficiency and combined severe aortic stenosis and incompetence and mitral incompetence who underwent a double valve replacement at our institution. he had a history of several bleeding episodes following minor surgery. previous factor viii levels were between 8 and 26%. using standard cardiopulmonary bypass, a double valve replacement with a 23 and 29 mm bileaflet prosthesis in aortic and mitral position, respectively, was performed. a high dose aprotinin regime was used (5.5 x 10 a iu). three doses of factor viii concentrate were given in the perioperative period, totalung 7000 1u until the 1st postoperative day. repeated measurements of the factor viii level were performed. the postoperative chest tube drainage was 350 rot. until the 4th postoperative day an additional dose of 3000 iu of factor viii was given to maintain a level of at least 30%. the obligatory anticoagulation was achieved initially with heparin i.v. in therapeutic dosage. due to a persistent 3rd degree av block a permanent pacemaker was inserted with additional 2000 iu of factor viii. on the 17th postoperative day warfarin was commenced aiming for an inr of 3.0 -3.5. the patient was discharged home therearer. he was trained to monitor his inr with a coagu chek device. no bleeding episode occurred during the first 3 months follow up. open heart surgery can be performed safely in patients with factor viii deficiency with the use of factor viii concentrates and monitoring of factor viii levels. coating of biomaterials was developed using synthetic polymers with incorporated anticoagulants. stents were coated with a thin layer consisting of a polylactide polymer containing peg-hirudin and a stable prostacyclin analogue. these materials were tested with a ,,human shunt model" using nonant/coagulated blood of healthy volunteers. within minutes uncoated stents were covered by fibrin and aggregated platelets, which could be seen macroscopically and by scanning electron microscopy; coated stents were free from coaguiation plugs. this observations were supported by analysis of coagulatiuon activation markers. unlike coated stents, uncoated stents revealed high levels (>detection limit) of tat complexes and prothrombin fragments (f1-2). in a series of experiments stents were tested in sheep. in 16 sheep stents (coated/uncoated patmaz-schatz stents) were ptaced by conventional techniques in the left anterior descending artery. anticoagulant therapy consisted of a heparin bolus and intravenously given aspirin before stent implantation. no ant/coagulation was given thereafter. existing data show hyperplasia in the area of uncoated stents which was reduced around coated stents (this study will be finished in january 1996). this coating technique with incorporated anticoagulants reduces thrombogenicity during the early and late phase of biomaterial implantation. studies concerning catheters, vascular prosthesis and oxygenators are in progress. the mechanical circulatory support (mcs) is a therapy for patients (pts) with endstage cardiac insufficiency. during mcs thrombeembolic events, due to the surface thrombogenicity of the implanted device, are feared complications. activated blood platelcts play a major role in this context. therefore, patient's platelet morphology was investigated. during the period of mcs, using the novacor left ventricular assist system n100, blood samples of 8 pts were observed by means of scanning electron microscopy (sem). blood was collected preoperatively and after implantation daily during the first week as well as weekly for the first 3 months. samples were drawn via an 18gauge cannula into caeodylic-acid buffered glutaraldehyde and platelets were prepared for morphological investigations. platelet alterations were classified as non activated, activated and aggregated, based on "shape change" morphology. additionally, the common blood coagulation parameters were evaluated. preoperatively, 15.0 + 4.6 % of activated platelets were found. within the first postoperative week, the mean level of activated platelets raised to 32.8 + 8.0 % (p<0.05). comparing short-(<30 days) vs. long-term (>30 days) mcs, a significant difference of activated ptatelets (overall mean values) could be seen (24.3 +_ 3.3 % vs. 34.8 _+ 3.4 %, p=0.004). during mcs a correlation between hemolysis and platelet aggregates, as well as the values of activated dotting time and activated platelets were observed. also, specific platelet deformations and damages appeared during mcs, which could not be found preoperatively. all pts with mcs showed alterations of their platelet morphology induced by the activation of the implanted synthetic material. with regard to the postoperative antithrombotic therapy, these observations should be taken into consideration. during extracorporeal circulation (ecc) the blood and its compenents are exposed to artificial surfaces and inflammatory respenses are activated, especially the complement, coagulation, fibrinolytic and kallikrein systems. furthermore leukocyte activation occurs and platelet function is impaired. these humoral and cellular systemic responses are known as the "pustperfusion syndrome" with clinical symptomes like lenkocytosis, increased capillary perraeability, accumulation of interstitial fluid and organ dysfunction. the impertance and even perhaps the existence of the damaging effects of cpb have been widely debated in the literature over the past 30 years. many efforts have been made to reduce traumatizing factors, e.g. the use of membrane instead of bubble oxygenators. recently, heparin-coated equipmen~ and tubings have been proposed to avoid excessive contact activation during cpb, the here presented study was designed to assess changes in coagulation and flbrinolytie activity in 20 patients undergoing cpb. in this regard we investigated coagulation parameters like fibrinogen, antithrombin, pmthrombin-fragments fl+2, thrombin-anthhmmhin complex, tissue-factor, fibrin-monomeres and parameters of the fibrinolytic system like tissue-plasminogen-activator, plasminantiplasmin-complex, d-dimers and plasminogen-activator inhibitor before, during and after cpb. the activation of the complement cascade was followed by measuring the concentration of c5a, c4 and c3c. the results demonstrate distinct alterations in above mentioned parameters. in spite of a high dose hepariulzation (act>450s) combined with an antifibrinolytic tw, atment an activation of the coagulation system was observed immediately after the onset of cpb followed by an activation of the fibrinolytic system. therefore further efforts should be done to develop new anticoagulatory regiments and improve the biocompatibility of materials used for cpb. during cardiopulmonary bypass blood is exposed to nonphysiologic conditions. the contact with artificial surfaces and mechanical stress result in a periopemtive response which includes activation of the complement, coagulation, fibrinolytic and kallikrein system, activation of nentrophils with degranulation and pmtease enzyme release, oxygen radical production and the synthesis of various proinflammatory cytokines. this so-called "pest-pump intlammatory response" has been linked to respiratory distress syndrome, renal failure and neurologic injmy. our goal was to investigate the time course of eytokine levels and the activation of leukozytes and platelets and to quantitate leucocyte subpepulatioas in 20 patients undergoing cpb. at different time points, pre, during and pest cpb, we determined the levels of interleukin (il) 113, il-2, il-4, il-6, il-8, il-10, tumor necrosis factor ¢z (tnf-a) and interferon "1' (ifn'--/) using elisa-techulques. lymphozyte subpepulations were characterized by flow cytometry and specific monoclonal antibodies against cd3 (pan t-cell marker), cd4 (surface antigen on t-helper cells), cd19 (surface antigen on b-cells), monocytes were determined by cd14 and platelets by cd41 (act. gpilb/llla) and cd42b (gp ib). single cell activation was analyzed using markers against cd25 (il-2 receptor), cd126 (il-6 receptor), hla-dr (mhc class ii), cd71 (transferrin receptor) and cd69 (activation inducer molecule), platelet activation was monitored with an antibody against cd62 (gmp-140). preliminary results revealed distinct increases in r,-6, il-8, and il-io following cpb whereas tnf-a and ifn--/levels were not significantly influenced. fttnhermore, activation of particular cell populations was observed. finally, our investigations should contribute to a better understanding of the complex humeral and cellular respenses induced by cpb and thus might help to develop new strategies to circumvent the negative impacts of cpb. optimal adjustment of anticoagulation in machine plasmapheresis is important for the quality of the prepared fresh frozen plasma (ffp) as well as for the safety of the donation. in the present study the suitability of prothrombin fragment ( ft+2 ) in the assessment of anticoagulation during plasmapheresis was investigated. matarlal and methods: 75 plasmapheresis procedures were performed on 25 donors (10 ~, 15o" ) using 3 different plasmapheresis machines (a 200, baxter; mcs 3p, haemonetics; pph 900, electromedics/medtronic). acid citrate dextrose formula a (acd-a) in a ratio to whole blood of 8 : 92 was used for anticoagulation. the concentration of fi+2 in the donor's blood was measured before and after plasmapheresis and in the prepared ffp. the actual acd-a volume used was also registered. results: there was a significant rise of the ft+2 -concentration in the donors blood after plasmapheresis with each of the three automatons: a 200:1.32 vs 1.14, p < 0.05; mcs 3p: 1.26 vs 0.98, p < 0.05; pph 900:1.20 vs 1.05, p < 0.05. the ffp prepared with each machine showed the following f~+2concentrations: 0.91± 0.18, 1.0:2 ± 0.17 and 0.93 ± 0.11 respectively. the difference between the groups was not significant. the elevation of the ft+2 -concentration in the donor's blood showed a negative correlation with the volume of the acd-a used. during 6 of the 75 procedures technical problems occurred (inadequate venous acces, occlusion of the citrate tube, reduced whole blood flow). after these procedures there was a marked elevation of f~+2 in the donors blood (2.74 ± 0.53), accompanied by an elevated f~+2 -concentration in the prepared ffp's. conclusion: these data show that ft+2 is a suitable parameter for the assessment of anticoagulation during plasmapheresis. several epidemiologic studies demonstrated that fibrinogen is an independent cardiovascular risk factor and should be considered for screening programs. prothrombin time derived fibrinogen (df) measurement combines the advantage of an established highly reproducible automated method with no additional reagents, except for calibration. several studies showed that the df values correspond well with the clanss method except in cases such as thrombolytic therapy in which the df results are higher. however, no results exist whether in patients with coronary heart disease with fibrinogen as a risk factor the df values are also comparable to the established clausss method. the aim of our study was to compare df values to clauss method results in cardiac patients, especially in patients before and after coronary bypass grafting (cab(]). measurements of df were performed on an acl 3000 (il) using the pt-fibrinogen-hs reagent. fibrinogen clanss method was done on the acl using fibrinogen c reagent (il) and on a kc4 (amelung) with fibrinogen a reagent (boehringer maanheim). for calibration we used the calibration plasma half volume (it.) with the fihrinogen concentration proposed by the manufacturer. plasma samples were obtained from 24 patients at admission before cabg and postoperatively up to 1 week, and from 23 healthy persons (staff). within assay imprecisious using normal and abnormal controls (il) were comparable with both methods showing cvs between 1.99 and 4.22 %. in normal healthy persons the medians of the df and the clanss method run on the acl were very similar (296 vs 302 rag/all), whereas kc4 values were about 10% lower (268 md/dl). in cabg patients at admission we found the same differences as in normals with the clanss method (acl: 363 vs kc4: 337rag/all), however the df values were siginficantly higher (median 418mg/dl). if we took a cutoff value of 320 mg/dl, as suggested by the results from the northwick park heart study, we would categorize into the high risk group 21 out of 24 patients using the df method, 20 with the clanss-acl method and 16 with the clanss-kc4 method, i.e. nearly 30% more patients were classified in the high risk group using the df method. postoperative samples showed the expected increases due to the acute phase response with the same magnitude of differences. because of its rapidity and reproducibility the df method is well suited for routine measurements, however, standardization remains an urgent task in order to avoid misinterpretation of results. for fibdnogen measurements in clinical laboratories, the two most widely used methods are the clotting time method according to clauss (cfib) and the sn called "derived" fibrinogen method (dfib) implemented in optical coagulometera with the fibrinogen concentration being derived flora the optical density of the fibrin clot in a standard prothrnmbin time (pt) assay. it is well known that under certain circumstances, e.g. in the presence of fibrin(ogen) degradation products (fdp), there is a discrepancy between the two methods with higher values for dfib than for cfib. yet the opposite discrepancy, i.e. fibrinogen values derived from the optical density of the clot grossly lower than values from dotting time assays, seems to be very rare and is poorly understood so far. the patient (male, 26 years) had ingested the esterase inhibitor parathion (e605) in an attempt o f suizide and was treated with high doses of atmpin. he had no clinical signs or history or family history of bleeding or thrombotic disorders. except for a very low pseudocholinesterase activity, all laboratory results were normal ineinding pt, afft, thrombin time, and factor xiii. pt and aptt did nnt differ between an optical coagulometer (electra 1000c, mla) and a mechanical one (kc..4, amelung). there was no evidence of disorders known to interfere with hemostasis like paraproteinemia or dyslipldemia. however, in all 7 blood samples received for dotting tests during a period of 7 days the macroscopic appearance of the fibrin clot was quite unusual (only slightly turbid/almost transparent) and there was a striking discrepancy between a very low or low dfib on the electra (pt reagent: thromboplastin is, dade) and a normal or high cfib (kc4; thrombin reagent, dade). on admission, values were 57 mgml (derived) vs. 275 mgldl (clauss). cfib rose to s42 mg]dl with dfib at 155 mg/dl in the last sample on day 7. ~ al! samples dfib was about 20 % (ls-23) of cf[b. when the patient's plasma was added m normal pooled plasma it caused, in a dose-dependent manner, values lower than predicted for dfib and values slightly higher than predicted for cfib. in the absence of data from additional (e.g. immunologic) methods the following principal possibilities (and combinations) have to be considered: 1) normal fibrinogen concentration and clot formation rate, but abnormal optical properties of the clot (cfib correct, dfib falsely tow); 2) normal optical properties of the clot, but accelerated clot formation and very low fibrinogen concentration (dfib correct, cfib falsely high). in either case, the molecular basis could be: a) a genetic or acquired molecular abnnrmality of fibrin/fibfinogen; b) an interfering substance. direct effects of the loxic agent parathion and/or the antidot drug atropin are not likely to be the cause since other patients, often with more severe parathion inmxicatian requiring higher doses of atmpin, showed normal optical density of the clot. we hope to perform a more in depth investigation of this abnormality in the future, including various methods, reagents, and instruments for fibrinogen measurement, a survey of the patient "s family, and studies of the molecular nature of the phenomenon. increased fibrinogen is known to be an independent predictor of subseqtmnt acut~ coronary syndromes. however. a multitude of methods for fibrinogen determination is available. there is a lack of standardisation among fibrinogen assays. in a family cohort study (patients'with combined hyperlipidaemia and f or hypemricaemia) fibrinogen was determined in plasma samples from 340 family members using a functional and an immunochemical assay. the fimctional assay according to clauss was performed on the analyser ca 5000 using the test fibrinogen a from boehringer. the immmmephelometric assay was performed on ~e behring nephelometer system using the reagent and standard from behring. a good similarity between both assays was obtained at low and high flbrinogen levels as well as in samples with increased c-reactive protein (crp). values obtained by both assays correlated similar with total cholesterol, ldl--cbelesterol and apolipeprntein b. the ratio functional fibrinagen / immlmochemial fibrinogen showed no dependence on cholesterol, t-pa, v wiuebrand factor and crp. release of two fibrinopeptides a from fibrinogen generates desaa-fibrin monomer, which rapidly aggregates, forming fibrin complexes. fibrin monomers can be detected in plasma samples after chemical desaggregation of fibrin complexes using thiocyanate by monoclonal antibody binding to the alpha-chain neo-n-termini generated by fibrinopeptide release. although postulated, an intermediate of fibrin formation, carrying one fibrinopeptide a and one fibrin alpha-chain neo-n-terminus has so far escaped analytical procedures. we have employed a monoctonal antibody specific for fibrin alpha-chain neo-n-terminus, mab 2b5, attached to magnetic microparticles, for isolation of fibrin-related material from plasma samples of patients with elevated soluble fibrin. the material was desorbed by sds-urea buffer and subjected to sds-page and immunoblotting. immunostaining with panspecific anti-fibrinogen and anti-fdp-e antisera showed a range of bands corresponding to fibrin monomers, and fibrin derivatives containing the fibrin e-domain. lmmunostaining with monoclonal anti-fibrinopeptide a antibody resulted in a doublet band corresponding in size to fibrin monomer. similar results were obtained with polyclonal antisera against fibrinopeptide a. for a more quantitative approach, desa-fibrin monomer was detected by an elisa procedure using mab 2b5 as capture and monoclonal anti-fibrinopeptide a antibody as tag. a sample with extremely high level of desaa-fibrin monomer, determined by elisa (enzymun®-test fm) was used for calibration, since reference material is not available. a correlation of r=o.g4 was found between desaa-fibrin monomer and relative desa-fibrin monomer levels. detection of desa-fibrin monomer required sample pretreatment with thiocyanate for desaggregadon of fibrin complexes. from these preliminary data it appears that desa-fibrin monomer accounts for a fairly constant proportion of soluble fibrin and is a polymerizing species. fibrinogen has been shown to be a major cardiovascular risk factor. especially for epidemiological studies, exact quantitation of fibrinogen in clinical plasma samples is of great imporance. fibrinogen levels are generally measured by clotting assay according to clauss, or by determination of derived fibrinogen values upon photometric measurement of prothrombin time (derfbg). the clotting assay has been shown to be influenced by high levels of soluble fibrin derivatives. the pt-derived fibrinogen levels appear rather convenient in clinical routine, since no additional reagents are needed. we have compared the clauss assay and derfbg with a turbidimetric fibrinogen assay using snake venom protease for fibrinopeptide release, performed in photometric autoanalyzers. d-direct antigen was measured in parallel using tinaqaant d-dimer lpia. results were correlated with total fibrinopeptide a release by thrombin, measured by elisa. a total of 484 samples were included, of which 29 samples (6 %) were recorded as above measurung range by derfbg. these samples encompassed a range of 5.90-10.40 g/l and 5.21-12.37 g/l in clauss, and turbidimetric assay, respectively. the range of values measured by derfbg assay was 0.72-9.14 g/i, corresponding to 0.26-11.00 gll and 0.24-10.48 g/1 in the clauss and turbidimetric assay, respectively. the correlation of derfbg with the clauss assay was re0.91, correlation with turbidimetric assay was r=0.92 for the values actually detected. the correlation between clauss and turbidimetric assay was r=0.93 for all values. there was no dependency of test results or inter-test variation upon d-direct. correlation graphs displayed a decreased test response of clauss assay in the high concentration range, resulting in an underestimation of fibrinogen concentration. the derfbg assay, in contrast, showed normal range values in samples from patients with fibrinotytic treatment and low fibrinogen levels in the other assays. correlation with fibrinopeptide a release was r=0.88 for clauss assay, r=0.89 for turbidimetric assay, and r=0.82 for derfbg. for clinical routine, derfbg appears to be applicable for all samples between 1.00 and 5.00 g/l with exclusion of samples from patients with fibrinolytic treatment or endogeneous hyperfibrinolysis. other samples may be analyzed by clotting assay or turbidimetric assay, although the latter appears to be more suited for measurement of high range samples. for inhibition of pk is 0.067 pmol/l the antifibrinolytic activity of the inhibitors was determined by measuring the lysis of radiolabelled human plasma clots• the compounds which inhibit plasmin and pk influence remarkably the streptokinase-induced clot lysis but not lysis induced by uk and tpa. surprisingly, inhibitors of uk and tpa do not influence clot lysis induced by uk or tpa. the structure-activity relationships for inhibition of ptasmin, uk, tpa and pk could help in the design of more potent inhibitors of fibrinotytic enzymes. uk inhibitors are of interest for the development of anti-invasiveness drugs, while plasmin/pk inhibitors could be prototypes of a "synthetic aprotinin". in the ecat angina pectoris study t-pa antigen was an indepcndem risk factor of subsequent acute coronary syndromes. pat indicates the risk bat depends on other known risk factors. it should be tested in 183 members of a family cohort study (patients with combined hyperlipidaemia and / or hyperuricaemia), if the active pal antigen or the whole pai antigen showed a stronger relation to t-pa and metabolic variables. the active pall antigen was determined using elisa actibind pat-1 (technoclone / lmmuno) , the whole pai-i antigen was measured using the f_lisa pat-1 (technoclone i immuno). t-pa activity was determined with the coaset t-pa from chromogenix, the tintelize tpa from biopool was the used test for determination of t-pa antigen. the active pat antigen showed a stronger correlation to t-pa activity and t-pa antigen than the whole pal antigen. circulating t-pa activity was influenced predominantly by the active pal antigen. both pat antigens were correlated in similar manner with metabolic variables, lipoproteins and b/vii. table: correlations of active and whole pal antigen (** p < 0,001) active pal antigen whole pal antigen active pat antigen 1,000 0,851 ** whole pal antigen 0,851 ** 1,000 t-pa activity -0,594 ** -0,492 ** bpa antigen 0,604 ** 0,497 ** body mass index 0,502 ** 0,462 ** triglycerides 0,452 ** 0,441 ** total cholesterol 0,252 ** 0,255 ** ldl-cholesterol 0,263 ** 0,264 ** hdl-cholesterol -0,357 ** -0,355 ** apolipoprotein b 0,428 ** 0,402 ** apolipoprotein a i -0,233 ** -0,211 ** the lower relationship of the whole pat antigen to t-pa is obviously caused by patient samples with high levels of whole pat antigen in contrast to normal values of active pat as well'as of t-pa. possibly, a high ratio of whole pai antigen / active pat antigen is caused by a raise of latent pal the main form of pat in the platelets. the clinical importance of an increased ratio whole pal antigen / active pal antigen remains under investigation. the cyclic antibiotics-polypeptides bacifracin a, bacilliquin from boci/lu~, licheniformis and gramicidin s from bocil/us brevis, var. (3. b., were used for investigation. we studied their influence on the fibrinoly~c and coagulation activity in vitro• me~hods. to solution of human plasmin (thrombin). containing 0.2 mg of protein (1 nih unff)/ml, the analyses' solution of antibiotics (0.1-8,0 mg) was added. then we defined the tlbrinolytlc activity of the mixes using azofibrin lysis, and fhrombin activity was determined according to the speed of fibrin clots formation from fibrinogen solution. results. in following table are submifled the results received in our laboratory {we also offer results of antibiotics influence on urokinase activity): ki, mm ki --the constant of inhibition. n. d. ~ in studied lirnils the inhibitor's activity was not observed. ---the inhibitor's activity was not define. i. --the inhibitor% activity was observed but ki not determined. +, +% +++ --effect of inhibffion (in rela*iive indexes). conc/us/on.~ the results received by us testify to the necessity of cautious approach to the use of antibiofics-polypeptides for various sorts of therapy in view of their possible influence on fibrinolytic and coagulation actlvlfy, of the organism. these results were used for preparation in our laboratory of biospeciflc sorbents containing c-ramicidin a, bacil}iquirt and gramicidin s.as ligands, they can reversibly bind thrombin, plasmin {plosminogen) and urokinase directly from crude exkacts. the enzymes are selectively eluted without substantial losses of specific activity in e yield of 60-90%. there is a great body of rather contradictory informations dealing with fibrinolysis in liver.. cirrhosis, which can be accelerated, normal or reduced, depending on the type of cirrhosis and investigation techniques (clot-lysis, fibrinolytic component measurements). our previous finding was, that in vitro plasma-clot lysis, induced by exogeneously added tpa or streptokinase proved to be reduced, and this had a good correlation with severity of the disease and the elevation of plasmatic yon willebrand factor levels. in vitro clo~/-[ lysis tests, induced by tpa were performed in 41 patients with alcoholic liver cirrhosis, utilising a microplate light-scattering assessment method. the tests were repeated using the same plasma samples in each patients with a microplate which was covered by cultured endothelial-cell monolayer (umbilical vein, huvec}. clot lysis speed proved to be 1.5-2 times slower with huvec milieu in the control group, while in the cirrhotic patients this inhibition was stronger and resulted in 5-fold reduction of lysis speed. our results suggest, that cirrhotic plasma is able to accelerate the release of fibrinolytic inhibitors from cultured endothelial cells, which phenomenon may also contribute to the complex alterations of in vivo fibrinolysis in cirrhotic patients. deep vein thrombosis (dvt) is a systemic disease with prolonged clinical manifectation. anticoagulation therapy in dvt is not completely effective. thrombolytic therapy may give rise to a systemic lytic state, the fibrinospesific agents (scu-pa and t-pa) have short half-lives in the circulation. we investigated the potency of the acylated plasminogen streptokinase activator complex (gbpg-sk) to deep vein clot dissolution as compared to well known sk and apsac both in v~tro and in vivo in the model of venous thrombosis in artherio-venous shunt in rats. it was shown in in vitro study that fibrinolytic activity of plasminogen activators mainly depends on their stability in plasma. stability studies carried out by incubating sk and pg-sk activator complexes in plasma with euglobulin precipitation . total fibrinolytic activity was measured by the fibrin plate method. gbpg-sk possessed the greatest stability in human plasma than apsac or sk because of its prolonged inactivation period (the deacylation half-life for gbpg-sk was 230 :e 21 rain in contrast with 73 -~ 6 min for apsac). the stability degree of two acylated thrombolytics (gbpg-sk and apsac) was in order to inverse proportion of their first order rate deacylation constants (2.9 • 10 -4 and 6.0 • 10-s sec-1 respectively). the fibrinolytic potency of sk, apsac and gbpg-sk was measured by 1251-labeled fibrin clot lysis in plasma and in vivo by lysis of the preliminary formed 1251-labeled fibrin clot inserted into the jugular vein. fibrinolytjc activity of acylated plasminogen activators gradually increased in time. under sk administration, the clot lysis came to the end by 2 hours while apsac and gbpg-sk haven't lost their activity for 5 -6 hours. gbpg-sk possessed significantly more prolonged fibrinolytic activity than apsac, the acyl-enzymes did not significantly influence on plasminogen,,.~2-anfiplasmin and fibrinogen levels in plasma according to their activity specific to fibrin-bound plasminogen. in opposite, sk produced a significant depletion of plasminogen, ~-2antiplasmin and fibdnogen levels in plasma. it seems, on the basis on in vitro and an animal experimentation, than apsac with its moderately fast deacylation rate is more suitable for rapid thrombolytic effect, but gbpg-sk with its slow deacylation rate is suitable for deep vein thrombosis, when the rapid thrombolysis is less critical. it's well known that the complete lysis of thrombi usually isn't observed at the thrombolytic therapy. at present study we have attempted to quantify the possible mechanism of fibrinolysis inhibition during the thrombolysis. 125i-labelled partially cross-linked fibrin clots of different volume (0.1-0.35 ml) were immersed in tris-hcl buffer (3 ml) containing plasmin (5-100 nm) at 37°c. the lysis rate was detected by counting of soluble fibrin degradation products (fdp). at all the eases lysis slowed down and stopped in 3 hs though clots dissolved up to only 60-85%. no irreversible inlaibition of plasmin caused by denaturation occur as was judged by the measurement of fibrinolytic activity at the diluted samples. however the increase of fdp concentration in surrounding buffer led to the reversible inhibition of fibrinolytic activity of plasmin up to 5% of baseline. the sds-page analysis under non-reduced conditions shown the acoumulation of high-molecular weight fdp at the surrounding buffer. the inhibition phenomenon could be connected with the specific binding of plasrnin with soluble fdp having exposed lysine residues and the subsequent removal of enzyme from fibrin surface. unexpectedly since the heterogeneous character of occurred reactions tile change of the clots surface area during lysis didn't affect the fibrinolysis kinetics in all the concentration intervals. to estimate the kinetic parameters the kinetic curves were linear in the coordinates [p1/t (l/t*ln(isl°{(lslo.lpi)). the obtained parameters were following: keat=l.36 min-l,km=l.33 ixm,kp=0.12 ~tm. the clinical trials have shown that fdp concentrations at the thrombolytic therapy of deep venous thrombosis and acute myocardial infarction usually was approximately in the range 0.05-0.2 ~tm. therefore the described phenomenon of fibrinolysis inhibition by formed fdp may take place during thrombolytic therapy. al. calatzis, an. calatzis, +m. klmg, +l. mielke, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology technische universit~.t monchen thrombelastography (teg) is an established method for the detection of fibrinolysis. fibfinolysis is usually determined when the teg amplitude decreases by more than 15% atter the maximum amplitude is reached. this takes a considerable amount of time (more than 30 minutes). our approach bases on the understanding of fibrinolysis as a process which runs in paraue[ to coagulation and is not exclusively subsidiary to it. the effect of fibrinolysis on the growing clot in the teg is shown by the comparison of two parallely performed teg measurements: exteg: teg measurement with standardised activation of the extrinsic system. apteg: exteg with in-vitro-fibrinolysis inhibition via aprotinin. exteg-reagent (ex): 1:2 dilution of innovin (recombinant thromboplastin reagent, dade) with aqua dest. apteg-reagent (ap): 5 parts innovin, 2 parts trasy[ol (aprotinin, bayer, i0.000 kie/ml), 3 parts aqua dest. test procedure: l0 p1 ex or ap + 300 ~l citrated blood (cb) + 50 lal cacl2-solution 0,15 m. the only difference of the two reagents is the addition of 20 kie aprotinin in the apteg, leading to an in-vitro fibrinolysis inhibition. the usage of disposable pins and cups (haemoscope, illinois, usa/e.m.s., vienna) is recommended for ensuring standardised conditions for both measurements. results and discussion: when there is a better clot formation in the apteg (corresponding to a lower so-cafled k-value) than in the exteg, fibdnolysis can be suspected. this technique requires only commercially available reagents and is easy to perform on conventional teg systems. due to the standardised coagulation activation with a thromboplastin reagent, fibrinolysis can be detected also when inhibitors like heparin are present in the circulation. according to our experience using this technique during liver transplantation, clinical relevant fibrinolysis can be detected as described in less than l0 minutes. many thromboembolic (massive pulmonary embolism, proximal deepvein thrombosis, etc.) and coronary diseases (infarction, acute phase, etc.) require fibrinolytic therapy to early recanalizafion. the application of the well-known or new thrombolytic agents needs the use of specific, simple and reproducible methods for the determination of fibdnolyfic activity. we suggest new methods for measuring the blood plasma concentrations of plasmin, plasminogen, antiplasmins, and urine urokinase activity. these methods involve the employment of chromogenic substrafe azofibrin (human fibrin, covalently labeled with p-diazobenzenesulfonic acid). method~. 0.2 ml of studied solution was added to 0,8 ml of azofibrin suspension in certain buffer (5-10 mg/ml) and the mixture incubated at 37 oc for 10-60 rain. after the end of incubation the mixture was filtered, the volume of solution brought up to 4 ml by 0.02 m naoh and the optical density was determined at 440 nm. resuffs. azofibrin can be used for quantitative determination of proteinases activity in search of new fibrinolytic means. for comparison the results of our studies fibrinolytic activity of some proteinases with the use of azofibrin are presented: activity. with an increase of pal and ldl-and a decrease of hdl-cholesterol concentrations k is concluded that the increased cardiovascular risk in diabetes meilitus was partly caused by a down regulation of the fibrinolytic system, increase of erythrocyte aggregation and plasma viscosity. also disturbances of lipid metabolism an abnormal whr seems to be of an additional atherogenous factor in dm. plasma concentrations of thrombin-anfithrombin-iii (tat), alpha-2antiplasmin-plasmin (app) complexes and ddimer were investigated in 50 patients treated with thrombolytic therapy for acute myocardial infarction (ami) either with streptokinase (n=24), urokinase (n=16) or recombinant t-pa (rt-pa, n=10). all patients received an intravenous heparin bolus of 5,000 iu on admission, which was followed at once by an infusion of 1,000 iu/hr for the next three days titrated to maintain the partial thromboplastin time at twice control value. tat, pap and ddimer were measured by enzyme immunoassay on admission, 1, 2, 4, 6, 8, 12, 24 hours and on day 3 and 7 after admission. groups did not differ significantly in regard to age, sex, delay and infarct location. on admission, no marker differed significantly between groups. thereafter, tat levels increased significantly exclusively in rtpa treated group. from 2 to 6 hours after admission, tat were significantly higher in rtpa treated patients than in streptokinase and urokinase treated group (p<0.02). however, during continous heparin infusion, which was started immediately after stop of thrombolytic therapy, in each group tat concentrations decreased below admission values. app were significantly higher only 1 hour after admission in the rt-pa group (p=0.03). ddimer did not differ signifieanfly between groups. our results demonstrate, that rtpa induces a hypercoagulable state, which may contribute to reocclusion after successful reopening of the infarctrelated coronary artery. the significant tat decrease during continous heparin infusion supports the concomitant use of thrombin inhibitors as adjunctive therapy with thrombolytlc treatment for ami. thus, in acute myocardial infarction patients, thrombin generation is markedly influenced by the thrombolytic agent used and concomitant heparin therapy. endothelium derived relaxing factor-no (edrf-no) plays a major role in regulation of vascular tonicity and also exerts platelet inhibitory action~ however, due to the chemical nature of edrf-no few is known about its production and activity as a general index or marker of vascular function in human diseases. one way to achieve this can be measurement of nitrate/nitrite excretion in the urine, which seems to reflect vascular edrf-no production. in this report a self-developed elisa method is described, which was used for this perpose. nitrate/nitrite urinary exretion proved to significantly decreased in insulin dependent and in non-insulin dependent diabetes mellitus as well after a comparison of the excretion values to other markers of angiopathy (yon willebrand factod soluble thrombomodulin, beta -thromboglobulin) it seems to be acceptable, that urinary nitrate/nitrite excretion can be a useful indicate of diabetic vascular disorders. two major concerns still accompany the application of prothrombin complex concentrates (pcc). viral safety has to be guaranteed and therefore several measures for virus inactivation or elimination are taken during the manufacturing process. the inherent risk of thrombo-embolic side effects has to be considered. to minimize these risks and to achieve good clinical efficiency the quality criteria for pcc's are under pending discussion. it is generally accepted that a modem pcc-preparation should contain all of the four coagulation factors in a well balanced proportion and that it should also contain protein c and protein s. additionally, the concentration of activated coagulation factors should be kept at a minimum. a present pcc-produedon process mainly consists of a qae-sephadex extraction of cryopeer plasma followed by a solvent/detergent virus inactivation step. further purification is achieved by subsequent chromatography on deae-sephamse. the aim of this study was to improve product quality by avoiding f viiactivation without implementing major changes to the production process. at the same time, a second virus eliminating step was added to the production process. it could be shown that speeding up the chromatographical process by switching the deae-sepharose-chromatography from a classical axial column to a radial chromatography resulted in a significant reduction of f viia-genemtion. mainly the reduction of contact time, resulting from the highest possible flow rates, leads to the wanted effect. the relation between f vii/f viia was 10 : 1 or more. in order to investigate the feasibility of virus filtration the eluate of the deae-sepharose column was filtered through a virus removing ultipor vffilter. the analysis of the solution before and after fillration showed that the filtration had no influence on coagulation factors activity, protein content, proteolytic activity etc. preliminary studies showed significant virus reduction values. in the past few years the problem of expediency of the treatment aimed at developing immunological tolerance in hemophil;a patients by way of complete removal of inhibitor with high doses of factor viii has been discussed in literature. we observed 121 patients with hemophilia. inhibitors to factor viii:c were revealed in 32.7 % of patients with hemophilia a and fo factor ix --in 1.6 % of patients with hemophilia b. the level of an inhibitor was not higher than 87 befhesda u/ml, that is those patients were not regarded as "high responders". a high incidence of inhibifors in young patients [from 7 to 26 years of age, 51.9 %) compared with older patients (from 27 to 40 years of age, 11.2 %) testifies to the probability of inhibitors development during treatment with modern concentrated preparation of factor viii, ix. inhibitor development in patients (40.5 %] in the course of antihemophilic concentrates transfusions is an evidence of alloimmunization of patients with proteins. the investigations show that in the course of transfusion therapy patients develop secondary immunodeficiency due to chronic antigenic stimulation of immune system with high doses of allogenic proteins. against the background of immunodeficiency patients with hemophilia develop complications of immune character: infections complications --53.9 %, aufoimmune processes --44.9 %, secondary tumours --1.2 %. plasmapheresis is the most rational method of removing inhibitor in patients with low level of inhibitor ("low responders", < 10 bu/ ml) and in patients with mean response. thus it should be noted that the treatment of patients aimed at developing immunological tolerance is not only expensive and economically unprofitable but also not indifferent fo the organism. in a recent multicenter study 73 previously untreated patientens (pups) with severe hemophilia a were treated with a recombinant factor viii concentrate (rfviii, recombinate©). during fviii treatment 21 (29%) developed inhibitors, 6 high titer (>5 bethesda units (bu)/ml), 4 low titer (<5 bu/ml) and 11 transient inhibitors. plasma samples from before treatment and during treatment but before inhibitor occurrence were available in 12 inhibitor patients. these plasma samples were analyzed by a highly sensitive immunoprecipitation (ip) assay for the presence of anti-tviii antibodies. in 9 (66%) a significant increase of anti-fv]]i antibodies was seen indicating the development of a clinical relevant inhibitor titer. this immune response occurred after 2 to 17 (median 5) exposure days (ed). in the same period only 3 out of 15 inhibitor patients showed a decreased in vivo recovery. in 16 pups who developed no inhibitors plasma samples from the entire treatment period were available. an immune response to rfviii treatment was seen in 7 pups after 2 to 43 ed (median 24 ed). the immune response was later and less pronounced in comparison to inhibitor pups before inhibitor occurrence. with the ip method the detection of an early immune response is possible which might be predictive for a later inhibitor development. the inclusion of the lip method should be considered for future multicenter pup studies. in the past anaphylactie reactions to plasma and plasma components have been a common complication of replacement therapy in patients with hemophilia a and b. we report on 3 severe bleeding episodes in 2 patients with hemophilia a and b, respectively. both patients had a history of life threatening anaphylactic reactions after exposure to different plasma derived clotting factor concentrations including intermediate purity factor viii-and factor ix-concentrate, respectively. high purity factor concentrates were tolerated well without any allergic side effects. a 67 years old patient with a moderate form of hemophilia a (f viii 4 %) had a history of severe immediate reactions with skin manifestations and bronchospasm after exposure to fresh frozen plasma, ctyoprecipitate and 3 different plasma derived factor viii-concentrates of intermediate purity. in all episodes pretreatment with corticosteroids and antihistamines was unsuccessfull in avoiding severe bronchospasm. replacement therapy with two different recombinant factor viii concentrates was tolerated well without any side effects. a 12 years old haemophiita b patient developed hypersensitivity reactions to prophylactic factor ix substitution, which could be overcome by using a factor ix .concentrate with improved purity. a recent recurrence of hypersensitmty under this treatment was finally overcome by the use of highly purified (monoclonal antibodies) factor ix concentrate. we conclude from these findings that high purity of factor concentrates, possibly due to the absence of soluble hla-antigens, are advantageous in patients disposed to allergic reactions. introduction: antibody formation against factor (f) viii remains one of the most severe complications of repeatedly transfused patients with haemophilia a. as reported previously in our study about the incidence of fviii inhibitors, we have observed a high incidence of fviii inhibitors among our haemophilia a patients. it is still not clear why certain haemophiliacs develop antibodies and others do not. a number of previous studies suggest that there is a genetic predisposition for the fviii inhibitor development. thus, the purpose of our study was to examine, if there is a correlation between fviii antibody-formation and genetically determined histoeompatibility antigen (hla) patterns in our haemophiliacs. patients and methods: hla-class i (a, b, c) and hla-class ii (dr, dq) typing was carried out for 51 respectively 44 multi-transfused paediatric haemophilia a patients (fviii:c activity < 5%), including 22 who had developed an antibody to fviii: 19 were high responders (> 5 bu), 3 were low responders (< 5 bu). hla-typing has been performed by a standurcl two-stage microlymphoc~.ftotoxicity procedure (drk frankfurt) using antisera with defiend hla-specifity (biotest diagnostica). results: we found an under-representation of hla-a2 in fviii inhibitor patients when compared with the subgroup without inhibitor. in regard to the hla-b and hla-c antigen frequencies there are no apparent differences between the groups. among the class ii antigens there were higher frequencies of dr1, drw52 and dqwl in the non-inhibitor group. however, the reduction in hla-a1, hla-cw5, hla-dqw3 respectively hla-dr4 frequency for inhibitor patients as reported previously could not be confirmed in our study. conclusion: so far it remains unclear if there is a significant association of a certain hla allels with the development of fviii antibodies. recombinant factor sq (r-viii sq, pharmacia) is a b-domain-deleted recombinant factor viii. it is formulated without albumin (hsa). the product has been shown to have in vitro and in vivo biochemical characteristics similar to a plasma derived full-length protein (p-viii). the international clinical trial programme was initiated in march 1993. pharmacokinetic studies have shown that the b-deleted r-viii sq should be given according to the same dosage principles as a full length p-viii. at present, the product is being tested in previously treated patients (ptps) and untreated patients (pups) with severe haemophilia a (viii:c < 2 %), both during long-term treatment (on demand therapy or prophylaxis) as well as during surgery. the long-term study in previously treated patients in germany was started in january 1994. thirteen patients have been included in 8 centers. all patients are still on treatment with r-viii sq, most of them receiving prophylactic treatment. global treatment efficacy has in general been considered excellent or good. no serious clinical adverse events related to the study product have been reported, nor have any inhibiting antibodies to factor viii or antibodies to mouse-lgg or cho-cell components developed in the patients. further results such as data on efficacy, half-life, recovery and safety will be presented in detail at the meeting. nowadays it is not sufficient to regard hemophilia only as hemorrhagic diafhesis of coagulation genesis, caused by deficiency or molecular anomalies of coagulation factor, without taking into account the immunity state. on examination of 125 patients (pts) (hemophilia a --110 pts, hemophilia b --11 pts, willebrandt's disease u 4 pfs) the development of immune complications was revealed in 34.4 %. chronic persistent hepatitis (3.2 %), chronic active hepatitis (3.2 %), herpes simplex (1.2 %), chlamidiosis (1.2 %), bacterial infection (7.2 %} were regarded as infectious complications. bacterial infections have a routine course due to preserved phagocytic function of neufrophils. and viral infections, whose ability to resistance is connected with t -cell link immunity, take on a chronic persistent course, mechanism of the development of autoimmune processes (autoimmune thrombocytopenic purpura --2.4 % of pts, immunocomplex disease --4.9 % of pts, the appearance of immune inhibifors --34.4 % of pts} is connected with the impairment of immunological surveillance over b -cells aufoimmune clones as a result of dysbalance in the system of t -lymphocyfes immunoregulatory subpopulations. lymphadenopathy and splenomegaly (4.9%) develop due fo benign proliferation of lymphoid tissue as a result of impairment of regulatory function of t -lymphocytes system, or they may be an evidence of virus infection. we observed one episode of acute leukemia. immune complications in hemophilia patients develop against the background of secondary immunodeficiency caused by chronic antigenic stimulation of patients' immune system with high doses of allogenic proteins, which plasma preparations contain. in immune complications hemophilia patients develop hemorrhages, whose pathogenesis is quite different from that caused by coagulation factor, so it should be taken into account in the course of treatment. control of hemophilia therapy classically was based on four parameters: life span expectancy of patients, orthopedic status (normal zero), pettersson score and social integration. oren, however, these parameters described an irreversible status with permanent damage particularly of the joints, especially when patients were grown-up. in order to establish risk-adapted therapy protocols to prevent hemophllic osteoarthropathies, quality control programs have to he set-up that allow for early adjustment of dosage and substitution frequency. here bleeding frequency is one the main parameters, being a clear hint for the possible development of a target joint. since 1988 we have established a computer database (haemopat) that contains data from all patients treated in our center. tables and graphs allow for early detection of increased bleeding tendency in a given joint, and accordingly for adjustment of therapy. the results of 8 years of measuring reasons of joint damage and not documenting the orthopathies as such will be demonstrated. parallelly a new program (haemopat win 1.0) will he introduced allowing for easier handling of data and their evaluation. this program will be used as of december 1995. in combination with a substitution calender to be filled in by all patients, in which factor concentrates, lot numbers, dosage, and date of administration will he constantly recorded, this program will extend our existing database in order to follow closely clinical and orthopedic parameters of each patient, and consequently acts as strict control of therapy quality. additionally, it provides sufficient data to fulfil any documentation needs, requested by medical authorities. the program will be available for all those interested free of charge. 2) kinderklinik der westf. wilhelms univ. mttuster 3-6) biotest pharma gmbh, dreieich haemoctin® sdh; the fviii sdh (sdh = solvent detergent and dry heat = 100 °c, 30 rain) from biotest pharma is a high purity (specific activity ~ 100) fviii concentrate manufactured from large human plasma pools. virus validation studies have shown virus inactivation/reduction (log 10) during the manufacturing process for lipid coated vints~ such as: h]v-1 > 16.2; psr > 16.8; vsv > 14,5; bvdv > 15.7; hcv > 4.5* and non enveloped vimsas such as: parvo** = 2.7; reo > 5.3*** and hav > 13.9. more than 50 hemophilia a patients (ptps = previously treated patients), baseline fviii activity < 1%, were included in an international drug monitoring study to follow their fviii inhibitur status. the hemophilia centers included were three centers from hungaria (helm pal children hospital and the national inst. of haematology, budapest and regional blood transfusion center, debrecen) and four centers from germany (two from berlin, one fraukfurffmain and one monster). patients were enrolled in the drug monitoring beginning aug. 1993. at the entry none of the patients had a detectable inhibitor. at the end of sept. 1995 there were no side effects or adverse events in connection with the use of haemuetin®. before the haemoctin drug monitoring study, the patients were treated with cryoprecipitate, or purified fviii products. inhibitor testing was done on patients plasma samples using the bethesda method. repeated fviii recovery determination at one time (between 12 to 24 hrs) after haemoctin® application demonstrated the expected recovery and normal half life time. none of the hemophilia a patients, treated with haemuetin® sdh developed a clinical relevant inhibitor. at the beginning of the stud)', the clinical efficacy of haemuetin® was studied in 16 hemophilia a patients and shown to give an in vivo recovery of 71 + 15 % by one stage assay and 77 + 17 % by a chromogenic assay. t ½ values were 13 + 2.8 and 12.7 + 3.2 hrs respectively. the study for the clinical efficacy of haemoctin® sdh was repeated in a group of 6 patients approximately two years later. although cd4 lymphocyte counts are known as reasonable predictors of prognosis in hiv infection, the cd4 count is not in all cases an infallible indicator of prognosis. therefore several serological markers are used to predict disease outcome, including beta-2 microglobulin (132m), immunoglobulin a (iga), lymphocyte counts (lymph) and others. in this study we followed a cohort of 23 haemophiliacs (19 with haemophilia a, 4 with haemophilia b) and 2 patients with severe von willebrands disease over a period of 28 months (mean, range: 22-34). testing for l~2m, igg, iga, igm, cd4 and cd8 cell counts (abs. and relat.), cd4/cd8 ratio, and absolute resp. relative leucocyte and lymphocyte counts was performed at least 3 times a year. at the same time clinical examinations and review of history were undertaken. mean of laboratory tests for every quarter of a year and significant changes during time of observation were calculated and correlated with clinical data. 1-4 5-8 9-12 13-16 17-20 21-24 cd41 440+956 344+924 278+925 302.-1:240 273.+.220 166+125 cd8 ~ 1165+474 1171+523 1236+1187 1017+439 930±412 873+478 1~2m z 2.0+0.6 3.0+1.0 3.0+1.2 3.0+1.1 3.5±1.0 3.5±1.3 lymph ~ 1.98+0.6 1.83+0.6 1.72+0.7 1.67±0.6 1.46±0.6 1.31±0.5 means/pl ± standard deviation means mg/l ± standard deviation during time ef observation we found significant changes of cd4 (abs. and relat.), abs. cd8 counts, cd4/cd8 ratio, f~2m, leucocytes and lymphocytes. the abs. cd4 and cd8 counts correlated clearly with lymphocytes und leucocytes counts but not with 1~2m. the prognostic value of the tested parameters is discussed by calculation of correlations with clinical data, anti-retroviral treatment and treatment of haemophilia. the availability of high purity factor concentrates has recently encouraged clinicians to use perioperative continuous infusion of fviii or fix to prevent or reduce bleeding in patients with haemophilia. in conliast to repeated highdose bolus injections, the continuous infusion trealment regime maintains constant coagulation factor activity at a level necessary for hemostasis, reducing the total cost of treatment by about 20% and preventing possible side effects of bolus doses. the new application mode, however, requires stable products which tolerate slow passage through an infusion device. our objective was to test in vitro the fviii concentrate immunate (stim plus) and the fix concentrate immi.ynine (stim plus) at room temperature, under conditions of long-term contact with polypropylene tubing in an infusion pump. infusion rates were chosen to mimic clinical situation. the control samples were not infused through the pump but were otherwise treated identically. test samples were drawn before and at 1, 4, 8, 24 and 48 hours after the onset of each infusion run. fviii (one-stage, two-stage and chromogenic assay) and fix (one-stage) activity were measured using immuno reagents. presence of activated factors were measured by napt'i', while flla, fxa, plasmin and pre-kallikrein activator were detected with specific chromogenic substrates. the data showed equivalent results between test and control samples with no loss of fviii or fix activity. the potencies of both immunate (stim plus) and immunine (stim plus) remained within 100 + 20% of labeued values within 48 hours after onset of infusion. in conclusion, immunate (stim plus) and immunine (st1m plus) are suitable for contiuous infusion when using automatic infusion device within applied test criteria. in htanans, circulating half-lives of asparaginase enzymes from e. coli and erwinia chrysanthemi vary within a wide range. moreover, half-lives differ not only among different e. coli strains but also among commercial e. coli preparations. to investigate the possible influence of two different sources of e. coil asparagmase (asn) preparations on the fibfinolytic system of leukemic children a prospective randomized study was performed correlating asn pharmacokiuetics (asn activity, asparagine depletion) with fibrinolytic parameters (plasminogen (plas), o.2-antiphismin (ct2ap), tissue-type plasminogen activator (t-pa), tissue type plasminogen activator inhibitor 1 (pal 1), d -i)imer (i)-d)). together with prednisono, vincristine and an anthracycline 20 children received i0000 iu-/m 2 asn medae r (originally purchased: kyowa hakko, kyogo japan) and 20 children 10000 iu/m 2 crasintin r (bayer, leverkusen, germany). blood samples for pharmacokinetic and coagulation analysis were drawn before the first asn administration and every third day whilst on medication. the results are shown in the 0.05 asn activity shows a negative correlation (spearman: rho/p) to plas (-,637/0.0003) and ct2ap (-, 751/0.0001). a positive correlation was found between asn activity and d -dimer formation (0.475/0.01). t-pa and pal 1 showed no relationship to asn activity. all children showed complete aspamgiue depletion at a detection limit of 0.1 um during the course of asn admiatstration. two thrombotic events occurred in the kyowa group, one of the distinctions between the two e. coli asn preparations administered ill this stndy is the absence of cystine in the kyowa asn, which also has a lower isoelectric point and a longer half-life than the bayer type a asn. with respect to this observations this may lead to longer inhibition of protein synthesis, which then may be the cause of a bigher rate of side effects. along with studies on asn pharmacokinoties dose recommendations need to be tailored to the specific asn preparation employed to ensure optimal antineoplastic efficacy while minimizing the hazard of complications. different types of coagulopathy in hepatic veno-occlusive disease (vod) and capillary leakage syn-drome (cls) after bone marrow transplantation w. ntimberger, s. eckhof-donovan, st. burdaeh and u. g6bel department for pediatric hematology and oncology, heinrich heine university medical center, diisseldoff, germany it is generally accepted, that cls, coagulation activation and refractoriness to platelet transfusions are part of the syndrome of hepatic vod. we assessed patients with either vod or cls or both vod and cls, in order to analyze the influence of either syndrome on different aspects of hemostasis. vod was diagnosed according to jones et al. [transplantation 44 (1987) 778]. diagnosis of cls was >_3% increase of body weight in the past 24 hours and non-responsiveness to furosemide [niirnberger et al., ann hematol 17 (1993) 67] . patients with vod, cls or both were compared to control patients without either diagnosis. eight patients suffered from both vod and cls, 5 patients only from vod, and 8 only from cls. 61 patients had neither syndrome and served as control population. activation of the coagulation system was assessed by increase of tat-complexes and/or increased consumption of at iil the hemostasis patterns were as follows: no. introduction: lung cancer goes along with coagulation activation and increased thromboembolic risk. acute phase reaction in cancer patients leads to elevated levels of c4b-binding protein (c4b-bp) followed by a shift from free to c4b-bp-bound protein s. we tried to find out whether there is a correlation between alterations of c4b-bp, protein c protein s system and interleukin 6 (il-6), which is one of the most potent inducers of hepatic acute phase reaction. patients: i. 25 patients with lung cancer; 2. control group: 11 patients in complete remission after lung cancer. methods: clotting methods: protein c and s activity; elisa tests: protein c antigen, tat-complexes, prothrombin fragments f i+2, il-6. electroimmuno-diffusion (laurell): free and total protein s, c4b-bp. results: tat-complexes and f i+2 were elevated in cancer patients. c4b-bp levels were slighthly increased (129±19 % of n.), protein s activity was 89±33 % of n. (control group: 108±23 % of n.). il-6 in lung cancer patients was 37.2252.9 pg/l (control: 27.2±8.2 pg/l). conclusion: one source of the hypercoagulable state in lung cancer patients is decreased protein s activity due to elevated c4b-bp levels. this is probably caused by hepatic acute phase reaction which is triggered by increased il-6 levels. these plasma levels correlate with levels of the tumor marker ca 125 and with the stage of the disease but correlations with patient outcome (disease recurrence and overall survival) have not previously been shown. plasma levels of d -dimer and ca 125 (determined by sandwich elisa assays} were measured prior to treatment in 36 women with figo stage t to iii ovarian cancer and correlated with tumor stage, relapse and overall survival over a mean follow -up period of 28 months (range 16 to 40 months). levels in 71 healthy women and 27 patients with benign ovarian disease served as controls. the occurrence of deep vein thrombosis in the cancer patients was also determined by impedance plethysmography that, when positive , was confirmed by contrast venography. preoperative d -dimer and ca i25 levels in ovarian cancer patients were statistically signfficantly higher than in controls. preoperative cut off values were calculated for the prediction of cancer relapse and survival for both measurements. d -dimer levels above a cut off level of 2060 ng/ml were statistically significantly associated with the rate of relapse but ca 125 levels were not. deep venous thrombosis occurred in 33 % of cases but there was no difference between properafive levels of d -dimer in patients who subsequently did versus did not develop deep vein thrombosis. high levels of d -dimer are associated with more advanced disease and with poor prognosis in patients with ovarian cancer. the high levels of d -dimer are a biologic feature of the malignancy itself that may be attributable, at least in part, to increased conversion of fibrinogen to fibrin in the tumor bed with subsequent degradation of fibrin by the fibrinolytic mechanism. thus d -dimer levels may serve as a marker for overall tumor burden as well as "disease activity". a high incidence of deep vein thrombosis exists in the course of the disease in ovarian cancer patients but preoperative levels of d -dimer are not predictive of this occurence. yon tempelhoff georg -friedrich, michael dietrich, dirk schneider, lothar heilmann. dept. obstet. gynecol. city hospital of ruesselsheim. -germany. an increase of plasminogen activator inhibitor activity (pai act.) in the plasma of cancer patients has been recently discribed. we have longitudinally investigated pai act. in 136 patients with primary breast cancer and compared the results with the outcome of malignancy. patients with untreated primary breast cancer and without proof of metastasis (t 1-4 n0-2 m0) were eligible for this study. in all patients coagulation tests including fibrinogen {method according to clauss), d -dimer (elisa} and pal act. (upa dependent inhibition test) were performed prior to primary operation, 6 months thereafter and at the time of cancer relapse. seventy -two healthy women and 43 patients with benign breast disease served as controls. during a mean follow -up of 32 + 16 months 34 patients (25 %) developed cancer recurrence and 13 (9.6 %) patients died. in all cancer patients preoperative levels of fibrinogen and pai act. were significantly higher compared to healthy women and to patients with benign breast disease. preoperatively only pal act. was significantly higher in patients with vs. without cancer recurrence (4.52 _+ 1.67 u/ml vs. 3.4 1 + 1.55 u/ml; p = 0.002). in patients with later recurrence pai a~t. significantly dropped 6 months after operation (p = 0.02) and was again significantly increased at the time of cancer recurrence (4.90 _+ 2.89; p = 0.001). a preoperative cut off value (calculated via cox model) of pai act. above 3.52 u/ml was significantly associated with the rate of relapse (tog rank: p = 0.0005) and in 70 % of patients who died of cancer preoperative pai act. were also above this cut off. impaired fibrinolysis in patients with breast cancer is significantly associated with the outcome of cancer. a monoclonal heparin antibody (mab) has been raised against native heparin using a heparin-bovine serum albumin conjugate prepared by reductive amination. for further analyses tyramine, which was covalently bound to low molecular mass heparin by endp0int attachment (malsch r et al: anal biochem 1994; 217: 255-264) , was labeled with 125-iodine at the aryl residue. the tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobuline igg. the mab recognized specifically intact heparin and heparin fractions. the lower detection limit of heparin preparations was 100 ng/ml. no cross reactivity of the mab occurred with other glycosaminoglycans such as heparan sulfate, dermatan sulfate, chondroitin sulfate a and c. oversulfated heparin showed lower affinity to the antibody hl.18 than 2-0-and 6-0-desulfated beparin. the method established for the purification of the mab was ammonium sulfate precipitation with followed dialysis. sds-page and high pressure capillary electrophoresis prooved the high purity of the received antibody. the biological activity of mab was tested by the chromogenic assay $2222 and remained stabile while purified. in conclusion, the present abstract describes an purified igg 1 monoclonal antibody directed against heparin and heparin fractions, which can be used for biological measurements. the concentration of heparin and dermatan sulfate in biological fluids is usually measured using radiolabeling. for this purpose aromatic compounds are usually used to insert radioactive iodine labeling at the saccharide backbone of the glycosaminoglycan. we developed methods for the specific labeling of hepann and dermatan sulfate at the terminal residue. tyramine was bound by reductive amination to the 2,5 anhydromannitosyl end of heparin, produced by nitrous acid degradation and confirmed by 13c.nm r spectroscopy. (anal biochem 217: 254-264, 1994) this method was also used to produce a low molecular mass dermatan sulfate (lmmd)derivative after partial deacatylation. in order to choose the proper method for evaluating the specific anticoagulant activity in the row of chitosan polysulphate (cp) samples with different degrees of pol3~merization and sulphation we applied to pharmaeapea article (a~) when assessing the ability of direct anticoagulants to depress the coagulability of recalcificated sheep blood (using the 3rd international heparin standard), and to measuring such acti¢ity as per pharmacokinetic model (a2). the model admits the "kinetics of cp elimination be linear in ease of intravenous injection to rabbits, as it is observed in heparin: ct=co exp(-i~ x t), where ct is cp concentration at the time moment t; co is cp concentration at the moment of injection; i~ is the elimination constant. besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific actidty a2 : t=kt ct + tin, where t is the time of clot formation at different tlme intervals after of cp injection; t~, is the time of clot formation prior to cp injection. t value was assessed in two tests: in blood coagulation time (bct) and in activated partial thromboplastin time (aptt). no correlation was observed between a1 and a2. at the same time the values of ifm and the period of semieliminatinn (tvz) with the use of the original method that were obtained with the help of the quantitative determination of cp in rabbit's blood taken at different time intervals after injection, showed a close correlation (1"=0,94 p<0,05) between the same parameters, obtained with the help of the of the pharmacokinetic model in bct test. thus, experimentally it was proved that the assumption of the linear elimination and the effect-dose dependence was true, which is necessary for a2 calculation. we recommend to use intravenous injection of the samples to animals with further assessment of the results according to the pliarmacokinetic model to calculate the specific anticoagulant activity in the row of chemically related potential direct anticoagulants. in this investigation we compared the biological activity of a low-molecular-heparm (lmw-heparin, mono embolcx®) after intravenous, subcutaneous and oral application in rats. sprague-dawly rats were anaesthetized by ketamine/diazepam and the blood samples were taken from the retro orbital sinuus. 150 axa u/kg body weight of the lmw-heparin were injected intravenously and subcutaneously to 10 rats each. between 3 minutes and 10 hours after injection serial blood samples were taken. 200 mg/kg (20.000 axa u/kg) body weight of the lmw-heparin were applicated orally using a stomach tube. blood samples were taken between 1 and 24 hours after oral application. the antifactor xa and antithrombin activities of the plasma samples were measured, using ehromogenic assays and the substances s 2222 and s 2238 (kabi vitmm). after i.v. injection the maximum axa and alia activities were 2.8 axa u/ml and 0.8 aiia u/nil respectively. after s.c. application the antifactor xa activity of the lmw-heparin showed a maximum of 0.5 axa u/ml atter 120 minutes. the antithrombin activity exhibited an eatiier maximum activity of 0.2 alia u/nil 60 minutes after injection. after the oral application no increase of the axa or alia activities was measured. the lmw-heparin has a high antifaetor xa and antithrombin activity after i.v. and s.c. injection. after oral application no activity of the lmw-heparin was measurable. these results implicate that fractionated heparin is not absorbed after oral application or is inactivated in the gastrointestinal tract. to improve the activity after oral application modified hepatins have to be synthesized. in an in vitro study the effect of various heparin derivatives (calciparin, fraxiparin, cy 222, cy 231, astenose, hexasaccharide, ssh 14) on thrombin-and adp-induced platelet aggregation as well as on adpmediated platelet activation in whole blood was investigated. all heparin derivatives caused a concentration-dependent inhibition of thrombin-induced aggregation of washed platelets. calciparin and astenose were found to be the most effective compounds with ic5o values of 0.67 and 1.2 p, mol/l, resp.; higher concentrations (5-30 times) were required for the other compounds. furthermore, the heparin derivatives were studied with regard to their potentiating effect on adp-induced platelet aggregation. in a concenwation range from 1 to 10 u/nil calciparin, fraxiparin, cy 222 and astenose led to a potentiation of the adpinduced aggregation whereas cy 231, hexasaccharide and ssh 14 did not show this effect. the increase in aggregation was associated with an increase in thromboxane a2 lbrmation. in addition, the effect of calciparin, fraxiparin, cy 222 and astenose on adp-induced platelet activation in whole blood was investigated by flow cytometric analysis using monoelonal antibodies to platelet surface receptors opiiia (cd-61) and p-selectin (cd-62). at concentrations that caused a maximum potentiation of adp-induced platelet aggregation these substances led to a strong increase of adp-mediated activation of platelets in whole blood. the effect was most pronounced when the blood was anticoagulated with calciparin and astenose, resp. in conclusion, the results suggest that the aggregation-promoting effect of heparin derivatives included in this study is dependent on the molecular weight and the degree of sulfation and is in part due to the generation of thromboxane. heparins are negatively charged polysaccharides and bind protamine forming a stable complex. here we report on the properties of microbeads (4.5 pro) coated by protamine. protamine chloride (0.16 ijm) was covalently bound to 0.5 mg paramagnetic tosyl..activated microbeads m-450 (dynal). the covalent binding of protamine was from 1.0 to 13,0 mg/g beads. protamine-dynabeads were produced in a phosphate buffer at different ph (7,0; 7,5; 8,0 and 8,5). the protamine-dynabeads produced ph 7.5 showed the best properties for flow cytometry analysis. in saline solution they bound lmm-heparin-tyramine-fitc (lmmh-tyr-fitc) dose dependently from 0.001 to 2 u/ml, whereas in plasma and blood they bound lmmh-tyr-fitc from 0.05 to 2 u/ml. dependent on the binding protocol, the microbeads also bind proteins unspecifically, i.e bovine serum albumine and protamine to a lower extent.the adsorbed proteins, however do not bind lmmh-tyr-fitc dose dependently. the saturation of the proteins on the beads was determined as their relative fluorescence intensity (rfi). in saline solution the saturation was measured at 380 rfi, in human plasma at 325 rfi and in whole blood at 252 rfi. using flow cytometry erythrocyctes, lymphocytes, monocytes and granulocytes were not bound to protamine dynabeads. these data demonstrate that protamine-dynabeads can be used to measure the concentration of lmmh-tyr-fitc in saline solution, plasma and blood because they do not bind to human blood cells. the present study was designed to investigate the anticoagulant action of inhaled low molecular weight (lmw)-heparin in healthy volunteers. 3,000 iu (group t), 9,000 iu (group 2), 27,000 iu (group 3) or 54,000 iu (group 4) lmw-heparin were given to 20 healthy volunteers each at 4 weeks intervals. in group 1 tissue iactor pathway inhibitor (tfpi) antigen and activity, 82222 chromogenic factor xa assay, heptest, aptt and thrombin clotting time (tot) remained unchanged during the 10 days observation period. in group 2 tfpi antigen and activity, aptt, tct and the $2222 method remained uneffected. heptest coagulation times were 18.7 + 2.0 before, 26.1 + 5.2 sec. 6 hrs and to 20.5 + 1.9 sec. 24 hrs after inhalation. in group 3 tfpi antigen increased from 74.1 + 13.9 to 80.5 + 14.2 ng/ml 3 hrs after inhalation. tfpi activity remained unchanged. $2222 method increased from 0.01 to 0.08 + 0.06 iu/ml 6 hrs after inhalation. heptest coagulation values were prolonged up to 42 _+ 7.6 s ec after 6 hrs and returned to normal within 72 hrs after inhalation. aptt and tct remained unchanged. after inhalation of 54,000 iu lmw-heparin, the following changes were observed: tfpi antigen increased to 103 +_. 17.9 ng/ml and normalized within 24 hrs. -i'fpi activity increased to 1.14 _+ 0.23 u 3 hrs after inhalation and was normal after 24 hrs. antifactor xa activity, as measured by s2222 method, increased to 0.343 + 0.196 u/ml after 6 hrs and was normal after 72 hrs. heptest coagulation values increased to 77.5 + 11.8 sec 6 hrs after inhalation and normalized after 144 hrs. aptt and tct did not change throughout the observation period. the data demonstrate a resorption of lmw-heparin by intrapulmonary route in man. no side effects were observed. recently we developed a tritium-labelled arachidonic acid ([3h]aa) release test with high sensitivity to membrane-toxic agents. the assay performed in u937 cells is intended to evaluate ehemicals, drugs and biomatefials with regard to their eytomembrane toxicity [kloeking et at. (1994) , toxicology in vitro 8, 775-777]. local irritation reactions are described in patients receiving therapeurieat dosages of lmw heparin. this fact prompted us to examine the following lmw hepafins and heparinoids for their membrane toxicity in u937 cells: reviparin-sodium, enoxaparine-sodium, mueopolysccharide polysulphate (mps), pentosan polysulfate sodium (pps), polysulfated bis-lactobionic acid amide derivatives lw10082 (aprosulate) and lw10086. for this purpose, [31--1]aa labelled u937 ceils were incubated with different concentrations of lmw heparins and heparinoids at 37°c for 1 hour. compared with untreated cells, the [~h]aa release of cells treated with 5 mg of the drugs was two times higher with reviparin sodium, three rimes higher with bis-lactobionic acid amide lw10086, five times higher with pentosan polysulfate, 20 times higher with ertoxaparine-sodlum, but it was equal to the control with mucopolysaccharide polysulphate. the rate of araehidonic acid release in response to a test chemical may therefore be used to assess the membrane-toxic effect of this substance and to predict its the inflammatory potential in the skin. semi-synthetic glyensaminoglycans (gags) with antithrombotic properties can be prepared from the e. coli k5 polysaecharide by coupled chemical and enzymatic methods. the molecular weight of these semi-synthetic gags can be adjusted to obtain products mimicking the molecular profile of a low molecular weight hepatm. in order to compare the biochemical and pharmacologic properties of a semi-synthetic gag (sr 80486a, sanofi/choay) with a commereiany available low molecular weight heparin, fraxiparine (sanofi, paris, france), valid biooheanical and pharmacologic methods were used. the molecular profile of this agent as determined by hplc exhibited a comparable distribution profile (mr=5.7 kda) in comparison to fraxiparine (ma=5.1 kda) . the anticoagulant properties of sr 80486a were comparable to fraxiparine in the aptt and heptest. however, in the usp assay, this agent showed slightly weaker activity. sr 80486a also exhibi~d comparable affinity to atffl and hcii. in comparison to fraxiparine, it produced a much weaker response in the hit screening system. in~ viv0 studies, sr 80486a preduecd strong dose-dependent antithrombotic actions in both the iv and sc studies in the rabbit jugular vein stasis thrombosis model (ed50=i 5-60 gg/kg). additionally, it also produced antithrombotic aefiorts in a rat jugular vein clamping model. the hemorrhagic effects of this agent were comparable to those of fraxipafine as measured in a rabbit ear blood loss model. intravenous administration of sr 80486a also revealed a comparable pharmaeokinetie behavior to fraxiparine. no abnomaiitias of the clinical chemistry (change in liver enzymes) and hematology profile (thrombocytopenia and lencecytosis, etc.) were noted in primates. at a dosage of i and 2.5 mg/kg iv, this agent also caused a release of functional tfpi which was comparable to the observed responses of other low molecular weight heparins. these studies suggest that sr 80486a is capable of producing similar pharmacologic effects as other low molecular weight heparms, however, additional optimization studies are required for demonslrating product equivalence. limited information on the comparative pharmacoldnetics of low molecular weight heparin (lmwh) is available on the data obtained from aptt, heptest, anti-xa and antmia assays. since these drugs are currently used for therapeutic indications using relatively high dosages and intravenous administration. aptt, heptest and antmia test may be valuable in the assessment of their effects. in order to investigate the relative pharmacokinetics of lmwh using apt'i', heptest, anti-xa and anti-iia methods, certoparin (sandoz, basel, switzerland) was administered to individual groups of healthy male volunteers (52-70 kg) via intravenous (30 mg) and subcutaneous (50 nag) routes in a crossover study. blood samples were drawn at 0, 5, 15, 30, 60, 90, 180, 360, 540 and 720 minutes. using a baseline pool plasma obtained from the same volunteers, calibration curves for each of the individual tests were constructed to extrapolate circulating levels of certoparin. a non-compartmental model using trapezoidal technique was used to obtain pharmacokinetic parameters such as t 1/2, vd, and clsys. in the intravenous studies, the t 1/2 was found to be dosedependent for aptt, heptest, anti-xa and antm]a. the auc, however, was significantly different for each test and was dose-dependent following the order: aptt<heptest<anti-xa<anti-iia. in the subcutaneous studies, 1the t 1/2 was both test and dose-dependent. the auc was also dose-dependent and followed the order: anti-xa>heptest>aptt>antmia. the clsys of the antma was much faster in comparison to the other tests. the clsys of the aptt and heptest was independent of dose. however, anti-xa clsys by this route was lower than other tests. the apparent vd followed the order aptt>antmia>heptest>anti-xa. the bioavailability of the certoparin as measured by various tests ranged from 81-119%. these studies suggest that beside providing pharmacokinetic data, aptt, heptest and anti-iia assays may provide useful data on thier safety and efficacy at high dosages. the immunological type of heparin associated thrombocytopenla (hat ii) is a severe complication of heparin treatment and is associated with arterial and venous thrombosis. only patients with absolute thrombocytopenia have prompted suspicion of hat in clinical practice. we report on a 44 year old male, who developed thromboembolic episodes after coronary angiography like reinfarction and thrombotic episodes of a. brachialis. fibrinolytic therapy combined with i.v. unffactionated heparin treatment was the therapy of choice and was followed by severe fua~er thromboembolic adverse effects. besides an impaired fibrinolytic response and elevated antiphospholipid anitbodies, we diagnosed hat type ii in hipa and elisa (stago-boehringer, marmheim). this special patient had platelet counts within a normal range, when developing the thromboembolic episodes. it appears that the normal platelet count during the thromboembolic episodes reflect a relative thrombocytopenia. from a clinical point of view we recommend the use of a lab panel to exclude hat type ii in patients with thromboembolic episodes under therapy with fractionated or unfractionated hepafin. platelet counts within a normal range are no absolute exclusion criterion for hat ii. low molecular weight heparins (lmwhs) are now commonly used for the prophylaxis of post-surgical thromboembolic complications. in this indication, lmwhs are administered as a single or twice a day subcutaneous regimen. usually these agents are administered at 30-40 mg total dose which is equal to 3000-4000 anti-xa (axa) iu. newer methods such as ehromogenic substrate based axa methods and the heptest clotting time can be used to determine the effects of lmwhs during the initial phases of prophylactic therapy. this may be useful in the elderly and weight compromised patients where a fixed dosage may not be optimal and may produce bleeding effects. similarly in the overweight patients, a fixed dose may not be efficacious. thus, monitoring of lmwhs in these patients may be useful in the optimization of their therapy. lmwhs are also used in the treatment of deep vein thrombosis using both intravenous and subcutaneous protocols. high dosages of up to 200 mg sc/day and infusions of up to 30 axa iu/kg/hr have been administered. in these conditions, the monitoring of the circulating lmwh levels may be useful in optimizing the dosage. we have modified the aca heparin (do_pont merck, wilmington, de) method to measure the lmwh levels in the plasma of patients treated with both the prophylactic and therapeutic dosage. owing to the required turnaround time, simple operation and reliable results, this method was found to be of value in the monitoring of these agents. this presentation provides an overview of the clinical application of various lmwhs with particular reference to the need of monitoring for their effects to optimize the clinical outcome. a double-blind, multicentric, controlled trial was performed in order to compare the antithrombotic efficacy and safety of single daily doses of 5000 ie anti-xa of low molecular weight heparin (lmwh) sandoz (certoparin) and 5000 ie unfractionated heparin (ufh) tid. in 288 patients undergoing elective total hip replacement blood samples were drawn before the first subcutaneous injection of lmwh or ufh resp., two hours after administration on the first and 7th postop, day and on the last day of prophylaxis (day 10-14), anti-xaactivity was measured by chromogenic substrate assay, heptest and aptt by clotting assays and tissue factor pathway inhibitor (tfpi) and heparin-pf4-antibodies by elisa techiques. as expected, the anti-xa-activity and the heptest values were significantly higher in the lmwh-group at all time points after administration of the drugs; the mean values of heptest were 35 sec in the ufh-and 75 sec in the lmwh-group respectively, the aptt was not different in both groups. at the end of prophylaxis positive antibodies to heparin-pf4complexes were detected ~n both groups; this however was not correlated with clinical thrombocytopenia. a detailed correlation between patients with deep vein thrombosis (dvt) and positive antibodies has still to be done (all patients were screened for asymptomatic dvt between day 10-14 by bilateral phlebography. tfpi was markedly increased in the lmwh-and only slightly elevated in the ufh-group; the differences are statistically significant. summarizing it can be concluded that antibodies to heparin-pf4complexes may occur without clinical symptoms of hepafin-induced thrombocytopenia type ii and that tfpi may play a sigificant role for the antithrombotic efficacy of ufh and lmwh. unfractienated heparin represents one of the most severe and frequent causes of drug-induced thrombooytopenia. heparin-indueed thrombocytopeala (hit) occurring early in therapy is often mild and serf-limited, appearing to be caused by a direct aggregant effect of heparin on platelets (hit type i). hit type ii, however, is immune-related an may result in absolute thrombocytopenia (platelet count <i50/~l) or a relative decrease in platelet count (?_50% reduction from baseline values). unlike most other immune thrombecytopenias in which bleeding complications often predominate, hit type ii may results in severe arterial and/or venous thromboses. the management of patients with hit type ii and thrombosis is often difficult. heparin must be stopped immediately. antithrombotic, fibfinolytic, or antiplatelet agents have been used in these situations, as have anticoagulants other than unfraetionated hepafirl plasmapheresis, used in other immune-related disorders, has also been occasionally reported. at our institution this intervention appears to provide significant elininal benefit to hit patients. several recent reports have noted that the heparin-dependent antibodies in hit type ii are directed against a complex of heparin bound to platelet factor 4 (pf4) and an elisa-based test system for hepafin-pf4 eomplexinduced antibodies (hpia; stago) has been develuped~ we report our experience in 3 patients with hit type ii in whom plasmapheresis was instituted and the antibody levels in blood were measured pre-and post-treatment to identify wkether circulating antibody titers decreased thus providing some explanation for the clinical benefit. the patients developed profound absolute thrombocytopenia (range, 3-21 k/~tl) and positive heparin-induced platetet aggregation studies following treamlent with uofractionated heparin. thromh0cytopenia induced by hepariu (hat-syndrome) typically appears several days after initiation of heparin therapy. this phenomenon is caused by antibodies of the lgg type which are induced by a heparin-pf 4-complex and which also activate platelets. in a recent study (n engl j med 1995; 332:1330-5) the hat-syndrom was diagnosed in 2.7 % (9 of 332) patients who had received unfractionated heparin (ufh) and in none of those 333 patients who had been treated with low-molecular-weight heparin (lmwh). 8 out of the 9 patients treated with ufh presented with at least one thrombotic event (7 times a venous, once an arterial event). the incidence of heparin-dependent igg antibodies (7.8 %) was higher in the ufh treated group than in patients treated with lmwh (2.2 %). our patient was a male caucasian white, 62 years of age, otherwise in good clinical condition with no severe diseases in his pmh. both parents, however, had suffered several thrombotic episodes. the patient was admitted to the hospital in september "95 with clinical signs of a deep vein thrombosis (dvt) of the right leg. the diagnosis was phiebographically confirmed. after having given informed consent the patient was treated with 5 cycles of r-tpa (loco-regional application) and 3 cycles of uhsk as part of a clinical trial (uhsk vs. rtpa, boehringer/m). lysis was successful with complete recanalisation of the veins. after completion of lysis the patient was further anticoagulated with coumarin overlapped by heparin (ufh). as a complication the patient suffered from a retroperitoneal and an upper gi4ract bleeding from esophagitis grade ii to hi while being on coumarin prophylaxis. therefore the patient was put on lmwh and then discharged with this prophylaxis. after less than two weeks the patient presented again to the hospital with a relapse of dvt in the same leg. phiebographically nearly all deep veins were occluded to a higher degree than before, i. e. also with concomitant pelvic vein affection and with hea~ 3, swelling of the whole leg. blood count showed a marked decrease of platelets to 27/nl. the platelet aggregation test demonstrated a hat-syndrome which was confirmed by the hipa-assay. the heparinoid orgaran® was not reactive in the assay. having seen a previous bleeding episode in this patient, we did not see an indication for another b'sis therapy. therefore we decided to put the patient on an intermediate dose of coumarin overlapped by hiradin (hat study, behring) instead of orgaran®, since hirudin allows a much more precise dose adjustment. after 10 days the patient was discharged with no further complications. risk factors for dvt have not been detected until now. to our knowledge this patient is one of the first cases of hat-syndrome induced by lmveh. under routine conditions aptt is the most frequently used test method for the surveillance of patients undergoing hepann therapy. in literature a 1,5 fold to 2,5 fold prolongation of the initial value is recommended for individual therapy a~ixatian. in contrast to the control of the enmarin therapy by means of prothrombin time measurement the individual hupafin sensitivity of the ~ reagents of this standard value hardly receives notice. commercially marketed ~ reagents differ from one another method and concentration of the activator as well as their phosphelipids. when choosing a reagent the user must only take into consideration the sensitivity against its factors, heparin and lupus but also the adalxability of the reagent towards the given measurement system ( software variability towards incubation time, sodimentalion problems in reagents with high kaolin concentration). reagents offering a high heparin sensitivity already are showing a prolongation of the coagulation time at low heparin dosage (0.1 -0.3 u/ml). in the therapeutic range (0.4 -0.8 u/ml) very often no linear relationship between prolongation of the aptr and heparin concentration level is found_ therefore very often coagulation times above the routine measuring range (180 s) can be observed in this range. moreover, various medical measuring devices differ in their ability to recogniso the retarded entry of coagulation. coagulation limes with different apq't reagents in correlation with the heparin concentration level can be found in the adjoined table. significantiy prolonged coagulation ti.~es of aptt lead to a reduction of the hel~ dose in clinical practice. due to the discrepancy between heparin concentmon level and the prolongation of the afrt in reagents with a non-linear heparin sensitivity a reduction of heparin dosage leads to an inmt~cient anticeagulalion and the risk of further thrombosis. therefore the producers should be asked to include precise directions on the heparin sensitivity of the aptt reagent in the therapeutic range in order to enable an optimal surveillance of the heparin therapy. a coordination between clinic and laboratory should be compulsory for the choosing of the aptt reagent. introduction: hat is a rare but sometimes fatal complication of therapy with unfractionated (ufh) and low molecular weight beparins (lmwh). we report on our experience with the clinical coume and management in 12 patients (pts). in all cases a severe decline of platelet count and in 11 cases a positive hipa test (= heparin-induced platelet antibodies) were documented. patients, clinical course and outcome: 8 female and 4 male patients (median age 68 years, range 22-78) were analysed in this retrospective study. 9 pts were referred from outside hospitals. all pts had been treated with ufh (5 pts additionally with lmwh) for prophylaxis (n = 9. in 2 pts pedoperatively) or treatment (n = 3) of venous thromboembolism (v'te). in 11 pts at least one vte occured during heparin therapy, in all cases prior to hat-diagnosis (vte: n = 13; putmonary embolism: n = 6; artenal embolism n = 1; thrombosis of sinus transversus n = 1). hat was suspected 16.5 days (median; range 12 -48) after first heparin application. hepadn therapy was discontinued immediately, at that time the median plateiet count was 35 g/i (range 10 -117). 9 pts were treated with orgaran r, followed by recombinant hirudin (r-hirudin, behdngwerke ag: clinical study) in 4 cases; 3 pts were treated exclusively with r-hirudin. plat~let count normalized in 9 pts within 3 days (median; range 1-10). one patient in the orgaran" group died on recurrent pulmonary embolism. 11 pts recovered without any further thromboembolic complication. conclusion: in our small series of 10ts hat was complicated with vte in 11/12 cases. in many pts hat was diagnosed strikingly late. for early diagnosis careful monitonng of platelet counts are mandatory dunng hepadn therapy, both orgaran ~ and r-hirudin seem to be safe and effective in preventing further thromboembolism, p 179 to prospectively evaluate coagulation or fibrinolytic activation in children long-term anticoagulation with lmwh was administered a longitudinal study was perfomed. within a 30 months period 48 children and adolescents (6 -19 years) suffering from malignant bone tumors (ewing's sarcoma or osteosarcoma) received enoxaparin (clexane r/rhone-poulenc rorer, france) for primary propylams. the majority of patients received 0.6 mg/kg bw enoxaparin 12 hours before major surgery (limb salavage). in addition, ten neonates and children received enoxaparin in the same dosage for secondary prophylaxis. in both groups therapy was adjusted to 0.2 -0.4 iu/ml anti xa activity. median (range) duration of therapy was performed for l0 (6 -24) weeks. before ('1"1), one (t2) and 3 weeks (t3) within the context of the present study an investigation on the physiology of clotting was conducted in three groups each consisting of 12 patients, who had to undergo a total end-prosthetic hip implantation (teph) for the first time. criterion for allocation to a particular group was type of intraoperative transfusion the patient received : infusion of whole blood, erythrocyte concentrate or fresh frozen plasma. at eight fixed times the activity or concentration of the components of the kallikrein-kinin and inhibitory systems was determined. the statistic evaluation ensued with the spss software package. for all three patient collectives an activation of both systems was to be observed with the teph-implantation whereby there were no statistically striking differences between the three groups. thus in the case of the kallikrein-like activity an intraoperative rise of over 37% was registered, while at the same point in time there was only a 4% reduction in the prekallikrein activity. postoperatively a 10% rise in the prekallikrein activity was to be observed. as a reserve inhibitor alpha 2-macroglobalin showed no significant change in activity during the period of observation whereas the acute phase proteins alpha 1-proteinase inhibitor and cl-esterase-inhibitor yielded a postoperative rise of 135% and up to 100%. the beta-factor xllainhibition showed a rise of 121% during the reconvalescent phase. the 65 year old female caucasian patient presented here, was admitted to a nearby district hospital in december '94 with the acute symptoms of pulmonary embolism. the source of the embolus could not be detected. high dose heparin was administered. pmi-i revealed a coronary artery disease and a peripheral artery occlusive disease of the left lower leg, yet blood pressure values of the lower ex~emities were only slightly below brachial pressures. the first episode of pulmonary embolism was followed by a second one two weeks later for which the patient received heparin again. following this treatment the platelet count decreased significantly to 10/ni. with an acute thrombosis of the lower caval vein and with leriche's syndrome, i. e. occlusion of the abdominal antra from the bifurcation down to the ileofemoral arteries bilaterally, the patient was transferred to our hospital. on initial examination we saw a patient in no acute distress, no dyspnea, blood pressure 120/80 mmhg, pulse 94/rain, no peripheral edema or disturbances of sensitivity. neither femoral, popliteal, nor pedic pulses were palpable on either side. a heparin associated thrombopenia was detected with the platelet aggregation assay and confirmed by the heparin induced platelet aggregation assay (hipaa). the autoantibodies targeting the underlying pf4-heparin complex were determined later by an elisa method in serum samples of the patient (asserachrom hia, diagnostica st,ago). a temporary anth&~r filter system was inserted transbmchially into the lower eaval vein and placed jnst above the thrombus. in the following days, 2 cycles of lysis therapy with 9 mil u streptokinase were administered taking six hours for each cycle. heparin was given in the mean time via the filter system in therapeutic dosages in order to prolong values 1.5 to 2 times the individual base line level. at the end of the second cycle, pulses were palpable inguinally, two hours later both popliteal and the right pedic pulse were present. the left leg showed better microperfusion than before. computer tumoglaphy did not reveal an)-thrombotic material in the descending aorta and the iliac arteries. for continuous anticoagulation after lysis we used the heparinoid orgaran® to overlap the period until full anticoagulation was achieved by coumarin. the heparinoid was tested before us non-reactlve in the hipaa. with this prophylaxis the platelet count increased to normal values. hat ii related thrombosis is the most severe complication of heparin therapy, especially in patients with pre-existing thromoembolic disorders (dvt, dic). we present to our knowledge the first case of hat ii during thrombolytic therapy (it) with simultaneous hepatin administration. at admission a 16 year old girl showed complete occlusion of the pelvic veins of the right leg (r common and external iliac v., common and proximal superficial femoral v.). as in children no prospective studies on the thrornbolysis of dvt with rt-pa are available, we started tt according to the frankfurt/m. regimen: rt-pa (actilyse) bolus injection 0.4 nag/kg bw followed by continuous infusion of 1 mg/kg bw/d; simultaneous low dose heparin 100 -200 u/kg bw/d. doppler ultrasonographie control on day nine after the beginning of tr revealed a further distal growth of the thrombus into the popliteal vein. at the same time at ir was decreased to 70% and there was a dramatic drop in platelet count to <50% of the baseline value (216 to 86 gpt/1). the diagnosis hat ]i was confirmed by positive hipa-test. the only heparin preparation not cross-reacting with the antibody was the lmw heparinoid orgaran. heparin therefore was replaced by orgaran: bolus injection 1,500 u/h followed by infusion of 200 u/h. rt-pa-therapy was not discontinued. at ri substitution was performed at levels <70%. under orgaran administration platelet count rose from 77 to 239 gpt/1 within 5 days. on day 12 complete reperfusion was achieved and rt-pa was discontinued ,on day 14. the patient was discharged from the hospital on prophylaxis with phenprocomnon in good general condition at admission thrombophilia parameters (protein c and s, apc) were within normal range. heparin induced thrombocgopenia (hit) is a serious but rare side effect of treatment with unfractionated heparin. patients with hit present with a rapid decrease in thrombocyte count and occurence of venous or arterial thrombosis under heparin treatment. patients with arterial occlusive disease who need surgical intervention are of high risk to develop rethrombosis especially during the first few postoperative days while they are under i.v. heparin-treatment. in our study we investigated prospectively all patients who were admitted to a surgical unit because of arterial occlusive disease and received surgical intervention. 15 patients were screened on the 3rd and 6th postoperative day for heparin-antiplatelet 4-antibodies, decrease in platelet count and thromboembolic complications. 3 patients developed antibodies against heparin-platelet factor 4. one of the 3 patients developed a venous thrombosis in the operated leg in combination with a decrease in thrombocyte count. we conclude that heparin-platelet factor 4-antibodies are often produced in patients under i.v. heparin treatment. only few of these patients actually suffer from clinically s3anptomatic hit. medizinische universit~itsklinik, institut far klinische biochemie und pathobiochemie, d-97080 wtirzburg, germany the important physiological and pathophysiological role of platetets in health and disease is well established. platelets are also the primary target of many vasoactive hormones, factors and drugs. in addition, platelets provide a very important model system for studying agonist-and antagonist-mediated signal transduction events (1,2). platelet activation by agonists is associated with shape change, extension of filopodia, generation of intracellular messengers, secondary agonists and activation of adhesion receptors and integrins including the fibrinogen receptor gp iib/iiia. activation of protein tyrosine ldnases and calcium-dependent protein kinases appear to be of critical importance for the mechanism of platelet activation. in contrast, platelet inhibition by vasoactive factors and drugs is often mediated by the camp and/or cgmp signal transduction cascades. recent data form our laboratory suggest that the focal adhesion protein vasp, a major target of the intracellular no/cgmp and prostacyclin/camp effector systems, is an important link between signal transduction pathways and elements controlling cell motility (3-6). the implications of the recent advances in platelet signal transduction research for understanding the physiology, pathophysiology and pharmacology of human platelets will be discussed. domain. however, in vivo truncated "i'po is much less potent than full length tpo due to its rapid clearance. this suggest that the c-terminal glycosylated domain acts to stabilize circulating tpo. tpo stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet producing megakaryocytes in vitro. in vivo tpo induces dramatic increa~ses in megakaryocyte number and platelet production in animals, indicating that tpo regulates both thrombopoiesis and megakaryocytopoiesis and is the primary regulator of physiological platelet production. this later notion is supported by the phenotype of ti~ and cmpl knockout mice in which platelet levels are only 10-20% of normal while other blood lineages are unaffected. in animal models of myetosuppresion ,and marrow transplant, tpo reduces the platelet nadir seen in the~se models and significantly accelerates platelet recovery, tpo also has profound effects on red cell recovery in these models. these results suggest that tpo will be clinically u~ful for the treatment of thrombocytopenia in caucer patients who undergo chemotherapy or bone marrow transplant. the haparg-study ( .~a.emostatic parameters as risk factors in healthy volunteers) was sponsored by the german ministry of research and started in 1984. a previous study ( pard, intern. angiology 5, 181-195,1986 ) had shown that spontaneously enhanced platelet aggregation as measured with the pat-ill-test and a high fibrinogen level were significant risk factors for new vascular occlusions in diabetic patients. it was the aim of the haparg study to establish if spontaneous plateletsggregation and other hemostatic variables are independent risk factors for vascular occlusions also in healthy volunteers. employees of hschst ag, frankfurt aged 40-65 years and personnel of the university of mainz, aged 30-60 y. entered this prospective study. besides anamnestic data on smoking, hypertension and diabetes blood pressure, the ankle/arm doppler -index and an ecg were recorded and serum cholesterol, hdl, ldl, triglycerides, uric acid and glucose were measured. 1885 men and 988 women entered the study and were followed for 4-6 years. during this period 53 vascular occlusions ocoured ( 36 coronary and 9 cerebral events and 8 peripheral vascular occlusions). only three of these events occured in women. besides age and gender as expected risk factors the multivariate logistic stepwise regression analysis revealed as significant risk factors slightly elevated levels of blood glucose (p=0.001), smoking (p=0.0013), an elevated diastolic blood pressure (p=0,018),followed by spontaneous aggregation (p = 0.017) and higher hdl-levels (p=0.034) as significant risk factors for men. higher fibrinogen levels just missed significance in this analysis (p=0.059). none of the other hemostatic parameters showed any significant correlation with the vascular events.to our knowledge this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. spontaneously enhanced platelet aggregation seems to be an independent risk factor and may be an indicator of an ongoing active atherosclerotic process. mild bleeding symptoms -petechia, prolonged bleeding after tooth extraction -were observed in a patient with normal coagulation tests but slightly disturbed phtelet function: decreased aggregation in response to adp, pal: and thrombin receptor-derived peptide (trap-6). platelet count, agglutination with both ristocetin and botrocetin and thrombininduced aggregation were in the normal range. an abnormal membrane glycoprotein detected in electrophoretic studies of the patient's platelet proteins was shown to be related to gp llxx by immunoblotting. the variant gp differed even from the largest normal polymorphic gp iba form a by an increase in molecular mass, platelets of the patient contain the variant and normal c form in equal amounts. the quantity of gp ib as detected by flow cytometry is normal. lmmunoblotting studies of proteolytic fragments of the abnormal gp indicated that the defect is located in the c-terminal part of the glycocalicin fragment. measurement of the length of glycocalicin molecules after glycerol-spraying rotary shadowing electron microscopy demonstrated two populations of molecules in the patient sample: a normal on with an average size of 470 angstr6m and a fraction with an average size 0f650 angstr6n~ preliminary results of molecular analysis of the gp iba gene in the patient confirm the localization of the genetic defect by protein studies. in summary, the patient is heterozygous for a hitherto undescdbed molecular variant ofgp ~a. barnes and co-workers have shown that simple triple-helical collagenlike peptides, comprising basically a repeat gly-pro-hyp structure, are highly platelet reactive in polymeric form. they additionally have shown that the activity does not involve gpla/lla (integdn c~z~l). platelets adhere under static conditions to these fibrillar peptides mainly in a bivalent-cation independent manner; the bivalent cation dependent adhesion can be inhibited by an antibody against gpflb/llla (integfin m~d33). however, platelets of a patient with type i glanzmann's thrombasthenia secreted their ¢z-granule-and dense body content just like normal controls while activated with the triple-helical gly-pro-hyp peptide. the lack of an essential involvement of cd36 was shown with cd36deficient platelets, which respond to the peptides as readily as normal platelets when fibdnogen binding and cd62 and cd63 expression were measured. to study the involvement of the yon willebrand factor (vwf) platetets from a patient with severe type 3 vwd (vwf not detectable in platelets and plasma) were investigated. the vwf deficient ptatelets responded quite normally to the fibritlar collagen-like peptide. addition of pudfied vwf to the vwf-deficient platelets before adding the paptide raised the platelet respond slightly we conclude that neither ~3~ integrin, nor m~ integdn, nor cd36, nor vwf are the pdmary platelet receptor for the collagen-like peptide. there is evidence that native low density lipoprotein (ldl) can be oxidatively modified in vivo and the oxidized ldl may contribute to thrombogenesis and atherogenesis by damaging endothelial cells, eliciting vasocontractiola and stimulating blood platelets. by means of biochemical methods, electron microscopy, reflection contrast microscopy and computer image analysis, we find that oxidized ldl, compared to native ldl, affects platelets at least in the following five aspects. 1) oxidized ldl alters platelet functional morphology in a time and dose dependent manner. 2) oxidized ldl induces secretion of serotonin from both morphological resting platelets and shape changed platelets. 3) oxidized ldl advances, accelerates and enlarges platelet adhesion. 4) oxidized ldl causes cytodamage in platelets and cultured endothelial cells. 5) oxidized ldl stimulates platelets and endothelium, which leads to a formation of microthrombi on the monolayer of human umbilical vein endothelial cells. further experiments show that oxidized ldl inhibits the activity of ca2+-atpase in purified plasma membranes of platelets, and therefore increases the cytosolic calcium level in platelets. these results clearly indicate that calcium signaling pathway is intimately involved in the mechanism of platelet activation induced by oxidized ldl. since oxidized ldl, a relevant platelet agonist, has been confirmed to be present in vivo, further studies on its role in platelet activation and plateletendothelium-interaction can help for better understanding thrombogenesis and atherogenesis. recantly physical parameters such as sheer stress, hypo-or byperoxic conditions and hyperthermia have been shown to modulate endothelial cell dependent fibrinolysis. in this study we investigated whether yet another physie~ stimulus namely hypothermia might have an impact on the expression of components of the fibdnolytic system of human endothelial cells in culture. confluent cultures of human umbilical vein endothelial cells (hlivecs) were exposed to gc ° for 10, 20, 40, 80, or 160 rain, respectively. control cultures were exposed to 37°c for the same time periods. thereafter medium was removed, fresh medium was added and the cells were incubated for 24 hours at 37°c conditioned media (cm) were harvested and plasminogen activator inhibitor-i (pal-i) antigen was determined by a specific elisa. our data showed that huvecs responded to exposure to 8°c with a time-dependent increase of pai-i in cm. maximum induction of pal-! antigen was seen after 20 minutes exposure to 8°c (pal-! antigen: 754_+.83 ng/105 cells for treated cells; 4202_17 ng/105 cells for control cells). the inereese in pal-1 antigen was also reflected on the level of specific mrna synthesis as evidenced by northern blotting which showed a twofold increase in pai-i specific mrna in huvecs exposed to 8°c as compared to control cells. in conclusion, our data gives evidence that hypothermic stress like several other forms of physiological or pathological stress, results in activation of endothelial ceils as evidenced by an increased expression of pai-i. our findings provide ~rther evidence that such activation of endothelial cells can be induced not only by biochemical mediators but also by physical stimuli. nitric oxide (no) is formed from intracellular l-arginine by two no synthase (nos) isoforms: a ca+ +-dependent constitutive form (cnos) and a ca+÷-independent inducible form (inos) which is expressed after challenge of cells with immunologic or inflammatory stimuli. endothelial cell derived no is an important signaling molecule inducing smooth muscle cell relaxation and platelet inhibition, however its role in cell proliferation is poorly understood. the present study investigates the pattern of nos isoforms and no production in actively proliferating low density human umbilical vein endothelial cells (ld-huvec) and in high density monolayers of quiescent cells (hd-huvec). ld-huvec but not hd-huvec expressed inos (measured as ca ++independent citrulline formation from l-arginine in cell lysates) with a maximal activity of 30-40 pmoles/min/mg protein, cnos (ca +÷dependent citrulline formation) was demonstrated in hd-huvec (0.02-0.2 pmoles/min/mg) but not measurable in ld-huvec lysates. lmmunoprecipitation of cell lysate with a mab against murine macrophage inos decreased inos activity of supernatant by 70% in ld-huvec. immunoprecipitation with an anti-ecnos mab did not modify inos activity in ld-huvec but abolished cnos activity in hd-huvec. no release (measured as nitrite + nitrate production in an 18 h incubation in the presence of 1 mm l-arginine) was 1.9 nmol/mg cell protein in the supernatant of hd-huvec whereas it was t0fold higher with ld-huvec. measurement of intracellular cgmp after stimulation of cells with i-2 #m ionomycin (a sensitive index of enos activity) showed a 400-800% increase of basal levels in hd-huvec but only 50-150% increase in ld-huvec. on the contrary, sodium nitroprusside-induced cgmp increase was similar in non-confluent and confluent cells. the expression of inos and the high no production in ld-huvec suggest a role of endogenous no in mediating cell proliferation. indeed, the nos inhibitors l-nmma and l-canavanine inhibited [~h]thymidine incorporation in ld-huvec by 60%. on the ++ nt no r uctlon ar x r d m other hand enos and ca -depende p od " e e p esse " differentiated huvec and correspond to the ability of the cells to regulate vascular tone and platelet activation. the processes of replication, maturation and motility of progenitor cells of different lineages in the bone marrow are tightly regulated, particulady by various cellular contacts and their modulation involving pericellular proteolysis. while mononuclear phagocytes and related cell lines are well characterized for surface localized plasminogen activation by receptor bound urokinase, megakaryocytes are poorly described in this respect. in the present study the expression of mrna of urokinase receptor in megakaryoblastic cell lines chrf-280, dami and meg01 was found at a low basal level and increased 5-10 fold after pma-treatment. urokinase receptor mrna levels remained differentiation dependent elevated for up to 5 days. functional analysis of upar expression on the cell surface was documented immunologically by facs-anatysis and on the functional level by direct binding of radio-labeled amine-terminal fragment of urokjnase and mirrawed the increase in urakinase recptar mrna level. as expected, antibody and ligand binding was sensitive towards treatment with phosphatidyl-inositol-specific phospholipase c, since the urokinase receptor belongs to the group of gpi-anchored receptors. in cultered megakaryocytes derived from bone marrow urokinase receptor mrna was demonstrated by rt-pcr, as well. these results indicate a differentiation-dependent increase of urokinase receptor expression on cells of megakaryoblastic cell lineage suggesting the participation of urokinase dependent events during maturation process and intravasation of these platelet precurors. regulation of am gene expression may be coupled to oxidative stress through redox sensitive transcriptional regulatory factors. the aim of this study was to investigate the influence of selected antioxidants on tnfa (100 u/ml, 6 h)-induced surface expression of elam-i, icam-i and vcam-i in human umbilical vein endothelial cells. am expression was determined by quantitative flow cytometry and immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase method). the following antioxidants were tested: pyrrolidine dithiocarbamate (pdtc), n-acetylcysteine (nac), glutathionemonoethyl ester (gshmee), probucol and the estradiol-derived scavestrogens j824, j861, j901. additionally, 17fi-estradiol was examined. the used concentrations were 1-5 mm for nac and gshmee and 1-100 ~m for all other substances. according to their influence on tnf-induced am expression, the tested antioxidants can be divided into three groups: 1) pdtc, nac and gshmee inhibit the expression of all three am, 2) j861 and j824 reduce vcam-i and icam-i and increase elam-i, 3) j901 and probucol show no effect. 17fl-estradiol elicits a potentiation of tnfa-induced expression of all three am. tnfa-induced vcam-i expression was most effectively inhibited by ptdc and j861 (100 em: 68% and 78% inhibition, respectively). the best inhibitors of icam-i induction were pdtc (100 ~m: 51% inhibition) and j824 (100 t~m: 47 % inhibition). j824 and j861 most effectively increased the elam-t induction (100/~m: 85 % and 62 %, respectively). the results confirm that antioxidant-sensitive mechanisms play a role in tnfct-induced am upregulation. it is suggested that icam-1-and vcam-l-expression is regulated differently from elam-i. the diversity of antioxidant effects might be due to distinct levels of redox control of transcription with an antioxidant-inhibited and an antioxidant-promoted step. heamophiliacs who continue to produce tilers of f.v11i inhibitors no higher than 5 to 10 bethesda units (bu) when given continuous f,viii replacement therapy are classified as low responders. studies carried out on low responders to assess the effect of immune tole~lcc therapy flit) in these patients. it was found that elimination ofiahibitors in low responders could be achieved using f.vlii at 20 to too u/kg given every 2 to 3 days over a period of 6 weeks. we consider that 1tt is justified in this group of patients. prevents rebooster effects and reduces elimimfion time of f.viii inhibitors. it also reduces the enhanced mortality in patients with inhibitors and has been shown to be cost-effective. in view of the improved prognosis, we recontmend starting itt in low respor, ders soon after the diagnosis has been made in order to minimize f.vill exposure before isfi.tiating el'. itr in low responders has a better success rate an.,d requires shorter u'eatment than itt in high responders. w.schramm, med.klin~, innenstadt, lmu, mfinchen following replacement therapie~ in hemphilm t_be developmem of inhibimrs to the p*ficat's deficient factor is the most serious sequelae. the incidence of inhibitors varies between 15 and 35 % of persons with hemophilia a. inhibitor develop, mostly in paticm~ with sovere bemoph/lia a, early in life and afmr few exposure days (9 -12 days). using the bethesdamethod the inh~itom can be derided in low (< 5 bu) and high msponder (>5 bu) hemopb~iacs with high fitcrs have ~ually serious ~ problems. they are resistent to mg,,flary replacement therapy, the ~ goal in the treawnent is to control severn acum bleedin~ and to eradicate the inlu'bitor perrmnanfly and to induce tolea'ance. in the tream'tmt of acute blcedings in patients with hlhibitors factor viii inhibitor bypassing ag~ts like activated prothro~ complex concenuxtes (feiba) or prothrombin complex concentrates (pcc) arc mostly used. the meehani~n of aefiou of theses concentrates is net fully investigated. their effect is usually related to the high coment of activated clotting factors ~d phosphoupids. since some years acdwated recombinant factor vii (f vii a) is used to treat patients with inl'dbitocs successfully in several clinical situations including surgery. in addition porcine factor vii1 is widely used in particular in the uk for the treatment of factor v].ii inhibitor patients and could demonstrade good clir3cal results, in case of life threatening bleedings a temporary reducfic~ of inhihitors could be. ~hieved by using extem*,ivc plasma exchange (protein a adsorption) and immune suppression with cyclophosphamid (~alm6 protocol). follow~g the first description by h. bmc~'~mn some modifications for tlm induction of irmnune tolerance in hemophilia a patients have ~en propet'ed. these schedule, can be derided into high, intemxxfime and low dosage roglrmms di:ffea'jng in the dosage of factor viii infused. successful rates about 70 to go % can be obtained with ~ and high dose regimens. but is has to be co~sidered that the~ expensive trea.t~nt regimens have a great physical and p .syc.hosocial impact to the benx~-li~s and thch" farm~e& the different immu~ mler-a.~ze mg~-'~ predominantly used in high rcsponder inhibitor. most of the patients with low concentrations of inhibitors cm be managed with factor viii in increased dosage. this is in agreement with the consensus recorrnr~rdadons for u'eatlncnt hemophiliacs in germany fi'orn 1994. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of eases, vk deficiency oceured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacental transport of vk in the perinatal period,following studies were carried out. t)hepaplastintest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlations were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastin test value of less than 120% of the normal adult value, the value of the hepaplastintest was less than 30 % of normal adult value in the cord venous blood° we also demonstrated that vk passed through the placenta but only in small qualities. 61hiv-negative patients (median age 4]yrs, 13-70}, formerly treated with non-virusinactivated coagulation products, underwent hepatologic examination, including afp screening and sonography. 31.suffer from severe, 20 from moderate or mild haemophilia a or b, 10 from other severe coagulation factor deficiencies. 52 had been treated with products of the swiss red cross (src) only (28 with small pool cryoprecipitate}, 4 with foreign products only, 5 with both src and foreign products. treatment intensity was variable with>20'000 iu/yr in 26,< 20'000 in ]6, < 1 treatment episode/yr in ]0, a total of only 1-3 treatment courses in 6 patients. 3 afibrinogenemic patients had prophylactic replacement therapy. hcv serology was positive in 56/6] patients (92%), in 47 with detectable hcv rna (77%). the 5 persons who escaped hcv infection, with normal alt-levels and without sonographic alterations, had low intensity treatment with small pool src preparations only. alt-levels were elevated in 33/56 anti-hcv positive patients (59%). 26/56 had abnormal sonographic findings (46%). there was a clear correlation between elevated alt-levels and abnormal sonographies: of 33 patients with elevated alt 23 had abnormal sonography, of 23 with normal alt 3 had abnormal sonography. 8 patients had liver cirrhosis (6 with clinically overt hepatopathy), 4 (4/56 = 1%!) with hepatocellu]ar carcinoma (hcc) with elevated afp-leveis. of these 4 patients 2 had intraarterial embolization with ]ipiodol-epirubicin; in 2 patients hcc diagnosis was made in a late stage. i patient with advanced liver cirrhosis underwent successful liver transplantation. 2 of the 6 patients with hepatopathy had severe haemophilia with temporary high alcohol intake, 4 had mild coagulatlon disorder with few treatment episodes. possible precipitating factors were coinfection with hbv, high alcohol consumation and first exposure to hcv contaminated blood products in an advanced age, but not intensive replacement therapy. very similar results for f vlll and vwf. since the factor viii level is kept steady above the level where there is an increased risk of haemorrhage, continuous infusion is haemostatically safer and more efficacious than bolus injections, another advantage is a progressive decrease of clearence during the first days after surgery which leads to a substantial reduction of factor concentrate consumption by avoiding the innecessary peaks of bolus injections. 22 children with severe form of haemophilia a undergoing elective surgery received continuous infusions with different plasma-derlved and recombinant f viii concentrates. before surgery, patients got bolus injections to raise the factor viii levels to more than 80 %. during continuous infusion factor viii levels were measured two to three times a day and the infusion rate of 4 to 5 iu/kg/h could be reduced on the second or third day to 2-3 iu/kg/h. the clinical efficacy was excellent with no bleeding events. in 5 children with vwd also undergoing elective surgery continuous infusions with humate pr were performed in the same way. no bleeding events were observed in these patients. none of the patients developed postoperative wound infections. the overall doses of f vtll concentrate 'were about 20-30 % lower than those required during replacement therapy with bolus doses. lg8 factor x frankfurt i : molecular and functional characterisation of a hereditary factor x defect (gla +25 lys) huhmann i., holler b., krinninger b., turecek p.l, richter g., scharrer i., forberg e., watzke h. univ. klinik f0r inhere med.i, abteilung for h~tmatologie und h~mostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrombin to thrombin. the congenital fx-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his p'fi (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular charactedsation of the genetic defect was performed by amplification of the eight exerts and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboll site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exert ii. fx was isolated from plasma of the propositus by monoq ion exchange chromatography. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intdnsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account. we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective in both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardiothoracic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardiopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, thrombin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64__+9 y). they lost 750+__460 ml blood (mean+sd) into the drains within the first 18 hours after end of surgery. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificity of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptidea (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n = 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other parameters were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibrin for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrin will provide further insights into fibrin(ogen) metabolism. heparin induced thrombocytopenia represents a multicomponent syndrome associated with the use of heparin and related drugs resulting in not only thrombocytopenia, but also arterial thrombosis of varying magnitude. the initial diagnosis ofthis syndrome is usually made by clinical observation and a drop in platelet count. conventional diagnostic methods include platelet aggregation responses to patient's serum and ~4c serotonin release in response to patient's serum, aggregation/agglutination of patient's platdets in response to heparins and the detection of patients anti-heparin platelet factor 4 (hpf4-ab) ned-antibodies by using elisa methodology. several other individualized methods are also used to demonstrate platelet activation. to test the diagnostic validity of the platelet aggregation (pa) 14c serotonin release (sr) and the relevance of hpf~-ab 340 serum samples collected from patients with clinically eunfwmed eases of lilt syndrome were compared in parallel in various assay systems. the diagnostic efficacy of these tests varied from 60-74% with the pa test providing better results than others. when the pa test was compared with serotonin release, a poor correlation was noted (r=0.47). in contrast, the correlation between the pa and hp4-ab was somewhat better (r=0.66). in another study, blood samples collected from 50 patients treated with ahigh dose low molecular weight beparin for two weeks (80 mg o.d.) were tested. 20 of these patients showed a high titre of hpf4.ab without any decrease of platelet count. none of these patients were found to be positive in the 14c serotonin release assay. a third study included blood samples from dvt patients administered with iv heparin infusion, high dose sc lmw heparin (certoparin) and iv lmw heparin for the management of dvt. none of these patient groups (20-34) exhibited any hit responses, hmvever, the incidence of high hpf4-ab titre was found to be 53% in heparin, 36% in patients with lmw heparin iv and 26% in lmw heparin sc groups. pa and sr studies revealed 8% and 12% false positive ~ respeetively. these studies clearty suggest that the currently available ~ for laboratory diagnosis of hit syndrome are of limited value, and caution should be exercised in the interpretation of the results obtained with these tests. heparin-induced thrombocytopenia (hit) is one of the major severe side effects during treatment with heparin. in postoperative medicine clinical studies demonstrated the prevalence of hit with unfractionated over fractionated heparins. few data are available from the non-ope1"ative medicine and from patients without thmmboembolism before heparinization. in a controlled prospective randomized study the safety and efficacy of low-dose heparin was compared with a lowmolecular-weight (lmw) heparin over 10 days in bedridden medical inpatients (haemostasis, in press). 1968 patients were randomized and controlled for the development of thrombocytopenia. thrombocytopenia was defined as a platelet count below 40.000lid at day 10. 4 patients developed thrombocytopenia in the heparin group and no patient in the lmwheparin group (p<0.05). none of the patients with thrombocytopenia developed a thromboembolic complication. in a second prospective case control study 90 patients with side effects on anticoagulants were treated with lmw-heparin once daily subcutaneously for a period of 1 month to 9 years. platelet count was performed every 1 to 3 months. none of these patients developed thrombocytopenia during heparinization with lmw-heparin. it is concluded that hit is a very rare complication in nonoperated bedridden medical patients. a decrease of platetet count may occur in about 0.5% of patients receiving low-dose heparin. the incidence of hit with thrombosis during low-dose heparin and of hit during lmw-heparin in non-operated patients is manyfold lower and remains to be determined. terminology: instead of the term "hemorrhagic disease of the newborn (hdn)" the term vkdb should be used, since neonatal bleeding is often not due to vkdeficieacy and vkdb may occur after the neonatal period (i.e. after 4 weeks). definition: vkdb is a bleeding disorder caused by reduced activity of vkdependent coagulation factors which responds to vk. diagnnsis: in a bleeding infant a prolonged pt (inr > 3.5) together with normal fibrinogen and platelet count is almost diagnostic of vkdb. the diagnosis is proven, if vk shortens the pt (after only 30-60 minutes) and/or stops bleeding. classification: classification by age of onset into early (< 24 h~. classic fdav 2-7) and lale form (> i week <6 months), and by etiology into idionathic and ~ec0nd~'y. in secondary vkdb in addition to breast feeding other factors can be demonstrated, such as poor intake or absorption of vk and increased consumption of vk. vk-prophylaxis: benefits: oral and intramuscular (i.m) vk (one dose of i nag) prevents equally well the classic form of vkdb. lm. vk appears to be more effective in preventing the late form (times 15-> 50). the protection achieved by single oral prophylaxis (times 3-5) is improved by triple oral vk (times 15-30). risks: because of poten[ial ri~l~ associated with extremely high levels of vk and the possibility of injection injury, i.m. vk has been questioned as the prophylaxis of choice for normal neonates. since vk is involved not only in coagulation but 'also in carboxviation with multiple effects, excessive deviations from the low physiologic concentrations, which prevail in the fully breast-fed healthy mature infant should be avoided. proposal: repeated (daily or weekly) small oral doses of vk are closer to physiologic conditions than single i.m. bolus doses, which expose neonates to excessively high vk levels. the incidence of intracranial vkdb can be reduced if the grave significance of warning signs is recognized (i.e, icterns, failure to thrive, feeding problems, minor bleeding, disease with cholostasis). whether or not the more reliable absorption of the new mixed mieellar (mm~ nrenaral~i0n of vk can reduce the protective oral dose of vk-.prophylaxis has to be evaluated. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of cases, vk deficiency occured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacentel transport of vk in the perlnatal period,followlng studies were carried out. 1)hepaplastlntest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlatlons were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastln test value of less than 120% of the normal adult value, the value of the bepaplastlntest was less than 30 % of normal adult value in the cord venous blood. we also demonstrated that vk passed through the placenta but only in small qualities. the point mutation g to a at nt 449 in exon v of the factor x gene (gin 102 to lys) has previously been found in two independent kindreds with fx deficiency. it occured in both families in an heterozygote state and was associated with two other genetic defect in the fx gene. we have identified another familiy in which this mutation occurs in a homozygote state. in this family the mutation is associated with the previously reported mutation gla 14 to lys which also occurs in a homozygote state. the pt and ptt of the proposita and her siter are markedly prolonged. the fx activity is reduced to <1% in the extrinsic system, to 30% in the intrinsic system and to 18 % after activation with rvv. the fx antigen is reduced to 20%. the coagulation profile of this family thus is identical with that of fx vorarlberg despite the fact that the fx vorarlberg kindred is only heterozygous for the mutation glal02 to lys. haplotype analysis could not rule out consanquinity with the fx vorarlberg kindred. these data suggest that the mutation at nt 449 which leads to a fairly dramatic amino acid change from glu to lys would indeed represent a polymorphism. to further address this question we cloned the fx gene in an expression vector (pcep 4) for transient expression in the human embryonic kidney cell line 293 and introduced the mutation at nt 449 by site directed mutagenesis. hereditary deficiency of factor ixa, a key enzyme in blood coagulation, causes hemophilia b, a severe x-chromosomelinked bleeding disorder; clinical studies have identified nearly 500 deleterious variants. the x-ray structure of porcine factor ixa shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for fx activation by phospholipid-bound flxa and cofactor villa. the 3.0 a resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (egf)-like modules; the n-terminal gla module is partially disordered. the catalytic module, with covalent inhibitor d-phe-pro-arg chloromethyl ketone, most closely resembles fxa but differs significantly at several positions. particularly noteworthy is the strained conformation of glu-388, a residue strictly conserved in known fixa sequences but conserved as gly among other trypsin-like serine proteinase. flexibility apparent in electron density together with modelling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fixa. most hemophilic mutation sites of surface fix residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary flxa-fvilla-fx complex structure: the stabilizing fvilla interactions force the catalytic modules together, completing flxa active site formation and catalytic enhancement. factor x frankfurt i molecular and functional characterisation of a hereditary factor x defect (gla +25 ---, lys) huhmann i., holler b., krinninger b., turecek pi., richter g., scharrer i., forberg e., watzke h.. univ. klinik ftlr innere medi, abteilung for h~matoiogie und hamostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrembin to thrombin. the congenital f×-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his ptt (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular characterisauon of the genetic defect was performed by amplification of the eight exons and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboti site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exon i1. fx was isolated from plasma of the propositus by monoq ion exchange chromatogrephy. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intrinsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account_ we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective ip both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardioth~)racic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardlopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, throm. bin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64+9 y). they lost 750+__460 ml blood (mean_+sd) into the drains within the first 18 hours after end of sur. gory. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificily of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptide a (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n= 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other paramelers were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibdn for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrln will provide further insighls into fibrin(ogen) metabolism. this study was conducted as a randomized parallel -group clinical trial comparing the safety and efficacy of a low molecular weight heparin {lmwh} -monoembolex sandoz and unfractionated standard heparin glfh) for the perioperative prevention of venous thromboembolie disease (dvt) following major surgms' in patients with gynecologic malignancy.. three hundred and twenty 4 women (six drop outsl werr randomized and received either 3 times daily 5000 [l" s.c. ul.'i-i (sandoz nuemberg germany] (n = 164) or once a day t5000 i~'v'. units s.c. monoembolex (n = 160) plus two placebo injections. heparin therapy was started the morning before opcrati(m and continued until the 7th postoperative day. up to the 10th poatop, day the incidence of dvt was 6.25 % (n = 10; incl. 7 pulmona~ embolisms pe) in the lmwh group and 6.10 % (n = 10; incl. 4 pe} in the ufh group. the overall incidence of clinically hemorrhagic wound complications was significantly decreased in the lmwh group 16.3 % (n = 2hi compared to the ufh group 26.8 % {n = 44; p < 0.0051. the incidence of major hemorrhagic episodes was 9.4 % in = 151 in the lmwh group and 14.0 %/n = 23) in the ufh group. this difference was not statisticauy significant. one case of fatal pe was observed in the lmwh -treated group. five women deaths in the lmwh group were observed during the study and 3 in the ufh group. this study demonstrates that the perioperative treaunent of low molecular weight heparins is more safety than standard heparins in gynecologic -oncologic patients undergoing major surge .ry. however, the incidence of thromboembohc complications is simmilar in both treatment regimes. to explore the effect of targeting an antithrombin to the surface of a thrombus, recombinant hirudin (hir) was covalently linked to the fab' fragment of fibrin-specific monoclonal antibody 59d8 (fab) resulting in a stable conjugate (hir-fab). in vitro, hir-fab was 9times more efficient than hir alone in inhibiting fibrin deposition on experimental clot surfaces in human or baboon plasma (p<0.01). to validate these results in vivo, hir-fab was compared to hir in a baboon model. the deposition of ill-in-labeled platelets onto a segment of dacron vascular graft present in an extracorporeal arteriovenous shunt was measured. blood flow rate was 40 ml/min. one hour local infusions of 4500 atu of either hir-fab or hir resulted in deposition of 0.16 x 109 and 2.17 x 109 plate!ets, respectively. equieffective dosages were 2000 atu hir-fab and 9000 atu hir resulting in deposition of 1.06 x 109 and 0.93 x 109 platelets, respectively. based on full dose response curves (n = 14), hir-fab was found to be > 4.5-fold more potent (based on activity) than hir. because of the small total amounts of antithrombins used and the short duration of these experiments, no significant systemic effects were observed. thus, fibrin-targeted recombinant hirudin prevents platelet deposition and thrombus formation more effectively than uncoupled hirudin in vitro and in an in vivo primate model. triabin, a 17 kda protein from the saliva of the assassin bug triatoma pallidipennis, is a new specific thrombin inhibitor (1). tt does not block the catalytic center but interferes with the anionbinding exosite of thrombin. the recombinant protein was produced with the baculovirus/insect cell system and used to study the inhibitory effect of triabin on thrombin-induced responses of human blood platelets and blood vessels. aggregation of platelets in tyrode's solution was measured turbidimetrically at 37°c. for the studies on blood vessels rings (2-3 mm) from small porcine pulmonary arteries were placed in organ baths for isometric tension recording. the integrity of the endothelium was assessed by the relaxant response to bradykinin. like hirudin, triabin inhibited the thrombin (0.1 u/ml)-induced aggregation of washed human platelets at nanomolar concentrations (ec50 = 2.6 nmol/l); whereas the adp-and collagen-induced aggregation were not suppressed. in pgf2c~-precontracted porcine pulmonary arteries, the thrombin (0.2 u/ml)-induced endothelium-dependent relaxation was inhibited by triabin in the same concentration range as found for inhibition of platelet aggregation. higher concentrations of triabin were required fo affect the contractile response of endothelium-denuded porcine pulmonary arteries to thrombin (1 u/ml). in all these assays, the inhibitory potency of triabin was dependent on the thrombin concentration used. these studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of the thrombin-mediated cellular effects. dept. of medicine, university hospital benjamin franklin, free university of berlin, dept. of medicine and dept. of surgery, heinrich-heine:university dusseldod after standardized training in home prothrombine estimation using the coaguchek system, 150 consecutive patients (p) who had st. jude medical aortic or mitral valve implantation were allocated to two random arms; 75 p were asked to control the inr themselves every third day. in the remaining 75 p anticoagulation was managed by the home physician without recommending an interval for these controls. all 150 p were monitored during the education period to a target therapeutic range of inr 3.5-4.0. p were asked to contact their home physician immediately if the inr was measured 0.5 below or above the target range (inr-corrider 3.0-4.5). all p had out-patient re-examinations every three months. thrombotic, thromboembelic and hemorrhagic complications were documented by the p using special documentation cards. the following findings were documented during the follow-up period: 4.49 0.9 0 the results of this randomized study demonstrate a significant improvement in the management of oral anticeagulation by home prothrombine estimation. significant (p<0.001) more inr measurements were found inside the target therapeutic range. moreover. bleeding and thromboembolic complications could be reduced (p = 0.038) in the study group with home prothrombine estimation. life-threatening thromboembolic and hemorrhagic complications were not observed in p who were on home prothrembine estimation, while three such events (2.72 %/year) were documented in group a. local vascular injury following ptca exposes circulating platelets to prodmmbogenic stimuli. by binding to platelet gp iiblliia fibrinogen crosslinks platelets, which represents the final common pathway of platelet aggregation. fradafiban (bibu52zw) is a non-peptide compound with effective, reversible inhibitory effects on fibrinogen binding to gp iib/ii/a on human platelets. in the first double-blinded, prospective phase ii study three escalating doses of bibu52zw as a continuous 24 h-i.v, infusion were tested in comparison to placebo in 65 patients with stable angina pectoris undergoing elective ptca. the mean receptor occupancy with 20rag, 40ms and 60ms per hour were 71.974, 84.5 % and 87.9% at 24 hours, respectively. as compared to placebo breeding time was significantly prolonged (7 vs 20rain) during fi-adaiiban infusion with a weak dose-dependency. platelet aggregation in platetet rich plasma ex vivo with collagen (2.0 and 4.0 gg/ml), adp (2.5 and 5.0 gmol/ml) or ca-ionophor a 23187 (2.5 and 5.0gg/ml) was significantly and dose-dependently inhibited as compared to placebo. using the two upper doses of fradafiban, we observed major bleeding complications in 8 patients requiring blood transfusions or vascular surreal repair. in these patients, too, maximal antiplatelet effects could be documented. these data sugest that bibu52zw is an effective fibrinogen receptor antagonist in patients. the requirement of ad hoc receptor occupancy determination or platelet function monitoring for safe and effective clinical use should be evaluated. in a placebo controlled interaction study 9 healthy volunteers were randomized to receive either a 24 hour infusion of peg-hirudin ( 0.02 mg/kg/h) after an i.v, bolus of 0.2 mg/kg + placebo, or 325 mg/day acetylsalicylic acid (asa) for three days followed by a placebo infusion or the peg-hirudin infusion + asa. each volunteer received all three treaments. there was a washout period of at least 14 days between the infusions. at short intervals aptt, activated clotting time (act), ecadntime (ect), alia-activity using the chromogenic substrate 2238, collagen-induced aggregation, platelet adhesion and platelet induced thrombin gene,ration time (pitt) were measured, bleeding time (simplate) was studied before drug administration, on day three before the infusion and 6 hours after start of the infusion.the infusion of peg-hirudin after 4 and 8 hours led to a mean hirudin plasma level of 1.8 pg/ml. asa markedly inhibited collagen induced aggregation as expected. the mean bleeding time was prolonged under the influence of peg-hirudin from 5.2 to 6.22 min, after asa from 5.8 -18.2 min and after the combination of peg-hirudin + asa from 5.4 -33.7 min. in each volunteer the bleeding time was longer under the combination than after asa alone. in two volunteers receiving peg-hirudin + asa the bleeding time measurement was stopped after 60 rain. none of the coagulation parameters or platelet function tests correlated with the prolongation of the bleeding time. however the bleeding time was excessively prolonged in those volunteers who had a marked prolongation under asa alone.the combination of hirudin at a higher dosage with asa probably is associated with a relative high risk of bleeding. either the hirudin dosage should be reduced if the combination seems feasabie or asa should be given after the end of hirudin treatment. fibrinogen with the sta/stago and the mla/dade systems correlated well, but neither system correlated well with the acl/il system. at iii, protein c, protein s, and anti-xa heparin assays using stago reagents performed as expected for normals and low abnormals on the sta. factor levels on the sta/stago system were less sensitive than factor levels obtained with the dade reagents on the mla or fibrometer. using the sta/stago system, thrombin time results correlated well with the aptt and heparin levels. the thrombin time was not associated with additional manipulation for assay preparation, nor any cross-contamination of reagent or sample, since on the sta reagents do not come into contact with tubing. the sta was not sensitive to hemolytic, icteric or lipernic samples for clotting assays artd showed the same sensitivity as the mla for chromogenic assays. the overall data comparisons, high throughput, minimal operator intervention for reagent/assay change and ease of operation warrant further evaluation of the sta hemostasis analyzer. a. wehmeier, d. s6hngen, c. rieth klinik for h#,matologie, onkologie und klinische immunologie der heinrich-heine-universit~it d0sseldorf hirudin selectively inhibits thrombin by direct interaction. because the effect of hirudin is independent of antithrombin iii and other factors, it seems an attractive alternative to current anticoagulants. however, it is uncertain whether hirudin influences plateletassociated thrombotic disorders and how it compares with conventional and lmw heparin. we investigated the effect of 2 recombinant hirudin preparations (rhein biotech, dt3sseldorf) on platelet function tests: in vitro bleeding time, adhesion to glass beads, aggregation in platelet-rich plasma and whole blood. hirudin was used in concentrations of 0.1-100 i.tg/mi, and was compared to trisodium citrate (0.38%), conventional heparin (50 iu/ml) or lmw heparin (fraxiparin, 500 iu/ml). both recombinant hirudins showed normal activity in thrombin neutralization tests, and prolongation of thrombin time and aptt. however, in vitro bleeding time was not prolonged by hirudin, but was more than doubled by addition of conventional and lmw heparins. platelet retention to glass bead columns was reduced by hirudin in a dose-dependent manner to about 40% but was more effectively reduced by both heparin preparations and citrate. hirudin had an inhibitory effect on p!atelet aggregation in prp induced by thrombin, collagen, and predominantly epinephrine but not adp and ristocetin. in whole blood, a small effect could only be observed with hirudin concentrations of >25 ~g/ml as compared to citrateanticoagulated blood. in summary, thrombin inhibition by recombinant hirudin has little effect on in vitro platelet function tests in comparison to heparins and calcium depletion. the role of endothelin (et), prostaglandins and the coagulation system in the pathogenesis of acute renal failure is still to be defined. in anaesthesized pigs the effects of i.v. infusion of et (3 /~g/kg) alone (group 1, n=6) and after pretreatment with the potent thrombin-inhibitor hirudin (0,5 mg/kg)(group 2, n=6) on haemodynamics, coagulation parameters (factor viii, antithrombin iii, precallicrein, fibrin monomers, aptt) and prostaglandins were investigated. plasma renin activity (pra)-, creatinine clearance-, urine volume-measurement and blood gas analysis were performed hourly. et-infusion caused an initial bp-reduction and marked hr-reduction followed by a transient bp-elevation and hr-reduction. activation of platelets can be directly measured by flow cytometry using monoclunal antibodies. in an in vitro study the effect of the thrombin inkibitors argatroban, efegatran, dup 714, recombinant hirudin and peghirudin on platelet activation induced by various agonists was studied in whole blood. blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregatiun of platelets. for anticoagulation of blood the thrombin inhibitors mentioned above were used at a final concentration of 10 ~tg/ml each. blood samples were then incubated at 37°c either with saline, r-tissue factor (rtf), arachidonic acid (aa), adenosine diphosphate (adp) or collagen. at definite times (1, 2.5, 5, 10 rain) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured by means of fluorescent monoclonal antibodies to platelet surface receptors gpiiia (cd-61) and p-selectin (cd-62). the agunists used induced a platelet activation of 37.4 + 15.2 % (rtf), 65.1 + 12.1% (aa), 19.3 + 7.4 % (adp) and 27.1 + 12.8 % (collagen). flow cytometric analysis showed that all thrombin inlaibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. in contrast, after induction of platelet activation with the other agonists an increased percent cd-62 expression was found showing a strong platelet activation with a maximum at the same times as in non-anticoagulated blood. in conclusion, the results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. the formation of active serine proteases including thrombin may be effectively inldbked by these agents. the observations further suggest that, while thrombin inkibitors may control serine proteases, these agents do not inhibit the activation ofplatelets mediated by other agonists. this work was supported by the grant bmft 07nbl01. animal experimental studies on the pharmacokinetics of peg-hirudin e. bucha, a. kossmehl, g. nowak max-pianck-gesellschaft e. v., arbeitsgruppe "pharmakologische h~imostaseologie", jena hirudin, when complexed with polyethylene glycol (peg), increases its molecular weight from 7 to 17 kda, thereby preventing extravasation of this drug. peg-hirudin is distributed almost only in the intravascular blood space. in addition, its increased molecular weight retards the renal elimination. the elimination half-life of hirudin in rats (58 + 12 min, as determined) is increased five-fold (244 ± 18 min). with the same hirudin dose applied, the blood level of hirudin is increased 19-fold, measured in the 13-elimination phase. in the urine of rats, 2 -3% of the hirudin activity were recovered following hirudin administration, but 48% could be detected after peg-hirudin had been applied. after subcutaneous administration of peg-hirudin, the trnaxwalue is reached at 380 rain (r-hirudin: 65 min); the cmax-value is increased 3-fold, compared to that of r-hirudin (2.5 pg/ml). 24 hours later, still one fifth of the maximum concentration (cma,) is present in the blood, and the renal elimination is still retarded. in the urine of rats, 12% of the hirudin activity applied were recovered in the 24-h urine sample. with intact renal function, following subcutaneous administration, peghirudin is abte to produce a constant blood level of hirudin over a long pedod. thrombin inkibitors such as r-hirudin (rh), argatroban (a), efegatran (e), and peghiradin (ph) are currently undergoing extensive clinical trials in such cardiovascular indications as ptca, ami, and treatment of unstable angina. a rapid assessment of the anticoagulant actions of these agents is, therefore, crucial to assure their efficacy and safety. currently, act and aptt are used to measure the anticoagulant effect of these agents. we have utilized a dry reagent technology based on the motion of paramagnetic iron oxide particles (plop) to measure the antithrombin effects of various thrombin inhibitors (cv diagnostics, raleight, nc). the heparin monitoring card has been modified to measure antithrombin agents in various anticoagulant ranges for (a)0 (e), (rh), and (ph). blood samples drawn from patients treated with (a) and (rh) have been evaluated and concentrations of these agents have been calculated using an external calibration curve. in the in vitro setting, citrated whole blood or citrated frozen plasma can be used to evaluate the anticoagulant effects of these agents. the results obtained are comparable to the act which is conventionally used for the monitoring of these agents. both (rh) and ( period. we would like to present a case of heparin-induced-thrombocytopenia (hit) in a 47 years old woman who underwend open heart surgery. she suffered from a combined aortic valve disease and leading stenosis. laboratory analysis showed constant low platelet counts (50/nl) without heparin application, so that an idiopathic thrombocytopenlc purpura was suspected. but platelets also decreased after heparin application. heparin-antibodies were found in the heparin induced platelet activation assay (hipaa). treatment with corticosteroids and immunoglobulines, respectively, showed no improvement but the patient unfortunately developed a pneumonia with legionetla pneumophila. therefore, the only suitable anticoagulant for the necessary aortic valve replacement was hirudin: a bolus injection of r-hirudin of 0,75 mg/kg b.w. was administered 10 min. bevore start of the extracorporal circulation (ecc), the heart-lung machine (hlm) was primed with 6 mg r-hirudin and another bolus of 5 mg of r-hirudin was administered. additionally 10 mg of r-hirudin was applicated to the cell-saver-reservoir. during the period of ecc ecarin clotting time and aptt values were taken every ten minutes for monitoring r-hirudin concentration. the postoperative anticoagulation was performed with a constant infusion of r-hirudin starting eight hours after the end of ecc and monitored by aptt. due to mechanical aortic valve the further anticoagulation was performed with phenprocoumon, starting 3 days postop. the therapy with hirudin showed no side-effects. hirudin, threrefore seems to be a suitable anticoagulant in patients with high risk for bleeding complications like this. doses fi:om 2-30 mg/kg gave similar post-op blood loss measurements without s dnseresponse (4-15 oc/kg) (less blood oozing than a historical heparin control but equivalent post-op blood loss; 10 q-3 ec/kg). doses >30 mg/kg showed more intra-op blood loss than the lowe~ doses, but equal post-.op blood loss. the bleeding time test was less elevated than for heparin. platelet counts and hematoerit did not vary except for hemodihition on pump. liver enzymes did not vary significantly pre-op to post. act values showed arg was eliminated (dose-dependently) by 1 hour post-op. dogs were hemodymamieally stable during the peri-operative period, and overall gave predictable responses to arg (as opposed to variable responses to heparin). in a substudy it was demonstrated that hypothermia did not affect the activity of arg, nor did varioos formnlations. this dose finding study strongly suggests that arg may be a safe and effective alternative to heparin for patients undergoing cpb. this is particularly important for the growing population of patients with hit who require cardiac surgery, for which no anticoagulant alternative is presently available. three recent clinical tdals with r-hirudin (timi 9, gusto 2 and hit) have shown that the risk of severe haemorrhagic side effects was strongly associated with high aptt-levels. the large intedndividual variability of the aptt and the lack of a linear dose-effect ratio, however, limits its value for reliable monitoring of the anticoagulant effect of hirudin since even severe overdosage due to impaired renal elimination may not be detected with this assay. we have therefore evaluated the ecadn clotting time (ect) as descdbed by nowak and bucha (thromb. haemost. 1993; 69: 1306) under conditions which allow conclusions on its reliability in the clinical situation.for this, citrated venous blood obtained from healthy volunteers, patients with unstable angina pectoris, and patients treated with marcumar was supplemented with different concentrations of peghirudin. measurements of aptt and ect were made in duplicate. in contrast to the aptt, the ect showed a close, linear relationship with peg-hirudin plasma concentrations in the range of 100 and 10 000 ng/ml. the lineadty of this relationship was not affected by the presence of unfractionated or low molecular weight hepadns in concentrations of up to 10 pg/ml. the ect was not affected by fibdnogen concentrations 60% below normal. a somewhat higher slope but no change in linearity was found in plasma from marcumar-patients with quick-values between 20 and 32%. no significant differences were found between values measured in citrated blood or plasma or using different coagulation timers. the most potent thrombin inhibitor containing a benzamidine moiety is napap (k i = 6 nmol/i). unfortunately, the pharmacokinetic properties (fast elimination by hepatic uptake and biliary excretion, poor enteral absorption) are unsuitable for the use of napap as an oral anticoagulant. the application of choice of a synthetic thrombin inhibitor would be the oral one, therefore, we looked for other lead structures. with the nc~-arylsulfonylated piperazides of 3-amidinophenylalanine we found a new group of derivatives which inhibit thrombin with ki-values in the nanomolar range. the piperazides exert anticoagulant activities with high selectivity, leaving activated protein c and components of the fibdnolytic system unaffected. in rats, the piperazides are rapidly eliminated from the circulation (tl/2 ~ 10 min) upon i.v. administration, too. after oral administration, the systemic bioavailability is low. upon intraduodenal administration of high doses widely varying blood levels were seen, depending on the mode of administration. to cladfy the importance of a possible hepatic first pass effect we studied in more detail the pharmacokinetics of the n~-(2naphthylsulfonyl)-3-amidinophenylalanine n'-acetylpiperazide in rats using hplc-analysis. like other benzamidines the piperazide is excreted via the bile to a high extent. enteral absorption rates of about 20 % are found after blocking the hepatic uptake and biliary excretion. hence, a hepatic first pass effect appears to be the main reason for low systemic bioavailability after orallenteral administration. at the same time, fast elimination from the circulation by hepatic uptake is the main problem for maintaining effective blood levels with benzamidines. therefore, the elucidation of the structural elements influencing the absorption and elimination processes of these types of inhibitors is necessary. the piperazides of 3-amidinophenylalanine bear the possibility to easily introduce a wide variety of substituents on the second nitrogen of the piperazine moiety. a 69-year-old female patient with diabetic nephropathy increasingly developed signs of allergisation combined with dyspnea, erythema, pruritus, and circulatory insufficiency two months after start of heparin-anticoagulated haemodialysis und initial surgical application of a double lumen venous catheter. in addition, growing thrombocytopenia was observed involving a drop in platelets by 50%, compared to the initial values. the haemodialytic efficiency was reduced by massive thrombosis of the dialyzer and subsequent repeated interruption of treatment. at the end of may 1995 heparin antibodies were detected and the hat diagnosis was confirmed. immediately afterwards, haemodialysis treatment was continued, applying hirudin as anticoagulant. using steam-stedlised haemophan dialyzers and 0.14 mg/kg r-hirudin (iketon, italy), the minimum therapeutic blood level of hirudin (0.4 pg/ml whole blood) was reached. this provided therapeutically relevant blood level conditions during a 4.5h haemodialysis. more than 80 regular haemodialyses were run without problems. in all hirudin-anticoagulated haemodialysis treatments the ecarin clotting time was used as the method of choice for bedside blood level and dosage control. after the 34th haemodialysis, the frequency was reduced from 3(4) to 2 haemodialyses a week. accordingly, the hirudin dose was increased to 0.2 mg/kg. the creatinine clearance increased continuously from initially 2.6 to 10.4 ml/min after the 13th week of hirudin-anticoagulated haemodialysis. platelet count and haemodialytic efficiency normalized. we could demonstrate that the regular use of hirudin as anticoagulant along with dialyzers impermeable to hirudin enables very good results in haemodialysis treatment in heparin-associated thrombocytopenia, hirudin is suited for use as anticoagulant in problem patients with hepadn-induced allergy when combined with a drug monitoring method fit for bedside use. capillary electrophoresis methods provide a fast measurement of proteins. thus we developed for pharmacokinetic measurements of r-hirudin and peg-hirudin capillary electrophoresis methods. for the measurement of r-hirudin we used fused silica capillary and a borate buffer. this buffer was used to detect r-hirudin, but could not be used to measure peg-hirudin. for simultaneous measurement we used a neutral capillary to prevent protein absorption to the capillary wall. the buffer was a 20 mm tricine buffer (ph = 8.0 field strength 500 v/cm). it resolved r-hirudin from peg-hirudin at 214 nm using reverse polarity. a linear correlation between the peak area and the concentration was found between 80 pgtml and 10 mg/ml for hirudin (r 2 = 0.99) and between 2,5 and 10 mg/ml for peg-hirudin (r2= 0.99) was found by coinspiking of human plasma and urine with r-hirudin and peghirudin the two proteins were completely resolved. a linear correlation between the peak area and the concentration was found. the method separates r-hirudin from peg-hirudin and may be applied to biological systems to measure the concentration of r-hirudin. triabin is a thrombin inhibitor from the saliva oft. pallidipennis structurally unrelated to any protease inhibitor known and which probably functions by an interaction with the anionbinding exosite of thrombin. we used sf9 insect cells infected with recombinant baculovirus to produce sufficient triabin for a detailed biochemical characterization. the activity of the protein purified from cell lysates was assessed in a fibrinogen clotting assay and was found to be similar to that of the natural protein. a 4-fold prolongation of thrombin-clotting time and aptt was achieved with 22 nm and 600 nm triabin, respectively. a kinetic analysis of the thrombin-catalyzed fibrinopeptide a release from fibrinogen showed that triabin is a tight-binding inhibitor. using the graphical method of dixon, the ki was determined to be 3 pm. introduction: thrombocytopenia is a common adverse effect of heparin therapy, in type ii hit platelet decrease induces severe complications. we here present two special cases of type ii hit. case report i: a 66 year old male patient with dvt of the left leg was treated with therapeutic doses of heparin. from the first to the 12th day of therapy, platelet count decreased from 122000 to 54000/ui. hit was confirmed by hipa-test, heparin therapy was s~opped and treatment with the heparinoid orgaran n was started. during the following days, arterial thromboses in the right a. femoralis occurred. several thrombe~tomies were not successful and although orgaran ~" was stopped because of suspected crossreactivity, amputation of the right leg could not be avoided. during the following days under hirudin-treatment platelet count normalized and no further complications occurred. case report 2: a 74 year old female patients suffering from hip fracture was treated by surgery with tep-operation and received prophylactic heparin treatment. after 6 days, platelet count decreased from initially 170000 to 8000/ul and dvt of the right leg was diagnosed. on the same day, severe bleeding into the left leg was observed and hemoglobin concentration was diminished to 7.8 g% (before surgery 16.0 g%). hit was confirmed by hipa te~t, heparin was stopped and treatment with orgaran started. thrombocyte count normalized and no further complications occured. conclusion: hit type ii can cause severe bleeding as well as thromboembolic complications. because of possible cross-reactivity between heparin and orgaran~,, hirudin should be given in hit patients. currently thrombin time (ti), aptt, activated clotting time (act) or anti ila -activity (alia), measured by a chromogenic substrate test are used to monitor hirudin treatment or prophylaxis. the "13" responds very sensitive to hirudin plasma levels end thus requires variable thrombin concentrations. aptt appears to be more adequate, however, it shows large interindividual variations and does not respond sensitive enough to higher hirudin concentrations. act is a simple whole blood clotting assay, but it is strongly influenced by the blood collection technique. the ecadn clotting time (ect) is a new clotting assay, recently described by nowak and bucha (thromb.haemost 1993, 69,1306) . it measures the clotting time of citrated blood or plasma after prothrombin activation by ecarin, a snake venom of echis carinatus. ec.t shows a linear dependence on different hirudin concentrations over a wide concentration range ( e.g. 0.1-5 pg/ml). in a clinical interaction study healthy volunteers were administered hirudin, asa or both. 15 male volunteers received an i.v. infusion of peg-hirudin (0.02 mg/kg/h) for 24 hours after an initial i.v. bolus of 0.2 mg/kg to compare the sensitivity and reliability of ect with aptt, l-r end act. the act was measured on the hemochron 801, usa, ect on a fibrin timer, aptf using the aptt lyophylized silica reagent by il and alia on an acl (il-milan) with the chromogenic substrate 2238. all tests were performed in duplicate. ect was more sensitive to different hirudin concentrations than aptt, or act. the ect results were better correlated with the alia-activity than ap'l"r and act. the lower detection range for ect is 0.05 pg/ml hirudin. ect is a very sensitive, simple and reliable test for the monitoring of hirudin treatment and prophylaxis. recombinant and synthetic inhibitors of thrombin such as hirudin, efegatran and argatroban are currently in various phases of clinical trials in several surgical and medical indications. the therapeutic effects of these agents are usually monitored by aptt whereas in cardiovascular indications, cefite act and hemotech® act are used. the reliability of both aptt and the act tests in predieting the safety of various thrombin inhibitors has been heavily debated. furthermore, some of these inkibiturs are administered simultaneously to heperinized or coumadinized patients and the obtained aptt and act results do not lady refleot the effects of these agents. fcafin is a snake venom derived fi'om echis carinatus which converts prothrombin into mesothrombin, targeting the arg~3-ile tm bond between the a and b chains of prothrombm. while thrombin inhibitors are capable of inhibiting mesothrombin, atiii/beparin complex does not have any effect. using purified ecarin, nowak and bucha (1993 thromb haemost 69:1306) proposed to assay hirudin. since thrombin inhibitors exhibit similar mechanisms of thrombin inhibition, ecarin clotting time (ect) was evaluated to test its diagnostic efficacy in various experimental and clinical settings. lyphilized eoarin was obtained from knoll ag, ludwigshafen, germany). concentration dependent clotting times for himdin, efegatran and argatroban were obtained in a range of 0-15 p.g/ml. all of the antithrombin agents produced a concentration dependent prolongation of ect and showed va~angpotendies inthe order ofefegatran> argatreban> hirudin, on a gravimetriebasis. on a molar basis, the anticoagulant order of potency was found to be hirudin> afegatran> argatroban. utilizing the ect, the effect of these inhibitors on patients undergoing bolus or infusion therapy, resulting in a concentration level of ~ 10 gt g/rnt, have been measured. unlike such global tests as pt and aptt, patients receiving simultaneous heparin or oral anticeagulants can be monitored for antithrombin specific prolongation ofthe ect. plasma samples from heparinized (aptt 45-90 sec) or coumadinized ('pt 15-25 see) patients, supplemented with argatroban or hiredin did not show any differences m the ect. a medified ecarm act comparable to the celite act has also been developed. initial results demonstrate that this test is not affected by aprotinin, heparin and reduction of the prothrorabin complexes in the inr range of 1.5 -3.0. these results indicate that ecarin based clotting times provide slx~etlie ~lts of circulating levels of thrombin inhibitors, which can provide reliable information to optimize their safe(y and efficacy. r-hirudin is a highly potent and selective inhibitor of the serine proteinase thrombin. after intravenous administration, r-hirudin is eliminated exclusively with the urine. its plasma half-life is very short, 1-2h. peg-hirudin is a derivative produced by coupling polyethylene glycol (peg) to a specially designed recombinant hirudin mutein. peg-coupling results in a considerable prolongation of the plasma half-life of peg-hirudin, compared to r-hirudin. after intravenous administration of r-hirudin into rats, a very small amount of ,,hirudin-like" activity (2 -4% of applied activity) was recovered in the urine. in contrast, after peg-hirudin had been administered, more than 30% of the applied activity could be recovered in rat urine. these results suggest differences in the renal metabolism of peg-hirudin and r-hirudin. within the scope of pharmacokinetie studies in rats we investigated the appearance of biologically active metabolites of peg-hirudin after kidney passage in urine. affinity chromatography on immobilised thrombin was used as a quick and gentle method in searching for biologically active hirudin metabolites in rat urine. but it had to be completed by anion-exchange and/or reversed-phase chromatography to ensure that all active metabolites were detected. the isolated biologically active metabolites were purified by reversed-phase hplc and were biochemieally characterized. in previously reported studies we found a hlrud n derivative consisting of the amino acids 1-50 as the main metabolite in rat urine following intravenous administration of r-hirudin. this metabolite was not detected in the urine after administration of peg-hirudin, confirming the suggestion of a different renal metabolism. carrageenans are high molecular weight sulfated polygalactans of plant origin (derived from red algae) with anticoagulant properties. in previous studies we investigated the anticoagulant activity of lambda-carrageenan, a highly sulfated type of carrageonans. unlike heparin, lambda-earrageenan exerts its anticoagulant activity primarily through direct inhibition of the serine proteinase thrombin. only a part of its antithrombin activity is indirectly mediated through antithrombin iii. to investigate relations between molecular weight and biological activities, tambda-carrageenan has been hydrolysed and fractionated. the molecular weight has been determined with the aid of size exclusion hplc using dextrans as molecular weight standards. the degree of sulfation has been determined by anion-exchange hplc. we have obtained low molecular weight lambdaearrageenans ranging from 10,000 dalton to 100,000 dalton with degrees of sulfation of 13 -17% and 33 -38%. the anticoagulant and antithrombin activity of low molecular weight carrageenans have been determined using coagulation assays and purified systems, and we have compared their activities with those of heparin and other sulfated polysaecharides. further, we have investigated the ability of lambda-carrageenan and its low molecular weight derivatives to inhibit the activity of human blood phagocytes. the activity has been determined by measuring the cellular chemiluminescence in a mieroplate himinometer using a himinol-dependent assay and zymosan as phagocytosis activating agent. we have used an assay in human whole blood and assays with isolated human mononuclear and polymorphnuclear cells. the anticoagulant activity and also the ability of carrageenans to inhibit the activity of human macrophages decrease with decreasing molecular weight and decreasing degree of sulfation. the natural ocouring, yellow pigment curcumin is the major component of tumeric and is commonly used as a spice and food-coloring agent. since curcumin has been reported to have anti-tumorpromoting, antithrombotic and anti-inflammatory properties, we studied, whether curcumin acts on the transcription factors ap-l(jun/fos) and nf-~:b in cultured endothelial cells (ec). when ec were cultured in the presence of curcumin, electrophoretic mobility shift assays (emsa) demonstrated, that binding of endogenous ap-1 to its dna recognition motif was suppressed. inhibition was due to direct interactions of curcumin with the dna-binding motif for ap-i. enhanced ap-1 binding, induced after tnfa stimulation of ec, was decreased in cells pretreated with curcumin. this resulted in reduced transcription and expression of tissue factor, known to be controlled by ap-f and nf-~b. nuclear run on assays proofed, that curcumin directly reduced the tnfa mediated transcription of genes, regulated by ap-1, as tf, endothelin-1 and c-jun. thus, curcumin did not only suppress apl(jun/fos)-binding, but also inhibited tnfa induced jun transcription, transient transfections with tissue factor promotor plasmids confirmed, that inhibition by curcumin was dependent on intact ap-i sites. beside its effect on ap-l-binding, curcumin reduced the radical dependent activation of nf-kb due to its antioxidant properties, however, this inhibition was indirect and less prominent. the relevance of the in vitro data was confirmed in vivo in mice bearing meth-a-sarcoma. when mice received curcumin before tnfa was injected, tumors showed reduced ap-1 activation. simultanously fibrin/fibrinogen deposition decreased, most probably due to reduced tissue factor expression. thus, curcumin inhibits ap-t activation and expression of endothelial genes controlled by ap-t in vitro and in vivo. (jung, 1991) . additionally, haemorhenlogical parameters (plasma viscosity, erythrocyte aggregation) were measured. in all patients aptt, bleeding time, platelet adhesiveness, von wiuebrand f~ctor and factor viii concentration and activity were determined. the patients with von willebrand disease showed characteristic morphological changes of capillary geometry. tortuosity of nailfold capillaries was markedly increased as well as the diameter of capillariez on the arterial and venous side. plasma viscosity was significantly low. multiple parameter analysis concerning to galen and gambino (1983) and using the parameters ,,plasma viscosity below 1.25 mpas", ,,torquation index higher than 5", ,,erythrocyte column diameter bigger than 16,9 gin" showed a positive predictive value of 100 %. capillary diameter and capillary tortuosity have a positive predictive value of 91,2 %. additionally, a reduction of the vasomotorie reserve and/or a decreased erythrocyte velocity in the capillaries below the reference range was found in most of the yon willebrand patients. it was quite remarkable, that 16 of 40 of the yon willebrand patients showed significant capillary bleedings. these findings confirm some former observations (e.g. o'brian 1950) and preliminary reports of our group (koscielny 1994). polymerase chain reaction (pcr)-based quantitation of mrna transcripts is an important tool in the investigation of the underlying molecular defects in inherited platelet disorders, such as the bernard-soulier syndrome. however, for the exact quantitation of mrna a number of methological requirements has to be met. first, a standard (s) mrna must be synthesized which is able to undergo the same processing as the target wild type (wt) mrna. secondly, the quantitation step following the pcr must differentially recognize standard and target dna, and thirdly, the assay must be precise with respect to both inter-and intraassay variability. in order to satisfy these requirements we constructed a s-gpib mrna which is identical to the wt-gpib mrna except a 13 bp long primer recognition site at its 5" end allowing differentiation between the pcr amplified wt-or s-gpib cdna through incorporation of a fluorescein or biotin labelled 5" primer. both standard and w[ gpib mrna showed identical amplification kinetics in the pcr reaction. the amplified dna was quantified using an dna binding assay. in this assay binding of amplified dna to gcn4 fusionprotein-coated microtiterplates is measured. since the gcn4 binding motif is incorporated into the wt-and s-gpib cdna through an identical 3" primer, competition between s-and wt-cdna during amplification has been analyzed. at a given concentration of 250 nm of gcn4. primer no competition between the sdna and wt-dna for the primer was observed during 25 pcr cycles. the sensitivity limit of the assay performed in this way was 250 amol wt-gpib~, dna, and intraassay variability reached from 1.66% to 6.72% calculated for 100 fmol and 5 fmol dna, respectively. to sum up, combination of rt-pcr with the amplified dna binding assay and usage of an internal standard mrna allows sensitive and accurate quantitation of gpiba mrna in human platelets. since upa and thrombin are main conrtibutors to the process of proliferation and migration of vascular smooth muscle cells (vsmc), which is part of the pathogenesis of atherosclerosis. we are currently assessing the role of spatial expression of upa and thrombin receptor (tr) on cells with human carotid artery plaques (n=10). we have used a double immunolabeling approach, combining anti-upa and anfi-tr antibodies. to identify the different cell types, we used the following antibodies: anti a-smooth muscle actin (a-sma) for smooth muscle cells, ulex europaeus agglutinin i (uea i) for endothelial cells, inflammation cell cocktail (cd68+cd45) for monocyte/macrophage and lymphocytes and an anti-proliferation cell nuclear antigen antibody (pcna) to stain proliferating cells. in the carotid atherosclerotic plaques, upa immunostaining was distributed focally, preferentially in the fibrous cap and some cells of the foam cell rich region (fcrr). it was present in distinct patterns: cytoplasmic staining. tr staining was distributed similar to upa staining. with double staining combining anti upa antibodies with anti-tr antibodies, cellular co-localisation of both upa and tr was demonstrated. these cells were identified as smooth muscle cells by -sma. inflammatory cells were mainly localized within the fcrr, they only stained for upa. in conclusion: our data demonstrates that upa and tr are coexpressed in vsmcs in human carotid artery atherosclerotic plaque tissue. we therefore conclude, that the mitogenic activity of upa is associated with the thrombin signalling pathway. in the proficiency test of the ,,deutsche gesellschaft flir klinische chemie" (dgkc) 1/95, 5 lyophilised plasma samples (immuno ag) were sent to participants: a normal plasma and 4 plasmas from persons under oral anticoagulation (oac-plasmas. inr 1.9 to 3.7). the participants (n=552) returned the pt times obtained and in most cases (n=355) also the isi value for the thromboplastin used (isi of pack insert). the inr was calculated using the pt of normal plasma and the isi of pack insert (method i). two additional methods for inr calculation were compared with method i. according to the concept of calibrated plasmas (houbouyan et al., t993), a calibration curve was constructed using the normal plasma and the 30ac-plasmas. the inr calculated using the pt •fn•rma¿ plasma and the laboratory-specific isi value given as 1/slope of the calibration curve (method ii) or was read off directly (method hi). for inr values, calculated by the 3 methods from the participants data (n=355), outlier elimination (2sd, iterative) was performed. the inr mean values for all 3 calculation models remain in a narrow range. using calibrated plasmas (method 1i and m), less outlier were eliminated and cv's obtained were smaller than using the conventional procedure ( i ). obviously, the inr inherited problems, such as accurate isi value, pt value of normal plasma and instrument/laboratory influences on isi, can be reduced using calibrated oac-plasmas. practical approach and educational considera-tions of home prothrombin time estimation a. bernardo, a. bernardo, c. halhuber herz-kreislauf-klinik, bad berleburg, germany specific training is necessary for the patient to achieve reliable and reproducible results in prolhrombin time measurement. the training scheme is based in many respects on experience with similar training courses for home control and management of diabetes and asthma. the education program is divided into a theoretical and a practical part. the theory part has group sessions of twenty patients of a time. the practical course is reduced to a maximum of five patients. the sessions are conducted by a medical doctor and by specialized medicaf/technical assistants. on average eight hours of theoretical education and two hours of practical training are sufficient. the contents of the theoretical lessons are: • need for anticoagulation after heart valve replacement, • potential interaction between anticoagulants and other medication, • accurate recording of the measured prothrombin time results, • techniques of prospective determination of the necessary amount of anticoagulant, • calculation of the individual doses, • potential pitfalls and mistakes, • corrections in case of over-and under-dosage, • early recognition of thromboembelic and/or bleeding complications. an alternative is a full-day intensive course which can be held during the weekend. our recently reported (1) observation that oral anticoagulant treatment causes an increase of heparin cofactor ii (hc ii) activity in plasma is now confirmed by a more extensive study. in 43 thrombophilic patients who were on vitamin k antagonist therapy (marcumar r) we found a median hc ii level of 142 % as compared to 119 % for 72 thrombophilic patients without any therapy (p < 0.002" ) and 104 % for 59 healthy controls (p < 0.001" ). moreover we observed that the increase of hc ii level was significantly correlated with increasing inr-values (r = 0.63, p < 0.001). follow-up observations on some patients showed, however, clear differences in the levels of hc ii activity after onset of vitamin k antagonist therapy. thus, some patients responded rapidly with a significant increase in activity ("strong responders") while others showed only slight changes ("weak responders"). in conclusion, the determination of hc ii activity may result in an improved estimation of the risk of bleeding, especially in high intensity treated patients (inr > 3.5). after intracoronary stent implantation an aggressive oral anticoagulation (oac) therapy is mandatory. to find out whether coagulation activation occurs after coronary stent implantation during high dose oac therapy markers of plasmatic coagulation and d-dimer were measured. patients 5 male patients (average age 57 years) were examined. blood samples were taken before and right after stent implantation and during the following week. patients got 30 mg phenprocoumon during the first three days and additionally heparin and acetylsalicylic acid (asa) were given. methods ptz, aptt, tz, protein c, tat-complexes, fi+2 and d-dimer were measured. results d-dimer levels increased steadily between day 0 and day 7. tatcomplexes showed a slight increase from day 0 (2.4 bg/i) to day 3 (15.3 ~tg/i). on day 7 tat levels were down again (2.0 p,g/l). fl+2 (day 0:1.0 ng/ml) also showed a slight increase on day 3 (1.3 ng/ml). protein c decreased steadily from day 0 (108%) to day 7 (15%). conclusion during the initial phase of oac therapy a coagulation activation is reported but no significant elevation of tat or fl+2 was found. this result shows that additional heparin and asa therapy was sufficient to avoid systemic coagulation activation. the increase of d-direct should be interpreted as a si~=m of local fibrinolytic reaction due to stent implantation. three methods for the determination of prothrombin time from capillary blood in patients under oral anticoagulation have been investigated. two methods were run on coaguchek® monitors (boehringer mannheim) from capillary whole blood. after fingerpuncture the first drop of blood was applied to the well of a coaguchek® test strip directly from the finger-tip, whereas the second drop was sucked into a non-anticoagulated plastic capillary (hirschmann) and immediately applied to the test strip -and vice versa to eliminate any influence of first and second drop of blood. the third method was hepato quick (boehringer mannheim) which was determined out of citrated capillary blood from an earlappuncture. 66 specimen of patients under oral anticoagulation were investigated. the method comparisons between each of the coaguchek® methods and the laboratory method show good results and the correlation between the coaguchek® methods is excellent. mean differences to the lab methods are -0.1 inr in both cases. no mean deviation was detectable between the coaguchek® methods. scattering of coaguchek® versus hepato quick was +/-0.6 inr in the range 1 to 4 inr except for three outliers and one patient with fluctuating results in the lab method which could not be resolved. introduction: haemorrhagic coumarin skin necrosis is a severe complication during initial phase of oral anticoagulant therapy. histological examination shows thrombotic occlusion of small vessels, but little is known concerning the pathophysiologic background of the bleeding component. recently, we described protein z deficiency in patients with bleeding complications of otherwise unknown origin. thus, we were prompted to measure protein z in patients with coumarin skin necrosis. patients: 4 patients (i man, 4 women; age: 35±10 years) suffering from haemorrhagic coumarin skin necrosis were examined. all patients had normal liver protein synthesis function, none was under oral anticoagulant treatment during this study. method: protein z antigen test, diagnostika stago, france. results: 4 out of the 5 patients examined had diminished protein z levels ( 700, 820, 1080, 1700 ug/l) in comparison to normals (2900 ug/l). in one of our patients, protein z was normal (3020 ug/l). conclusion: low protein z levels are additional risk factors for haemorrhagic coumarin skin necrosis. oral anticoagulant therapy is the treatment of choice in patients with need for long-term anticoagulation. since oral anticoagulants interfere with the function of vitamin k, it is not clear whether stable oral anticoagulation can be achieved in patients with need for continous substitution of fat-soluble vitamins including vitamin k. we report about a 59-year-old man who had experienced progressive hypertrophic obstructive cardiomyopathy over the preceeding 21 years. atrial fibrillation has been first diagnosed 18 years ago. latter on, recurrent ischemic attacks and embolism of the right arteria iliaca occurred. in 1993 the patient received extirpation of the ileum and subtotal amputation of the jejunum because of mesenteric infarction. the resulting short bowel syndrome requires continous substitution of fat-soluble vitamins. since vitamin k free preparations of fat-soluble vitamins for parenteral use are not available, prophylaxis of thrombosis has been performed with unfractionated hepadn. as a consequence of the longterm treatment with hepadn the patient developed severe osteoporosis. therefore, the decission 1:o discontinuate heparin therapy and initiate oral anticoagulation has been made. because of its shorter halflife warfarin (coumadin) was used instead of dicoumarol. over a 4 weeks lasting induction phase inr values were controlled daily. a dosage regime starting with '10 mg warfarin at the day of vitamin application (day 1) followed by 3.75 mg on day 2 and 1.25 mg on days 3, 5, and 6, respectively, was found to be optimal to maintain inr values within the target range (inr: 2.0-3.0). in order to minimize the risk of hemorrhage the vitamin administration was changed to the subcutaneous route. during an observation period of 6 months neither any bleeding or thrombotic complications nor a vitamin deficiency occurred. these data indicate that stable oral anticoagulation can be achieved despite extreme variation of vitamin k plasma levels. portable monitors for home monitoring of inr are well established for adults on oral anticoagulants. patient's compliance is improved as well as long term outcome. experience concerning accuracy of the procedure in children is limited. 32 inr determinations were performed in parallel from venous and capillaryblood samples of an infant on phenprocoumon, starting at the age of 4 months. the coaguchek® monitor from boehringer mannheim was used. choosing an arbitrary range of agreement of ,qnr 0.5 for both determinations, 81% of the measurements were within the defined range. 5/6 outliers were due to low inr resulting from difficulties in capillary blood sampling. the degree of agreement increased when the procedure was performed at least once a week. in conclusion: inr determination with a portable monitor may be helpful in home monitoring oral an.ticoagulant therapy in young children. a dose adjustment should be done only on the base of inr determination of venous blood -if it is considered the gold standard -to avoid over-anticoagulation. a stable anticoagulation is one of the most difficult tasks in attending patients with heart-valve-prosthesis. if prothrombin times are out of the therapeutic range, the risk of bleeding or thromboembolism increases disproportionately. for this reason any improvement in anticoagulant control and/or management can have far reaching consequences in decreasing complications, in extending longevity and in improving quality of life. for the first time a clinical trial was started in 1986 and continues until today at the cardiac rehabilitation center bad berleburg, germany with patients mainly after heart valve replacement. the patients were trained to measure their own prothrombin time and to adjust their own dosage of the oral anticoagulant. within six years 600 patients were trained: 216 patients could be followed up with regard to their selfdetermined prothrombin times. the results were within the therapeutic range in 83.1% of the measurements (n=14.812) taken by the patients themselves. on average, the patients who determine their prothrombin time themselves did so at a weekly interval. neither major bleeding nor thromboembolic complications could be observed in the 205 patient-years of home prothrombin estimation. it is to be hoped that the usual rate of complications can be reduced when patients determine their prothrombin time themselves at a close interval, resulting in more constant values in the therapeutic range and slight corrections of the anticoagulant dose. home prothrombin estimation promises better quality of life and has a considerable potential to achieve this goal. circulating plasma thrombomodulin (tm) is a novel endothelial cell marker, which may reflect endothelial injury. tm acts as thrombin receptor which neutralises the fibrin-forming effect of thrombin, and also accelerates the formation of the anticoagulant protein c/s pathway. tm therefore belongs to the anticoagulant defence system against thrombosis. increased tm levels have been described in various diseases such as ards, thromboemboembolic diseases, ttp, diabetes, le and cml reflecting alterations of the vascular system at the endothelial level. to find out to what extent cardiac catheterisation imtates vascular endothelium, tm concentrations (stago, asnieres, france: x 10 3 iu/ml) were investigated prospectively in 58 infants and children (three days -16 years). blood samples were drawn before the intervention, immediately at the end and 24 h later, snap frozen (-70 °c) and investigated serially in dublicate six weeks -3 months later. the results (median and range values) are shown in the enhanced tm concentrations immedately after the operative intervention, followed by normalisation within 24 h, indicates that cardiac catheterisation in pediatric patients rather leads to a short lasting irritation of the vascalar endothelium than to severe irreversible endothelial damage. recently in an al=wl" based method dahlb~ick et al described in vitro resistance to the anticoagulant effect of activated protein c (apc) in thrombophilic adult patients. apcr is in the majority of cases associated with the arg 506 gin point mutation in the factor v gene. concerning the special properties of the neonatal hemostatic system (low vitamin k dependent coagulation factors, physiological prolongation of the pt and aptf) we adjusted this ap'it based method (chromogenix, m~,lndal, sweden) to neonatal requirements: apcr was measured in 120 healthy infants according to dahlb~ck. the results were expressed as apc-ratios: clotting time obtained in a 1:1, 1:5 and 1:11 dilution with factor v deficient plasma (instrumentation laboratory munich. germany) using the apc/caci2solution divided by clotting time obtained with cac12 in the same i:1, 1:5 and 1:11 dilution. in addition, plasma of 24 neonates with septicaemia were investigated and data of 18 infants aged birth -three months with arg 506 gin +/-were shown. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mnl i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. results are shown in the 1.6(1.4-1.95) neonates and infants were considered to be apcr when the aptt ratio was < or = 2. concerning the special properties of the neonatal hemostatic system, our data show concordance with the pcr method in neonates and infants only, when the aptt based method was performed in the i: 11 plasma dilution. case report: we report on an 8-year old boy with severe hemophilia b and frequent screaming at night. eeg showed spike wave activity, starting from the temporal lobe, but generalizing within seconds. complex partial seizures were diagnosed and therapy with carbamazepine was initiated. as no improvement was seen nmr was performed. this revealed lesions within the right frontal cortex. higher doses of carbamazepine were not succcssfull as was therapy with phenytoin and pfimidone respectevely. the patient is now treated with carbamazepine and valproate. he still suffers from one short seizure per day. because of his seizures we started prophylactic replacement therapy with 600 i.e. factor ix twice per week. discussion: in 1992 wilson et al. first detected brain abnormalities in 25 of 124 children and adolescents with hemophilia a or b who were negative for immunodeficieney virus (1). the most common findings (14/25 patients) were small, focal, nonhemorrhagic white matter lesions of high signal intensity on t2weighed images. similar lesions have been reported in children with sickle cell cerebral infarction (2) . only three of these 14 patients had seizures, all of those having a documented history of intracranial hemorrhage. our patient has similar lesions as those described by wilson et al. but no history of intracranial hemorrhage is documented. even if tuberous sclerosis might be a differential diagnosis, we think that the abnormalities are related to hemophili a or its treatment, because the patient has no further signs of this disorder. conclusions: 1. in patients with hemophilia and seizures nmr might be useful as a high sensitive method for the detection of gray and white matter changes. 2. further studies should be initiated to determine the prevalence of pathological conditions in the brain of hemophiliac patients. disseminated intravascular coagulation (dic) is a rare, but foudroyant disease occuring in gram-negative sepsis like meningococcal septicemia. despite the avallibility of potent antibiotics, mortality in mertingococcal disease remains high ( about 10 % ), rising to 40 % in patients presenting with severe shock and consecutive dic. as the clinical course and the severity of manifestations of systemic meningococcal infections varies there is a need for early diagnosis of the infection and stage of coagulopathy in order to reduce the high mortality rate. few and rapidly available parameters are needed to classify the wide spectrum of clinical and laboratory findings in patients with dic. the parameters include partial thmmboplastin time, pmthmmbin time, plasma levels of fibrinogen, fibrin monomers and dimers, fibrin degradation products and the thrombocyte count. monitoring the course of hemostaseologicai findings in 26 pediatric patients with systemic meningococeal infections we observed a change of coagulation parameters as early as in the first stages of the infection: a prolongation of partial thromboplastin time to an average of 69. 1 sec (range 22 -150 sec, norreal 30 -45 sec), a decrease of prothrombin time to 45.7 % (range 13 -71 %, normal 70 -100 %) and of antithrombin iii to an average level of 16. 8 u/ml (normal 20 -29 u/ml ) was found 1 to 4 (-6) hours after admission. the consecutive development of hemostaseological parameters mentioned above permitted to define the stage of coagulopathy and thus to induce a stage related therapy. primary treatment consisted in control of shock by liquid substitution, compensation of metabolic acidosis, correction of clotting disorders ( at iii and heparin in stage of pre-dic ; at iii and fresh frozen plasma in case of advanced dic ) and treatment with g-lactam antibiotics ( e. g. cefotaxime or ceftriaxone ). an early assessment of the coagulation disorders in meningococcal disease can be based on few coagulation parameters, thus an appropriate treatment may be arranged to prevenl the patient from a fatal outcome of meningococcai septicemia and protect him from the development of a waterhouse-friderichsen-syndrome. this study was designed to prospectivdy evaluate coagulation and flbrinolyfie activation in 60 children (neonate -16 years) during cardiac catheterisation with low dose flush heparin (10 iu/ml saline). aptt (instrumentation laboratory: see), anti xa activity (xa; chromogenix: iu/ml), prothrombin fragment ft.2 (f1.2; behring werkc marburg: nmol/l) and d -dimer formation (d-d; bnhring werke/vhrburg: ug/l) were investigated before (t1), at the end (t2) and 24 h after cardiac catheterisation (t3). in addition, to evaluate the influence of inherited thrombophilia in all patients resistance to activated protein c (apcr), protein c, protein s and antithrombin were investigated. during catheterisation median (range) hepadn was administered in a total dose of 60 (17-206) iu/kg bw. in addition infants < 6 months of age (arterial catheterisatiun only) or patients with known thrombophilia received 300 -400 iu/kg hepafin for fmther 24 hours. the results (median and range) are shown in the ft.2 was sigificanfly elevated above the pediatric boundary immediately after the intervcation and nearly reached baseline values 24 h later. in contrast no cfinically relevant fibrinolytic activation was seen: d -dimer formation increased within the pediatric boundary immediately after the catheter and returned to basdine levels 24 h later. three children showed resitance to apc. tn one child stroke occurred before. not knowing the result of apcr in the remaining two patients only one neonate received further prophylactic heparin. the third neonate without heparin prophylaxis suffered from venous occlusion within two days after the intervenfon~ in addition, no protein c, protein s or antithrombin deficiencies were found. although administration of low dose flush heparinisation during cardiac cathetefisation could not prevent short -term coagulation activation, no thrombotic events occurred in children without inherited thrombophilia. if fnrther prophylactic hepariuisation in children with a~r, protein c, protein s or antithrombin deficiencies may prevent vascular occlusion requires a more intensive study. a.sandvoss, w.eberl, m.b0rchert introduction: capillary leakage, edema and hypovolemia are common complications in preterm infants especially if birth weigth is below 1.500 g. septicemia, asphyxia and immaturity seem to be most important risk factors. to determine the influence of c 1-esterase inhibitor (cilna) in preventing contact phase and complement activation we investigated c11na concentrations in normal and symptomatic preterm infants. methods: activity of cilna were measured by chromogenic substrate method (behringwerke), cilna concentration with radial immunodiffusion (behringwerke,germany). results: cllna-activity in asymptomatic preterm infants (n= 14) was 65+/-15% of normal at birth. healthy newborns showed activities of 80+/-20%. cilna reached normal adult values 2-4 days after birth. preterm infants with respiratory distress syndrome(n = 14) showed lower activity on day 2-5, patients with additional septicemia (n=15) had decreasing c1 ina-activities in the first three days of life. individual course of cllna-activity and thrombocyte count correlated in the group with irds with and without septicemia. in children with capillary leakage onset of diuresis went parallel with raising cllna-activity. markers of contact phase (f xlla) and complement activation (c 5al were investigated in single cases and evidence for involvement of both systems was found. conclusion: contact activation and complement system play an important role in capillary leakage in preterm infants. cilna regulates both systems. activity of cilna correlates with clinical course, substitution therapy is possible and may improve outcome of these critical ill patients. antiphospholipid antibodies (apa) interfere with hemostasis probably by inhibition of protein c or prothrombinase complex. thereby, apa might lead to thrombosis or increased bleeding. however, incidence and clinical importance of apa has not yet been investigated in children. therefore, we assayed plasma samples of 220 children, aged 0,1 to 19 years (mean 7 years) by elisa detecting igg-and lgm-antibodies directed against eardiolipin, phosphatidyl serine and phosphatidic acid. in patients with increased bleeding, thrombophilia or prolonged clotting tests a detailed coagulation analysis was performed. according to their diagnosis children were devided into 5 groups: i. autoimmune diseases, ii. infections, iii. metabolic diseases, iv. other diseases, v. healthy children. results: apa were found in 69/220 patients. in the respective groups we demonstrated apa in the following proportions: 1. lgg-isotype: activitiy of c1 esterase inhibitor (c11na) is reduced in preterm infants especially if birth weigth is below 1.500 g and respiratory distress syndrome and/or septicemia is present. capillary leakage with generalized edema, hypovolemia and hypotension is resulting in imbalance between inhibition and activation of contact phase and complement system. iln four patients we investigated seven courses of substitution ;with commercial c1 esterase inhibitor preparation (berinertr,behringwerke), case reports are given. all patients had clinical symptoms of capillary leakage, all had septicemia accompanied by either respiratory distress, disiseminated intravascular coagulation or mutiple organ failure. jefficiacy of substitution therapy is dose related, supranormal iactivities of cilna are necessary, reflecting raised consumption of inhibitor in ongoing disease. clinical effects on diuresis, catecholamine need and especially on thrombocyte counts are demonstrated. or arterial thromboembolic event in children e. lenz, c. heller, w. schr6ter*, w. kreuz johann w. goethe-universit~itskinderklinlk, frankfurt a. main, germany * georg-augast-universit/itskinderidinik, g/3ttingen, germany venous thrombosis as well as arterial thrombo-occlusive events are rarely observed in childhood, but can lead to life-threatening situations and longterm sequelae in these patients. after the initial stage of treatment (thrembolysis or thrombectomy) the pediatrician has to decide how to efficiently prevent re-thrombosis in the individual patient. anticoagulation after venous thrombosis is generauy recommended for 6 months after the event; if an underlying thrombophilic condition has been detected in the patient anticoagulation has to be considered lifelong. when evaluating antithrombotic therapies for children it is of importance to consider whether the anticoagulatory effect is mainly necessary in the venous or arterial vessel system. the hemorrhagic risk and side effects of the different anticoagulatory preparations have to be taken into account, especially when treating small children. only limited experiences exist concerning the suitability of the preparations for long-term anticoagulation in children and general recommendations on the ideal dosage in pediatric patients are still missing. we want to disscuss different types of anticoagulants (such as coumarins, unfractionated heparin, low molecular weight heparin (lmwh) and inhibitors of platelet aggregation) their mode of action, their suitability for pediatric patients and their side effects and relevance of these side effects especially in children. from the experience in our own pediatric patients, we would like to report on the indications, which can be given to administer these different preparations, the dosage regimen we recommend and the laboratory tests to monitor save and efficient re-occlusion prophylaxis in our patients. in this context we would like to present our data on 8 patients with either thrombosis or arterial infarction due to a thrombophilic condition, who had all contraindicatioas to oral anticoagulation by coumarins. because prophylaxis for re-thrombosis was mandatory in these patients, lmwh was given for long-term anticoagulation in a dally subcutaneous dosage of 100-150 anti-xa u/kgbw. monitoring was done by anti-xa-test (0,4-0,8 anti-xa u/ml). under this regimen none of the patients developed re-thrombosis or bleeding complications. alopecia was seen as a side-effect. this study was designed to prospectively evaluate coagulation and fihrinolytic activation after cardiopulmonary bypass with aprotinin (2x17000 u/kg bw) in 42 infants and children aged 0.1 -15 years, and to correlate these findings to the clinical outcome. prothrombin fragment f 1.2 (f1.2; behring werke marburg: nmol/l), antithrombin-serinesterase -complex (atm; stago: ng/ml), d -dimer formation (d-d; behring werke marburg: ug/l), tissue-type-plasminogen activator ag (t-pa; chromogenix: ng/ml), plasminogen activator inhibitor 1 antigen (pai; chromogenix: ng/ml) and cl-inhibitor (c1; behring werke marburg: x 10-3 g/l) were investigated before the operation (t1), at the end of the operation (t2), and on postoperative days 1 (t3), 4-6 (t4) and 7-9 (t5), respectively. the results are shown in the table (median and median absolut deviation): t1 t2 t3 t4 "1"5 nv fi.2 0.9 +/-0.5 1.7 +/-0.9 1.4+/-1 1.8+/-0.8 1.6+/-0. the platelet (pl) function defect induced by thrombolytic agents has been attributed either to the degradation of pl surface receptors or to the anti-aggregatory effect of fgdps. in contrast to other plasminogen activators scu-pa is intimately inked with pl: they can rapidly incorporate exogenous seu-pa, release it upon stimulation and bind the proenzyme. recently we have reported that exposure of prp to recombinant scu-pa (2.5-t00 um) in timed interval 1-30 min resulted in dose-dependent inhibition of pl aggregation. timecourse changes of the process were followed by the biexpotential kinetics: a rapid initial inhibition during the first 3-5 rain with the moderate suppression of pl aggregation in the 30 min period. when tcu-pa (25-100 nm) was exposed to prp in the same conditions dose-and time-dependent inhibition of pl aggregation was also observed. since the effect was obtained no earlier than t0 min after exposure of tcu-pa to prp, and the threshold dose was higher. comparable inhibition of pl aggregation was obtained with 25nm of scu-pa versus 100nm of tcu-pa and the llbrinogen depletion by the end of the 30 min period was 2% and 30% respectively. it's likely that tcu-pa and its precursor have different mechanisms of action on the pl aggregatory function. in a recent study we have shown that recombinant rscu-pa inhibits platelet (pl) aggregation in prp. to exclude the possible influence of rscu-pa/plasma interfere on this process the aggregation of washed pls was under the investigation. pls were washed according to modified mustard's method, suspended in buffer and adjusted to 250,109/1. the resuspended pls were exposed to 5-100 nm of rscu-pa for 30 min at 370(;. at time points 3, 5, 15 and 30 min the aggregation with 0.6 iu/ml of thrombin was measured. it was found that the exposure of pls to rscu-pa (20-100 nm) for 3 man resulted in marked inhibition of their aggregation. since after 15-30 man of incubation with 20-50 nm of rscu-pa the inhibitory effect on pl aggregation became less pronounce or even disappeared. when 5 nm of rseu-pa was used the inhibition of pl aggregation became significant only by 15 rain of exposure period and didn't change for 30 man of investigation. the observed results may be cormeeted with uptake of rscu-pa by pls from surrounding buffer as well as with individual variations of pl response to the same concentration of rscu-pa. loss of glycosylation may result in a reduced platelet (p) survival and perhaps altered function. we analyzed the structural and functional effect of specific deglycosylation (combinations of n/o-glycosidase and neuraminidase treatment) of p and isolated p gpib. washed and formaldehyde-fixed p were digested as follows: 1) with neuraminidase (0.125u/ml) + o-glycosidase (3.1mu/ml) + n-glycosidase (1.25u/ml), 2) with neuraminidase alone (0.2u/ml), 3) with n-glycosidase (2u/rnl) and 4) with neuraminldase (0.2u/ml) + o-glycosidase (5mu/ml). all reactions were performed in the presence of protease inhibitors (pmsf, leupeptin, sbti), after washing x2 the p and identically treated controls were analyzed by flowcytometry with the antibodies 6di (mab: a-gpib), 7i-l2 0vlab: a-gpiiia), and the lectins wheat germ agglutinatinln (wga, for neunac) and peanut agglutinin (pna, for [3dgal(1-3)-galnac) which confirmed effective and specific deglycosylation by the respective enzymes (but gave only minor differences with 6di and 7h2). the botrocetin (13) and ristocetin (r)induced agglutinations showed arer treatment 1) (all enzymes) a full inhibition of r-induced agglutination but only a mildly reduced b-induced agglutination (70% of normal). treatment 2 and 3 (neuraminidase alone, and n-glycosidase alone) affected both agglutinations only mildly (70-80% of normal).treatrnent 4) (o-deglycosylation) however showed a major inhibition of r-agglutination down to 30%, while b-agglutination interestingly was almost fully retained. the results of the rotary shadowing electron microscopy of purified gpib suggested a collapse of the normally stretched, glycosylated, gplb, not only after the treatment with all three glycosidases, but also .after o-deglycosylation alone. we conclude that oglycosylation is most important for ristocetin-induced platelet-von willebrand factor-interaction and responsible for the typical stretched shape. the phenomenon of in vitro platelet aggregation and consequent pseudothrombocytopenia (ptcp) in the presence of calciumchelatization by na-edta and sodium-citrate was studied in blood samples of a patient. initial platelet counts electronically measured were 20000/ul blood anticoagulated with na-edta and sodium-citrate. normal platelet counts were found in heparin-anticoagulated blood and in capillary blood. immunoglobulines of the igg and igm subclass were identified in the patients plasma. by incubation of the patient's serum with platelets of healthy individuals, platelet-clumping occurred in the presence of na-edta and sodium-citrate but not in the presence of heparin. the platelet membrane glycoproteins (gp) hb/llia, ix and iiia/vnr g-chain were involved in the antigen antibody reaction as demonstrated by specific antibodies and flow-cytometry. on platelet surface permanent calcium-exchange and -replacement is dependent on external calcium concentration. calcium depletion induced by calcium chelators as na-edta and sodium-citrate might conformationally change platelet surfaces and induce formation of neoantigens. the decrease of gp llb/illa platelet surface antigen to 10% (normal >75%) indicated the important role of the gp iib/iiia receptor at ptcp. the saliva of tdatoma pallidipennis, a triatomine bug, was found to contain a protein called "pallidipin", that specifically inhibits collageninduced platelet aggregation but not adhesion or shape change. to investigate the mechanism of action of recombinant pallidipin the influence on platelet fibdnogen binding after activation by collagen type i in different concentrations was measured by flow cytometry. the same concentrations of pallidipin that inhibited the couagen-induced platelet aggregation completely did not cause any inhibitory effect on fibdnogen-binding in the prp from the same donor measured contemporaryly. collagen type i-induced platelet aggregation of cd36-deficient platelets from two different unrelated blood donors was inhibited by the same concentration of pallidipin that inhibited aggregation of control platelets. there was no inhibition of collagen-induced fibdnogen-binding in the cd36-deficient platelets as well. pallidipin did not cause inhibition of collagen-induced membrane expression of cd62 and cd63 of control and cd36-deflcient platetets as measured by flow cytometry. however eadier studies had shown an inhibition of collagen-induced atp and {3tg secretion by pallidipin. therefore we compared the effect of pallidipin in unstirred and stirred prp samples. while pallidipin had no effect in unstirred samples it showed strong inhibition of ptg secretion in stirred samples. we therefore conclude that pallidipin does not act on collagen-induced aggregation through cd36 and that the inhibition is a post fibdnogenbinding event. pallidipin does not influence the first steps in secretion, which are independent from cytoskeleton and platelet-platelet contact, but inhibits the following steps. 17-hydroxy-wortmannin does not inhibit the transport of 1nm-gold labelled fibrinogen in resting platelets. e. morgenstem, b. kehrel and k.j. clemetson medical biology, saarland univ., homburg, germany, haemostasis research, univ. muenster, germany and theedor-kocher-lnstitut, univ. bern, switzerland. wortmannin, an inhibitor of phosphoinositide 3-kinase and of myosin light chain kinase blocks reactions of the activated platelet. to obtain informations about the role of the contractile cytoskeleton in receptor-mediated transport of resting platelets, the effect of 17-hydroxy-wodmannin (hw) on the endocytosis of fibrinogen from the surface of resting platelets was studied. gel filtered platelets (gfp) were incubated for 10 min at 37°c with hw (3x10-6m) or with iloprost. controls and gfp preincubated with hw or ilopmst were incubated with 1.4nm-gold labelled fibrinogen molecules (fg-au; final concentration 40p.g/ml) at 37°c. the experiments were stopped after 5 or 30 min by rapid freezing. after freeze substitution in acetone with 4% osmiumtetroxide, sedal sections were prepared. the sections were examined after incubation with ascorbic acid (5% in h20) for 30 rain at 20°c (to reduce metallic osmium) and silver-enhancement using danscher's (1981) method (to visualize the fg-au). examination of adp stimulated platelets in the presence of 40fg/ml fg-au shows that the ligand is able to mediate aggregation. the examination reveals, that fg-au was present in a low density on the platelet surface, in higher density in the surface connected system (scs), in coated pits and vesicles and separated smooth vesicles (representing endosomes?) as well as in the matrix of alpha-granules. after 30rain, the number of labeled granules was increasing. labels on the surface and on the mentioned cytoplasmic membranes were observed during the whole period of incubation. hw or iloprost did not alter the resting gfp and the mentioned qualitative ultrastructural findings in both preparations did not show differences to the controls. we conclude from the results with hwthat the regular contractile function of the cytoskeleton is not necessary to transport the fg-au in resting platelets. methods: edta anticoagulated whole blood was incubated with thiazole orange and analyzed with a flow cytometer. young platelets were defined by having a high fluorescence from thiazole orange (normalized to platelet size). platelets were also incubated with fluorescent antibodies to gpib, gp lib/ilia and gmp-140 (two colour method). results: surface expression of gpib was the same in young and older platelets. results for gp lib/ilia and gmp-140 (in resting and activated platelets) will be presented. conclusion: young platelets can easily be detected using thiazole orange and flow cytometry. there is no differential expression for gpib. further results will be presented. the influence of erythrocyte and thrombocyte content on the release of atp by different agents in whole blood specimens was tested. the measurement had been performed in the lumi-aggregometer using the principle of the luciferin-luciferase reaction. altogether 39 blood samples were diluted gradually before induction of the release reaction by arachidonic acid (1,25 mmol/i final concentration), adp (30 ijmol/i) and collagen (1,0 and 5,0 tjg/ml). the peak of the obtained curves was transformed into percent values of the maximal deflection by the undiluted sample (= peak in relation) and into atp concentrations (= absolute peak) after testing the atp standard in parallel for each dilution step separately. the peak in relation increases by increasing dilution with all inducers. it was identic with the atp standard and with collagen, somewhat lower with arachidonic acid and much higher by adp. a luminescence-optical effect may influence all these results. the absolute peak decreases by dilution under arachidonic acid and collagen as it was expected by the decreasing thrombocyte content of the samples. under induction by adp no decrease of the absolute peaks by increasing dilution of the samples was abserved. this can be explained only by liberation of atp from the erythrocytes. the atp standard is essential for the quantification of the release reaction. adp doesn't suit for it. collagen with a final concentration of 1 pg/ml was proven as the best inducer. platelet aggregation induced by several agents has been photometrically investigated in disc shaped rotating cuvettes coated with vessel wall tissues obtained from human umbilical cord, either endothelium or smooth muscle cells or extracellular matrix or combinations of them. in addition, effects of endothelium incubated with several cytokines on platelet aggregation have been studied. endothelial cells strongly inhibited aggregation depending on their cell count and the concentration of the inducer. smooth muscle cells showed the same effect but very less marked. in presence of extracellnlar matrix spontaneous aggregation occured. endothelium could inhibit this spontaneous aggregation when present in the same cuvette, smooth muscle cell could not. incubation of endothelium with several cytokines increased its anti-thombotic properties. for example, at a platelet count of 3x105/id in the prp, 10 -6 m adp led to maximal aggregation in uncoated cuvettes, in presence of 5,5x106 endothelial cells aggregation was completely abolished, in presence of 2,75x10 "6 cells aggregation was decreased to 40%. smooth muscle cells diminished the aggregation effect of 0,1 nih thrombin to 67% when only one side of the cuvette was coated and to 63% when both sides were coated. endothelium could not inhibit aggregation induced by 2,5 x 10 -6 m adp but endothelium incubated with 500 u/ml tnf-a or 30 u/ml intedeukin-lfl or lmm l-nitro-arginin for 24 h did completely inhibit aggregation. platelets become sticky and adhere to surfaces or to another without contracting and secreting. during maturation of megakaryocytes finally platelets lost their genomic nuclear message. only mitochondrial dna of platelets can be identified. we focused our attention on the impact of mitochondrial dna and the mitochondrial transscriptive mechausisms during platelet activation in normals. materials and methods: leucocyte free (nagentte chamber, flow cytometric analysis) platelet rich plasma or platelet concentrates a_~er hemapheresis were filtered by pall 100 leucocyte filters. the influence of different anticoagulants (commercially available sarstedt tubes containing citrate, heparim edta and 500 atu/ml hirudin wacker) was examined. activation was due to a 60 nun. hemapheresis procedure ( 3-5fold increase of cd 62, cd 63) and ex rive stmaulation due to 4 niy u/ml thrombin, 0.025 m cac12 or combmatious. the guanidiurn method for total rna preparation were used according to t. brown: current protocols in molecular biology 4.21-4.9.14,1991. different primers of mitochondrial genome (e.g. cytochrome b and atpase) were prepared using pcr and mitochondrial transscription was examined using northern-blot-technique. results: 1., there is less activation of mitochondrias using hirudin anticoagnlation, but a 2fold increase of mitochoindrial rna content in heparinized samples. 2., stimulation with thrombin leas to an increase to 5.5 e-l0 rna btg/platelet, compared to 4.7 -4.8 e-10 rna ~tg/platelet under unstimulated conditions.. conclusion: there is evidence for the importance of platelets mitochondrial dna and mitochondrinl transsefiption in regulation of cytosceleton and platelet activation. thrombospondin-1 (tsp-1) is a large homotrimeric glycoprotein originally identified as a platelet alpha-granule component. the investigation of its putative role in a variety of pathophysiologies like haemostatic disturbance, malignancy and wound healing requires specific laboratory reagents. monoclonal antibodies are one of the most powerful of these reagents. therefore, we purified human tsp-1 from thrombin-stimulated platelets using affinity chromatography to generate monoclonal antibodies in mice. a subclass igg 1 monoclonal antibody designated 48.42 was purified from ascitic fluid and further characterised. western blot experiments demonstrated that this antibody reacted only with the unreduced molecule whereas the tsp-1 subunit chain was not recognised. no cross-reactivities with human fibrinogen, fibronectin, vitronectin and von willebrand factor were found. preliminary results indicate that the monoclonal antibody 48.42 can be used to investigate tsp-1 function in several assays including immunocytochemistry and cell adhesion as has been demonstrated for hl-60 cells. in addition, a sandwich enzyme immunoassay was developed using goat-antihuman tsp-1 igg and derivatised monoclonal antibody 48.42 (peroxidase, biotin) as a sensitive method for detection of tsp-1 in human body fluids. in the following study the expression of the platelet antigen (cd62p) and the leukocyte antigen (cdllb) were measured in whole blood, in addition to platelet-leukoeyte adhesion (rosette formation) by means of multicolour fluorescent labelling (cd45, cd14, cd42a). the measurements were carded out both in freshly drawn whole blood which had been antieoagulated with different agents, and in stirred samples of whole blood under controlled conditions (37°c, 1000 rpm, different stirring times). the results are presented as the percent positive events in each gate (platelets, leukocytes -pmnl, monocytes, lymphocytes and rosettes -plateletpositive events in the pmnl, monocyte and lymphocyte gates), whose mean fluorescence is given in addition to an index comprising the product of the percent positive events and their mean fluorescence. stirring (max 15 rain) induced an increase of cd62p on the platelet surface of ca. 10%, without any change in the mean fluorescence. under these conditions increased cdllb on pmnl and monoeytes could be detected. an increase in the rosette formation could also be measured (greater index), in that the percent of monocytes which were platelet-positive increased with no change in the mean fluorescence of the positive events, whereas pmnl showed an increased mean fluorescence, but not an increased number, of platelet-positive events. the time-dependent changes in rosette formation on stirring could be further increased by addition of adp. these results show that it is possible to measure rosette formation, and also the influence of effector agents (inhibitors or activators of platelets or leukocytes) on rosette formation, in whole blood using flow eytometry. 17 itp patients undergoing splenectomy were observed after 1-30 years following operation and divided into 2 groups. first group consisted of 8 patients with normal platelets count and absence of haemorrhagic syndrome. second group was formed of 9 itp-patienfs with episodes of thrombocytopenia recovery following certain time period after splenectomy. in the aim to study the cellular immunity there were carried out immunophenotypical investigations of blood samples using immunofluorescence method with monoclonal antibodies application. the increase of b-cells, expressing cd22, cd37, hla-dr-antigen has been revealed in the 2nd group. quantity of srfc, cd3 +, cd5 + cells in the blood of recovered patients was lower than in patients of the first group. this group was also characterized by statistically significantly increased level of cd4 + cells while the cd4/cd8 ratio was equal to 1.0 :i: 0.3 % (0,5 + 0,1% in patients of the second group, respectively, p>o,05}. also the relatively high expression of activating antigens in patients with thrombocytopenia recovery after splenectomy was stated. among infectious complications in all patients observed were predominantly found various types of throat infection, mainly with unsatisfactory treatment possibilities. we have observed the opsi-syndrome in 2 patients, being featured with marked tiredness, breath loss, intolerance of hard physical working, diminished ability to maintain physical activity. extracellular matrix (ecm) produced by human endothelial cells closely resembles the vascular subendothellal basal lamina in its organization and chemical composition. thus it contains collagens, fibroneetin, von witlebrand factor, thrombospondin, fibrinogen, vitronectin, laminin and heparin-sulphate. platelets carry different receptors on their membrane surface with specific binding capacities for one or more of these extracellular matrix proteins, such as glycoprotein (gp) iibiiia, gp ib/ix and gpiiib. incubation of platelets with ecm results in platelet adhesion, degranulation, prostaglandin synthesis and aggregation. we studied patients whose platelets showed either a receptor defect in gpiibiiia or gpiiib or a storage pool disease. adhesion experiments were performed using siliconised glass, collagen coated surfaces, immobilized fibrinogen as well as human subendothelial matrix. platelet adhesion of patients with thrombasthenia glanzmann (receptor defect of gpiibiiia) resulted in a total lack of binding to silieonised glass and immobilized fibfinogen. adhesion to collagen was almost normal in spite of the fact that only single platelets sticked to the surface and no microaggregates were observed. the adhesion to ecm was diminished and also no aggregates were detected. patients with a receptor defect in gpiiib showed normal platelet adhesion to siliconised glass and immobilized fibrinogen but binding to collagen and ecm was markedly reduced, while platelets with a storage pool defect sticked to siliconised glass but failed to adhere to ecm. by centrifugation of citrate blood (250 x g, 10 min) erythrocytes and leucocytes go to the bottom, whereas plasma and thrombocytes stream in the upper part of the probe. so the thrombocyte count doubbles in the platelet rich plasma in contrast to the platelet count in the whole blood volume. if the thrombocytes are more or less activated, they adhaere on erythrocytes, leucocytes or aggregate end are not able to stream upwards. the quotient between thrombocyte counts in prp and whole blood is a measure for thrombocyte activation. we chequed the value of this screening in different groups of patients with arterial occlusions disease (aod), chronical venous disease (cvd), diabetes mellitus (dm] and in healthy control persons (control). variation coefficient of the method is 3.7 (prp) and 4.4 (tc) respectively (coulter counter). differences to the control group are significant. changes in the patient groups in dispensaires follow up 5 years are also significant. nicardipin -induced immunthrombocytopenia p. eichler 1, c. hinrichs 2 , g greinacher l i.institut fur immunologic und transfusionsmedizin, ernst-moritz-arndt-universitat greifswald, 2. deister-s0ntel-klinik, bad m0nder drug-dependent immune-thrombocytopenias are a rare but clinically important variant of immune-thrombocytopenias. patients are at risk to suffer from severe bleeding complications. especially in patients receiving multiple drugs, diagnosis of drug-dependent immune-thromboeytopenia is often difficult. we report the case of a 71 year old male patient who received allopurinol, captopril, digitoxin, furosemid, and nieardipin. the patient presented with hematomas (pit. count < 10 g/l) and later developed bone marrow dysplasia. in an elisa using whole platelets and patient serum, a weak reactivity in the presence of furosemid, but a stronger reactivity in the presence of nicardipin (antagonil, ciba-geigy) could be demonstrated. the reaction pattern is given in the the enzyme-immunological determination of soluble fibrin (sf) proved to be highly sensitive and specific. this sf-elisa detected fibrin hacking fibrinopeptide a (fpa) via the monoclonal antibody 2t35 specific for the neoepitope generated on the aa-chain after the split of fpa. lill et al. recently introduced a new assay modification which utilizes the same antibody as the old one but takes advantage of a pretreatment of plasma specimens with kscn. this strong chaotropic ion is used to dissociate the various fibrin complexes possibly hiding fibrin epitopes. it was the aim of this study, therefore, to compare the two sf-elisa modifications (with and without kscn-pretreatment of specimens) . in order to examine the dynamics of thrombin-induced fibrin(ogen) metabolism we made course observations in patients with a certain form of septicemia. both assay modifications detected fibrin(ogen) derivatives which differed considerably in kinetics (n= 160 samples from 10 courses). the former sf-elisa (no kscn) correlated well with prothrombin fragments, thrombin-antithrombin !11 -complexes and with the release of fibrinopeptide a ( r > 0.96, n= 151). results of the new sf-elisa with kscn pretreatment of patients' plasma, however, correlated conspiciously well with d-dimer levels (r > 0.94) but distinctly less with the markers of thrombin generation (-0.12 < r < 0.29). this good correlation with d-dimer levels was unaccountable since the d-dimer maximum occured significantly later than the peak of markers of thrombin generation (p < 0.05). therefore, kscnpretreatment of fibrin specimens seems to lead to a change in the specificity of the fibrin assay despite usage of the same catching antibody. different half-iifes of differently composed fibrin complexes should be considered in trying to explain the findings. nevertheless, the results of the former assay without kscn-treatment correlated much better with the well-known dynamics of thrombin-induced fibrin generation during hemostasis activation than the data from the new assay modification. consequently, further examinations are necessary to specify the effect of kscn on soluble fibrin complexes and the resulting assay specificity. a rapid assay for the determination of the primary hemostasis potential (php) of whole blood has been developed (kundu et al, 1995) from the original method of kratzer and born. the new system employs a disposable test cartridge which holds the sample (citrated whole blood) and all components for the tests at the same time. the test procedure is very simple. the cartridge is loaded with -500 p.l citrated whole blood and is inserted into the platelet function analyzer (pfa 100aaw). the test is started automatically after a preincubation phase of 2.5 rain. the reaction starts with the contact of the whole blood and the capillary which is connected with a collagerdephinephrin coated membrane with a small aperture inside the test cartridge. under constant negative pressure the sample is aspirated and through the contact ofplatelets and vwf with collagen adherence and aggregation begins. the adhesion and aggregation process leads to the formation of a platelet plug which obstructs the flow through the aperture. the result of the php is reported as closure time (ct). additional parameters such as bleeding volumes are possible as well. first results show good reproducibility, normal values in the range of up to 150 sec. and a good discrimination of healthy donors from patients with congenital or acquired platelet dysfunctions. the system detects aspirin induced thrombocyte function defects and von willebrand disease. in ease of an abnormal result in the collagerdepinephrin system a second type of cartridge with a collagerdadp coating can be employed. in the majority of cases aspirin induced dysfunctions are normalized and could thus detect aspirin use. the proposed system may be a valuable tool for routine assessment of the primary hemostasis potential in a routine citrate blood sample laboratory. inducing mental stress in 20 young healthy male volunteers aged 20 to 40 ),ears with no previous history of thmmbophilia or a hemorrhagic diathesis was performed by a first time parachute descent from an altitude of 1000 meters. the purpose of this investigation was to find out whether there are any changes in the corpuscular and plasmatic fractions of peripheral blood. we were especially interested in elucidating changes in the procoagulatory and/or fibrinolysis systems. venous blood samples were obtained directly before and directly after the jump. flight time from the departure of the airplane to the landing of the parachutists was approximately 20 minutes. the maximum time that elapsed between the two blood withdrawals were 45 minutes. in a preliminary study with different voinnteem, certain fluid imbalances had been observed. absolute numbers of leukoeytes (6.9 vs. 9. l/n0, erythrocytes (4.6 vs. 5.1/pl), and platelets (246 vs.276/nl) significantly increased (p < .001), as well as the hemoglobin concentration from 145 to 156 g/l (p < .018). even though fluid imbalances before and after the jump had practically been excluded by measuring nearly identical hematoerit values (.41 vs..42), we noticed a marked drop in aptr (27 vs. 23 sec) and a significant increase in factor viii ~tivity. as a direct stress response, we found a rise in fibrinogen concentration (2.4 vs. 2.8 g/l) which is one of the shortest acting acute phase proteins. concerning reactive fibrinolysis, d-dimers showed an increase in concentration from 115 lag/l to still normal values of 192 lag/l, which was not significant due to low numbers of values (p = .086). we observed similar changes in fibrin monomers and prothrombin fragments fl+2. from other investigations on the kinetics of the activation of the procoagulatory system we know that maximum activil7 is not reached until 24 hours after initiation of activation.these investigations studied perioperative changes in different kind of operations which served as a control group concerning the degrees of tissue damage and resulting coagulation disturbances. to better understand these phenomena we plan to induce mental stress in a laboratoq' environment to further exclude unknow~a influences on the mechanisms which can activate the procoagulatory and fibrinolytic systems. triodena (t) 30/40/30 ug ee, 50/70/100 ug gestodene) were tested for their effect on hemostatic parameters. three groups (n=20) of healthy female volunteers were treated for 6 months with one of these oc. blood was taken before treatment (day 24-28 of pretreatment cycle, 0) and on days 18-22 of the 3 ~ (i) and 62 (ii) treatment cycle. indications of an activation of blood coagulation and fibrinolysis were detected as the plasma levels of prothrombin fragment f i+2 and of fibrin split product d-dimer and plasmin antiplasmin complexes were found elevated during treatment. the following main regulatory components of blood coagulation, activators and inhibitors, were investigated: factor vii antigen fviiag, fvii clotting activity fviie, circulating activated factor vii cfviia and antithrombin 3 at3 activity, total protein s antigen tps-ag, free protein s antigen fps-ag, protein s activity psact, circulating thrombomodulin etm fviiag, fviie and cfviia significantly increased during treatment; cfviia: 0: c 32.4 mu/ml a prethrombotic condition characterized by elevated levels of circulating soluble fibrin has been claimed to be a predisposing factor for accumulation of coronary thrombotic material in acute myocardial infarction. the present study includes 161 patients with clinical suspicion of myocardial infarction. blood samples were drawn by the primary care physician, upon arrival in the hospital, and after 2, 6, 12, and 24 hours of hospital stay. patients with myocardial infarction were identified by typical course in 12 lead ecg, and upon sequential determination of troponine t, myoglobin, ck, and ck-mb. patients with primary cpr were excluded from evaluation. soluble fibrin was measured by enzymun®-test fm (boehringer mannheim). patients with acute myocardial infarction display soluble fibrin levels within the normal range (< 5 ~tg/ml) during the initial two hours after onset of symptoms. there was no significant difference between patients with myocardial infarction and patients with coronary heart disease without myocardial infarction. slightly elevated levels were found in patients with atrial fibrillation, reflecting intracardiac fibrin formation. in patients without fibrinolytie treatment, a slight increase of soluble fibrin levels with a maximum after approximately 8 hours is observed. most patients with fibrinolytic treatment display a considerable increase in soluble fibrin, with maximum levels immediately after infusion of the fibrinolytic agent. four patients with pulmonary embolism showed soluble fibrin levels in the range of 40-300 [.tg/ml, which remained in the same range during the entire observation period. in conclusion, circulating soluble fibrin is not increased in patients with acute myocardial infarction and does not appear to be a predictor of acute coronary events. high levels of soluble fibrin in patients with fibrinolytic therapy may reflect release of fibrin from thrombotic material, but also de novo generation of fibrin due to release of active thrombin from thrombi not necessarily located in the coronary vessels. detection of elevated levels of soluble fibrin in patients with acute chest pain should result in careful examination for signs of pulmonary embolism or aortic aneurysm. the possibility to determine activated coagulation factors opens the question if data provide evidence of an activated coagulation or fibrinolysis and if this has a prospective value. we investigated patients with confirmed thrombosis, postsurgical septieaemia and also after liver transplantation. in all patients factor viia, xii, xiia and also the fibrinolytic parameters t-pa, pai-1, pap, plasminogen and a2-ap were determined. in addition, f1+2 and apc-resistance with heterocygote factor v-leiden-mutation and confirmed thrombosis. we found increased factor viia which showed partly also an increased fl+2. patients with other pathological results such as a reduced t-pa and/or increased pai-1 showed a low incidence of elevations in factor vii or f1+2. the activation of factor xii seems to be of minor importance in patients with thrombosis. a different picture is found in septic and transplanted patients. obviously factor xii-activation is of major importance in this group. a deterioration of the clinical symptoms is correlated with an increased factor xiia which is paralleled by a decrease of factor xiiactivity. the investigation of fibrinolysis parameters such as pai-1 and pap demonstrate a fibrinolytic disturbance of the balance. statistically significant are differences in septicaemic patients both in the surgical and in the internistical group in contrast to polytrauma patients. in patients with liver transplantations significant changes are apparently related to rejection of the transplanted organ together with a deterioration of the clinical picture. the possibility to detect activated coagulation factors may be a tool to detect changes in the hemostasis system at an early stage and to use this for an improved therapy. control of long-term oral anticoagulation is usually performed by serial determinations of the prothrombin time. however, the assessment of effective anticoagulation versus the potential risk of bleeding complication is difficult to achieve. molecular markers of blood coagulation activation might add valuable information in individual cases. we investigated 48 patients with thromboembolic manifestations (deep vein thrombosis n=22, pulmonary embolism n= 13, myocardial infarction n= 13) for one year beginning with admission to the hospital. tat, prothrombin fragments f 1 +2, d-dirner and fibrin monomer concentrations were analysed. all markers were significantly increased at the time of initiation of anticoagulant therapy thus reflecting a prethrombotic situation. patients suffering from venous thromboembolism demonstrated higher concentrations of tat and f 1 +2 in comparison to myocardial infarction (34.6 vs 12.3 pg/1, p=o.009; 2.8 vs 1.3 nmol/i, p=0.0025). f 1 +2, tat and d-dimer concentrations decreased gradually over the first 14 days of anticoagulant therapy reaching values within the established normal ranges in all cases. f 1 +2 and tat concentrations reflect the activity of the coagulation system during long-term anticoagulation whereas analysis of fibrin monomer yielded partly controversial results. we conclude that f 1 + 2 and tat appear to be superior to fibrin monomer for the individual control of oral anticoagulant therapy. the influence of thyroid failure on haemostasis is controversial. mainly hypoceagulable states have been described in clinically overt hypothyroidism. since hypothyroidism has been associated with an increased risk of atherosclerosis, we studied a wide range of haemostatic factors in untreated female patients with subclinical (b, n=42, age 59+13) or overt (c, n=8, age 55-zcj) hypothyroidism, as well as in hypothyroid women under 1"4 treatment (d, n=8, age 57+9) and euthyroid controls (a, n=80, age 50+14). simple screening tests (prothrombin time, activated partial thromboplastin time, fibdnogen), procoagulant factors (fvii, fviii, von willebrand factor), coagulation inhibitors (antithrombin ill, hepadn cofactor ii, protein c, protein s) and fibdnolytic factors (plasminogen, antiplasmin, plasminogen activator inhibitor, tissue plasminogen activator) were measured. results factor vii activity (vii:c), factor vii antigen (vii:ag) and their ratio were found increased in hypothyroid patients. factor viii activity showed the same tendency, whereas von willebrand factor ramained unchanged, as did all other parameters with exception of free protein s, which declined in overt hypothyroidism and in t4 treated subjects. these differences tended to diminish after exclusion of 26 women with estrogen replacement therapy for menopause, but the ratio vii:cnii:ag, as well as fvii:c still remained significantly higher in hypothyroid patients. conclusions: subclinical and overt hypothyroidism are associated with significantly higher levels of factor vii:c and vii:ag. the disproportionate increase in vii:c compared to vll:ag, as shown by their ratio, might reflect the presence of activated factor vii (vila), which in turn indicates a hypercoagulable state. this pattern becomes more pronounced with the concomitant estrogen replacement after menopause. exocytosis following platelet activation leads to translocation of cd62p (p-selectin), cd63, and thrombospondin, from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. we here report detectability of these molecules preformedprior to platelet activation -inside the cytoplasm of resting platelets. two different methods are compared, i. e. using either methanol or the fix&perm kit (an der grub) for cell membrane permeabilization. in addition, interleukin(il)-ice is shown to be present in platelet cytoplasm after methanol treatment, but not after permeabilization using fix&perm. whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining. our data demonstrate the feasibility of the methods presented for the detection of intracellular platelet molecules. this technique should also provide a means for estimating the relative quantity of intracellular platelet antigens, provided the permeabilization procedure does not lead to antigen leakage or destruction. physical exercise activates the clotting as well as the fibrinolytic system as indicated in numerous investigations of exercise by running and by bicycle ergometer but not by swimming. the positive effect of an endurance training in coronary sport groups is induced also by influences on the hemostatic system. the influences are suppression of the clotting activation by the acute exercise and by an increased fibrinolysis response. different hemostatic parameters, therefore, were analyzed before and after swimming of male coronary patients (n=33; median ag~ 61 years, achieved heart rate: 68/min). indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 among the coronary patients (tat from 2,1 to 3,4 pg/1; fi+2 from 0,92 to 1,1 nmol/1). the degree of clotting activation among the coronary patients was less than that observed in a group of young volunteers in a former investigation. this must be explained by existence of the coronary heart disease or by the higher age in the patient group. indicating an activation of fibrinolysis t-pa activity increased significantly in coronary patients (from 0,14 to 0,5 iu/ml) resulting in an unchanged balance between coagulation and fibrinolysis. from this findings of the hemostatic systems no increased risk of the coronary patients by swimming can be derived. a prerequisite, however, are precautions l±ke to devoid exercise in the anaerobic range, exclusion of major heart failure and of cardiac arrhythmias before begirming of the swim training. the principle of the fontan operation consists in anastomosing the right atrium to the pulmonary arteria, thus bypassing the right ventricle and using the only functional single ventricle as a pump for the systemic circulation. there are only few data about the influence of the changes in hemodynamics on coagulation and fibrinolysis. we investigated the coagulation system in 20 children and young adults aged 4 to 21 years in a general examination 4 to 61 months after fontan procedure. besides other abnormalities of the coagulation system, there were significantly increased values for the thrombin-antithrombin-iii-complex (tat) in 12 patients (60%). as a marker for an activation of the fibdnolytic system we found elevated plasmin-alpha2-antiplasmin-(pap-) levels in 14 patients (70%). less frequently, the concentrations for the prothrombin-fragments 1 and 2 (f1 and 2) (7 patients, 35%) or the d-dimer (2 patients, 10%) were increased. we didn't find significant differences in a clot-lysis-assay between fontanoperated patients and an age-matched control group. there was no significant correlation between activation of coagulation and clinical situation or diameter of the pulmonary arteria. whether the present data can help to estimate the risk for a thrombo-embolic complication following fontan procedure, still has to be investigated. the results of the clot-lysis-assay suggest, that for lysis of thrombi the same dose of rt-pa should be used as for other patients. a 2nd generation functional protein s assay p. van dreden* and e. adema** * serbio, gennevilliers france, ** boehringer mannheim, tutzing germany a second generation protein s test was developed with improved sensitivity to protein s and better reagent stability. the test result was found to be unaffected by apc-resistence (10 patients, heterozygote for the mutation with a apti' + apc ratio between 1.4 and 1.9), heparin up to 2 iu/ml and f viii activity between 1 and 250%. in the test, diluted sample is mixed with protein s deficient plasma, activated factor v, activated protein c, phospholipids and an intrinsic pathway activator. this mixture is incubated for 3 minutes. during this time, the activated protein c inactivates part of the f va. the extend of f va inactivation depends on the protein s concentration. after 3 minutes caci2 is added and the time untill clot formation is measured. the clotting time is a linear function of the protein s concentration between 10 and 140% protein s. for the three preproduction lots the difference in dotting time between 10 and 100% protein s was 43-54 seconds. this compares to 30-40 seconds typically obtained with the old test. within run precision (n= i0 on sta) is cv= 2 -7% on the basis of protein s. day to day precision (n=10 on sta) was found to be cv= 4 -11%, again calculated on the basis of protein s concentration. the cv of 11% was obtained for an avk plasma with 13% protein s; it corresponds to a standard deviation of only 1.5% in protein s. the insensitivity to interferences, in particular apc-resistence and better precision and stability are expected to improve the quality/reliability of a protein s determination. in this study we evaluated the use of hormonal contraception on the parameters protein c, protein s and pal. samples from 71 women with, without hormonal contraception and in menopause were assayed by coagulometric (protein s clotting test (behdngwerke, marburg, frg) or chromogenic methods (protein c activity test and pal reagent from behringwerke, marburg, frg) in double determination and were compared with the reference ranges. in addition thromboplastin time (thromborel s reagent) and fibrinogen (multifibrin) from behringwerke, marburg, frg, and aptt (actin fs reagent from dade corp., unterschlei6heim, frg) were determined. in women using hormonal contraceptives (p<0,01) and in menopause (p<0,05) protein s activity was significantly reduced compared to other women (<45 years) while protein c acitivity did not change. in menopausal women a higher susceptibility to thrombosis was supported by an increase of aptt (p<0,05) and fibronogen (p<0,01). while there was no change for pal, plasminogen was significantly lower in women using hormonal contraceptives and in menopause (p<0,05). we could not observe a higher turnover of coagulation and fibdnolytjc system with hormonal contraception. noteworthy was the occurence of low (<200 mg/dl) and borderline fibrinogen (max. 220 mg/dl) in 40,9% of women res. in 22,8% of women (together with borderline aptt) who had an individuell risk for arterial disease. protein s protein c fibdno~en aptt plasminog~ without hcc 109,1-+13,6 78,3-+14,1 255,0-+14,0 36, [2] [3] [4] [5] 4 24, [0] [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] 2 with hcc 85, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] 2 78, 5±12, 0 253, 8.+24, 1 35, [2] [3] [4] 8 14, 9 menopause 90, 3+97, 6 87, 4±41, 4 307, 05:57, 6 39, [8] [9] [10] 0 12, 6 hcc= hormonal contraception hemostatic parameters in a patient undergoing bone marrow and subsequent liver transplantation due to veno-occlusive disease c. salat 1, , e. holler t,3, hi. kolbl, 3, b. reinhardt l, r. pihusch 1, p. g0hring 2, s. poley 2, e. hiller 1 l=med. klinik iii, 2 = institut flit klin. chemie, klinikum grosshadern der ludwig-maximilians-universit~tt mfinchen, 3=h~tmatologikum der gsf a 40 year old patient suffering from all received allogeneic bone marrow transplantation (bmt). after an uncomplicated early posttransplant period the patient was dismissed after 4 weeks. a bilirubin rise with subsequent liver failure was observed during the following weeks. according to biopsy proven hepatic veno-occlusive disease (vod) liver transplantation was performed on day 79. unfortunately the patient died on day 140 due to aspergillosis. we monitored levels of protein c (pc) and s (ps) as well as pall during the pre-and posttranspiant period. pal1 level was normal (<43 ng/ml) during the first 4 weeks after bmt but increased with the manifestation of vod (317.5 ng/ml on day 47). it reached its peak immediately before liver transplantation (547.6 ng/ml) and returned to normal levels within the next few days. pc levels which were normal before bmt decreased prior to clinical diagnosis of vod and were normal after liver transplantation. ps levels lay within the normal range at all timepoints. vwf was elevated before bmt (240%) and remained relatively stable during the whole investigatonal period ranging from 170 to 260%. it is assumed that vod is initiated by an endothelial cell injury -possibly due to radiochemotherapy -and subsequent hypercoagulability. our results indicate that the "endothelial cell marker" vwf is not helpful in predicting vod. the kinetics of the investigated parameters underline the significance of pc and pai-1 as described by others and our group earlier, whereas ps does not seem to play a role in the pathogenesis of vod. the budd-chiari syndrome (bcs) is characterized by hepatic venous outflow obstruction that may be caused by the precipitation of a thrombus. it frequently coseggregates with other major diseases like myoloproliferative diseases or defects in the haemostatic system (antiprotein c and protein s deficiencies e.g.). only recently, the factor v leiden mutation (fvlm) has also been associated with bcs. we hypothesized that defects in the thrombo-modelling associated anticoagulant pathways (tmaap) are a major risk factor for the precipitation of bcs. we screened our cohort of 27 patients (pts) with bcs for the presence of defects in the tmaap and identified 3 pts with protein s deficiency (psd). these pts were screened for the three point mutations in exon 1 (codon-25; ins t), exon 15 (codon 636; a-->t) and in intron 10 (g-->a + 5) of the ps alpha-gene that have been demonstrated by bertina et al to coseggregate with psd. restriction enzyme analysis and confirmation-sensitive gel electrophoresis for the detection of single-base differences in doublestranded pcr-products were employed. all living family members of the indicator pts were also screened for heterogeneties in the three point mutation as described. no single abnormality in these genes despite presence of pbd in those family members was found. in addition, pts and family members were also screened for fvlm. one pt and two of his family members, in addition to psd, were subject to fvlm. the other two lots and their family members were not subject to fvlm. in contrast to the first family, despite psd, those two pts suffered from morbus crohn and acute myeloid leukaemia as risk factors for bcs. we conclude: psd is one major risk factor for the precipitation of bcs. to precipitate this disease, one additional risk factor is required. psd may be caused by genomic defects in the protein s gene other than those described by bertina. only a few publications describe a thromboembolic disease due to dramatically reduced protein s levels being associated with viral or bacterial infections, autoimmune mechanisms are suspected but the aetiopathogenesis is still under discussion. we report on a 5 year old boy who developed purpura fulminans of the left leg during varicella infection. on the fourth day of infection the disease started with pain and haemorrhagic efflorescence localized at the left taft. on admission the boy suffered from a purpura fulminans with central necrosis measuring 15x8 era. suspecting a hereditary thrombophilic disease we started therapy with protein c concentrate and recombinant tissue type plasminogen activator. the fellowing coagulation investigation showed a severe deficiency of protein s (total protein s-antigen < 5 u/ml, free antigen not measurable) in combination with factor v leiden mutation. other thrombophilie and coagulation parameters did not show deviation from normal range. after 4 weeks we saw a slight improvement of the total protein s antigen up to 50 u/ml. the free protein s antigen was still undetectable. during the following weeks the patient recovered slowly and the protein s activity and antigen normalized. because of skin necrosis thromboembolie prophylaxis was initiated with low molecular weight heparin (fragmin®, 100 ie/kgbw/die) and continued for 6 months. under this therapy there were no further thromboembolic events. these results suggested an autoimmune protein s deficiency in a patient suffering from chickenpo×. an analyses of autoantibodies at the time of diagnosis showed a slight increase of the antieardiolipin antibodies (igg 16,1 iu/ml, igm 15,1 iu/ml) which normalized during hospitalisation. we suspect an antibody to protein s probably caused by similar presented viral antigens. we suppose that autoimmune mechanism during different infections in combination with a heterzygous apc-resistance may be a potential risk factor for developing thrombotic disease. in the central nervous system mrna encoding for prothrombin and thrombin receptor is present and astroglial cells in culture process and secrete thrombin. moreover, effects of thrombin on brain cells including change of neudte outgrowth and astrocyte shape are described, but the molecular mechanisms are unclear. we investigated the effects of human <z-thrombin and a six amino acid thrombin receptor activating peptide (trap-6, sfllrn) on [ca2+]i, phosphoinositide hydrolysis and protein kinase c activation in the rat glioma c 6 cell line which is a widely used in vitro model for studying glial functions. stimulation of c 6 cells with both c{-thrombin and trap-6 resulted in transient [ca2+]i mobilization, [3h]inositol phosphate response and activation of the pkc isoenzymes c{,i],7,5, and s. these results suggest that ~-thrombin and trap-6 activate at least partially the same intraceltular signaling pathways in rat glioma c 6 cells which is an evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma c 6 cells. further investigations in this field are in progress including both receptor binding studies and more detailed analysis of thrombin receptor mediated signaling network and the cellular response of thrombin induced transmembranal signal transduction pathways in c 6 glioma. thrombin is a potent mitogen in vascular smooth muscle cells (smc). the mitogenic effect of bovine thrombin and human thrombin receptor-activating peptide (hutrap-14) was investigated in bovine coronary artery smooth muscle cells. confluent cells (passage 4-8) were incubated for24 h in serum-free medium with thrombin (10 nm) or trap (10-100 pm). the mitogenic response was assessed using [sh]thymidine incorporation. results demonstrated that thrombin induced a three-to fourfold increase in [3h]thymidine incorporation compared to the control, thus being as active as the potent mitogen pdgf-bb (20 ng/ml). the mitogenic effect of thrombin was dependent on its proteolytic activity. when the active site of thrombin was irreversibly inhibited by d-phe-pro-arg-chloromethylketone (ppack) no increase in [°h]thymidine incorporation was observed even at the high concentration of 250 nm ppackthrombin. thrombin-induced [3h]thymidine incorporation was significantly reduced by the reversible direct thrombin inhibitor nc£-(2naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (napap, lpm) . in contrast to thrombin trap-14 at a concentration up to 100 pm did not increase [3h]thymidine incorporation in the absence or presence of the aminopeptidase inhibitor amastatin (10 ijm). in order to demonstrate whether hutrap caused a stimulation of the receptor and signal transduction in bovine smc the activation of protein kinase c (pkc) was measured as translocation of the cytosolic to the membrane bound fraction. it was found that both thrombin (10 nm) and trap (10 pm) induced a translocation of pkc after 20 rain. in conclusion, thrembin and trap activated pkc in coronary smooth muscle cells but only thrombin acted as a mitogen. endothelial cells once stimulated with thrombin are resistant to subsequent stimulation directly after the first. but after a recovery period of about 90 min the cells are sensitised again for activation by thrombin. in our experiments this resensitisation couldn't be inhibited by actinomycin d or cycloheximide nor by the phosphatase inhibitor ocadaic acid. therefore an intracellularly pool of thrombin receptors has been proposed possibly co-localised with golgi vesicles. brefeldin a (bfa) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organisation of the golgi apparatus. because of this we examined the effects of bfa on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. the vwf release from weibei-patade vesicles was used as physiological parameter of activation of thrombin receptors by thrombin. the addition of bfa (20 pg/ml -20 ng/ml) to endothelial cells blocked the receptor response regeneration, whereas the first stimulation was not influenced by bfa at the concentrations used. these results give further evidence that the proposed storage pool of thrombin receptors is iocalised in vesicles of the golgi apparatus. randomized comparison of double bolus reteplase (r-pa) and front-loaded alteplase (rt-pa) in patients with acute myocardial infarction (rapid ii) c. bode, rw. smalling, j. kalbfleisch, g. berg, dj. odenheimer, e. bshm, wd weaver and the rapid ii investigators. med. klinik iii, university of heidelberg, germany the study was undertaken to assess the efficacy of a double bolus regimen of 10 + 10 megaunits of reteplase, a recently developed deletion mutant of tissue-type plasminogen activator, relative to front-loaded (gusto-regimen) alteplase. there was no age limit and patients were recruited up to 12 hrs after onset of symptoms. the primary endpoint "patency at 90 minutes" was centrally assessed in a blinded fashion. infarct related coronary artery patency (timi grade 2 or 3) and complete patency (timi grade 3) at 90 min for reteplase vs. alteplase were 83.4% vs. 73.3% (p=0.03) and 59.9% vs. 45.2% (p=0.01), respectively. at 60 minutes, the incidence of both, patency and complete patency was also significantly higher in retep ase vs. aiteplase treated patients: 81.8% vs. 66.1% (p=0.03). and 51.2% vs. 37.4% (p=0.01). reteplase treated patients required fewer additional coronary interventions within the first 6 hrs of treatment (13.3% vs. 26.5%, p=0.01). there were no significant differences between reteplase vs. alteplase regimens in 35 day mortality (4.1% vs. 8.4%), severe bleedings (12.4% vs 9.7%) • or hemorrhagic stroke (1.2% vs. 1.8%). reteplase, when given as a double bolus of 10+10 mu to patients with acute myocardial infarction, achieves significantly higher rates of early patency and requires fewer acute coronary interventions than front-loaded alteplase without an apparent increased risk of complications. lysis therapy with uhsk of deep vein thrombosis (dvt) of the lower extremity commoniy results in a relatively high patency rate. in our patients, patency was achieved in approx. 70-80%. these results were supported by several studies. in dvt of the pelvic veins, however, this kind of aggressive therapy is excluded due to the risk of iatrogenically induced pulmonary embolism. on the other hand, the need for an effective therapy is evident because of the usually occtnring complete occlusion of the pelvic veins and the following severe clinical symptoms 0phlegnmsia cerulca dolens etc.). until september "95, 44 consecutive patients (16 males, mean age 41 ranging from 18 to 63 years) with dvt of pelvic veins were included in this study. after clinical and phiebographic/computer tomographic (ct) diagnosis, a temporary filter system was placed intravenously either by a wambmchial (34 pat.) access route, lransfemorally (6 pat_) or wansjngularly (4 pat.) under mdiological control. the open filter was positioned above the thrembus in the lower caval vein: 26 times an antbeor system, 11 times an anglocor and 10 times a cook filter were used. in the following three days up to 3 cycles of lysis therapy with 9 mli u of streptokinase were administered, each cycle lasted six hours. heparin was given simultanously via the filter system in therapeutic dosages in order to prolong alrit values 1.5 to 2 times the individual base line level. effective lysis was controlled by a second phlebography/ct, and the filter system removed through the introducer sheath valve. after lysis therapy all patients were put on coumarin with overlapping heparinization. successful lysis (complete recaanlisation without remaining thrombi and lysis of more than 50 per cent of the original thrombus) could be achieved in 62% (29 patients). in two cases a fatal pulmonary embolism occurred. 5 times great parts of a thrombus were captured by the filter and could be removed with the system. due to an insufficient boparin therapy we detected a newly developed thrombosis of the lower cavul vein twice after lysis. this complication could be resolved by additional administration of streptokinase. in nearly all cases we found extensive hematomas on the catheter insertion sites. 4 patients with transfemoral applications had to be transfitsed with red packed cells due to retroperitoneal hematoma. one filter system was broken after use. lysis of dvt of the pelvic veins with uhsk and with the protection of a removable temporary filter system situated in the lower caval vein enables us to carry out an effective therapy. the filter systems, however, have to be ameliorated in order to better prevent fatal pulmonary embolism. heparin should be administered at least in the remainder of the day of lysis. the insertion site should be the enbital vein because compression for bleeding complication is easily feasible at this location. we report about seven of our patients (pts.) with bcs treated by fibrinolytic therapy (5 female, 2 male, age 27, 6 years + 8). only one case was of unknown origin while in four pts. myeloproliferative syndromes, in one protein c deficiency and in another one morbus behcet was detected in five pts. chronical and in two acute bcs was diagnosed. as complications were found: in three pts• thrombosis of vena iv.) cava, in one ofv. porta and v. lienalis, in one more of v• porta and v• cava. we treated just one patient with urokinase (3 x 800.000 iu/d for 18 days). four pts. were treated with rt-pa in concentrations of 25 to 50 mg/d (for a period of up to 14 days) and continuous heparin infusion. high doses of rt-pa (80 to 120 mg/d for one or four days) were given in one case of acute and one of chronic bcs. partial (after high-dose rt-pa nearly complete) recanalisation of thrombotic hepaticel veins was only achieved in the two acute cases of bcs. no significant recanalisation was proven in hepatical veins of chronic bcs. three times complete fibrinolysis of v. cava thrombosis (two after highdose therapy) and one partial lysis could be attained. in one patient partial lysis of v. porta and in another one of v. lienalis was found. furthermore complete recanatisation of v. porta followed local rt-pa lysis during surgical intervention. in our two pts. with acute bcs significant improvement of hepatical venous flow could be measured after fibrinolytic therapy• in chronic bcs hepatical venous flow did not improve while the complication of v. cava thrombosis could eventually be treated successfully. probably due to fibrinolytic therapy one case of esophageal varix bleeding occurred. the benefit of flbrinolytic therapy of bcs seems to be influenced by the dur~ion of the disease• according to our results fibrinolytic therapy should be considered in cases of acute bcs as well as in the above mentioned thrombotic complications• recent studies suggest that transport of thrombolytic agents through the clot can play a major role in determining the reperfusion time needed for thrombolysis. as the mechanisms of this phenomena are rather unclear we studied fibrin (0.4-1.5 ~tm) clot lysis by plasmin (30 nm) presented outside or inside the clot in purified system (tris-hc1 buffer, 37°c). the clots was formed by addition of cac12 (30 mm), thromboplastin (1 u/ml) and human thrombin (6 nih/ml)t the lysis degree was determined either by counting of radiolabelled fibrin degradation products or by time of total lysis. obtained kinetic parameters of this reaction: kest=99.4 rain -1, km=i.9 ~tm (plasmin inside clot); kcat=l.36 rain -1, km=i.3 lzm (plasmin outside clot). this data indicate that eatalytie efficiency of plasmin from inside the clot is 50-fold higher than from outside as measured from kcat/km. furthermore we studied plasma clot (0.25 ml) lysis in original plasma (3 ml) containing sk (5-100 nm) and seu-pa (5-100 rim) in the final concentrations from inside or outside the clot. the results are presented in the the similar results have been got when the fibrin clots were formed with batroxobin instead of thrombin. it may be concluded that fibfinolysis is a surface-dependent process. it's likely that the higher level of plasminogen activators in blood would prevent thrombus formation, while physiological clots would stay resistant. for the improvement of the thrombolytic therapy in children more clinical data are needed. therefore we report on our experiences with rt-pa presenting the clinical course in 11 patients (age range: 7 days to 16 years) with venous thrombosis (n=9) and purpura fulminans by meningococcosis (n=2). the patients were treated with rt-pa (actilyse r) for 10,5 hours to 13 days. the venous thromboses were localized in lilac/femoral veins (n=4), brachiocephalic/jugular/subclavian veins (n=3) and the cava superior vein (n=l). central venous catheters were causative in 2 cases. the dosage range was between 0.2 to 0.5 mg/kg for the initial dose and 0.8 to 2.0 mg/kg/d for the subsequent continous infusion. ten patients received simultaneously heparin. complete clot dissolution occured in 6 cases, partial clot dissolution in 1 patient. in 4 patients with a history of the first clinical signs of thrombosis of 3 weeks or more the lysis was not successful. concerning the side effects only small bleeding episodes on former venipuncture sites were observed in 5 patients. systemic effects such as the decrease of the plasma flbrinogen concentration were rare too. in conclusion, the thrombelysis with rt-pa presents an effective and safe therapy in children at: the rt-pa dosage used. in case of a history of the thrombotic event of more than 3 weeks a thrombotytic therapy should not be carried out. d.dimer is widely used in clinical situations associated with thrombotic diseases. the most popular application is the exclusion diagnosis of deep-veinthrombosis and pulmonary-embolism and the assay is performed in emergency. d.dimer is also frequently measured for evaluating the thrombotic risk in the post-operative period. quantitative, flexible and sensitive assays are required. we have developed a new automatic method which works on the coagulation walk-away instrument, sta. this assay was developed with two monocloual antibodies specific for d.dimer coated on microlatex particles and it uses an immuno-turbidimetric technology. absorbance is measured at 540 nm and is a direct relationship of d.dimer concentrations. the assay works with 50 ~tl undiluted plasma and is performed in less than 10 minutes. the dynamic range is from 0.2 to 16 [~g/ml and no prozone effect is observed up to concentrations higher than 100 ~tg/ml. the detection threshold is of 0.2 [lg/ml. the intra-assay reproducibility is below 5% and the inter-assay reproducibility is below 6%. recovery studies of purified d.dimer added to normal plasma were between 94 and 105%. no interference of heparin, bflirubine, lipids or of rheumatoid factor up to concentrations of 100 u/ml is observed. there is no effect of high fibrinogen concentrations (> 10 g/l). when compared with conventional elisa techniques (asserachrom ddi), the assay demonstrated a correlation coefficient of 0.97 on 131 samples from normal individuals and hospitalised patients with elevated d.dimer concentrations. slope was of 0.97 and intercept was of -0.07. this new assay offers a full flexibility for individual testing as the calibration curve is stable for at least one week on the instrument. it is then well adapted for all the applications of d.dimer measurements in coagulation laboratories. 16 children between an age of 3 days and 11 months ( median 6 weeks ) with thrombotic or embolic occlusion of major vessels were treated with rt-pa for thrombolysis. the affected vessels were both sided renal veins or one sided renal vein and v. cava inf. in 8 cases, the v. cava superior in 3, the v. cava inf. plus renal veins plus aorta in 1, the left ventdcle in 1, the aorta in 1, the a. femoralis in 1 and the v. portae in 1 case. 10 out of 16 occlusions were associated with an indwelling catheter. underlying dieseases were sepsis (4), prematudty (3), vitiurn (2), asphyxia (1), short bowel syndrome (1), hus (1), diabetes (1), cmv (1), exsiccosis (1) and m. hirschsprung (1). thrombolysis was performed with an bolus of rt-pa (0.1-0.2 mg/kg) followed by continuous infusion (0.8-2.4 (-9) mg/kg/24h, median 1.8 mg/kg/24h). low dose hepadn (100 ie/kg/24h) was given dudng full dose hepadn (aptt 1,5-2 times normal) after the thrombolysis. in 5 pts. rt-pa was administered locally through the catheter and in 11 cases systemically. in 13 patients the vessels could be recanalised completely, in 2 partially, in 1 patient the therapy had to be discontinued. in 2 vessels a reocclusion occurred. bleedings were noted in three patients, all from recent venous puncture sites. the results encouraged us to start a multi-canter trial which has been approved by the ethical committee and is open for recrural. the aim is to compare efficacy and safety of rt-pa with urokinase, the only recommended standard in the management of critical major vessel obstruction in newborns and infants. the design is a randomised, notblinded trial with a cross-over option after three days in cases without success. study end points are recanalisations, major bleedings and number of cross-overs. inclusion criteria are age under 1 year, lifethreatening vessel obstruction, age of thrombus up to 10 days, no precaeding fibdnolytic therapy. exclusion cdteda are cerebral hemorrage, pedventricular leukomalacia, surgery dudng the last 7 days and cns injuries during the last 2 months. although our knowledge on inherited thrombotic coagulation disorders has greatly expanded within the last years, there are still man}, patients with recurrent venous thrombosis in whom no obvious predasposition can be identified.thus we decided to include also so-called rare defects associated with thrombosis in our routine thrombophilia screening programme, such as fxii deficiency. fxii is an important element m the intrinsic pathway of fibrinolysis and there is evidence for an insufficient fibrinolytic activity in fxii deficient pts..up to date only few and controversial data exist about the frequency of fxii deficiency in pts. with thrombophilia. cons~uently the aim of our study was to evaluate the association between fxii deficiency and juvenile venous thrombosis in a great population. patients and methods: 1554 pts. (851 female, 703 male, aged i to 61 ys, median age 38.2 ys) with venous thromboembolism before the age of 45 ys were studied. one-stage clotting activity assay of fxii (fxii:c) was performed on acl using fxii deficient plasma from instrumentation laborato~. fxii antigen concentration (fxii:ag) was measured by electroimmundiffusion using reagents from behfingwerke, enzym research respectively. the normal ranges are tl~. routine reference values obtained m our labratory from 80 healthy subjects (40 males, 40 females, median age 26.2 ys); 95% range: fxii:c 53-135%, fxii:ag 57-132%). results: 122/1554 pts.were classified as fxi1 deficient (f 60, m 62), giving a prevalence of 7.8%. severe fxii deficiencies with fxii:c below 1% were observed in 7 pts..ll5 pts= proved to have moderate fxii deficiency with fxihc ranging lrom 2 to 51% and fxii:ag ranging from 1 to 53%. in none of them inherited deficiencies of other well established thrombophila risk factors could be detected. none of the fxii deficient pts. had positive lupus anticoagulant tests. familial fxii deficiency was found m 9 cases. discussion and conclusion: the precedences of fxii deficiency amongpts, with venous thromboembolism was previously described to be 7.5-10%. supporting these data, we have shown a praevalence of fxii deficiency of 7.8 %. in comparison to the frequency of other well established thrombophila risk factors we consequently have observed a relatively high prevalence of fxii deficiency m our study group.these data, from the largest such study reported, strongly indicate that fxii deficiency may not be a rare deficiency and may be more frequently associated with thrombosis than currently suspected. we describe a family with an exceptionally rare, i.e. plasminogen, deficiency, combined with subnormal activities of coagulation factor xii (hageman factor). the first thromboembolic event, pulmonary embolism in the proposita was diagnosed at age 35. since that time, 'spontaneous' venous thromboembolic events verified by phlebography and perfusion/ventilation lung scan recurred once every year despite oral coumarin therapy, whose intensity varied over an exceptionally wide range despite tight control the patient was repeatedly given succesful thrombolytic therapy with streptokinase or recombinant tissue plasminogen activator. her plasma plasminogen chromogenic activity was 51-59 % compared to a normal plasma pool (reference range 70-130 %), plasminogen antigen was diminished to the same extent. the patient's factor xii exhibited only 28-55 % activity in a factor-deficient plasma assay as compared to a normal plasma pool. other known risk factors for recurrent venous thromboembolism were not present : no evidence of malignancy, no obvious precipitating events, normal values of antithrombin iii, protein c, protein s, fihrinogen, thrombin time, platelets, lupus-like anticoagulant, aptt prolongation after addition of activated protein c. the proposita's mother had died at age 60 from pulmonary embolisnt no coagulation studies are available. the proposita's sister was first diagnosed deep leg vein thrombosis at age 17, since that time recurrent episodes of venous thromboembolism have been diagnosed also in an other hospital. this sister's plasminogen activity was 120%, but factor xii activity was reduced to 55 %. three brothers of the proposita were examined, too, all in their 3rd decade of life. none of them recalled symptoms of or treatment for thromboembolic disease. in one brother, factor xii activity was normal (100-105 %), but plasminogen only about 50 %. in the 2nd brother, factor xii was very variable (64, 42 and 94 %), plasminogen was in the lower normal range, in the 3rd brother, factor xii was about 50 % (repeatedly), plasminogen was normal. current knowledge about the risk of thromboembolism with both enzymes is limited, the optimal management remains controversial. msrgit serbsn,maria cucuruz,dan madras,carmen petrescu, natalie rosiu,rodica costa iii rd psediatric clintc,universtt v of medicine, the unsatisfactory efficiency of entihepetitis b vaccination in our haemsphiliscs suggested the control of the immune status in 52 hiv negative patients,by establishing through flowcitomstrie with monoclonsl antibodies the lymphocyte subsets (cd3,cd4,cds,cd&/cd8 ratio and cd19) and by seric tmmunoglobulins levels; the immunological parameters have been correlated with the serological markers of hepatitis infections (hay, hbv,hcv ebd hdv) as well as on dependence with the treatment (blood,plasma,crysprecipitate,fector viii/ix concentrate) and the quantity of their consumption (ui/k9 weight/yesr).the interpretation of the results pointed out • significant lower level of cd3,cd& (p<o,o01) st the group of hsemophiliscs treated with blood,plasma and cryopraclpitate; sdditionsly,in this group of patients it was found s significant drop of the cd19 counts (p< 0,001) without an immunoglobulin-deficiency. the same behaviour was found at the group of haemophiliacs carriers of hb~ and hcv markers.the superpositisn of hepatitis infections on the treatment with blood and native blood products in most of the cases has let unanswered the question related to the mechanism of the immunodeficiency: the hepatitis b or c infections or the negative impact of the itarative important alloantigenlc load ? illumination with l~tm methylenblue (mb) is used for inactivation of enveloped viruses in human plasma. via a photooxidative mechanism viral structures as well as plasmaproteins especially fibrinogen can be damaged. fibrinogen is an important mediator in the cell-cell interaction of platelets. the four rgd sequences (arg-gly-asp) of fibrinogen can be recognized by the activated fibrinogenreceptor gphb/iiia . after binding to gpiib/iiia one molecule fibdnogen can link two platelets. the aim of this study was to investigate the influence of mb treatment on the fibrinogen activity in human plasma. furthermore we were interested to demonstrate if altered fibrinogen inhibit platelet aggregation by blocking the gpiib/]iia receptor. human plasma was treated with 1-50~m mb. after illumination fibdnogen was isolated by fl-alanin precipitation. purified fibdnogen was analysed by one-and twodimensional gelelectrophoresis. influence of modified fibrinogen on platetet aggregation was measured with gelfiltrated platelets. possible receptorblockade were examined in an 125j-fibrinogen competition assay. densitometric scanning of reduced sds-pages showed a decrease of the a-subunit as well as an increase of high molecular aggregates with increased mb concentrations. this effect was not detectable for fibrinogen isolated from plasma with 1bm mb. this results were confirmed by two-dimensional gelelectrophoresis. ibm mb treatment showed no receptor blockade in aggrement with normal platelet aggregation after stimulation with adp and fibrinogen. proposed validation of a quantitive quality-assured pcr method f. dorner, j. eibl, g. zerlauth immuno ag we have developed a highly sensitive and reliable quantitative method, based on the polymerase chain reaction (pcr) and on the reverse transcriptase polymerase chain reaction (rt-pcr), to screen for the nucleic acids of human immunodeficiency virus type 1 (hiv-i), hepatitis b virus (hbv) and hepatitis c virus (hcv) in plasma and plasma products. the detection of pcr products is carried out by laser induced fluorescence, the procedure is therefore termed "laser induced fluorescence pcr" (lif-pcr). the procedure allows the precise quantitation of genome equivalents due to the use of two calibrators that are co-amplified by the same set of primers as the target template in one and the same test tube. a high degree of reliability is achieved by co-precipitation of the extract, co-amplification and co-determination of the calibrators together with the genome equivalents to be determined. in this report we present validation data for the lif-pcr and discuss them in the light of the recently published ich-guidelines on the validation of analytical procedures. taking into account the pecularitles of the replacement therapy of haemcphiliacs in romania the study aimed at evaluating the dimension and profile of blood-born infections in these patients; 142 hsemophlllacs,elasslfled in 3 groups: a-ulth no exposure to blood or bloodproducts, b-treated ~ith factor-concentrates and c-ulth plasma,cryoprecipitate + factor-concentrates in their therapy have been investigated (elisa method) for serological markers of hepatitis a,b,c end d, cvtomegalovirus (cmv) and hiv i and hiu 9 infection. the infections profile of our haemophiliacs~hes some peculsrities. the hepatitis infection is the most extended,lnvolving more than 75% of the patients. the frequency is for hepatitis -bz,z~ , u -"/~,~ , g -bb,7% end o -24% : the multiple infection was diagnosed in more than 50% of cases. the prevalence uas significantly hlgher in the group c. we studied blood count and relevant coagulation system parameters of autologous blood donors. indications for autologous blood donation were divided in 4 categories: a group of diverse (a, n=13), reduction mammoplasty (b, n:5), prostatectomy (c, n=io) and total hip replacement (d, n=74). we performed bleod count (bc), thromboplastin time (tt), partial thromboplastin time (aptt), fibrinogen (fib), f vhi act, vwf rco, vwf ag, f xiii, at iii, d-dimer-fragments (dd) and fibrinmonomers (fm). all parameters were analyzed as initial value, bc, tt, aptt, fib, f xiii and at iii were measured additionally at each donation. the percentage of initial pathologic data was (1) were included in the study. the inclusion criterittm was: an endegen(y.~ red cell reserve (ercr) of less than 400 ml in men and less than 540 ml in women. ercr is the volume of blood that can be withdrawn up to tim hematocrit of 0.34. the selected dosages of rhepo (200, 400 and 800 iu/kg body weight biweekly over 4 weeks) were based on the calculated blood volume and the ercr. a unit of whole blood, separated into an autologens red cell concentrate and one unit of fresh frozen plasma, was collected when ]mnoglobin concentration was at least 11.5g/100 ml (or hematocrit was at least 0.34) up to an autologons predeposit of 800 ml red cell volume (rcv). additionaly, platelet concentrations and serum ferritin levels were measured~ results: at the initial clinical examination the ercr was 333 ml in women and 307 in men. this resulted in a weekly dosage of 2x32.000 iu rlaepo in women and 2x25.000 iu rhepo in men. 7 patients donated 5 red cell each (858 ml rcv on an average) within 19.1 days. the targeted rcv could not be reached in 3 patients because the operation was reschednled. the collection from one patient had to be aborted because of angina pcetoris complains. tic following side-cffczts occurred: episodic hypcrtejlsion (2 tim~), chest pain (5), fafigne (8), inflnenza-lihe syndrome (3). platelet count increased from initially 203 4-68 x 10'//d (i00 %) during therapy to a maximum level of 260 + 93 10v~ (128 %) without any clinical signs of thrombeembolism. in the control group (autologous blood donation weekly 3 or 4 times without erythrupoiedn stimulation, n=12) platelets increased from initially 230 __+ 73 10'/fi (100%) to 255 + 71 10v#i (110%) i1]aximlzm colldttsions: the adlninist/'ation of lhepo in the above described manner to autologous donors undergoing heart surgery is well tolerated. it allows to collect a sufficient number of autologous blood units and the preparation of a predeposit at short notice. essential thrombocythemia is a chronic myeloproliferative disease with a benign clinical course that is mainly complicated by microcirculatory or thrombotic vascular disorders. it is at present uncertain which patients need (lifelong) therapy and whether cytoreduction is necessary. we retrospectively evaluated vascular complications in 128 patients (33 men, 95 women) with a median age of 61 years in relation to therapy. three major treatment groups were analysed: 17 patients treated with acetylsalicylic acid (ass) alone, 29 patients given alkylating agents, and 62 patients treated with interferon alpha (ifn). there was no significant number of patients treated with hydroxyurea. ifn-treated patients were younger (median age 54 years) than patients in the other groups, but platelet counts before therapy were similar (median 0.84-1.13 x 1012/i). before therapy, 47% of patients treated with ass, 45% treated with alkylating drugs, and 32% treated with ifn had bleeding events, microcirculatory or thrombotic complications. dudng therapy, median platelet count was 0.9 x 1012/i in patients on ass, 0.68 x 1012/i in patients treated with alkylants, and 0.48 x 1012/i in ifn-teated patients. the rate of patients with complications during therapy was 18% (9.5%/patient year) for ass, 14% (6.3%/patient year) for patients on alkylating drugs, and 15% (6.2°/dpatient year) for ifn-treated patients. we conclude that all three treatment regimes seemed to have a beneficial effect on the incidence of vascular complications in essential thrombocythemia. the results give a rough estimate of complication rates to be expected and may form the basis for randomized clinical trials. adhesion of activated platelets to leukocyte* has been shown to be mediated by at least two molecules on the platelet surface: p-selectin (cd62) and fibrinogen (fg) bound to the gp iib/iiia complex. interaction of platelets with teukocytes via cd62 is believed to stimulate the production of reactive oxygen species (ros) in leukocyte*. the role of platetet-bound fg as a signalling molecule in platelet-leukocyte interaction is controversially discussed: stimulatory as well as inhibitory effects on leukocyte ros production has been described. in the present study we measured ros production in undiluted samples of citrated whole blood (wb) as well as in suspensions of erythrocytes and leukocytes in platelet-rich plasma (platelet-rich wb) or platelet-poor plasma (ptatelet-poor wb). ros production was determined by measuring luminol-enhanced chemiluminescence (cl). stirring wb samples at 1,000 rpm for 10 min resulted in an activation of leukocyte* (increase of cl, upregulation of cd1 lb) and platelets (platelet aggregation, upregulation of cd62). stirring-induced cl was significantly higher in platelet-poor wb when compared to platelet-rich wb. higher cl in plateletpoor wb than in platelet-rich wb was also found when adp (moderate stimulation of platelets and leukocyte*) or fmlp (strong stimulation of leukocyte*) but not when pma (strong platelet and leukocyte stimulation) was added. dextran sulphate that is known to block cd62-mediated interaction ofplatelets with leukocyte* caused a dose-dependent inhibition of stirring-induced cl in wb samples. in contrast, when binding of fg to the platelet surface was blocked by rgds or the fg receptor antagonist gr144053, stirring-induced cl, but not cl in unstirred samples, was significantly enhanced. gr144053 also reduced adhesion of platelets to neutrophils, but did not affect cd62 exposure. the results indicate that interaction of platelets with leukocyte* in whole blood via cd62 and fg may have opposite effects on leukocyte ros production: stimulation by cd62 and inhibition by platelet-bound fibrinogen. late reocclusive processes represent one of the most commonly seen serious adverse events after coronary angioplasty. the pathophysiology of both of these processes is multifunctional and represens complex time dependent pathophysiologic events involving both the humeral and cellular processes. to test the effects of low molecular weight heparin (certoparin, sandoz ag, numberg germany) against late occlusive processes, four groups (groups a-d) of rats (7-10) were anesthetized and the external common carotid arteries were exposed. all groups received a 200 u/kg of unfraetionated heparin through the femoral vein. a 2f balloon emboleetomy catheter was inserted into the left carotid artery and passed throug the left common carotid artery leading to the aortic arck vascdar injury was induced by repeatedly (x3) inflating the catheter balloon and withdrawing it to the bifurcation. following this procedure, two hours after, equlgravimetric amounts of various agents were administered via sc route to each of the individual groups. group a was administered with certoparin od for 21 days. group b received a lower low molecular weight heprin (mr 3.4 kda). group c received unfraetionated heparin and group d served as a control. on day 21, perfusion fixation was performed on all animals. the injured and contralateral carotid arteries were excised for histelogic examinatior~ in comparison to the saline control, varying degrees of the inhibition of myointimal proliferation following arterial injury was noted in all groups. however, the degree of inhibition followed the order: heparin<certopann<llmwh_ in contrast to heparin treated group (c), the other groups a, b, and d did not exhibit any hemorrhagic lesions. however, degree of perivascular fibrosis, hemorrhage and inflammation were observed at the infusion site. in comparison to heparin, certoparin and its lower low molecular weight derivatives exhibit better efficacy in reducing balloon angioplasty induced restenosis. the observed efficacy of these agents may be due to better bioavailability and sustained duration of action. in the present paper the binding of heparin to granulocytes of human and animal species was analyzed and compared with the binding to monocytes and lymphocytes. a low-molecularmass heparin (lmmh) was covalently bound to tyramine (tyr) and subsequently tagged with fluorescein-5-isothiocyanate (fitc) by endpoint attachmenc. binding to leukocytes was quantified using flow cytometry analysis. the leukocyte populations were identified by their light scatter properties in man by cd4, cd13, and cd14 antibodies, in mice by cd 4 and gr 1 antibodies, and in rat by cd 45, ed 9, and rp 3 antibodies. granulocytes, monocytes and lymphocytes of all species bound dose-dependently lmm-heparin. the binding of lmmh-tyr-fitc ~to granulocytes was higher in human, rat, rabbit, dog, and pig as compared to monocytes and lymphocytes. mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. binding of lmmh-tyr-fitc was reversible in the presence of unlabeled heparin or lmmh. more than 99% of human, rat, rabbit, dog and sheep granulocyte populations could be'distinguished from monocytes and lymphocytes by their fluorescence intensity after incubation with lmmh-tyr-fitc. this separation was not obtained for mouse and pig granulocytes. the data present evidence of a specific heparin binding to granulocytes of many species and indicate the significance of fluorescent labeled lmm-heparin for biological investigations. background and purpose: ptca is an effective treatment for restoring coronary artery pat~cy; however, acute thrombotic rencclusion is a drawback of the procedure. currem prevention of rencclusion relies on antiplatelet agmts (aspirin) and antithrombin agents 0aeparin). in the present study we examined the time course of changes in blood coagulation and fibrinolytic function following ptca in patients with ischaemic heart disease. 20 patients were randomly assigned to receive either standard therapy (250 nag aspirin p.o.; 5.000 iu hepafin i.e. bolus, and 5-10.000 iu heparin intra-proeadurally)(n=10), or adjunctive therapy (aspirin, hepann; 100 mg sodium pmtosan polysulphate, spp intra-cortmarily and subcutaneously) (n=10). the sensitive markers of thrombin activation (thrombin-amithrombin iii complex ('rat), prothrombin fragmem f 1+2 (pl+2), d-dimer) and of fibrinolytic activation (plasmin-antiplasmin complex (pap), tissue type plasminogan activator (t-pa) and plasminogen activator inhibitor (pal)-i activities) were measured before, immediately after, and 6 hs after ptca. results: we found elevated mean levels of tat and fl+2 in both groups during the procedure, while plasma coneentrafiuns of d-dimer and pap remained in normal range. activity of pai-i was not elevated compared to normal comrol. the t-pa activity obtained immediately post-ptca showed significant increase in patients given spp. primary angiologic success was achieved in 80% of both groups. one patient in each group had recurrent ischaemia and emergency interventions (angioplasty, stent, bypass surgery) were performed. one patient receiving adjunctive therapy died in cardiogenic shock within 24hs arer ptca. major bleeding was observed in one patient given spp. conclusion: no significant intracoronary haemostatic activation occurred in the majority of patients during and up to 6hs after ptca. treatment with spp was associated with enhanced fibrinolytic activity. the benefits and risks of adding spp to heparm and aspirin should be evaluated by further studies, in july 19,¢1 the paul ehdich institute became responsible for quality and safety of bt~d and blood products. since ~his time, batch m~se of ppsb and factor ix prapamtions were carded out regularly, whereas ihe obligatop i testlng of all other products derived frem pooled plasma will be starling in janualy 1996. the european pharmacopoeia does .or contain directions for the quality control of ppsb preparations. therefore, ifua ~ coneenning factor ix clotting concentrates are applied, ircludmg the non activated ptt and the classical tkrombln test these m~ods are not su'rlable for reliable exclusion of ituombogenk~b/ ,of pp,sfl, because vlla actlvry is not d~. a hllhedo unsolved problem is the high sensitivity of file classical thrombin test often not correlating with ~ data of thrombogenlci~. in ordm td es~abl~ an effective quality conlrd protocol, different methods for the qual~ative detennlna~on of ila, vlla. ixa, and xa (chrornogenic, fluorege~c clolfing reactions) wera instaued. in agreement with the [deralure, considerable differences were found among the ppsb pr~ons registered in germany. cedain amounts of activated do~ng racers may be well tole~ted in redp/ents. ~no are in a steady state condition. however, in severely ill patienls, eg. post operatively, tissue factor may be expressed which pofenllatesthe dsk of ~aclor vlta. in the case of a certain ppsb preparation hlgh ¢onceatratio~ of acthrated cto~ng factors, especially ~vated factor vii, cotmlaled with fatal comlffcal~ns, evaluating ~ expe~ concenlralbn limits for adjvated prothrombin co~np~.x factors as prerequisite for ~e batch r~ease have to be applied. aprotinin is used as a hemostatic agent in cardiopulmonary bypass surgery and other indications where hemostatic compromise results in hemorrhagic manifestations. the mechanism by which this agent produces hemostatic effects is not known, however, inhibition of serine proteasas such as plasmin, kallikrein and dastase and modulataion of platelet receptors have been considered as possible target sites. to determine the effect of aprotinin on tissue factor mediated activation of human platelets, whole blood samples were drawn at baseline and 24 hours aider aspirin (650 mg po) ingestion (n=5). blood samples were supplemented with saline and aprotinin (40 ug/ml) and tissue factor activation was studied at 200 pg/ral. platelet activation was investigated using gtycoprotein ffla and gmp 140 specific antibodies on a flow cytometer (epic coulter). tissue factor was found to produce the activation of both the baseline and asp' "mnized platelets. aprotinirt augmented the tissue factor activation of platelets in both the control and aspirin supplemented groups. however, the relative increase in the aspifinized group was much monger (25% of the baseline). these results suggest that aprotinin augments the tissue factor mediated activation of platelets in both the normal and aspirinized individuals. while these effects may result in hemostatic restoration in aspirin compromised patients, in normal individuals the clinical implications of this effect remain to be clarified. , 10-20 (group 2) or >20 years (group 3) duration. anticoagulated whole blood was incubated with fluorescent antibodies to gpib and gmp-140 (two colour method) and analyzed with a flow cytometer. thrombomodulin, f1+2, protein s, 13-thromboglobulin were measured according to standard procedures. results: surface expression of gmp-140 was not different in groups 1 to 3, however, there was a tendency to higher acitvation in group 1 (<10 years iddm). results for thrombomodulin, f1+2, protein s, 13-thromboglobulin will also be presented. conclusion: though it did not reach statistical significance, platelet acitvation seems to be more important during early diabetes. this wilt be correlated with endothelial and plasmatic activation markers. in our clinic four patients with hiv-related thrombocytopenia were treated with a lot of gammagard (93f21abllf), which later turned out to be hcv contaminated. before infusion all patients were negative for hcv antibodies and hcv rna. 2 to 8 months after infusion 2/4 patients, who suffered from arc at the time of hcv infection with cd4 counts >100/pl, seroconverted, whereas in the two other patients, who suffered from aids with cd4 counts below 100/pl, there was no seroconversion. in all cases hcv rna was found. genotyping with inno-lipa (innogenetice) showed hcv genotype l(b) in all patients. liver enzymes and hcv rna copies were measured repeatedly over a period of one year after infection. the 2 patients with arc showed a strong increase of hcv rna titre during the first 3 to 4 months after infection, followed by a rapid decrease within the next months. in the patients with aids hcv rna copies increased moderately within the first 4 to 6 months, followed by a slow decrease. elevation of liver enzymes was mild in the aids patients and seems to be independent from the hcv rna titre. in the arc patients liver enzymes changed parallel to hcv rna titers with a delay of 2 to 3 months. the course of hiv infection was only slightly influenced by the acute hepatitis c as measured by cd4 counts, i%2microglobulin and hiv rna copies. introduction:mechanisms underlying ischemia/reperfusion injury have been thoumughly investigated in experimental models. leucocytes appear to play a main role through production of cytokines and overexpresssion of adhesion molecules. in experimental animals, administration of monocional antibodies (mab) recognizing cd18 can reduce organ injury following ischemia/repedusion. no data, however, have been reported concerning clinical ischemia situations. patients and methods:we investigated expression of cdt8, cd1 la, cdf l b and cd1 lc in granulocytes, monocytes and lymphocytes from peripheral blood of five patients undergoing elective hand surgery. the tourniquet was applied on the upper arm and heparinized samples from cubital veins were obtained before and at the end of ischemia. control samples were drawn from the nonischemic contralataral arm with the same timing, duration ot ischemia ranged between sixty and one hundred minutes (80~16). whole blood samples were incubated with specific, fluorochmme labelled antibodies and analyzed by fluorocytometry (facscan, becton dickinson, san jose, ca). mean fluorescence intensity (mfi), quantitatively reflecting surface expression of the indicated markers was evaluated for the individual cell populations. data were compared by the paired student's t-test, p<0,05 was evaluated as significant. results:mfi for all markers was comparable in all cell populations in samples obtained before ischemia from both arms. in contrast, expression of cd18 was significantly enhanced in granulocytes (321_+50 vs. 189_+38), monocytes (653-+54 vs. 426+122) and lymphocytes (299_+45 vs. 228-+36) from samples derived from the ischamic arm, as compared with the nonischemic arm, as measured at end of ischemia. at the same time, an increase of cdf lb on granulocytes (500~_342 vs. 213+150) and monocytes (533+359 vs.237-+206) but not on lymphocytes was found, no modifications of cdlta and cdttc expression could be observed. there was no correlation between duration of ischemia and quantitative expression of these markers, conclusions:our data indicate that relatively short ischemia periods induce an increased expression of ~2" integrins adhesion molecules on leucocytes. these results suggest, at close similarity with findings from expodmental models, that overexpression of adhesion molecules might play an important role in the induction of ischemia/reperfusion injury, in humans. in patients suffering from chronic inflammatory bowel diseases, such as morbus crohn and colitis ulcerosa, we observe massive, sometimes barely staunchable bleedings. hereby, the deficiency of coagulation factors, especially of factor xiii in plasma is established. ttowever the influence of factor xiii on the pathomechanism of the underlying disease is still under discussion. therefore we studied the f xiii content in the intestinal mucosa. an immunohistochemicat method was developed using commercially available antibodies against f xiii subunit-a, the detection of mucosal factor xiii depends on the amount of chromogen bound to the antibody-horseradish-peroxidase complex. with this method, it is possible to locate but not to quantify f xili in the intestinal tissue. therefore we developed an elisa-metbod in homogenized intestinal tissue, using commercially available antibodies. its precision was validated using a standard curve with commercially available factor xiii preparations (fibrogemmin®). the detection limit of this method is > 0.05 i.u. f xiii/ml of tissue solution. freezed dried intestinal tissue (lmg) was homogenized in 1 ml buffer using a potter. specimens of the large bowel revealed f xiii values of 0,21 + 0,0038 i.u. (x __+ sd), tissue solution. with this method it is possible to quantify tissue-bound faxtor xiii. studies are in progress to elucidate the content of f xiii in the intestine of patient's suffering from infammatory bowel diseases in order to contribute data to the pathomechanisms of f xiii deficiency. in a previous double-blind, controlled trial we were able to show that aprotinin administration has significantly contributed to reduce periand postoperative bleeding complications without increasing the risk of thromboembotic complications. the question arises whether this beneficial effect may be associated with its effects on intraoperative fibrinolysis. therefore, 20 patients were treated with or without aprotinin (2 million kiu loading dose over 15 minutes followed by 500,000 kiu per hour), and citrated blood samples were obtained at the following time points: before operation, after induction of the anesthesia, at the beginning of operation, intraoperatively when the femur shaft was implanted, and 24 hours postoperatively. the determinations of plasmin/antiplasmin-complexes, d-dimers, thrombin/antithrombin iii-complexes, and prothrombinfragments 1 +2 were performed by means of test kits from behring, germany (enzygnostrpap micro, enzygnost r d-dimer testkit, enzygnost r tat micro and enzygnost r f 1 +2 respectively). -all markers of activated fibrinolysis and blood coagulation were significantly increased in the groups with and without aprotinin treatment, the highest activities to be seen when the femur shaft was implanted. however, the values of pap and d-directs of the aprotinin group were below the values of the control group until the end of operation. the markers of activated coagulation showed the opposite effect, however the differences between the two groups were not significant. as expected, the aptt was significantly prolonged in the aprotiningroup. the aprotinin treatment was also associated with a significantly lower blood loss in these patients. -concluding it can be said it is not clear whether the blood saving effect of aprotinin may be exclusively attributed to its antiplasmin activity since the differences of the fibrinolysis parameters were not statistically significant. further blood samples should be analysed between the implantation of the femur shaft and the end of operation. in our laboratory large amounts of human prothrombin are required (30-50 mg/week). as we try to produce meizothrombin and meizothrombin-des-fragment-1 from human prothrombin and to apply it as an antidote for hirudin, the classical adsorption to barium sulphate or aluminum hydroxide from human plasma cannot be used. commercially available human prothrombin is expensive and of an unacceptable quality for our applications. in most of these batches we found small amounts of factor x and prothrombin activation products. we now developed a procedure to isolate prothrombin from "prothrombin complex concentrates" (ppsb-250-bulk, drk-blutspendedienst nds.). the concentrate also contains fac-tor vii, factor ix, and factor x. the prothrombin had to be separated from these factors. the concentrate we used contained amounts of other proteins and activation products of prothrombin (e.g. prethrombin-1) as well. for the preparation of prothrombin from ppsb we used anion exchangechromatography (resource-q ®) on an fplc ®. we applied dissolved ppsb directly or after buffer exchange on sephadex g-25 onto the column at room temperature. the prothrombin was eluted with an naci-gradient in trisodium citrate buffer, ph 7.0. the buffer conditions are similar to the conditions used in the preparation of ppsb. the quality of the prothrombin so obtained was sufficient for most of our experiments. a second purification step on ion-exchange resulted in a 99% pure product devoid of contaminating factor activities and activation intermediates as examined with coomassie and silver stained sds-page electrophoresis and assays for factor x. this prothrombin contained full enzymatic activity and its activation by specific snake venom prothrombin activators showed the known activation products. we are now able to isolate the amounts of pure prothrombin required for preclinical investigations. most of the commercially available lmwhs such as enoxaparin, fraxiparin, and fragrnin are prepared by chemical methods which can result in desulfation and other chemical modifications of the internal structure leading to differences in the pharmacologic effects. on the other hand, tiactionated lmwhs retain their native characteristics and are structurally similar to heparin. in addition, the oligosaocharide sequence responsible for atiii binding is not modified. physical methods such as gamma irradiation (~co) have been used to fi'agment sulfated glycosaminoglyeans yielding fragraents without chemical modifications (deambrosi et at. in : biomedical and biotechnological advances in industrial polysaccharides, pp. 45-53). utilizing this technique, depolymerized heparius exhibiting different molecular weights can be obtained. this communication reports on the biochemical and pharmacologic effects of several such depolymerized heparins to demonstrate the molecular weight dependence on biologic activity. fragments exhibiting molecular weights of 5, 7, 8, and 9 kda were prepared by exposing concentrated heparin solutions to a rectilinear gamma ray beam at intermittent doses of 2.5 to 25 mrad under controlled temperatures. unlike the chemically depolymerized heparins, these fractions did not exhibit any decrease in charge density or atiii affinity. in routine assays for heparin, a clear cut molecular weight dependance on the anticoagulant and antiprotease actions was observed. on a gravimetric basis, these agents produce superior antithrombotic actions in comparison to chemically depolymerized derivatives. these studies suggest that gamma irradiation can be used to prepare lmwhs which retain their molecular integrity and therefore may prove to exhibit a more comparable biologic profile to hepari~ futthermore, lmwhs produced by gamma irradiation lack the usual double bond fommtion which requires the use of additives which can alter the product profile. university hospital, dept. of angiology, frankfurt a.m., germany introduction: thromboembolic disease constitutes a major clinical problem and among others a defective fibrinolytic system has been suggested as a predisposing factor for the development of thrombosis. the plasma fibrinolytic system can be impaired by inherited deficiencies of plasminogen defective release from the wessel wall tissue plasminogen activator (t-p'a) or by high ptusma levels of regulatory proteins, such as plasmino-8en. activator inhibilors (pal). the aim ....... of the present study w~s to eshmate the prevalence of decreased fibnnolyl~c actwlty m young pls. with thrombophilia. patients: a great population of 884 pts. (fenmle 478, male 406; age 21-61 ys median 39.8 ys) with venous thromtx~emolism before the age of 45 years were investigated in regard to their plasma fibrinolytie system. in none of them well established thrombophilia risk factors could be identified previously. methods: plasminogen ~behdngwerke), pai-1 activity (ehromogenic assay, biopool), pal-i anugen coneentration (elisa, biopool), t-pa activity (chromogenic assay, biopool) and antigen concentration (elisa, biopool) were measured before and after venous oeclusion.vo was performed z 12 month after the last thromboembolic epi~xle. 24 healthy subjects (median age 24.7 ys) served as controls. results." 24 pts.(2.7%) were classified as plasminogen deficiencies (activity and antigen). 142 pts.(16%) had significantly elevated levels of pal activity (up to 120 u/ml) and pal antigen (up to 90 ng/ml). none of the pts. with high pal levels had laboratory signs of acute phase reaction. low t-pa activity could be demonstrated and confirmed in 121 pts., aecordingto a prevalence of 13.6% (range: 0-2.7 u/ml; reference limils: 2.8 -21.8 u/ml). however, there was a significant negative correlation between t-pa activity and pal values. in 67 pts. (55.4%) the low t-pa activity was associated with increased pal levels whereas the t-pa antigen concentration was normal. a parallel reduction of t-pa activity and t-pa antigen (range: 0.35-3.5 ng/ml; reference limits: 3.6 -21.0 ng/ml) were determined repeatedly in 54 pts. (f 23, m 31, median age 39 ys). thus, the prevalence of a defective t-pa release was 6.1% in our study group. conclusion." in comparison to the frequency of inherited deficiencies of other well established thrombophila risk factors we have observed a relativel~ high prevalence of diminished t-pa activity, elevation of pal respectively in our study group. our data strongly indicate that besides t-pa and pal acuvity, antigen concentration for both parameters should be determined in pts. with thrombophilia. the antithrombotic and anticoagulant effect of the supersulfated low molecular weight heparin ssh 14 was studied after i.v. and s.c. administration in rats. thrombus formation in the jugular vein was induced by i.v. injection of activated human serum and following stasis for 20 rain and was assessed by a thrombus score ranging from 0 (no thrombus formation) until 3 (complete thrombus formation). ssh t4 injected either 10 min (i.v.) or 30 rain (s.c.) before thrombus induction caused a dose-dependent antithrombotic effect in a range from 0.25 to 2 mg/kg i.v. and 1 to 4 mg/kg s.c. there were clear differences in the antithromboric effectiveness between female and male animals, i.e, in female rats antithrombotically effective doses were lower than in male rats (edh0 after i.v. injection in females 0.35 mg/kg, in males 0.9 mg/kg). the sex differences were confirmed in studies on the time course of the antithrombotic effect. after i.v. injection of fully effective doses (2 mg/kg i.v. and 4 mg/kg s.c., resp.) the antithrombotic effect disappeared after 8 h in female or after 4 h in male rats. for studies on the anticoagulant action blood was drawn from the femoral artery and after centrifugation global clotting assays were performed in plasma. similar to its antithrombotic action ssh 14 also caused doseand sex-dependent anticoagulant effects. the most sensitive assays were the aptt and the heptest; thrombin time and prothrombin time were less or not influenced by ssh 14. in conclusion, ssh 14 was found to be an effective anticoagulant and antithrombotic agent in experimental studies in rats. at present there is no explanation for the clear sex differences found in this species. venous thromboembolic disease is the most frequent complication in patients undergoing total knee replacement therapy. patients and methods: after informed consent 3x30 patients were included in an open randomized clinical study and the incidence of venous thromboembolisrn was examined using different regimes for heparin prophylaxis (30 patients received fraxiparin 36 rag once daily, 30 patients clexane 40 once daily and 30 patients 7500 u calciparin twice daily). there were no differences between the groups concerning age, sex, body weight, risk factors, surgeons, decrease in hemoglobin~ and requirements for blood products. pre surgery, day 1, day 5-7 phiebograms were performed and also tat, dimers, fl+2 prothrombin fragments were examined. results: 1., dvt in 26 patients (28.9%). dvt in 5/30 patients under calciparm prophylaxis, 8/30 patients under fraxiparin and 13/30 patients under clexane treatment. 2., low speciflty (3.4%) of dimers and tat (24%) for detecting a dvt in these special patients undergoing knee replacement therapy, elevated fi+2 fragments in the dvt group at ti and t2 vs the patients without dvt (t1 dvt: 3.24+-1.8 vs. 1.6+-0.3 -p= 0.0042). 3, only 8/26 patients (31%) with dvt had clinical signs of thrombosis. conclusions: 1., there is an increase of thrombin gneeration measured by tat and dimers after knee replacement therapy. there are further studies with more patients necessary to confirm that fl+2 prothrombin fragments can discriminate between patients with and without dvt from a clinician's point of view. 2., phlebographicauy confimled dvt in almost 30% of our patients demonstrate the high thromboembolic risk in these patients. von willebrand's disease (vwd) type 2 is characterized by absence of high molecular weight muitimers. qualitative changes in the structure of the molecule might be associated with enhanced binding of von willebrand factor (vwf) to platelet glycoprotein lb. therefore in some patients vwd type 2 is associated with severe thrombocytopenia. here, we report on a 9 year old boy who presented with severe purpura and platelet counts about 20000/gl at the age of 2 years. thrombocytopenia did not respond to corticosteroids. a normalized platelet count of short duration was observed after high-dose immunoglobulins. in addition, increase of platelets was seen after anti-d treatment. thus, although platelet associated antibodies were not detected, thrombocytopenia seemed to be caused by an autoimmune mechanism. despite platelet counts above 50000/gl, the patient experienced severe bleedings with a significant decrease of hemoglobin levels. therefore, he needed several transfusions. coagulation analysis revealed vwd. application of ddavp lead to a normalization of partial thromboplastin time (ptt) and an increase of factor viii with subsequent cessation of bleeding symptoms. recently, vwd was typed 2 by lack of high molecular weight multimers. in conclusion, we report a case with vwd type 2 responding to ddavp. however it is unclear, whether thrombocytopenia is part of the vwd type 2 or of autoimmune origin. since autoimmune antibodies have not been detected, the effect of immunoglobulin treatment might be explained by blockade of enhanced binding of vwf to glycoprotein lb. von willebrad disease (vwd) with a prevalence of 0,8% (ruggeri 1994, rodeignere 1987) seems to be the most frequent inherited hemostatic disorder. • the diagnostic criteria for vwd are clinical picture, family hostory, laboratory findings: bleeding time, partial tromboplastine time (ptt), level of factor viii:e, vwf, vwf:ag, ristocetin induced platelets aggregation (ripa) and multim~-analysis.the diagnosis ofvwd is occasionally difficult, especially in early childhood because the laboratory data may vary due to time of investigation, as well as abnormalities may not be present in all sub-types the aim of this study was the evaluation of diagnostic approach to vwd in childhood and diagnostic reliability of all available laboratory tests. all previously mentioned laboratory tests have been done on our own material (51 child who satisfied all criteria for vwd, 23 boys and 28 girls, 1-9 years old) except mulfimer analysis which was unavailable in some cases. majority of laboratory tests proved to be highly specific and necessary for diagnosis. however, the diagnostic reliability of fviii:c and adhesion of platelets is much lower in mild cases in comparison to total sample, while ptt is an unvaiied test. the most specific screening test for vwd is vwf which diagnostic reliability is almost 1,00. the optimal strategy to establish general diagnosis of mild forms ofvwd is use of vwf and vwf:ag plus ripa if necessary and multimer analysis to classify variant types. we report on a new multimeric structural defect of vwf detected in a german family (two sisters and their three children): all members of the family who presented to our outpatient clinic had an increased spontanous bleeding tendency (moderate or strong hematoma, epistaxis, menorrhagia). prolonged bleeding could be observed after surgical procedures (adenotomia, tooth extraction) and after trauma (laceration). wound heeling was impaired in two cases. clotting assays showed slightly prolonged apti" and a mild decrease of f viii:c, vwf:ag and vwf:rcof levels. collagen binding activity was within normal ranges. bleeding time (simplate i) was slightly prolonged. the analysis of the multimeric structure in plasma showed quantitative and qualitative abnormalities: all multimers were detectable; the structure of vwf was reproducably abnormal in all family members so that the defect must be caused genetically. the thmmbocytic vwf showed neither qualitative nor quantitative alterations. minirin@ (ddavp) was administered as a test dose of 0,3 ~tg/kg bw in 100 ml 0,9% nacl-solution i.v. to evaluate efficacy and tolerance: clotting assays showed normalization of a_vrt, f viii:c, vwf:ag, vwf:rcof in plasma and shortening of bleeding time in three cases. an insufficient rise of vwf:ag and vwf:rcof levels could be observed in one case. one patient had no rise of f vm:c but a corrected bleeding time. multimeric analysis showed no structural change. the administration of ddavp was well tolcrated in all cases. the existance of all multimers in plasma and the normal collagen binding activity suggest that the structural abnormalities of vwf in this family does not cause functional defects so that the defect could be classified as a type i vwd. the response to ddavp was only partially effective. mild von willebrand disease (vwd) is far the most frequent congenital bleeding tendency. its diagnosis is very helpful in pre-operative check-up in order to avoid bleeding complications during surgery. following post-operative periods or monitoring the management of haemorrhagic episodes in vwd patients is also strongly recommended. current methods involve complex technologies, are time consuming and require large series. these assays lack the expected flexibility for rapid individual testing in patients. a new and flexible assay which works on the fully automatic walk-away coagulation instrument, sta, has been developed for these applications (liatest vwf). the technology is an immuno-turbidimetric method using mierolatex particles coated with rabbit polyelonal antibodies specific for vwf. the assay has a dynamic range from 2 to 420% yon willebrand factor (vwf) concentration, it works with a 2 fold dilution of tested plasma (50 td) and it offers a calibration established with the nibsc international standard. the total assay time is of less than 10 minutes and the detection threshold is of 2% there is no prozone effect up to concentrations higher than 1,000% vwf. intra-assay reproducibility is < 4% and inter-assay one < 5%. in dilution studies a mean recovery of 98% was obtained. in a study on 55 plasma samples from norma~ individuals, patients with high vwf concentrations, and vwd, comparison with the elisa technique demonstrated a correlation coefficient of 0.997 with a slope of 0.978 and an intercept of 3.30. in the low assay range too, a good agreement was obtained with the elisa. we conclude that liatest vwf is a reliable, flexible, sensitive, and rapid automated assay which fits well the vw'f assay applications in coagulation laboratories. fibrinolysis, the process during which the active enzyme plasmin is generated in a regulated and localised way, is -in a classical understanding -responsible for the dissolution of blood clots formed in a vessel. for this activity, t-pa is generally assumed to be the most important plasminogen activator and its activity, is regulated by enzyme kinetic mechanisms dependent on the presence of fibrin. with this background t-pa is used for thrembolytic therapy with great success. however, data from t-pa knockout mice indicate that t-pa might not be responsible for inhibiting the spontaneous development of intravsacular thrombi but only for dissolution of fibrin formed upon a coagulation challenge. in contrast, u-pa, generally assumed to be important for extravascular proteolytie activity on activated or tumour cells, seems to lead to the development of spontaneous fibrin formation in a mouse knockout model. on the other hand, the major plasminogea activator inhibitor pal-i seems not only to regulate intravascular fibrinolysis but seems to also be important for the progression of vascular diseases (neointima formation is e.g. increased in a pai-1 knockout model, but increased levels of pai-1 seem to predict reocclusion after angioplasty). in addition to their functioning as enzymes and inhibitors, components of the fibrinolytic system seem also to be involved in signalling processes in tumour and other cells. the u-pa/u-pa-receptor system could be shown to function as a chemotactic system and to elicit a migratory and mitogenle response in monoeytes and tumour ceils as well as in vascular cells. for such a response activation of tyrosine kinases of the sre-family might be responsible in some cell lines, but other signal transduction pathways e.g. involving caveolae and the starprotein can not be excluded. there seems to be a further important role of components of the fibrinolytic system which involves serine protease inhibitors (serpins): serpins have homologies to hormone binding proteins and cleavage of serpins by their target enzymes not only leads to inactivation of the enzyme but also to a possible release of bound hormones from the serpins. from these data clearly the relevance of any regulation of the fibrinolytie, system depends on the specific function of the system to be dealt with. in addition to "fibrin binding", "receptor mediated" and "genetic control" (e.g. 4g vs. 5g in the pai-i promotor) also "signal transduction" and "hormone delivery" are distinct functions of the system with specific regulation. plasmatic for both, healthy persons as well as for patients with angina pectoris it could be shown that increased values of plasma fibrinogen, factor viic and vwf:ag are significantly associated with the risk to suffer an acute myocardial infarction or cardiac sudden death. the same holds for tpa:ag. however, a group analysis in quintiles reveals that particularly low tpa:ag values are connected with a particularly low coronary risk. unexpectedly also the acute phase protein crp is positively associated with increased coronary risk. for clinical purposes these factors have already been included into coronary risk scores in order to improve the individual risk prediction in combination with lipids and other risk factors. the assessment of the pathophysiological significance of these observations remains at dispute. 4 pathways are discussed: 1. the assumption that increased plasma values of those factors indicate increased coagulation activity could so far not be established in prospective studies. 2. both vwf:ag and tpa:ag are produced in endothelial cells. an increase of their plasma level could therefore indicate increased endothelial cell functions which accompanies progressive atheromatosis. the risk association of the two acute phase proteins crp and fibrinogen could be interpreted analogously. 3. first prospective studies favour the assumption of a genetic determination to an increased production of coagulation proteins in persons at particular coronary risk. it could also be shown that there is a certain dependance of the gene-polymorphism for co-and 13-fibrinogen chains from the coronary risk. 4. even slightly elevated concentrations of fibrinogen and/or vwf:ag may influence the quality of a coronary thrombus both by increased physical stability and by reduced fibrinolytic lysibility. this could mean that an early coronary clot under these conditions could more readily develop to a stable, occlusive thrombus. a newborn with pronounced bleeding tendency had a prothrombin (prth) deficiency below 2.8% in a clotting assay. both parents had activities of 71% and 69%, respectively. however, the immunological determination ofprth by elisa revealed normal concentrations in all family members (93%-101%). furthermore, thrombin generation as investigated by a chromogenic assay using ecarin for activation of prth was normal as well. activation of prth by fxa was investigated by reealcificafion of the plasma samples and further analyzed for prth and its derivatives produced. although clotting times still were different, finally, normal levels of fl+2 and tat were generated as determined by elisa. western blot analysis using polyclonal (rabbit) antibodies to prth and a monclonal antibody specific to human thrombin, revealed different patterns of prth degradation products. tat was only weakly visible in the serum of the mother and nearly absent in the child.the mobility of prothrombin and thrombin was different compared to normals indicating a lower molecular weight. after reduction of disulfide bridges a higher molecular weight of thrombin was observed compared to normals indicating an insufficient cleavage ofprth and formation ofprethrombin 2. these observations let suggest that prothrombin marburg is a deletion mutant lacking the cleavage region arg 320-ile321. upon cleavage by factor xa only prethrombin 2 is formed under liberation of fl+2. this prethrombin 2 is able to cleave chromogenic substrates in the ecarin assay. probably, prethrombin 2 forms a complex with atiii which is detected by elisa, but unstable under denaturing conditions as in the western blot. as a major complication of haemophilia a treatment, up to 30% of the severely affected patients develop antibodies to substituted factor viii. investigating 133 patients and considering the data of further 231 patients of the haemophilia database, we could show, that risk of inhibitor developement depends on the patient's mutation type. patients with more severe gene defects, like intron 22 inversions, stop mutations or large deletions had a risk of about 35% for inhibitor developement, which was about 7 times higher than for missense mutations or small deletions. besides an influence of mutation type, we investigated other parameters e. g. immune response genes (i-ila-genotype) and clinical aspects (treatment onset and frequency, type of concentrate) that might also affect inhibitor formation. to exclude any effect of mutation type, we focussed on 72 patients with an intron 22 inversion. hla-typing showed that some t-ila-alleles (dqb0602, bt) occurred more otten and others (dqa103, dqb603, dr13, c2) less frequent in inhibitor patients. treatment onset, frequency and type of concentrate apparently do not affect inhibitor incidence. the results presented here, prove that inhibitor development is considerably influenced by the mutation type. this supports the hypothesis that patients with severe molecular defects have no endogenous factor viii protein and that substituted factor viii represents a foreign protein, leading to an immune response, e. g. the production of alloantibodies. in addition, the immune response seems to be modified by the hla-genotype. however oar findings (in terms of genotype and treatment parameters) can only explain part of the inhibitor pathogenesis. it is still unsolved why substituted factor viii does not lead to a recognizable immune response in 2/3 of the patients with severe molecular factor viii gene defects. consequently other factors, probably concerning the antenatal phase, must be involved. viia in the treatment of patients with inhibitors against factor viii or ix: a german/swiss/austrian multi~center trial d. ellbriiek*, i. scharrer**, j. dethling***, and the rfviia study group *section h~mostaseology, university ulm **dept. of angiology, jwg-university hospital frankfurt a.m. ***novo nordisk, mainz administration of activated recombinant factor vii (rfviia) can by-pass the fvnlwlx pathway and offers an alternative treatment for patients with antibodies (inhibitors) against these factors. from november 1994 to october 1995, a total of 25 bleeding episodes and 10 surgical interventions in 18 patients were treated with rfviia in a phase iiib multicenter trial. diagnosis was hemophilia a (n = 15) or b (n=l) with inhibitor, and acquired inhibitor against factor viii (n=2). various serious bleeds, from complicated joint and gingival bleeds to lifethreatening psoas bleeds, have been treated. operations have been tooth extractions, radiosynovectomy, implantation and explantadon of porth-acaths and one adenotomy. dose regimen was 90-120/zg/kg bw every two to three hours until clinical improvement, with subsequent dose reduction. results: for bleeding episodes, response to rfviia after 24 hours was effective in 72%, partially effective in 12 9"0, ineffective in 129"o and not evaluable in 1 (4%) of the patients. two of the three treatment failures were associated with very long dosage intervals of rfviia. the third patient was in a critical situation with artificial high pressure respiration and polytransfusion because of a hematothorax, and suffered a terminal intracerebral bleed. the efficacy of rfviia for surgery was very good. response to treatment was independent of antibody titer. no signs of dic or activation of coagulation were noted. conduslon: in our experience, rfviia is an efficient and safe treatment for inhibitor patients with acute bleeding episodes. it should be investigated, whether rfviia can be an alternative treatment also for the hometreatment situation. successful immunetolerance therapy of f vih-inhibitor in children after changing from high to intermediate purity f vih concentrate w. kreuz, j. joseph-$teiner, d. mentzer, g. auerswald*, t. beeg, s. becker zentrum der kinderheilkunde, j. w. goethe-universit~itj frankfurt am main *professor hess kinderklinik bremen introduction: inhibitor to f viii is the most severe complication in treatment of patients with haemophilia a. the incidence of f viii inhibitors is estimated to range between 15-33%. several authors reported that the immunetolerance therapy (itr) of f viii-inhibitors can be induced with high dose f viii concentrate. objective: this presentation will show data of four children with haemophilia a and f viii inhibitor (high responder), who had an unsuccessful lit with high dose f viii concentrate (high purity) in the first step. f viii concentrate was changed to an intermediate purity product (haemate hs®) in the subsequent course of h't. all patients received bleeding prophylaxis with an activated-prothrombin-complex-concentrate (feiba®). results: median age was 13 (9-18) months, when the inhibitor was first detected. in all four patients the f viii inhibitor titre increased under immunetolerance treatment with f viii concentrate (high purity) in the first step of therapy. after changing the f viii concentrate (intermediate purity) the inhibitor titres decreased continuously after a rebooster effect to 0be within months. median duration of f viii inhibitor elimination time (until first testing of 0 be) was 3 (2-5) months. in all patients the f viii inhibitor was successfully eliminated. until now all patients are under prophylactic treatment with f viii concentrate and had no positive inhibitor testing since. median observation time since the first testing of 0 be is 14 (4-60) months. conclusion: different studies concerning immunetolerance treatment have been successful with f viii concentrates of different purity. according to our experience in these four presented patients, we assume that probably not the purity of the f viii concentrate is important for the induction of immunetoleranee, rather than the type of f viii presentation in the used concentrate. the used preparation (haemate hs®) is a f viii concentrate with high concentration of vwf, which is known to be important for the protection of f viii against degradation by proteases. this may be a mechanism for a prolonged antigen presentation to the immunesystem and thus may have a positive impact on the outcome ot itr. long scale trials are needed to prove the above assumptions. thrombasthenia glanzmann is a disease affecting platelet function because of a partial or total lack of glycoprotein (gp) ilbllla expression or a modification of this complex. since the receptor dysfunction goes along with reduced or absent platelet aggregation and adhesion, it causes bleeding complications in case of injury. here we report about a 60 years old women, who suffered since early childhood from a severe bleeding disorder. life threating bleeding complications occured after tooth extraction and after abdominal surgery. analysis of the patients platelets revealed normal values for the platelet count, whereas their volume showed to be increased (11 fl). clot retraction was diminished to 17%. platelet adhesion to siliconised glass and human subendothelial matrix was reduced, as was the spreading of the platelets. adp (i#m) induced platelet aggregation was inhibited, while collagen-, ristocetin-and thrombin-induced aggregation showed to be normal. cross immunelectrophoresis resulted in an atypical peak of gpiibllla with reduced electrophoretic mobility. in the electroimmunoassay according to laurell 14% of gpiibllla was detected. moreover we observed a markedly diminished 125j-fibrinogen binding. sequence analysis of the gpiib and gpiila cdna after pcr amplification unraveled a g2508 --, a transition in gpiib, substituting gly805 --* glu. the structure/function relationship of this mutation has still to be investigated. we report two new abnormal fibrinogen variants, denoted as bem iv and milano xi, both having an exchange of arginine to histidine in position 16 of the ac~-chain. routine coagulation studies revealed prolonged thrombin and reptilase clotting times, low plasma fibrinogen concentrations determined by a functional assay but normal fibrinogen levels measured by the immunological assay. the onset of turbidity increase following addition ofthrombin to purified fibrinogen was markedly delayed in both variants. release of fibrinopeptide b by thrombin, measured by reversed phase hplc, was normal whereas only one half amount of normal fibrinopeptide a was released. in addition to normal fibrinopeptide a, an abnormal fibrinopeptide a* was cleaved from both dysfunctional fibrinogens. the structural defect was determined by asymmetric pcr and direct sequencing of a gene fragment coding for the nh2-terminus of the aachain. both variants were found to be heterozygous for the transition g to a at nucleotide position 1203, leading to the substitution actl6 arg-->his, resulting in a delayed fibrin polymerization. the simple assay permits detection of the most common amino acid substitutions occuring in the nh2-terminus of the ac~-chain of the functionally abnormal fibrinogen variants. protein c inhibitor (pci) a member of the serpin family is also known as plasminogen activator-3 (pal-3). pci was first described as a component of human plasma, regulating the activity of activated protein c and other sedne proteases of the human coagulation and fibdnolysis system. since then pci was found to be present in extra-plasmatic systems also. high concentrations of pci were detected in human seminal plasma suggesting a role for pci in human fertility. significant concentrations of pci mrna and antigen were located in lysosomes of proximal tubular kidney cells suggesting an intracellular function for pci in this environment. in this study we present evidence that pci is also present in human pancreas. rna from human pancreas was reverse transcribed and pcr amplified. the resulting pci cdna was identical with pci cdna from human liver. ~p labeled antisense rna probes used in in situ hybridization experiments with human pancreas tissue sections showed that pci rna was located in the acinar ceils. pancreatic fluid was analyzed by sds-page and immunoblotting. using monospecific antibodies directed against human plasma pci, a 57 000 mw protein band was observed which comigrated with purified human plasma pci. our results show that pancreas cells contain a significant concentration of pci mrna. this message is localized in the secretory acinar cells. therefore we conclude that pci antigen found in pancreatic fluid is likely to originate in the pancreas. the role of pancreatic pci is unknown at present. however, since thrombosis and systemic hypercoagulable states are known complications of pancreatic diseases our results and in vitro experiments by others showing that pci can inhibit pancreatic enzymes such as chymotrypsin and trypsin indicate that pci may be part of the inhibitor potential which protects pancreatic tissue from auto degradation. these inhibitors normally prevent the release of active pancreatic proteases into the vasculature or microcirculation where destabilization of the coagulation balance and subsequent thrombus formation could occur. institute for clinical chemistry and laboratory diagnostics and *clinic for cardiology, universi w of duesseldorf p-selectin (cd 62p, the former granule membrane protein 140 or gmp 140) is an integrated membrane protein of platelets and endothelial cells. under inactivated conditions it is stored in the alpha granules of platelets and in the weibei-palade bodies of endothelial cells. endothelial cells covering atherosclerotic plaques show an increased expression of p-selectin. 13-thromboglobulin (13-tg), which is also expressed from the alpha granules of platelets during adhesion or aggregation, is regarded as a marker of platelet activation in vivo. coronary thrombosis plays a central role in the pathogenesis of acute coronary syndromes. we therefore analysed cd 62p and 13-tg in acute coronary syndromes, healthy subjects (hs, n=l i), patients with stable angina pectoris (sap, n=20), unstable angina pectoris (uap, n=l 2) and acute myocardial infarction (ami, n= 12). plasma samples were obtained by using ctad vacutainer tubes (0.109 m na~-citrate, theophylline, adenosine dipyridamole). patients with cad showed significantly increased plasma concentrations of cd 62p (hs: 98+20 versus sap: 133+38ng/ml, p<0.05; versus uap: 128+28 ng/ml, p<0.01; versus ami: 144+72 ng/ml, p<0.05) independent of the severity of clinical symptoms. in comparison only patients with ami showed significant higher 8-tg concentrations compared with hs (hs: 30+20 versus ami: 39+14ng/ml, p<0.05). although the cd 62p plasma concentrations showed no relationship to the clinical severity, hence there was a positive correlation between cd 62p (r=0.47; p< 0.001; n=55) to the severity of cad classified as i, 2, 3 vessel disease. it is concluded that elevated cd 62p concentrations are correlated with the severity of cardiovascular disease. cd 62p is not suitable for differential diagnosis of acute coronary syndromes, because it is elevated independently of the clinical status of the patients. the involvement of platelets in the pathogenesis of acute myocardial infarction may be indicated by the increased 13-tg concentrations. iklinik nr herz-, thorax-und herznahe gef&schirurgie und 2institut x~tr klinische chemie und laberatodumsmedizin der universint regensburg an increased blood loss following surgery with extracorporeal circulation (ecc) contributes to the morbidity and mortality. postoperative haemorrhage following ecc has been related to a platelet function defect and the activation of the blood dotting and fibrinulytic system. we investigated platelet surface antigen expression and parameters indicating activation of the clotting and fibrinolytic cascade to assess the predictive potential of these variables for increased blood loss after ecc. g0 patients referred for coronary bypass gra~ing with no history of a bleeding disorder and normal routine clotting tests were included. on the day prior to surge~ and immediately upon arrival on the intensive care unit blood samples were drawn. the surface expression of glycoprotein (gp) lib-ilia, gp lb, and p-selectin was meamred with and without in vitro stimulation with adenosine diphosphate (adp) using whole blood flow cytomet~y. platelet counts and platelet factor 4 (pf4), as well as, routine clotting tests were performed. activation of the clotting and fibrinolytic system were judged from thrombin-antithrombin-iii complex fiat), fibrinogen fig), d-dimers (dd), cc2-antiplasmin (tz2a), prothrombin fragment 1+2 (fl+2),and tissue plasm~ activator (t-pa). blood loss fxom chest tubes was measured hourly until removal of drains. following ecc the levels of pf4, tat, dd, o~2a, fl+2, and dd were sigulticnatly increased (p<0.0001) compared to baseline values. gp iib-iila, gp ib, p-selectin, platelet count, and fg were significantly reduced (p<0.0001). analysis of variance (anova) revealed that postoperative values of gp ib (p<0.0001), dd (p<o.05), fg (p<0.01),and platelet count (10<0.05) had a significant influence on blood loss. none of the preoperative data was significantly associated with increased blood loss. a subgroup of patients who bled more than the mean + 1 sd (14 cases) was formed_ in logistic regression analysis gp ib expression (p=0.01) and dd (p=0.007) were of value in identifying bleeders. the sensitivity of the equation was 56%, the stx~ificity 97% and the positive predictive value 82%. although the reasons for an increased blood loss following ecc are multifactorial, we were able to identify a small number parameters with a significant infl~nco. the combination of the expression of yon willebrand receptor on the platelet su_rfaco and d-dimer levels in plasma was successful in predicling an increased blood loss following heart surgery. this may be helpful to define indications for platelet transfusion and flesh frozen plasma. the most widely distributed receptor for the fc-region of igg is the fcreceptor iia (fcyriia). for this receptor a functionally relevant polymorphism of amino acids argmhis is known. we previously showed hit-antibodies to bind to platelets via feyriia and now whether clinical manifestation of hit is associated with the fe'/riia polymorphism. therefore, we developed a rapid two step allelle-specific pcr (asp) method for genotyping the foyriia. with control samples the asp was compared with the time consuming and complicated allele-specific oligonucleofide hybridization technique, the phenotype expression of fctriia (using the moabs iv.3 and 41h16) and the monocyte stimulation assay. complete concordance between the assays was found. we used the asp to determine the allelic distribution of the inst. physiol. chem., marburg; *)univ. of virginia, charlottesville, the functional heterogeneity of blood platelets is clinically significant and may contribute to the pathogenesis of cardiovascular and atherosclerotic diseases l). in order to understand the biochemical basis of the functional heterogeneity of platelets, we have investigated the signal transducing pathway of platelet activation a) in terms of guanylate cyclase and phosphodiesterase activities and b) at the level of protein phosphorylation and the receptor-effector coupling via g i-and gs-proteins in functional heterogeneous blood platelet subpopulations separated according to their densities. furthermore, we analyzed the adp-ribosylation of rhoa, which is a lowmolecular weight gtp-binding protein and is thought to be involved in the platelet "shape change" reaction and aggregation. aggregation was enhanced in low-density platelets (sp i) and less inhibited when cgmp was increased by no, compared to high-density platelets (spiii). sp i possessed a lower basal level of cgmp and exhibited a smaller increase in cgmp after stimulation with no. cyclic amp-dependent phosphodiesterase activity was higher in sp i compared to spiii, whereas cgmp-dependent phosphodiesterase activity was similar in sp i and sp iii. sp i, when compared to sp iii platelets, showed a higher degree of prephosphorylation of proteins in the range of 47, 32-34, and 20-22 kda. after thrombin stimulation, the phosphorylation of these proteins was similar in the subpopulations. pertussis toxin,induced adp-ribosylation of the 40-41 kda gi-protein was highest in sp i, whereas cholera toxin induced adpribosylation of the gs-protein and botulinum c3 exoenzyme-induced adpfibosylation of rhoa were highest in sp iii. the findings suggest that a) the higher aggregability in sp i compared to sp iii may be caused by a poorer ability of guanylate cyclase to form cgmp and a higher activitiy of campdependent phosphodiesterase and that b) the adp-ribosylation of g-protein subunits and rhea may contribute to the functional heterogeneity in the subpopulation leading to the greater ability of sp i for aggregation. 1) opper et al., atherosclerosis, 113, 211-217, 1995. causes of accelerated atherogenesis in non-insulin-dependent diabetes mellitus (niddm), known to be associated with increased plasma activity of plasminogen activator inhibitor type 1 (pal-l) as well as with increased plasma concentrations of proinsulin and insulin, remain enigmatic. this study was designed to determine whether proinsulin and insulin increase pal-1 in vivo thereby potentially accelerating atherogenesis. to simulate hyper(pro)insulinemia seen with niddm, we infused equimolar concentrations of insulin (1 iu/kg), proinsulin, c-peptide, or placebo, all endotoxin-free, i.v. over 1 h in 53 conscious rabbits maintained euglycemic. plasma pal-1 did not increase with placebo (n=16) or c-peptide (n=4), but did with proinsulin and insulin peaking in 3 h at 3.5-and 3.7-fold (n=16 and n=17, p<0.o02 for each) in parallel with pal-1 protein quantified by reverse fibrin autography. the increases were specific as reflected by unchanged activities of o~-antiplasmin and t-pa. pat-1 gene expression (pal-1 mrna) increased with proinsulin and insulin by 2.1-and 2.1-fold in 3 h in aorta, 1.9-and 2.4-fold in liver (p<0.05 for each), but not in heart, lung, spleen, or kidney and returned to baseline in 24 h (n=4 each). in situ hybridization localized the increased pal-1 mrna in aorta to endothelium (n=3 each). thus, both proinsulin and insulin directly augment gene expression of pal-1 in arterial endothelium in vivo, thereby increasing plasma pal-1 activity, attenuating endogenous fibrinolysis, and potentially accelerating atherogenesis. the sarco-endoplasmic-retikulum ca+÷atpases (serca) that sequester calcium into intracellular stores, represent the most important mechanism to maintain cytosolic calcium levels low and to return to a resting level after activation. recent studies in platelets and rat endothelial cells have identified two forms belonging to the serca 2b and serca 3 types. the monoclonal antibody pl/im 430 raised to human platelet intracellular membranes has been shown to recognise the serca 3 ÷+ isoform and to inhibit ca sequestration in human platelets. the aim of this study was to identify ca atpase distribution in human umbilical vein endothelial cells (huvec) and human polymorphonuclear leukocytes (pmnl). polyclonal antibodies with known specificity towards serca 2b (r 2/88) and serca 3 (89/3) in addition to pl/im 430 were used in histochemical studies of permeabilised cells and in western blotting experiments using cell lysates and mixed membranes. using the alkaline phosphatase anti-alkaline phosphatase (apaap) method r 2/88 and 89/3 reacted strongly with proteins in permeabilised huvec and pmnl, however pl/im 430 showed good staining only with pmnl. in western blotting experiments r 2/88 recognised proteins with molecular sizes of 100 kda (known to be the parent ca++atpase) and 70 kda (which may represent antibody recognised degradation product), in both huvec and pmnl. 89/3 ÷+ recognised bands at 100 kda (parent ca atpase), 74 kda and 46 kda in both huvec and pmnl membranes. in agreement with histochemical results pl/im 430 recognised proteins (100, 74 and 46 45 + + kda) only in pmnl membranes. in ca transport studies pl/im 430 ÷+ inhibited ca uptake in membranes prepared from pmnl but not in huvec. the distinct reaction of pl/im 430 with pmnl and huvec suggests the possible presence of distinct sub-isoforms of serca 3 with the pmnl and platelet proteins being similar and different to the serca 3 isoform present in huvec. microvascular endothelial cell (ec) lines with a limited life span of 2-3 months in vitro were established from human bone marrow, skin or solid tumor tissue. homogeneity of the lines was verified by flow cytometric analysis and cytokine expression patterns. the cultured ec constitutively expressed hla-class i antigens, cd44, procollagen type i, cd62e, cd34, and receptors for granulocyte colony stimulating factor (g-csf). endothelial cells constitutively produced il-6 and il-8 as well as tpa and pai-i as determined by elisa. in the presence of 5 iu/ml of exogeneous il-la, g-csf was secreted at high amounts (lng/ml) and gm-csf at low amounts (15pg/ml) in confluent monolayers and during over night incubation. these ec potently bound to isolated neutrophilic granulocytes. when granulocytes were stimulated by either 10~ 4 or 10 .7 m formyl-methionyl-leucyl-phenylanaline (fmlp) to produce oxygen radicals, the preincubation with a single cell suspension of ec caused oxygen radical scavenging. a ratio of 1 : 100 ec to neutrophilic granulocytes resulted in a 50 __+ 10% inhibition of fmlp-induced and luminol-enhanced chemiluminescence in vitro. this effect was probably due to the constitutive nitrogen oxide (no) production by ec. unexpectedly, preincubation of ec with 50 iu/ml of interferon-y (ifn-y) to increase no-secretion did not alter the extent of the oxygen scavenging effect measured at different ratios of ec to neutrophilic granulocytes. according to the surface antigen expression pattern, identical ifn-f treatment upregulated adhesion receptors on ec. concomitantly, binding of neutrophils to ec was significantly increased. thus, the net oxygen-scavenging effect by ec appears to be stable under the condition of ifn-y stimulation which indicates maintenance of ec function and effective neutralizition of non-specific oxygen radical damage. moreover, the established test system can be useful to investigate the selective contribution of other inflammatory cytokines, adhesion molecules, macrophages and platelets on ec function in vitro. atherosclerosis is characterized by intimal migration and proliferation of smooth muscle cells (smcs), by macrophage infiltration and extracellular matrix remodeling. circumstantial evidence suggests that urokinasetype plasminogen activator (upa) may be relevant in these processes. this study examines upa expression and upa antigen localization in human coronary arteries with ,carious degrees of atherosclerotic lesions. representative segments of macroscopically normal areas (mnas), early lesions (els) and fibrous plaques (fps) were processed in parallel for in situ hybddization and immunohistochemical analysis. in mnas, upa was detected both in the luminal endothelium and in celocalization with ~-actin-positive smcs throughout the tunica media. in els, upa mrna and antigen were substantially reduced in the luminal endothelium. however, cellular upa expression was detected in the subendothelial area in colocalization with tissue-infiltrating cd68positive macrophages. in the deeper intimal and in the medial layers strong upa expression was observed in association with ~-actinpositive smcs, with most of the upa-positive cells being located in areas with a high proliferative activity as detected by pcna staining. in fps, upa mrna was widely expressed in macrophage-containing areas within the fibrous cap and at the periphery of the necrotic core of the lesions. in addition, upa antigen was identified in acellular areas of the necrotic core regions. compared to the diseased intima of these lesions, the adjacent media stained only weakly for upa. in conclusion, this study demonstrates an enhanced expression and synthesis of upa by intimal smcs and macrophages in advanced atherosclerotic lesions of human coronary artedes these findings suggest a role for upa in the progression of the coronary atherosclerosis. adenosine produced by vascular cells, platelets and other tissues has been proposed to be a vasodilator, inhibits platelet aggregation, stimulates proliferation and migration of endothelial cells as well as exhibits antiinflammatory effects. adenosine of endothelial origin intra-and extracellularly formed and highly concentrated at the luminal surface may constitute an important antiaggregatory mechanism, which is part of the well-known antithrombogenicity of an intact endothelial lining. it was the aim of this study to investigate whether adenosine could influence the fibfinolytic potential of endothelial cells. when confluent human umbilical vein endothelial cells were incubated with adenosine (0.01-10mm) a significant dose dependent decrease in plasminogen activator inhibitor type-1 (pai-i) antigen production by this cells was seen as compared to control. this effect by adenosine was also time dependent and seen after 2 hours of incubation with adenosine. as determined by elisa, pai-i antigen in conditioned media of endothelial cells incubated with 10mm adenosine decreased to 57% as compared to control. pai-i antigen in extracelullar matrix of these cells was also reduced by incubation with adenosine. these results were also reflected on the level of specific mrna as determined by northern blotting. pai-i mrna decreased in endothelial cells after incubation for 4 hours with adenosine (10mm) to 77% (2.2kb) and 85% (3.2kb) of control. in conclusion our data demonstrate that adenosine can downregulate the expression of pai-1 in cultured endothelial cells thereby increasing the profibrinolytic capacity of these cells. this effect of adenosine might contribute to its antithrombotic potential in addtion to its known antiaggregatory effect. sowohl die effizienz als such die komplikationen einer antikoagulantien-dauerbehandlung sind in erster linie yon der gore und engmaschigkeit der gednnungskontrellen abh~ngig seit 1986 wird die schulung zur gerinnungs-selbstkontrolte in der herz-kreistauf-klinik bad bedebarg in gruppen eingeobt. die theoretische und praktische anleitung erfolgt in 8 stunden durch eine in der patientenschuiung erfahrene arzthelfedn (oder krankenschwested-pfleger) und einen arzt. die praktische schulung wird durch einen patientennahen theoretischen tell erg~nzt. verglichen mit konventionell ~berwachten oral antikoagulierten patienten (ca. 50% der tpz-werte liegen im theapeutischen bereich) erreichten 216 quickwert-selbstbestimmer, daf$ 83,1% yon 14 812 tpz-werten im therapeutischen bereich lagen (tabelle 1 ). schwerwiegende thromboernbolische ereignisse oder blutungskomplikatienen wurden nicht beobachtet. durchschnittlich kontrolliert der quickwert-selbstbestimmer seine gerineungswerte w6chentlich, itmgstens im zweiwochen-abstand. ob sich blutungs-und thremboembolierisiko langfristig senken lassen bei gleichze~iger verbesserung yon prognose und lebensqualit~it, bedarf des nachweises im rahmen einer randomisierten, kontrollierten und prospektiven studie. tabelle i verteilun~l der selbstbestimrnten gednnur ~swerte zeitraum 1986 zeitraum -1988 zeitraum 1986 zeitraum -1990 zeitraum 1986 zeitraum -1992 to evaluate the potential benefit of an optimized oral anticoagulation management with inr measurements performed by the patients themselves, two prospective randomized studies have been completed. the first study (n=60) was performed to evaluate the accuracy of inr self-testing (inr-st) compared with standard laboratory controls as well as the improvement of anticoagutation monitoring by inr-st, e.g. the percentage of measurements found inside the target therapeutic inr-range. the 231 parallel measurements revealed a high accuracy of inr-st with a 4.5% median difference between parallel controls of single measurements. in addition, inr-st resulted in a significant increase of measurements inside the target therapeutic range (from 54 to 80%) in the entire group. however, inr-st performed every 4 weeks did not result in a significant improvement, while selfcontrols every week (from 44 to 84%) and every 2 days (from 51 to 84%) did. in a second study (150 patients) the incidence of bleeding and thromboembolic complications were evaluated in patients who either had standard anticoagulation measurements o[ performed inr-st. during a median follow-up of 1.5 years in the group with standard oral anticoagulation management an incidence for bleedings of 10.9% per year and for thromboembolic complications of 3.6% per year was found, while in the group of patients with inr-st bteedings occured with an incidence of 4.5% per year and thromboembolisms with 0.9% per year. it can be concluded that inr-st can be performed with high accuracy by the patients themselves. inr-st results in more frequent controls of the inr and consequently in significantly longer time periods where the patients are inside the optimal target therapeutic inr range. the more accurate anticoagulation management resulted in our study group in a significantly lower incidence of thromboembolic and hemorrhagic complications. the cell biology of tissue factor (thromboplastin) h. prydz, the biotechnology centre of oslo, norway tissue factor (tf,thromboplastin) is the most potent trigger of blood clotting knuwn.lts presence in tissue extracts has been known since before the turn of the century.erwin chargaff (of dna fame) first described that tf consisted of two parts (a protein and a lipid moiety), a fact that was independently rediscovered in the early sixties by deutsch and lechner in vienna and by myself in oslo. in 1973 tf was first purified and shown to be an integral membrane protein of mr about 50 kda (this was a slight overestimate,recent studies indicate 46-48 kda). in 1987 four groupes independently cloned tf edna, later also genomic dna. the structure of tf suggested that it was a membrane spanning receptor, with an extracellular part of about 219 amino acids folding into two domains remotely like the domains of the ig-superfamiliy, and more specifically the eytokine receptors. tf is synthesized to a varying degree in many tissues, but not normally in tissues in contact with blood. however, during acute inflammatory situations and under the influence of a number of agents (immune cnmplexcs, lectins, cytuklnes,bacterial components, complement components etc) monucytes and vascular endothelial cells are induced to synthesize tf. another possible way tu get tf into contact with blood is thruugh trauma, surgical ur otherwise. the fact that tf is inducible has created a lot uf interest in its cell biology. we reported recently that binding of factor viia tu tf un the cell surface elicited a marked increase in intracenular ca spikes, thus rendering the final proof that tf not only luoks like a receptor but really is one, since all the three essential criteria are fulfilled: tf has a transmembrane segment and a cytoplasmic tail tf has a high specificity/high affinity ligaud (factor vii and factor x) tf induces an intracellular signal upon ligand binding recent results regarding tf as a receptor and regarding its intracellular traffic will be reported. both f.ix and f.x are activated by the tf/f.viia complex. we have used a tf-initiated model system to examine the roles played by f.ixa and f.xa in initiating coagulation. the in vitro model contained a cellular tf source; plasma concentrations of f.ii, v, viii, ix and x, tissue factor pathway inhibitor and antithrombin iii. we tested the ability of exogenously added f.ixa or f.xa to substitute for tf/viia activity in initiating platelet activation and thrombin generation. f.xa was 10 times more potent than f.ixa in initiating platelet activation, but f.ixa was more potent in promoting iia generation. in the presence of tf/viia, zymogen f.x was required both for platelet activation and iia generation, while f.ix was only required for iia generation. thus, tf/viia-activated f.ixa and f.xa have distinct physiological roles. the main role of f.xa is to activate platelets by producing an initial small amount of iia. the process of platelet activation is much more efficient if this initial iia is produced on the tf-bearing surface. f.ixa, by contrast, enhances iia generation on platelets by providing f.xa for platelet surface prothrombinase assembly. we conclude that activation of both f.ix and f.x by tf/viia is essential for efficient initiation of coagulation. furthermore, f.x activated on the surface of a tf-bearing cell isnot functionally equi.valent to f.x activated on the platelet surface. therefore, f.x activation by tf/viia does not compensate for a lack of f.ix or viii in hemophilia because the f.xa activated by tf/viia is located on the wrong surface for efficient thrombin generation. our results emphasize that the location, of an activated coagulation factor is a critical determinant of its role in hemostasis. the x-ray crystallographic structure of the fviia-tf complex located the areas within fviia in contact with tf to the first egflike and the protease domains. high-affinity binding of fviia to tf depends on ca2+ and previous studies suggest that ca2+ binding to a site in the protease domain is required. however, using surface plasrnon resonance we have shown that removal of the n-terminal gla domain and hydrophobic stack (hs) from fviia results in a decreased affinity for tf. in search for the mechanism by which regions in fvua not in direct contact with tf affect the affinity, various techniques have been employed. a plausible model is one in which the gla domain and hs optimize the interaction with tf by affecting regions that mediate the association with tf. using gel filtration we demonstrated that the hydrodynamic radii of des(1-38)-fviia and, especially, fviia decreased upon ca2+ binding, whereas des(1-44)-fviia was virtually unaffected. this suggested that the gla domain and the hs interact in a ca2+-dependent manner with other regions in fviia, presumably the protease domain, making fviia more globular. using cd spectroscopy, we could detect ca2+-induced changes in the secondary structure of fviia, all assignable to an increased helical content of the gla domain. the amidolytic activity of fviia in the absence of tf has a ca2+ dependency that suggests involvement of the gla domain in obtaining full activity. this activity was very sensitive to guanidine hydrochloride (c50<0.5 m) and its quenching corresponded to unfolding of the ca2+-loaded gla domain as measured by changes in the trp fluorescence of fviia. based on these results it is conceivable that the gla domain and perhaps the hs of fviia interacts with the protease domain and that this globular form mediates the initial contact with "it. several studies have indicated a profound role for fvii(a) in arterial thrombogenesis. in the present study we quantified the inhibitory effect of dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone recombinant fviia (degr-rfviia) on acute thrombus formation at medium-sized artery blood flow conditions characterized by a wall shear rate of 650 s -1. native human blood was drawn directly from an antecubital vein by a pump into a heparin coated mixing device where degr-rfviia (0.9 -880 nmol/l final plasma concentration) or buffer were mixed homogeneously with the flowing blood. subsequently, the blood entered a parallel-plate perfusion chamber in which a thrombogenic surface consisting of immobilized tf and phospholipids was positioned. fibrin deposition, platelet-fibrin adhesion and platetet thrombus volume triggered by this surface were measured by morphometry. degr-rfviia inhibited these thrombus parameters in a dose dependent manner with ic50 values between 0.5 and 0.7 nmol/l the plasma levels of thrombin-antithrombin m complexes were inhibited at an ic50 of 0.6 nmot/l since the concentration of fvii and fviia in normal plasma is about 10 and 0.1 nmol/l, respectively, theic50 values indicate that degr-rfviia is a very efficient inhibitor of thrombus formation in this model. thus, inhibition of the extrinsic pathway of coagulation may represent and entirely new and effective arterial anti-thrombotic approach. the anticoagulant agent from medicinal leeches, hirudin, is presently gaining popularity as an antithrombotic drug. the design of recombinant forms of the minipretein and their availability led to the clinical use of this naturally occurring thrombin inhibitor. extensive studies in experimental animals have shown that due to its pronounced and specific antithrombin effect hirudin is an antithrombotic agent of high quality. apart from its anticoagulant effect, hirudin is pharmacodynamically inert and is well tolerated in vivo. brain abnormalities in male children and adoleescents with hemophilia: detection with mr imaging mri of sickle cell cerebral infarction in addition, portions of the preparations were incubated with 1.4nm-gold labelled fibrinegen molecules (fg-au; final concentration 4gp_g/ml) for 10 see at 37°c. the expenments were stopped al~er 3 or 10 rain by glutaraldehyde fixation. serial sections were prepared and examined after silver-enhancement using danscher's (1981) method to visualize the fg-au. hwdid not alter the resting gfp. after .3,rain, activated hw-platelets changed their shape moderately but in contrast to the controls did not aggregate. moreover, the conatdcting cytoskeleton that centralizes platelet organelles was not formed. fg-au was found on the surface of the control platelets, in high density in the contact spaces between the aggregated platelets and internalized in the surface connected system. only a very small number of hwtreated cells had fg-au on their surface and showed internalization. 10 rain after adp most of the platelets in both preparations had retumod to a discospherecytic shape it is confirmed that hw inhibits the platetet aggregation and in addition, 1) the formation of the contractile cytnskeleton as well as 2) the binding and interalization of the used ligand fibrinogen santoso 2 l.institute for immunology and transfusion medicine, emst-moritz-arndt-university greifswald; 2. institute for transplant vasculopathy in cardiac allografts is the leading limitation to long-term survival of cardiac transplant recipients. activation of the coagulation mechanism by adherent monocytes expressing tissue factor (tf) is expected to be involved in the development of transplant atherosclerosis. in this in vitro study the effect of the immunosuppressant cyclosporine a (csa) on monocyte tf expression and the regulation of the tf gene transcription was analysed. csa was shown to inhibit the induction of monocyte procoagulant activity (pca) by decreasing lipopolysaccharide (lps)-induced tissue factor (tf) expression in monocytes. csa exerted a dosedependent inhibitory effect on monocyte tf expression at concentrations (1,5 txmol/l csa: 50 % inhibition) not decreasing the cellular protein synthetic rate. addition of csa during lpsstimulation prevented activation of the nuclear factor (nf) ~b as demonstrated by electrophoretic mobility shift assay. western blot analysis confirmed reduced nuclear translocation of nf-~b in the presence of csa. inhibition of the lps-induced nf-~b activation by csa resulted in a reduced tf gene transcription as demonstrated by rt-pcr-analysis. thus, cyclosporine a prevents tf expression by inhibiting the induction of tf gene transcription in activated monocytes. csa may therefore provide a therapeutical tool acting not only as a direct immunomodulating agent, but also by influencing local coagulation activation. the n-glyean pattern of recombinant human coagulation factors ii (rf-i~ and ix (rf-ix), derived from both transfected chinese hamster ovary (cho) cells and african green monkey (vero) cells, and produced at industrial scale were analyzed by binding to carbohydrate specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. human plasma-derived coagulation factors 1i (hpf-i1) and ix (hpf-ix) exhibited complex-type glycan structures with carbohydrate chains capped with c~(2-6)sialic acid. terminal galactose-b(1-4)-n-acetylglucosamine units were detected in hpf-ix. both cho cell-derived rf-]i and rf-ix exhibited complot-type glycosylafion and contained ~2-3)-sialio acid in addition to terminal galactose-b(1-4)-n-acetylglucosamine. vero cell-derived if-ix exhibited a complex-type glycan structure sinailar to that of cho cell derived rf-ix. in contrast, rf-li produced by vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "highmarmose" structures and complex-type glycosylation containing et(2-3)-sialie acid. galaetose-b(1-4)-n-acetylglucosamine structures and a low concentration of ct(2-6)-sialic acid were detected in both micro-heterogeneity fractions of veto cell-derived rf-li. although different in the'tr carbohydrate structures, coagulation factors ii and ix obtained recombinantly from both transformed cho-cells and vero-cells exhibited coagulation activities comparable to the plasma-derived proteins. exposure of tissue factor (tf) to flowing blood, as a consequence of vascular injury, leads to tf/f.viia-initiated coagulation which may play a key role in triggering intravascular thrombosis. in order to evaluate the potential advantages of tf pathway blockade over existing therapy, the antithrombotic and the anticoagulant .effects of a monoclonal anti-rabbit tf antibody (ap-1) and heparin were compared in a rabbit arterial thrombosis model. thrombosis was induced by a local injury to the carotid artery and the recurrence of thrombus formation was followed by monitoring the cyclic flow variations (cfv) of the arterial blood flow. rabbits received intravenously either ap-1, heparin or vehicle, and cfv were monitored on line for 120 min. activated partial thromboplastin time (aptt), and activated clotting time (act) were measured at the end of the experiment control ap-i heparin <lmg/kg* 0.2mg/kg/hr 0.8mg/kg/hr intraabdominallretroperitoneal, cns) on a named-patient basis. later also mild/moderate bleeds, predominantly joint bleeds were included.in 80-90% of the bleeds novoseven was found to be hemostatically efficacious. also in major surgery (hip-and knee-replacement including a bilateral total condylar arthroplasty, herniotomy) hemostatic efficacy has been found to be 94% since rfvlla is proteotytically active only in complex with tf the risk for systemic activation of the coagulation system should be minimal. also very few side-effects have been seen.doses between 75-120 tjg/kg have been used, which initially have to be given at 2 hours intervals. to achieve satisfactory hemostasis both an initial dose high enough to induce immediate hemostasis and an appropriate dose interval ensuring a plasma level of fvli:c well above the hemostatic lower level for a period of time, the length of which is dependent on the type of bleeding, are important. accordingly, the highel the initial dose the more robust with regard to dose interval will the treatment be. acute myocardial ischemia is usually triggered by coronary thrombosis. heparin (hep), along with aspirin, is commonly used for the treatment of unstable angina (ua) and myocardial infarction (mi). hirudin (hiq is a potent antitbrombin agent which may offer therapeutic advantages over standard hep for the treatment ua and mi. the oasis phase i pilot study conducted in 24 canadian centres, has evaluated the safety, feasibility and efficacy of hir (hbw 023, behringwerke ag, marburg) compared to hep in 601 patients with mi or suspected non q-wave mi. patients (95 % received aspirin) were randomized to a 72 hr infusion of either hep (5000 u bolus followed by 1200 u/hr; n=253), or low dose hir (0.2 mg/kg bolus then 0.10 mg/kg/hr; n=175) or medium dose lair (0.4 ms/ks bolus then 0.15 mg/kg/hr; n=173). at seven days of follow-up the incidence of major bleeds was 0.4 % in the heparin group, 0.6 % low dose hit, 1.2 % medium dose lair (p=ns), minor bleeds were 10.3 %, 17.1% and 19.1% respectively (p---0.008 hep vs med dose); there were no hemorrhagic strokes. the incidence of the primary outcome of cardiovascular death, new mi and severe recurrent ischemia (refractory or severe angina) was 17.0 %, 15.4 % and 8.7 % respectively (p---0.014 hep vs reed dose). there was also a trend towards lower rates of coronary artery bypass surgery in reed dose hir compared to hep. in a 200 patient substudy, levels of thrombin antithrombin complex, fibfinopeptide a and d-dimer were all suppressed to a greater extent by mr compared to hep. these results indicate that hirudin may be a more effective agent for the treatment of acute coronary syndromes, but more information on safety and efficacy is needed from larger randomized trials. full clinical and coagulation resuks will be presented. hat is caused by an immunologic response to heparin. affected patients need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory confirmed hat (hipa-test) with r-hirudin (behringwcrke ag, marburg). treatment regimens were: ai acute hat: 0.4 mg/kg b.w., continuous infusion 0a5 mg/kg b.w/h (51 patients); a2 acute hat with thrombolysis: bolus 0.2 mg/kg b.w., continuous infusion 0.1 mg/kg b.w./h (5 patients); b: proph, treatment: continuous infusion 0. i mg/kg b.w.pa (18 patients); c: during cardio-pulmunary bypass. primary objective was: increase in platelet counts or maintenance of normal baseline values during effective anticoagulation. secondary objectives were the incidence of new thromboembolic complications (tecs), major hemorrhage, limb amputations and deaths. results were compared with a historical control of 120 ix-at patients, all confirmed by the hipa test, but treated before rhirudin was available (danaparoid, marcumar, no anticoagulation). primary efficacy analysis: respunders ai: 74%o, a2: 60%, b: 44%, c: 50%, total: 65%. all adverse events beside bleeding complications had been judged to be caused by the underlying disease and not directly related to r-hirndin. proportion of patients with event in the obse~wation period; death: a1 5.9%, a2 0%, b 16.774, c 0%, total: 7.3%, during rhirudin treatment: 1.2%; new tec: ai 5.9%, a2 0%, b 27.8%, c 0%, total 9.8%, during rhirudin: 2.4%; limb amputation: ai 3.9%, a2 0%, b 5.6°/0, c 0%, total: 3.7%, during rbirudin: 3.7% eaapoiat we conclude, that r-hirndin is an effective anticoagulant in hat typa ii patients allowing rapid re, coved" of platelet counts. compared to a historical control the r-hirudin group had a strongly reduced incidence of combined endpuint of mortality, limb amputation and new tecs. major bleedings occurred slightly more frequent than in the historical control. however, the dosage used in regimen b might have been too low, as flds group revealed an effective anticoagulatiun and increase in platelet counts in only 43% and concomitantly had the higl)est incidence of new tecs. key: cord-004894-75w35fkd authors: nan title: abstract date: 2006-06-14 journal: eur j epidemiol doi: 10.1007/s10654-006-9021-1 sha: doc_id: 4894 cord_uid: 75w35fkd nan the organisers of the european congress of epidemiology 2006, the board of the netherlands epidemiological society, and the board of the european epidemiology federation of the international epidemiological association (iea-eef) welcome you to utrecht, the netherlands, for this iea-eef congress. epidemiology is a medical discipline that is focussed on principles and methods of research on causes, diagnosis and prognosis of disease, and establishing the benefits and risks of treatment and prevention. epidemiological research has proven its importance by contributions to the understanding the origins and consequences of diseases, and has made major contributions to the management diseases and improvement of health in-patients and populations. this meeting provides a major opportunity to affirm the scientific and societal contributions of epidemiological research in health care practice, both in clinical medicine and in public health. during this meeting major current health care problems are addressed alongside methodological issues, and the opportunities and challenges in approaching them are explored. the exchange of ideas will foster existing co-operation and stimulate new collaborations across countries and cultures. the goal of this meeting is to promote the highest scientific quality of the presentations and display advanced achievements in epidemiological research. our aim is to offer a comprehensive and educational programme in the field of epidemiological research in health care and public health, and create room for discussions on contemporary methods and innovations from the perspective of policy makers, professionals and patients. above all, we want to stimulate open interaction among the congress participants. your presence in utrecht is key to an outstanding scientific meeting. the european congress of epidemiology 2006 is organised by epidemiologists of utrecht university, under the auspices of the iea-eef, and in collaboration with the netherlands epidemiological society. utrecht university, founded in 1634, is the largest university in the netherlands and harbours the largest academic teaching hospital in the netherlands. the epidemiologists from utrecht university work in the faculties of medicine, veterinary medicine, pharmacy and biology. the current meeting was announced through national societies, taking advantage of their newsletters and of the iea-eef newsletter. in addition, avoiding the costs and disadvantages of the traditional journal advertisements and leaflets, information about the congress was disseminated via an internet mailing list of epidemiologists, which was compiled from, among other, the meeting in porto in 2004, the european young epidemiologist network (http://higiene.med.up.pt/eye/) and several institutions and departments. many of the procedures followed this year were based on or directly borrowed from the stimulating iea-eef congress in porto in 2004. publication in an international journal of large circulation of the congress programme and abstracts selected for oral and poster presentation, signifies the commitment of the organisers towards all colleagues that decided to present their original work at our meeting, and is intended to promote our discipline and to further stimulate the quality of the scientific work of european epidemiologists. and methods to the objectives and quality of its description; presentation of results; importance of the topic; and originality. a final rating was given on a 0-10 point scale. the two junior epidemiologists independently evaluated each abstract. based on ratings of the juniors, the senior epidemiologist gave a final abstract rating. the senior reviewer decided when juniors disagreed, and harnessed against untoward extreme judgements of the juniors. based on the judgement by the seniors abstracts with a final rating of 6 or higher were accepted for presentation. next, in order to shape the scientific programme according to scientific and professional topics and issues of interest for epidemiologists, members of the scientific programme committee grouped the accepted abstracts in major thematic clusters. for these, topics, keywords and title words were used. within each cluster, abstracts with a final rating of 8 or higher, as well as abstracts featuring innovative epidemiological approaches were prioritised to be programmed as an oral presentation. the 642 submitted abstracts had an average final rating of 6 (sd=1). in total 148 abstracts (23%) with a final rating of 5 or lower were rejected. because of the thematic programming some abstracts with a final rating of 8 or higher will be presented as posters, while few with a final rating of 7 are programmed as oral presentation. there were 345 abstracts (54%) accepted and programmed for poster presentation; each poster will be displayed for a full day. in total 149 abstracts (23%) are accepted for oral presentation. these are programmed in 29 parallel sessions. based on the topics of their abstracts the oral sessions were arranged into 7 themes, notably epidemiology of diseases, methods clinical & population research, burden of disease, high risk populations, growth and development, public health strategies, translational epidemiology. sessions from one theme never are programmed parallel. in table 1 we present the submitted and accepted abstracts (oral or poster) according to the distribution of country of origin. in table 2 submitted abstracts are displayed according to their topic, as classified by the authors using the topic long list of the submission form. the scientific programme committee convened in a telephone meeting by the end of the summer of 2005 and decided on the above programming process. the scientific programme committee was informed on the result of the programming process by the end of april 2006. fifteen abstracts were submitted for the eye session work in progress. of these 3 abstracts were selected for oral presentation and thereby nominated for the eye award. in total, 10 abstracts were submitted in relation to the student award, of which 6 were programmed for oral presentation and thus nominated. during the congress authors of poster presentations may name themselves as candidate for the poster award. during the closing ceremony the winners of the student award 2006 and the poster award 2006 will be announced. these awards are an initiative of the netherlands epidemiological society that will fund them in 2006. according to the iea rules expenses of congress participation for applicants from low-income countries will be covered. the board of the iea-eef will select a maximum of 10 candidates; their travel and registration expenses will be (partly) covered from the congress budget. in order to stimulate participation form as many as possible junior researchers and young epidemiologists the congress budget covers registration fee reduction for undergraduate (msc) participants and eye members. this also holds for the registration fee reduction of iea-eef members and nes members. it is 11 years ago (1995) that the iea regional european meeting was held in the hague, the marcon 348 wei 352 leray 353 gehring 354 leray 356 spallek 362 greving 367 laan 372 brussee 379 brussee 383 mayde groot 385 hoefman 386 goettsch 387 posters ordered by abstract number 11 olawuyi 19 de vries 21 uotinen 22 smits 26 gimeno 28 houben 29 de vries 30 de vries 31 ten berg 34 diaconu 36 diaconu 46 streppel 47 belo 48 sauvaget 51 koopman 71 kilsztajn 76 pembrey 81 schmidt 85 kretzschmar 102 scholtens 109 defraye 116 medronho 117 eljedi 124 van de garde 127 mello 128 hosper 129 feleus 134 bayingana 135 de wit 139 stolk 169 teixeira 171 teixeira 175 verhoef 180 capon 185 de boer 186 lazarevska 198 terschu¨ren 201 khosravi boroujeni 202 molag 208 mikolajczyk 209 luijsterburg 216 stolk 219 mirabelli 236 barreto 242 curzio 262 pereira 268 van gageldonk-lafeber 284 van nispen 287 roobol 294 mokkink 295 rava 303 haukka 304 jansen 307 de kraker 313 bogers 322 donalisio 325 behrens 329 borders 332 melis 347 pac 364 muller 365 van den hooven 369 van der sande 373 van den berg 395 novoa 410 van den boogaard 411 vannoord 412 koedijk 414 kuczerowska 420 giorgi rossi 432 vannoord 433 baussano 434 agabiti 457 bierma-zeinstra 460 van wier 464 faustini 465 jarrin 477 juhl 484 miguel 485 maira 497 lindert 508 van den berg 509 mierzejewska 520 fonseca cardoso 525 martens 536 cotton 539 corte 542 ursoniu 544 vernic 545 boer 563 ruskamp 568 szurkowska 572 bijkerk 574 fonseca cardoso 576 mazur 595 ahrens 598 dijkstra 600 ajdacic-gross 603 ajdacic-gross 604 lucas 607 santos 634 gielkens-sijstermans 639 tobi 643 de kraker proteomics and genomics are supposed to be related to epidemiology and clinical medicine, among other because of the putative diagnostic usefulness of proteomics and genomics tests. hence, clinical and sometimes even public health applications are promised by basic sciences. it is debated whether such promises and subsequent expectation are fulfilled. what are at meaningful and consequential examples of current findings in proteomics, genomics and similar approaches in biomedical research? are they different from the ''classic'' tools and frameworks of clinical epidemiology? in the context of proteomics and genomics, etiologic studies, primary prevention, epidemiological surveillance and public health are concerned with the influence of environmental exposures on gene expression and on the accumulation of genetic alterations. proponents and advocates of proteomics and genomics have suggested that their products can yield clinically useful findings, e.g., for early diagnosis, for prognosis, for therapeutic monitoring, without always needing to identify the proteins, peptides or other 'biomarkers' at stake. do we feel comfortable with this ''black-box'' reasoning, i.e. do we question the role of pathophysiological and mechanistic reasoning in clinical medicine? how much sense does it make for epidemiology to play with and scrutinize proteomics and genomics approaches in epidemiology and clinical medicine? what are at present (and in the near future) the main biological, clinical and public health implications of current findings in these research fields? in this plenary session these and other questions regarding the place and role of proteomics and genomics in clinical epidemiological research are discussed from different perspectives. infection diseases: beneficial or disaster for man? infectious diseases pose an increasing risk to human and animal health. they lead to increasing mortality, in contrast to the situation fifty years ago when new control measures still provided hope of overcoming many problems in the future. improved hygiene, better socio-economic circumstances, vaccination and use of antibiotics has led to a gradual decline of tuberculosis, rheumatic fever, measles and mumps in western societies over the last five decades. paradoxically, absence of exposure to infectious agents has a major impact as well. the decline in infectious disease risk is accompanied by a gradual increase of allergic and autoimmune diseases and this association is believed to be causal. exposure to infectious agents from early on in life can markedly boost an individual's natural resistance and hence influence the individual's reaction to future exposure to both biological and non-biological antigens. in this plenary session we want to emphasise both aspects of the effect of infectious agents on human and animal health. evidence based medicine in health care practice and epidemiological research p. glasziou & l. bonneux evidence-based medicine is defined as the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. proponents of evidence-based medicine maintain that coming form a tradition of pathophysiological rationale and rather unsystematic clinical experience, clinical disciplines should summarize and use evidence concerning their practices, by using principles of critical appraisal and quantitative clinical reasoning. for this they should convert clinical information needs into answerable questions, locate the available evidence, critically appraise that evidence for its validity and usefulness, and apply the results of the best available evidence in practice. applying the principles of evidence-based medicine implies improvement of the effectiveness and efficiency of health care. therefore, evidence-based medicine has commonalties with clinical medical and epidemiological research. for integration of evidence-based medicine into health care practice the challenge is to translate knowledge from clinical medical and epidemiological research, for example in up to date practice guidelines. the limitations of using evidence alone to make decisions are evident. the importance of the values and preference judgments that are implicit in every clinical management decision are also evident. critics of evidence based medicine argue that applying best available research evidence in practice in order to improve the effectiveness and efficiency of health care contradicts with the importance of the values and preference judgments in clinical management decisions. in this plenary session we want to contrast these and other viewpoints on evidence based medicine in health care practice. statistical topics i: missing data prof. dr. t. stijnen this workshop will be an educational lecture on missing data by professor stijnen from the department of epidemiology and biostatistics of the erasmus mc rotterdam, the netherlands. every clinical or epidemiological study suffers from the problem of missing data. in practice mostly simple solutions are chosen, such as restricting the analysis to individuals without missing data or imputing missing values by the mean value of the individuals with observed data. it is not always realised that these simple approaches can lead to considerable bias in the estimates and standard errors of the parameters of interest. in the last 10 to 15 years much research has been done to better methods for dealing with missing values. in this workshop first an overview will be given of the methods that are available and their advantages and disadvantages will be discussed. most attention will be given to multiple imputation, to date generally considered as the best method for dealing with missing values. also the available software in important statistical packages such as spss and sas will be shortly discussed. prof. dr. b. van hout suppose that one wants to know how often individuals have a certain characteristic, and suppose that one doesn't have any knowledge -any knowledge at all -how often this is the case. now, suppose that one starts by checking 10 individuals and only finds 1 individual with this characteristic. than the probability that the 11' th individual has the characteristic is 1/6. the fact that this is not 1/10 (although it will be close to that if the numbers of observations increase) may be counter-intuitive. it will become less so, when realising how it is obtained from the formal integration of the new information with the complete uncertainty beforehand. this formal integration, with a prior indicating that it is as likely to be 1/100 as it is to be 50/100 as it is 99/100, and with 9 negative and 1 positive observation -is by way of bayes rule. the italian mathematician, actuary, and bayesian, bruno de finetti (1906 -1985 , estimated that it would take until the year 2020 for the bayesian view of statistics to completely prevail. the purpose of this workshop is to not only convince the attendants that this is appealing outlook but also to aid the workshop participants in realising this prediction. after a first introduction of the work of reverend bayes, a number of practical examples are presented and the attendant is introduced in the use of win-bugs. a first example -introducing the notion of noninformative priors -concerns a random effects logistic regression analysis. second, the use of informative priors, is illustrated (in contrast with non-informative priors) using an analysis of differences in quality of life as observed in a randomised clinical trial. it will be shown how taking account of such prior information changes the results as well as showing how such information may increase the power of the study. in a third example, it will be shown how winbugs offers a powerful and flexible tool to estimate rather complex multi-level models in a relatively easy way and how to discriminate between various models. within this presentation some survival techniques (or stress control techniques) will be presented for when winbugs starts to spit out obscure error-codes without giving the researcher any clue where to search for the reason behind these errors. communicating research to the public h. van maanen, prof. dr. ir. d. kromhout, a. aarts most researchers will at some point in their career face difficulties in communicating research results to the public. whereas most scientific publications will pass by the larger public in silence, now and then a publication provokes profound interest of popular press. interest from the general public should be regarded as positive. after all, public money is put into research and a researcher has a societal responsibility of spreading new knowledge. however, often, general press interest is regarded upon as negative by the researcher. the messages get shortened, distorted or ridiculed. whose responsibility is this misunderstanding between press and researchers? should a researcher foresee press reaction and what can be done to prevent negative consequences? is the press responsible background and relevance: patients with a carotid artery stenosis, including those with an asymptomatic or moderate stenosis, have a considerable risk of ischemic stroke. identification of risk factors for cerebrovascular disease in these patients may improve risk profiling and guide new treatment strategies. objectives and question: we cross-sectionally investigated whether carotid stiffness is associated with previous ischemic stroke or tia in patients with a carotid artery stenosis of at least 50%. design and methods: patients were selected from the second manifestations of arterial disease (smart) study, a cohort study among patients with manifest vascular disease or vascular risk factors. arterial stiffness, measured as change in lumen diameter of the common carotid arteries during the cardiac cycle, forms part of the vascular screening performed at baseline. the first 420 participants with a stenosis of minimally 50% in at least one of the internal carotid arteries measured by duplex scanning were included in this study. logistic regression analysis was used to determine the relation between arterial stiffness and previous ischemic stroke or tia. results: the risk of ischemic stroke or tia in the highest quartile (stiffest arteries) relative to the lowest quartile was 2.1 (95% ci 1.1-4.1). these findings were adjusted for age, sex, systolic blood pressure, minimal diameter of the carotid artery and degree of carotid artery stenosis. conclusion and discussion: in-patients with a ‡50% carotid artery stenosis, increased common carotid stiffness is associated with previous ischemic stroke and tia. measurement of carotid stiffness may improve selection of high-risk patients eligible for carotid endarterectomy and may guide new treatment strategies. background (and relevance): patients with advanced renal insufficiency are at increased risk for adverse cardiovascular disease (cvd) outcomes. objectives and question: the aim was to establish whether impaired renal function is an independent predictor of cvd and death in an unselected high-risk population with cvd. design and methods: the study was performed in 2784 patients with cvd. primary outcomes were all vascular events and all cause death. during a median follow-up of 39 months, 291 patients had a vascular event (10%) and 266 patients died (9.5%). results: the adjusted hazard ratio (hr) of an estimated glomerular filtration rate <60 vs >90 ml/min per 1.73 m2 was 1.8 (95% ci 1. 2-2.7) for vascular events and 1.4 (95% ci 0.9-2.2) for all cause death. for stroke as a separate outcome it was 2.7 (95% ci 1. 2-5.8) . subgroup analysis according to vascular disease at presentation or the risk factors hypertension, diabetes and albuminuria had no influence on the hr's. conclusion and discussion: renal insufficiency is an independent risk factor for adverse cvd events in patients with a history of vascular disease. renal function was a particularly important factor in predicting stroke. the presence of other risk factors hypertension, diabetes or albuminuria had no influence on the impact of renal function alone. background and relevance: patients with hypertension have an increased case-fatality during acute mi. coronary collateral (cc) circulation has been proposed to reduce the risk of death during acute ischemia. objectives and question: we determined whether and to which degree high blood pressure (bp) affects the presence and extent of cc-circulation. design and methods: cross-sectional study in 237 patients (84% males), admitted for elective coronary angioplasty between january 1998 and july 2002. collaterals were graded with rentrop's classification (grade 0 -3). ccpresence was defined as rentrop-grade ‡1. bp was measured twice with an inflatable cuff-manometer in seated position. pulse pressure was calculated by systolic blood pressure (sbp) )diastolic blood pressure (dbp). mean arterial pressure was calculated by dbp + 1/3*(sbp-dbp). systolic hypertension was defined by a reading ‡140 mmhg. we used logistic regression with adjustment for putative confounders. results: sbp (odds ratio (or) 0.86 per 10 mmhg; 95% confidence-interval (ci) 0.73-1.00), dbp (or 0.67 per 10 mmhg; 95% ci 0.49-0.93), mean arterial pressure (or 0.73 per 10 mmhg; 95% ci 0.56-0.94), systolic hypertension (or 0.49; 95% ci 0.26-0.94), and antihypertensive treatment (or 0.53; 95% ci 0.27-1.02), each were inversely associated with the presence of cc's. also, among patients with cc's, there was a graded, significant inverse relation between levels of sbp, levels of pulse pressure, and collateral extent. conclusion and discussion: there is an inverse relationship between bp and the presence and extent of cc-circulation in patients with ischemic heart disease. background and relevance: silent brain infarcts are associated with decreased cognitive function in the general population. objectives and question: we examined whether this relation also exists in patients with symptomatic arterial disease. furthermore, we compared cognitive function of patients with stroke or tia, with cognitive function of patients with symptomatic arterial disease at other sites in the arterial tree. design and methods: an extensive screening was done in 336 consecutive patients participating in the second manifestations of arterial disease (smart) study, including a neuropsychological test. inclusion diagnoses were cerebrovascular disease, symptomatic coronary artery disease, peripheral arterial disease, or abdominal aortic aneurysm. mri examination was performed to assess the presence of silent infarcts in patients without symptomatic cerebrovascular disease. the patients were assigned to one of three categories according to their patient history and inclusion diagnosis: no stroke or tia, no silent infarcts (n = 220; mean age 57 years); no stroke or tia, but silent infarcts present (n = 33; mean age 65 years); stroke or tia at background and relevance: patients with manifest vascular disease are at high risk of a new vascular event or death. modification of classical risk factors is often not successful. objectives and question: we determined whether the extra care of a nurse practitioner (np) could be beneficial to the cardiovascular risk profile of high-risk patients. design and methods: randomised controlled trial based on the zelen design. 236 patients with manifestations of a vascular disease and who had ‡2 modifiable vascular risk factors were prerandomised to receive treatment by a np plus usual care or usual care alone. after 1 year, risk factors were re-measured. primary endpoint was achievement of treatment goals for risk factors. results: of the pre-randomised patients, 95 of 119 (80%) in the intervention group and 80 of 117 (68%) in the control group participated in the study. after a mean follow-up of 14 months, the patients in the intervention group achieved significantly more treatment goals than did the patients in the control group (systolic blood pressure 63% versus 37%, total cholesterol 79% vs 61%, ldl-cholesterol 88% vs 67%, and bmi 38% vs 24%). medication use was increased in both groups and no differences were found in patients' quality of life (sf-36) at followup. conclusion and discussion: treatment delivered by nps, in addition to a vascular risk factor screening and prevention program, resulted in a better management of vascular risk factors compared to usual care alone in vascular patients after 1 year follow-up. were used as non-invasive markers of vascular damage and adjusted for age and sex if appropriate. results: the prevalence of the metabolic syndrome in the study population was 45%. in pad patients this was 57%; in chd patients 40%, in stroke patients 43% and in aaa patients 45%. patients with the metabolic syndrome had an increased mean imt (0.98 vs. 0.92 mm, p-value <0.01), more often a decreased abpi (14% vs. 10%, p-value 0.06) and increased prevalence of albuminuria (20% vs. 15%, p-value 0.03) compared to patients without this syndrome. an increase in the number of components of the metabolic syndrome was associated with an increase in mean imt (p-value for trend <0.001), lower abpi (p-value for trend < 0.01) and higher prevalence albuminuria (p-value for trend < 0.01). conclusion and discussion: in patients with manifest vascular disease the presence of the metabolic syndrome is associated with advanced vascular damage. background (and relevance): in patients with type 2 diabetes the progression of atherosclerosis is accelerated, as observed by the high incidence of cardiovascular events. objectives (and question): to estimate the influence of location and extent of vascular disease on new cardiovascular events in type 2 diabetes patients. design and methods: diabetes patients (n = 669), mean age 59 years, with and without prior vascular disease were followed through 1996-2004 (mean follow-up 3 years). patients with vascular disease (n = 399) were classified according to symptomatic vascular location, and number (extent) of locations. we analyzed occurrence of new (non)-fatal cardiovascular events using cox proportional hazards models and kaplan-meier analysis. results: multivariate-adjusted hazard ratios (hrs) were comparable in diabetes patients with cerebrovascular disease (hr 3.2; 95% ci 1.3-7.4), coronary heart disease (hr 3.3; 1.5-7.1) and peripheral arterial disease (hr 2.6; 1.1-6.3), compared to those without vascular disease. multivariate-adjusted hr was 2.7; (1. 3-5.9 ) in patients with one vascular location and 4.8; (2.1-10.9) in those with ‡2 locations. the 3-year risks were respectively 11.6% (7.4-15.7) and 26.7% (16. 8-36.7) . conclusion and discussion: diabetes patients with prior vascular disease have an increased risk of cardiovascular events, irrespective of symptomatic vascular location. cardiovascular risk increased with the number of locations. data emphasize the necessity of early and aggressive treatment of cardiovascular risk factors in diabetes patients. background (and relevance): despite recent advances in medical treatment, cardiovascular disease (cvd) is still health problem number one in western societies. a multifactorial approach with the aid of nurse practitioners (nps) is beneficial for achieving treatment goals and reducing events in patients with manifest cvd. objectives (and question): in the self-management of vascular patients activated by internet and nurses (spain) pilot study, we want to implement and test a secure personalized website with additional treatment and coaching of a np for hypertension, hyperlipidemia, diabetes mellitus, smoking and obesity in patients with clinical manifestations of cvd. design and methods: 50 interesting patients are going to use the secure patient-specific website. before the use of the web-application, risk factors are measured. realistic treatment goal(s) for elevated risk factors based on current guidelines are made and appointments how to achieve the treatment goal(s) are determined between the patients and the np in a face to face contact. patients can enter his/ her own weight or a new blood pressure measurement for instance, besides the regular exchange information with the responding np through e-mail messages. the np personally replies as quick as possible and gives regular but protocol driven feedback and support to the patient. the risk factors are remeasured after six months. conclusion and discussion: the spain study is aimed to implement and test a patient specific website. secondary outcome is the change in cardiovascular risk profile. the pre-post measurements of risk factors and the amount of corresponding e-mail messages between the patient and the np enhances the feasibility of this innovative way of risk factor management. background (and relevance): modification of vascular risk factors has been shown to be effective in reducing mortality and morbidity in patients with symptomatic atherosclerosis. nevertheless, reduction of risk factors in clinical practice is difficult to achieve and maintain. objectives (and question): in the risk management in utrecht and leiden evaluation (rule) study, a prospective, comparative study, we assess the effects of a multidisciplinary vascular screening program on improvement of the cardiovascular risk profile and to compare this to a setting without such a program that provides current standard practice in patients referred for cardiovascular disorders. design and methods: patients with diabetes mellitus, coronary artery disease, cerebrovascular disease, or peripheral arterial disease (150 per disease category in each hospital) referred by the general practitioner will be enrolled, started january 2005. at the umcu, patients need to be enrolled in the vascular screening program or will be identified through the hospital registration system. at the lumc patients will be identified through the hospital registration system. risk factors will be measured in the two hospitals at baseline and one year after their initial visit. a risk function will be developed for this population based on data of the whole cohort. analysis will be performed on the two comparison groups as a whole, and on subgroups per disease category. changes in risk factors will be assessed with linear or logistic regression procedures, adjusting for differences in baseline characteristics between groups. conclusion and discussion: the rule study is aimed to evaluate the added value of a systematic hospital based vascular screening program on risk factor management in patients at high risk for vascular diseases. background: signs of early cerebral damage are frequently seen on mri scans in elderly people. they are related to future manifest cerebrovascular disease and cognitive deterioration. cardiovascular risk factors can only partially explain their presence and progression. evidence that inflammation is involved in atherogenesis continues to accumulate. chronic infections can act as an inflammatory stimulus. it is possible that subclinical inflammation and chronic infections play a role in the pathogenesis of early cerebral damage. objectives (and question): to unravel the role of inflammation and chronic infection in the occurrence and progression of early cerebral damage in patients with manifest vascular disease. design and methods: 1200 participants of the smart study with manifest vascular disease underwent an mr investigation of the brain between may 2001 and december 2005. starting in january of 2006 all patients are invited for a second mr of the brain after an average follow-up period of four years. both at baseline and after follow-up all cardiovascular risk factors are measured and blood samples are stored to assess levels of inflammatory biomarkers and antibodies against several pathogens. occurrence and progression of early cerebral damage is assessed by measuring the volume of white matter lesions, the number of silent brain infarctions, cerebral atrophy, aberrant metabolic ratios measured with mr spectroscopy and cognitive function at baseline and after follow-up. the relation between inflammation, chronic infection and the occurrence and progression of early cerebral damage will be investigated using both crosssectional and longitudinal analysis. abstract 13 monocyte chemoattractant protein 1 (mcp-1) polymorphism and susceptibility for coro-nary collaterals j.j. regieli 1,2 , j. koerselman 2 , ng sunanto 1,2 , m. entius 1 , p.p. de jaegere 1 , y. van der graaf 2 , d.e. grobbee 2 , p.a. doevendans 1 1 heart lung institute, dept of cardiology 2 clinical epidemiology, julius center for health sciences and primary care, utrecht, netherlands background (and relevance): collateral formation is an important beneficial condition during an acute ischemic event. a marked interindividual variability in high risk patients is seen, but at present the basis for this variability is unclear and can not be explained solely by environmental factors. a genetic factor might be present that could influence coronary collateral formation. objectives (and question): we have analyzed the association between a single nucleotide polymorphism in mcp-1 and the formation of coronary collaterals in patients admitted for angioplasty. mcp-1 has been suggested to play an important role in collateral development. design and methods: this study involved 226 caucasian patients who were admitted for coronary angioplasty. coronary collateral development was defined angiographically as rentropgrade ‡1. polymorphisms in the promoter region of mcp-1 ()2518) were identified by pcr and allele specific restriction digestion. this method allows identification of individuals with either aa, ag or gg at mcp-1 position )2518. statistical analysis was performed using a x2-test, unconditional logistic regression, likelihood ration and a wald's test. results: we could genotype 224 of the 226 patients. coronary collaterals (rentropgrade >1) were found in 84 patients. the allele frequency for aa, ag and gg was 53.1%, 40.2% and 6.7%, respectively. the dis-tribution of mcp-1 genotypes in subjects without collaterals was in hardy weinberg equilibrium. we found that individuals with g allele (24%) were more likely to have collaterals than those with homozygous aa (or 1.66, 95% ci 0.96 to 2.87) adjusted for potential confounders. linear regression shows that the allele g increased the likelihood for collateral presence with a factor 1.41. conclusion and discussion: this study provides evidence for a role for genetic variation of mcp-1 gene in the occurrence of coronary collaterals in high risk patients. 2001 until september 2003 patients with recently established clinically manifest atherosclerotic disease with >2 modifiable vascular risk factors were selected for the study. the mean self-efficacy scores were calculated for vascular risk factors (age, sex, vascular disease, weight, diabetes mellitus, smoking behavior, hypercholesterolemia, hypertension, and hyperhomocysteinemia). results: diabetes, overweight, and smoking, but none of the other risk factors, were significantly associated with the level of self-efficacy in these patients. patients with diabetes had lower self-efficacy scores (3.0) for exercise and controlling weight (3.7) than patients without diabetes (4.2 p = 0.05) and (4.1 p = 0.01) respectively. overweight patients scored low on controlling weight (3.8 and 3.5 p<0.001) and choosing healthy food (3.7 and 3.3 p = 0.01) than patients who were on a healthy weight (4.3 and 4.0). conclusion and discussion: patients with vascular diseases appear to have high levels of self-efficacy regarding medication use (4.8), exercise (4.1), and controlling weight (4.0). in patients with diabetes, overweight and in smokers, self efficacy levels were lower. practice implications: in nursing care and research on developing self-efficacy based interventions, lower self-efficacy levels can be taken into account for specific vascular patient groups. background (and relevance): little is known about the role of serum uric acid in the metabolic syndrome and increased risk of cardiovascular disease. we investigated the association between uric acid levels and the metabolic syndrome in a population of patients with manifest vascular diseases and whether serum uric acid levels conveyed an increased risk for cardiovascular disease in patients with the metabolic syndrome. design and methods: this is a nested case-cohort study of 431 patients originating from the second manifestations of arterial disease (smart) study. all patients had manifest vascular diseases, constituting peripheral artery disease, cerebral ischemia, coronary artery disease and abdominal aortic aneurysm. analyzing the relationship of serum uric acid with the metabolic syndrome, age, sex, creatinine clearance, alcohol and diuretics were considered as confounders. investigating the relationship of serum uric acid levels with the risk for cardiovascular disease, values were adjusted for age and sex. results: the metabolic syndrome was present in 50% of the patients. serum uric acid levels in 214 patients with metabolic syndrome were higher compared to 217 patients without (0.36 ± 0.08 mmol/l vs. 0.32 ± 0.09 mmol/l). serum uric acid concentrations increased with the number of components of the metabolic syndrome (0.30 mmol/l to 0.38 mmol/l) adjusted for age, sex, creatinine clearance, alcohol and use of diuretics. increased serum uric acid concentrations showed to be independently associated with the occurrence of cardiovascular events in patients without the metabolic syndrome (age en sex adjusted hr: 1.9, 95% ci 0. 9-3.9) , contrary to patients with the metabolic syndrome (adjusted hr: 1.3, 95% ci 0.6 -2.7). conclusion: elevated serum uric acid levels are strongly associated with the metabolic syndrome, yet are not linked to an increased risk for cardiovascular disease in patients with the metabolic syndrome. however, in patients without the metabolic syndrome elevated serum uric acid levels are associated with increased risk for cardiovascular disease. the objective of this study is to investigate the overall and combined role of late-life depression, prolonged psychosocial stress exposure, and stress hormones in the etiology of hippocampal atrophy and cognitive decline. design and methods: as part of the smart study, 1200 participants with manifest vascular disease underwent an mri of the brain between may 2001 and december 2005. in a subsample of 800 subjects, cognitive function and depressed mood were assessed. starting in january 2006, all patients are invited for a follow-up mri of the brain. at this follow-up measurement, minor and major depression, hypothalamic-pituitary-adrenal (hpa) axis function indicated by salivary cortisol, psychosocial stress exposure indicated by stressful life events early and later in life, and cognitive functioning will also be assessed. the independent and combined effects of late-life depression, (change in) hpa-axis activity, and psychosocial stress exposure on risk of hippocampal atrophy and cognitive decline will be estimated with regression analysis techniques adjusting for potential confounders. introduction the netherlands epidemiological society advocates according to good epidemiological practice, that research with sound research questions and good methodology should be adequately published independent of the research outcomes. although reporting bias in clinical trials is fully acknowledged, failure to report outcomes or selective reporting of outcomes in non-clinical trial epidemiological studies is less well known, but most likely occurs as well. in this mini-symposium the netherlands epidemiological society wants to give attention to this phenomenon of not publishing research outcomes, to encourage publication of all outcomes of adequate research. different scopes to this subject will be addressed: the background, an example of occurrence, initiatives to possibly avoid it and an editor's point of view. selective reporting of outcomes in clinical studies (reporting bias) has been described to occur frequently. therefore a registration of clinical trials is started which enables to address this problem in the future since occurrence of not publishing negative or adverse outcomes can be investigated with this registration. in non-clinical epidemiological studies the failure to report outcomes or selective reporting of outcomes most likely occurs as well, but is less studied and reported. again studies with negative outcomes or no associations are the ones most likely not to be reported. the most important obstacles for not publishing no or negative associations are tradition and priorities of researchers and journals. the reviewers might play a role in this as well. the netherlands epidemiological society advocates according to good epidemiological practice, that research with sound research questions and good methodology should be adequately published independent of the research outcomes. however, reality occurs not to be accordingly. therefore we would like to give attention to this phenomenon of not publishing research outcomes in non-trial-based epidemiological studies, to encourage publication of all outcomes of adequate research. in this mini-symposium, firstly the effects of failure or selective publishing of outcomes on subsequent meta-analysis in a non-clinical research setting will be demonstrated. afterwards, initiatives to promote and improve publication of observational epidemiological research will be addressed, the editor's point of view on this phenomenon will be given and finally concluding remarks will be given. background: there are several reasons for suspecting reporting bias in time-series studies of air pollution. such bias could lead to false conclusions concerning causal associations or inflate estimates of health impact. objectives: to examine time-series results for evidence of publication and lag selection bias. design and methods: all published time-series studies were identified and relevant data extracted into a relational database. effect estimates were adjusted to an increment of 10lg/m 3 . publication bias was investigated using funnel plots and two statistical methods (begg, egger). adjusted summary estimates were calculated using the ''trim and fill'' method. the effect of lag selection was investigated using data on mortality from 90 us cities and from a european multi-centre panel study of children. results: there was evidence of publication bias in a number of pollutant-outcome analyses. adjustment reduced the summary estimates by up to 20%. selection of the most significant lag increased estimates by over 100% compared with a fixed lag. conclusion and discussion: publication and lag selection bias occurs in these studies but significant associations remain. presentation and publication of time-series results should be standardised. background: selective non-publication of study outcomes hampers the critical appraisal and appropriate interpretation of available evidence. its existence could be shown empirically in clinical trials. observational research often uses an exploratory approach rather than testing specific hypotheses. results of multiple data analyses may be selected based on their direction and significance. objectives: to improve the quality of reporting of observational studies. to help avoid selective non-publication of study outcomes. methods: ''strengthening the reporting of observational studies in epidemiology (strobe)'' is an international multidisciplinary initiative that currently develops a checklist of items recommended for the reporting of observational studies (http:// www.strobe-statement.org). results: strobe recommends to avoid selective reporting of 'positive' or 'significant' study results and to base the interpretation on main results rather than on results of secondary analyses. discussion: strobe cannot prevent data dredging, but it promotes transparency at the publication stage. for instance, if multiple statistical analyses were performed in a large dataset to identify new exposure-outcome associations, authors should give details and not only report significant associations. strobe could have a ''feedback effect'' on study quality since, ideally, researchers think ahead when a study is planned and consider points that are essential for later publication. good publishing practice begins with researchers considering (1) whether an intended study can bring added value, irrespective its result, (2) and whether its methodology is valid to pick up positive and negative outcomes equally well. when reporting (3) they should adequately discuss the significance of a negative result (4) and be as eager to publish negative results as positive ones. as to editors, intentional bias in relation to study results is considered editorial malpractice, whatever its motivation. unintentional bias may be more frequent but will not easily be noticed, also by editors. editorial responsibility implies several levels (accepting for review, choice of reviewers, assess their reviews, decision making, and a repeated process in case of resubmission). various designs for process evaluation can be considered. evaluation will be more difficult for journals with few professional support. collaboration between journals can help, and may also avoid 'self evaluation bias'. in line with registering of randomized trials, registers for observational study protocols could facilitate monitoring for bias and searching unpublished results. but practicalities, methodological requirements, and bureaucratic burden should not be underestimated. in principle, in an era of electronic publishing every study can be made widely accessible widely also if not 'accepted', by editors or authors themselves. however, this would need huge changes in culture of authoring and reading, editorial practice, publishing business, and scientific openness. background: high circulating levels of insulin-like growth factor-i (igf-i), a mitogenic and anti-apoptotic peptide, have been associated with increased risk of several cancer types. objective: to study circulating levels of igf-i and igf binding protein-3 (igfbp-3) in relation to ovarian cancer risk. design and methods: within the european prospective investigation into cancer and nutrition (epic), we compared levels of igf-i and igfbp-3 measured in blood samples collected at baseline in 214 women who subsequently developed ovarian cancer (37 women diagnosed before age 50) and 388 controls. results: the risk of developing ovarian cancer before age 50 ('premenopausal' was increased among women in the middle or top tertiles of igf-i, compared to the lowest tertile: or = 2.69 [95% ci: 0.87 8.35 ], and or = 3.20 [95% ci: 1.01 -10.2], respectively (p trend = 0.06). results were adjusted for bmi, previous hormone use, fertility problems and parity. adjustment for igfbp-3 levels slightly attenuated relative risks. in older women we observed no association between igf-i, igfbp-3 and ovarian cancer risk. discussion and conclusion: in agreement with the only other prospective study in this field (lukanova et al, int j cancer, 2002) , our results indicate that high circulating igf-i levels may increase the risk of premenopausal ovarian cancer. background: the proportion of glandular and stromal tissue in the breast (percent breast density) is a strong breast cancer risk factor. insulin-like growth factor 1 (igf-1) is hypothesized to influence breast cancer risk by increasing breast density. objectives: we studied the relation between premenopausal circulating igf-1 levels and changes in breast density over menopause. design and methods: mammograms and blood samples of 684 premenopausal participants of the prospect-epic cohort were collected at baseline. a second mammogram was collected after these women became postmenopausal. we determined serum igf-1 levels. mammographic density was assessed using a computer-assisted method. changes in percent density over menopause were calculated for quartiles of igf-1, using linear regression, adjusted for age and bmi. results: premenopausal percent density was not associated with igf-1 levels (mean percent density 0.43 in all quartiles). however, women in the highest igf-1 quartile showed less decrease in percent density over menopause (1st quartile: )0.094 vs 4th quartile: )0.059, p-trend = 0.02). this was mostly explained by a stronger decrease of total breast size in women with high igf-1 levels. conclusion and discussion: women with high igf-1 levels show a lower decrease of percent density over menopause than those with low igf-1 levels. background: body mass index (bmi) has been found to be associated with risk of colon cancer in men, whereas weaker associations have been reported for women. reasons for this discrepancy are unclear but may be related to fat distribution or use of hormone replacement therapy (hrt) in women. objective: to examine the association between anthropometry and risk of colon cancer in men and women. design and methods: during 6.1 years of followup, we identified 984 cases of colon cancer among 368,277 subjects free of cancer at baseline from 9 european countries. results: bmi was significantly related to colon cancer risk in men (rr per kg/ m2, 1.05; 95%-ci 1.02-1.08) but not in women (rr 1.02; 1.00-1.04; p interaction = 0.04), whereas waist-hip-ratio (whr) was equally strong related to risk in both genders (rr per 0.1, men, 1.24; 95%-ci 1.05-1.46; women, 1.24; 1.10-1.39; p interaction = 0.92). the positive association for whr was not apparent among postmenopausal women who used hrt. conclusions: abdominal obesity is an equally strong risk factor for colon cancer in both sexes and whr is a disease predictor superior to bmi in women. the association may vary depending on hrt use in postmenopausal women; however, these findings require confirmation in future studies. background: fruits and vegetables are thought to protect against colorectal cancer. recent cohort studies, however, have not been able to show a protective effect. patients & methods: the relationship between consumption of vegetables and fruit and the incidence of colorectal cancer within epic was examined among 453,158 subjects of whom 1808 developed colorectal cancer. a multivariate cox proportional hazard model was used to determine adjusted cancer risk estimates. a calibration method based on standardized 24-hour dietary recalls was used to correct for measurement errors. results: after adjustment for potential confounding and exclusion of the first two years of follow-up, the results suggest that consumption of vegetables and fruits is weakly, inversely associated with risk of colorectal cancer (hr 1.00, 0.85, 0.85, 0.86, 0.79, for quintiles of intake, 95% ci upper quintile 0.65-0.97, p-trend 0.06), with each 100 gram daily increase in vegetables and fruit associated with a statistically borderline significant 3% reduction in colorectal cancer risk (hr 0.97; 0.94-1.00). linear calibration strengthened this effect. further subgroup analyses will be presented. conclusion: findings within epic support the hypothesis that increased consumption of fruits and vegetables may protect against colorectal cancer risk. a diverse consumption of vegetables and fruit may influence the risk of gastric and oesophageal cancer. diet diversity scores (dds) were calculated within the epic cohort data from >520,000 subjects in 10 european countries. four scores, counting the number of ffq-based food-items usually eaten at least once in two weeks, were calculated to represent the diversity in the overall vegetable and/or fruit consumption. after an average follow-up of 6.1 years, 400 incident cases of gastric and oesophageal cancer were observed. cox proportional hazard models were used to compute tertile specific risks, stratified by follow-up duration, gender and centre and adjusted for total consumption of vegetables and fruit and potential confounders.preliminary findings suggest that, compared to individuals who eat from only 5 or less vegetable sub-groups, individuals who usually eat from eight different subgroups, have a reduced gastric cancer risk (hr 0.68; 95% ci 0.45-1.05). in comparison to all others, individuals who usually eat only the same fruit may experience an elevated risk (hr 1.34; 95% ci 0.95-1.89). these findings from the epic study suggest that a diverse consumption of vegetables may reduce gastric and oesophageal cancer risk. subjects with a very low diversity in fruit consumption may experience higher risk. g. steindorf 1 , l. friedenreich 2 , j. linseisen 1 , p. vineis 3 , e. riboli 3 for the epic group 1 german cancer research center, heidelberg, germany 2 alberta cancer board, alberta, canada 3 imperial college london, great-britain background: previous research on physical activity and lung cancer risk, conducted predominantly in males, has yielded inconsistent results. objectives: we examined this relationship among 416,277 men and women from the epic-cohort. design and methods: during 6.3 years of follow-up we identified 607 men and 476 women with incident primary lung cancer. detailed information on recreational, household and occupational physical activity, smoking habits, and diet was assessed. relative risks (rr) were estimated using cox regression. results: we did not observe an inverse association between occupational, recreational or household physical activity and lung cancer risk either in males or in females. we found a modest reduction in lung cancer risk associated with sports in males and cycling in females. for occupational physical activity, lung cancer risk was increased for unemployed men (rr = 1.55; 95% confidence interval 1.18-2.03) and men with standing occupations (rr = 1.34; 1.01-1.78) compared with sitting professions. conclusion: our study shows no convincing protective associations of physical activity with lung cancer risk. discussion: it may be speculated that the elevated risks for occupational physical activity could reflect the higher probability that manual workers are exposed to industrial carcinogens compared to workers having sitting/office jobs. purposes: epidemiological research almost always means using data and, increasingly, human tissue as well. the use of these resources is not free but is subject to various regulations, which differ in the european countries on several important aspects. usually these regulations have been determined without involvement of active epidemiological researchers or patient organisations. this workshop will address the issues involved in these regulations in the european context. it will serve the following purposes: -to provide arguments and tools and to exchange best practices for a way out of the regulatory labyrinths especially in cross european research projects; -to provide a platform for epidemiologists and patient groups to discuss their concerns about impediments for epidemiological research with other parties, like data protection authorities. targeted audience: the mini symposium is primarily meant for epidemiologists, but provides an excellent opportunity to meet and discuss with other stakeholders, like from patient groups, data protection authorities, the european commission etc. as well. therefore program allows for extra time for discussion. the other stakeholders will be explicitly invited. a special 'day ticket' is available to satellite symposium epidemiology and the seventh eu research framework over the last few years the seventh eu research framework has been drafted. it is now rapidly moving towards the first calls for proposals. previous eu research programmes and frameworks have been criticised because they are considered to include too few possibilities for epidemiological research and public health research. this satellite-symposium will provide an outline of the research framework and inform researchers about the current state of affairs of the seventh eu research framework. special focus will be on the possibilities for epidemiology and public health research. 30-14.35 welcome by our host prof. jan willem coebergh, rotterdam, introduction, international and national regulations on the use of data and tissue or research in europe, different approaches to: evert-ben van veen l.l.m. (medlawconsult, the netherlands) -'identifiability' of data -consent for using data and tissue for research the tubafrost code of conduct to exchange data and tissue across europe. 14. 50-15.05 data and tissuebanking for research in denmark: a liberal approach the danish approach to use patient data for epidemiological research, cooperation of the danish data protection authority, the danish act of 2004 to use anonymous but coded tissue for research based on an opt-out system, first experiences hans storm ph.d. (copenhagen, denmark) 15. 05-15.20 estonian data protection act: a disaster for epidemiology the story of the birth of the act, implementing the european data protection directive and of its consequences reveal political and administrative incapability resulting in gradual vanishing of register-based epidemiological research. background: non-invasive assessment of atherosclerosis is important. most of the evidence of coronary calcium has been based on images obtained by electron beam ct (ebct). current data suggest that ebct and multi-slice ct (msct) give comparable results. since msct is more widely available than ebct, information on its reproducibility is relevant. objective: to assess inter-scan reproducibility of msct and to evaluate whether reproducibility is affected by different measurement protocols, slice thickness, cardiovascular risk factors and technical variables. design: cross-sectional study. materials and methods: the study population comprised 76 healthy postmenopausal women. coronary calcium was assessed in these women twice at two separate visits using msct (philips mx 8000 idt 16). images were made using 1.5 and 3.0 mm slice thickness. the agatston, volume and mass scores were assessed. reproducibility was determined by mean differences, absolute mean differences and intra-class correlation coefficients (iccc). results: the reproducibility of coronary calcium measurements between scans was excellent with iccc of > 0.98, and small mean and absolute mean differences. reproducibilility was similar for 1.5 as for 3.0 mm slices, and equal for agatston, volume and mass measurements. conclusion: inter-scan reproducibilility of msct is excellent, irrespective of slice thickness and type of calcium parameter. background: it has been suggested that the incidence of colorectal cancer is associated with socioeconomic status (ses). the major part of this association may be explained by known lifestyle risk factors such as dietary habits. objective: to explore the association between diet and ses measured at area-based level. methods: the data for this analysis were taken from a multi-centre case-control study conducted to investigate the association between some environmental, genetic factors and colorectal cancer incidence. the townsend scores (as deprivation index) were categorized into fifths. a linear regression analysis was used to estimate difference in mean of each continuous variable of diet by deprivation fifth. results: the mean of processed meat consumption in the most deprived area was higher compared to the mean of that in the most affluent areas (mean difference = 5.5, 95% ci: 3.94, 7.05). by contrast, the mean of vegetables and fruits consumption in the most deprived areas was lower than that in the affluent areas. conclusion: our findings suggest that lifestyle factors are likely to be related to ses. thus any relation between ses and colorectal cancer may direct us to seek for the role of different life style factors in aetiology of this cancer. background: the reason for the apparent decline in semen quality during the past 50 years is still unexplained. objective: to investigate the effect of exposure to cigarette smoke in utero on the semen quality in the male offspring. design and methods: in this prospective follow-up study, 350 adult sons of mothers, who during pregnancy provided information about smoking and other lifestyle factors, are sampled in six strata according to prenatal tobacco smoke exposure. each man provides a semen sample, a blood sample, and answers a questionnaire, which is collected in a mobile laboratory. external quality assessment of semen analysis is performed twice a year. results: until now, a total of 265 men have been included. the participation rate is 52%. the percentage of men with decreased sperm concentration (<20 mill/ml) is 23%. the unadjusted median (25-75% percentile) sperm concentration in the non-exposed group (n = 90) is 49 (23-86) mill/ml compared to 33 (12-63) mill/ml among men exposed to >19 cigarettes per day in fetal life (n = 26 aim: to estimate the prevalence of overweight and obesity, and their effects in physical activity (pa) levels of portuguese children and adolescents aged 10-18 years. methods: the sample comprises 12669 subjects (6489 females-6180 males) attending basic/secondary schools. the prevalence of overweight and obesity was calculated using body mass index (bmi), and the cut-off points suggested by cole et al. (2000) . pa was assessed with the baecke et al. (1982) questionnaire. proportions were compared using chi-square tests and means by anova. results and conclusions: overall, 17.1% were overweight (females = 16.4%; males = 17.8%) and 3.3% were obese (females = 3.0%; males = 3.6%). prevalence was similar across age and gender. bmi changed with age (p<0.001), and a significant interaction between age and gender was found (p = 0.001): whereas bmi in males increased with aging, in females increased up to 16 years and stabilized onwards. males showed significantly higher values of pa (p<0.001). both genders had a tendency to increase their pa until 16-17 years. a significant interaction between age and gender (p = 0.006) points out different gender patterns across age: pa increased with aging in males but in females started to decline after 16 years. no significant differences in pa were found between normal weight, overweight and obese subjects (p = 0.352). background: atherosclerosis is an inflammatory process. however, the relation between inflammatory markers and extent and progression of atherosclerosis remains unclear. objectives: we studied the association between c-reactive protein (crp) and 5 established measures of atherosclerosis. design and methods: within the rotterdam study, a population-based cohort of 7,983 persons over age 55, we measured crp, carotid plaque and intima-media thickness (imt), abdominal artery calcification, ankle-brachial index (abi) and coronary calcification. using ancova, we investigated the relation between crp and extent of atherosclerosis. we studied the association between progression of extra coronary atherosclerosis (mean follow-up period: 6.4 years) and crp using multinomial regression analysis. results: crp levels were positively related to all measures of atherosclerosis, but the relation was weaker for measures based on detection of calcification only. crp levels were associated with severe progression of carotid plaque (multivariable adjusted odds ratio: 1.5, 95% ci: 0.9-2.5), imt (1.7, 1.0-2.8) and abi (1.7, 1.1-2.6). no relation was observed with progression of abdominal artery calcification. conclusion and discussion: crp is related to extent and progression of atherosclerosis. the relation seems weaker for measures based on detection of calcification only, indicating that calcification of plaques might attenuate the inflammatory process. background: maternal stress during pregnancy has been reported to have an adverse influence on fetal growth. the terrorist attacks of september 11, 2001 on the united states have provoked feelings of insecurity and stress worldwide. objective: our aim was to test the hypothesis that maternal exposure to these acts of terrorism via the media had an unfavourable influence on mean birth weight in the netherlands. design and methods: in a prospective cohort study, we compared birth weights of 1885 dutch neonates who were in utero during the attacks with those of 1258 neonates who were in utero exactly 1 year later. results: in the exposed group, birth weight was lower than in the non-exposed group (difference, 48 g, 95%ci 13.6, 82.9, p = 0.006). the difference in birth weight could not be explained by tobacco use, maternal age, parity or other potential confounders, nor by shorter pregnancy durations. conclusion: these results provide evidence supporting the hypothesis that exposure of dutch pregnant women to the september 11 events via the media has had an adverse effect on the birth weight of their offspring. objective: asian studies suggested potential reduction in the risk of pneumonia among patients with stroke on ace-inhibitor therapy. because of the high risk of pneumonia in patients with diabetes we aimed to assess the effects of ace-inhibitors on the occurrence of pneumonia in a general, ambulatory population of diabetic patients. methods: a case-control study was performed nested in 142,175 patients with diabetes. cases were defined as patients with a first diagnosis of pneumonia. for each case, up to 4 controls were matched by age, gender, practice, and index date. current ace-inhibitor use was defined within a time-window encompassing the index date. results: ace-inhibitors were used in 12.7% of 4,719 cases and in 13,7% of 15,322 matched controls (crude or: 0.92, 95% ci 0.82 to 1.01). after adjusting for potential confounders, ace-inhibitor therapy was associated with a reduction in pneumonia risk (adjusted or: 0.72, 95% ci 0.64 to 0.80). the association was consistent among different relevant subgroups (stroke, heart failure, and pulmonary diseases) and showed a strong dose-effect relationship (p<0.001). conclusions: use of ace-inhibitors was significantly associated with reduced pneumonia risk and may apart from blood pressure lowering properties be useful in prevention of respiratory infections in patients with diabetes. background: progressive decline in serum levels of testosterone occurs with normal aging in both men and women. this is paralleled by a decrease in physical performance and muscle strength, which may lead to disability, institutionalization and mortality. objective. we examined whether low levels of testosterone were associated with three-year decline in physical performance and muscle strength in two population-based samples of older men and women. methods: data were available for 623 men in the longitudinal aging study amsterdam (lasa) and 1172 men and 1278 women in the health, aging, and body composition (health abc) study. levels of total testosterone and free testosterone were determined at baseline. physical performance and grip strength were measured at baseline and after three years. results: total and free testosterone were not associated with change in physical performance or muscle strength in men. in women, low levels of total testosterone (<=20 ng/dl) increased the risk of decline in physical performance (p = 0.03), and low levels of free testosterone (<2 pg/ ml) of decline in muscle strength (p = 0.03). conclusion: low levels of total and free testosterone were associated with decline in physical performance and muscle strength in older women, but not in older men. background: obesity and physical inactivity are key determinants of insulin resistance, and chronic hyperinsulinemia may mediate their effects on endometrial cancer (ec) risk. aim: to examine the relationships between prediagnostic serum concentrations of cpeptide, igf binding protein (igfbp)-1 and igfbp-2, and ec risk. methods: we conducted a case-control study nested within the epic prospective cohort study, including 286 incident cases of ec, in pre-and post-menopausal women, and 555 matched control subjects. odds ratios (or) and 95% confidence intervals (ci) were calculated using conditional logistic regression models. results: in fasting women (>6 h since last meal), serum levels of c-peptide, igfbp-1 and igfbp-2 were not related to risk. however, in nonfasting women (6 h or less since last meal), ec risk increased with increasing serum levels of c-peptide ( background: tobacco is the single most preventable cause of death in the world today. tobacco use primarily begins in early adolescent. objective: to estimate the prevalence and evaluate factors associated with smoking among high school going adolescents in karachi, pakistan. methods: a school based cross sectional survey was conducted in three towns of karachi from january through may 2003. two-stage cluster sampling stratified on school types was employed to select schools and students. self-reported smoking status of school going adolescents was our main outcome in analysis. results: prevalence of smoking (30 days) among adolescents was 13.7%. multiple logistic regression model showed that after adjustment for age, ethnicity and place of residence, being student of a government school (or=1.6; 95% ci: 1.0-2.7), parental smoking (or = 1.7; 95% ci: 1.1 -2.8), uncle (or = 1.7; 95% ci: 1. 2-2.8) , peer smoking (or = 6.2; 95% ci: 3.9 -9.9) and spending leisure time outside home (or = 3.9; 95% ci 1. 2-13.2) were significantly associated with adolescents smoking. conclusion: a 13.7% prevalence of smoking among school going adolescents and influence of parents and peers in initiating smoking in this age group warrant the need for effective tobacco control in the country especially among the adolescents. background: individual patient data meta-analyses (ipd-ma) have been proposed to improve subgroup analyses that may provide clinically relevant information. nevertheles, comparison of the effect estimates of ipd-ma and meta-analyses of published data (map) are lacking. objective: to compare main and subgroup effect estimates of ipd-ma and map. methods: an extended literature search was performed to identify all ipd-ma of randomized controlled trials, followed by a related article search to identify maps with a similar domain, objective, and outcome. data were extracted regarding number of trials, number of subgroups, effect measure, effect estimate and their confidence intervals. results: in total 16 ipd-ma and 18 map could be included in the analysis. twentyfive main effect estimates could be compared; of which 22 were in the same direction. although over 100 subgroups were studied in both ipd-ma and map, only 18 effect estimates could be compared; 17 were in the same direction. subgroup analyses in map most often related to trial characteristics, whereas subgroup analyses in ipd-ma were related to patient characteristics. conclusion: comparable ipd-ma and map report similar main and subgroup effect estimates. however, ipd-ma more often study subgroups based on patient characteristics, and thus provide more clinically relevant information. patients with diabetes have an increased risk of a complicated course of community-acquired lower respiratory tract infections. although influenza vaccination is recommended for these persons, vaccination levels remain too low because of conflicting evidence regarding potential benefits. as part of the prisma nested casecontrol study among 75,000 persons recommended for vaccination, we studied the effectiveness of single and repeat influenza vaccination in the subgroup of adult diabetic population (9, 238) during the 1999-2000 influenza a epidemic. case patients were hospitalized for diabetes, acute respiratory or cardiovascular events, or died and controls were sampled from the baseline cohort. after control for age, gender, health insurance, prior health care, medication use and co-morbid conditions logistic regression analysis showed that the occurrence of any complication (131 hospitalizations, 61 deaths) was reduced by 56% (95% confidence interval 36% to 70%). vaccine effectiveness was similar for those who received the vaccine for the first time and for those who received an earlier influenza vaccination. although we did not perform virological analysis or distinguish type i from type ii diabetes we conclude that patients with diabetes benefit substantially from influenza vaccination independent of whether they received the vaccine for the first time or received earlier influenza vaccinations. background: construction workers are at risk of developing silicosis. regular medical evaluations to detect silicosis preferably in the pre-clinical phase are needed. objectives: to identify the presence or absence of silicosis by developing an easy to use diagnostic model for pneumoconiosis from simple questionnaires and spirometry. design and methods: multiple logistic regression analysis was done in 1291 dutch construction workers, using chest x-ray indicative for pneumoconiosis (ilo profusion category > 1/1) as the reference standard (prevalence 2.9%). model calibration was assessed with graph and the hoshmer-lemeshow goodness of fit test; discriminative ability using area under receiver operating characteristic curve (auc); and internal validity using bootstrapping procedure. results: age > 40 years, current smoking, high exposure job title, working > 15 years in the construction industry, 'feeling unhealthy', and standardized residual fev1 below )1.0 were selected as predictors. the diagnostic model showed a good calibration (p = 0.5) and discriminative ability (auc 0.81; 95% ci 0.74 to 0.85). internal validity was reasonable (correction factor of 0.82 and optimism corrected auc of 0.76). conclusions: and discussion: our diagnostic model for silicosis showed reasonable performance and internal validity. to apply the model with confidence, external validation before application in a new working population is recommended. background: artemisinin based combination therapy (act) reduces microscopic gametocytaemia, the malaria parasite stage responsible for transmission from man to mosquito. as a result, act is expected to reduce the burden of disease in african populations. however, molecular techniques recently revealed high prevalences of gametocytaemia below the microscopic threshold. our objective was to determine the importance of sub-microscopic gametocytaemia after act treatment. methods: kenyan children (n=528) aged 6 months -10 years were randomised to four treatment regimens. gametocytaemia was determined by microscopy and pfs25 real-time nucleic acid sequence-based amplification (qt-nasba). transmission was determined by membrane feedings. findings: gametocyte prevalence at enrolment was 89.4% (219/245) as determined by pfs25 qt-nasba and decreased after treatment with act. membrane feedings in randomly selected children revealed that the proportion of infectious children was up to fourfold higher than expected when based on microscopy. act did not significantly reduce the proportion of infectious children but merely the proportion of infected mosquitoes. interpretation: sub-microscopic gametocyte densities are common after treatment and contribute considerably to mosquito infection. our novel approach indicates that the effect of act on malaria transmission is much smaller than previously suggested. these findings are sobering for future interventions aiming to reduce malaria transmission. background: adequate folate intake may be important in the prevention of breast cancer. factors linked to folate metabolism may be relevant to its protective role. objectives: to investigate the association between folate intake and breast cancer risk among postmenopausal women and evaluate the interaction with alcohol and vitamin b12 intake. methods: a prospective cohort analysis of folate intake among 62,739 postmenopausal women from the e3n french cohort who completed a validated food frequency questionnaire in 1993 was conducted. during 9 years follow-up 1,814 cases of pathology-confirmed breast cancer were documented through followup questionnaires. nutrient intakes were categorized in quintiles and energy-adjusted using the regression-residual method. cox modelderived relative risks (rr) were adjusted for known risk factors for breast cancer. results: the multivariate rr comparing the extreme quintiles of folate intake was 0.76 (95% ci 0.65-0.88; p-trend=0.005). after stratification, the association was observed only among women whose alcohol consumption was above the median (=6.2 g/day) and among women who consumed =6.5lg/day of vitamin b12. however, tests for interaction were not significant. conclusions: in this population, high intakes of folate were associated with decreased breast cancer risk; alcohol and vitamin b12 intake may modify the observed inverse association. background: the simultaneous rise in the prevalence of obesity and atopy in children has prompted suggestions that obesity might be a causal factor in the inception of atopic diseases. objective: we investigated the possible role of ponderal index (kg/m 3 ) as marker for fatness at birth in early childhood atopic dermatitis (ad) in a prospective birth cohort study. methods: between november 2000 and november 2001, mothers and their newborns were recruited after delivery at the university of ulm, germany. active follow-up was performed at the age of 12 months. results: for 872 (82%) of the 1066 children included at baseline, information on physician reported diagnosis of ad was obtained during follow-up. incidence of ad was 12.4% at the age of one year. mean ponderal index at birth was 24.7 kg/m 3 . risk for ad was higher among children with high ponderal index at birth (adjusted or for children within the third and fourth compared to children within the second quartile of ponderal index: 2.14; 95% ci 1. respectively) background: the relationship between duration of breastfeeding and risk of childhood overweight remains inconclusive, possibly in part caused by using never breastfeeding mothers as the reference category. objectives: we assessed the association between duration of breastfeeding and childhood overweight among ever breastfed children within a prospective birth cohort study. methods: between november 2000 and november 2001 all mothers and their newborns were recruited after delivery at the university of ulm, germany. active follow-up was performed at age 24 months. results: among 855 children (80% of 1066 children included at baseline) with available body mass index at age two 72 (8.4%) were overweight. whereas 76 children (8.9%) were never breastfed, 533 (62.3%) were breastfed for at least six months, and 322 (37.7%) were exclusively breastfed for at least six months. compared to children who were exclusively breastfed less than three months, the adjusted or for overweight was 0.8 (95% ci 0.4; 1.5) in children who were exclusively breastfed for at least three but less than six months and 0.4 (95% ci 0.2; 0.9) in children who were exclusively breastfed for at least six months. conclusion: these results highlight the importance of prolonged breastfeeding in the prevention of overweight in children. background: in africa, hiv and feeding practices influence child mortality. exclusive breastfeeding for 6 months (bf6) and formula feeding (ff) when affordable are two who recommendations for safe feeding. objective: we estimated the proportion and the number of children saved with each recommendation at population level. design and methods: data on 31 sub-saharan countries were analysed. we considered saved a child remaining hiv-free and alive after two years of life. a spreadsheet model based on a decision tree for risk assessment was used to calculate this number according to six scenarios that combine the two recommendations without and with promotion then with promotion and group education. results: whatever the country, the number of children saved with bf6 would be higher than with ff. overall, without promotion, 52 315 ( background: farming has been associated with respiratory symptoms as well as protection against atopy. effects of different farming practices on respiratory health in adults have rarely been studied. objectives: we studied associations between farming practices and hay fever and current asthma in organic and conventional farmers. design and methods this cross-sectional study evaluated questionnaire data of 1205 conventional and 593 organic farmers. associations between health effects and farm exposures were assessed by logistic regression. results: organic farmers reported slightly more hay fever than conventional farmers (9.3% versus 6.9%, p = 0.07). however, organic farming was no independent determinant for hay fever in multivariate models including farming practices and potential confounders. livestock farmers who grew up on a farm had a five-fold lower prevalence of hay fever than crop farmers without farm childhood (or 0.2, 95% ci 0.1-0.5). use of disinfectants containing quaternary ammonium compounds was positively related to hay fever (or 2.4, 95% ci 1.1-5.1). no effects of farming practices were found for asthma. conclusion and discussion: our study adds to the evidence that a farm childhood in combination with current livestock farming protects against allergic disorders. this effect was found for both organic and conventional farmers. background: although a body mass index (bmi) above 25 kg/m 2 is clearly associated with an increase in mortality in the general population, the meaning of high levels of bmi among physically heavily working men is less clear. methods: we assessed the association between bmi and mortality in a cohort of 19513 male construction workers, aged 25-64 years, who underwent an occupational health examination in wu¨rttemberg (germany) during 1986-1992 and who were followed over a 10 years period. covariates considered in the proportional hazard regression analysis included age, nationality, smoking status, alcohol consumption, and comorbidity. results: during the follow-up 802 deaths occurred. there was a strong u-shaped association between bmi and all-cause mortality, which was lowest for bmi levels between 25 and 35 kg/m 2 . this pattern persisted after exclusion of the first years of follow-up and control for multiple covariates. compared with men with a bmi < 25.0 kg/m 2 , the relative mortality was 0.76 (95% confidence interval: 0,64-0,91), 0.75 (0.59-0.97) and 1.00 (0.62-1.62) for bmi ranges 25-29.9, 30-34.9 and = 35.0 kg/m 2 . conclusion and discussion: bmi levels commonly considered to reflect overweight or moderate obesity in the general population may be associated with reduced mortality in physically heavily working men. background: colonoscopy with removal of polyps may strongly reduce colorectal cancer (crc) incidence and mortality. empirical evidence for optimal schedules for surveillance is limited. objective. to assess risk of proximal and distal crc after colonoscopy with polypectomy. design and methods: history and results of colonoscopies were obtained from 540 cases and 614 controls in a population-based case-control study in germany. risk of proximal and distal crc according to time since colonoscopy was compared to risk of subjects without previous colonoscopy. results: subjects with previous detection and removal of polyps had a much lower risk of crc within four years after colonoscopy (adjusted odds ratio 0.38, 95% confidence interval 0.18-0.78), and a similar risk as those without colonoscopy in the long run. within four years after colonoscopy, risk was particularly low if only single or small adenomas were detected. most cancers occurring after polypectomy were located in the proximal colon, even if polyps were found in the sigma or rectum only. conclusion and discussion: our results support suggestions that surveillance colonoscopy after removal of single and small adenomas may be deferred to five years and that surveillance should include the entire colorectum even if only distal polyps are detected. background: a population-based early detection programme for breast cancer has been in progress in finland since 1987. recently, detailed information about actual screening invitation schemes in 1987 -2001 has become available in electronic form, which enables more specific modeling of breast cancer incidence. objectives: to present a methodology for taking into account historical municipality-specific schemes of mass screening when constructing predictions for breast cancer incidence. to provide predictions for numbers of new cancer cases and incidence rates according to alternative future screening policies. methods: observed municipality-specific screening invitation schemes in finland during 1987-2001 were linked together with breast cancer data. the incidence rate during the observation period was analyzed using poisson regression, and this was done separately for localized and nonlocalized cancers. for modeling, the screening programme was divided into seven different phases. alternative screening scenarios for future mass-screening practices in finland were created and an appropriate model for incidence prediction was defined. results and conclusion: expanding the screening programme would increase the incidence of localized breast cancers; the biggest increase would be obtained by expanding from women aged 50-59 to 50-69. the impacts of changes in the screening practices on predictions for non-localized cancers would be minor. background: new screening technologies are implemented to routine screening in increasing numbers, with limited evidence on their effectiveness. randomised evaluation of new technologies is encouraged but rarely done. objective: to evaluate in a randomised design whether the effectiveness of an organised cervical screening programme can be improved by means of new technologies. methods: since 1999 150,000-170,000 women have been invited annually to a randomised multi-arm trial ran within the finnish organised cervical screening programme. the invited women are randomly allocated to three study arms of different primary screening tests: conventional cytology, automation-assisted cytology and, since 2003, human papillomavirus (hpv) testing. up to 2005, we have gathered information on 185,000 screening visits in the automation-assisted arm and 4,653 in the hpv arm, and we have compared the results to conventional screening. results: automation-assistance resulted in a slightly increased detection of precancers, but the efficacy based on interval cancers is not known. results on hpv screening suggest higher detection of precancers and cancers compared to conventional screening. conclusion: evidence of higher effectiveness of new screening technologies is needed, especially when changing the existing screening programmes. the multi-arm trial shows how these technologies can be implemented to routine in a controlled manner. introduction: nodules and goitres are important risk factors for thyroid cancer. as the number of diagnosed cases of thyroid cancer is increasing, the incidence of such risk factors has been assessed in a french cohort of adults. methods: the su.vi.max (supple´mentation en vitamines et mine´raux antioxydants) cohort study included 12741 middle-aged adults followed-up during eight years. incident cases of goitres and nodules have been identified retrospectively by scheduled clinical examinations and spontaneous consultations by the participants. cox proportional hazards modeling was used to identify factors associated to thyroid diseases. results: finally, 160 incident cases of nodules and goitres were identified among 4,002 subjects free of thyroid diseases at inclusion. after an average follow-up of 7 years, the incidence of goitres and nodules was 2.2% in 45-60 year old men, 4.9% in 35-44 year old women and 7.4% in 45-60 year old women. identified associated factors were age, low urinary thiocyanate level and oral contraceptive use in women, and high urinary thiocyanate level and low urinary iodine level in men. conclusion: estimated incidences are consistent with those observed in other countries. the protective role of urinary thiocyanate in both men and women and, in women, oral contraceptives deserve further investigation. background: various statistical methods for outbreak detection in hospital settings have been proposed in the literature. usually validation of those methods is difficult, because the long time series of data needed for testing the methods are not available. modeling is a tool to overcome that difficulty. objectives: to use model generated data for testing sensitivity and specificity of different outbreak detection methods. methods: we developed a simple stochastic model for a process of importation and transmission of infection in small populations (hospital wards). we applied different statistical outbreak detection methods described in the literature to the generated time series of diagnosis data and calculated and the sensitivity and specificity of different methods. results: we present roc curves for the different methods and show how they depend on the underlying model parameters. we discuss how sensitivity and specificity measures depend on the degree of underdiagnosis, on the ratio of admitted colonised patients to colonisation resulting from transmission in the hospital, and on the frequency of testing patients for colonisation. conclusions: modeling can be a useful tool for evaluating statistical methods of outbreak detection especially in situation where real data is scarce or its quality questionable. associated with higher mammographic density and breast pain, has been increased which has bearing on screening performance. objective: we compared the screening performance for women aged 49-69 years with dense and lucent breast patterns in two time periods and studied the possible interaction with use of hrt. methods: data were collected from a dutch regional screening programme for women referred in 1994-1995 (n = 642) and 2001-2002 (n = 107) . in addition, we sampled controls for both periods that were not referred (n = 1927 and n = 212 resp.) and women diagnosed with an interval cancer. mammograms were digitised and computer-assisted methods used to measure mammographic density. among other parameters, sensitivity was calculated to describe screening performance. results: screening performance has improved slightly, but the difference between dense and lucent breast patterns still exists (e.g. sensitivity 62% vs. 78%). hrt use has increased; sensitivity was particularly low (38%) in the group of women with dense breast patterns on hrt. discussion: in conclusion, the detrimental effect of breast density and the interaction with hrt on screening performance warrants further research with enlargement of the catchment area, more referred women, interval cancers and controls. background: population based association studies might lead to false-positive results if possibly underlying population structure is not adequately accounted for. to assess the nature of the population structure some kind of cluster analysis has to be carried out. we investigated the use of self-organizing maps (soms) for this purpose. objectives: the two main questions concern identification of an either discrete or an admixed population structure and identification of the number of subpopulations involved in forming the structured population under investigation. design and methods: we simulated data sets with different population models and included varying informative marker and map sizes. sample sizes ranged from 200 to 2400 individuals. results: we found that a discrete structure can easily be accessed by soms. a near to perfect assignment of individuals to their population of origin can be obtained. for an admixed population structure though, soms do not lead to reasonable results. here, even the correct number of subpopulations involved can not be identified. conclusion: in conclusion, soms can be an alternative to a model-based cluster analysis if the researcher assumes a discrete structure but should not be applied if an admixed structure is likely. background: little is known about the combined effect of duration of breastfeeding, sucking habits and malocclusion in the primary dentition. objectives: we studied the association of breastfeeding and non-nutritive sucking habits on malocclusion on the primary dentition. design and methods: a cross-sectional study nested in a birth cohort was carried out in pelotas, brazil. a random sample of 359 children aged 6 was examined and their mothers interviewed. the foster and hamilton criteria were used to define anterior open bite (aob) and posterior cross bite (pcb). information regarding breastfeeding and non-nutritive sucking habits was collected from birth to 6 years-old. poisson's regression analysis was used. results: non-nutritive sucking habits between 12 months and 4 years of age (pr3.5 [2.3;5.4] ) and digital sucking at 6 years of age (pr1.5[1.1;2.1]) were risk factors for aob. breastfeeding for less than 9 months (pr7.6[1.5; 39.5] ) and the regular use of a pacifier between 12 months and 4 years of age (pr7.5[1.3;44.3]) were the risk factors for pcb. for pcb an interaction was identified between lack of breastfeeding and the use of a pacifier. conclusion: lack of breastfeeding and longer non-nutritive sucking habits during early childhood were the main risk factors for malocclusion in primary dentition. background: recent, dramatic coronary heart disease (chd) mortality increases in beijing, can be mostly explained by adverse changes in risk factors, particularly total cholesterol and diabetes. it is important for policy making to predict the impact of future changes in risk factors on chd mortality trends. objective: to assess the potential impact of changes in risk factors on numbers of chd deaths in beijing from 1999 to 2010, to provide evidence for future chd strategies. design: the previously validated impact model was used to estimate the chd deaths expected in 2010 a) if recent risk factor trends continue or b) if levels of risk factors reduce. results: continuation of current risk factor trends will result in a 48% increase in chd deaths by 2010, (almost half being attributable to increases in total cholesterol levels). even optimistically assuming a 1% annual decrease in risk factors, chd deaths would still rise by 21% because of population ageing. conclusion: a substantial increase in chd deaths in beijing may be expected by 2010. this will reflect worsening risk factors compounded by demographic trends. population ageing in china will play an important role in the future, irrespective of any improvements in risk factor profiles. background: since smoking cessation is more likely during pregnancy than at other times, interventions to maintain quitting postpartum may give the best opportunity for a long-time abstinence. it is still not clear what kind of advice or counseling should be given to help prevent the relapse postpartum. objectives: to identify the factors, which predispose women to smoking relapse after delivery. design and methods: the cohort study was conducted in 2004 and 2005 in public maternity units in lodz, poland. the study population consisted of pregnant women between 32-36 weeks of pregnancy who have quit smoking no later than three months prior to participation in the study. smoking status was verified using saliva cotinine level. women were interviewed twice: during pregnancy and three months after delivery. results: within three months after delivery about half of women relapsed into smoking. the final model identified the following risk factors for smoking relapse: having partner and friends who smoke, quitting smoking in late pregnancy, and negative experiences after quitting smoking such as dissatisfaction with weight, nervousness, irritation, loosing pleasure. conclusion. this study advanced the knowledge of the factors that determine smoking relapse after delivery and provided preliminary data for future interventions. introduction: it remains difficult to predict the effect of an particular antihypertensive drug in an individual patient and pharmacogenetics might optimise this. objective: to investigate whether the association between use of angiotensin converting enzyme (ace)-inhibitors or ß-blockers and the risk of stroke or myocardial infarction (mi) is modified by the t-allele of the angiotensinogen m235t polymorphism. methods: data were used from the rotterdam study, a population-based prospective cohort study. in total, 4093 subjects with hypertension were included from july 1st, 1991 onwards. follow-up ended at the diagnosis of mi or stroke, death, or the end of study period (january 1st, 2002) . the drug-gene interaction and the risk of mi/stroke was determined with a cox proportional hazard model (adjusted for each drug class as time-dependent covariates). results: the interaction between current use of ace-inhibitors and the angiotensinogen m235t polymorphism increased the risk of mi (synergy index (si) = 4.00; 95% ci:1.32-12.11) and non-significant increased risk of stroke (si = 1.83; 95% ci:0.95-3.54). no interaction was found between current use of ß-blockers and the agt m235t polymorphism on the risk of mi or stroke. conclusion: subjects with at least one copy of the 235t allele of the agt gene might have less benefit from ace-inhibitor therapy. [2.4-6.9 ] to 1.5 [1.1-2.1] in those without ms-idf and 1.9 [1.5-2.6] with ms-idf. ms-ncep had no effect. conclusion and discussion: although cardiovascular disease was self-reported, we conclude that the higher prevalence of cardiovascular disease is partly accounted for by marked differences in the prevalence of metabolic syndrome. the ms-idf criteria seem better for defining metabolic syndrome in ethnic groups than the ms-ncep criteria. background: selenium is an essential trace mineral with antioxidant properties. objective: to perform meta-analyses of the association of selenium levels with coronary heart disease (chd) endpoints in observational studies and the efficacy of selenium supplements in preventing chd in randomized controlled trials. methods: we searched medline and the cochrane library from 1966 through 2005. relative risk (rr) estimates were pooled using an inversevariance weighted random-effects model. for observational studies reporting three or more categories of exposure we conducted a dose-response meta-analysis. results: twenty-five observational studies and 6 clinical trials met our inclusion criteria. the pooled rr comparing the highest to the lowest categories of selenium levels was 0.85 (95% confidence interval 0.74-0.99) in cohort studies and 0.43 (0.29-0.66) in case-control studies. in dose-response models, a 50% increase in selenium levels was associated with a 24% (7-38%) reduced risk of coronary events. in randomized trials, the rr comparing participants taking selenium supplements to those taking placebo was 0.90 (0.75-1.09). conclusion: selenium levels were inversely associated with the risk of chd in observational studies. the randomized trials findings are still inconclusive. these results require confirmation in randomised controlled trials. currently, selenium supplements should not be recommended for cardiovascular prevention. 140 background propensity score analysis (psa) can be used to reduce confounding bias in pharmacoepidemiologic studies of the effectiveness and safety of drugs. however, confidence intervals may be falsely precise because psa ignores uncertainty in the estimated propensity scores. objectives: we propose a new statistical analysis technique called bayesian propensity score analysis (bpsa). the method uses bayesian modelling with the propensity score as a latent variable. our question is: does bpsa yield improved interval estimation of treatment effects compared to psa? our objective is: to implement bpsa using computer programs and investigate the performance of bpsa compared to psa. design and methods: we investigated bpsa using monte carlo simulations. synthetic datasets, of sample size n = 250, 1000, 4000, were simulated by computer. the datasets were analyzed using bpsa and psa and we estimated the coverage probability of 80% credible intervals. results the estimated coverage probabilities ranged from 78% to 84% for bpsa, and from 42% to 82% for psa, with simulation standard errors less than 2%. background: several factors associated with low birth weight, such as smoking and body mass index (bmi) do not explain all ethnic differences. this study investigates the effects of working conditions on birth weight among different ethnic groups. methods: questionnaire data, filled in 2 weeks after prenatal screening, was used from the amsterdam born children and their development (abcd) study (all pregnant women in amsterdam [7/1/03-7/3/04 (n = 12.381), response 8267 (67%)]. ethnicity (country of birth). was dichotomised into dutch and non-dutch. working conditions were: weekly working hours, weekly hours standing/walking, physical load and job-strain (karasek-model). only singleton deliveries with pregnancy duration = 37 weeks were included. results: although only 39.4% of the non-dutch women worked during first trimester (79.6% of the dutch women), they reported significantly more physical load (15.1% vs 9.0%), more hours standing/walking (10.6% vs 6.3%) and more high job-strain (10.2 vs 5.7). linear regression revealed that only high job-strain lowered significantly birth weight (non-dutch: 159 gram and dutch: 75 gram). after adjusting for confounders (gender, parity, smoking, maternal length, maternal bmi and education), this was only significant in the non-dutch group (112 vs. 30 gram). conclusion: job-strain has more effect on birth weight in non-dutch compared to dutch women. background: in 2004 panama population was estimated in 3.2 million habitants, from which three millions lived in malaria endemic areas. until january 26 1998 malaria control activities were accomplished under a vertical structure. objective: to evaluate the evolution of malaria control in panama, before and after the decentralization of the malaria program. design and methods: average (standard deviation) of the program indexes are described for the last decades. the correlation between positive smears index and per capita cost of the program is analyze. results: in the 1960's the average (standard deviation) positive smears index per 100 habitants was 3.3% (1.8); in the 1970's: 0.4% (0.5); in the 1980's: 0.13% (0.1); in the 1990's: 0.32% (0.2); and in the first five years of 2000: 1.7% (1.1). after the decentralization of the program was accomplished in 1998, the positive smears index increased 5.3 fold. the average per capita cost involved in malaria control activities per decade ranged between 0.19 y 1.25 us dollars and presented a determination coefficient of 0.42 in the reduction of the positive smears index. discussion: the decentralization had significant detrimental implications in the control program capabilities. background: notification rates of new smear-positive tuberculosis in the central mountainous provinces (26/100,000 population) are considerably lower than in vietnam in general (69/100,000 population). this study assessed whether this is explained by low case detection. objective: to assess the prevalence and case detection of new smear-positive pulmonary tuberculosis among adults with a prolonged cough in central mountainous vietnam. design and methods: a house-to-house survey of adults 15 years or older was carried out in 12 randomly selected districts in three mountainous provinces in central vietnam in 2003. three sputum specimens were microscopically examined of persons reporting a cough of 3 weeks or longer. results: the survey included 68,946 persons with a response of 95%. a cough of 3 weeks or longer was reported by 1,298 (1.9% 95% ci 1. 8-2.2) persons. of these, 18 were sputum smear-positive of whom 2 had had anti-tuberculosis treatment. the prevalence of new smear-positive tuberculosis was 27/100,000 population (95% ci 11-44/100,000 population). the patient diagnostic rate was 1.6 per person-year, suggesting that the case notification rate as defined by who was 76%. conclusion: low tuberculosis notification rates in mountainous vietnam are probably due to low tuberculosis incidence. explanations for low incidence at high altitude need to be studied. background: although patients with type 2 diabetes (dm2) have an increased risk of urinary tract infections (utis), not much is known about predictors of a complicated course. objective: this study aims to develop a prediction rule for complicated utis in dm2 patients in primary care. design and methods: we conducted a 12-month prospective cohort study, including dm2 patients aged 45 years or older from the second dutch national survey of general practice. the combined outcome measure was defined as the occurrence of recurrent cystitis, or an episode of acute pyelonephritis or prostatitis. results: of the 6,343 dm2 patients 46% was male and mean age was 67 years (sd 11). incidence of the outcome was 3 per 100 patient years (n = 179). predictors were age, male sex, number of physician contacts, incontinence of urine, cerebro vascular disease or dementia and renal disease. the area under the receiver-operating curve (auc) was 0.77 (95% ci 0.73 to 0.80). subgroup analyses for gender showed no differences. there is an increased early postoperative mortality (operation risk) after elective surgery. this mortality is normally associated with cardiovascular events, such as deep venous thrombosis, pulmonary embolism, and ischemic heart diseases. our objective was to quantify the magnitude of the increased mortality and how long the mortality after an operation persists. we focused on the early postoperative mortality after surgery for total knee and total hip replacements from the national registries in australia and norway, which cover more than 95% of all operations in the two nations. only osteoarthritis patients between 50 and 80 years of age were included. a total of 244.275 patients remained for analyses. smoothed intensity curves were calculated for the early postoperative period. effects of risk factors were studied using a nonparametric proportional hazards model. the mortality was highest immediately after the operation ($1 deaths per 10.000 patients per day), and it decreased until the 3rd postoperative week. the mortality was virtually the same for both nations and both joints. mortality increased with age and was higher for males than for females. a possible reduction of early postoperative mortality is plausible for the immediate postoperative period, and no longer than the 3rd postoperative week. background/objectives: single, modifiable risk factors for stroke have been extensively studied before, but their combined effects were rarely investigated. aim of the present study was to assess single and joint effects of risk factors on stroke and transitoric ischemic attack (tia) incidence in the european prospective investigation into cancer and nutrition (epic)-potsdam study. methods: among 25538 participants aged 35-65 years at baseline 100 total stroke cases and 112 tia cases occurred during 4.3 years of follow-up. relative risks (rr) for stroke and tia related to risk factors were estimated using cox proportional hazard models. results: after adjustment for potential confounders rr for ischemic stroke associated with hypertension was 2.32 (95% ci, 1.35-3.99) and for tia 1.14 (95% ci 0.76-1.71). the highest rr for ischemic stroke (rr 5.12, 95% ci 1.49-17.6, p trend<0.0001) and tia (rr 3.08, 95% ci 1.00-9.44, p trend=0.024) were observed among participants with 4 or 5 modifiable risk factors. 58.5 % of ischemic strokes and 26.2% of tia cases were attributable to hypertension, diabetes mellitus, high alcohol consumption, hyperlipidemia, and smoking. conclusion: almost 60% of ischemic stroke cases could be explained by classical modifiable risk factors. however, only one in four tia cases was attributable to those risk factors. background: the investigation of genetic factors is gaining importance in epidemi-ology. most relevant from a public health perspective are complex diseases that are characterised by complex pathways involving gene-gene-and gene-environment-interactions. the identification of such pathways requires sophisticated statistical methods that are still in their infancy. due to their ability in describing complex association structures, directed graphs may represent a suitable means for modelling complex causal pathways. objectives: we present a study plan to investigate the appropriateness for using directed graphs for modelling complex pathways in association stud-ies. design and methods: graphical models and artificial neural networks will be investigated using simulation studies and real data and their advantages and disadvantages of the respective ap-proaches summed up. furthermore, it is planned to construct a hybrid model exploiting the strengths of either model type. results and conclusions: the part of the project that concerns graphical models is being funded and ongoing. first results of a simulation study have been obtained and will be presented and discussed. a second project is currently being applied for. this shall cover the investigation of neural networks and the construction of the hybrid model. this study investigates variations in mortality from 'avoidable' causes among migrants in the netherlands in comparison with the native dutch population. data were obtained from population and mortality registries in the period 1995-2000. we compared mortality rates for selected 'avoidable' conditions for turkish, moroccan, surinamese and antillean/aruban groups to native dutch. we found slightly elevated risk in total 'avoidable' mortality for migrant populations (rr = 1.13). higher risks of death among migrants were observed from almost all infectious diseases (most rr>3.00) and several chronic conditions including asthma, diabetes and cerebro-vascular disorders (most rr>1.70). migrant women experienced a higher risk of death from maternity-related conditions (rr = 3.37). surinamese and antillean/ aruban population had a higher mortality risk (rr = 1.65 and 1.31 respectively), while turkish and moroccans experienced a lower risk of death (rr = 0.93 and 0.77 respectively) from all 'avoidable' conditions compared to native dutch. control for demographic and socioeconomic factors explained a substantial part of ethnic differences in 'avoidable' mortality. conclusion: compared to native dutch, total 'avoidable' mortality was slightly elevated for all migrants combined. mortality risks varied greatly by cause of death and ethnicity. the substantial differences in mortality for a few 'avoidable' conditions suggest opportunities for improvement within specific areas of the healthcare system. warmblood horses scored by the jury as having uneven feet will never pass yearly selection sales of the royal dutch warmblood studbook (kwpn).to evaluate whether the undesired trait 'uneven feet' influences performance, databases of kwpn (n = 62234 horses) and knhs (n = 16015 show jumpers, n = 24269 dressage horses) were linked through the unique number of each registered horse. using a proc glm model of sas was investigated whether uneven feet had effects on age at first start and highest performance level. elite show jumpers with uneven feet start at 7.2 years and dressage horses 9.4 years of age, which is a significant difference (p<0.05) with elite even feet horses (8.0 respectively 9.0 years). at their maximum level of performance horses with even feet linearly scored in show jumping 31.1 at regular and 116.8 at elite level (29.0 resp. 105.7 with uneven feet), while in dressage horses scores were 38.0 at regular and 139.7 at elite level (35.5 resp. 136.7 with uneven feet).the conformational trait 'uneven feet' appears to have a significant effect on age at first start, while horses with even feet demonstrate a higher maximal performance than horses with uneven feet. objectives: to identify children with acute otitis media (aom) who might benefit more from treatment with antibiotics. methods: an individual patient data meta-analysis (ipdma) on six randomized trials (n = 1643 children). to preclude multiple testing, we first performed a prognostic study in which predictors of poor outcome were identified. subsequently, interactions between these predictors and treatment were studied by fixed effect logistic regression analyses. only if a significant interaction term was found, stratified analyses were performed to quantify the effect in each subgroup. results: interactions were found for: age and bilateral aom, and otorrhea. in children less than 2 years with bilateral aom, a rate difference (rd) of )25% (95% ci )30; )20%) was found, whereas in children aged 2 years or older with unilateral aom the rd was )4% (95% ci )6; )2%). in children with and without otorrhea the rd were )36% (95% ci )45; )27%), and )14% (95% ci )17%; )11%). conclusion: although there still are many areas in which ipdma can be improved, using individual patient data appear to have many advantages especially in identifying subgroups. in our example, antibiotics are beneficial in children aged less than 2 years with bilateral aom, and in children with otorrhea. major injuries, such as fractures, are known to increase the risk of venous thrombosis (vt). however, little is known of the risk caused by minor injuries, such as ankle sprains. we studied the risk of vt after minor injury in a population-based case-control study of risk factors for vt, the mega-study. consecutive patients, enrolled via anticoagulation clinics, and control subjects, consisting both of partners of patients and randomly selected control subjects, were asked to participate and filled in a questionnaire. participants with cancer, recent plastercasts, surgery or bedrest were excluded from the current analyses. 270 out of 2207 patients (12.2%) and 200 out of 4467 controls (4.5%) had suffered from a minor injury resulting in a three-fold increased risk of vt (odds ratio adjusted for age and sex 3.2; 95% confidence interval 2.7-3.9) compared to those without injury. the risk was highest in the first month after injury and was no longer increased after 3 months. injuries located in the leg increased the risk five-fold, while those located in other body parts did not increase the risk. these results show that minor injuries in the leg increase the risk of vt. this effect appears to be temporary and mainly local. introduction: in southeast asia, dengue was considered a childhood disease. in the americas, this disease occurs predominantly in older age groups, indicating the need for studies to investigate the immune status of the child population, since the presence of antibodies against a serotype of this virus is a risk factor for dengue hemorrhagic fever (dhf). objective: to evaluate the seroprevalence and seroincidence of dengue in children living in salvador, bahia, brazil. methods: a prospective study was carried out in a sample of children of 0-3 years by performing sequential serological surveys (igg/ dengue). results: seroprevalence in 625 children was 26.6%. a second survey (n = 289 seronegative children) detected an incidence of 33.2% and no difference was found between males and females or according to factors socioeconomic analyzed. conclusion and discussion: these results show that, in brazil, the dengue virus circulates actively in the initial years of life, indicating that children are also at great risk of developing dhf. it is possible that in this age group, dengue infections are mistaken for other febrile conditions, and that there are more inapparent infections in this age group. therefore, epidemiological surveillance and medical care services should be aware of the risk of dhf in children. since 1996, in the comprehensive cancer centre limburg (cccl) region, all women aged 30-60 years are invited to participate in the cervical cancer screening programme once every five years. we had the unique opportunity to link data from the cervical screening programme and the cancer registry. we studied individual pap smear testing and participation in the screening programme preceding the diagnosis of cervical cancer. all invasive cases of cervical cancer of women aged 30-60 years in the period 1996-2003 were selected. subgroups were based on results of the pap smear and invitation and participation in the screening programme. time interval between screening and detection of tumours was calculated. in 1996-2003, the non-response rate was 18%. in total, 211 invasive cervical cancer cases were detected of which 54 were screening and 32 interval carcinomas. in the group of women who were invited but did not participate and women who were not invited, respectively 64 and 61 tumours were detected. these tumours had a higher stage compared to screening carcinomas. in the cccl region, more and higher stage tumours were found in women who did not participate in the screening compared to women with screening tumours. background: pcr for mycobacterium tuberculosis (mtb) has already proved to be a useful tool for the diagnosis and investigation of molecular epidemiology. objectives: evaluation of pcr assay for detection of mycobacterium tuberculosis dna as a diagnostic aid in cutaneous tuberculosis. design and methods: thirty paraffinembedded samples belonging to 28 patients were analyzed for acid fast bacilli. dna was extracted from tissue sections and pcr was performed using specific primers based on is 6110 repeated gene sequence of mtb. results: two of the tissue samples were positive for acid fast bacilli (afb). pcr was positive in eight samples from six patients. amongst them, two were suspected of having lupus vulgaris confirmed histopathologically, whom their entire tests were positive. accounting histopathology as gold standard, the sensitivity of pcr in this study was determined as 75%. conclusion: from 8 cases of skin tuberculosis diagnosed by histopathology, 6 were positive by pcr technique, which shows the priority of previous method to molecular technique. discussion: pcr assay can be used for rapid detection of mtb from cutaneous tuberculosis cases, particularly when staining for afb is negative and there is a lack of growth on culture or when fresh material has not been collected for culture. background: recent epidemiological studies used automated oscillometric blood pressure (aod) devices that systematically measure higher blood pressure values than random zero sphygmomanometer devices (rzs) hampering the comparability of the blood pressure values between these studies. we applied both a random zero and an automated oscillometric blood pressure device in a randomized order in an ongoing cohort study. objectives: the aim of this analysis was to compare the blood pressure values by device and to develop a conversion algorithm for the estimation of blood pressure values from one device to the other. methods: within a randomized subset of 2365 subjects aged 45-75 years, each subject was measured three times by each device (hawskley random zero and omron hem-705cp) in a randomized order. results: the mean difference (aod-rzs) between the devices was 3.9 mmhg and 2.6 mmhg for the systolic and diastolic blood pressure respectively. linear regression models including age, sex, and blood pressure level can be used to convert rzs blood pressure values to aod blood pressure values and vice versa. conclusions: the results may help to better compare blood pressure values of epidemiological studies that used different blood pressure devices. a form was used to collect relevant perinatal clinical data, as part of a european (mosaic) and italian (action) project. the main outcomes were mortality and a variable combining mortality and severe morbidity at discharge. the cox proportional hazards and logistic regression models were used, respectively, for the two outcomes. results: twenty-two of percent of vpbs were among fbms. comparing to control group, the percentage of babies below 28 weeks and plurality was statistically significant higher among babies of fbms: 52% vs. 41.7 and 28.3% vs. 16.0%. adjusting for potential confounders, no association for mortality among immigrant group was found, whereas a slightly excess of morbidity-mortality was observed (odd ratio, 1.36; 95% cis 0.99-1.88). conclusions: the high proportion of vpbs among fbms and the slight excess observed in morbidity and mortality indicate the need to improve the health care delivery for the immigrant population. background: high-risk newborns have excess mortality, morbidity and use of health services. objectives: to describe re-hospitalizations after discharge from an italian region. design and methods: the population study consisted of all births with <32 weeks' gestation discharged alive from the twelve neonatal intensive care units in lazio region during 2003. the perinatal clinical data was collected as part of a european project (mosaic). we used the regional hospital discharge database to find hospital admissions within 12 months, using tax code for record linkage. data were analyzed through logistic regression for re-hospitalization. results: the study group included 361 children; among these, 111 (30.7%) were re-hospitalized; overall, 206 readmission were observed. the median total length of stay for re-admissions was 5 d. the two most common reasons for re-hospitalization were respiratory (37.8%) and gastrointestinal (16.5%) disorders. the presence of a severe morbidity at discharge (odd ratio 2.03: 95% cis 1.20-3.42) and male sex (odd ratio 1.97; 95% cis 1.24-3.15) predicted re-hospitalization in multivariate model. conclusions: almost one out three preterm infants was re-hospitalized in the first 12 months. readmissions after initial hospitalization for a very preterm birth could be a sensitive indicator of quality of follow-up strategies in high risk newborns. background: self-medication with antibiotics may lead to inappropriate use and increase the risk of selection of resistant bacteria. in europe the prevalence varies from 1/1000 to 210/1000 respondents. self-medication may be triggered by experience with prescribed antibiotics. we investigated whether in european countries prescribed use was associated with self-medication with antibiotics. methods: a population survey was conducted in 19 european countries with 15548 respondents completing the questionnaire. multivariate logistic regression analysis was used to study the relationship between prescribed use and self-medication (both actual and intended) in general, for a specific symptom/disease or a specific antibiotic. results: prescribed use was associated with selfmedication, with stronger effect in northern/western europe (odds ratio 7.6, 95% ci 4.2-13.6) than in southern (2.1, 1.2-3.6) and eastern europe (1.9, 1.3-2.7). prescribing of a specific antibiotic increased the probability of self-medication with the same antibiotic. prescribing for a specific symptom/disease increased the likelihood of self-medication for the same symptom/disease. the use of prescribed antibiotics and actual self-medication were both determinants of intended self-medication in general and for specific symptoms/diseases. conclusions: routine prescribing of antibiotics increases the risk of self-medication with antibiotics for similar ailments, both through the use of leftovers and buying antibiotics directly from pharmacies. background: in 2003 the american national kidney foundation published a guideline based on opinion and observational studies which recommends tight control of serum calcium, phosphorus and calcium-phosphorus product levels in dialysis patients. objectives: within the context of this guideline, we explored associations of these plasma concentrations with cardiovascular mortality risk in incident dialysis patients. design and methods: in necosad, a prospective multi-centre cohort study in the netherlands, we included 1629 consecutive patients new on haemodialysis or peritoneal dialysis between 1997 and 2004. risks were estimated using adjusted time-dependent cox regression models. results: mean age was 60±15 years, 61% was male, and 64% was treated with haemodialysis. cardiovascular mortality risk was significantly higher in haemodialysis patients (hr: 1.5; 95% ci: 1.1 to 2.1) and in peritoneal dialysis patients (hr: 2.4; 1.3 to 4.2) with elevated plasma phosphorus levels when compared to patients who met the target. in addition, having elevated plasma calcium-phosphorus product concentrations increased cardiovascular mortality risk in haemodialysis (hr: 1.5; 1.1 to 2.1) and in peritoneal dialysis patients (hr: 2.2; 1.3 to 3.8). conclusion: application of the current guideline in clinical practice is warranted since it reduces cardiovascular mortality risk in haemodialysis and peritoneal dialysis patients in the netherlands. background: urologists are increasingly confronted with requests for early detection of prostate cancer in men from hereditary prostate cancer (hpc) families. however, little is known about the benefit of early detection among men at increased risk. objectives: we studied the effect of biennial screening with psa in unaffected men from hpc families, aged 50-70 years, on surrogate endpoints (test and tumour characteristics). methods: the netherlands foundation for the detection of hereditary tumours holds information on approximately 140 hpc families. here, 172 nonaffected men from these families were included and invited for psa testing every 2 years. we collected data on screening history and complications related to screening. results: in the first round, serum psa was elevated (3 ng/ml or greater) in 39 of 172 men screened (23%). further diagnostic assessment revealed 14 patients with prostate cancer (8.1%). compared to population-based prostate cancer screening trials, the referral rate is equal but the detection rate is twice as high. discussion: in conclusion, the results of prostate cancer screening trials will not be directly applicable to screening in hpc families. the balance between costs, side-effects and potential benefits of screening when applied to a high-risk population will have to be assessed separately. background: in industrialized countries occupational tuberculosis among health care workers (hcws) is re-emerging as a public health priority. to prevent and control tuberculosis transmission in nosocomial settings, public health agencies have issued specific guidelines. turin, the capital of the piedmont region in italy, is experiencing a worrying rise of tuberculosis incidence. here, hcws are increasingly exposed to the risk of nosocomial tuberculosis transmission. objectives: a) to estimate the sex-and age-adjusted annual rate of tuberculosis infection (arti) (per 100 person-years [%py]) among the hcws, as indicated by tuberculin skin test conversion (tst) conversion, b) to identify occupational factors associated with significant variations in the arti, c) to investigate the efficacy of the regional preventive guidelines. design and methods: multivariate survival analysis on tst conversion data from a dynamic cohort of 2185 hcws in turin, between 1998 and 2004. results: the overall estimated arti was 2.4 (95% ci: 1.9-3.0) %py. the risk of tst conversion significantly differed among workplaces, occupations, and age of hcws. the guidelines implementation was associated with an arti reductions of 1.35 (95% ci: 1.33-1.27) %py. conclusions: we identify occupational risk categories for targeting surveillance and prevention measures and assessed the efficacy of the local guidelines. background: a positive family history (fh) of breast cancer is an established risk factor for the disease. screening for breast cancer in israel is recommended annually for positive-fh women aged = 40 y and biennially for average-risk women aged 50-74 y. objective: to assess the effect of having a positive breast cancer fh on performing screening mammography in israeli women. methods: a cross-sectional survey based on a random sample of the israeli population. the study population consists of 1,605 women aged 40-74 y and telephone interviews were used. logistic regression models identified variables associated with mammography performance. results: a positive fh for breast cancer was reported by 153 (9.5%) participants. performing a mammogram in the previous year was reported by 43.1% and 24.7% of the positive and negative fh subgroups, respectively (p<0.0001). rates increased with age. among positive fh participants, being married was the only significant correlate for a mammogram in the previous year. conclusions: over 60% and around 55% of high-risk women aged 40-49 y and = 50 y, respectively, are inadequately screened for breast cancer. screening rates are suboptimal in average-risk women too. discussion: national efforts should concentrate on increasing awareness and breast cancer screening rates. to evaluate the association between infertility, infertility treatments and breast cancer risk. methods: a historical prospective cohort with 5,788 women who attended 5 israeli infertility centers between 1964 and 1984. their medical charts were abstracted. breast cancer incidence was determined through linkage with the national cancer registry. standardized incidence ratios (sirs) and 95% confidence intervals were computed by comparing observed cancer rates to those expected in the general population. additionally, in order to control for known risk factors, a casecontrol study nested within the cohort was carried out as well based on telephone interviews with breast cancer cases and controls matched by 1:2 ratio. results: compared to 115.2 expected breast cancer cases, 131 were observed (sir = 1.1;non-significant). risk for breast cancer was higher for women treated with clomiphene citrate (sir = 1.4; 95% ci 1.0-1.8). similar results were noted when treated and untreated women were compared, and when multivariate models were applied. in the nested case-control study, higher cycle index and treatment with clomiphene citrate were associated with significantly higher risk for breast cancer. conclusions: clomiphene citrate may be associated with higher breast cancer risk. smoking is a strong risk factor for arterial disease. some consider smoking also as a risk factor for venous thrombosis, while the results of studies investigating the relationship are inconsistent. therefore, we evaluated smoking as a risk factor for venous thrombosis in the multiple environmental and genetic assessment of risk factors for venous thrombosis (mega) study, a large population-based case-control study. consecutive patients with a first venous thrombosis were included from six anticoagulation clinics. using a random-digit-dialing method a control group was recruited in the same geographical area. all participants completed a questionnaire including questions on smoking habits. persons with known malignancies were excluded from the analyses, leading to a total of 4086 patients and 2567 controls. current and former smoking resulted in a small increased risk of venous thrombosis (ors adjusted for age, sex and bmi) (or-current:1.5 ci95:1.3-1.7, or-former:1.2 ci95:1.0-1.4). an increasing amount and duration of smoking was associated with an increase in risk. the highest risk was found among young (lowest tertile:18 to 41 yrs) current smokers; twenty or more pack-years resulted in a 3.3-fold increased risk (ci95:2.2-5.0). in conclusion, smoking results in a small increased risk of venous thrombosis, with the greatest relative effect among young heavy smokers. objective: to explore whether the observed association between silica exposure and lung cancer was confounded by exposure to other occupational carcinogens, we conducted a nested case-control-study among a cohort of male workers in 29 chinese mines and potteries. methods: 511 lung cancer cases and 1879 matched controls were selected. exposure to respirable silica as well as relevant occupational confounders were evaluated quantitatively based on historical industrial hygiene data. the relationship between silica exposure and lung cancer mortality was analyzed by conditional logistic regression analysis adjusted for exposure to arsenic, polycyclic aromatic hydrocarbons (pahs), radon, and smoking habit. results: in a crude analysis adjusted for smoking only, a significant trend of increasing risk of lung cancer with exposure to silica was found for tin, copper/iron miners, and pottery workers. however, after the relevant occupational confounders were adjusted, no association can be observed between silica exposure and lung cancer mortality (pro mg/m3-year increase of silica exposure: or = 1.01, 95% ci: 0.99 -1.02). conclusion: our results suggest that, the observed excess risk of lung cancer among silica exposed chinese workers is more likely due to exposure to other occupational carcinogens such as arsenic and pahs rather than due to exposure to respirable silica. background: modelling studies have shown that lifestyle interventions for adults with a high risk of developing diabetes are costeffective. objective: to explore the cost-effectiveness of lifestyle interventions for adults with low or moderate risk of developing diabetes. design and methods: the short-term effects of both a community-based lifestyle program for the general population and a lifestyle intervention for obese adults on diabetes risk factors were estimated from international literature. intervention costs were based on dutch projects. the rivm chronic diseases model was used to estimate long-term health effects and disease costs. costeffectiveness was evaluated from a health care perspective with a time horizon of 70 years. results: intervention costs needed to prevent one case of diabetes in 20 years range from 1,000 to 5,000 euro for the community program and from 5,000 to 21,000 euro for the intervention for obese adults. cost-effectiveness was 3,000 to 3,500 euro per quality adjusted life-year for the community program and 3,500 to 5,500 for the lifestyle intervention. conclusion: a lifestyle intervention for obese adults produces larger individual health benefits then a community program but, on a population level, health gains are more expensively achieved. both lifestyle interventions are cost-effective. background: in barcelona, the proportion of foreign-born patients with tuberculosis (tb) raised from 12.8% in 1999 to 35,2% in 2004. objective: to determine differences in infection by country of origin among contacts investigated by the tb programme in barcelona from 2000-2004. design and methods: data were collected on 1198 cases and their 4638 contacts. generalized estimating equations were used to obtain the risk of infection (or and 95% ci) to account for potential correlation among contacts. results: contacts of foreign born cases were more infected than contacts of natives patients (41% vs 20%, p<0.001) factors related to infection among contacts of foreign cases were inner city residency (or:1.8,95% ci: 1.2-2.8) and sputum smear positivity of the case (or:1.9,95% ci: 1. 2-2.8) and male contact (or:1.4,95% ci: 1.0-1.9), but not daily contact (or:1.1,95% ci:0.8-1.6) among natives cases, inner city residency (or:2.0,95% ci: 1.2-3.2), sputum smear positivity (or:2.2,95% ci: 1.5-3.1) and daily exposure (or:2.3,95% ci: 1.7-2.9) increased risk of infection. conclusion: contacts immigrant tb cases have a higher risk of infection than contacts of natives cases, however daily exposure to an immigrant case was not associated with a greater risk of infection. this could be explained by the higher prevalence of tb infection in their country of origin. background: an inverse association between birthweight and subsequent coronary heart disease (chd) has been widely reported but has not been formally quantified. we therefore conducted a systematic review of the association between birthweight and chd. design and methods: seventeen studies including a total of 144,794 singletons that had reported quantitative or qualitative estimates of the association between birthweight and chd by october 2005 were identified. additional data from two unpublished studies of 3801 individuals were also included. in total, the analyses included data on 4210 non-fatal and 3308 fatal coronary heart disease events in 147,009 individuals. results: the mean weighted estimate for the association between birthweight and chd incidence was 0.84 (95% ci 0.81-0.88) per kg of birthweight. overall, there was no significant heterogeneity between studies (p = 0.09) or evidence of publication bias (begg test p = 0.3). fifteen studies were able to adjust for some measure of socioeconomic position, but such adjustment did not materially influence the association: 0.85 (95% ci 0.81-0.90). discussion: these findings are consistent with one kilogram higher birthweight being associated with 10-20% lower risk of subsequent chd, but the causal nature of this association remains uncertain and its direct relevance to public health is likely to be small. objective: diabetes has been reported to be associated with a greater coronary hazard among women compared with men with diabetes. we quantified the coronary risk associated with diabetes by sex by conducting a meta-analysis of prospective cohort studies. methods: studies reporting estimates of the relative risk for fatal coronary heart disease (chd) comparing those with and without diabetes, for both men and women were included. results: 37 studies of type-2 diabetes and chd among 447,064 individuals were identified. the summary relative risk for fatal chd, diabetes versus not, was significantly greater among women than men: 3.50 (95% ci 2.70 to 4.53) versus 2.06 (95% ci 1.81 to 2.34), p<0.0001. after excluding eight studies that had only adjusted for age, the sex risk difference was substantially reduced, but still highly significant (p = 0.003). the pooled ratio of the relative risks (female: male) from the 29 multiple-adjusted studies was 1.46 (95% ci 1.14 to 1.88). conclusions: the relative risk for fatal chd associated with diabetes is 50% higher in women than in men. more adverse cardiovascular risk profiles among women with diabetes, combined with possible treatment disparities that favour men, may explain the greater excess coronary risk associated with diabetes in women. background: malaria in sri lanka is strongly seasonal and often of epidemic nature. the incidence has lowered in recent years which increased the relevance of epidemic forecasting for better targeting control resources. objectives: to establish the spatio/temporal correlation of precipitation and malaria incidence for use in forecasting. design and methods: de-trended long term (1972 de-trended long term ( -2001 monthly time series of malaria incidence at district level were regressed in a poisson regression against rainfall and temperature at several lags. results: in the north and east of sri lanka, malaria seasonality is strongly positively correlated to rainfall seasonality (malaria lagging one or two months behind rainfall). however, in the south west, no significant (negative) correlation was found. also in the hill country, no significant correlation was observed. conclusion and discussion: despite high correlations, it still remains to be explored to what extent rainfall can be a used as a predictor (in time) of malaria. observed correlation could simply be due to two cyclical seasonal patterns running in parallel, without causal relationship. e.g. similarly, strong correlations were found between temperature and malaria seasonality at 9 months time lag in northern districts, but causality is biologically implausible. background: few studies assessed the excess burden of acute respiratory tract infections (rti) among preschool children in primary care during viral seasons. objective: to determine the excess of rti in preschool children in primary care attributable to influenza and respiratory syncytial virus (rsv). methods: we performed a retrospective cohort study including all children aged 0-5 years registered in the database of the utrecht general practitioner (gp) network. during 1998 during -2002 , gps recorded episodes of acute rti. surveillance data of influenza and rsv were obtained from the weekly sentinel system of the dutch working group on clinical virology. viral seasons and base-line period were defined as the weeks with respectively more than 5% and less than 1% of the yearly number of isolates of influenza or rsv. results: on average 329 episodes of rti were recorded per 1,000 child years (95% ci:321-337). notably more consults for rti occurred during influenza-season (rr 1.74, 95% ci:1.63-1.86) and rsv-season (rr 2.27, 95% ci:2.14-2.42) as compared to base-line period, especially in children younger than two years of age. conclusion: substantial excess rates of rti were demonstrated among preschool children in primary care during influenza-season and particularly during rsvseason, notably in the younger age group. background: many cancer patients who have already survived some time want to know about their prognosis, given the precondition that they are still alive. objective: we described and interpreted population-based conditional 5-year relative survival rates for cancer patients. methods: the longstanding eindhoven cancer registry collects data on all patients with newly diagnosed cancer in the southeastern part of the netherlands (2.4 million inhabitants). patients aged 25-74 years, diagnosed between 1985 and 2002 and followed up until january 1, 2005 were included. conditional 5-year relative survival was computed for every additional year survived. results: for many tumours conditional 5-year relative survival approached 90-100% after having survived 5-10 years. however, for stomach cancer and hodgkin's lymphoma conditional 5-year relative survival increased to only 80-90% and for lung cancer and non-hodgkin's lymphoma it did not exceed 70-80%. initial differences in survival at diagnosis between age and stage groups disappeared after having survived for 5-10 years. conclusion: prognosis for patients with cancer changes with each year survived and for most tumours patients can considered to be cured after a certain period of time. however, for stomach cancer, lymphoma's and lung cancer the odds for death remains elevated compared to the general population. background: systematic review with meta-analysis, now regarded as 'best evidence', depends on availability of primary trials and on completeness of review. whilst reviewers have attempted to assess publication bias, relatively little attention has been given to selection bias by reviewers. method: systematic reviews of three cardiology treatments, that used common search terms, were compared for inclusion/exclusion of primary trials, pooled measures of principal outcomes and conclusions. results: in one treatment, reviews included 18, 11, 19, 28, 27 and 35 trials. there was little overlap: of 35 trials in the last review only 5, 4, 6, 13 and 3 were included by others. reported summary effects ranged from (most effective to least significant); mortality relative risk 0.29 (0.12, 0.70) in 4 trials to 0.96 (0.85, 1.09) in 23, and in one morbidity measure; standardised mean difference from 0.28 (0.10, 0.47) in 11 trials (822 patients) to 0.01 ()0.02, 0.04) in 10 (3960 patients). reviewers' conclusions ranged from 'highly effective' to 'no evidence of effect'. conclusions: these examples illustrate strong selection bias in published meta-analyses. post hoc review contravenes one important principal of science 'first the hypothesis, then the test'. selection bias by reviewers may affect 'evidence' more than does publication bias. in the context of a large population based german case control study examining the effects of hormone therapy (ht) on breast cancer risk, we conducted a validation study comparing ht prescription data with participants' self-reports for data quality assurance. included were 224 cases and 225 controls aged 50-74 years, stratified by age and hormone use. study participants provided detailed information on ht use to trained interviewers, while gynecologists provided prescription data via telephone or fax. data were compared using proportion of agreement, kappa, intraclass correlation coefficient (icc), and descriptive statistics. overall agreement for ever/never use was 88.2%, while agreement for ever/never use by type of ht was 80.6%, 80.3%, and 90.5% for mono-estrogen, cyclical, and continuous combined therapy, respectively. icc for duration was high (0.82 (95% ci: 0.77-0.85)), as were the iccs for age at first and last use (0.88(95% ci: 0.85-0.91) and 0.98 (95% ci: 0.97-0.98), respectively). comparison of exact brand name resulted in perfect agreement for 50.2% of participants, partial agreement for 29.3%, and no agreement for 20.7%. higher education and shorter length of recall were associated with better agreement. agreement was not differential by disease status. in conclusion, these self-reported ht data corresponded well with gynecologists' reports. background: legionnaires' disease (ld) is a pneumonia of low incidence. however, the impact of an outbreak can be substantial. objective: to stop a possible outbreak at an early stage, an outbreak detection programme was installed in the netherlands and evaluated after two years. design: the programme was installed nationally and consisted of sampling and controlling of potential sources to which ld patients had been exposed during their incubation period. potential sources were considered to be true sources of infection if two or more ld patients (cluster) had visited them, or if available patients' and environmental strains were indistinguishable by amplified fragment length polymorphism genotyping. all 39 municipal health services of the netherlands participated in the study. the regional public health laboratory kennemerland sampled potential sources and cultured samples for legionella spp. results: rapid sampling and genotyping as well as cluster recognition helped to target control measures. despite these measures, two small outbreaks were only stopped after renewal of the water system. the combination of genotyping and cluster recognition lead to 29 of 190 (15%) patient-source associations. conclusion and discussion: systematic sampling and cluster recognition can contribute to ld outbreak detection and control. this programme can cost-effectively lead to secondary prevention. -up (1990-2002) , 1896 primary invasive breast cancers occurred. results: compared with hrt never-use, use of estrogen alone was associated with a significant 1.4-fold increased risk. the association of estrogen-progestagen combinations with breast cancer risk varied significantly according to the type of progestagen: while there was no increase in risk with estrogen-progesterone (rr 1.0 [0.8-1.3]), estrogen-dydrogesterone was associated with a significant 1.3-fold increase, and estrogen combined with other synthetic progestins with a significant 1.8-fold increase. although the latter type of hrt involves a variety of different progestins, their associations with breast cancer risk did not differ significantly from one another. rrs did not vary significantly according to the route of estrogen administration (oral or transdermal/percutaneous). conclusion and discussion: progesterone rather than synthetic progestins may be preferred when an opposed estrogen therapy is to be prescribed. additional results on estrogen-progesterone are needed. background: although survival of hodgkin's lymphoma (hl) is high (>85%), treatment may cause long-term side-effects like premature menopause. objectives: to assess therapy-related risk factors for premature menopause (age <40) following hl. design and methods: we conducted a cohort-study among 387 female 5year hl-survivors, aged <31 at diagnose and treated between 1965 and 1995. patients were followed from first treatment until june 2001, menopause, death, or age 40. cumulative dose of various chemotherapeutic agents as well as radiation fields were studied as risk factors for premature menopause. cox-regression was used to adjust for age, year of treatment, smoking, bmi, and oral contraceptive-use. results: after a median follow-up of 11.4 years, 130 (34%) women reached premature menopause. overall 20 women (5%) were treated with chemotherapy only, 115 (30%) with radiotherapy only and 252 (65%) with both radio-and chemotherapy. exposure to procarbazine ), cyclophosphamide (hr 3.6 [2.1-5.9] ) and irradiation of the ovaries ]) were associated with significant increased risks for premature menopause. for procarbazine a dose-response relation was observed. procarbazine-use has decreased over time. conclusion: to decrease the risk for premature menopause after hl, procarbazine and cyclophosphamide exposure should be minimized and ovarian irradiation should be avoided. background: casale is an italian town where a large asbestos cement plant was active for decades. previous studies found increased risk for mesothelioma in residents, suggesting a decreasing spatial trend with distance from the plant. objective: to analyse the spatial variation of risk in casale and the surrounding area ($100,000 inhabitants) focussing on non-occupationally exposed individuals. design/methods: population-based case-control study including pleural mesotheliomas diagnosed between 1987 and 1993. information on the 97 cases and 250 controls comprised lifelong residential and occupational history of subjects and their relatives. nonparametric tests of clustering were used to evaluate spatial aggregation. parametric spatial models based on distance between the longest-lasting subject residence (excluding the last 20 years before diagnosis) and the source enabled estimation of risk gradient. results: mesothelioma risk appeared higher in an area of roughly 9-11 km radius from the source. spatial clustering was statistically significant (p = 0.003) and several clusters of cases were identified within casale. risk was highly related to the distance from the source; the best fitting model was the exponential decay with threshold. conclusion/discussion: asbestos pollution has increased dramatically the risk of mesothelioma in the area around casale. risk decreases slowly with the square of distance from the plant. malaria control programmes targeting malaria transmission from man to mosquito can have a large impact of malaria morbidity and mortality. to successfully interrupt transmission, a thorough understanding of disease and transmission parameters is essential. our objective was to map malaria transmission and analyse microenvironmental factors influencing this transmission in order to select high risk areas where transmission reducing interventions can be introduced. each house in the village msitu-wa-tembo was mapped and censused. transmission intensity was estimated from weekly mosquito catches. malaria cases identified through passive case detection were mapped by residence using gis software and the incidence of cases by season and distance to river were calculated. the distribution of malaria cases showed a clear seasonal pattern with the majority of cases during the rainy season (chisquare = 62.3, p<0.001). living further away from the river (p = 0.04) was the most notable independent protective factor for malaria infection. transmission intensity was estimated at 3.4 (95% ci 0.7 -9.9) infectious bites per person per year. we show that malaria in the study area is restricted to a short transmission season. spatial clustering of cases indicates that interventions should be planned in the area closest to the river, prior and during the rainy season. background: the effectiveness of influenza vaccination of elders has been subject of some dispute. its impact on health inequalities also demands epidemiological assessments, as health interventions may affect early and most intensely better-off social strata. objectives: to compare pneumonia and influenza (p&i) mortality of elders (aged 65 or more years old) before and after the onset of a largescale scheme of vaccination in sao paulo, brazil. methods: official information on deaths and population allowed the study of p&i mortality at the inner-city area level. rates related to the period 1998 to 2002, during which vaccination coverage ranked higher than 60% of elders were compared with figures related to the precedent period (1993) (1994) (1995) (1996) (1997) . the appraisal of mortality decrease used a geo-referred model for regression analysis. results: overall p&i mortality reduced 26.3% after vaccination. also the number of outbreaks, the excess of deaths during epidemic weeks, and the proportional p&i mortality ratio reduced significantly after vaccination. besides having higher prior levels of p&i deaths, deprived areas of the city presented a higher proportional decrease of mortality. conclusion: influenza vaccination contributed for an overall reduction of p&i mortality, while reducing the gap in the experience of disease among social strata. background: alcohol's first metabolite, acetaldehyde, may trigger aberrations in dna which predispose to developing colorectal cancer (crc) through several distinct pathways. our objective was to study associations between alcohol consumption and the risk of crc, according to two pathways characterized by mutations in apc and k-ras genes, and absence of hmlh1 expression. methods: in the netherlands cohort study, 120,852 men and women, aged 55-69 years, completed a questionnaire on risk factors for cancer in 1986. case-cohort analyses were conducted using 575 crc cases with complete data after 7.3 years of follow-up, excluding the first 2.3 years. gender-specific adjusted incidence rate ratios (rr) and 95% confidence intervals (ci) were estimated. results: neither total alcohol, nor beer, wine or liquor consumption was clearly associated with the risk of colorectal tumors lacking hmlh1 expression or harboring a truncating apc mutation and/or an activating k-ras mutation. in men and women, total alcohol consumption above 30 g/day was associated with an increased risk of crc harboring a truncating apc and/or activating k-ras mutation, though not statistically significant. (rr:1.37 (95% ci: 0.8-2.5) in men, rr: 1.88 (95% ci: 0.8-4.6) in women). in conclusion, alcohol consumption is not involved in the studied pathways leading to crc. background: educational level is commonly used to identify social groups with increased prevalence of smoking. other indicators of socioeconomic status (ses) might however be more discriminatory. objective: this study examines to what extent smoking behaviour is related to other ses indicators, such as labour market position and financial situation. methods: data derived from the european household panel, which includes data on smoking for 11 european countries. we selected data for 101,312 respondents aged 25-60 years. the association between ses indicators and smoking prevalence was examined through logistic regression analyses. results: preliminary results show that, in univariate analysis, all selected ses indicators were associated with smoking. higher rates of smoking in lower social groups were observed in all countries, except for women in some mediterranean countries. in multivariate analyses, education retained an independent effect on smoking. no strong effect was observed for labour market position (occupational class, employment status) or for income. however, smoking prevalence was strongly related to economic deprivation and housing tenure. conclusion: these results suggest that different aspects of people's ses affect their smoking behaviour. interventions that aim to tackle smoking among high-risk groups should identify risk groups in terms of both education and material deprivation. objective: we investigated time trends in overweight and leisure time physical activities (ltpa) in the netherlands since 1980. intra-national differences were examined stratified for sex, age and urbanisation degree. design and methods: we used a random sample from the health interview survey of about 140 000 respondents, aged 20-to-69 years. self-reported data on weight, height and demographic characteristics were gathered through interviews (yearly) and data on ltpa were collected by selfadministered questionnaires . linear regression was performed for trend analyses. results: during 1981-2004, mean body mass index (bmi) increased by 1.0 kg/m2 (p = 0.001). trends were similar across sex and urbanisation degrees. in 20-to-39 year old women, mean bmi increased more (1.7 kg/m2; p = 0.05) than in older women. concerning ltpa, no clear trend was observed during 1990 observed during -97 and 2001 observed during -04. however, in 2001 year old women spent $ 150 min/wk less on ltpa compared to older women, while this difference was smaller during 1990-97. conclusions: mean bmi increased more in younger women, which is consistent with the observation that this group spent less time on ltpa during recent years. although the overall increase in overweight could no´t be explained by trends in ltpa, physical activity interventions should target the younger women. background: prediction rules combine patient characteristics and test results to predict the presence of an outcome (diagnosis) or the occurrence of an outcome (prognosis) for individual patients. when prediction rules show poor performance in new patients, investigators often develop a new rule, ignoring the prior information available in the original rule. recently, several updating methods have been proposed that consider both prior information and information of the new patients. objectives: to compare five updating methods (that vary in extensiveness) for an existing prediction rule that preoperatively predicts the risk of severe postoperative pain (spp). design and methods: the rule was tested and updated on a validation set of 752 new surgical patients (274 (36%) with spp). we estimated the discrimination (the ability to discriminate between patients with and without spp) and calibration (the agreement between the predicted risks and observed frequencies of spp) of the five updated rules in 283 other patients (100 (35%) with spp). results: simple updating methods showed similar effects on calibration and discrimination as the more complex methods. discussion and conclusion: when the performance of a prediction rule in new patients is poor, a simple updating method can be applied to improve the predictive accuracy. about two million ethnic germans (aussiedler) have resettled in germany since 1990. analyses with a yet incomplete follow-up of a cohort of aussiedler showed a different mortality compared to russia and germany. objectives: we investigated whether the mortality pattern changed after a complete follow-up and whether residential mobility after resettlement has an effect on mortality. we established a cohort of 34393 aussiedler who moved to germany between 1990 and 2001. we calculated smr for all causes, external causes, cardiovascular deaths and cancer in comparison to german rates. results: with a complete follow-up, the cohort accumulated 248798 person years. overall, 1726 deaths were observed (smr 0.85, 95% ci: 0.81-0.89). smr for all external causes, all cancer and cardiovascular deaths were 0.84, 0.85 and 0.79, respectively. increased number of moves within germany was associated with increased mortality. conclusion and discussion: the mortality in the cohort is surprisingly low, in particular for cardiovascular deaths. there is a mortality disadvantage from external causes and for some selected cancers. this disadvantage is however not as large as would be expected if aussiedler were representative of the general population in fsu countries. mobility as an indicator for a lesser integration will be discussed. background: breast cancer screening (bcs) provides an opportunity to analyze the relationship between lymph node involvement (ln), the most important prognostic factor, and biological and time dependent characteristics. objective: our aim was to assess those characteristics that are associated with ln in a cohort of screen-detected breast cancers. methods: observational population study of all invasive cancers within stage i-iiia detected from 1996 to 2002 through a bcs program in catalonia (spain). age, tumor size, histological grade, ln status and screening episode (prevalent or incident) were analyzed. pearson chi-square or fisher's exact test, mann-whitney test and stratified analyses were applied, as well as multiple logistic regression techniques. results: twenty nine percent (95% ci 21.7-35.4%) out of 168 invasive cancers had ln and 37.5% were prevalent cancers. in the bivariate analysis, tumor size and age were strongly associated to ln (p< 0.010) while grade was related to ln only in incident cancers (p = 0.027). grade was associated with tumor size (p = 0.005) and with screening episode (p = 0.013). adjusting for screening episode and grade, age and tumor size were independent predictors of ln. conclusion: in conclusion, age and tumor size are independent predictors of ln. grade emerges as an important biological factor in incident cancers. background: the evidence regarding the association between smoking and cognitive function in the elderly is inconsistent. objectives: to examine the association between smoking and cognitive function in the elderly. design and methods: in 2003, all 740 participants of a population-based cohort study aged 70 years or older were eligible for a telephone interview on cognitive function using validated instruments, such as the telephone interview of cognitive status (tics). information on smoking history was available from questionnaires administered in 2002. we estimated the odds ratios (or) of cognitive impairment (below 25th percentile) and the corresponding 95% confidence intervals (ci) by means of logistic regression adjusting for age, sex, alcohol consumption, body mass index, physical exercise, educational level, depressive symptoms and co-morbidity. results: in total, 465 participants were interviewed and had complete information on smoking history. former smokers had a lower prevalence of cognitive impairment (oradjusted = 0.61;95% ci:0.33-1.13) compared with never smokers, but not current smokers (oradjusted = 0.96;95% ci:0.34-2.70). conclusion: there is no association between current smoking and cognitive impairment in the elderly. discussion: the lack of association between current smoking and cognitive impairment is in line with previous non-prospective studies. the inverse association with former smoking might be due to smoking cessation associated with co-morbidities. background: many studies have reported late effects of treatment in childhood cancer survivors. most studies, however, focused on only one late effect or suffered from incomplete follow-up. objectives: we assessed the total burden of adverse events (ae), and determined treatment-related risk factors for the development of various aes. methods: the study cohort included 1362 5-year survivors, treated in the emma childrens hospital amc in the netherlands between 1966-1996. aes were graded for severity by one reviewer according to the common terminology criteria adverse events version 3.0. results: medical follow-up data were complete for 94.3% 5-year survivors. median follow-up time was 18 years. almost 75% of survivors had one or more aes, and 24.6% had even 5 or more aes. of patients treated with rt alone, 55% had a high or severe burden of aes, while this was only 15% in patients treated with ct alone. radiotherapy (rt) was associated with the highest risk to develop an ae of at least grade 2, and was also associated with a greater risk to develop a medium to severe ae burden. conclusions: survivors are at increased risk for many health problems that may adversely affect their quality of life and long-term survival. background: studies in the past demonstrated that multifaceted interventions could enhance the quality of diabetes care. however many of these studies showed methodological flaws as no corrections were made for patient case-mix and clustering or a nonrandomised design was used. objective: to assess the efficacy of a multifaceted intervention to implement diabetes shared care guidelines. methods: a cluster randomised controlled trial of 2097 patients with type 2 diabetes was conducted at 40 general practises (n = 1714) and one outpatient clinic (n = 383). in primary care, facilitators analysed barriers to change, introduced structured care, gave feedback and trained practice staff. at the outpatient clinic, an abstract of the guidelines was distributed. case-mix differences such as duration of diabetes, education, co-morbidity and quality of life were taken into account. results: in primary care, more patients in the intervention group were seen on a three monthly basis (85.7% versus 69.4%, p<0.001) and their hba1c was statistically significant lower (6.9±0.9 versus 7.0±0.1, p<0.01). however, significance was lost after correction for case-mix (p = 0.6). change in blood pressure and total cholesterol was not significant. we were unable to demonstrate any change in secondary care. conclusion: multifaceted implementation did improve the process of care but left cardiovascular risk unchanged. background: we have performed a meta-analysis including 43 studies on the diagnostic accuracy of mr-mammography in patients referred for further characterization of suspicious breast lesions. using the bivariate diagnostic meta-analysis approach we found an overall sensitivity and specificity of 0.90 and 0.73, respectively. the aim of the present analysis was to detect heterogeneity between studies. materials and methods: seventeen study and population characteristics were separately included in the bivariate model to compare sensitivity and specificity between strata of the characteristics. results: both sensitivity and specificity were higher in studies performed in the united states compared to non-united states studies. both estimates were also higher if two criteria for malignancy were used instead of one or three. only specificity was affected by the prevalence of cancer: specificity was highest in studies with the lowest prevalence of cancer in the study population. furthermore, specificity was affected by whether the radiologist was blinded for clinical information: specificity was higher if there was no blinding. conclusions: variation between studies was notably present across studies in country of publication, the number of criteria for malignancy, the prevalence of cancer and whether the observers were blinded for clinical information. objective: the aim of this project is to explore variation in three candidate genes involved in cholesterol metabolism in relation to risk of acute coronary syndrome (acs), and to investigate whether dietary fat intake modifies inherent genetic risks. study population: a case-cohort study is designed within the danish 'diet, cancer and health' study population. a total of 1500 cases of acs have been identified among 57,053 men and women who participated in a baseline examination between 1993-1997 when they were aged 50-64 years. a random sample of 1500 participants will serve as 'control' population. exposures: all participants have filled out a detailed 192-item food frequency questionnaire and a questionnaire concerning lifestyle factors. participants were asked to provide a blood sample. candidate genes for acs have been selected among those involved in cholesterol transport (atp-binding cassette transporter a1, cholesterol-ester transfer protein, and acyl-coa:cholesterol acyltransferase 2). five single nucleotide polymorphisms (snps) will be genotyped within each gene. snps will be selected among those with demonstrated functional importance, as assessed in public databases. methods: statistical analyses of association between genetic variation in the three chosen genes and risk of acs. explorations of methods to evaluate biological interaction will be of particular focus. background: c-reactive protein (crp) has been shown to be associated with type 2 diabetes mellitus. it is unclear whether the association is completely explained by obesity. objective: to examine whether crp is associated with diabetes independent of obesity. design and methods: we measured baseline characteristics and serum crp in 5954 non-diabetic participants of the rotterdam study and followed them for a mean of 9.8 years. cox regression was used to estimate the hazard ratio. in addition, we performed a meta-analysis on 10 published studies. results: during follow-up, 658 participants developed diabetes. serum crp was significantly and positively associated with the risk to develop diabetes. the risk estimates attenuated but remained statistically significant after adjustment for obesity indexes. age, sex and body mass index (bmi) adjusted hazard ratios (95% ci) were 1.90(1.43-2.52) for the fourth quartile, 1.66(1.25-2.20) for the third quartile, and 1.51(1.16-2.01) for the second quartile of serum crp compared to the first quartile. in the meta-analysis, weighed age, sex, and bmi adjusted risk ratio was 1.44(1.16-1.78), for the highest crp category (>2.6 mg/l) compared to the reference category (<0.5 mg/l). conclusion: our findings shows that the association of serum crp with diabetes is independent of obesity. background: effectiveness of screening can be predicted by episode sensitivity, which is estimated by interval cancers following a screen. full-field digital or cr plate mammography are increasingly introduced into mammography screening. objectives: to develop a design to compare performance and validity between screen-film and digital mammography in a breast cancer screening program. methods: interval cancer incidence was estimated by linking 721 000 screening visits from 1991-2001 at an individual level to the files of the cancer registry in finland. these data was used to estimate the study size requirements for analyzing differences in episode sensitivity between screen-film and digital mammography in a randomized setting. results: the two-year cumulative incidence of interval cancers per 100 000 screening visits was estimated to be 300. to allow the maximum acceptable difference in the episode sensitivity between screenfilm and digital arm to be 20% (80% power, 0.05 significance level, 1:1 randomization ratio, 85% attendance rate), approximately 240 000 women need to be invited. conclusion: only fairly large differences in the episode sensitivity can be explored within a single randomized study. in order to reduce the degree of non-inferiority between the screen-film and digital mammography, meta-analyses or pooled analyses with other randomized data are needed. session: socio-economic status and migrants presentation: oral. background: tackling urban/rural inequalities in health has been identified as a substantial challenge in reforming health system in lithuania. objectives: to assess mortality trends from major causes of death of the lithuanian urban and rural populations throughout the period of 1994-2004. methods: information on major causes of death (cardiovascular diseases, cancers, external causes, and respiratory diseases) of lithuanian urban and rural populations from 1994 to 2004 was obtained from lithuanian department of statistics. mortality rates were age-standardized, using european standard. mortality trends were explored using the logarithmic regression analysis. results: overall mortality of lithuanian urban and rural populations was decreasing statistically significantly during 1994-2004. more considerable decrease was observed in urban areas where mortality declined by 1.7% per year in males and 2.2% in females, compare to the decline by 1.2% in males and 1.7% in females in rural areas. the most notable urban/rural differences in mortality trends with unfavourable situation in rural areas were estimated in mortality from stoke, breast cancer in females, and external causes of death (traffic accidents and suicides). background: recent studies indicate that depression plays an important role in the occurrence of cardiovascular diseases (cvd). underlying mechanisms are not well understood. objectives: we investigated whether low intake of omega-3 fatty acids (fas) is a common cause for depression and cvd. methods: the zutphen elderly study is a prospective cohort study conducted in the netherlands. depressive symptoms were measured with the zung scale in 332 men, aged 70-90 years, and free from cvd and diabetes in 1990. dietary factors were assessed with a cross-check dietary history method. results: compared to high intake (= 156.2 mg/d), low intake (<58.9 mg/d) of omega-3 fas, adjusted for demographics and cvd risk factors, was associated with an increased risk of depressive symptoms (or 2.20; 95% ci 1.06-4.58) at baseline, and non-significantly with 10-year cvd mortality (hr 1.14; 95% ci 0.67-1.95). the adjusted hr for a 5-point increase in depressive symptoms for cvd mortality was 1.13 (95% ci 1.01-1.26), and did not change after additional adjustment for omega-3 fas. conclusion: low intake of omega-3 fas may increase the risk of depression. our results, however, do not support the hypothesis that low intake of omega-3 fas explains the relation between depression and increased risk of cvd. background: during the last decades the incidence of metabolic syndrome has risen dramatically. several studies have shown beneficial effects of nut and seed intake on components of this syndrome. the relationship with prevalence of metabolic syndrome has not yet been examined. objectives: we studied the relation between nut and seed intake and metabolic syndrome in coronary patients. design and methods: presence of metabolic syndrome (according to international diabetes federation definition) was assessed in 904 stable myocardial infarction patients (77% men) aged 60-80 years, as part of the alpha omega trial. dietary data were collected by food-frequency questionnaire. results: the prevalence of metabolic syndrome was 42%. median nut and seed intake was 3.82 g/day (interquartile range, 1.53-8.33 g/day). intake of nuts and seeds was inversely associated with the metabolic syndrome (prevalence ratio: 0.84; 95% confidence interval: 0.59-1.19, for >10 g/day versus <1 g/day), after adjustment for age, gender, dietary and lifestyle factors. the prevalence of metabolic syndrome was 31% lower (p = 0.050) in men with a high nut and seed intake compared to men with a low intake, after adjustment for confounders. conclusion and discussion: intake of nuts and seeds may reduce the risk of metabolic syndrome in stable coronary patients. background: in epidemiology, interaction is often assessed by adding a product term to the regression model. in linear regression the regression coefficient of the product term refers to additive interaction. however, in logistic regression it refers to multiplicative interaction. rothman has argued that interaction as departure from additivity better reflects biological interaction. hosmer and lemeshow presented a method to quantify additive interaction and its confidence interval (ci) between two dichotomous determinants using logistic regression. objective: our objective was to provide an estimation method for additive interaction between continuous determinants. methods and results: from the abovementioned literature we derived the formulas to quantify additive interaction and its ci between one continuous and one dichotomous determinant and between two continuous determinants using logistic regression. to illustrate the theory, data of the utrecht health project were used, with age and body mass index as risk factors for diastolic blood pressure. conclusions: this paper will help epidemiologists to estimate interaction as departure from additivity. to facilitate its use, we developed a spreadsheet, which will become freely available at our website. background: the incidences of acute myocardial infarction (ami) and ischemic stroke (is) in finland have been among highest in the world. accurate geo-referenced epidemiological data in finland provides unique possibilities for ecological studies using bayesian spatial models. objectives: examine sex-specific geographical patterns and temporal variation of ami and is. design and methods: ami (n = 205,213) and is (n = 115,383) cases in 1991-2003 in finland, localized at the time of diagnosis according to the place of residence address using map coordinates. cases and population were aggregated to 10 km x10 km grids. full bayesian conditional autoregressive models (car) were used for studying the geographic incidence patterns. results: the incidence patterns of ami and is showed on average 70% (95% ci 50-85%) common geographic variation and significantly the rest of the variation was disease specific. there was no significant difference between sexes. the patterns of high-risk areas have persisted over the years and the pattern of is showed more annual random fluctuations. conclusions: although ami and is showed rather similar and temporally stable patterns, significant part of the spatial variation was disease specific. further studies are needed for finding the reasons for disease specific geographical variation. most studies addressing socio-economic inequalities in health services use fail to take into account the disease the patient is suffering from. the objective of this study is to compare possible socioeconomic differences in the use of ambulatory care between 2 distinct patient groups: diabetics and patients with migraine. data was obtained from the belgian health interview surveys 1997, 2001 and 2004. in total 4381 patients with self reported diabetes or migraine were identified. in a multilevel analysis the probability of a contact and the volume of contacts with the general practitioner and/or the specialist were studied for both groups in relation to educational attainment and income. adjustment was made for age, sex, subjective health and comorbidity at the individual level, and doctors' density and region at district level. no socio-economic differences were observed among diabetic patients. among patients with migraine we observed a higher probability of specialist contacts in higher income groups (or 1,6;95% ci 1,1-2,4) and higher educated persons (or 1,6;95% ci 1,2-2,2), while lower educated persons tend to report more visits with the general practitioner. to correctly interpret socio-economic differences in the use of health services there is need to take into account information on the patient's type of disease. background: the suitability of non-randomised studies to assess effects of interventions has been debated for a long time, mainly because of the risk of confounding by indication. choices in the design and analytical phase of non-randomised studies determine the ability to control for such confounding, but have not been systematically compared yet. objective: the aim of the study will be to quantify the role of design and analytical factors on confounding in non-randomised studies. design and methods: a meta-regression analysis will be conducted, based on 41 cohort and 8 case-control studies analysed in a recent cochrane review on influenza vaccine effectiveness against hospitalisation or death in the elderly. primary outcome will be the degree of confounding as measured by the difference between the reported effect estimate (odds ratios or relative risks) and the best available estimate (nichol, unpublished data) . design factors that will be considered include study design, matching, restriction and availability of confounders. statistical techniques that will be evaluated include multivariate regression analysis with adjustment for confounders, stratification and, if available, propensity scores. results the rsults will be used to develop a generic guideline with recommendations how to prevent confounding by indication in non-randomised effect studies. the wreckage of the oil tanker prestige in november 2002 produced a heavy contamination of the coast of galicia (spain). we studied relationships between participation in clean-up work and respiratory symptoms in local fishermen. questionnaires including details of clean-up activities and respiratory symptoms were distributed among associates of 38 fishermen's cooperatives, with postal and telephone follow-up. statistical associations were evaluated using multiple logistic regression analyses, adjusted for sex, age, and smoking status. between january 2004 and february 2005, information was obtained from 6,780 fishermen (response rate 76%). sixty-three percent had participated in clean-up operations. lower respiratory tract symptoms were more prevalent in clean-up workers (odds ratio (or) 1.73; 95% confidence interval 1.54-1.94). the risk increased when the number of exposed days, number of hours per day, or number of activities increased (p for linear trend <0.0001). the excess risk decreased when more time had elapsed since last exposure (or 1.45 (1.28-1.64) and 2.16 (1.91-2.45) for more and less than 17 months, respectively; p for interaction <0.05). in conclusion, fishermen who participated in the clean-up work of the prestige oil spill show a prolonged dosedependent increased prevalence of respiratory symptoms one to two years after the beginning of the spill. background. hpv testing has been proposed for cervical cancer screening. objectives: evaluating the protection provided by hpv testing at long intervals vs. cytology every third year. methods: randomised controlled trial conventional arm: conventional cytology. experimental arm: in phase 1 hpv and liquid-based cytology. hpv-positive cytology-negatives referred for colposcopy if age 35-60, for repeat after one year if age 25-34. in phase 2 hpv alone. positives referred for colposcopy independently of age. endpoint: histologically confirmed cervical intraepithelial neoplasia (cin) grade 2 or more. results: overall 94,323 women were randomised. preliminary data at recruitment are presented. overall, among women aged 35-60 years relative sensitivity of hpv versus conventional cytology was 1.43 (95% c.i. 1.09-1.88) and relative positive predictive (ppv) value was 0.59 (95% c.i. 0.45-0.76). among women aged 25-34 relative sensitivity of hpv vs. conventional cytology was 1.58 (95% c.i. 1.03-2.44) during phase 1 but 3.79 (95% c.i. 2.26-6.35) during phase 2. conclusions: hpv testing increased cross-sectional sensitivity, but reduced ppv. in younger women data suggest that direct referral of hpv-positives to colposcopy results in relevant overdiagnosis of regressive lesions. measuring detection rate of cin at the following screening round will allow studying overdiagnosis and the possibility of longer screening intervals. background: plant lignans are present in foods such as whole grains, seeds, fruits and vegetables, and beverages. they are converted by intestinal bacteria into the enterolignans, enterodiol and enterolactone. enterolignans possess several biological activities whereby they may influence carcinogenesis. objective: to study the association between plasma enterolignans and the risk of colorectal adenomas. colorectal adenomas are considered to be precursors of colorectal cancer. design and method: the case-control study included 532 cases with at least one histologically confirmed colorectal adenoma and 503 controls with no history of any type of adenoma. associations between plasma enterolignans and colorectal adenomas were analyzed by logistic regression. results: associations were stronger for incident than for prevalent cases. when only incident cases (n = 262) were included, high compared to low enterodiol plasma concentrations were associated with a reduction in colorectal adenoma risk after adjustment for confounding variables. enterodiol odds ratios (95% ci) were 1.00, 0.69 (0.42-1.13), 0.60 (0.37-0.99), 0.53 (0.32-0.88) with a significant trend (p = 0.01) through the quartiles. although enterolactone plasma concentrations were 10fold higher, enterolactone's reduction in risk was not statistically significant (p for trend = 0.09). conclusion: we observed a substantial reduction in colorectal adenoma risk among subjects with high plasma concentrations of enterolignans, in particular enterodiol. background: smoking is a risk factor for tuberculosis diseases. recently the question was raised if smoking also increases the risk of tuberculosis infection. objective: to assess the influence of environmental tobacco smoke (ets) exposure in the household on tuberculosis infection in children. design and methods: a crosssectional community survey was done and information on 1373 children was obtained. tuberculosis infection was determined with a tuberculin skin test (tst) (cut-off 10 mm) and information on smoking habits was obtained from all adult household members. univariate and multivariate analyses were performed, and odds ratio (or) was adjusted for the presence of a tb contact in the household, crowding and age of the child. results: ets was a risk factor for tuberculosis infection (or: 1.92, 95% ci: 1.27 -2.92) when all children with a tst read between two and five days were included. the adjusted or was 1.64 (95% ci: 1.03 -2.61). in dwellings were a tuberculosis case had lived the association was strongest (adjusted or 5.67, 95% ci: 1.26 -25.65). conclusion and discussion: ets exposure seems to be a risk factor for tuberculosis infection in children. this is of great concern considering the high prevalence of smoking and tuberculosis in developing countries. background and objective: to implement a simulation model to analyze demand and waiting time (wt) for knee arthroplasty and to compare a waiting list prioritization system (ps) with the usual first-in, first-out (fifo) system. methods: parameters for the conceptual model were estimated using administrative data and specific studies. a discrete-event simulation model was implemented to perform 5-year simulations. the benefit of applying the ps was calculated as the difference in wt weighted by priority score between disciplines, for all cases who entered the waiting list. results: wt for patients operated under the fifo discipline was homogeneous (standard deviation (sd) between 1.3-1.7 months) with mean 18.7. wt under the ps had higher variability (sd between 10.6-11.6) and was positively skewed, with mean 7.6 months and 10% of cases over 24 months. when wt was weighted by priority score, the ps saved 6.6 months (95% ci 6.4-6.9) on average. the ps was favorable for patients with priority scores over 50, but penalized those with lower priority scores. conclusions: management of the waiting list for knee arthroplasty through a ps was globally more effective than through fifo, although patients with low priority scores were penalized with higher waiting times. background: we developed a probabilistic linkage procedure for the linking of readmissions of newborns from the dutch paediatrician registry (lnr). 15% of all newborns (30.000) are admitted to a neonatal ward. the main problems were the unknown number of readmissions per child and the identification of admissions of twins. objective: to validate our linking procedure in a double blinded study. design and methods: a random sample of admissions from 200 children from the linked file has been validated by the caregivers, using the original medical records. results: response was 97%. for admissions of singletons the linkage contained no errors except for the small uncertain area of the linkage. for admissions of multiple births a high error rate was found. conclusion and discussion: we successfully linked the admissions of singleton newborns with the developed probabilistic linking algorithm. for multiple births we did not succeed in constructing valid admission histories due to too low data quality of twin membership variables. validation showed alternative solutions for linking twin admissions. we strongly recommend that linkage results should always be externally validated. background: salmonella typhimurium definitive phage type (dt) 104 has emerged as an important pathogen in the last two decades. a 10-fold increase in cases in the netherlands during september-november 2005 prompted an outbreak investigation. objective: the objective was to identify the source of infection to enable preventive measures. methods: a subset of outbreak isolates was typed by molecular means. in a case-control study, cases (n = 109) and matched population controls (n = 411) were invited to complete self-administered questionnaires. results: the molecular typing corroborated the clonality of the isolates. the molecular type was similar to that of a recent s. typhimurium dt104 outbreak in denmark associated with imported beef. the incriminated shipment was traced after having been distributed sequentially through several eu member states. sampling of the beef identified s. typhimurium dt104 of the same molecular type as the outbreak isolates. cases were more likely than controls to have eaten a particular raw beef product. conclusions: our preliminary results are consistent with this s. typhimurium dt104 outbreak being caused by contaminated beef. our findings underline the importance of european collaboration, traceability of consumer products and a need for timely intervention into distribution chains. background: heavy-metals may affect newborns. some of them are presenting tobacco smoke. objectives: to estimate cord-blood levels of mercury, arsenic, lead and cadmium in newborns in 2 areas in madrid, and to assess the relationship with maternal tobacco exposure. design and methods: bio-madrid study obtained 115 cord-blood samples from recruited trios (mother/father/ newborn). cold-vapor atomic absorption spectrophotometry (aas) was used to measure mercury and graphite-furnace aas for the other metals. mothers answered a questionnaire including tobacco exposure. median, means and standard errors were calculated and logistic regression used to estimate or. results: median levels for mercury and lead were 7.5 mg/l and 13.7 mg/l. arsenic and cadmium were undetectable in 82% and 47 % of samples. preliminar analysis showed a significant association of maternal tobacco exposure and levels of arsenic (or:3.59;95% ci:1. 11-11.55 ), cadmium (or:2.24;95% ci:1.04-4.79), and lead (or:3.83;95% ci:1.41-10.36). smoking in pregnancy was associated to arsenic (or:3.90;95% ci:1.17-12.96), while passive exposure was more related to lead (or:3.61;95% ci:1.26-4.79) and cadmium (or:2.06;95% ci:0.91-4.97). conclusion: madrid newborns have high cord-blood levels of mercury. tobacco exposure in pregnancy might increase levels of arsenic, cadmium and lead. background: road traffic accidents (rta) are the leading cause of death for young. rta police reports provide no health information other then the number of deaths and injured, while health databases have no information on the accident dynamics. the integration of the two databases would allows to better describe the phenomenon. nevertheless, the absence of common identification variables through the lists makes the deterministic record linkage (rl) impossible. objective: to test feasibility of a probabilistic rl between rta and health information when personal identifiers are lacking. methods: health information came from the rta integrated surveillance for the year 2000. it integrates data from ed visits, hospital discharges and deaths certificates. a deterministic rl was performed with 149 police reports, where the name and age of deceased were present. results of the deterministic rl was then used as gold standard to evaluate the performance of the probabilistic one. results: the deterministic rl resulted in 141 (94.6%) linked records. the probabilistic rl, where the name was omitted, was capable to correctly identify 130 (87.2%). conclusions: performance of the probabilistic rl was good. further work is needed to develop strategies for the use of this approach in the complete datasets. background: overweight constitutes a major public health problem. the prevalence of overweight is unequally distributed between socioeconomic groups. risk group identification, therefore, may enhance the efficiency of interventions. objectives: to identify which socioeconomic variable best predicts overweight in european populations: education, occupation or income. design: european community household panel data were obtained for 9 countries (n = 52,855). overweight was defined as a body mass index >= 25 kg/m2. uni-and multivariate logistic regression analyses were employed to predict overweight in relationship to socioeconomic indicators. results: major socioeconomic differences in overweight were observed, especially for women. for both sexes, a low educational attainment was the strongest predictor of overweight. after control for confounders and the other socioeconomic predictors, the income gradient was either absent or positive (men) or negative (women) in most countries. similar patterns were found for occupational level. for women, inequalities in overweight were generally greater in southern european countries. conversely, for men, differences were generally greater in other european countries. conclusion: across europe, educational attainment most strongly predicts overweight. therefore, obesity interventions should target adults and children with lower levels of education. background: because incidence and prevalence of most chronic diseases rise with age, their burden will increase in ageing populations. we report the increase in prevalence of myocardial infarction (mi), stroke (cva), diabetes type ii (dm) and copd based on the demographic changes in the netherlands. in addition, for mi and dm the effect of a rise in overweight was calculated. methods: calculations were made for the period 2005-2025 with a dynamic multi-state transition model and demographic projections of the cbs. results: based on ageing alone, between 2005 and 2025 prevalence of dm will rise from 550.000 to 870.000 (+58%), prevalence of mi from 310.000 to 365.000 (+18%), stroke prevalence from 185.000 to 290.000 (+57%) and copd prevalence from 55 455.000 to 540.000 (+19%). a continuation of the dutch (rising) trend in overweight prevalence would in 2025 lead to about 940.000 diabetics (+70%). a trend resulting in american levels would lead to over 1 million diabetics (+90%), while the impact on mi was much smaller: about 375.000 (+20%) in 2025. conclusion: the burden of chronic disease will substantially increase in the near future. a rising prevalence of overweight has an impact especially on the future prevalence of diabetes background: there has been increasing concern about the effects of environmental endocrine disruptors (eeds) on human reproductive health. eeds include various industrial chemicals, as well as dietary phyto-estrogens. intra-uterine exposures to eeds are hypothesized to disturb normal foetal development of male reproductive organs and specifically, to increase the risk of cryptorchidism, hypospadias, testicular cancer, and a reduced sperm quality in offspring. objective: to study the associations between maternal and paternal exposures to eeds and the risks of cryptorchidism, hypospadias, testicular cancer and reduced sperm quality. design and methods: these associations are studied using a case-referent design. in the first phase of the study, we collected questionnaire data of the parents of 231 cases with cryptorchidism, 329 cases with hypospadias and 742 referent children. in the second phase, we will focus on the health effects at adult age: testicular cancer and reduced sperm quality. in both phases, we will attempt to estimate the total eed exposure of parents of cases and referents at time of pregnancy through an exposure-assessment model in which various sources of exposure, e.g. environment, occupation, leisure time activities and nutrition, are combined. in addition, we will measure hormone receptor activity in blood. background: eleven percent of the pharmacotherapeutic budget is spent on acid-suppressive drugs (asd); 3% of patients are chronic user. most of these indications are not conform to dyspepsia guidelines. objectives: we evaluated the implementation of an asd rationalisation protocol among chronic users, and analysed effects on volume and costs. method: in a cohort study 2871 patients from 158 gp's with protocol were compared to a control group of 8120 patients from 267 gp's without. prescription data of 2002-2004 were extracted from agis health database. a log-linear regression model compared standardised outcomes of number of patients that stopped or reduced asd (>50%) and of prescription volume and costs. results: gp's and patients in both groups were comparable. 7% in the intervention group had stopped; 6% in the control group (p<0.01). the volume had decreased in another 11% of patients; 8% in control group (p<0.001). compared to the baseline data in the control group (100%) the adjusted or of volume in the intervention group was 98.2%. the total costs adjusted or was 97.5%. the implementation significantly reduced the number of chronic users, and substantially dropped volume and costs. active intervention from insurance companies can stimulate rationalisation of prescription. background/objective: today, 20% of lung cancers are resectable (stage i/ii). 5-year survival is therefore low (15%). spiral computed tomography (ct) screening detects more lung cancers than chest x-ray. it is unknown if this will translate into a lung cancer mortality reduction. the nelson trial investigates whether 16detector multi-slice ct reduces lung cancer mortality with at least 25% compared to no screening. we present baseline screening results. methods: a questionnaire was sent to 550,000 men and women. of the 150,000 respondents, 30,500 high-risk current and former smokers were invited. until december 22, 2005, 15,530 of them gave informed consent and were randomised (1:1) in a screen arm (ct in year 1, 2 and 4) and control arm (no screening). data will be linked with the cancer registry and statistics netherlands to determine cancer mortality and incidence. results: of the first 5,700 baseline ct examinations 82% was negative (ct after one year), 16% indeterminate (ct after 3 months) and 2% positive (referral pulmonologist). seventy percent of detected tumours were resectable. conclusion/discussion: ct screening detects a high percentage of early stage lung cancers. it is estimated that the nelson trial is sufficiently large to demonstrate a 25% lung cancer mortality reduction or more. background: due to diagnostic dilemmas in childhood asthma, drug treatment of young children with asthmatic complaints often serves as a trial treatment. objective: to obtain more insight into patterns and continuation of asthma medication in children during the first 4 years of life. design: prospective birth cohort study methods: within the prevention and incidence of asthma and mite allergy (piama) study (n = 3,291 children) we identified 125 children using asthma medication in their first year of life. results: about 80% of children receiving asthma medication before the age of one, discontinued use during follow-up. continuation of therapy was associated with male gender (adjusted odds ratio [aor] 3.6, 95% confidence interval [ci]: 1.6-8.2), a diagnosis of asthma (aor 2.9, 95% ci: 1.3-6.3) and receiving combination or controller therapy (aor 2,6, 95% ci: 1.1-6.1). conclusion: patterns of medication use in preschool children support the notion that both beta2-agonist and inhaled corticosteroids are often used as trial medication, since 80% discontinues. the observed association between continuation of therapy and both an early diagnosis of asthma and a prescription for controller therapy suggests that, despite of diagnostic dilemmas, children in apparent need of prolonged asthma therapy are identified at this very early age. background: this study explored the differences in birthweight between infants of first and second generation immigrants and infants of dutch women, and to what extent maternal, fetal and environmental characteristics could explain these differences. method: during 15 months all pregnant women in amsterdam attending their first prenatel screening were asked to fill out a questionnaire (sociodemographic status, lifestyle) as part of the amsterdam born children and their development (abcd)-study; 8267 women (67%) responded. only singleton deliveries with pregnancy duration = 37 weeks were included (n = 7209). results: infants of all first and second generation immigrant groups (surinam, the antilles, turkey, morocco, ghana, other countries) had lower birthweights (range: 3227-3529 gram) than dutch infants (3548 gram). linear regression revealed that, adjusted for maternal height, weight, age, parity, smoking, marital status, gestational age and gender, infants of surinamese women (1st and 2nd generation), antillean and ghanaian women (both 1st generation) were still lighter than dutch infants (93.7, 166.7, 113.1, and 128.0 grams respectively; p<0.05). conclusion: adjusted for maternal, fetal and environmental characteristics infants of some immigrant groups had substantial lower birthweights than infants of dutch women. other factors (like genetics, culture) can possibly explain these differences. introduction: missing data is frequently seen in cost-effectiveness analyses (ceas). we applied multiple imputation (mi) combined with bootstrapping in a cea. objective: to examine the effect of two new methodological procedures of combining mi and bootstrapping in a cea with missing data. methods: from a trial we used direct health and non-health care costs and indirect costs, kinesiophobia and work absence data assessed over 12 months. mi was applied by multivariate imputation by chained equations (mice) and non-parametric bootstrapping was used. observed case analyses (oca), where analyses were conducted on the data without missings, were compared with complete case analysis (cca) and with analyses when mi and bootstrapping were combined after 10 to 30% of cost and effect data were omitted. results: by the cca effect and cost estimates shifted from negative to positive and cost-effectiveness planes and acceptability curves were biased compared to the oca. the methods of combining mi and bootstrapping generated good cost and effect estimates and the cost-effectiveness planes and acceptability curves were almost identical to the oca. conclusion: on basis of our study results we recommend to use the combined application of mi and bootstrapping in data sets with missingness in costs and effects. background: since the 1980s, coronary heart disease (chd) mortality rates have halved with approximately 50% of this decrease being attributable to medical and surgical treatments. objective: this study examined the cost-effectiveness of specific chd treatments. design and methods: the impact chd model was used to calculate the number of life-years-gained (lyg) by specific cardiology interventions given in 2000, and followed over ten years. this previously validated model integrates data on patient numbers, median survival in specific groups, treatment uptake, effectiveness and costs of specific interventions. cost-effectiveness ratios were generated as costs per lyg for each specific intervention. results: in 2000, medical and surgical treatments together prevented or postponed approximately 1,635 chd deaths in patients aged 25-84 years; this generated approximately 13,645 extra life years. aspirin and beta-blockers for secondary prevention of myocardial infarction and heart failure, and spironolactone for heart failure all appeared highly cost-effective (<e1,500 per lyg). less cost effective, however, were revascularisation for chronic angina (cabg surgery e12,970 and angioplasty e14,865 per lyg), or statins for primary prevention (e11,475 per lyg) or for community patients with angina (e11,220 per lyg). conclusions: cost effectiveness ratios generally favoured simple medical treatments for myocardial infarction, secondary prevention, and heart failure. objective: is population-based primary prevention more favourable than secondary prevention (risk factor reduction in coronary heart disease (chd) patients)? methods: the previously validated im-pact model was used to integrate data describing chd patient numbers, specific treatment uptake levels, trends in major risk factors, and the mortality benefits of these risk factor changes in healthy people and in chd patients. results: approximately 2,530 fewer deaths were attributable to reductions in the three major risk factors between 1985 and 2000. declining smoking prevalence resulted in approximately 685 fewer deaths: 395 in healthy people and 290 in known chd patients. decreasing population total cholesterol concentrations resulted in approximately 1,340 fewer deaths attributable to dietary changes (1,250 in healthy people and 90 in chd patients) plus 265 fewer deaths attributable to statin treatment (45 in people without chd and 220 in chd patients). decreasing mean population blood pressure resulted in approximately 170 fewer deaths attributable to secular falls in blood pressure (150 in healthy people and 20 in chd patients) plus approximately 70 fewer deaths attributable to antihypertensive treatments in people without chd. conclusions: compared with secondary prevention (620 fewer deaths), primary prevention (1,910 fewer deaths) achieved a three-fold larger reduction in chd deaths. background: carotenoid intake has been positively associated with plasma concentrations in different populations. however, the influence of body mass index (bmi) on this association is mostly unknown. objectives: we explored the relationship between intake of carotenoids and vitamin c, and their plasma concentrations by bmi categories in spanish elderly people. desgin/methods: a dietary interview using a 135-item food-frequency questionnaire was conducted with 240 men and 293 women, 65 and older, participating in the eureye study in spain. blood samples were collected for measurement of carotenoids and vitamin c. correlation coefficients (r) between energy adjusted nutrient intakes and plasma concentrations were calculated by categories of bmi, adjusting for sex, age and smoking. results: significant correlations between acarotene, ß-carotene, lycopene, ß-cryptoxanthin and vitamin c intakes, and plasma concentrations were observed, r=0.21;0.19;0.17;0.20,0.41, respectively (p<0.05). correlations for carotenoids changed substantially by bmi categories, with the highest correlations for a-carotene, ß-carotene, and, to a lesser extension, ß-cryptoxanthin and lycopene, in subjects with bmi<25 (0.39;0.34;0.25;0.22 respectively), and the lowest, in subjects with bmi=30 (0.08;0.19;0.18;0.19 respectively). correlations for vitamin c remained unchanged by bmi. conclusions: our data suggest that carotenoids in plasma may be good markers of dietary intake in elderly subjects with lower bmi. background: reduced heart rate variability (hrv) is associated with an increased mortality, morbidity and worse prognosis of cardiovascular diseases (cvd). there is a lack of population-based data to examine the distribution of hrv, its association with cardiovascular risk factors (cvrf), and its role as a potential mediator of the effect of established risk factors on cvd. objective: to study the distribution of hrv and its association with cvrf in an elderly eastern german population. design and methods: this analysis is based on data of 1336 participants (45-83 years) of an ongoing cross-sectional study. time-and frequency-domain measures of hrv were computed. their association with cvrf was determined by linear regression modelling. results: age-and heart rate-adjusted standard deviation of the durations of nn intervals and high frequency power was significantly higher in women. significant inverse associations of timeand frequency-domain hrv with anthropometric parameters, triglycerides, blood pressure, prevalent diabetes and age were observed in both sexes. there was a graded inverse association of hrv with age except for the oldest age-group. discussion/conclusion: established cvrf are associated with a reduced hrv. therefore reduced hrv could be a mediator between cvrf and cvd, which will be examined in a follow-up study. background: aalen's model and cox's proportional hazard model postulate different relationship between hazard and covariates and the subject matter seldom suggests which of the models are to be preferred. actually the assumption of constant effects, either additive or multiplicative, may be incorrect especially in cancer survival analysis. objectives: the aim of the study is to analyse survival for a breast cancer cohort where typical prognostic factors lack of proportionality. design and methods: the cohort consists of 2700 invasive breast cancer cases from turin cancer registry within a high resolution study in co-operation with the breast cancer screening program, diagnosed from 1997 through 2000, aged 40-79. an additive-multiplicative cox-aalen model with age at diagnosis, categorized tumour size (t) and nodal status (n) is fitted. results: the prognostic role of age at diagnosis, t and n are confirmed. proportionality assumption holds only for age (hazard ratio = 1.03, p-value<0.0001). t, particularly the category referring to 'extended to chest wall or skin' versus 'less than 2 cm', has a significant time-varying effect (p-value = 0.02) after 12 months since diagnosis. discussion: more flexible regression tools than cox model which is routinely used for analyzing cancer survival are needed. cox-aalen proves to be an appealing alternative. background: the major software packages for meta-analysis of causal -notably intervention -research are generally too expensive for developing countries. moreover, interactive educational software for students and teachers is largely unavailable. we recently developed the 'mix' program, an easy-to-maintain, interactive, and educational meta-analysis add-in for excel, available as free download at http://www.mix-for-meta-analysis.info. objectives: to formally establish whether the program's numerical and graphical output is comprehensive and accurate enough for scientific standards. design and methods: the study was designed as a software validation. data sets, primarily intervention studies, with substantially different features were analyzed by three investigators with mix and stata, using the latter program as golden standard. additionally, a feature (software options) comparison was made with modern programs for meta-analysis, such as cma 2.0, metawin 2.1, weasyma 2.2, and revman 4.2. results: the results of the validation project will be fully reported at the congress. preliminary results indicate that the mix program's output is comparable to stata output. it distinguishes itself from other software by the variety of graphical output, the excel-based interface, and free availability. conclusion and discussion: the mix program appears to provide valid output and may be an attractive, feature-rich alternative to existing meta-analysis software. background: an ankle-arm index (aai) <0.9 of systolic blood pressure (sbp) as marker of peripheral arterial disease is a predictor of coronary and carotid artery disease. the present gold standard examination with doppler-ultrasound and sphygmomanometric cuff is subject to observer bias and requires intensive training. for epidemiologic studies, valid and easy-to-use examination methods are needed. objective: to validate the easy-to-use omron hem-705cp for sbp measurements in the ankle and for aai determination in epidemiologic studies. design/methods: in a population-based sample of 112 subjects (45-80 years), arm and ankle sbp measurements by the omron hem-705cp and corresponding aai were compared with those using a hand-held doppler and sphygmomanometric cuff. bland-altman plots of sbp and aai differences, validation criteria for use in clinical practice and diagnostic values for the detection of an aai<0.9 were employed. results: omron measured higher sbp than doppler. sbp and aai differences increasing towards higher sbp levels. specificity, sensitivity and negative predictive value for the detection of aai<0.9 were >99% (positive predictive value was 67%). conclusion: omron fails the validation criteria for ankle sbp measurement. however, the ease of use of the device could outweigh the inaccuracy if used as a screening tool for aai<0,9 in epidemiologic studies. background: associations exist between: 1) parental birth weight and child birth weight; 2) birth weight and adult psychopathology; and 3) maternal psychopathology during pregnancy and birth weight of the child. this study is the first to combine those associations. objective: to investigate the different pathways from parental birth weight and parental psychopathology to child birth weight in one model. design and methods: depression and anxiety scores on 6,507 mothers and 4,764 fathers during 20 weeks pregnancy and birth weights from 6,116 children were available. path analyses with standardized regression coefficients were used to evaluate the different effects. results: in the unadjusted path analyses significant effects existed between: maternal (r = .17) and paternal birth weight (r = .13) and child birth weight; maternal birth weight and maternal depression (r=).05) and anxiety (r=).06); and maternal depression (r = .06) and anxiety (r = .06) and child birth weight. after adjustment for confounders, only maternal (r = .10) and paternal (r = .08) birth weight and maternal depression (r=).02) remained significantly related to child birth weight. conclusion after adjustment maternal depression, and not anxiety, remained significantly related to child birth weight. discussion future research should focus on the different mechanisms of maternal anxiety and depression on child birth weight. background: most patients with peripheral arterial disease (pad) die from coronary artery disease (cad). non-invasive cardiac imaging can assess the presence of coronary atherosclerosis and/or cardiac ischemia. screening in combination with more aggressive treatment may improve prognosis. objective: to evaluate whether a non-invasive cardiac imaging algorithm, followed by treatment will reduce the 5-year-risk of cardiovascular events in pad patients free from cardiac symptoms. design and methods: this is a multicenter randomized controlled clinical trial. patients with intermittent claudication and no history of cad are eligible. one group will undergo computed tomography (ct) calcium scoring. the other group will undergo ct calcium scoring and ct angiography (cta) of the coronary arteries. patients in the latter group will be scheduled for a dobutamine stress magnetic resonance imaging (dsmr) test to assess cardiac ischemia, unless a stenosis of the left main (lm) coronary artery (or its equivalent) was found on cta. patients with cardiac ischemia or a lm/lm-equivalent stenosis will be referred to a cardiologist, who will decide on further (interventional) treatment. patients are followed for 5 years. conclusion: this study assesses the value of non-invasive cardiac imaging to reduce the risk of cardiovascular events in patients with pad free from cardiac symptoms. background: hpv is the main risk factor for cervical cancer and also a necessary cause for it. participation rates in cervical cancer screening are low in some countries and soon hpv vaccination will be available. objectives: aim of this systematic review was to collect and analyze published data on knowledge about hpv. design and methods: a medline search was performed for publications on knowledge about hpv as a risk factor for cervical cancer and other issues of hpv infection. results: of individual studies were stratified by age of study population, country of origin, study size, publication year and response proportion. heterogeneity was described. results: knowledge between 27 included studies varied substantially. thirteen to 57% (closed question) and 0.6 to 1.9% (open question) of the participants knew about hpv as a risk factor for cervical cancer. women had consistently better knowledge on hpv than men. there was confusion of hpv with other sexually transmitted diseases. conclusion and discussion: studies were very heterogeneous, thus making comparison difficult. knowledge about hpv infections depended on the type of question used, gender of the participants and their professional background. education of the general public on hpv infections needs improvement, specially men should also be addressed. background: influenza outbreaks in hospitals and nursing homes are characterized by high attack rates and severe complications. knowledge of the virus' specific transmission dynamics in healthcare institutions is scarce but essential to develop cost-effective strategies. objective: to follow and model the spread of influenza in two hospital departments and to quantify the contributions of the several possible transmission pathways. methods: an observational prospective cohort study is performed on the departments of internal medicine & infectious diseases and pulmonary diseases of the umc utrecht during the 2006 influenza season. all patients and regular medical staff are checked daily on the presence of fever and cough, the most accurate symptoms of influenza infection. nose-throat swabs are taken for pcr analysis for both symptomatic individuals and a sample of asymptomatic individuals. to determine transmission, contact patterns are observed between patients and visitors and patients and medical staff. results/discussion: spatial and temporal data of influenza cases will be combined with contact data in a mathematical model to quantify the main transmission pathways. among others the model can be used to predict the effect of vaccination of the medical staff which is not yet common practice in the studied hospital. background: the long term maternal sequelae of stillbirths is unknown. objectives: to assess whether women who experienced stillbirths have an excess risk of long term mortality. study design: cohort study. methods: we traced jewish women from the jerusalem perinatal study, a population-based database of all births to west jerusalem residents (1964 -1976 who gave birth at least twice during the study period, using unique identity numbers. we compared the survival to 31-12-2004 of women who had at least one stillbirth (n = 569) to that of women who had only live births (n = 24108) using cox proportional hazards models. results: during a median follow up of 36.2 years, 77 (13.5%) mothers with stillbirths died compared to 1,483 (6.2%) unexposed women; crude hazard ratio (hr) 2.15 (95% ci: 1.71-2.70). the mortality risk remained significantly increased after adjustments for sociodemo-graphic variables, maternal diseases, placental abruption and preeclampsia (hr: 1.42, 95% ci: 1.12-1.80). stillbirth was associated with increased risk of death from cardiovascular (adjusted hr:1.95, 0.99-3.84), circulatory (1.77, 1.07-2.92) and genitourinary (4.59, 1.41-14.91) causes. conclusions: the finding of increased mortality among mothers of stillbirths joins the growing body of evidence demonstrating long term sequelae of obstetric events. future studies should elucidate the mechanisms underlying these associations. resilience is one of the essential characteristics of successful ageing. however, very little is known about the determinants of resilience in old age. our objectives were to identify resilience in the english longitudinal study of ageing (elsa) and to investigate social and psychological factors determining it. the study design was a crosssectional analysis of wave 1 of elsa. using structural equation modelling, we identified resilience as a latent variable indicated by high quality of life in the face of six adversities: ageing, limiting long-standing illness, disability, depression, perceived poverty and social isolation and we regressed it on social and psychological factors. the latent variable model for resilience showed a highly significant degree of fit (weighted root mean square resid-ual=0.035). determinants of resilience included good quality of relationships with spouse (p = 0.002), family (p = 0.028), and friends (p< 0.001), good neighbourhood (p<0.001), high level of social participation (p<0.001), involvement in leisure activities (p = 0.003); perception of not being old (p<0.001); optimism (p = 0.041), and high subjective probability of survival to an older age (p<0.001). we concluded that resilience in old age was as much a matter of social engagement, networks and context as of individual disposition. implications of this on health policy are discussed. background: there is extensive literature concluding that ses is inversely related to obesity in developed countries. several studies in developing populations however reported curvilinear or positive association between ses and obesity. objectives: to assess the social distribution of obesity in men and women in 3 middle-income countries of eastern and central europe with different level of economic development. methods: random population samples aged 45-69 years from poland, russia and czech republic were examined between 2002-2005 as baseline for prospective cohort study. we used body-mass index (bmi) and waist/hip ratio (whr) as obesity measures. we compared age-adjusted bmi and whr for men and women by educational levels in all 3 countries. results: the data collection was concluded in summer 2005. we collected data from about 29,000 subjects. lower ses increased obesity risk in women in all 3 countries (the strongest gradient in the czech republic and the lowest in russia), and in czech men. there was no ses gradient in bmi in polish men and positive association between education and bmi in russian men. conclusions: our findings strongly agree with previous literature showing that the association between ses status and obesity is strongly influenced by overall level of country economic development. background: inaccurate measurements of body weight, height and waist circumference will lead to an inaccurate assessment of body composition, and thus of the general health of a population. objectives: to assess the accuracy of self-reported body weight, height and waist-circumference in a dutch overweight working population. design and methods: bland and altman methods were used to examine the individual agreement between self-reported and measured body weight and height in 1298 overweight workers (67% male; mean age 43.9 +/) 8.6 years; mean body mass index [bmi] 29.5 +/) 3.4 kg/m2). the accuracy of self-reported waistcircumference was assessed in a subgroup of 250 persons (70% male; mean age 44.1 +/) 9.2 years; mean bmi 29.6 +/) 3.0 kg/ m2), for whom both measured and self-reported waist circumference was available. results: body weight was underreported by a mean (standard deviation) of 1.4 (1.9) kg, body height was on average over-reported by 0.7 (1.5) cm. bmi was on average underreported by 0.68 (0.8) kg/m2. waist-circumference was overreported by 1.1 (4.0) cm. the overall degree of error from selfreporting was between 0.4 and 2.3%. conclusion and discussion: self-reported anthropometrics seem satisfactorily accurate for the assessment of general health in a middle-aged overweight working population. the incidence of breast cancer and the prevalence of metabolic syndrome are increasing rapidly in chile, but this relationship is still debated. the goal of this study is to assess the association between metabolic syndrome and breast cancer before and after menopause. a hospital based case-control study was conducted in chile during 2005. 170 cases with histologically confirmed breast cancer and 170 age matched controls with normal mammography were identified. metabolic syndrome was defined by atpiii and serum lipids, glucose, blood pressure and waist circumference were measured by trained nurses. data of potential confounders such as, obesity, socioeconomic status, exercise and diet were obtained by anthropometric measures and a questionnaire. odds ratios (ors) and 95% confidence intervals (cis) were estimated by conditional logistic regression stratified by menopause. in postmenopausal women, a significant increase risk of breast cancer was observed in women with metabolic syndrome (or = 1.90, 95% ci = 1.00-3.60). the elements of metabolic syndrome strongly associated were high levels of glucose and hypertension. in conclusion, postmenopausal women with metabolic syndrome had 90% of excess risk of breast cancer. these findings support the theory that there is a different risk profile of breast cancer after and before menopause. background: physical exercise during pregnancy has numerous beneficial effects on maternal and foetal health. it may, however, affect early foetal survival negatively. objectives: to examine the association between physical exercise and spontaneous abortion in a cohort study. design and methods: in total, 92,721 women recruited to the danish national birth cohort in early pregnancy, provided information on amount and type of exercise during pregnancy and on possible confounding factors. 3,187 women experienced foetal death before 23 gestational weeks. hazard ratios for spontaneous abortion in four periods of pregnancy ()10, 11-14, 15-18, and 19-22 weeks) according to amount (min/week) and type of exercise, respectively, were estimated using cox regression. various sensitivity analyses to reveal distortion of the results from selection forces and information bias were made. results: the hazard ratios of spontaneous abortion increased stepwise with amount of physical exercise and were largest in the earlier periods of pregnancy (hrweek11-14 = 3.4 (ci 2.7-4.3) for 420 min/week, compared to no exercise). weight bearing types of exercise were strongest associated with abortion, while swimming showed no association. these results remained stable, although attenuated, in the sensitivity analyses. discussion: handling of unexpected findings that furthermore challenge official public health messages will be discussed. hemodialysis (hd) patients with a low body mass index (bmi) have an increased mortality risk, but bmi changes over time on dialysis treatment. we studied the association between changes in bmi and all-cause mortality in a cohort of incident hd patients. patients were followed until death or censoring for a maximum follow-up of 7 years. bmi was measured every 6 months and changes in bmi were calculated over each 6-mo period. with a time-dependent cox regression analysis, hazard ratios (hr) were calculated for these 6-mo changes on subsequent mortality from all causes, adjusted for the mean bmi of each 6-mo period, age, sex and comorbidity. 712 men and 494 women were included (age: 64?14 years, bmi: 25.1?4.6 kg/m2, 7-y mortality: 75%). a loss of bmi>5% was independently associated with an increased mortality risk (hr: 1.83, 95%-ci: 1.41-2.37), while a loss of 1-5% showed no difference (hr: 0.89, 0.69-1.14) compared to no change in bmi ()1% to +1% change). a gain in bmi of 1-5% showed beneficial (hr: 0.67, 0.51-0.88), while a gain of bmi>5% was not associated with a survival advantage (hr: 1.02, 0.69-1.50). in conclusion, hd patients with a decreasing bmi have an increased risk of all-cause mortality. background: within the tripss-2 project, impact of clinical guidelines (gl) on venous thromboembolism (vte) prophylaxis was evaluated in a large italian hospital. gl were effective in increasing the appropriateness of prophylaxis and in reducing vte. objectives: we performed a cost-effectiveness analysis by using a decision-tree model to estimate the impact of the adopted gl on costs and benefits. design and methods: a decision-tree model compared prophylaxis cost and effects before and after gl implementation. four risk profiles were identified (low, medium, high, very high). possible outcomes were: no event, major bleeding, asymptomatic vte, symptomatic vte and fatal pulmonary embolism. vte patients risk and probability of receiving prophylaxis were defined using data from the previous survey. outcome probabilities were assumed from literature. tariffs and hospital figures were used for costing the events. results: gl introduction reduced the average cost per patient from e 190 to 165 ()13%) with an increase in terms of event free patients (+4%). results are particularly relevant in the very high risk group. conclusion: the implementation of locally adapted gl may lead to a gain in terms of costs and effects, in particular for patients at highest vte risk. background: assisted reproductive techniques are used to overcome infertility. one reason of success is the use of ovarian stimulation. objectives: compare two ovarian stimulation protocols, gonadotropin-releasing hormone agonists/antagonists, assessing laboratorial and clinical outcomes, to provide proper therapy option. identify significant predictors of clinical pregnancy and ovarian response. design and methods: retrospective study (agonist cycles, 203; antagonist cycles, 177) including ivf/intracytoplasmic sperm injection cycles. multiple logistic and regression models, with fractional polynomial method were used. results: antagonist group exhibited lower length of stimulation and dose of recombinant follicle stimulating hormone (rfsh), higher number of retrieved and fertilized oocytes, and embryos. agonist group presented thicker endometrium, better fertilization, implantation and clinical pregnancy rates. clinical pregnancy has shown positive correlation with endometrial thickness and use of agonist; negative correlation with age and number of previous attempts. retrieved oocytes shown positive correlation with estradiol on day of human chorionic gonadotrophin (hcg) and use of antagonist; negative correlation with rfsh dose. conclusion: patients from antagonist group are more likely to get more oocytes and quality embryos, despite those from agonist group are more likely to get pregnant. background: prevalence studies of the metabolic syndrome require fasting blood samples and are therefore lacking in many countries including germany. objectives: to narrow the incertitude resulting from extrapolation of international prevalence estimates, with a sensitivity analysis of the prevalence of the metabolic syndrome in germany using a nationally representative but partially non-fasting sample. methods: stepwise analysis of the german health examination survey 1998, using the national cholesterol education program (ncep) criteria, hemoglobin a1c (hba1c), non-fasting triglycerides and fasting time. results: among 6666 participants aged 18-79 years, the metabolic syndrome prevalence was (i) 13.6% with 13.3% inconclusive cases using the unmodified ncep criteria, (ii) 17.6% with 9.4% inconclusive cases using hba1c > 6.1% if fasting glucose was missing, (iii) 23.8% with 0.6% inconclusive cases when additionally using non-fasting triglycerides = 75th percentile stratified by fasting time, and (iv) 21.2% to 23.8% with <1% inconclusive cases using different cutoffs (hba1c 6.1%, non-fasting triglycerides 200 and 250 mg/dl). discussion: despite a lower prevalence of obesity in germany compared to the us, the prevalence of the metabolic syndrome is likely to be in the same order of magnitude. this analysis may help promote healthy life styles by stressing the high prevalence of interrelated cardiovascular risk factors. background: epidemiologic studies that directly examine fruits and vegetables (f&v) consumption and other lifestyle factors in relation to weight gain are sparse. objective: we examined the associations between the f&v intake and 10-y weight gain among spanish adult people. design/methods: the study was conducted with a sub-sample of 214 healthy people aged 15 y and over at baseline in 1994, who participated in a population-based nutrition survey in valencia-spain. data on diet, lifestyle factors and body weight (direct measurement) were obtained in 1994 and 2004. information on weight gain was available for 187 participants in 2004. results: during the 10-y period, participants tended to gain on average 4.61 kg (median = 3.6 kg). in multivariate analyses, participants with the highest tertile of f&v intake at baseline had a 65% of lower risk of gaining =3.6 kg compared with those who had the lowest intake tertile after adjustment for sex, age, education, smoking, tv-viewing, bmi, and energy intake (or = 0.35;95% ci:0.15 0.84;p-fortrend = 0.02). for every 100 g/d increase in f&v intake, the or was reduced by 14% (or = 0.86;0.75-0.99;p-trend=0.036). tvviewing at baseline was positively associated with weight gain, or for-1 h-increase=1.32 (1.01-1.71;p-trend=0.04). conclusions: our findings suggest that a high f&v intake and low tv-viewing may reduce weight gain among adults. background: diabetic patients develop more readily atherosclerosis thus showing greatly increased risk for cardiovascular disease. objective: the heinz nixdorf recall-study is a prospective cohort-study designed to assess the prognostic value of new risk stratification methods. here we examined the association between diabetes, previously unknown hyperglycemia and the degree of coronary calcification (cac). methods: a population-based sample of 4,814 men and women aged 45-74 years was recruited from three german cities between 2000-2003. baseline examination included amongst others a detailed medical history, blood analyses and electron-beam tomography. we calculated adjusted prevalence ratios (pr), adjusting for age, smoking, bmi and 95%-confidence intervals (95% ci) with log-linear binomial regression. results: the prevalence of diabetes is 8.4% (men: 9.8%, women: 6.7%), for hyperglycemia 5.7% (men: 8.1%, women: 3.4%). prevalence ratio for cac in male diabetics without overt coronary heart disease is 1.87 (95% ci: 1.28-2.72), for those with hyperglycemia 1.62 (1.09-2.46). in women the association is even stronger: 2.62 (1.78-3.87) with diabetes, 1.92 (1.11-3.31) with hyperglycemia. conclusion: the data support the concept of regarding diabetic patients as being in a high risk category meaning >20% hard events in 10 years. furthermore, even persons with elevated blood glucose levels already show higher levels of coronary calcification. background: birth weight is associated with health in infancy and later in life. socioeconomic inequality in birth weight is an important marker of current and future population health inequality. objective: to examine the effect of maternal education on birth weight, low birth weight (lbw, birth weight<2,500 g), and small for gestational age (sga) background: in clinical practice patient data are obtained gradually and health care practitioners tune prognostic information according to available data. prognostic research does not always reproduce this sequential acquisition of data: instead, 'worst', discharge or aggregate data are often used. objective: to estimate prognostic performance of sequentially updated models. methods: cohortstudy of all very-low-birth-weight-babies (<1500 g) admitted to the study neonatal icu <2 days after birth (984 eligible from 1991 to 2002) and followed-up until 2 years old (7.8% lost-to-follow-up). main outcomes: disabling cerebral palsy at 2 years (37, 3.8%) or death (194, 19 .7% )95% in the first 4 weeks). main prognostic determinants: neonatal cerebral lesions identified with cranial ultrasound (us) exams performed per protocol on days 2, 7, 28 and at discharge. logistic regression models were updated with data available at these different moments in time during admission. results: at days 2, 7 and 28 respectively, main predictor (severe parenchymal lesion) adjusted odds ratio: 18, 31 and 37; us model c-statistic: 0.69, 0.75 and 0.80. discussion: prognostic models performance in neonatal patients improved from inception to discharge, particularly for identification of the high risk category. time of data acquisition should be considered when comparing prognostic instruments. in epidemiological longitudinal studies one is often interested in the analysis of time patterns of censored history data. for example, how regular a medication is used or how often someone is depressed. our goal is to develop a method to analyse time patterns of censored data. one of the tools in longitudinal studies is a nonhomogeneous markov chain model with discrete time moments and categorical state space (for example, use of various medications). suppose we are interested only in the time pattern of appearance of a particular state which is in fact a certain epidemiological event under study. for this purpose we construct a new homogeneous markov chain associated with this event. the states of this markov chain are the time moments of the original nonhomogeneous markov chain. using the new transition matrix and standard tools from markov chain theory we can derive the probabilities of occurence of that epidemiological event during various time periods (including ones with gaps). for example, probabilities of cumulative use of medication during any time period. in conclusion, the proposed approach based on markov chain model provides a new way of data representation and analysis which is easy to interpret in practical applications. background: tuberculosis (tb) cases that belong to a cluster of the same mycobacterium tuberculosis dna fingerprint are assumed to be consequence of recent transmission. targeting interventions to fast growing clusters may be an efficient way of interrupting transmission in outbreaks. objective: to assess predictors for large growing clusters compared to clusters that remain small within a 2 years period. design and method out of the 10567 culture confirmed tb patients diagnosed between 1993 and 2004, 4783 (45%) had unique fingerprints while 5784 were part of a cluster. of the clustered cases 673 were in a small (2 to 4 cases within the first 2 years) and 83 in a large cluster (more than 4 cases within the first 2 years). results independent risk factors for being a case within the first 2 years of a large cluster were non-dutch nationality (or = 6.38 95% ci [1. 38-29.55 background: passive smoking causes adverse health outcomes such as lung cancer or coronary heart disease (chd). the burden of passive smoking on a population level is currently unclear and depends on several assumptions. we investigated the public health impact of passive smoking in germany. methods: we computed attributable mortality risks due to environmental tobacco smoke (ets). we considered lung cancer, chd, stroke, copd and sudden infant death. frequency of passive smoking was derived from the national german health survey. sensitivity analyses were performed using different definitions of exposure to passive smoking. results: in total, 3301 deaths every year in germany are estimated to be caused by exposure to passive smoking at home (women 2293, men 1008). most of these deaths are due to chd (2148) and stroke (774). additional consideration of passive smoking at workplace increased the number of deaths to 3864. considering any exposure to passive smoking and also active smokers who report exposure to passive smoking increased the number of deaths further. conclusions: passive smoking has an important impact on mortality in germany. even the most conservative estimate using exposure to ets at home led to a substantial number of deaths related to passive smoking. des daughters have a strongly increased risk of clear-cell adenocarcinoma of the vagina and cervix (ccac) at a young age. longterm health problems, however, are still unknown. we studied incidence of cancer, other than ccac, in a prospective cohort of des daughters (desnet project). in 2000 13,674 questionnaires were sent to des daughters registered at the des center in utrecht. also, informed consent was asked for linkage with disease registries. for this analysis, data of 12,219 responders and nonresponders were linked to palga, the dutch nationwide network and registry of histo-and cytopathology. mean age at the end of follow-up was 41.7 years. a total of 244 incident cancers occurred. increased standardized incidence rates (sir) were found for vaginal/vulvar cancers (sir = 4.1, 95% confidence interval (95% ci) 1.4-9.7), melanoma (sir = 1.9, 95% ci 1.4-2.6)) and breast cancer (sir=1.2, 95% ci 1.0-1.4) as compared to the general population. no increased risk was found for invasive cervical cancer, possibly due to effective screening. results for breast and cervical cancer are consistent with the sparse literature. the risk of melanoma might be due to surveillance bias. future analyses will include non-invasive cervical cancer, stage specific sirs for melanoma and adjustment for confounding (sister control group) for breast cancer. background: contact tracing plays an important role in the control of emerging infectious diseases in both human and farm animal populations, but little is known yet about its effectiveness. here we investigate in a generic setting for well-mixed populations the dependence of tracing effectiveness on the probability that a contact is traced, the possibility of iteratively tracing yet asymptomatic infectives, and delays in the tracing process. methods and findings: we investigate contact tracing in a mathematical model of an emerging epidemic, incorporating a flexible infectivity function and incubation period distribution. we consider isolation of symptomatic infected as the basic scenario, and determine the critical tracing probability (needed for effective control) in relation to two infectious disease parameters: the reproduction ratio under isolation and the duration of latent period relative to the incubation period. the effect of tracing delays is considered, as is the possibility of single-step tracing vs. iterative tracing of quarantined invectives. finally, the model is used to assess the likely success of tracing for influenza, smallpox, sars, and foot-and-mouth disease epidemics. conclusions: we conclude that single-step contact tracing can be effective for infections with a relatively long latent period or a large variation in incubation period, thus enabling backwards tracing of super spreading individuals. the sensitivity to changes in the tracing delay varies greatly, but small increases may have major consequences for effectiveness. if single-step tracing is on the brink of being effective, iterative tracing can help, but otherwise it will not improve much. we conclude that contact tracing will not be effective for influenza pandemics, only partially for fmd epidemics, and very effective for smallpox and sars epidemics. abstract: infections of highly pathogenic h5n1 avian influenza in humans underline the need for tracking of the ability of these viruses to spread among humans. here we propose a method of analysing outbreak data that allows determination of whether and to what extent transmission in a household has occurred after an introduction from the animal reservoir. in particular, it distinguishes between onward transmission from humans that were infected from the animal reservoir (primary human-to-human transmission) and onward transmission from humans who were themselves infected by humans (secondary human-to-human transmission). the method is applied to data from an epidemiological study of an outbreak of highly pathogenic avian influenza (h7n7) in the netherlands in 2003. we contrast a number of models that differ with respect to the assumptions on primary versus secondary human-to-human transmission. session: mathematical modelling of infectious diseases presentation: oral. usually models for the spread of an infection in a population are based on the assumption of a randomly mixing population, where every individual may contact every other individual. however, the assumption of random mixing seems to be unrealistic, therefore one may also want to consider epidemics on (social) networks. connections in the network are possible contacts, e.g. if we consider sexually transmitted diseases and ignore all spread by other than sexual ways, the connections are only between people that may have intercourse with each other. in this talk i will compare the basic reproduction ratio, r0 and the probability of a major outbreak of network models and for randomly mixing populations. furthermore, i will discuss which properties of the network are important and how they can be incorporated in the model. in this talk a reproductive power model is proposed that incorporates the following points met when an epidemic disease outbreak is modeled statistically: 1) the dependence of the data is handled with a non-homogeneous birth process. 2) the first stage of the outbreak is described with an epidemic sir model. soon control measures will start to influence the process. these measures are in addition to the natural epidemic removal process. the prevalence is related to the censored infection times in such a way that the distribution function, and therefore the survival function, satisfies approximately the first equation of the sir model. this leads in a natural way to the burr-family of distributions. 3) the non-homogeneous birth process handles the fact that in practice, with some delay, it is the infected that are registered and not the susceptibles. 4) finally the ending of the epidemic caused by the measures taken is incorporated by modifying the survival function with a finalsize parameter in the same way as is done in long-term survival models. this method is applied to the dutch classical swine individual and area (municipality) measures of income, marital and employment status were obtained. there were 9,011 suicides and 180,220 controls. after controlling for compositional effects, ecological associations of increased suicide risk with declining area levels of employment and income and increasing levels of people living alone were much attenuated. individual-level associations with these risk factors were little changed when controlling for contextual effects. we found no consistent evidence that associations with individual level risk factors differed depending on the areas characteristics (cross-level interactions). this analysis suggests the ecological associations reported in previous studies are likely to be due in greater part to the characteristics of the residents in those areas than area-influences on risk, rather than to contextual effects. were found to be at higher risk. risk was significantly greater in women whose first full-term pregnancy was at age 30 or more (or 7.79, ). in addition, more than 5 full-term pregnancies would be expected to correlate with an increase in the risk (x2 111.12, p<0.05). in multivariate analysis, history of breast feeding is a significant factor in decreasing risk (or 0.68, 95% ci 0. 12-0.97 the euregion meuse-rhine (emr) is an area with different regions, regarding language, culture and law. organisations and institutions received frequently signals about an increasing and region-related consumption of addictive drugs and risky behaviour of adolescents. as a reaction 11 institutions from 4 regions of the emr started a cross-border cooperation project 'risky behaviour adolescents in the emr'. the partners intend to improve the efficiency of prevention programmes by investigating the prevalence and pre-conditional aspects related to risky behaviour, and creating conditions for best-practice-public-health. the project included two phases: study. two cross-border (epidemiological) studies where realized: a quantitative study of the prevalence of risky behaviour (46000 pupils) and a qualitative study mapped preconditional aspects of risky behaviour and possibilities to preventive programmes. implementation. this served bringing about recommendations on policy level as well as on prevention level. during this phase the planning and realisation of cross-border preventionprogrammes and activities started. there is region-related variance of prevalence in risky-behaviour of adolescents in de emr. also there are essential differences in legislation and regulation, (tolerated) policy, prevention structures, political and organizational priorities and social acceptance toward stimulants. cross-border studies and cooperation between institutions have resulted in best-practice-projects in (border) areas of the emr. abstract background: beta-blockers increase bone strenght in mice and may reduce fracture risk in humans. therefore, we hypothesized that inhaled beta-2 agonists may increase risk of hip fracture. objective: to determine the association between daily dose of beta-2 agonist use and risk of hip fractures. methods: a case-control study was conducted among adults who were enrolled in the dutch phar-mo database (n = 950,000). cases (n = 6,763) were patients with a first hip fracture. the date of the fracture was the index date. four controls were matched by age, gender and region. we adjusted our analyses for 10 indicators of asthma/copd severity, and for disease and drug history. results: low daily doses (dds) (<400 ug albuterol eq.) of beta2-agonists (crude or 1.2, 95% ci 0.8-1.8) did not increase risk of hip fracture, in contrast to high dds (>1600 ug albuterol eq., crude or 2.0, 95% ci 0.1.5-2.7). after extensive adjustment for indicators of the severity of the underlying disease, (including corticosteroid intake), fracture risk in the high dd group decreased to 1.5 (95% ci 1.1-2.1). conclusions: high dds of beta-2 agonists are linked to increased risk of hip fracture. extensive adjustments for the severity of the underlying disease is important when evaluating this association. abstract salivary nitrate arises from ingested nitrate and is the main source of gastric nitrite, a precursor of carcinogenic n-nitroso compounds. we examinated the nitrate and nitrite levels in saliva of children who used private wells for their drinking water supply. saliva was collected in the morning, from 150 children aged 7-16 years. control group (n = 50) drank water containing 0.03-15.5 mg/l (milligrams/litre) nitrate. exposure groups consisting of subjects (n = 50) who used private wells with nitrate levels in drinking water below 50 mg/l (mean ± standard deviation 20.05 ± 11.42 mg/l) and above 50 mg/l (n = 50) (141.32±80.56 mg/l) respectively. the nitrate and nitrite of saliva samples was determined by high performance liquid chromatographs method. the values of nitrate in saliva samples from exposed groups ranged between 4.57 to 25.94 mg/l (15.33±8.88 mg/l). for control groups, the levels of 0.89 to 14.57 mg/l (7.18±4.54 mg/l) were registered. no differences between levels of salivary nitrite from control and exposed groups were found. regression analysis on water nitrate concentrations and salivary nitrate showed significant correlations. in conclusion, we estimate that salivary nitrate may be used as biomarkers of human exposure to nitrate. abstract disinfection of public drinking water supplies produces trihalomethanes. epidemiological studies have associated chlorinated disinfection by-products with cancer, reproductive and developmental effects. we studied the levels of trihalomethanes (chloroform, dibromochloromethane, bromodichloromethane, bromoform) in drinking water delivered to the population living in some urban areas (n=20). the water samples (n=150) were analysed using gas chromatographic method. assessment of exposure to trihalomethanes in tap water has been on monitoring data collected over 2-12 months periods and that we averaged over entire water system. analytical data revealed that total trihalomethanes levels were higher in the summer: mean ± standard deviation 72.07±42.88lg/l (micrograms/litre). these organic compounds were present in the end of distribution networks (9.87±5.87lg/l). it is noted that, sometimes, we found high concentrations of chloroform exceeding the sanitary norm (100lg/l) in tap water (maximum value 41.65lg/l). results of sampling programs showed stronger correlations between chlorine and trihalomethanes value (correlation coefficient r = 0.821 to 0.952, credible 95%interval). in conclusion, the population drank water with the law concentration of trihalomethanes, especially chloroform. abstract objective: to determine the validity of a performance-based assessment of knee function, dynaport[rsymbol] kneetest (dpkt), in first-time consulters with non-traumatic knee complaints in general practice. methods: patients consulting for nontraumatic knee pain in general practice aged 18 years and older were enrolled in the study. at baseline and 6-months follow-up knee function was assessed by questionnaires and the dpkt; a physical examination was also performed at baseline. hypothesis testing assessed cross-sectional and longitudinal validity of the dpkt. results: a total of 87 patients were included for dpkt of which 86 were available for analysis. the studied population included 44 women (51.2%), median age was 54 (range 18-81) years. at follow-up, 77 patients (89.5%) were available for dpkt. only 3 out of 11 (27%) predetermined hypotheses concerning cross-sectional and longitudinal validity were confirmed. comparison of the general practice and secondary care population showed a major difference in baseline characteristics, dynaport knee score, internal consistency and hypotheses confirmation concerning the construct validity. conclusion: the validity of the dpkt could not be demonstrated for first-time consulters with non-traumatic knee complaints in general practice. measurement instruments developed and validated in secondary care are not automatically also valid in primary care setting. abstract although animal studies have described the protective effects of dietary factors supplemented before radiation exposure, little is known about the lifestyle effects after radiation exposure on radiation damage and cancer risks in human. the purpose of this study is to clarify whether lifestyle can modify the effects of radiation exposure on cancer risk. a cohort of 40,000 japanese atomic-bomb survivors, for whom radiation dose estimates were currently available, had their lifestyle assessed in 1980. they were followed during 20 years for cancer incidence. the combined effect of smoking, drinking, diet and radiation exposure on cancer risk was examined in additive and multiplicative models. combined effects of a diet rich in fruit and vegetables and ionizing radiation exposure resulted in a lower cancer risk as compared to those with a diet poor in fruit and vegetables and exposed to radiation. similarly, those exposed to radiation and who never drink alcohol or never smoke tobacco presented a lower oesophagus cancer risk than those exposed to radiation and who currently drink alcohol or smoke tobacco. there was no evidence to reject either the additive or the multiplicative model. a healthy lifestyle seems beneficial to persons exposed to radiation in reducing their cancer risks. abstract background: clinical trials have shown significant reduction in major adverse cardiac events (mace) following implantation of sirolimus-eluting (ses) vs. bare-metal stents (bms) for coronary artery disease (cad). objective: to evaluate long-term clinical outcomes and economic implications of ses vs. bms in usual care. methods: in this prospective intervention study, cad patients were treated with bms or ses (sequential control design). standardized patient and physician questionnaires 3, 6, and 12 months following implantation documented mace, disease-related costs and patient quality of life (qol). results: 602 patients treated with ses (mean age 63±9, 87% male), 295 with bms (mean age 65±10, 79% male). there were no significant baseline differences in cardiovascular risk factors and severity of cad. after 12 months, 18% ses vs. 30% bms patients had suffered mace (p<0.05). initial hospital costs were higher with ses than with bms, but respective 12month follow-up direct and indirect costs were lower (5,052±642 vs. 6,052±590 euro and 753±459 vs. 2,013±422 euro, p = ns). overall, disease-related costs were similar in both groups (ses 11, 765±827, bms 11, 826±760, p = ns) . differences in qol were not significant. conclusions: as in clinical trials, ses patients experienced significantly fewer mace than bms patients during 12-month follow-up with similar overall costs and qol. abstract background: meta-analyses that use individual patient data (ipd-ma) rather than published data have been proposed as an improvement in subgroup-analyses. objective: to study 1) whether and how often ipd-ma are used to perform subgroup-analyses 2) whether the methodology used for subgroup-analyses differs between ipd-ma and meta-analyses of published data (map) methods: ipd-ma were identified in pubmed. related article search was used to identify map on the same objective. metaanalyses not performing subgroup-analysis were excluded from further analyses. differences between ipd-ma and map were analysed, reasons for discrepancies were described. we recently developed a simple diagnostic rule (including 7 history and physical findings plus d-dimer assay results) to safely exclude the presence of deep vein thrombosis (dvt) without the need for referral in primary care patients suspected of dvt. when applied to new patients, the performance of any (diagnostic or prognostic) prediction rule tends to be lower than expected based on the original study results. therefore, rules need to be tested on their generalizability. the aim was to determine the generalizability of the rule. in this cross-sectional study, 532 primary care patients with suspicion of dvt were prospectively identified. the 8 rule items were obtained from each patient plus ultrasonography as reference standard. the accuracy of the rule was quantified on its discriminative performance, sensitivity, specificity, negative predictive value, and negative likelihood ratio, with accompanying 95% confidence interval. dvt could be safely excluded in 21% (23% in the original study) of the patients, without referral. none of these patients had dvt (0.7% in the derivation population). in conclusion, the rule appears to be a safe diagnostic tool for excluding dvt in patients suspected of dvt in primary care. abstract background: long-term exposure to very low concentrations of asbestos in the environment and relation to incidence of mesothelioma contributes to insight into the dose-response relationship and public health policy. aim: to describe regional differences in the occurrence mesothelioma in the netherlands in relation to the occurrence in the asbestos polluted area around goor and to determine whether the increased incidence of pleural mesothelioma among women in this area could be attributed to environmental exposure to asbestos. methods: mesothelioma cases were selected in the period 1989-2002 from the netherlands cancer register (n = 4781). for the women in the region goor (n = 30) exposure to asbestos due to occupation, household or environment was verified from the medical files, the general practitioner and next-of-kin for cases. results: in goor the incidence of pleural mesothelioma among women was 5-fold increased compared with the netherlands and among men 2fold. of the additional 19 cases among women, 11 cases were attributed to the environmental asbestos pollution and in 4 cases this was the most likely cause. the average cumulative asbestos exposure was estimated at 0.1 fiber-years. . temporal trends and gender differences were investigated by random slope analysis. variance was expressed using median odds ratio (mor). results: ohcs appeared to be more relevant than administrative areas for understanding physicians' propensity to follow prescription guidelines (mor_ohc = 2.7 and mor_aa = 1.5). conclusion and discussion: as expected, the intervention increased prevalence and decreased variance, but at the end of the observation period practice variation remained high. these results may reflect inefficient therapeutic traditions, and suggest that more intensive interventions may be necessary to promote rational statin prescription. abstract background: mortality rates in ska˚ne, sweden have decreased in recent years. if this decline has been similar for different geographical areas have not been examined closely. objectives: we wanted to illustrate trends and geographical inequities in all cause mortality between the 33 municipalities in ska˚ne, sweden from 1970 to 2000. we also aimed to explore the application of multilevel regression analysis (mlra) in our study, since it is a relatively new methodology when describing mortality rates. design and methods: we used linear mlra with years at the first level and municipalities at the second to model direct age-standardized rates. temporal trends were examined by random slope analysis. variance across time was expressed using intra-class correlation (icc). results: the municipality level was very relevant for understanding temporal differences in mortality rates (icc = 26 %). in average, mortality decreased by 34/10 ù 4 along the study period but this trend varied considerably between municipalities, geographical inequalities along the years were u-shaped with lowest variance in the 80s (var = 26). conclusion: mortality has decreased in ska˚ne but municipality differences are increasing again. mlra is a useful technique for modelling mortality trends and variation among geographical areas. abstract background: ozone has adverse health effects but it is not clear who is most susceptible. objective: identification of individuals with increased ozone susceptibility. methods: daily visits for lower respiratory symptoms (lrs) in 6 general practitioner (gp) offices in the north of the netherlands (1989-2000, 38000 patients) were related to daily ozone levels in summer. ozone effects were estimated for patients with asthma, copd, atopic dermatitis, and cardiovascular diseases (cvd) and compared to effects in patients without these diseases. generalized additive models adjusting for trend, weekday, temperature, and pollen counts were used. results: the mean daily number of lrs-visits in summer in the gp-offices varied from 1.3 to 8.5. mean (sd) 8-hour maximum ozone level was 62.6 (30.5) lg/m3. rrs (95% ci) for a 90lg/m3 increase (from 5th to 95th percentile) in the mean of lag 0 to 4 of ozone for patients with/ without disease are: abstract asthma is a costly health condition, its economic effect is greater than that estimated for aids and tuberculosis together. following global initiative for asthma recommendations that require more data about the burden of asthma, we have determined the cost of this illness from 1998-2002. an epidemiological approach based on population studies was made to estimate global as well as direct and indirect costs. data were obtained mainly from the national health ministry database, the national statistics institute of spain and the national health survey. the costs were averaged and adjusted to 2002 e. we have found a global burden (including private medicine) of 920 million e. indirect and direct costs account for 35,4 and 64,6%.the largest components within direct costs were pharmaceutical (29.6%), primary health care systems (8,4%), hospital admissions (5.2%) and hospital non-emergency ambulatory visits (3.2%). within indirect costs, total cessation of work days (19.4%), permanent labour incapacity (8.5%) and early mortality (3.1%) costs were the main components. pharmaceutical cost is the first component as in most studies from developed countries, followed by primary health care systems unlike some reports that consider hospital admissions in second place. finally, direct costs represent 1.3% of the total health care expenditure. abstract background: it is well known that fair phenotypical characteristics are a risk factor for cutaneous melanoma. the aim of our study was to investigate the analogous associations between phenotypical characteristics and uveal melanoma. design/methods: in our casecontrol study we compared incident uveal melamom patients with population controls to evaluate the role of phenotypical characteristics like iris-, hair-and skin color and other risk factors in the pathogenesis of this tumor. a total of 455 patients and 827 controls matched on sex, age and region were interviewed. conditional logistic regression was used to calculate odds ratio (or) and 95% confidence intervals (95% ci). results: risk of uveal melanoma was increased among people with light iris color (or = 1.9 95% ci 1.4-2.7) and light skin color was slightly associated with an increased risk of uveal melanoma (or 1.4 95% 1.1-1.4). hair color, tanning ability, burning tendency and freckles as a child showed no increased risk. results of the combined analysis of eye-and hair color, burning tendency and freckles showed that only light iris color was clearly associated with uveal melanoma risk. conclusion: among potential phenotypical risk factors only light iris-and skin color were identified as risk factor for uveal melanoma. abstract background: between-study variation in estimates of the risk of hcv mother-to-child transmission (mtct) and associated risk factors may be due to methodological differences or changes in population characteristics over time. objective: to investigate the effect of sample size and time on risk factors for mtct of hcv. design and methods: heterogeneity was assessed before pooling data. logistic regression estimated odds ratios for risk factors. results: the three studies included 1633 mother-child pairs born between 1992 and 1998, 346 born between 1986 and 2000, and 1758 between 1999 and 2003 . there was no evidence of heterogeneity of the estimates for maternal hcv/hiv co-infection and mode of delivery (q = 2.16, p = 0.34 and q = 1.42, p = 0.49, respectively). in pooled analysis the proportion of hcv/hiv co-infected mothers significantly decreased from 54% before 1994 to 10% since 2002 (p<0.00001). the pooled adjusted odds ratios for maternal hcv/hiv co-infection and elective caesarean section delivery were 2.80 (95%ci 1.99-3.95), p<0.001 and 1.11 (95%ci 0.79-1.57), p = 0.53 respectively. there was no evidence that the effect of risk factors for mtct changed over time. conclusion: although certain risk factors have become less common, their effect on mtct of hcv has not changed substantially over time. abstract background: the need to gain insight into prevailing eating pattrerns and their health effects is evident. objective: to identify dietary patterns and their relationship with total mortality in dutch older women. methods: principal components analysis on 22 food groups was used to identify dietary patterns among 5,427 women (60-69 y) included in the dutch epic-elderly cohort (follow-up $8.2 y). mortality ratios for three major principle components were assessed using cox proportional hazard analysis. results: the most relevant principal components were a 'mediterranean-like' pattern (high in vegetable oils, pasta/rice, sauces, fish, and wine), a 'traditional dutch dinner' pattern (high in meat, potatoes, vegetables, and alcoholic beverages) and a 'healthy traditional' pattern (high in vegetables, fruit, non-alcoholic drinks, dairy products, and potatoes). in 44,667 person years 277 deaths occurred. independent of age, education, and other life style factors only the 'healthy traditional' pattern score was associated with a lower mortality rate, women in the highest tertile experienced a 30 percent reduced mortality risk. conclusion: from this study a healthy traditional dutch diet, rather than a mediterranean diet, appears beneficial for longevity and feasible for health promotion. this diet is comparable to other reported 'healthy' or 'prudent' diets that have been shown to be protective. parents of 413 (aged 0-4) and 294 (aged 5-15) children were sent a questionnaire, as were 113 adolescents (aged 10-15). to assess validity, generic outcome instruments were included (infant toddler quality of life questionnaire (itqol) or the child health questionnaire (chq) and the euroqol-5d). response rate was 48-53%. internal consistency of hobq and boq-scales was good (cronbach's alpha's >0.7 in all but two scales). test-retest results showed no differences in 70-92% of scales. high correlations between hobq-and boq-scales and conceptually equivalent generic outcome instruments were found. the majority of hobq (7/10) and boq scales (11/12) showed significant differences between children with a long versus short length of stay. the dutch hobq and boq can be used to evaluate functional outcome after burns in children. the study estimated caesarean section rates and odds ratios for caesarean section in association with maternal characteristics in both public and private sectors; and maternal mortality associated with mode of delivery in the public sector, adjusted for hypertension, other disorders, problems and complications, as well as maternal age. results: the caesarean section rate was 32.9% in the public sector, and 80.4% in the private sector. the odd ratio for caesarean section was 2.6 (95%ci: 2.6-2.7) for women with 12 or more years of education. the odd ratio for maternal mortality associated with caesarean section in the public sector was 3.3 (95%ci:2.6-4.3). conclusion and discussion: sao paulo presented high caesarean section rates. caesarean section compared to vaginal delivery in the public sector presented higher risk for mortality even when adjusted for hypertension, other disorders, problems and complications, as well as maternal age. we show that serious bias in questionnaires can be revealed by bland-altman methods but may remain undetected by correlation coefficients. we refute the argument that correlation coefficients properly investigate whether questionnaires rank subjects sufficiently well. conclusions: the commonly used correlation approach can yield misleading conclusions in validation studies. a more frequent and proper use of the bland-altman methods would be desirable to improve epidemiological data quality. abstract screening performance relies on quality and efficiency of protocols and guidelines for screening and follow-up. evidence of low attendance rates, over-screening of young women and low smear specificity gathered by the early 1990's in the dutch cervical cancer screening program called for an improvement. several protocols and guidelines were redefined in 1996, with emphasis on assuring that these would be adhered to. we assessed improvement since 1996 by changes in various indicators: coverage rates, follow-up compliance and number of smears. information on all cervix uteri tests in the netherlands registered until 31st march 2004 was retrieved from the nationwide registry of histo-and cytopathology (palga). five-year coverage rate in the age group 30-60 years rose to 77%. the percentage of screened women in follow-up decreased from 19% to 3%. fourteen percent more women with abnormal smears were followed-up, and the time spent in follow-up decreased. a 20% decrease in the annual number of smears made was observed, especially among young women. in conclusion, the 1996 changes in protocols and guidelines, and their implementation have increased coverage and efficiency of screening, and decreased the screening-induced negative side effects. similar measures can be used to improve other mass screening programmes. abstract background: it is common knowledge that in low endemic countries the main transmission route of hepatitis b infection is sexual contact, while in high endemic regions it is perinatal transmission and horizontal household transmission in early childhood. objectives: to get insight into what determines the main transmission route in different regions. design and methods: we used a formula for the basic reproduction number r0 for hepatitis b in a population stratified by age and sexual activity to investigate under which conditions r0 > 1. using data extracted from the literature we investigated how r0 depends on fertility rates, rates of horizontal childhood transmission and sexual partner change rates. results: we identified conditions on the mean offspring number and the transmission probabilities for which perinatal and horizontal childhood transmission alone ensures that r0 > 1. those transmission routes are then dominant, because of the high probability for children to become chronic carriers. sexual transmission dominates if fertility is too low to be the driving force of transmission. conclusion: in regions with high fertility rates hepatitis b can establish itself on a high level of prevalence driven by perinatal and horizontal childhood transmission. therefore, demographic changes can influence hepatitis b transmission routes. abstract background: the artificial oestrogen diethylstilboestrol is known to be fetotoxic. thus, intrauterine exposure to other artificial sex hormones may increase the risk of fetal death. objective: to study if use of oral contraceptive 4 months prior to or during pregnancy is associated to an increased risk of fetal death. design and methods: a cohort study of 92 719 pregnant women who were recruited into the danish national birth cohort during the years 1996-2002 and interviewed about exposures during pregnancy, either during the first part of their pregnancy (n = 90 167) or following a fetal loss (n = 2552). cox regression analyses with delayed entry were used to estimate the risk of fetal death. results: in total 1102 (1.2%) women took oral contraceptives during pregnancy. use of combined oestrogen and progesterone oral contraceptives (coc) or progesterone only oral contraceptives (poc) during pregnancy were not associated with increased hazard ratios of fetal death compared to non-users, hr 1.01 (95% ci 0.71-1.45) and hr 1.37 (95% ci 0.65-2.89) respectively. neither use of coc nor poc prior to pregnancy was associated with fetal death. conclusion: use of oral contraceptive 4 months prior to conception or during pregnancy is not related to an increased risk of fetal death. abstract background: few studies have been performed to assess if water fluoridation reduces social inequalities among groups of different socioeconomic status, and none of them was conducted in developing countries. objectives: to assess socioeconomic differences between brazilians towns with and without water fluoridation, and to compare dental caries indices among socioeconomic strata in fluoridated and non-fluoridated areas. design and methods: a countrywide survey of oral health performed in 2002-3 and comprising 34,550 children aged 12 years provided information about dental caries indices in 249 brazilian towns. socioeconomic indices, the coverage and the fluoride status of the water supply network of participating towns were also appraised. multivariate regression models were performed. inequalities in dental outcomes were compared in towns with and without fluoridated tap water. results: better-off towns tended to present a higher coverage by the water supply network, and were more inclined to add fluoride. fluoridated tap water was associated with an overall improved profile of caries, concurrent with an expressively larger inequality in the distribution of dental disease. conclusion: suppressing inequalities in the distribution of dental caries requires an expanded access to fluoridated tap water; a strategy that can be effective to foster further reductions in caries indices. objective: to investigate the role of family socioeconomic trajectories from childhood to adolescence on dental caries and associated behavioural factors. design and methods: a population-based birth cohort was carried out in pelotas, brazil. a sample (n=888) of the population of subjects born in 1982 were dentally examined and interviewed at aged 15. dental caries index, care index, toothbrushing, flossing, and pattern of utilization of dental services were the outcomes. these measures were compared among four different family income trajectories. results: adolescents who were always poor showed, in general, a worse dental caries profile, whilst adolescents who never were poor had a better dental caries profile. adolescents who had moved from poverty in childhood to nonpoverty in adolescence and those who had moved from non-poverty in childhood to poverty in adolescence had similar dental profiles to those who were always poor except for pattern of utilization of dental services which was higher in the first group. conclusion: poverty in at least one stage of the lifespan has a harmful effect on dental caries, oral behaviours and utilization of dental services. we assessed contextual and individual determinants of dental caries in the brazilian context. a country-wide survey of oral health informed the dental status of 34,550 twelve-year-old schoolchildren living in 250 towns in 2003. a multilevel model fitted the adjustment of untreated caries prevalence to individual (socio-demographic characteristics of examined children) and contextual (geographic characteristics of participating towns) covariates. being black (or = 1.6; 95% ci: 1.5-1.7), living in rural areas (or = 1.9; 1.7-2.0) and studying in public schools (or = 1.7; 1.6-1.9) increased the odds of having untreated decayed teeth. the multilevel model identified the fluoride status of water supplies (ß=)0.3), the proportion of households linked to the water network (ß=)0.3) and the human development index (ß=)0.2) as town-level covariates of caries experience. better-off brazilian regions presented an improved profile of dental health, besides having a less unequal distribution of dental treatment needs between blacks and whites, rural and urban areas, and public and private schools. dental caries experience is prone to socio-demographic and geographic inequalities. monitoring contrasts in dental health outcomes is relevant for programming socially appropriate interventions aimed both at overall improvements and at the targeting of resources for groups of population presenting higher levels of needs. abstract background: ultraviolet radiation (uvr) is the main cause of nonmelanoma skin cancer but has been hypothesised to protect against development of prostate cancer (pc). if this is true, skin cancer patients should have lower pc incidence than the general population. objectives: to study the incidence of pc after a diagnosis of skin cancer. design methods: using the eindhoven cancer registry, a cohort of male skin cancer patients diagnosed since 1970 (2565 squamous cell carcinoma (scc), 9295 basal cell carcinoma (bcc) and 1419 melanoma (cm)) was followed up for incidence of invasive pc. observed incidence rates of pc amongst skin cancer patients were compared to those in the reference population, resulting in standardised incidence ratios (sir). results: scc (sir 0.88 (95%ci: 0.64; 1.2)) and bcc (sir 0.79 (95%ci: 0.65;0.94)) showed a decreased incidence of pc, cm did not. patients with bccs occurring in the chronically sun-exposed head and neck area (sir 0.79 (95%ci: 0.64; 0.97) had significantly lower pc incidence rates. conclusion discussion: although numbers of scc and cm were too small to obtain unequivocal results, this study partly supports the hypothesis that uvr protects against pc and also illustrate that cm patients are different from nmsc patients in several aspects. abstract introduction: hypo-and hyperthyroidism have been associated to various symptoms and metabolic dysfunctions in men and women. incidences of these diseases have been estimated in a cohort of middle-aged adults in france. methods: the su.vi.max (sup-ple´mentation en vitamines et mine´raux antioxydants) cohort study included 12741 volunteers followed-up for eight years since 1994-1995. the incidence of hypo-and hyperthyroidism was estimated retrospectively from scheduled questionnaires and the data transmitted by the subjects during their follow-up. factors associated to incident cases have been identified by cox proportional hazards models. results: among the 5166 subjects free of thyroid dysfunction at inclusion, 95 incident cases were identified. after an average follow-up of 7.5 years, the incidence of hyper-and hypothyroidism was 0.5% in men, 2.3% in 35-44 year old women, and 3.6% in 45-60 year old women. no associated factor was identified in men. in women, age and alcohol consumption (>15 grams/day) increased the risk of hypo-or hyperthyroidism, while a high urinary thiocyanate level in 1994-1995 would be a protective factor. conclusion: the incidences of hypo and hyperthyroidism observed in our study as well as the associated risk factors found are in agreement with the data of studies performed in other countries. abstract background: lung cancer is the most frequent malignant neoplasm world-wide. in 2000, the number of new lung cancer cases was estimated at 1.2 million, which makes over 12% of all new cases of neoplasm registered all round the globe. it is also the leading cause of cancer deaths. objective: the objective of this paper is to provide a systematic review of life-related factors for lung cancer risk. methods: data sources were medline from january 1950 to december 2005, title in the field. search terms included: lung cancer, tobacco smoke, education, diet, alcohol consumption or physical activity terms. book chapters, monographs, relevant news reports, and web material were also reviewed to find articles. results: the results of the literature review suggest that smoking is a major, unquestionable factor of lung cancer risk. exposure to environmental tobacco smoke (ets) and education could also play a role in the occurrence of the disease. diet, alcohol consumption and physical activity level are other important but less extended determinants of lung cancer. conclusions: effective prevention programs against some of the life style-related factors for lung cancer, especially against smoking must be developed to minimize potential health risks and prevent the future cost of health. stedendriehoek twente and south (n = 179), additional data (co-morbidity, complications after surgery and follow-up) were gathered. cox-regression analyses were used. results: the proportion resections declined from 80% of patients <60 to 30% of patients aged > = 80 years, whereas primary radiotherapy increased from 7% to 36%. in the two regions 25 patients (14%) underwent resection. co-morbid conditions did not influence the choice of the therapy. 75% had complications. postoperative mortality was 20%. in multivariate analysis, only treatment had an independent effect.two year survival was 58% for patients undergoing surgical resection and 27% for those receiving radiotherapy (p<0.05). conclusion: number of co-morbid conditions did not influence choice of treatment, postoperative complications, and survival in patients with nsclc > = 80 years. the epidemiology of oesophageal cancer has changed in recent decades. the incidence has increased sharply, mainly comprising men, adenocarcinoma and tumours of the lower third of the oesophagus. the eurocare study suggested large variation in survival between european countries, primarily related to early mortality. to study potential explanations, we compared data from the rotterdam and thames cancer registry. computer records from 18,320 patients diagnosed with oesophageal cancer in the period 1993-2003 were analysed by age, gender, histological type, tumour subsite, period and region. there was a large variation in resection rates between the two regions, 31% for rotterdam versus 14% for thames (p<0.001). resection rates were higher for men, younger patients, adenocarcinoma and distal tumours. postoperative mortality (pom) was defined as death within 30 days of surgery and was 7.4% on average. pom increased with age from 3.3% for patients younger than 60 years to 12.6% for patients older than 70 years. pom was significantly lower in high-volume hospitals (>20 operations per year), 8.7% versus 2.4% (p<0.001). this study shows a large variation in treatment practice between the netherlands and the united kingdom. potential explanations will need to be studied in detail. abstract russia has experienced tremendous decline in life expectancy after break up of the ussr. surprisingly, im has also been decreasing. less is known on the structure of im in different regions of russia. the official im data may be underestimated partly due to misreporting early neonatal deaths (end) as stillbirths (sb). end/sb ratio considerably exceeding 1:1 indicates misreporting. we present the trends and structure of im in arkhangelsk oblast (ao), north-west russia from 1980 to 2004 as obtained from the regional statistical committee. im decreased from 22.3 to 10.1 per 1000 live births. cause-specific death rates (per 100,000) decreased from 251 to 7.0 for infectious diseases, from 745 to 56 for respiratory causes, from 141 to 56 for traumas, from 384 to 285 for inborn abnormalities but did not change for conditions of the perinatal period (501 in both 1980 and 2004) . the end/sb ratio increased from 1 to 1.5. in 2004, im from infections and respiratory causes in the ao are much lower than in russia in general. the degree of misreporting end as sb in the ao is lower than in russia in general. other potential sources of underestimation of im in russia will be discussed. abstract background: epidemiological studies that investigated malocclusion and different physical aspects in adolescents are rare in the literature. objective: we studied the impact of malocclusion on adolescents' self-image regardless of other physical aspects. design and methods: a cross-sectional study nested in a cohort study was carried out, in pelotas, brazil. a random sample of 900 15 yearsold adolescents was selected. the world health organization (1987) criteria were used to define malocclusion. interviews about self-reported skin colour and appearance satisfaction were administered. the body mass index was calculated. gender, birth weight and socioeconomic characteristics were obtained from the birth phase of the cohort study. poisson regression models were performed. results: the prevalence of moderate or severe malocclusion was 31.6% [95%ci 28.5; 34.7] in the whole sample without significant difference between boys and girls. a higher statistically significant difference of appearance dissatisfaction was identified in girls (46.5%) than in boys (29.8%). a positive association between malocclusion and appearance dissatisfaction was observed only in girls, after adjusting for other physical and socioeconomic characteristics. conclusions: malocclusion influenced appearance dissatisfaction only in young women. abstract background: factors for healthy aging with good functional capacity and those which increase the risk of death and disability need to be identified. objectives: we studied the prevalence of low functional capacity and its associations in a small city in southern brazil. design and methods: a population based cross sectional study was carried out with a random sample size of 345 elderly people. a home-applied questionnaire including socioeconomic, demographic, house conditions, socioeconomic self-perception characteristics was applied. the low functional capacity was defined as the difficulty in the performance of 6 or more activities or inability to carry out 3 of those activities according to scale proposed by rikli and jones. descriptive statistics, association using chi-square test as well as the multiple logistic regression analysis were performed. abstract introduction: assessment of trichiuriasis spatial distribution is important to evaluate sanitation conditions. our objective was to identify risk areas for the trichuris trichiura infection. methods: cross sectional study was held in 19 census tracts of duque de caxias county, rio de janeiro, brazil. collection and analysis of fecal specimens and a standardize questionnaire were carry out in order to evaluate socio-economic and sanitation conditions in a sample of 1,546 children between 1 and 9 years old. geoestatistics techniques were used to identify risk areas for trichiuriasis. results: the mean age of the studied population was 4.4 years old, which 52% were females and 48% were males. the prevalence of trichuris trichiura in the sample was 17%. children whose mothers studied for 4 years or less had odds ratio (or) = 1.9 than children whose mothers studied for more than 4 years old. children who were living in houses without water supply had or = 2.6 comparing to children living in houses with water supply. the spatial analysis identified risk areas for infection. conclusion: the results show association between socio-economic conditions and the proliferation of trichuris trichiura infection. the identification of risk areas can guide efficient actions to combat the disease. abstract background: refuge life and diabetes mellitus can affect the healthrelated quality of life (hrqol). objective: to assess how both aspects influence hrqol of the diabetic refugees in gaza strip. methods: overall 591 subjects filled a self-administered questionnaire including world health organization quality of life questionnaire (whoqol-bref) and some socio-demographic information. the sample consisted of three frequency matched groups for gender and sex, 197 each. first group were refugees with diabetes mellitus, second refugees without diabetes and third diabetes patients with no refugee history. the response rate was 87% on average. global score consisting of all four domains of who-qol-bref was dichotomized by the value of 50 and logistic regression was used for the analysis. results: crude odds ratios (or) for lower quality of life were 17.2 (95% ci 10.4-28.3) for diabetes refugees compared to diabetes non-refugees and 19.4 (11.7-32. 2) compared to non-diabetes refugees. after adjusting for age, gender, education, employment, income status and number of persons depending on the respondents or was 11.2 (6.1-20.5) and 35.3 (17.7-70.4), respectively. additionally, adjusting for length of diabetes and complications reduced the or to 5.6 (2.4-13.4) for diabetes refugees compared to diabetes non-refugees. conclusion: quality of life is highly reduced in refugees with diabetes. abstract background: pesticides have a significant public health benefit by increasing food production productivity and decreasing diseases. on the other hand, public concern has been raised about the potential health effects of the exposure to pesticides on the developing fetus and child. objectives: to review the available literature to find an epidemiological studies dealing with the exposure to pesticides and children health. design and methods: epidemiological studies were identified during search of the literature basis. following health effects were taking into account: adverse reproductive and developmental disorders, childhood cancer, neurodevelopmental effects and the role of pesticides as endocrine disrupters. results: pesticides were associated with wide range of reproductive disorders. the association between exposure to pesticides and the risk of childhood cancer and neurodevelopmental effects was found in several studies. epidemiological studies have been limited by luck of specific pesticide exposure, exposure based on job title, small size of examined groups. conclusions: in the light of existing although still limited evidence of adverse effects of pesticide exposure it is necessary to reduce the exposure. the literature review suggests a great need to increase awareness of people who are occupationally or environmentally exposed to pesticides about its potential negative influence on their children. in order to match local health policy more with the needs of citizens, the municipal health service utrecht started the project 'demand-orientated prevention policy'. one of the aims was to explore the needs of the utrecht citizens. the local health survey from 2003 contained questions about needs of information and support with regard to disorders and lifestyle. do these questions about needs give other results compared to questions about prevalence of health problems? in total 2284 utrecht citizens aged 16 to 54 years returned the health questionnaire (response rate 55%). most needs were observed on subjects concerning overweight and mental problems, and were higher among women, moroccans, turks, low educated people and citizens of deprived areas. the prevalence of disorders and unhealthy lifestyles did not correlate well to the needs (majority correlation coefficients: <0,30). most striking, of the utrecht population 30% were smokers and 20% excessive alcohol drinkers, while needs related to these topics were low. furthermore, higher needs among specific groups did not always correspond to higher prevalences of related health problems in these groups. these results show the importance of including questions about needs in a health survey, because they add additional information to questions about prevalences. abstract background: recent studies associated statin therapy with better outcome in patients with pneumonia. because of an increased risk of pneumonia in patients with diabetes we aimed to assess the effects of statin use on pneumonia occurrence in diabetic patients managed in primary care. methods: we performed a case-control study nested in 142,174 patients with diabetes. cases were defined as patients with a diagnosis of pneumonia. for each case, up to 4 controls were matched by age, gender, practice, and index date. patients were classified as current statin user when the index date was between the start and end date of statin therapy. results: statins were currently used in 1.1 % of 4,719 cases and in 2.1% of 15,322 controls (crude or: 0.51, 95% ci 0.37-0.68). after adjusting for potential confounders, statin therapy was associated with a 51% reduction in pneumonia risk (adjusted or: 0.49, 95% ci 0.35-0.68). the association was consistent among relevant subgroups (stroke, heart failure, and pulmonary diseases) and independent of age or use of other prescription drugs. conclusions: use of statins was significantly associated with reduced pneumonia risk in diabetic patients and may apart from lipid lowering properties be useful in prevention of respiratory infections. abstract introduction: cigarette smoking is the most important risk factor for copd development. therefore, smoking cessation is the best preventive measure. aim: to determine the beneficial effect of smoking cessation on copd development. methods: incidence of copd (gold stage > = 1) was studied in smokers without copd who quitted or continued smoking during 25 yr of followup. we performed logistic regression analyses on pairs of observations. correlations within a subject and time, and time between 2 successive surveys were taken into account. abstract objectives: to describe the prevalence and severity of dental caries in adolescents of the city of porto, portugal, and to assess socioeconomic and behavioral covariates of dental caries experience. methods: a sample of 700 thirteen-year-old schoolchildren underwent dental examination. results from the dental examination were linked to anthropometric information and to data supplied by two structured questionnaires assessing nutritional factors, sociodemographic characteristics and behaviors related to health promotion. dental caries was appraised in terms of the dmft index, and two dichotomous outcomes, one assessing the prevalence of dental caries (dmft = 1); the other assessing the prevalence of a high level of dental caries (dmft = 4). results: consuming soft drinks derived from cola two or more times per week, attending a public school, being girl and having parents with low educational attainment were identified as risk factors both for having dental caries and for having a high level of dental caries. conclusion: the improvement of oral health status in the portuguese context demands the implementation of polices to reduce the frequency of sugar intake, and could benefit from an overall and longstanding expansion of education in society. abstract background: migrant mortality does not conform to a single pattern of convergence towards rates in the host population. to better understand how migrant mortality will develop, there is a need to further investigate how the underlying behavioural determinants change following migration. objective: we studied whether behavioural risk factors among two generations of migrants converge towards the behaviour in the host population. design and methods: cross-sectional interview-data were used including 299 moroccan and 476 turkish migrants, aged 15-30. questions were asked about smoking, alcohol consumption, physical inactivity and weight/height. age-adjusted prevalence rates among first and second generation migrants were compared with prevalence rates in the host population. results: converging trends were found for smoking, physical inactivity and overweight. for example, we found a higher prevalence of physical inactivity in first generation turkish women as compared to ethnic dutch (or = 1.78(1.22-2.62)), whereas among second generation no differences were found (or = 0.82(0.57-1.17)). however, this trend was not found in all subgroups. additionally, alcohol consumption remained low in all subgroups and did not converge. conclusion and discussion: behavioural risk factors in two generations of migrants seem to converge towards the prevalence rates in the host population. although, some groups and risk factors showed a deviant pattern. abstract background/relevance: arm-neck-shoulder complaints are common in general practice. for referral in these complaints, only guidelines exist for shoulder complaints and epicondylitis. besides, other factors can be important. objective: what factors are associated with referral to physiotherapy or specialist in non-traumatic armneck-shoulder complaints in general practice, during the first consultation? design/methods: 31 general practitioners (gps) recruited consulters with new arm, neck or shoulder complaints. data on complaint-, patient-, gp-characteristics and management were collected. the diagnosis was categorised into: shoulder specific, epicondylitis, other specific or non-specific. multilevel analyses (adjustment for treating gp) were executed in procgenmod to assess associated variables (p<0.05). results: during the first consultation, 23% was referred for physiotherapy and 5% for specialist care. indicators of reference to physiotherapy were: long duration of complaint, recurrent complaint and gp located in a little/not urbanised area. while having shoulder specific or other specific diagnoses was negatively associated. indicators of reference to specialist care were: having other specific diagnosis, long duration of complaint, musculoskeletal co-morbidity, functional limitations and consulting a less experienced gp. conclusion/discussion: most referrals were to physiotherapy and only a minority to specialist care. mainly diagnosis and other complaint variables indicate on 'who goes where'. besides gp-characteristics can play a role. abstract background: the ruhr area has for 170 years been a synonym for a me´gapolis of heavy industry with a high population density. presently, 40% of the population of the state of north rhine-westphalia live there, i.e. more than five million people. objectives: for the first time, social and health indicators of nrw's health indicator set were brought together for this me´gapolis area. design and methods: new standard tables were constructed for the central area of 'ruhr-city' including seven cities with more than 2000 inhabitants/km2 and the peripheral zone with eight districts and cities. for the pilot phase, four socio-demographic and four health indicators were recalculated. comparability of the figures was achieved by age standardization. the results obtained were submitted to a significance test by identifying 95% confidence intervals. results: the centre of 'ruhr-city' is characterised by elderly, unemployed, foreign, low-income citizens living closely together. infant mortality lies above nrw's average, male life expectancy is 1.34 years lower and female life expectancy 0.90 years lower than life expectancy in nrw (without 'ruhr-city'). several avoidable deaths' rate in the ruhr area are significantly higher than the average in nrw. specific intervention strategies are required to improve the health status in 'ruhr-city'. abstract background: general practitioners (gps) have a fundamental role to play in tobacco control, since they reach a high percentage of the target population. objectives: to evaluate specific strategies to enhance promotion of smoking cessation in general practice. design and methods in a cluster-randomized trial, 82 medical practices were randomized following a 2·2 factorial design. 577 patients aged 36-75 years who smoked at least 10 cigarettes per day (irrespective of their intention to stop smoking) were recruited. the intervention included (ti) the provision of a two-hour physician group training in smoking cessation methods plus direct physician payments for every participant not smoking 12 months after recruitment; and (tm) provision of the same training plus direct participant reimbursements for pharmacy costs associated with nicotine replacement therapy or bupropion treatment. results: in the mixed logistic regression model, no effect was identified for intervention ti (odds ratio (or) = 1.26, 95% confidence interval (ci) 0.65-2.43), but intervention tm strongly increased the odds of cessation (or = 4.77, 95% ci 2.03-11.22). conclusion and discussion: the cost-free provision of effective medication along with improved training opportunities for gps may be an effective measure to enhance smoking cessation promotion in general practice. in europe, little research on international comparison of health surveys has been accomplished, despite a growing interest in this field. smoking prevalence is chosen to explore data comparability. we aim to illustrate methodological problems encountered when comparing data from health surveys and investigate international variations in smoking behaviour. we examined a sample 89.754 individuals aged 16 and more, from six european health surveys performed in 2000-2002. problems met during the comparisons are described. we took the example of current smoking as an indicator allowing a valid comparison of the prevalences. the differences in age and sex distribution between countries were adjusted through direct standardisation. additionally, multivariate analysis will assess variations in current smoking between countries, when controlling for sex, age, and educational level. methodological problems concern comparability of socioeconomic variables. the percentage of current smokers varies from 29% to 43%. smoking patterns observed by age groups, sexes and educational level are similar, although rates per country differ. further results will determine if the variations in smoking related to socioeconomic status are alike. this international comparison of health surveys highlights methodological problems encountered when comparing data of several countries. furthermore, variations in smoking may call for adaptations in public health programs. from research it appears that adolescent alcohol use in the achterhoek is much higher than in the rest of the netherlands and rapidly increasing. excessive alcohol use has consequences for health and society. parents play an important role in preventing excessive adolescent alcohol use, but are not aware of the problem and consequences. for this reasons the municipalities in the achterhoek launch an alcohol moderation programme, starting with a regional media campaign to increase problem awareness among parents. the objective of this study is to assess the impact of this media campaign in the achterhoek. three successive independent cross-sectional telephone surveys, interviewing approximately 350 respondents each, will be conducted before, during and after the campaign. respondents will be questioned on knowledge and awareness of excessive adolescent alcohol use, its consequences and the role child raising can play. also the reach and appreciation of the different activities of the campaign will be investigated. results: of the surveys before and during the implementation will be known by may 2006. with these first findings the unawareness of the problem among parents and partly the reach and appreciation of the campaign can be assessed. abstract background: obesity is a growing problem, increasingly so in children and adolescents. overweight is partly 'programmed' during pregnancy, but few comprehensive studies looked prospectively into the changes of body composition and metabolic factors from birth. objectives: the aim of the population-based birth-cohort study within gecko is to study the etiology and prognosis of overweight and the metabolic syndrome during childhood. design and methods: the gecko drenthe will be a population based observational birth-cohort study, which includes all children born from april 2006 to april 2007 in drenthe, one of the northern provinces of the netherlands. during the first year of life, the study includes repeated questionnaires, extensive anthropometric measurements and blood measurements at birth (cord blood) and at the age of eleven months. results: the number of babies born in the drenthe province is about 5.500 per year. the results from a feasibility study conducted in february 2006 will be presented. conclusion: gecko drenthe is a unique project that will contribute to the understanding of the development of obesity in childhood and its tracking into adulthood. this will enable early identification of children at risk and opens the way for timely and tailored preventive interventions. abstract background: tunisia is facing an epidemiologic transition with the extension of chronic diseases that share common risk factors. obesity is a leading risk factor and happens to occur frequently in early life. objective: to study the prevalence and the risk factors of obesity and overweight among urban schoolchildren in sousse, tunisia. methods: cross sectional study of a tunisian sample of schoolchildren aged between 13 and 17 years living in the urban area of sousse, tunisia. a representative sample of 1600 school children selected by multistage cluster sampling procedure. measurements: weight and height, blood pressure measured by electronic system, fasting blood lipids. questionnaire assessment was used for family history of cardiovascular disease, smoking habits, physical activity and diet. abstract background: quality of life (qol) measurements are acknowledged as very important in the evaluation of health care. objectives: we studied the validity and the reliability of the hungarian version of the whoqol-bref among people living in small settlements. method: a questionnaire-based cross-sectional study was conducted in a representative sample (n = 814) of persons aged 35 years and over in south-east-hungary, in 2004. data were analysed by the spss 13.0. the internal consistency was evaluated using cronbach's alpha; for comparison of the qol scores amongst the various groups the two-tailed t-tests were used; convergent validity was assessed by spearman coefficients. results: the male:female ratio was 48.3 to 51.7%, and the average age 59.68 (sd:14.37) years. the domain scores were 14.14 (sd:3.08) for the physical, 14.19 (sd:2.42) for psychological, 14.15 (sd:3.03) for the social, and 14.01 (sd:2.08) for the environment domains. the cronbach's alpha values ranged from 0.73 to 0.87 across domains. the whoqol-bref seemed to be suitable to distinguish healthy and unhealthy people. the scores for all domains correlated with the self-evaluated health, and overall quality of life (p<0.01). conclusion: our study supported that the whoqol-bref provided a valid, reasonable and useful determination of the qol of people living in hungarian villages. abstract background: further than a cardiovascular disease, arterial hypertension (aht) is the main cardiovascular risk factor. in spain, the aht prevalence reaches 47%, placed in the third position after germany and finland in affecters percentage. although its high morbi-mortality, the aht is a forecast factor. the treatment's objective (pharmacological and life style modifications) of hypertensive patients is not only to reduce blood pressure levels to optimum levels but also to treat all modifiable vascular risk factors. objective: economic impact evaluation of direct costs due to aht pathology (cie9-mc 401-405) in spain in 2003, according to autonomous region. design and methods: descriptive and transversal study of costs estimation in the period between january to december 2003 in spain according to autonomous region. the study is based on data available from the national health ministry database and the national statistics institute of spain. results: the national health service assigned 2000 million e to aht treatment. 73,4% of the total cost is owe to pharmaceutical service expenses, 23,2% to primary health care and a 3,4% to hospital admissions. conclusion and discussion: the costs generated by aht are mainly due to the pharmaceutical service. the costs distribution is modified according to the geographical region. abstract background: over the last decades, for low-stage cervical cancer less surgical treatment and for high-stage cervical cancer chemoradiotherapy was recommended in the national guidelines. objectives: to describe changes and variation in treatment and survival in cervical cancer in the regions of the comprehensive cancer centre stedendriehoek twente (cccst) and south (cccs) in the netherlands. design and methods. 1736 newly diagnosed cervical cancer cases were selected from both cancer registries in the period 1989-2002. patient characteristics, tumour characteristics, treatment and follow-up data were collected from the medical records. results: in figo stages ia2-ib2 the percentage hysterectomy decreased from 90% in 1989 -1993 to 77% in 1999 -2002 (p<.05) and survival improved comparing 1989 -1993 with 1999 -2002 . figo stages iii-ivb had mostly received radiotherapy only (60%). no differences in survival between years of diagnosis were found. in the cccs-region more chemoradiotherapy was given in these stages (11% versus 5% in the cccst-region in the whole period). conclusion and discussion:. abstract background: the reason for the increased prevalence of depression in type 2 diabetes (dm2) is unknown. objective: we investigated whether depression is associated with metabolic dysregulation or that depression is rather a consequence of having dm2. methods: baseline data of the utrecht health project were used. subjects with cardiovascular disease were excluded. 4,203 subjects (age: 38.0 +/) 12) were classified into four mutually exclusive categories: normal fasting plasma glucose (fpg < 5.6 mmol/l), impaired fpg (> = 5.6 and < 7.0 mmol/l), undiagnosed dm2 (fpg > = 7.0 mmol/l), and diagnosed dm2. depression was defined as either a score of 25 or more on the depression subscale of the symptom check list-90 or use of antidepressants. results: subjects with impaired fasting glucose and undiagnosed dm2 had no increased prevalence of depression. diagnosed dm2 patients had an increased prevalence of depression (or = 2.11 (1.08-4.11)) after adjustment for gender, age, body mass index, smoking, alcohol consumption, physical activity, education level and number of chronic diseases. conclusions: our findings suggest that depression is not related to disturbed glucose homeostasis. the increased risk of depression in diagnosed dm2 only, suggests that depression is rather a consequence of the psychosocial burden of diabetes. abstract background: breast-conserving surgery (bcs) followed by radiotherapy (bcs-rt) is a safe treatment option for the large majority of patients with tumours less than 5 cm. aim: the use of bcs and bcs-rt in pt1 (?2 cm) and pt2-tumours (2-5 cm) was investigated in the netherlands in the period 1990 and 2001. methods: from the netherlands cancer registry patients were selected with invasive pt1 (?2.0 cm) or pt2 (2.1-5.0 cm) tumours, without metastasis at time of diagnosis. trends in the use of bcs and rt after bcs were determined for different age groups and regions. results: in the period 1990-2001 52,937 pt1-tumours and 36,285 pt2-tumours were diagnosed. the %bcs in pt1-tumours increased in all age groups. it remained lowest in patients 80 years and older (32% in 2001). in pt2-tumours a decrease was observed in patients 80 years and older (from 23% to 17% in 2001). in both pt1 and pt2tumours the %bcs-rt increased in patients 80 years and older to respectively 59% and 44%. between regions and hospitals large differences were seen in %bcs and %bcs-rt. conclusion: multidisciplinary treatment planning, based on specific guidelines, and patient education could increase the use of bcs combined with rt in all age groups. abstract this is a follow-up study on the adverse health effects associated with pesticide exposure among cut-flower farmers. survey questionnaires and detailed physical and laboratory examinations were administered to 114 and 102 respondents, respectively, to determine pesticide exposure, work and safety practices, and cholinesterase levels. results showed that pesticide application was the most frequent activity associated with pesticide exposure, and entry was mostly ocular and dermal. majority of those exposed were symptomatic. on physical examination, 90 or 88.2 % of those examined were found to have abnormal peak expiratory flow rate (pefr). eighty-two percent had abnormal temperature, followed by abnormal general survey findings (e.g. cardiorespiratory distress). 51% had cholinesterase levels below the mean value of 0.7 ? ph/hour, and 25.5% exhibited a more than 10% depression in the level of rbc cholinesterase. certain hematological parameters were also abnormal, namely hemoglobin, hematocrit, and eosinophil count. using pearson's r, factors strongly associated with illness due to pesticides include using a contaminated piece of fabric to wipe sweat off (p.=0.01) and reusing pesticide containers to store water (p.=0.01). the greatest adverse effect of those exposed is an abnormal cholinesterase level which confirms earlier studies on the effect of pesticides on the body. objectives: this pair-study was performed to find out the rate of spontaneous abortions in female workers exposed to organic solvents from the wood-processing industry. methods: the level of organic solvents was assessed within the workplaces during a 10year period. 366 exposed female workers from the wood-processing industry were examined. the occupational and non-occupational data associated with their fertility were obtained by applying a standard epidemiological computed questionnaire. the reference group consisted of female workers non-exposed to hypo-fertilizing agents, residing in the same locality. the rate of spontaneous abortions was evaluated in both groups as an epidemiological fertility indicator. results: within the studied period, the organic solvents levels exceeded several time the maximal admissible concentrations in all workplaces. the long-term exposure to organic solvents caused a significant increase in rate of spontaneous abortions compared to the reference group (p<0.05). the majority of abortions (85%) have happened in the first trimester of pregnancy. conclusions: the long-term exposure to organic solvents may cause low fertility on female workers because of the spontaneous abortions. it is advised to reduce the organic solvents level in the air of all workplaces, as well as to stop working the pregnant women in exposure to organic solvents. abstract introduction: rio de janeiro city (rj) presents a fast aging of the population with changes in morbi-mortality. cardiovascular diseases are the first cause of death in elderly population. more than a half of ischemic heart diseases (ihd) cases occur in aged people (> 60 years old). objective: describe the spatial distribution of ihd mortality in the elderly population in rj and associations with socio-demographics variables. methods: data were gathered from information on mortality system of the ministry of health and the 2000 demographic census of the foundation of the brazilian institute for geography and statistics. the geographic distributions of the standardized coefficient of mortality due to ihd and socio-demographics variables, by districts, in 2000 were analyzed in arcgis 8.0. spatial autocorrelation of ihd was assessed by the moran and geary indices. a conditional autoregressive model was used to evaluate the association between idh and socio-demographics variables. results: association between idh mortality and income, educational level, family type and to possess computer, videocassette and microwave was found. conclusion: spatial analysis of the idh mortality and socio-demographics factors influence are fundamental to subsidize more efficient public policies in sense to prevention and control of this important injury of health. abstract purpose: to evaluate the prognostic impact of isolated loco-regional recurrences on metastatic progression among women treated for invasive stage i or ii breast cancer (within phase iii trials concerning the optimal management of breast cancer). patients and methods: the study population consisted of 3,602 women primary surgically treated for early stage breast cancer, enrolled in eortc trials 10801, 10854, or 10902, by breast conservation (55%) and mastectomy (45%) with long time of follow-up (median: 10.2 range: 0. 2-17.4 ). data were analysed in a multi-state model by using multivariate cox regression models, including a state-dependent covariate. results: after the incidence of the loco-regional recurrence, a positive nodal status at baseline is a significant prognostic risk factor for distant metastases. the effects of the young ages at diagnosis and larger tumour size, become less significant after the incidence of loco-regional recurrences. the presence of a locoregional recurrence in itself is a significant prognostic risk factor for distant metastases after loco-regional recurrences. the effect of the time to the loco-regional recurrence is not a significant prognostic factor. conclusion: the presence of local recurrence is an important risk factor for outcome in patients with early breast cancer. abstract background: the relationship between the antral follicles and ovarian reserve tests (ort) to determine ovarian response in ivf is extensively studied. we studied the role of follicle size distribution in the response on the various orts in a large group of subfertile patients. methods: in a prospective cohort study, female patients were included if they had regular ovulatory cycles, subfertility for >12 months, > = 1 ovary and > = 1 patent ovarian tube. antral follicles were counted by ultrasound and blood was collected for fsh, including a clomiphene challenge test (ccct), inhibin b, and estradiol before and after administration of puregon [rsymbol] . (efort test). statistical analysis was performed using spss 12.0 for windows. results: of 740 eligible patients, 489 participated. mean age was 32.6 years and mean duration of subfertility was 21.7 months. age, baseline fsh, ccct and efort correlated with the number of small follicles (2-5 mm) but not with large follicles (6-10 mm). regression analysis confirmed that the number of small follicles and average follicle size contributed to ovarian response after correction for age, while large follicles did not. conclusion: small antral follicles are responsible for the hormonal response in ort and may be suitable to predict ovarian response in ivf. abstract background: dengue epidemics account annually for several million cases and deaths worldwide. the high endemic level of dengue fever and its hemorrhagic form (dhf) correlates to extensive house infestation by aedes aegypti and multiple viral serotype human infection. objective: to describe dengue incidence evolutionary patterns and spatial distribution in brazil. methods: it is a review study that analyzed serial case reports registered since 1981 until 2003. results: it was shown that defined epidemic waves followed the introduction of every serotype (den 1 to 3) , and reduction in susceptible people possibly responded for downward case frequency. conclusions and discussion: an incremental expansion of affected areas and increasing occurrence of dhf with high lethality were noted in recent years. in contrast, efforts based solely on chemical vectorial combat have been insufficient. moreover, some evidence demonstrated that educational action do not permanently modify population habits. in this regard it was stated that while vaccine is not available, further dengue control would depend on potential results gathered from basic interdisciplinary research and intervention evaluation studies, integrating environmental changes, community participation and education, epidemiological and virological surveillance, and strategic technological innovations aimed to stop transmission. abstract background: patient participation in treatment decisions can have positive effects on patient satisfaction, compliance and health outcomes. objectives: study objectives were to examine attitudes regarding participation in decision-making among psoriasis patients and to evaluate the effect of a decision-aid for discussing treatment options. methods: a 'quasi experiment' was conducted in a large dermatological hospital in italy: a questionnaire evaluating the decision-making process and knowledge on treatments was selfcompleted by a consecutive sample of 231 psoriasis patients after routine clinical practice and by a second sample of 171 patients exposed to a decision-board. results: in routine clinical practice 67.9% of patients wanted to be involved in treatment decisions, 28.4% wanted to leave decisions entirely to the doctor and 3.7% preferred making decisions alone. 17.9% and 25.3% of the control and decision-board group had good knowledge level. at multivariate analysis good knowledge on treatments increased the likelihood of preferring an active role (or = 2.21; 95%ci 1.3-3.9; p = 0.006). the decision-board only marginally improved patient knowledge and doctor-patient communication. conclusion and discussion: in conclusion, large proportions of patients want to participate in decision-making, but insufficient knowledge can represent a barrier. further research is needed for developing effective instruments for improving patient knowledge and participation. abstract background: the only available means of controlling infections caused by the dengue virus is the elimination of its principal urban vector (aedes aegypti). brazil has been implementing programs to fight the mosquito; however, since the 1980's the geographic range of infestation has been expanding steadily, resulting in increased circulation of the virus. objective: to evaluate the effectiveness of the dengue control actions that have been implemented in the city of salvador. methods: prospective design, serologic inquiries were made in a sample population of residents of 30 urban 'sentinel areas'.the seroprevalence and one year seroincidence of dengue are estimated and the relationship between intensity of viral circulation and the standards of living and vector density is analysed. results: there were high overall seroprevalence (67.7%) and seroincidence (70.6%) for the circulating serotypes (denv-1 and denv-2). the effectiveness of control measures appears to be low, and although a preventable fraction of 29.7% was found, the incidence of infections in these areas was still very high (55.4%). conclusions and discussions: it is necessary to revise the technical and operational strategies of the infection control program in order to attain infestation levels that are low enough to interrupt the circulation of the dengue virus. this study investigates the difference in cancer mortality risks between migrant groups and the native dutch population, and determines the extent of convergence of cancer mortality risks according to migrants' generation, age at migration and duration of residence. data were obtained from the national population & mortality registries in the period 1995-2000 (75860 person years, 173461 cancer deaths). we used poisson regression to compare the cancer mortality rates of migrants originating from turkey, morocco, surinam, netherlands antilles/aruba to the rates for the native dutch. results: all-cancer mortality among all migrant groups combined was significantly lower compared to the native dutch population (rr=0.55 ci: 0.52-0.58). mortality rates for all cancers combined were higher among 2nd generation migrants, among those with younger age at migration, and those with longer duration of residence. this effect was particularly pronounced in lung cancer and colorectal cancer. for most cancers, mortality among 2nd generation migrants remained lower compared to the native dutch population. surinamese migrants showed the most consistent pattern of convergence of cancer mortality. conclusions: the generally low risk of cancer mortality for migrants showed some degree of convergence but the cancer mortality rates did not yet reach the levels of the native dutch population. abstract background: legionnaires' disease (ld) is a notifiable disease in the netherlands. ld cases are reported to authorities for national surveillance. supplementary, a national ld outbreak detection program (odp) is installed in the netherlands. these two registration systems have their own information exchange process and databases. objectives: surveillance systems are known to suffer from incompleteness of reported data. co-existence of two databases creates the opportunity to investigate accuracy and reliability in a national surveillance system. design and methods: comparison was made between the outcome 'diagnosis by culture' in both databases and physical presence of legionella strains in laboratories for 174 patients. accuracy is described using the parameters sensitivity and correctness. for reliability, cohen's kappa coefficient (?) was applied. results: accuracy and reliability were significantly higher in the odp database, but not optimal in both databases when compared to data in laboratories. the odp database was moderately reliable (? = 0.56; 95%ci 0.43-0.69), the surveillance database slightly (? = 0.14; 95%ci 0.02-0.27). conclusion: our findings suggest that diagnostic notification data concerning ld patients are most accurate and reliable when derived directly from diagnostic laboratories. discussion: involvement of data-entry persons in outbreak detection results in higher reliability. unreliable data may have considerable consequences during outbreaks of ld. the aim of the study was to investigate the medical students' plans to emigrate, quantify the scale of migration in the near future and to build a profile of the possible emigrants. data were collected based on anonymous questionnaire delivered to random group of 367 medical students (katowice). we used the binary logistic regression and multivariate analysis to identify the differences between groups preferring to go abroad or stay in poland. 85% respondents confirmed that considerate the emigration; 35.3% of them declared they are very likely to move and further 10.7% is certain. 23,9% of those considering emigration confirmed having taken practical steps towards moving. binary logistic regression showed no difference between people who were certain or almost certain to go and those who were not considering going for most baseline characteristics: hometown size, socio economic background and having family tradition of the medical profession (p = 0.19). only marks' mean differentiates between the two groups: 4.01 for those who will definitely stay vs 3.7 for students who will definitely move (p = 0.02). the multivariate analysis gave similar results. conclusions: most of the students consider the emigration, but the declarations of will to departure are more frequent among those with the worse marks. abstract background: falls incidence in home resident elderly people varies from 30% to 40%. falls induce loss of self-sufficiency and increase mortality and morbidity. objectives: to evaluate falls incidence and risk factors in a group of general practice elderly patients. design : prospective cohort study with 1 year follow-up methods: eight hundreds elderly (>75 years) were visited by 18 practitioners for a baseline assessment. information on current pathologies and previous falls in the last six months was collected. functional status was evaluated using: short portable mental state questionnaire, geriatric depression scale, activities of daily living (adl), instrumental activities of daily living, total mobility tinetti score. falls were monitored through 2 phone-interviews at 6 and 12 months. data were analyzed through logistic regression. results: twenty-eight percent of the elderly fell in the whole period. sixty percent of falls were not reported to the practitioner. independent predictors for falls were adl score (adl<5: or = 1.88; 95% ci 1.04-3.38) and previous falls (or = 1.60; 95% ci 1.02-1.52). tinetti score was significantly associated to falls only in univariate analysis. conclusions: practitioners can play a key-role in identifying at-risk subjects and managing prevention interventions. falls monitoring and a continuous practice of comprehensive geriatric assessment should be encouraged. abstract background: oral health represents an important indicator of health status. socio-economic barriers to oral care among elderly are considerable. in the lazio region, a public health program for oral rehabilitation was implemented to offer dentures to elderly people with social security. objectives: to compare hospitalisation between elderly enrolled in the program and a control group. design and methods: for each elderly enrolled in the program living in rome, three controls, matched for sex and age, were selected from rome municipality register. hospital admissions in the two-year period before enrollment were traced by record-linkage with the hospital discharge register. results: totally, 2,935 admissions occurred. the annual admissions rate was 218 per 1000 elderly among controls and 329 in the program group (incidence rate ratio: irr = 1.50; 95% ci 1.40-1.63). when comparing diagnosis-specific rates, significant excesses were observed in the program group for respiratory diseases ( abstract background: herpes simplex virus (hsv) type 1 and 2 are important viral sexually transmitted diseases (sti) and can cause significant morbidity. in the netherlands, data about prevalences in the general population are hampered. objective: description of the seroprevalences of hsv-1 and hsv-2 and associated factors in the netherlands. design and methods: a population based serum bank survey in the netherlands with an age-stratified sample was used (1995) (1996) . antibodies against hsv-1 and hsv-2 were determined using elisa. a questionnaire was used to get information on demographics and risk factors. a logistic regression adjusting for age and full multiple regression were done to establish risk factors. results: questionnaires and sera were available for 7166 persons. both hsv-1 and hsv-2 seroprevalence increased with age. seroprevalence of hsv-1 was 59.5% and was amongst others associated with female sex and being divorced. seroprevalence of hsv-2 was 8.4% and was amongst others associated with being divorced and a history of sti. conclusion: seroprevalence is higher in certain groups like teenagers, women, divorced people and those with a history of sti. prevention should be focused on those groups. more research is needed on prevention methods, which can be used in the netherlands, like screening or vaccination. abstract background: frequently, statistically significant prognostic factors are reported with suggestions that patient management should be modified. however, the clinical relevance of such factors is rarely quantified. objectives: we evaluated the accuracy of predicting the need for invasive treatment among bph patients managed conservatively with alpha1-blockers. methods: information on eight prognostic factors was collected from 280 patients treated with alpha1-blockers. with phm regression coefficients a risk score for retreatment was calculated for each patient. the analyses were repeated on 1000 groups of 280 patients sampled from the original case series. these bootstrap results were compared to the original results. results: three significant predictors of retreatment were identified. the 20% of patients with the highest risk score had an 18-month risk of retreatment of only 20%. analyses of less than half of all the bootstrap samples resulted in the same three significant prognostic factors. the 20% of patients with the highest risk score in each of the 1000 samples experienced a highly variable risk of retreatment of 0% to 42%. conclusions: four of the five high risk patients would be overtreated with a modified policy. internal validation procedures may warn against the invalid translation of statistical significance into clinical relevance. background: e-cadherin expression is frequently lost in human epithelium derived cancers, including bladder cancer. for two genetic polymorphisms in the region of the e-cadherin gene (cdh1) promoter, a reduced transcription has been reported: a c/a single nucleotide polymorphism (snp) and a g/ga snp at 160 bp and 347 bp, respectively, upstream of the transcriptional start site. objective: we studied the association between both polymorphisms and the risk of bladder cancer. methods: 174 patients with bladder cancer and 326 population controls were genotyped for the )160 c/ a and the )347 g/ga promoter polymorphisms using pcr-rflp. results: a significantly increased risk for bladder cancer was found for a allele carriers compared to the homozygous c allele carriers (or 1.58; 95% ci: 1.06-2.35). the risk for the heterozygous and homozygous a allele carriers, was increased approximately 1.5 and 2-fold, respectively. the association was stronger for more aggressive tumors. we did not find any association between the )347 g/ga snp and bladder cancer. conclusion: this study indicates that the )160 c/a snp in the e-cadherin gene promoter is a low-penetrance susceptibility factor for bladder cancer. background: health problems, whether somatic, psychiatric or accident-related cluster within persons. the study of allostatic load as a unifying theme (salut) aims to identify risk factors that are shared by different pathologies and that could explain this clustering. studying patients with repetitive injuries might be helpful to identify risk factors that are shared by accident-related and other health problems. objectives: to study injury characteristics in repetitive injury (ri) patients as compared to single injury (si) patients. methods: the presented study included 196 ri patients and 558 si patients. medical records provided information about injury characteristics and patients were asked for possible causes and context. results: ri patients suffered significantly more from contusions than si patients (25% vs 16%). regarding the context, si patients were significantly more injured in traffic (28% vs 23%). in both groups most injuries were attributed to 'mere bad luck' (ri 44%, si 49%), closely followed by 'clumsiness or inattention' (ri 39%, si 44%). ri patients pointed out aggression or substance misuse significantly more often than si patients (17% vs 7%). conclusion: ri patients seem to have more 'at risk' behavior (i.e. aggression, impulsivity), which will increase their risk for psychiatric health problems. abstract background: breastfeeding may have a protective effect on infant eczema. bias as a result of methodological problems may explain the controversial scientific evidence. objective: we studied the association between duration of breastfeeding and eczema when taking into account the possible influence of reverse causation. design and methods: information on breastfeeding, determinants and outcomes at age one year was collected by repeated questionnaires in 2834 mother infant-pairs participating in the koala study (535 cases of eczema). to avoid reverse causation, a periodspecific-analysis was performed in which only 'at risk' infants were considered. results: no statistically significant association between the duration of breastfeeding (>12 weeks versus formula feeding) and the risk of eczema in the first year was found (or 2.07 95%ci 0.69-6.22). after excluding from the analysis all breastfed infants with symptoms of eczema reported in the same period as breastfeeding, also no statistical significant association was found for the duration of breastfeeding and eczema between 4 and 12 months (or 1.45 95%ci 0.34-6.25). conclusion and discussion: in conclusion, no evidence was found for a protective effect of breastfeeding duration on eczema. this conclusion was strengthened by risk period-specific-analysis which made the influence of reverse causation unlikely. abstract background: the internet can be used to meet health information needs, provide social support, and deliver health services. the anonymity of the internet offers benefits for people with mental health problems, who often feel stigmatized when seeking help from traditional sources. objectives: to identify the prevalence of internet use for physical and mental health information among the uk population. to investigate the relationship of internet use with current psychological status. to identify the relative importance of the internet as a source of mental health information. design and methods: self-completion questionnaire survey of a random sample of the uk population (n = 1800). questions included demographic characteristics, health status (general health questionnaire), and use of the internet and other information sources. results: 59% of internet users had sought health information online, and 18% had sought mental health information. use was higher among those with current psychological problems. only 12% of respondents identified the internet as one of the most accurate sources of mental health information, compared with 24% who identified it as one of the sources they would use. conclusions: health service providers must recognise the increasing use of the internet in healthcare, even though it is not always regarded as being accurate. abstract old age is a significant risk factor for falls. approximately 45% of people older than 80 are falling at least once a year, mostly in the own homes. resulting hip fractures cause at least partial immobility in 40-50% of the affected persons. almost 20% are sent to nursing homes afterwards. in mecklenburg-west pomerania, ageing of the population proceeds particularly fast. to prevent falls and the loss of independent living a falls prevention module was integrated in a community-bbased study conducted in cooperation with a general practitioner (gp). in the patients homes' a trained nurse performed a test to estimate the falls risk of each patient and a consultation how to reduce risk, e.g. eye sight check, gymnastic exercise. in the feasibility study 11 (55%) out of 20 patients (average age 74 years), agreed to a visiting of each room of their homes in search for tripping dangers. the evaluation was assisted by a standardized, computer-based documentation. the prevention module received a considerable acceptance despite the extensive home visiting. within one month the patients started to transfer advice into practice. during the first follow up visits of the nurse three patients reported e.g. to have started gymnastics and/or wear stable shoes. abstract background: the emergence of drug resistant m. tuberculosis (mtb) is an increasing problem in both developed and developing countries. objectives: investigation of isoniazid (inh) and rifampin (rif) susceptibility patterns among mtb isolates from patients. design and methods: in total 80 sputum samples were collected. smears were prepared for acid fast staining and all the isolates were identified as m. tuberculosis by preliminary cultural and biochemical tests. the isolates were examined for inh and rifampin resistance using conventional mic method and pcr technique by using specific inh (kat g) and rifampin (rpo b) resistant primers. results: seven isolates were resistant to both inh and rifampin by mic method. in pcr technique, 5 and 6 out of 7 above mentioned strains showed resistant to inh and rifampin respectively. coclusion: the epidemiology of drug resistance is 8.7% in region of study which is significant. discussion: conventional mic method despite being time consuming is more sensitive for evaluation of drug resistance, however, pcr as a rapid and sensitive technique is recommended additionally to conventional method for having quicker results to start treatment and disease control management. abstract background and objectives: we studied in literature which design characteristics of food frequency questionnaires (ffqs) influence their validity to assess both absolute and relative levels of energy intake in adults with western food habits, and to rank them according to these intakes. this information is required in harmonizing ffqs for multi centre studies. design and methods: we performed a review of studies investigating the validity or reproducibility of ffqs, published since 1980. the included studies validated ffqs against doubly labeled water (for energy expenditure) as a gold standard, or against food records or 24 hour recalls for assessing relative validity (for energy intake). the design characteristics we studied were the number of food items, the reference period, the administration mode, and inclusion of portion size questions. results: and conclusion: for this review we included 35 articles representing the validation of 37 questionnaires. three questionnaires were validated against dlw, ten against urinary n and 25 against 24-hour recalls or food records. in conclusion a positive linear relationship (r = 0.57, p<0.0001) was observed between the number of items on the ffq and the reported mean energy intake. details about the influence of other design characteristics on validity will be discussed at the conference. abstract background: high ethanol intake may increase the risk of lung cancer. objectives: to examine the association of ethanol intake with lung cancer in epic. design and methods: information on baseline and past alcohol consumption, lifetime tobacco smoking, diet, and anthropometrics of 478,590 participants was collected between 1992 and 2000. cox proportional hazard regression was used to examine the association of ethanol intake at recruitment (1119 cases) and mean lifelong ethanol intake (878 cases) with lung cancer. results: non-consumers at recruitment had a higher lung cancer risk than low consumers (0.1-4.9 g/day) [hr = 1.22, 95% ci 0.99-1.50]. lung cancer risk was lower for moderate ethanol intake at recruitment (5.0-14.9 g/day) compared with low intake (hr = 0.76, 95% ci 0.63-0.90); no association was seen for higher intake. compared with lifelong low consumers, lifelong non-consumers did not have a higher lung cancer risk (hr = 1.01, 95% ci 0.67, 1.50) but lifelong moderate consumers had a lower risk (hr = 0.80, 95% ci: 0.66-0.97). lung cancer risk tended to increase with increasing lifelong ethanol intake (=60 vs. 0.1-4.9 g/ day hr = 1.29, 95% ci: 0.93-1.74). conclusion: while lung cancer risk was lower for moderate compared with low ethanol intake in this study, high lifelong ethanol intake might increase the risk. abstract background: one of the hypotheses to explain the increasing prevalence of atopic diseases (eczema, allergy and asthma) is imbalance between dietary intake of omega-6 and omega-3 fatty acids. objectives: we evaluated the role of perinatal fatty acid (fa) supply from mother to child in the early development of atopy. design and methods: fa composition of breast milk was used as a marker of maternal fa intake and placental and lactational fa supply. breast milk was sampled 1 months postpartum from 312 mother-infant pairs in the koala birth cohort study, the netherlands. the infants were followed for atopic symptoms (repeated questionnaires on eczema and wheezing) and sensitisation at age 2 (specific serum ige against major allergens). multivariate logistic regression analysis was used to adjust for confounding factors. results: high levels of omega-3 long chain polyunsaturated fas were associated with lower incidence of eczema in the first year (odds ratio for the highest vs lowest quintile 0.31, 95% confidence interval 0.12-0.81; trend over quintiles p = 0.007). wheeze and sensitisation were not associated with breast milk fa composition. conclusion and discussion: the results support the omega-6/3 hypothesis. we suggest that anti-inflammatory activity of omega-3 eicosanoid mediators is involved but not allergic sensitisation. abstract background: acute myocardial infarction (ami) is among the main causes of death in italy and is characterized by high fatality associated with a fast course of the disease. consequently timeliness and appropriateness of the first treatment are paramount for a positive recovery. objectives: investigate the differences among italian regions of ami first treatment and in-hospital deaths. design and methods: following the theoretical care pathway (from onset of ami to hospitalization and recovery or death), regional in-hospital deaths are decomposed into the contributions of attack rate, hospitalization and in-hospital fatality. hospital discharges, death and population data are provided by the official statistics. results: generally in northern and central regions there is an excess of observed in-hospital deaths, while the opposite occurs in southern regions. conclusion: in northern and central regions the decomposition method suggests a more frequent and severe illness, generally accompanied by a higher availability of hospitals; exceptions are lombardia and lazio, where some inefficiencies in the hospital system are highlighted. in most southern regions the decomposition confirms a less frequent and less severe illness; exceptions are campania and sicilia, where only the less severe patients reach the hospital and then recover, while the others die before reaching the hospital. abstract background: atherosclerotic lesions have typical histological and histochemical compositions at different stages of their natural history. the more advanced atherosclerotic lesions contain calcification. objective: we examined the prevalence of and associations between calcification in the coronary arteries, aortic arch and carotid arteries assessed by multislice computed tomography (msct). methods: this study was part of the rotterdam study,a population-based study of subjects aged 55 years and over. calcification was measured and quantified in 600 subjects. correlation coefficients were computed using spearman's correlation coefficients. results: the prevalence of calcification increased with age throughout the vascular bed. in subjects aged 80 and over, up to 100% of men had calcification in the coronary arteries and up to 100% of women had calcification in the aortic arch. in men, the strongest correlation was found between calcification in the aortic arch and the carotid arteries (r=0.60, p<0.001). in women, the relations were somewhat lower, the strongest correlation was found between calcification in the coronary arteries and the carotid arteries (r = 0.47, p<0.001). conclusion and discussion: in conclusion, the prevalence of calcification was generally high and increased with increasing age. the study confirms the presence of strong correlations between atherosclerosis in different vessel beds. abstract background: health status deteriorates with age and can be affected by transition from active work to retirement. objective: to assess the effect of retirement on age related deterioration of health. methods: secondary analysis of the german health survey (bundesgesundheitssurvey 1998) and california health interview survey (chis 2003) . subjective health was assessed by a single question regarding respondent's health status from 1 = excellent to 5 = poor. locally weighted regression was used for exploratory analysis and b-splines for the effect estimation in regression models. results: subjective health decreased in an obviously non-linear manner with age. in both cases the decrease could be reasonably approximated by two linear segments, however the pattern was different in the german and california sample. in germany, the change point of the slope describing deterioration of health was located at 59. abstract objective: to assess the effectiveness of physiotherapy compared to general practitioners' care alone, in patients with acute sciatica. design, setting and patients: a randomised clinical trial in primary care with a 12-months follow-up period. 135 patients with acute sciatica (recruited 2003 -2004) were randomised in two groups: 1) intervention group received physiotherapy (active exercises), and 2) control group received general practitioners' care only. main outcome measures the primary outcome was patients' global perceived effect. secondary outcomes were severity of leg and back pain, severity of disability, general health and absence from work. the outcomes were measured at 3, 6, 12 and 52 weeks after randomisation. results: at 3 months follow-up, 70% of the intervention group and 62% of the control group reported improvement (rr 1.1; 95% ci 0.9 to 1.5). at 12 months follow-up, 79% of the intervention group and 56% of the control group reported improvement (rr 1.4; 95% ci 1.1; 1.8). no significant differences in secondary outcomes were found at short-term or long-term follow-up. conclusion: at 12 months follow-up, evidence was found that physiotherapy added to general practitioners' care is more effective in the treatment of patients with acute sciatica than general practitioners' care alone. abstract background: only little is known about the epidemiology of skin melanoma in the baltic states. objectives: the aim of this study was to provide insights into the epidemiology of skin melanoma in lithuania by analyzing population-based incidence and mortality (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) time trends and relative survival based on 3485 skin melanoma. methods: we calculated age-standardized incidence and mortality rates (cases per 100,000) using the european standard population and calculated period estimates of relative survival. for the period 1993-2002, 76% of all registered cases were checked by reviews of the medical charts. results: about 97% of the 2013 cases of the period 1993-2002 were reported to the cancer registry indicating a high quality of cancer registration of skin melanoma in lithuania. the incidence rates increased from 1978 (men: 1.7, women: 2.3) to 2002 (men: 5.0, women: 7.0). mortality rates increased from 1990 (men: 1.2, women: 1.7) to 2002 (men: 2.3, women: 2.2). relative 5-year relative survival rates among men were 10% lower than among women. the overall difference in survival is mainly due to a more favorable survival among women aged 60-74 years. conclusions: overall prognosis is less favorable among men most likely due to diagnoses at later stages. abstract background: only little is known about the epidemiology of skin melanoma in the baltic states. objectives: the aim of this study was to provide insights into the epidemiology of skin melanoma in lithuania by analyzing population-based incidence and mortality (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) time trends and relative survival based on 3485 skin melanoma. methods: we calculated age-standardized incidence and mortality rates (cases per 100,000) using the european standard population and calculated period estimates of relative survival. for the period 1993-2002, 76% of all registered cases were checked by reviews of the medical charts. results: about 97% of the 2013 cases of the period 1993-2002 were reported to the cancer registry indicating a high quality of cancer registration of skin melanoma in lithuania. the incidence rates increased from 1978 (men: 1.7, women: 2.3) to 2002 (men: 5.0, women: 7.0). mortality rates increased from 1990 (men: 1.2, women: 1.7) to 2002 (men: 2.3, women: 2.2). relative 5-year relative survival rates among men were 10% lower than among women. the overall difference in survival is mainly due to a more favorable survival among women aged 60-74 years. conclusions: overall prognosis is less favorable among men most likely due to diagnoses at later stages. abstract background: multifactorial diseases share many risk factors, genetic as well as environmental. to investigate the unresolved issues on etiology of and individual susceptibility to multifactorial diseases, the research focus must move from single determinantoutcome relations to modification of universal risk factors. objectives: the aim of the lifelines project is to study universal risk factors and their modifiers for multifactorial diseases. modifiers can be categorized into factors that determine the effect of the studied risk factor (eg gen-expression), those that determine the expression of the studied outcome (eg previous disease), and generic factors that determine the baseline risk for multifactorial diseases (eg age). design and methods: lifelines is carried out in a representative sample of 165.000 participants from the northern provinces of the netherlands. apart from questionnaires and clinical measurements, a biobank is constructed (blood, urine, dna). lifelines will employ a three-generation family design (proband design with relatives), which has statistical advantages, enables unique possibilities to study social characteristics, and offers practical benefits. conclusion: lifelines will contribute to the understanding of how disease-overriding risk factors are modified to influence the individual susceptibility to multifactorial diseases, not only at one stage of life but cumulatively over time: the lifeline. abstract background: obesity-related mortality is a major public health problem, but few studies have been conducted on severely obese individuals. objectives: we assessed long-term mortality in treatment-seeking, severely obese persons. design and methods: we enrolled 4837 persons in six centres for obesity treatment in four italian regions, with body mass index (bmi) at first visit => 40 kg/m2 and age => 18. after exclusion of duplicate registrations and persons with missing personal or clinical data, 4662 persons were followed up; as 164 (3.5%) could not be traced, 4498 persons (972 men, 3526 women) were retained for analysis. results: there were 484 (153 men, 331 women) deaths; the standardized mortality ratios (smrs) and 95% confidence intervals were 278 (236-326) among men and 210 (188-234) among women. mortality increased with increasing bmi, but the trend was not monotonic in men. lower smrs were observed among persons recruited more recently. excess mortality was inversely related to age attained at follow-up. conclusions and discussion: the harmful, long-term potential of severe obesity we documented confirms observations from studies carried out in different nutritional contexts. the decrease in mortality among most recently recruited persons may reflect better treatment of obesity and of its complications. abstract background: in finland, every cancer patient should have equal access to high quality care provided by the public sector. therefore no regional differences in survival should be observed. objectives: the aim of the study was to find any regional differences in survival, and to elaborate whether possible differences could be explained, e.g., by differences in distributions of prognostic factors. design and methods: the study material consisted of 159,000 patients diagnosed in 1993 to 2000 with cancer at one of the 15 major primary sites. the common closing date was 31 dec. 2001. finland was divided into five university hospital regions. stage, age at diagnosis and sex were used as prognostic factors. the relative survival rates for calendar period window, 1998-2001, were tabulated using period method and modelled. results: survival differences between the regions were not significant for most primary sites. for some sites, the differences disappeared in the modelling phase after adjusting for the prognostic factors. for a few of the primary sites (e.g., carcinoma of the ovary), regional differences remained after modelling. conclusion: we were able to highlight certain regional survival differences. ways to improve the equity of cancer care will be considered in collaboration with the oncological community. abstract background: the prevalence of cardiovascular disease (cvd) is extremely high in dialysis patients. disordered mineral metabolism, including hyperphosphatemia and hypercalcaemia, contributes to the development of cvd in these patients. objectives: to assess associations between plasma calcium, phosphorus and calciumphosphorus product levels and risk of cvd-related hospitalization in incident dialysis patients. design and methods: in necosad, a prospective multi-centre cohort study in the netherlands, we included 1629 consecutive patients new on haemodialysis or peritoneal dialysis between 1997 and 2004. risks were estimated using adjusted time-dependent cox regression modeling. results: mean age was 60±15 years, 61% was male, and 64% was treated with haemodialysis. cvd was the cause of hospitalization in 159 haemodialysis patients (26% of hospitalizations) and in 60 peritoneal dialysis patients (22%). most common cardiovascular morbidities were peripheral vascular disease and coronary artery disease in both patient groups. in haemodialysis patients risk of cvd-related hospitalization increased with elevated plasma calcium (hazard ratio: 1.8; 95% ci: 1.1 to 2.9) and calcium-phosphorus product levels (1.8; 95% ci: 1.2 to 2.7). in peritoneal dialysis patients, we observed similar effects that were not statistically significant. conclusion: tight control of plasma calcium and calcium-phosphorus product levels might prevent cvd-related hospitalizations in dialysis patients. abstract background: nurses are at health risk due to the nature of their work. analysis of morbidity among nurses was conducted to provide insight concerning the relationship between their occupational exposure and health response. methods: self reported medical history, was collected from an israeli female-nurses cohort (n = 395, 50+ years old) and their siblings (n = 180, age matched +/)7 years) using a structured questionnaire. to compare disease occurrence between the two groups we used chi-square tests and hazard ratio (hr) was calculated by cox-regression to account for age of onset. results: cardiovascular diseases were more frequent among the nurses compared to the controls: heart diseases 14.2% vs. 6.1, p = .0052 (hr=2.02, p = .0329); hypertension 43.8% vs. 23.3%, p<.0001 (hr=1.78, p = .0008). the frequency of hyperlipidemia was 40.3% among the nurses, and only 12.2% among the controls. (hr=3.31,p = .0001). for the following chronic diseases the occurrence were significantly higher among the nurses and the hrs were significantly higher than 1: thyroid, hr=2.21; liver, hr=10.37. total cancer and diabetes rates were similar in the groups (hr$1). conclusions: the results suggest an association between working as a nurse and the existence of risk factors for cardiovascular diseases. the specific related determinants of their work should be further evaluated. abstract background: early referral (er) to a nephrologist and arteriovenous fistulae as first vascular access (va) reduce negative outcomes in chronic dialysis patients (cdp). objectives: to evaluate the effect of nephrologist referral timing and type of the first va on mortality. design and methods: prospective cohort study of 2260 incident cdp notified to lazio dialysis registry (italy) in 2002-2004. late referral (lr) was a patient not referred to nephrologists within 6 months before starting dialysis. we dichotomized va as fistulae versus catheters. to estimate mortality hazard ratios (hr) a multivariate cox model was performed. results: we observed 22.3% lr subjects and 24.8% catheters as first va; proportion of catheters was 41.2% vs. 20.1% (p<0.001) for lr and er, respectively. we found a higher mortality hr for patient with a catheter as first va both for er (hr = 1.58; 95%c.i. = 1.22-2.06) and lr (hr = 2.56; 95%c.i. = 1.92-3.43); the interaction between referral and va was slight significant (p = 0.13). conclusions: the originality of our study was to investigate the influence of nephrologist referral timing and va on cdp mortality using a population registry, area-based: we found that a catheter as first va has an independent effect for mortality and modifies the effect of referral timing on this outcome. abstract patients with idiopathic venous thrombosis (vt) without known genetic risk factors but with a positive family history might carry yet unknown genetic defects. to determine the role of unknown hereditary factors in unexplained vt we calculated the risk associated with family history. in the multiple environmental and genetic assessment of risk factors for vt (mega) study, a large population-based case-control study, we collected blood samples and questionnaires on acquired risk factors (surgery, immobilisation, malignancy, pregnancy and hormone use) and family history of 2463 patients and 2926 control subjects. overall, positive family history was associated with an increased risk of vt (or (95% ci): 2.4 (2.0-2.9)), especially in the absence of acquired risk factors (or (95% ci): 2.8 (2.2-3.6) ). among participants without acquired factors but with a positive family history, prothrombotic defects (factor v leiden, prothrombin 20210a, protein c or protein s deficiency) were identified in 80 out of 236 (34%) patients compared to 22 out of 143 (15%) control subjects. after excluding participants with acquired or prothrombotic defects, family history persisted as a risk factor (or (95% ci): 2.4 (1.7-3.3)). in conclusion, a substantial fraction of thrombotic events is unexplained. family history remains an important predictor of vt. abstract background: alcohol may have a beneficial effect on coronary heart disease (chd) through elevation of high-density lipoprotein cholesterol (hdlc) or other alterations in blood lipids. data on alcohol consumption and blood lipids in coronary patients are scarce. objectives: to assess whether alcohol consumption and intake of specific types of beverages are associated with blood lipids in older subjects with chd. design and methods: blood lipids (total cholesterol, hdlc, ldl cholesterol, triglycerides) were measured in 1052 myocardial infarction patients aged 60-80 years (78% male), as part of the alpha omega trial. intake of alcoholic beverages, total ethanol and macro and micronutrients were assessed by food-frequency questionnaire. results: seventy percent of the subjects used lipidlowering medication. mean total cholesterol was 5.14 mmol/l and hdlc was 1.28 mmol/l. in men but not in women, ethanol intake was positively associated with hdlc (difference of 0.095 mmol/l for =15 g/d vs. 0 g/d, p = 0.022) after adjustment for diet, lifestyle, and chd risk factors. also, liquor consumption was weakly positively associated with hdlc in men (p = 0.042). conclusion and discussion: moderate alcohol consumption may elevate hdlc in (treated) myocardial infarction patients. this is probably due to ethanol and not to other beneficial substances in alcoholic beverages. session: posters session 3: july 1 2006 presentation: poster. abstract objective: early detection and diagnosis of silicosis among dust exposed workers is based mainly on the presence of rounded opacities on radiographs. it is thus important to examine how reliable the radiographic findings are in comparison to pathological findings. methods: a systematic literature search via medline was conducted. validity of silicosis detection and its influence on risk estimation in epidemiology was evaluated in a sensitivity analysis. results: 4 studies on comparison between radiographic and pathological findings of silicosis were identified. the sensitivity of radiographic diagnosis of silicosis (ilo 1/1) varied between 39% and 71%, and specifity between 60% and 99%. under the realistic assumption of silicosis prevalence between 2% and 8% in dust exposed workers, 23% to 56% of silicosis identified may be falsely diagnosed. the sensitivity analysis indicates that invalid diagnostics alone may lead to the finding of an increased risk of lung cancer among patients with silicosis. it may also lead to findings of 1% to 4% of radiographic silicosis even when there is no case of silicosis. however, the risk of silicosis could also be underestimated if the prevalence of silicosis exceeds 10%. conclusion: epidemiological studies based on patients with silicosis should be interpreted with caution. abstract introduction: epidemics of dengue occurring in various countries have stimulated investigators to seek innovative ways of improving current knowledge on the issue. objective: to identify the characteristics of spatial-temporal diffusion of the first dengue epidemic in a major brazilian city (salvador, bahia). methods: notified cases of dengue in salvador in 1995 were georeferenced according to census sector (cs) and by epidemiological week. kernel density estimation was used to identify the spatial diffusion pattern. results: of the 2006 cs in the city, cases of dengue were registered in 1400 (70%). spatial distribution showed that in 1995 practically the entire city had been affected by the virus, with a greater concentration of cases in the western region, comprising cs of high population density and predominantly horizontal dwellings. conclusion and discussion: the pattern found showed characteristics of a contagious diffusion process. it was possible to identify the epicenter of the epidemic from which propagation initiated. the speed of progression suggested that even if a rapid intervention was initiated to reduce the vector population, it would probably have little effect in reducing the incidence of the disease. this finding confirms the need for new studies to develop novel technology for prevention of this disease. abstract background: knowing the size of drug user hidden populations in a community is important to plan and evaluate public health interventions. objectives: the aim of this study was to estimate the prevalence of opiate and cocaine users in liguria region by using the covariate capture-recapture method applied to four data sources. methods: we performed a cross-sectional study in the resident population aged 15-54 years (780.995 people at 2001 census). during 2002 individual cases identified as opiate or cocaine primary users were flagged by four sources (drug dependence services, social service at prefectures, therapeutic communities, hospital discharges). poisson regression models were fitted, adjusting for dependence among sources and for heterogeneity in catchability among categories of the two examined covariates: age (15-29 and 30-54 years) and gender. results: the prevalence of opiate or cocaine users was 2,0% (95% c.i., 1,5 -2,8%) and 2,1% (95% c.i.= 0,6 -8,5%) respectively. conclusions: the estimated prevalence of opiate and cocaine users is consistent with that found in inner london: 1.6% and 1.9% respectively (hickman m.,2004; hope v.d., 2005) . the covariate capture-recapture method applied to four data sources allowed identifying a large cocaine-using population and resulted appropriate to determine drug user hidden populations. abstract background: in 2002-2004 we performed a population based diabetes screening programme. objectives: to investigate whether the yield of screening is influenced by gp and practice characteristics. methods: a questionnaire containing items on the gp (age, gender, employment, specialty in diabetes, applying insulin therapy) and the practice (setting, location, number of patients from ethnic minority groups, specific diabetes clinic, involvement of practice assistant and practice nurse in diabetes care, cooperation with a diabetes nurse) was sent to 106 general practitioners (gps) in 79 practices in the southwestern region of the netherlands. multiple linear regression analysis was performed. outcome measure was the ratio screen detected diabetic patients/known diabetic patients per practice (sdm/kdm). results: sdm/kdm was independently associated with higher age of the gp (regression coefficient 0.20; 95% confidence interval 0.07 to 0.34), urban location ()4.60; )6.41 to )2.78) and involvement of the practice assistant in diabetes care (2.27; 0.49 to 4.06) . conclusion: a lower yield of screening, assumably reflecting a lower prevalence of undiagnosed diabetes, was found in practices of younger gps and in urban practices. a lower yield was not associated with an appropriate practice organization nor with a specialty of the gp in diabetes. session: posters session 3: july 1 2006 presentation: poster. background: since few years increased incidence rates for childhood cancer were reported from industrialized countries. these findings were discussed controversial, because increases could be caused by changing of potential risk factors. objectives: the question is: are observed increasing rates due to actual changes in incidence rates or mainly caused by changes in registration practice or artefacts? methods: for europe, data from the accis project (pooled data from 32 european population-based cancer registries, performed at iarc, lyon; responsible: e. steliarova-foucher) (1978) (1979) (1980) (1981) (1982) (1983) (1984) (1985) (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) , and for germany, data of the german childhood cancer registry available from1980 onwards were used. results: accis data (based on 75,000 cases) show significantly increased data with an overall average annual percentage change of about 1 % and it is seen for mainly all diagnostic subgroups. for germany, increases are seen for neuroblastoma (due to screening programmes) and brain tumours (due to improved registration). for acute leukaemia the observed increase is explained by changes in classification. conclusion and discussion: the increased incidence for europe can only partly be explained by registration artefacts or improved diagnostic methods. the observed patterns suggest that an actual change exists. in germany, from 1980 till now the observed increased rates could be explained by artefacts. abstract suicide is the fourth most common cause of death among working age finns. among men socioeconomic status is strongly and inversely associated with suicide mortality, but little is known about socioeconomic differences in female suicide. we studied the direct and indirect effects of different socioeconomic indicators -education, occupation-based social class and income -on suicide among finnish women aged 25-64. also the effect of main economic activity was studied. we used individual level data from the 1990 census linked to the death register for the years 1991-2001. altogether over 14 million person-years were included and 2137 suicides were committed. age-adjusted rii conducted using poissonregression model was 1.72 (95% ci 1.47-2.03) for education, 1.97 (1.68-2.30) for social class and 2.13 (1.82-2.49) for income. however, almost all of the effect of education was mediated by social class. fifteen per cent of social class was explained by education and 40 per cent was mediated by income. the effect of income on suicide was mainly explained by economic activity. in conclusion, net of other indicators occupation-based social class is a strong determinant of socioeconomic differences in female suicide mortality, and actions aimed at preventing female suicide should target this group. abstract c-reactive protein levels (crp) in the range between 1 and 5 mg/ l independently predict the risk of future cardiovascular events. besides being a marker of atherosclerotic processes, high-normal crp levels may also be a sign of a more pronounced response to everyday inflammatory stimuli. the aim of our study is to assess the association between response to everyday stimuli and the risk of myocardial infarction. we will perform a population based case-control study including a total of 600 persons. cases (n = 100) are first myocardial infarction (mi) patients. controls (n = 100) are partners of the patients. offspring of the mi patients (n = 200) are included because disease activity and the use of medication by the mi patients may influence the inflammatory response. in order to assess the inflammatory response in mi patients the mean genetically determined inflammatory response in the offspring will be assessed and used as a measure for the inflammatory response in the mi patients. the offspring is free of disease and medication use. partners of the offspring (n = 200) are the controls for the offspring. influvac vaccine will be given to assess crp concentration, i.e. inflammatory response, before and after the vaccination. abstract background:. ischemic heart disease risk may be influenced by long-term exposure to electromagnetic fields (emf) in vulnerable subjects, but epidemiological data is inconsistent. objectives: we studied whether the long-term occupational exposure to emf is related to an increased myocardial infarction (mi) risk. design and methods: we conducted a prospective case-control study, which involved 1042 mi cases and 2341 controls. emf exposure in 48 cases and 63 controls was assessed subjectively. the effect of emf exposure on mi risk was estimated using multivariate logistic regression. results: after adjustment for age, smoking, blood pressure, body mass index and psychological stress the odds ratios for emf exposure <10 years was 2.74; 95 % ci 0.62-12.07, for emf exposure 10-20 years -2.08; 95 % ci 0.91-4.76 and for emf exposure >20 years -2.05; 95 % ci 1.24-3.39. conclusion: longterm occupational exposure to emf may increase the risk of mi. our crude estimates of emf exposure might have impact on excess risk because of nondifferential misclassification in assigning exposure. abstract background: it has been suggested that noise exposure is associated with ischemic heart disease risk, but epidemiological evidence is still limited. objectives: we studied whether road traffic noise exposure increases the risk of myocardial infarction (mi). design and methods: we conducted population-based prospective casecontrol study, which involved 1042 mi cases and 2341 controls. we measured traffic-related noise levels at the 117 electoral districts and linked these levels to residential addresses. we used multiple logistic regression to assess effect of noise exposure on mi risk. results: after adjustment for age, smoking, blood pressure, body mass index, and psychological stress the risk of mi was higher for the men exposed to 70-75 dba ( background: some studies have suggested that patients, depressed following acute myocardial infarction (mi), experience poorer survival. however, i) other studies show no significant association, when adjusted for recognized prognostic indicators and ii) some 'natural' responses to mi may be recorded in questionnaires as indicators of depression. method: depression was assessed in mi patients, by interview on two measures (gwb and sf36) 1-2 weeks after discharge, clinical data were abstracted from patients' medical records and vital status was assessed at 2-4 years. survivals of depressed, marginally depressed and normal patients were calculated by kaplan meier method and comparisons made by log rank and cox proportional hazard modelling. results: crude survival at 3 years in 2137 patients was higher for depressed and marginally depressed (13%) than for normals (10%), although not significantly. in multivariate analysis, four patient characteristics contributed significantly to survival: age (p<0.001), previous mi (<0.001), diabetes (<0.001) and sex (<0.05): other potential explanatory variables, including hypertension, infarct severity and depression were excluded by the model. abstract background: the low coronary heart disease (chd) incidence in southern europe could result in lower low density lipoprotein cholesterol oxidation (oxldl). objective. the aim of this study was to compare oxldl levels in chd patients from several european countries. methods: a cross-sectional multicenter study included 796 stable chd male subjects aged 25 to 74 years from northen (finland and sweden), central (germany), and southern europe (greece and spain). lipid peroxidation was determined by plasma oxldl. results: the score of adherence to mediterranean diet, antioxidant intake, alcohol intake, and lipid profile were significantly associated with oxldl. oxldl levels were higher in northern (60.9 u/l) than in centre (54.4 u/l) and southern populations (53.8 u/l), p = 0.01, in the adjusted models. the probability of northern europe to have the highest oxldl levels was 95.5%, and 98.9, % in logarithm of triglyceride-adjusted and fully adjusted models, respectively. the probability of this order to hold after adjustment for country was 78.4%. conclusion: a gradient on lipoperoxidation from north to central and southern europe is very likely to exist, and parallels that observed in the chd mortality, and incidence rates. southern populations may have more favourable environmental factors against oxidation than northern europe. abstract background: whereas socioeconomic status (ses) has been established as a risk factor for a range of adverse health outcomes, little literature exists examining socio-economic inequalities in the prevalence of congenital anomalies. objectives: to investigate the relationship between the ses and the risk of specific congenital anomalies, such as neural tube defects (ntd), oral clefts (oc) and down's syndrome (ds). design and methods: a total of 485 cases of congenital anomaly and 970 non-malformed control births were collected between 2002 and 2003 from the italian archive of the certificates of delivery care. as a measure of ses, cases and controls were given a value for a 4 level deprivation index. data were analysed using a logistic regression model. results: we found 31 cases of dtn, 287 cases of sof and 167 cases of ds. the risk of having a baby with ntd was significantly higher for women of low ses (or = 2.7;c.i.: 1.1-7. 3), as well as for oc (or = 1.7; c.i.:1.3-2.3). no significant evidence for ses variation was found for ds. conclusion and discussion: our data suggest risk factors linked to ses, such as nutritional factors, lifestyle, and access to health services, may play a role in the occurrence of some malformations. abstract background: general practitioners (gp) are well-regarded by their patients and have the opportunity to play an active role in providing cessation advice. objectives: this study was run to examine whether a public health programme based on a carefully adapted programme of continuing education can increase gps' use of cessation advice and increase the success rates of such advice. methods: the particular context due to a randomization of gp leads us to consider a cluster randomization trial. marginal models, estimated by gee and mixed generalized linear models are used for this type of design. results: the cessation rate is relatively high for all smokers enrolled in the trial (n = 1075): a total of 313 smokers were ex-smokers at one year (29.1%). patients who were seen by trained gps were more likely to successfully stop smoking than those seen by the control gps (31.3% vs 24.4%). motivated subjects, aged over 40, lower had anxiety scores, and confidence in their ability to stop smoking, were predictive of successful cessation at one year follow-up. conclusions: cluster analysis indicated that factors important to successful cessation in this population of smokers are factors commonly found to influence cessation. abstract background: and purpose conventional meta-analysis showed no difference in primary outcome for coronary bypass surgery without (offpump) or with (onpump) the heart-lung machine. secondary outcome parameters such as transfusion requirements or hospitalization days favored offpump surgery. combined individual data analysis improves precision of effect estimates and allows accurate subgroup analyses. objective: our objective is to obtain accurate effect estimates for stroke, myocardial infarction, or death, after offpump versus onpump surgery, by meta-analysis on pooled individual patient data. method and results: bibliographic database search identified eleven large trials (>100 patients). the obtained data for 9 trials data included 1933 patients. primary endpoint was composite (n = 117), secondary endpoints were death (n = 34), stroke (n = 24) and myocardial infarction (n = 75). hazard ratio for event-free survival after offpump vs onpump (95% ci) was: composite endpoint 0.94(0.66;1.36), death 1.12(0.57;2.20) myocardial infarction 1.07(0.68;1.69), stroke 0.84(0.38;1.88). after stratification for diabetes, gender and age the results slightly favored offpump for high-risk groups. hazard ratios remained not statistically significant. conclusion: no clinical or statistical significant differences were found for any endpoint or subgroup. offpump coronary bypass surgery is at least equal to conventional coronary bypass surgery. offpump surgery therefore is a justifiable option for cardiac surgeons for cardiac bypass surgery. in 1996-1997 an outbreak of pertussis occurred, mostly among vaccinated children. since then the incidence has remained high. therefore, a fifth dose with acellular booster vaccine for 4-yearolds was introduced in october 2001. the impact of this vaccination on the age-specific pertussis incidence was assessed. mandatory notifications and hospitalisations were analysed for 2002-2004 and compared with previous years. surveillance data show 'epidemic' increases of pertussis in 1996, 1999, 2001 and 2004 . the total incidence/100,000 in 2004 (59.8) was higher than in the previous epidemic year 2001 (50.0). nevertheless, the incidence of notifications and hospitalisations in the age-groups targeted for the booster-vaccination had decreased with respectively 72% and 86% compared to 2001. in contrast, the incidence in adolescents and adults almost doubled. unlike other countries that introduced a pre-school booster, the incidence of hospitalised infants <6 months also decreased (31% compared with 2001). as expected, the booster-vaccination for 4-year-olds has decreased the incidence among the target-population itself. more importantly, the decreased incidence among infants <6 months suggests that transmission from siblings to infants has also decreased. in further exploration of the impact of additional vaccination strategies (such as boostering of adolescents and/or adults) this effect should not be ignored. abstract acute respiratory infections (ari) are responsible for considerable morbidity in the community, but little is known about the presence of respiratory pathogens in asymptomatic individuals. we hypothesized that asymptomatic persons could have a sub clinical infection and so act as a source of transmission. between 2000 and 2003 all patients with ari who visited their sentinel general practitioner were reported to estimate the incidence of ari in dutch general practices. a random selection of them (cases) and an equal number of asymptomatic persons visiting for other complaints (controls) were included in a case-control study. nose/ throat swabs of participants were tested for a broad range of pathogens. the overall incidence of ari was 545 per 10,000 person years, suggesting that in the dutch population an estimated 900,000 persons annually consult their general practitioner for respiratory complaints. viruses were detected in 58% of the cases, ?-haemolytic streptococci group a in 11% and mixed infections in 3%. besides, pathogens were detected in approximately 30% of controls, particularly in the youngest age groups. this study confirms that most ari are viral and supports the reserved policy of prescribing antibiotics. furthermore, we demonstrated that asymptomatic persons might be a neglected source of transmission. abstract background: the baking and flour producing industries in the netherlands agreed on developing a health surveillance system to reduce the burden of and improve prognosis of occupational allergic diseases. objectives: to develop and validate a diagnostic model for sensitization to wheat and fungal amylase allergens, as triage instrument to detect occupational allergic diseases. design and methods: a diagnostic regression model was developed in 391 bakers from a cross-sectional study with ige serology to wheat and or amylase allergens as the reference standard. model calibration was assessed with hoshmer-lemeshow goodness of fit test; discriminative ability using area under receiver operating characteristic curve (auc); and internal validity using bootstrapping procedure. external validation was conducted in 200 other bakers. results: the diagnostic model consisted of four questionnaire items (history of asthma, rhinitis, conjunctivitis, and work-related allergic symptom) showed good calibration (p = 0.7) and discriminative ability (auc 0.73; 95% ci 0.67 to 0.79). internal validity was reasonable (correction factor of 0.85 and optimism corrected auc of 0.70). external validation showed good calibration (p = 0.9) and discriminative ability (auc 0.73; 95% ci 0.63 to 0.83). conclusions and discussion: this easily applicable diagnostic model for sensitization to flour and enzymes shows reasonable diagnostic accuracy and external validation. abstract background: in the netherlands the baking and flour producing industries (3,000 small bakeries, 80 industrial bakeries, and 50 flour manufactures) agreed to reduce the high rate (up to 30%) of occupational related allergic diseases. objectives: to conduct health surveillance for early detection of occupational allergic diseases by implementing a diagnostic model as triage instrument. design and methods: in the preparation phase, a validated diagnostic regression model for sensitization to wheat and or a-amylase allergens was converted into score chart for use in occupational health practice. two cut off points of the sum scores were selected based on diagnostic accuracy properties. in the first phase, a questionnaire including the diagnostic predictors from the model was applied in 10.000 bakers. surveillance simulation was done in 4194 bakers recently enrolled in the surveillance. workers with high questionnaire scores were referred for advanced medical examination. results: implementing the diagnostic questionnaire model yielded 59%, 23%, and 18% bakers in the low, intermediate, and high score groups. workers with high scores showed the highest percentage of occupational allergic diseases. conclusions and discussion: with proper cut off points for referral, the diagnostic model could serve as triage instrument in health surveillance to early detect occupational allergic diseases. abstract background: the prevalence of cardiovascular risk factors in spain is high but myocardial infarction incidence is lower than in other countries. objective: to determine the role of basic lipid profile on coronary heart disease (chd) incidence in spain. methods: a cohort of 5,732 healthy spanish individuals aged 35 to 74 years was followed for 5 years. the end-points were fatal and non-fatal myocardial infarction, and angina. results: the 180 participants who developed a coronary end-point were significantly older (62 vs 56), more often diabetic (30% vs 16%), smoker (39% vs 24%) and hypertensive (65% vs 44%) than the rest, and their average total and hdl-cholesterols (mg/dl) were: 233 vs 232 (ns) and 47 vs 54, (p<0.001), respectively. chd incidence among individuals with low hdl levels (<40 in men/<46 in women) was higher than in the rest: 11.7&aeyear-1 vs 7.3&aeyear-1 (p<0.05) in men, and 8.8&aeyear-1 vs 3.2&aeyear-1 (p<0.001) in women. hdl-cholesterol was the only lipid related variable significantly associated with chd: hazard ratio for 1 mg/dl increase was 0.98 (95% ci:0.96-0.99) in men, and 0.96 (95% ci:0.93-0.98) in women, after adjusting for classical risk factors. conclusion: hdl-cholesterol is the only classical lipid variable associated with chd incidence in spain. abstract background: it is widely recognized that health service interventions may reduce infant mortality/imr rate which usually occurs alongside with economic growth. however, there are reports showing that imr decrease under adverse economic and social conditions, indicating the presence of other unknown determinants. objective: this study aims to analyze temporal tendency of infant mortality in brazil during a recent period (1980 to 1998) of economic crisis. methods: temporal series study using data from the mortality information system, censuses (ibge) and epidemiological information (funasa). applying arima -autoregressive integrated moving average, it was described series parameters and, spearman correlation coefficients were used to evaluate the association between infant mortality coefficient and some determinants. results: the infant mortality showed a declining tendency ()59.3%) and strong correlation to the majority of the indicators analyzed. however, only correlation between infant mortality coefficient and total fecundity and birth rates differed significantly within decades. conclusions/discussion: fecundity variation was responsible to the persistence of mortality decline during the eighties. in the next period those indicators of life conditions, mostly health care, could be more important. abstract background: across european union (eu) member states, considerable variation exists in the structure and performance of surveillance systems for communicable disease prevention and control. objectives: the study aims to support the improvement of surveillance systems of communicable diseases in europe while using benchmarking for the comparison of national surveillance systems. design and methods: surveillance systems from england & wales, finland, france, germany, hungary and the netherlands were described and analysed. benchmarking processes were performed with selected criteria (e.g. case definitions, early warning systems). after the description of benchmarks, best practices were identified and described. results: the six countries have in general wellfunctioning communicable disease control and prevention systems. nevertheless, different strengths and weaknesses in could be identified. practical examples for best practice from various surveillance systems demonstrated fields for improvement. conclusion and discussion: benchmarking national surveillance systems is applicable as a new tool for the comparison of communicable disease control in europe. a gold standard of surveillance systems in various countries is very difficult to achieve because of heterogeneity (e.g. in disease burden, personal and financial resources). however, to improve the quality of surveillance systems across europe, it will be useful to benchmark surveillance systems of all eu member states. abstract background: therapeutic decisions in osteoarthritis (oa) often involve trade-offs between accepting risks of side effects and gaining pain relief. data about the risk levels patients are willing to accept are limited. objectives: to determine patients' maximum acceptable risk levels (marls) for different adverse effects from typical oa medications and to identify the predictors of these risk attitudes. design and methods: marls were measured with a probabilistic threshold technique for different levels of pain relief. baseline pain and risk levels were controlled for in a 2x2 factorial design. clinical and sociodemographic characteristics were assessed using a selfadministered questionnaire. results: for 196 subjects, marls distributions were skewed, and varied by level of pain relief, type of adverse effect, and baseline risk level. given a 0% baseline risk, for a 2-point (0-10 scale) pain benefit the mean (median) marls were 3.0% (0%) for heart attack/stroke; 5.7% (0%) for stomach bleed; 13.4% (4.5%) for hypertension; and 23.4% (10.5%) for dyspepsia. most clinical and sociodemographic factors were not associated with marls. conclusion: subjects were willing to trade substantial risks of side effects for pain benefits. this study provides new data on risk acceptability in oa patients that could be incorporated into practice guidelines for physicians. background: several independent studies have shown that single genetic determinants of platelet aggregation are associated with increased ihd risk. objectives: to study the effects of clustering prothombotic (genetic) determinants on the prediction of ihd risk. design and methods: the study is based on a cohort of 17,357 women, aged 49 to 70 years, who were followed from 1993 to 1999. during this period, there were 200 women with registered ihd (icd9 410-414) . a nested case cohort analysis was performed to study the relation of plasma levels vwf and fibrinogen, blood group genotype and prothrombotic mutations in the gene of a2b1, gpvi, gpib and aiibb3 to ihd. results: blood group ab, high vwf concentrations and high fibrinogen concentrations were associated with increased incidence of acute ihd. when the effects of blood group ab/o genotype, plasma levels fibrinogen and vwf were clustered with a score, there was a convincing relationship between a high prothrombotic score and increased incidence of acute ihd: the full-adjusted hr (95% confidence interval) was 3.2 (1.4-6.7) for women with the highest score when the lowest score was taken as reference. conclusions: clustering of prothrombotic markers is a major determinant of increased incidence of acute ihd. abstract background: studies have revealed heart rate variability (hrv) was a predictor of hypertension; however its 24h-recording has not been analysed with the 24-hour ambulatory blood pressure. objective: we studied the relationship between hrv and blood pressure. methods: hrv and blood pressure were measured by 24-hour ambulatory recordings, in randomly selected population without evidence of heart disease. cross-sectional analyses were conducted in 571 women and 372 men (mean age: 65.64±0.79). hrv values, measured by the standard deviation of rr intervals (sdnn), were compared after logarithmic transformation between the blood pressure levels (135/85 mmhg), using analysis of variance. stepwise multiple-regression was performed to assess on sdnn the cumulative effects of systolic and diastolic blood pressure, clinical obesity, fasting glycaemia, c-reative protein, treatments, smoking and alcohol consumption. results: sdnn was lower in hypertensive men and women (p<0.05), independently of drug treatments. after adjustment for factors associated with hypertension, sdnn was no more associated with hypertension, but with obesity, glycaemia and c-reative protein in both genders. sdnn was negatively associated with diastolic blood pressure in men (p = 0.03) in the multivariate approach. conclusion: whereas blood pressure levels were not related to the sdnn in the multivariate analysis, diastolic blood pressure contributed to sdnn in men. it has been proposed that n-3 fatty acids may protect against the development of allergic disease, while n-6 fatty acids may promote its development. in the piama (prevention and incidence of asthma and mite allergy) study we investigated associations between breast milk fatty acid composition of 158 allergic and 107 non allergic mothers and allergic disease (doctor diagnosed asthma, eczema or hay fever) in their children at the age of 1 year and at the age of 4 years. in children of allergic mothers prevalences of allergic disease at age 1 and at age 4 were relatively high if the breast milk they consumed had a low content (wt%) of n-3 fatty acids and particularly of n-3 long chain polyunsaturates (lcps), a low content of trans fatty acids, or a low ratio of n-3lcps/n-6lcps. the strongest predictor of allergic disease was a low breast milk n-3lcps/n-6lcps ratio (odds ratios (95% ci) of lowest vs highest tertile, adjusted for maternal age, parity and education: 2.80 (1.07 to 7.28) for allergic disease at age 1 and 2.86 (1.09 to 7.52) for allergic disease at age 4). in children of non allergic mothers no statistically significant associations were observed. abstract background/relevance: to find out about the appropriateness of using two vision related quality of life instruments to measure outcome of visually impaired elderly in a mono-and multidisciplinary rehabilitation centre. objective/design: to evaluate sensitivity of the vision quality of life core measure (vcm1) and the low vision quality of life questionnaire (lvqol) to measure changes in vision related quality of life in a non-randomised followup study. methods: visually impaired patients (n=319) recruited from 4 ophthalmology departments administered questionnaires at baseline (2000) (2001) (2002) (2003) , 3 months and 1 year after rehabilitation. person measures were analysed using rasch analyses for polytomous rating scales. results: paired sample t-tests for the vcm1 showed improvement at 3 months (p = 0.02; effect size = 0.12 and p = 0.003; effect size=0.15) for the monodisciplinary and the multidisciplinary groups respectively. at 1 year only the multidisciplinary group showed improvement on the vcm1 (p = 0.03; effect size = 0.12). on the lvqol, no significant improvement or deterioration was found for both groups. discussion: although, vcm1 showed improvement in vision related quality of life over time, the effect sizes appeared to be quite small. we conclude that both instruments lack sensitivity to measure changes. another explanation is that rehabilitation did not contribute to quality of life improvements. abstract background: the natural history of asthma severity is poorly known. objective: to investigate prognostic factors of asthma severity. methods: all current asthmatics identified in 1991/93 in the european community respiratory health survey were followed up and their severity was assessed in 2002 by using the global initiative for asthma categorization (n = 856). asthma severity was related to baseline/follow-up potential determinants by a multinomial logistic model, using intermittent asthmatics as reference category for relative risk ratios (rrr). results: patients in the lowest/highest levels of severity at baseline had an 80% likelihood of remaining in a similar level. severe persistent had a poorer fev1%predicted at baseline, higher ige levels (rrr=2.06;95% ci:1.38-3.06), higher prevalence of chronic cough/phlegm (4.90;2.18-11.02) than intermittent asthmatics. moderate persistent showed similar associations. mild persistent were similar to intermittent asthmatics, although the former showed a poorer control of symptoms than the latter. subjects in remission had a lower probability of an increase in bmi than current asthmatics (0.86;0.75-0.97). allergic rhinitis, smoking, respiratory infections in childhood were not associated with severity. conclusion: asthma severity is a relatively stable condition, at least for patients at the two extremes of the severity spectrum. high ige levels and persistent cough/phlegm are strong markers of moderate/severe asthma. abstract background: thyroid cancer (tc) has a low, yet growing, incidence in spain. ionizing radiation is the only well established risk factor. objectives: this study sought to depict the municipal distribution of tc mortality in spain and to argue about possible risk factors. design and methods: posterior distribution of relative risk for tc was computed using a single bayesian spatial model covering all municipal areas of spain (8, 077) . maps were plotted depicting standardised mortality ratios, smoothed municipal relative risk (rr) using the besag, york and mollie`model, and the distribution of the posterior probability that rr>1. results: from 1989 to 1998 a total of 2,538 tc deaths were registered in 1,041 municipalities. there was a higher risk of death in some areas of canary islands, galicia and asturias. abstract igf-i is an important growth factor associated with increased breast cancer risk in epidemiological and experimental studies. lycopene intake has been associated with decreased cancer risk. although some data indicate that lycopene can influence the igfsystem, this has never been extensively tested in humans. the purpose of this study is to evaluate the effects of a lycopene intervention on the circulating igf-system in women with an increased risk of breast cancer. we conducted a randomized, placebo-controlled cross-over intervention study on the effects of lycopene supplementation (30 mg/day, 2 months) in pre-menopausal women with 1) history of breast cancer (n = 29) and 2) high familial breast cancer risk (n = 47). drop-out rate was 21%. mean igf-i and igfbp-3 concentrations after placebo were 175.8±51.2 ng/ml and 2.54±0.42 mg/ml respectively. lycopene supplementation did not significantly alter serum total igf-i (mean lycopene effect: )1.4 ng/ml; 95% ci: )8. 2-5.4 ) and igfbp-3 ()0.002 mg/ml; )0.052-0.056) concentrations. dietary energy and macronutrient intake, physical activity, body weight, and serum lycopene concentrations were assessed, and are currently under evaluation. in conclusion, this study shows that 2 months lycopene supplementation has no effect on serum igf-system components in a high risk population for breast cancer. abstract introduction: patients who experience burden during diagnostic tests may disrupt these tests. the aim was to describe the perception of melanoma patients with lymph node metastases of the diagnostic tests. methods: patients were requested to complete a self-administrated questionnaire. experienced levels of embarrassment, discomfort and anxiety were calculated, as well as (total) scores for each burden. the non-parametric friedman test for related samples was used to see if there was a difference in burden. results: the questionnaire was completed by 34 patients; response rate was 87%. overall satisfaction was high. in total 26% felt embarrassment, 27% discomfort and 39% anxiety. overall, 31% felt some kind of burden. there was no difference in anxiety between the three tests. however, patients experienced more embarrassment and discomfort during the pet (positron emission tomography) scan (p = 0.027 and p = 0.002). conclusion: overall levels of burden were low. however, patients experienced more embarrassment and discomfort during the pet scan, possibly as a result of lying immobile for a long time. the accuracy, costs and patients upstaged will probably be the most important to determine the additional value of fdg-pet and ct, but it is reassuring to know that only few patients experience severe or extreme burden. abstract gastric cancer (gc) is the second most frequent cause of cancer death in lithuania. some intercultural aspects of diet that is related to the outcome could be the risk factors of the disease. the objective of the study was to assess an associations between risk of gc and dietary factors. a case-control study included 379 cases with diagnose of gc and 1137 controls that were cancer and gastric diseases free. a questionnaire used to collect information on possible risk factors. the odds ratios (or) and 95% confidence intervals (ci) estimated by the conditional logistic regression model. after controlling for possible confounders that were associated with gc, use of salted meat (or = 2.21; 95% ci = 1.43-3.42; >1-2 times/week vs. almost never) smoked meat (or = 1.79; 95% ci = 1.23-2.60; >1-4 times/week vs. less), smoked fish (or = 1.70; 95% ci = 1.13-2.53; >1-2 times/week vs. less) was associated with increased risk of gc. higher risk of gc was associated with frequent use of butter, eggs and noodles. while frequent consumption of carrots, cabbages, broccolis, tomatoes, garlic, beans decreased the risk significantly. the data support a role of salt processed food and some animal foods in increasing the risk of gc and plant foods in reducing the risk of the disease. abstract background: standards for the evaluation of measurement properties of health status measurement instruments (hsmi), including explicit criteria for what constitutes good measurement properties, are lacking. nevertheless, many systematic reviews have been performed to compare and select hsmi, using different criteria to judge the measurement properties. objectives: (1) to determine which measurement properties are reported in systematic reviews of hsmi and how these properties are defined, (2) which standards are used to define how measurement properties should be assessed, and (3) which criteria are defined for good measurement properties. methods: a systematic literature search was performed in pubmed, embase and psychlit. articles were included if they met the following inclusion criteria: (1) systematic review, (2) hsmi were reviewed, and (3) the purpose of the review is to identify all measurement instruments assessing (an aspect of) health status and to report on the clinimetric properties of these hsmi. two independent reviewers selected the articles. a standardised data-extraction form was used. preliminary results: 103 systematic reviews were included. conclusions: large variability in standards and criteria used for evaluating measurement properties was found. this review can serve as basis for reaching consensus on standards and criteria for evaluating measurement properties of hsmi. abstract residential exposure to nitrogen dioxide is an air quality indicator and could be very useful to assess the effects of air pollution on respiratory diseases. the present study aims at developing a model to predict residential exposure to no2, combining data from questionnaires and from local monitoring stations (ms). in the italian centres of verona, torino and pavia, participating in ecrhs-ii, no2 concentrations were measured using passive samplers (ps-no2) placed outside the kitchen of 340 subjects for 14 days. simultaneously, average no2 concentrations were collected from all the mss of the three centres (ms-no2). a multiple regression model was set up with ps-no2 concentrations as response variable and questionnaire information and ms-no2 concentrations as predictors. the model minimizing the root mean square error (rmse), obtained from a ten fold cross validation, was selected. the model with the best predictive ability (rmse=12.20), had as predictors: ms-no2 concentrations, season of the survey, centre, type of building, self-reported intensity of heavy vehicle traffic. the correlation coefficient between predictive and observed values was 0.79 (95% ci: 0.75-0.83). in conclusion, this preliminary analysis suggests that the combination of questionnaire information and routine data from the mss could be useful to predict the residential exposure to no2. abstract background: currently only 18% of dutch mothers comply with the who recommendation to give exclusive breastfeeding for at least six months. therefore, the dutch authorities consider policies on breastfeeding. objectives: quantification of the health effect of several breastfeeding policies. methods: a systematic literature search of published epidemiological studies conducted in the general 'western' population. based on this overview a model is developed. the model simulates incidences of 11 diseases of mother and child depending on the duration that mothers breastfeed. each policy corresponds to a distribution in the duration of breastfeeding. the health effects of each policy are compared to the present situation. results: breastfeeding has beneficial health effects on both the short and the long term for mother and child. the longer the duration of breastfeeding, the larger is the effect. most public health gain is achieved by introducing breastfeeding to all newborns rather than through a policy focusing just on extending the lactation period of women already breastfeeding. conclusion: breastfeeding has positive health effects. policy should focus preferentially on encouraging all mothers to start with breastfeeding. abstract background: constant increase of international trade and travel activities has risen the significance of pandemic infectious diseases worldwide. the 2002/2003 sars outbreak rapidly spread from china to 28 countries, from which 9 were located in europe. in order to control and prevent pandemic infections in europe, systematic and effective public health preparation by every member state is essential. method: supported by the european commission, surveys focusing on national sars (september 2003) and influenza (october 2005) preparedness were accomplished. a descriptive analysis was undertaken to identify differences in european infectious disease policies. results: guidelines and guidance for disease management were well established in most european countries. however, the application of control measures, like e.g. measures for mass gatherings or public information policies, had varied among member states. discussion: the results show that european countries are aware of preparing for pandemic infections. yet, the effectiveness of certain control measures is analysed insufficiently. further research and detailed knowledge about factors influencing international spread of diseases is required. 'hazard analysis of critical control points' (haccp) will be applied to evaluate national health response in order to provide comprehensive data for recommendations to european pandemic preparedness. abstract background: influenza is still an important problem for public health. knowing its space-time evolution is of special interest in order to carry out prevention plans. objectives: to analyze the geographical diffusion of the epidemic wave in extremadura. methods: the influenza incidence absolute rates in every town have been calculated, according to the registered cases per week in the compulsory disease declaration system. continuous maps have been represented using a geographical interpolation method (inverse distance weighting (idw) was applied with weighting exponents of 2). results: the 2004/05 season began in the 40th week of 2004, with a small influenza incidence. there have been concrete cases in those towns until the 46th week. punctual areas diffusion in the north and southwest of the region between the 46th and the 51st weeks. the highest incidence appeared between the 52nd week of 2004 and the 3rd of 2005. influenza cases started to decrease in the northwest and north of the region from the 3rd week of 2005, till the 10th week, when most of the cases were found in the southwest. conclusion: there is a space-time diffusion of influenza, due to the higher population density. we propose to analyze these data combining temperature information. abstract background: acute lower respiratory tract infection (lrti) can cause various complications leading to morbidity and mortality notably among elderly patients. antibiotics are often given to prevent complications. to minimise costs and bacterial resistance, antibiotics are only recommended in case of pneumonia or in patients at serious risk for serious complications. objective: we assessed the course of illness of lrtis among dutch elderly primary care patients and assessed whether gps were inclined to prescribe antibiotics more readily to patients at risk for complications. methods: we retrospectively analysed medical data from 3,166 episodes of lrti among patients? 65 years of age presenting in primary care to describe the course of illness. the relation between prescriptions of antibiotics and patients with risk factors for a complicated course was assessed by means of multivariate logistic regression. results: in episodes of acute bronchitis antibiotics were more readily prescribed to patients aged 90 years or older. in exacerbations of copd or asthma gps favoured antibiotics in male patients and when diabetes, neurological disease or dementia was present. conclusion: gp's do not take all high risk conditions into account when prescribing antibiotics to patients with lrti despite recommendations of national guidelines. abstract background: the putative association between antidepressant treatment and increased suicidal behaviour has been under debate. objectives: to estimate the risk of suicide, attempted suicide, and overall mortality during antidepressant treatments. design and methods: study cohort consisted all subjects without non-affective psychosis, hospitalized due to a suicide attempt during the years 1997 -2003 , followed up by using nationwide registers. main outcome were completed suicides, attempted suicides, and mortality. main explanatory variable was antidepressant usage. results: 470 suicides, 4411 suicide attempts and 1263 deaths were observed. when the effect of background variables was taken into account, the risk of suicide attempts was increased markedly during antidepressant treatment (rr for selective serotonin reuptake inhibitors or ssri 1.34, 1.19-1.50) compared with no antidepressants. however, the risk of completed suicides was not increased. a lower mortality was observed during ssri use (rr 0.67, 0.54-0.84), which was mainly attributable to decrease in cardiovascular deaths. conclusion and discussion: in this suicidal high risk cohort the use of any antidepressant is associated with an increased risk of suicide attempts, but not with the increased risk of completed suicide. antidepressants and, especially, ssri use is associated with a substantial decrease in cardiovascular deaths and overall mortality. abstract background: the quattro study is a rct on the effectiveness of intensified preventive care in primary health care centres in deprived neighbourhoods. additional qualitative research on the execution of interventions in primary care was considered necessary for the explanation of differences in effectiveness. objectives: our question was: can we understand rct outcomes better with qualitative research? design and methods: an ethnographic design was used. in their daily work we observed 2 researchers for 8 months 2 days a week, and 4 practice nurses for 5 days each. two other practice nurses were interviewed. all transcribed observations were analysed thematically. results: from the rct showed differences in effectiveness among the centres and that intensified preventive care provided no additional effect compared to structural physical measurements. ethnographic results show that these differences are due to variations in execution of the intervention among the centres. conclusion: in conclusion ethnographic analysis showed that differences in execution of intervention lead to differences in rct outcomes. the rct conclusion 'no additional effect' is problematic. discussion as variations in primary care influence a rcts' execution they create methodological problems for research. to what extent can additional qualitative research improve rct research. abstract background: acute myocardial infarction is the most important cause of morbidity from ischemic heart disease (ihd) and is the leading cause of death in the western world. objectives: to assess the benefits and harms of 'dan shen' compound for acute myocardial infarction. methods: we searched the cochrane controlled trials register on the cochrane library, medline, embase, chinese biomedical database and the chinese cochrane centre controlled trials register. we included randomized controlled studies lasting at least 7 days. main results: eleven studies with 1196 participants in total were included. seven studies compared the mortality in routine treatment plus 'dan shen' compound and single routine treatment. one trial compared the arrhythmia in routine treatment plus 'dan shen' compound injection and single routine treatment. two trials compared the revascularization in routine treatment plus 'dan shen' compound injection and single routine treatment. conclusions: evidence is insufficient to recommend the routine use of 'dan shen' compound because of the small number of included studies and their low quality. no well designed randomized controlled trials with adequate power to provide a more definitive answer have been conducted. in addition, the safety of 'dan shen' compound is unproven, though adverse events were rarely reported. abstract antimicrobial resistance is emerging. to identify the scope of this threat and to be able to take proper actions and evaluate these, monitoring is essential. the remit of earss is to maintain a comprehensive surveillance system that provides comparable and validated data on the prevalence and spread of major invasive bacteria with clinically and epidemiologically relevant antimicrobial resistance in europe. since 2000, earss collects routine antimicrobial susceptibility test (ast) results of invasive isolates of five indicator bacteria, tested according to standard protocols. in 2004, ast results for 80,000 isolates were provided by 800 laboratories, serving 1200 hospitals, covering 100 million inhabitants in 30 countries. through a biannual questionnaire denominator information was collected. the quality of ast results of laboratories was evaluated by the yearly external quality assessment. currently, earss includes all member and candidate states (29) of the european union, plus israel, bosnia, bulgaria and turkey. participating hospitals treat a wide range of patients and laboratory results are of sufficient validity. earss identified antimicrobial resistance time trends and found a steady increase for most pathogen-compound combinations. in conclusion, earss is a comprehensive system with sufficient quality to show that antimicrobial resistance is increasing in europe and threatens health-care outcomes. abstract introduction: since chloroform has been detected in drinking waters, the number of studies has increased to identify the presence of trihalomethanes (thms) in drinking waters, as well as to establish the possible effects they may have population health. objectives: to determine thms levels in the water distributing network in the city of valencia. design and methods: over a one-year period, 16 points of the drinking water distributing netowrk have undergone sampling at 1 week intervals. the concentration of these pollutants was determined by gas chromatography. results: our results showed greater concentrations of the species substituted by chlorine and bromine atoms (dichlorobromomethane and dibromochloromethane) in the range of 10-20z lg/l for both, 0-10 lg/l for trichloromethane and between 0-5 lg/l for tribromomethane. an increase in thms concentration was observed in those points near the sea, although they did not exceed the legal limit of 100lg/l. conclusion: we established two areas of concentration of these species in water: high and average, according to their proximity to the sea. abstract background: childhood cancer survivors are known to be at increased risk for second malignancies. objectives: we studied longterm risk of second malignancy in 5-year survivors, according to therapy and follow-up interval. methods: the risk of second malignancies was assessed in 1368 5-year survivors of childhood cancer treated in the emma children's hospital amc in amsterdam and compared with incidence in the general population of the netherlands. complete follow-up till at least january 2001 was obtained for 91.9% of the patients. the median follow-up time was 16.8. results: sixty second malignancies were observed against 5.4 expected, yielding a standardized incidence ratio (sir) of ). the absolute excess risk (aer) was 3.2 per 1,000 persons per year. the sir appeared to stabilize after 15 years of follow-up, but the absolute excess risk increased with longer follow-up (aer follow-up > = 25 years of 8.24). patients who were treated with radiotherapy experienced the greatest increase of risk. conclusions: in view of the quickly increasing background rate of cancer with ageing of the cohort, it is concerning that even after more than 20 years of follow-up the sir is still increased, as is the absolute excess risk. the chek2*1100delc germline variant has been shown to increase susceptibility for breast cancer and could have an impact on breast cancer survival. this study aimed to determine the proportion of chek2*1100delc germline mutation carriers, and breast cancer survival and tumor characteristics, compared to non-carriers in an unselected (for family history) breast cancer cohort. women with invasive mammary carcinoma, aged <50 years and diagnosed in several dutch hospitals between 1973 and 1995, were included. for all patients, paraffin embedded tissue blocks were collected for dna isolation (normal tissue), and subsequent mutation analyses, and tumor revision. in 1479 breast cancer patients, 54 (3.7%) chek2*1100delc carriers were detected. chek2*1100delc tumors characteristics, treatment and patient stage did not differ from those of non-carriers. chek2*1100delc carriers had 2 times increased risk of developing a second breast cancer compared to non-carriers. with a mean follow up of 10 years, chek2*1100delc carriers had worse recurrence free and breast cancer specific survival than non-carriers. in conclusion, this study indicates a worse breast cancer outcome in chek2*1100-delc carriers compared to non-carriers. the extension of the presence of the chek2*1100delc germline mutation warrants research into therapy interaction and possibly into screening of premenopausal breast cancer patients. abstract background: for primary or secondary prevention (e.g. myocardial infarction) hormone therapy (ht) is no longer recommended in postmenopausal women. however, physicians commonly prescribe ht to climacteric women as a treatment of hot flashes/night sweats. objective: to assess efficacy and adverse reactions of ht in climacteric women with hot flashes (including night sweats). methods: for our systematic review (sr), we searched databases (medline, embase, cochrane) for randomized controlled trials, other srs and meta-analyses, published 1999 to 2004. the quality of the studies was assessed using checklists corresponding to the study type. results: we identified 18 studies of good/excellent quality. they included predominantly caucasian women and lasted 3-12 months. in all studies, ht showed a reduction of 75-95% in the number of hot flashes, which was significantly better than placebo. most common adverse events of ht were uterine bleeding and breast pain/tenderness. cardiovascular diseases and neoplasms were reported only sporadically. conclusions: ht is highly effective in treating hot flashes in climacteric women. however, to assess serious adverse events longer studies (including also non-caucasian women) are needed, as there are only sparse data available. abstract igf-i is an important growth factor, and has been associated with increased colorectal cancer risk in both prospective epidemiological and experimental studies. however, it is largely unknown which lifestyle factors are related to circulating levels of the igf-system. studies investigating the effect of isoflavones on the igf-system have thus far been conflicting. the purpose of this study was to evaluate the effects of isoflavones on the circulating igf-system in men with high colorectal cancer risk. we conducted a randomized, placebo-controlled, cross-over study on the effect of a 2-month isoflavone supplementation (80 mg/day) on igf levels in 40 men with a family history of colorectal cancer or a personal history of colorectal adenomas. dropout rate was 5%, and all but 3 men were more than 80% compliant. isoflavones supplementation did not significantly alter serum total igf-i ()1.6%; 95%ci: )9.3 -6.1) and igf binding protein 3 (+0.7%, 95%ci: )3.5 -4.8) concentrations. other covariables, e.g. dietary energy and macronutrient intake, physical activity, and body weight, are currently under evaluation. in conclusion, this study shows that a 2-month isoflavone supplementation has no effect on serum igf-system components in men with high colorectal cancer risk. abstract background/objective: eurociss-european cardiovascular indicators surveillance set project, funded under the health monitoring programme of european commission, aims developing health indicators and recommendations for monitoring cardiovascular diseases (cvd). methods: prioritise cvd according to their importance in public health; identify morbidity and mortality indicators; develop data collection and harmonizing recommendations; describe data collection, validation procedures and discuss their comparability. population (geographical area, age, gender), methods (case definition, icd codes), procedures (record linkage, validation), morbidity indicators (attack rate, incidence, case fatality) collected by questionnaire. results: the main outcome was the inventory of acute myocardial infarction (ami) populationbased registers in the 18 european partner countries: 8 countries have no register, 10 regional, 4 of which also national. registers differ for: icd codes (only ami or also acute and subacute ischemic forms), ages (35-64, 35-74, all) , record linkage (probabilistic, personal identification number), calendar years, validation (monica, esc/acc diagnostic criteria). differences make morbidity indicators difficult to compare. conclusion: new diagnostic criteria led to a more exhaustive definition of myocardial necrosis as acute coronary syndrome (acs). given the high burden of ami/acs, efforts are needed to implement population-based registers in all countries. application of recommended indicators, validated through standardized methodology, will provide reliable, valid and comparable data. abstract objective: the objective of this paper was to compare and discuss the use of odd ratios and prevalence ratios using real data with complex sampling design. method: we carried out a cross-sectional study using data obtained from a two-stage stratified cluster sample from a study conducted in 2001-2002 (n = 1,958) . odds ratios and prevalence ratios were obtained by unconditional logistic regression and poisson regression, respectively, for later comparison using the stata statistical package (v. 7.0). confidence intervals and design effects were considered in the evaluation of the precision of estimates. two outcomes of a cross-sectional study with different prevalence were evaluates: vaccination against influenza (66.1%) and self-referred lung disease (6.9%). results: in the high-prevalence scenario, using prevalence ratios the estimates were more conservative and we found narrower confidence intervals. in the low-prevalence scenario, we found no important differences between the estimates and standard errors obtained using the two techniques. discussion: however, it is the researcher's task to choose which technique and measure to use for each set of data, since this choice must remain within the scope of epidemiology. abstract background: in italy coronary heart disease chd mortality has been falling since the 1970s. objective: to examine how much of the fall between 1980 and 2000 could be attributed to trends in risk factors, medical and surgical treatments. methods: a validated model was used to combine and analyse data on uptake and effectiveness of cardiological treatments and risk factor trends. published trials, meta-analyses, official statistics, longitudinal studies, surveys are main data sources. results: chd mortality fell by 41% in men and 43% in women aged 25-84; 42,930 fewer deaths in 2000. approximately half mortality fall was attributed to treatments in patients and half to population changes in risk factors: in men, mainly improvements in cholesterol (39%) and smoking (33%) rather than blood pressure (6%). in women 1/3 mortality fall attributable to improvements in cholesterol (31%) and blood pressure (5%); adverse trends in smoking ()14%). adverse trends also in bmi ()2% in both genders) and diabetes ()4% in men; )0.5% in women). conclusion: half chd mortality fall was attributable to risk factors reductions, principally cholesterol in men and women and smoking in men; in women rising smoking rates generated substantial additional deaths. a comprehensive strategy promoting primary prevention is needed. objective: to investigate the efficacy of ni in the post exposure prophylaxis (pep), i.e. in persons who had contact with an influenza case. design and methods: we conducted a systematic electronic data base review for the period between 1999 and 2004. studies were selected and graded by two independent reviewers. the proportion of influenza-positive patients was chosen as primary outcome. for all analyses fixed effect models were used. weighted relative risks (rr) and 95% confidence intervals (ci) were calculated on an intention-to-treat basis. results: 5 randomized controlled trials (n=3,663) were included in the analysis. zanamivir and oseltamivir were effective against an infection with influenza (rr=0.23, 95% ci 0.15-0.36 and rr=0.20, 95% ci 0.11-0.34, respectively). prophylactic efficacy was comparable in the subgroup of persons who had contact with an index case with lab-confirmed influenza (4 studies, all ni, rr=0.18, 95% ci 0.12-0.28). conclusions: the available evidence suggests that ni are effective in the pep of influenza. discussion: results have to be interpreted with caution when transferred into general medical practice because study populations mainly included young and healthy adults without chronic diseases. abstract an important risk factor of breast cancer, mammographic breast density (mbd) is inversely associated with reproductive factors (age at first childbirth, and lactating). as pregnancy and lactating are highly correlated, whether this decline is induced by pregnancy or lactating is still unclear. we hypothesize that lactation reduces mbd independent of age at first pregnancy and parity. a study was done on 4051 women in the third sub-cohort of the dom project who had complete data regarding lactating, dy, had a child but varied by duration of lactating. multiple logistic regression analysis was done using dy (yes/ no) as outcome variable. explanatory variables added into the model were age, bmi, parity and age at first childbirth. a significant univariate relation was seen between lactating of the first child and dy. or 0.83 (ci 95% 0.73; 0.94). adjusted for explanatory variables, the or changed to 0.89 (ci 95% 0.73; 1.08). lactating seems to contribute independently to the reduction of mbd over and above pregnancy itself. given the limitations of the dichotomous dy ratio scores, additional studies will address which part; either glandular mass or fat tissue is responsible for the observed relation which will be measured from mammograms to be digitized. abstract background: alcohol consumption is common, but little is known about whether drinking patterns vary across geographic regions. objectives: to examine potential disparities in alcohol consumption across census regions and urban, suburban, and rural areas of the united states. design and methods: the data source was the national epidemiologic survey on alcohol and related conditions, an in-person interview of approximately 43,000 adults. the prevalence of abstinence and, among drinkers, the prevalences of heavy and daily drinking were calculated by census region and metropolitan status. multivariate logistic regression analyses were conducted to test for differences in abstinence and per drinker consumption after controlling for confounders. results: the odds of abstinence, heavy, and daily drinking varied widely across geographic areas. additional analyses stratified by census region revealed that rural residents in the south and northeast as well as urban residents in the northeast had higher odds of abstinence. rural residents in the midwest had higher odds of heavy drinking. conclusion and discussion: heavy alcohol consumption is of particular concern among drinkers living in the rural areas of the united states, particularly the rural midwest. other nations should consider testing for similar differences as they implement policies to promote safe alcohol consumption. abstract background: long-term exposure to particulate air pollution (pm) has been suggested to accelerate atherogenesis. objective: we examined the relationship between long-term exposure to traffic emissions and the degree of coronary artery calcification (cac), a measure of atherosclerosis. methods: in a population based, crosssectional study, distances between participants' home addresses and major roads were calculated with a geographic information system. annual mean pm2.5-exposure at the residence was derived from a small scale geostatistical model. cac, assessed by electronbeam computer tomography, was modelled with linear regression by proximity to major roads, controlling for background pm2.5air pollution and individual level risk factors. results: of 4424 participants 499 lived within 150 m of a major road. background-pm2.5 ranged from 16.1 to 26.8 lg/m3 (mean 22.8). mean cacvalues were strongly dependent on age, sex and smoking status. reducing the distance to major roads by 50% leads to increases in cac by 10.1% (95%ci 0.6-20.5%) in the unadjusted model and 5,5% (95%ci )2. 2-13.8 ) in the adjusted model. stronger effects (adjusted model) were seen in men (7,4%, 95%ci )3.9-20.1) and male non-smokers (10,5%, 95%ci )3.2-13.0). conclusions: this study provides epidemiologic evidence that long-term exposure to traffic emissions is an important risk factor for coronary calcification. abstract background: this polymorphism has been associated risk factor levels and in one study with a reduced risk of acute myocardial infarction (ami). yet, the risk relation has not been confirmed. objectives: we investigated the role of this polymorphism on occurrence of ami, coronary heart disease (chd) and stroke in healthy dutch women. design and methods: a case-cohort study in a prospective cohort of 15236 initially healthy dutch from1993 until january 1st 2000. results: we applied a cox proportional hazards model with an estimation procedure adapted for case-cohort designs. a lower ami (n=71) risk was found among carriers of the ala allele (n=364) compared with those with the more common pro12pro genotype (hazard ratio=0.51; 95% ci, 0.26 to 1.00). no relation was found for chd (n=211;hr 0.82; 95% ci, 0.58-1.17) and for stroke (n=74;hr 1.41; 95% ci, 0.85-2.33). in our data little evidence was found for a relation between pparg2 and risk factors. conclusion and discussion: this study shows the pro12ala polymorphism in pparg2 gene is modestly related to a reduced risk of ami in our study. no statistically significant relation was found for chd and stroke. abstract background: pseudo cluster randomization was used in a services evaluation trial because individual randomization risked contamination and cluster randomization risked selection bias due to expected treatment arm preferences of recruiting general practitioners (gps). gps were randomized in two groups. depending on this randomization, participants were randomized in majority to one study arm: intervention:control/80:20 or intervention:control/ 20:80. objectives: to evaluate internal validity of pseudo cluster randomization. have gps treatment arm preferences? what is the effect on allocation concealment and selection bias? design and methods: we compared the baseline characteristics of participants to study selection bias. using a questionnaire, gps indicated their treatment arm preferences on a visual analogue scale (vas) and the allocation proportions they believed were used to allocate their patients over treatment arms. results: gps preferred allocation to the intervention (vas 14.5 (sd 15.6); 0-100: 0 indicates strongly favoring the intervention arm). after recruitment 67% of gps estimated a randomization ratio of 50:50 was used. the participants showed no relevant differences at baseline. conclusion and discussion: gps profoundly preferred allocation to the intervention group. few indications of allocation disclosure or selection bias were found in the dutch easycare trial. pseudo cluster randomization proofs to be a valid randomization method. abstract background: epidemiological studies rely on self-reporting to acquire data on participants, although such data are often limited in reliability. the aim here is to assess nuclear magnetic resonance (nmr) based metabonomics for evaluation of self-reported data on paracetamol use. method: four in-depth 24-hour dietary recalls and two timed 24-hour urine collections were obtained for each participant in the intermap study. a 600 mhz 1h nmr spectrum was acquired for each urine specimen (n = 9,943). training and test sets involved two strata, i.e., paracetamol metabolites yes or no in the urine spectra, selected from all 17 population samples by a principal component analysis model. the partial least squares-discriminant analysis (pls-da) model based on a training set of 110 samples was validated by test set (n = 620). the model correctly predicted stratum for 575 of 587 samples (98%) after removal of 33 outliers not fitting the model, sensitivity 95.4%, specificity 100%. this model was used to predict paracetamol status in all intermap specimens. it identified 384 participants (8.2%) who underreported analgesic use, of whom 63 underreported analgesic use in both clinical visits. conclusion: nmr-based metabonomics can be used as a tool to enhance reliability of self-reported data. abstract background: in patients with asthma, the decline in forced expiratory volume in one second (fev1) is accelerated compared with non-asthmatics. objective: to investigate long-term prognostic factors of fev1 change in asthmatics from the general population. methods: a cohort of 667 asthmatics (20-44 years-old) was identified in the frame of the european community respiratory health survey (1991/93), and followed up in 1998/2002. spirometry was performed on both occasions. the annual fev1 decrease (?fev1) was analysed by multi-level regression models, according to age, sex, height, bmi, occupation, familiarity of asthma, hospitalization for asthma (baseline factors); cumulative time of inhaled corticosteroid (ics) use and annual weight gain during the follow-up; lifetime pack-years smoked. results: when adjusting for all covariates, ics use for >2 years significantly reduced ?fev1, with respect to non-users, of 10.4(95%ci: 1.6-19.2) ml/year. ?fev1 was 9.9(0.5-19.4) ml/year lower in women than in men. it increased by 0.7(0.2-1.2) ml/year for every additional year in patient age and by 7.2(3.3-11.0) ml/year for every additional kg/year in the rate of weight gain. conclusion: long-term ics use (>2 years) seems to be associated with a reduced ?fev1 over a 9-year followup. body weight gain seems a crucial factor in determining lung function decrease in asthmatics. abstract background: effectiveness of screening can be predicted by episode sensitivity, which is estimated by interval cancers following a screen. full-field digital or cr plate mammography are increasingly introduced into mammography screening. objectives: to develop a design to compare performance and validity between screen-film and digital mammography in a breast cancer screening program. methods: interval cancer incidence was estimated by linking 721 000 screening visits from 1991-2001 at an individual level to the files of the cancer registry in finland. these data were used to estimate the study size requirements for analyzing differences in episode sensitivity between screen-film and digital mammography in a randomized setting. results: the two-year cumulative incidence of interval cancers per 100 000 screening visits was estimated to be 300. to allow the maximum acceptable difference in the episode sensitivity between screen-film and digital arm to be 20% (80% power, 0.05 significance level, 1:1 randomization ratio, 85% attendance rate), approximately 240 000 women need to be invited. conclusion: only fairly large differences in the episode sensitivity can be explored within a single randomized study. in order to reduce the degree of non-inferiority between the screen-film and digital mammography, meta-analyses or pooled analyses with other randomized data are needed. according to the literature up to 70% of colorectal cancers worldwide is preventable by dietary change. however the results of the epidemiologic studies are not consistent across the countries. the objective of the study is to evaluate the role of dietary nutrients on colorectal cancer risk in poland. the hospital-based case-control study was carried out in 2000-2003. in total, 239 histologically confirmed cancer cases and 548 controls were recruited. adjustment for age, sex, education, marital status, multivitamin use, alcohol consumption, cigarette smoking, family history and energy consumption was done by logistic regression model. low tertile of daily intake in the control group was defined as a reference level. the lower colorectal cancer risk was found in cases with high daily intake of dietary fiber (or = 0,62; 95%ci: 0,42-0,91) and vitamin e (or = 0,67; 95%ci: 0,46-0,99). on the other hand, an increased risk for high monosaccharides consumption was observed. the risk pattern wasn't changed after additional adjustment for physical activity and body mass index. the results of the present study support the protective role of dietary fiber and some antyoxidative vitamins in the etiology of colorectal cancer. additionally they suggest that high consumption of monosaccharides may lead to elevated risk of investigated cancers. abstract assessment of nutrition is very difficult in every population, but in children there's additional question if child can properly recognize and recall foods that have been eaten. the aim of this study was to assess if dietary recall administered to adolescents can be used in epidemiological studies on nutrition. subjects were 100 children, 9-15 years old, and they caretakers. 24-h recall was used to evaluate children's nutrition. both, child and caretaker were asked to recall all products, drinks and dishes eaten by child during the day before recall. the statistical analyses were done separately for each meal. we have noticed statistically significant differenced for intake of energy and almost all nutrients from the lunch. the observed spearman rank correlation coefficients between child and his caretaker ranged from 0.39 for vitamin c up to 0.71 for intake of carbohydrates. only calcium intake (146.16 vs. 123.52 mg/day) differentiated groups for the breakfast and b-carotene for the supper. the study showed that the recall with adolescents could be helpful source of data for the research in the population aspect. however, one shouldn't use such data for the examination of the individual nutritional habit of children, especially information about dinner can be biased. abstract background: acute bronchitis is one of the most common diagnoses made by primary care physicians. in addition to antibiotics, chinese medicinal herbs may be a potential medicine of choice. objectives: this review aims to summarize the existing evidence of comparative effectiveness and safety of chinese medicinal herbs for treating uncomplicated acute bronchitis. methods: we searched the cochrane central register of controlled trials, medline, embase, chinese biomedical database and etc. we included randomised controlled trials comparing chinese medicinal herbs with placebo, antibiotics or other western medicine for treating uncomplicated acute bronchitis. at least two authors extracted data and assessed trial quality. main results: four trials reported the time to improvement of cough, fever, and rales; two trials reported the proportion of patients with improved signs and symptoms; thirteen trials analyzed the data of global assessments of improvement. one trial reported the adverse effect during treatment. conclusions: there is insufficient quality data to recommend the routine use of chinese herbs for acute bronchitis. the benefit found in individual studies and this systematic review could be due to publication bias and study design limitations. in addition, the safety of chinese herbs is unknown, though adverse events are rarely reported. design and methods: patients with a definite ms and classified as dead or alive at 1st january 2004 were included in this retrospective observational study. influence of demographic and clinical variables was assessed with kaplan meier and cox methods. standardised mortality ratios were computed to compare patients' mortality with the french general population. results: a total of 1935 patients were included (623 men, 1322 women). the mean age at ms onset was 31 +/) 10 years and the mean follow-up duration was 15 +/) 9 years (29260 patients-years). by 2004, 85 deaths occurred (3 per 1000 patients-years). male gender, progressive course, polysymptomatic onset and high relapse rate were related to a worse prognosis. ms did not increase the number of deaths in our cohort compared to the general french population (75 expected), except for highly disabled patients (58 observed, 18 expected). conclusion: this study gave precise insights on mortality in multiple sclerosis in west france. mattress dust. methods: we performed nested case-control studies within ongoing birth cohort studies in germany, the netherlands, and sweden and selected approximately 180 sensitised and 180 non-sensitised children per country. we measured levels of bacterial endotoxin, ß(1->3)-glucans, and fungal extracellular polysaccharides (eps) in dust samples collected on the children's mattresses. results: combined across countries, higher amounts of dust and higher endotoxin, ß(1->3)-glucans, and eps loads of mattress dust were associated with a significantly decreased risk of sensitization to inhalant allergens, but not food allergens. after mutual adjustment, only the protective effect of the amount of mattress dust remained significant [odds ratio (95% confidence interval) 0.57 (0.39-0.84)]. conclusion: higher amounts of mattress dust might decrease the risk of allergic sensitization to inhalant allergens. the effect might be partly attributable to endotoxin, ß(1->3)-glucans, and eps. it is not possible to distinguish with certainty, which component relates to the effect, since microbial agents loads are highly correlated with amount of dust and with each other. abstract background: postmenopausal hormone therapy (ht) increases mammographic density, a strong breast cancer risk factor, but effects vary across women. objective: to investigate whether the effect of ht use on changes in mammographic density is modified by polymorphisms in the estrogen (esr1) and progesterone receptor (pgr) genes. design and methods: information on ht use, dna and two consecutive mammograms were obtained from 795 ht users and 781 never ht users of the dutch prospect-epic and the english epic-norfolk cohorts. mammographic density was assessed using a computer-assisted method. changes in density between mammograms before and during ht use were analyzed using linear regression. results: a difference in percent density change between ht users and never users was seen in women with the esr1 pvuii pp or pp genotype (2.24%; p<0.01), but not in those with the pp genotype (0.90%; p = 0.47). similar effects were observed for the esr1 xbai and the pgr +331 g/a polymorphisms. the pgr progins polymorphism did not appear to make women more susceptible to the effects of ht use. discussion and conclusion: our results suggest that specific polymorphisms in the esr1 and pgr genes may make women more susceptible to the effects of ht use on mammographic density. abstract background: there is a paucity of data on the cancer risk of turkish migrant children in germany. objectives: to identify cancer cases of turkish origin in the german childhood cancer registry (gccr) and to compare the relative incidence of individual cancers among turkish and non-turkish children. design and methods: we used a name algorithm to identify children of turkish origin among the 37,259 cancer cases below 15 years of age registered since 1980. we calculated proportional cancer incidence ratios (pcir) stratified for sex and time period. results: the name algorithm performed well (high sensitivity and specificity), and 1774 turkish childhood cancers were identified. overall, the relative frequency of tumours among turkish and non-turkish children is similar. there are specific sites and cancers for which pcirs are different; these will be reported during the conference. conclusion: our study is the first to show differences in the relative frequency of cancers among turkish and non-turkish children in germany. discussion: case control studies could help to explain whether observed differences in the relative frequency of cancers are due to differences in genetic disposition, lifestyle or socio-economic status. mutations in the netherlands cohort study on diet and cancer. data from 120,852 participants, 528 cases and 4,176 subcohort members were analysed from a follow-up period between 2.3 to 7.3 years after baseline. adjusted gender-specific incidence rate ratios (rr) and 95% confidence intervals (ci) were calculated over tertiles of folate intake in case-cohort analyses. high folate intake did not reduce overall colon cancer risk. however, in men only, it was inversely associated with apc[csymbol] colon tumours (rr 0.58, 95% ci 0.32-1.05 for the highest versus the lowest tertile of folate intake), but positively associated with apc+ colon tumours (highest vs. lowest tertile: rr 2.77, ci 1.29-5.95). folate intake was neither associated with overall rectum cancer risk, nor with rectum cancer when apc mutation status was accounted for. we observed opposite associations between folate intake and colon cancer risk with or without apc mutations in men, which may implicate a distinct mutated apc pathway mediated by folate intake in men. abstract background and objectives: ten years after completion of the first serum bank of the general population to evaluate the long-term effects of the national immunisation programme (nip) a new serum collection is desirable. the objective is to provide insight into age-specific estimates of the immunity to childhood diseases and estimates of the incidence of infectious diseases with a frequent sub clinical course. design and methods: a two-stage cluster sampling technique was used to draw a nationwide sample. in each of five geographic regions, eight municipalities were randomly selected proportionally to their size. within each municipality, an age-stratified sample of 380 individuals (0-79 yr) will be drawn from the population register. in addition eight municipalities will be selected with lower immunization coverage to obtain insight into the immune status of persons who often refuse vaccination on religious grounds. furthermore over sampling of migrants will be performed to study whether their immune status is satisfactory. participants will be asked to fill in a questionnaire and to allow blood to be taken. extra blood will be taken for a genetic study. results and conclusion: the design of a population-based serum collection aimed at the establishment of a representative serum bank will be presented. abstract background: during the last decade, the standard of diabetes care evolved to require more intensive management focussing on multiple cardiovascular risk factors. treatment decisions for lipidlowering drugs should be based on cholesterol and blood pressure levels. objectives: to investigate the influence of hba1c, blood pressure and cholesterol levels on subsequent intensification of lipid-lowering therapy between 1998-2004. design and methods: we conducted a prospective cohort study including 2,373 type 2 diabetes patients who had at least two consecutive annual visits to a diabetes nurse. treatment intensification was measured by comparing drug regimes per year, and defined as initiation of a new drug class or dose increase of an existing drug. results: between 1998-2004, the prevalence of lipid-lowering drug use increased from 12% to 39%. rates of intensification of lipid-lowering therapy remained low in poorly controlled patients (12% to 28%;tc/hdl ratio>6). intensification of lipid-lowering therapy was only associated with tc/hdl ratio (age-adjusted or = 1.6;95%ci 1.5-1.7) and this association became slightly stronger over time. blood pressure was not found to be a predictor of the intensification of lipid-lowering therapy (or = 1.0). conclusion: hypercholesterolemia management intensified between 1998-2004, but therapy intensification was only triggered by elevated cholesterol levels. more attention for multifactorial risk assessment is needed. abstract background: there are no standard severity measures that can classify the range of illness and disease seen in general practice. objectives: to validate new scales of morbidity severity against age, gender, deprivation and poor physical function. design and methods: in a cross-sectional design, morbidity data for consulters in a 12-month period was linked to their physical function status . there were 9003 english older consulters (50 years +) and 7753 dutch consulters (18 years +). consulters for 116 morbidities classified on four gp-defined ordinal scales of severity ('chronicity', 'time course', 'health care use' and 'patient impact on activities of daily living') were compared to consulters for morbidity other than the 116, by age-groups, gender, and dichotomised deprivation and physical function scores. results: for both countries, on all scales, there was an increasing association between morbidity severity and older ages, female gender, more deprivation (minimum p<0.05) and poor physical function (all trends p<0.001). the estimates for categories, for example, within the 'chronicity' scale was ordered as follows: 'acute' (unadjusted odds ratio 1.4), 'acute-on-chronic' (1.8), 'chronic' (2.2) and 'life-threatening' (5.4). conclusions: new validated measures of morbidity severity indicate physical health status and offer the potential to optimise general practice care. hospitalization or death. calibration and discriminative capacity were estimated. results: among 445 episodes of lrti in elderly patients with dm, 68 endpoints occurred (attack rate 15%). reliability of the model was good (goodness-of-fit test p = 0.54). the discriminative properties of the original rule was acceptable (area under the receiver-operating curve (auc): 0.79, 95% ci: 0.72 to 0.86). conclusion: the prediction rule for the probability of hospitalization or death derived from an unselected elderly population with lrti appeared to have acceptable discriminative properties in diabetes patients and can be used to target management of these common diseases. confounding by indication is a major threat to the validity of nonrandomized studies on treatment effects. we quantified such confounding in a cohort study on the effect of statin therapy on acute respiratory disease (ard) during influenza epidemics in the umc utrecht general practitioner research database among persons aged > = 50 years. the primary endpoint was a composite of pneumonia or prednisolone-treated ard during epidemic, non-epidemic and summer seasons. to quantify confounding, we obtained unadjusted and adjusted estimates of associations for outcome and control events. in all, 22,638 persons provided 130,558 persons-periods, statin therapy was used in 5.3% and in 3,333 person-periods an outcome event occurred. without adjustments, statin therapy was not associated with the primary endpoint during influenza epidemics (relative risk [rr] 1.02; 95% confidence interval [95%ci]: 0.83-1.25). after applying multivariable generalized estimating equations (gee) and propensity score analysis the rrs were 0.72 (95% ci: 0.57-0.90) and 0.69 (95% ci: 0.59-0.89). the findings were consistent across relevant strata. in non-epidemic influenza and summer seasons the rr approached 1.0 while statin therapy was not associated with control event rates. observed confounding in the association between statin therapy and acute respiratory outcomes during influenza epidemics masked a potential benefit of more than 30%. abstract background: despite several advances in the treatment of schizophrenia, the currently available pharmacotherapy does not change the course of illness or prevent functional deterioration in a substantial number of patients. therefore, research efforts into alternative or adjuvant treatment options are needed. in this project, called the 'aspirine trial', we investigate the effect of the antiinflammatory drug acetylsalicylic acid as an add-on to regular antipsychotic therapy on the symptoms of schizophrenia. objectives: to objective is to study the efficacy of acetylsalicylic acid in schizophrenia on positive and negative psychotic symptoms, immune parameters and cognitive functions. design and methods: a randomized placebo controlled double-blind add-on trial of 80 inpatients and outpatients with schizophrenia, schizophreniform or schizoaffective disorder is performed. patients are 1:1 randomized to either 3 months 1000 mg acetylsalicylic acid per day or 3 months placebo, in addition to their regular antipsychotic treatment. all patients receive pantoprazole treatment for gastroprotection. participants are recruited from various major psychiatric hospitals in the netherlands. the outcomes of this study are 3-month change in psychotic and negative symptom severity, cognitive function, and several immunological parameters. status around 45 participants have been randomized. no interim analysis was planned. abstract background: congenital cmv infection is the most prevalent congenital infection worldwide. epidemiology and outcome are known to vary with socio-economic background, but few data are available on epidemiology and outcome in a developing country, where the overall burden of infectious diseases is high. objective: to determine prevalence, riskfactors and outcome of congenital cmv infection in an environment with high infectious disease burden methods: as part of an ongoing birth cohort study, baby and maternal samples were collected at birth, and tested with an inhouse pcr for the presence of cmv. standardised clinical assessment were performed by a paediatrician. placental malaria was also assessed. follow-up is ongoing till the age of 5 years. preliminary results: the prevalence of congenital cmv infection was 36/700 (5.1%). the infected children were more often first born babies (47.4% vs 21.5%, p<0.001). while no seasonality was observed, placental malaria was more prevalent among congenitally infected children (25.0% vs 11.3%,p = 0.04). there were no symptomatic babies detected. conclusion: this prevalence of congenital cmv is much higher than reported in industrialised countries, in the absence of obvious clinical pathology. further follow up is needed to assess impact on response to vaccinations, growth, and morbidities. of wheeze or cough at night in the first 3 years. data on respiratory symptoms and dda were collected by yearly questionnaires. in total, 193 symptomatic children with and 916 without an early dda were included in the study population. results: fifty-one percent of the children with and 29% of the children without an early dda had persistent respiratory symptoms at age 6. persistence of symptoms was associated with parental atopy, eczema, nose symptoms without a cold, or a combination of wheeze and cough in the first 3 years. conclusions: monitoring the course of symptoms in children with risk factors for persistent symptoms, irrespective of a diagnosis of asthma, may contribute to early recognition and treatment of asthma. little is known about the response mechanisms of survivors of disasters. objective: to examine selective non-response and to investigate whether attrition has biased the prevalence estimates among survivors of a disaster. design and methods: a longitudinal study was performed after the explosion of a fireworks depot in enschede, the netherlands. survivors completed a questionnaire 3 weeks (t1), 18 months (t2) and 4 years post-disaster (t3). prevalence estimates resulting from multiple imputation were compared with estimates resulting from complete case analysis. results: non-response differed between native dutch and nonwestern immigrant survivors. for example, native dutch survivors who participate at t1 only were more likely to have health problems at t1 such as depression than native dutch who participated at all three waves (or = 1.9, 95% ci: 1. 2-2.9) . in contrast, immigrants who participated at t1 only were less likely to have depression at t1 (or = 0.2, 95% ci: 0.1-0.6). conclusion and discussion: among native dutch survivors, the imputed estimates of t3 health problems tended to underestimated than the complete case estimates. the imputed t3 estimates among immigrants were unaffected or somewhat overestimated than the complete case estimates. multiple imputation is a useful statistical technique to examine whether selective non-response has biased the prevalence estimates. session: posters session 3: july 1 2006 presentation: poster. background: several epidemiologic studies have shown decreased colon cancer risk in physically active individuals. objectives: this review provides an update of the epidemiologic evidence for the association between physical activity and colon cancer risk. we also explored whether study quality explains discrepancies in results between different studies. methods: we included cohort (male n = 16; female n = 10) and case-control studies (male n = 13; female n = 12) that assessed total or leisure time activities in relation to colon cancer risk. we developed a specific methodological quality scoring system for this review. due to the large heterogeneity between studies, we refrained from statistical pooling. results: in males, the cohort and case-control studies lead to different conclusions: the case-control studies provide strong evidence for a decreased colon cancer risk in the physically active while the evidence in the cohort studies is inconclusive. these discrepant findings can be attributed to either misclassification bias in cohort or selection bias in case-control studies. in females, the small number of high quality cohort studies precludes a conclusion and the case-control studies indicate an inverse association. conclusion: this review indicates a possible association of physical activity and reduction of colon cancer risk in both sexes but the evidence is not yet convincing. abstract background/objectives: radiotherapy after lumpectomy is commonly applied to reduce recurrence of breast cancer but may cause acute and late side effects. we determined predictive factors for the development of late toxicity in a prospective study of breast cancer patients. methods: late toxicity was assessed using the rtog/ eortc classification among 418 women receiving radiotherapy following lumpectomy after a mean follow-up time of 51 months. predictors of late toxicity were modelled using cox regression in relation to observation time, adjusting for age, bmi and biologically effective dose in the maximum at the skin. results: 133 (31.8%) patients presented with telangiectasia and 28 (6.7%) patients with fibrosis. we observed a strong association between development of telangiectasia and fibrosis (p<0.01). increasing patient age was a risk factor for telangiectasia and fibrosis (p for trend 0.02 and 0.04, respectively). boost therapy (hazard ratio (hr) 4.2, 95% ci 1.7-10.7) and acute skin toxicity (hr 1.6, 95% ci 1.0-2.6) significantly increased risk of telangiectasia. risk of fibrosis was elevated among patients with atopic diseases (hr 2.2, 95% ci 1.0-4.7). discussion: our study revealed several risk factors for late complications of radiotherapy. further understanding of differences in response to irradiation may enable individualized treatment and improve cosmetic outcome. doctor-diagnosed asthma and respiratory symptoms (age 6) were available for 311 (rint) and 125 (no) children. results: the discriminative capacities of rint and exhaled no were statistically significant for the prediction of doctor-diagnosed asthma, wheeze (rint only) and shortness of breath (rint only). due to the low prevalence of disease in this general population sample, the positive predictive values of both individual tests were low. however, the positive predictive value of the combination of increased rint (cutoff 1.05 kpa.l-1.second) and exhaled no (cut-off 10 ppb) was 38% for the prediction of doctor-diagnosed asthma, with a negative predictive value of 97%. combinations of rint or exhaled no with atopy of the child showed similar results. conclusions: the combination of rint, exhaled no and atopy may be useful to identify high-risk children, for monitoring the course of their symptoms and to facilitate early detection of asthma. abstract background: in 1992 a cargo aircraft crashed into apartment buildings in amsterdam, killing 43 people, and destroying 266 apartments. an extensive, troublesome aftermath followed with rumours on toxic exposures and health consequences. objectives: we studied the long-term physical health effects of occupational exposure to this disaster among professional assistance workers. design and methods: in this historical cohort study we compared the 334 firefighters and 834 police officers who were occupationally exposed to this disaster (i.e. who reported one or more disasterrelated tasks) with their nonexposed colleagues (n = 194, and n = 634, respectively), using regression models adjusted for background characteristics. data collection took place from january 2000 to march 2002, and included various clinical blood and urine parameters (including blood count and kidney function), and questionnaire data on occupational exposure, physical symptoms, and background characteristics. the overall response rate was 71%. results: exposed workers reported various physical symptoms (including fatigue, skin and musculoskeletal symptoms) significantly more often than their nonexposed colleagues. in contrast, no consistent significant differences between exposed and nonexposed workers were found regarding clinical blood and urine parameters. discussion and conclusion: this epidemiological study demonstrates that professional assistance workers involved in a disaster are at risk for long-term unexplained physical symptoms. abstract background and objectives: recent studies indicate that women with cosmetic breast implants have significantly increased risk of suicide. reasons for elevated risk are not known. it is suggested that women with cosmetic breast implants differ in their characteristics and have more mental problems than women of general population. aim of this study was to find out possible associations between physical or mental health and postoperative quality of life among finnish women with cosmetic breast implants. design and methods: information was collected from patient records of 685 women and structured questionnaires mailed to 470 women of the same cohort. data was analysed by using pearson chi square testing and logistic regression modelling. results: although effects of implantation on postoperative quality of life in different areas were mainly reported as positive or neutral, 12 % of the women reported decreased state of health. postoperative dissatisfaction and decreased quality of life were significantly associated with diagnoses of depression (p = 0.01) and local complication called capsular contracture (p<0.001). conclusion: our results are consistent with previous results finding most of the cosmetic breast surgery patients satisfied after implantation. however, this study brings new information on associations between depression, capsular contracture and decreased quality of life. abstract cancer and its treatments often produce significant persistent morbidities that reduce quality of life (qol) in cancer survivors. research indicates that both, physical exercise and psycho-education might enhance qol. therefore, we developed a 12-week multidisciplinary rehabilitation program that combines physical training with psycho-education. the aim of the present multicenter study is to determine the effect of multidisciplinary rehabilitation on qol as compared to no treatment and, additionally, to physical training alone. furthermore, we will explore which variables are related to successful outcome (socio-demographic, disease related, physiological, psychological and environmental characteristics). 225 participants are needed to detect a medium effect. at present, 170 cancer survivors are randomised to either the multidisciplinary or physical rehabilitation program or a 6-month waiting list control group. outcome assessment will take place before, halfway, directly after, 3 and 9 months following the intervention by means of questionnaires. physical activity will be measured before, halfway and directly after rehabilitation using maximal and submaximal cycle ergometer testing and muscle strength measurement. effectiveness of multidisciplinary rehabilitation will be determined by analysing changes between groups from baseline to post-intervention using multiple linear and logistic regression. positive evaluation of multidisciplinary rehabilitation may lead to implementation in usual care. continuous event recorders (cer) have proven to be successful in diagnosing causes of palpitations but may affect patient qol and increase anxiety. objectives: determine qol and anxiety in patients presenting with palpitations, and to evaluate the burden of the cer on qol and anxiety in patients presenting to the general practitioner. methods: randomized clinical trial in general practice. the short form-36 (sf-36) and state-trait anxiety inventory (stai) were administered at study inclusion, 6-weeks and 6months. results: at baseline, patients with palpitations (n = 226) reported lower qol and more anxiety than a healthy population for both males and females. there were no differences between the cer arm and usual gp care at 6-weeks. at 6-months the usual care group (n = 92) showed minimal qol improvement and less anxiety compared to the cer group (n = 103). type of diagnosis did not account for any of these reported differences. conclusion: anxiety decreases and qol increases in both groups at 6-weeks and 6-month follow-up. hence it is a safe and effective diagnostic tool, which is applicable for all patients with palpitations in the general practice. abstract background: clinical benefits of statin therapy are accepted, but their safety profiles have been under scrutiny, particularly for the most recently introduced statin, rosuvastatin, relating to serious adverse events involving muscle, kidney and liver. objective: to study the association between statin use and the incidence of hospitalizations for rhabdomyolysis, myopathy, acute renal failure and hepatic impairment (outcome events) in real life. methods: in 2003 and 2004, 10,147 incident rosuvastatin users, 37,396 incident other statin users and 99,935 patients without statin prescriptions from the pharmo database of >2 million dutch residents were included in a retrospective cohort study. potential cases of hospitalization for myopathy, rhabdomyolysis, acute renal failure or hepatic impairment were identified using icd-9-cm codes and validated using hospital records. results: there were 26 validated outcome events in the three cohorts including one case each of myopathy (other statin group) and rhabdomyolysis (non-treated group). there were no significant differences in the incidence of outcome events between rosuvastatin and other statin users. discussion: this study indicated that the number of outcome events is less than 1 per 3000 person years. rosuvastatin does not lead to an increased incidence of rhabdomyolysis, myopathy, acute renal failure and hepatic impairment compared to other statins. the aim: the aim of the study was to assess the influence of insulin resistance (ir) on the coronary artery disease (cad) occurrence in middle aged women with normal glucose tolerance (ngt) material and methods: in 1998-2000 year 986 women aged 35-55, participants of the polish multicenter study on diabetes epidemiology were examined. anthropometric, biochemical (fasting lipids, fasting and after glucose load plasma glucose and insulin) and blood pressure determinations were performed . ir was defined as the matsuda index (irmatsuda) below the lower quartile of the irmatsuda distribution in ngt population the questionnaire examination of the lifestyle, present and past diseases was performed. results: ir was observed in 31 % of all examined women and in 17.9% with ngt. cad was diagnosed in 10, 3% of all examined women and in 6,9% of those with ngt. the relative risk of cad related to ir in ngt and normotensive women was 2,90 (95% ci:1,07-7,88) (p<0.05). regular menstruation was observed in 41,1% of cad women. irmatsuda was not different for cad menstruating and non menstruating women (respectively 9,31±1,01 and 9,70±1,68). conclusion: in middle aged, normotensive and normal glucose tolerant women ir seems to be an important risk factor of cad abstract background: in germany, primary prevention at population level is provided by general practitioners (gp). little is known about gps' strategies to identify patients at high risk for vascular diseases using standardised risk scores. objectives: we studied gp attitudes and current practice in using risk scores. methods-a cross-sectional survey was conducted among 744 gps in north rhine-westphalia, germany, using mailed self-administered questionnaires on attitudes and current practice in identification of patients at high risk for vascular diseases. results: in 2005, 350 gps participated in the study. 69.0% of gps stated to know the framingham-score, 81.3% the procam-score and 29.4% the score-score. 47.4% of gps reported regular use of standardised risk scores to identify patients at high risk for vascular diseases, most frequently procam-score (79.7%), followed by score-score (6.8%) and framingham-score (6.8%). main reasons for not using standardised risk scores were assumed rigid assessment of individual patients' risk profile (51.9%), time-consuming appliance (35.2%) and higher confidence in own work experience (17.3%). conclusion: use of standardised risk scores to identify patients at high risk for vascular diseases is common among gps in germany. however, more educational work might be useful to strengthen gps' belief in the flexible appliance of standardised risk scores in medical practice. among epilepsy patients than in general population, but effects of specific antiepileptic drugs on birth rate are not well known. objectives: to estimate birth rate in epilepsy patients on aed treatment or without aeds and in a population-based reference cohort without epilepsy. design and methods: patients (n = 20, 101) with reimbursement for aeds for the first time between 1985 and 1994 and information on their aed use, were identified from the databases of social insurance institution of finland. reference cohort without epilepsy (n = 29,828) and information on live births were identified from the finnish population register centre. the analyses were performed using poisson regression modelling. results:birth rate was decreased in epilepsy patients in relation to reference cohort without epilepsy in both genders regardless of aed use. in relation to untreated patients, women on any of the aeds had non-significantly lower birth rates. among men, birth rate was decreased in men on oxcarbazepine (rr = 0.52, 95% ci = 0.32,0.84), but was not clearly lower among those on carbamazepine (rr = 0.86,95% ci = 0.66,1.13) or valproate (rr = 0.78,95% ci = 0.55,1.11) when compared to untreated patients. conclusion: our results suggest that birth rate is decreased among epilepsy patients on aeds, more so in men. abstract background: hereditary hemochromatosis (hh), characterised by excessive iron absorption, subsequent iron storage and progressive clinical features, can when diagnosed at an early stage be successfully treated. high prevalence of the c282y-mutation on the hfe-gene in the hh patient population may motivate genetic screening. objectives: in first-degree relatives of c282y-homozygotes we studied the gender and age -related biochemical penetrance of hfe-genotype to define a high-risk population eligible for screening. design and methods: one-thousand-six first-degree family members of 280 probands with clinically overt hfe-related hh from five medical centres in the netherlands were approached. data on levels of serum iron parameters and hfe-genotype were collected. elevation of serum ferritin was defined using the centre-specific normal-values by age and gender. results: among the 610 participating relatives, highest serum iron parameters were found in male c282y-homozygous siblings aged >55 years: 97% had elevated levels of serum ferritin. generally, male gender and increased age are related with higher iron values. discussion and conclusion: genetic screening for hh is most relevant in male and elderly first-degree relatives of patients with clinically overt hfe-related hh, enabling regular investigations of iron parameters in homozygous individuals. abstract background: nosocomial infection causes increased hospital morbidity and mortality rates. although handwashing is known to be the most important action in its prevention, adherence of health care workers to recommended hand hygiene procedures is extremely poor. objective: evaluation of compliance of hand hygiene recommendations in health care workers of a tertiary hospital in barcelona after a course on hand hygiene was given to all nurses in the hospital during the previous year. methods: by means of nondeclared observation, compliance (handwashing or disinfecting, not solely glove exchange) of recommendations given by the center for disease control related to opportunities for hand hygiene was registered, in procedures of diverse risk level for infection, both in physicians and nurses. results: in 1288 opportunities for hand hygiene carried out by 254 health care workers compliance of recommendations was 19.9%. adherence differed between wards (68.9% in intensive care units, 17.8% in medical wards and 4.3% in surgical wards) and slightly between health care workers (21.4% in physicians, 19.2% in nurses). discussion: in conclusion, after one year of an intervention on education, adherence to hand hygiene recommendations is very low. these results enhance the need of reconsidering the type of interventions implemented. type of comorbidity affects qol most. objectives: we studied whether qol differed in subjects with dm with and without comorbidities. in addition, we determined differences in type of comorbidity. design and methods: cross-sectional data of 193 dm patients, participants of a population-based dutch monitoring project on risk factors for chronic disease (morgen) were analyzed. qol was measured by the short form 36. we compared the means of 8 subdimensions for dm patients with one comorbidity (cardiovascular diseases (cvd), musculoskeletal diseases (msd) and asthma/copd) to dm patients without this comorbidity, by regression analyses adjusted for age and sex. results: the prevalences of cvd, msd and asthma/copd were 17.1%, 26.4%, and 46.6%. all comorbidities were associated with lower qol, especially for physical functioning. the mean difference (95% ci) was 13. abstract background: the extent or increase of ueds is suggested repeatedly, but never before the scientific literature was systematic studied. objectives: a systematic appraisal of the worldwide incidence and prevalence rates of upper extremity disorders (ueds) available in scientific literature was executed to gauge the range of these estimates in various countries and to determine whether the rates are increasing in time. design and methods: studies that recruited at least 500 people, collected data by using questionnaires, interviews and/or physical examinations, and reported incidence or prevalence rates of the whole upper-extremity including neck, were included. results: no studies were found with regard to the incidence of ueds and 13 studies that reported prevalence rates of ueds were included. the point prevalence ranged from 1.6-53%; the 12months prevalence ranged from 2.3-41%. one study reported on the lifetime prevalence (29%). we did not find evidence of a clear increasing or decreasing pattern over time. it was not possible to pool the date, because the definitions used for ueds differed enormously. conclusions: there are substantial differences in reported prevalence rates on ueds. main reason for this is the absence of a universally accepted way of labelling or defining ueds. abstract background: the absolute number of women diagnosed with breast cancer increased from 7,899 in 1989 to 11,687 in 2003 in the netherlands. likewise, the age standardized rate increased from 100.1 to 120.7 per 100,000 women. besides the current screening programme, changes in risk profile could be a reason for the increased incidence. objective: we studied the changes in breast cancer risk factors for women in nijmegen. methods: in the regional screening programme in nijmegen, almost 20,000 women aged 35-64 years filled in a questionnaire about risk factors in [1975] [1976] . similar questions were applied in the nijmegen biomedical study in 2002, where 2345 women of 35-64 year participated. the median age in both studies was 50 years. results: the frequency of a first-degree relative with breast cancer was 9.9% and 11.1% in 1975 and 2002, respectively . none of the other risk factors, as the age of women at 1st birth (26.3% respectively 27.1%), nulliparity (19.2% resp. 20.6%), age at menarche (13.1% resp. 13.2%), age at menopause (47.2% resp. 49.2%) and obesity (11.9% resp. 11 .4%), changed in time. conclusion: the distribution of risk factors hardly changed, and is unlikely to explain the rise in breast cancer incidence from 1989 onwards. abstract background: a single electronic clinical history system has been developed in the bac (basque autonomous community) for general use for all health centres, thus making it possible to collect information online on acute health problems as well as chronic ailments. method: the prevalence of diabetes, high blood pressure and copd (chronic obstructive pulmonary disease) was estimated using icd-9-cm diagnosis performed by primary care physicians. an estimate was also made of the prevalence of cholesterolemia based on the results of analyses requested by physicians. results: in 2004, 441,097 patients (out of a total population of 2,082,587) were assessed for serum cholesterol levels. based on this highly representative sample, it was estimated that 13.3% had serum cholesterol levels above 250 mg/dl. the prevalence of diabetes mellitus in people over the age of 29 was 6.4%. the prevalence of high blood pressure in people over 13 was 25%. discussion: the primary care database makes it possible to access information on problems related to chronic illnesses. knowing the prevalence of diabetes patients enables doctors to analyse all aspects related to services used by the diabetic population. it also makes it possible to monitor analytical data in real time and evaluate health service outcomes. examinations were used to asses risk factors for diabetes. cases (n = 101) were matched on age and sex to controls (n = 709) who were not treated with antidiabetic drugs. logistic regression was used to calculate odds ratios (or). results: the or of incident diabetes for acei-use versus non-acei use was 3.63 (95%ci :1.93-6.88). for ace dd homozygotes the or was 0.57 (95%ci:0.07-4.47) and for ace-i allele carriers 5.72 (95%ci:2.77-11.81). the interaction or was 10.05 (95%ci:1.13-89.38). the agt and at1r genotypes did not modify the association between acei use and diabetes. abstract background: lignans have antioxidant and estrogen-like activity, and may therefore lower cardiovascular and cancer risk. objective: we have investigated whether intake of four plant lignans (lariciresinol, pinoresinol, secoisolariciresinol, matairesinol) was inversely associated with coronary heart disease (chd), cardiovascular diseases (cvd), cancer, and all-cause mortality. design: the zutphen elderly study is a prospective cohort study in which 570 men aged 64-84y were followed for 15 years. lignan intake was estimated using a recently developed database, and related to mortality using cox proportional hazards analysis. results: median total lignan intake in 1985 was 977lg/d. beverages such as tea and wine, vegetables, bread, and fruits were the major lignan sources. total lignan intake was not related to mortality. however, matairesinol was inversely associated with chd, cvd, cancer, and all-cause mortality. multivariate adjusted rrs (95% ci) per sd increase in intake were 0.72 (0.53-0.98) for chd, 0.83 (0.69-1.00) for cvd, 0.81 (0.65-1.00) for cancer, and 0.86 (0.76-0.97) for allcause mortality. conclusions: total lignan intake was not associated with mortality. the intake of matairesinol was inversely associated with mortality from chd, cvd, cancer, and all-causes. we can not rule out that this is due to an associated factor, such as wine consumption. abstract despite the drastic increase in the amount of research into neighbourhood-level contextual effects on health, studies contrasting these effects between different domains of health within one contextual setting are strikingly sparse. in this study we use multilevel logistic regression models to estimate the existence of neighbourhood-level variations of physical health functioning (pcs) and mental well-being (ghq) in the helsinki metropolitan area and assess the causes of these differences. the individual-level data are based on a health-survey of 40-60 year old employees of the city of helsinki (n = 4313, response rate 68%). the metropolitan area is divided into 53 neighbourhoods, which are characterised using a number of area-level indicators (e.g. unemployment rate). our results show moderate but systematic negative effect of indicators of neighbourhood deprivation on physical functioning, whereas for mental health the effect is absent. these effects were strongest for proportion of manual workers; odds ratio for poor physical functioning was 0.8 for respondents living in areas with low proportion of manual workers. part of this effect was mediated by differences in health behaviour. analyses on cross-level interactions show that individual-level socioeconomic differences in physical health are smallest in most deprived areas, somewhat contradicting the results of earlier studies. abstract background: the second-eye cataract surgery is beneficial, nevertheless, there is a considerable proportion of unmet needs. objective: to estimate the proportion of second-eye cataract surgery in the public health system of catalonia, and explore differences in utilisation by patients' gender, age, and region of residence. methods: a total of 154,215 senile cataract surgeries performed between 1999 and 2002 were included. proportions observed were adjusted through independent logarithmic regression models for each study factor. results: the proportion of second-eye surgery showed an increasing trend (r2 59.4%) from 23.8% (95% ci 21.6; 26.1) in november 2000 to 32.5% (95% ci 31.4; 33.6) in december 2002, and its projection to 5 years was 35,7% (95% ci 33.6; 37.7). the proportion of second-eye surgery was 2% (95% ci 0.9; 3.1) greater in women than in men. patients 80 years or older had a lowest proportion (20.7%; 95% ic 20.2; 21.3), which nevertheless increased during the period, unlike that of patients aged less than 60 years. differences among regions were moderate and decreased throughout the period. conclusions: if the observed trends persist, there will be a substantial proportion of unmet need for bilateral surgery. we predict greater use of second-eye surgery by older patients. abstract background: persistence with bisphosphonates is suboptimal which could limit prevention of fractures in daily practice. objectives: to investigate the effect of long term persistent bisphosphonate usage on the risk of osteoporotic fractures. methods: the pharmo database, including drug-dispensing and hospital discharge records for > two million subjects in the netherlands, was used to identify new female bisphosphonate users >50 years from jan '96 -jun '03. persistence with bisphosphonates was determined using the method of catalan. a nested matched case-control study was performed. cases had a first hospitalization for an osteoporotic fracture (index-date). controls were matched 10:1 to cases on year of inclusion and received a random index-date. the association with fracturerisk was assessed for one and two year persistent bisphosphonate use prior to the index-date. analyses were adjusted for di fferences in patient characteristics. results: 14,760 bisphosphonate users were identified and 541 had a hospitalization for osteoporotic fracture during follow-up. one year persistent bisphosphonate use resulted in a 26% lower fracture rate (or 0.74; 95% ci 0.57-0.95) whereas two year persistent use resulted in a 32% lower rate (or 0.68; 95% ci 0.47-0.96). conclusion and discussion: these results emphasize the importance of persistent bisphosphonate usage to obtain maximal protective effect of treatment. abstract background: in 1997 the who recommended all countries to add hepatitis b (hbv) vaccination to their national immunization programs. the netherlands is a low hbv endemic country and therefore adopted a vaccination policy targeted towards high-risk groups. methods: during 2004, epidemiological data and blood samples were collected from all reported patients with an acute hbv infection. a fragment of the s-gene was sequenced and phylogenetically analysed to clarify transmission patterns between risk groups. results: of 295 hbv cases reported, 60% was infected through sexual contact (34% homo-/bisexual, 23% heterosexual). for 158 patients samples were available for genotyping. phylogenetic analysis identified 6 genotypes: a(64%), b(3%), c(3%), d(21%), e(5%) and f(4%). of men who have sex with men (msm), 86% were infected with genotype a. among heterosexuals, all genotypes were found. in many cases, genotypes b-f were direct or indirect related to countries abroad. only 1 injecting drug user was found (genotype a). conclusion: genotype a is predominant in the netherlands, including most of the msm. migrant hbv carriers play an important role in the dutch hbv epidemic. genotyping provides insight into the spread of hbv among highrisk groups. this information will be used to evaluate the vaccination policy in the netherlands. abstract background: excess weight might affect the perception of both physical and mental health in women. objective: to examine the relationship between body mass index (bmi) and hrqol in women aged 18-to 60-year-old in a rural zone of galicia. design and methods: population-based cross-sectional study covering 1321 women, personally interviewed, from 14 villages. hrqol was assessed with sf-36 questionnaire, through personal interviews. each scale of sf-36 was dichotomised in suboptimal or optimal hrqol using previously defined cut-offs. odds ratios (or) obtained from logistic regression summarize the relationship of bmi with each scale, adjusting for sociodemographic variables, sedentary leisuretime, number of chronic diseases and sleeping hours. results: a 14.7% of women were obese (bmi = 30 kg/m2) and 31.2% overweight 9 kg/m2) . frequency of suboptimal physical function was higher among overweight women (adjusted or:1.76; 95% ci:1.27-2.45) and obesity (adjusted or:2.10;95% ci:1.40-3.16). furthermore, obese women had higher frequency of suboptimal scores on the general health scale (adjusted or:1.64; 95% ci:1.10-2.44). no differences were observed regarding mental health scores among women with different bmi categories. conclusion: in women from rural villages, overweight is associated with worse hrqol in physical function and general health. abstract background: pneumococcal vaccination among elderly is recommended in several western countries. objectives: we estimate the cost-effectiveness of a hypothetical vaccination campaign among the 65+ general population in lazio region (italy). methods: a cohort was followed during a 5 years timeframe. we estimated the incidence of invasive pneumococcal disease, in absence of vaccine, based on actual surveillance and hospital data. the avoided deaths and cases have been estimated from literature according to trial results. health expenditures included: costs of vaccine program, inpatient and some outpatient costs. cost-effectiveness was expressed as net healthcare costs per episode averted and life-year gained (lyg) and was estimated at baseline and in deterministic and stochastic sensitivity analyses. all parameters were age-specific and varied according to literature data. results: at baseline net costs per event averted and lyg at 2001 prices were, respectively, e34,681 (95% ci: e28,699-e42,929) and 23,361 (95% ci: e16,419-e38,297). in the sensitivity analysis, bacteraemic pneumonia incidence and vaccine effectiveness increased the net cost per lyg by 131% and 218% in the worst-case scenario, and decreased it to e4,249 in the best-case. conclusions: the intervention was not cost saving. the uncertainties concerning invasive pneumococcal disease incidence and vaccine effectiveness make the cost-effectiveness estimates instable. spain 1989 -1998 abstract background: spatial data analysis can detect possible sources of heterogeneity in spatial distribution of incidence and mortality of diseases. moreover small area studies have greater capacity to detect local effects linked to environmental exposures. objective: to estimate the patterns of cancer mortality at municipal level in spain using smoothing techniques in a single spatial model. design and methods: cases were deaths due to cancer, registered at a municipal level nation-wide for the period 1989-1998. expected cases for each town were calculated using overall spanish mortality rates and standard mortality ratios were computed. to plot the maps, smoothed municipal relative risks were calculated using besag york and mollie`model and markov chain monte carlo simulation methods. as an example maps for stomach and lung cancer neoplasms are shown. results: it was possible to obtain the posterior distribution of relative risk by a single spatial model including 8077 towns and the 46398 adjacencies. maps showed the singular patterns for both cancer locations. conclusion: the municipal atlas allows to avoid edge local effects, improving the detection of spatial patterns. discussion: bayesian modelling is a very efficient way to detect spatial heterogeneity by cancer and other causes of death. abstract background: little is known about the impact of socioeconomic status (ses) on outcomes of surgical care. objectives: we estimated the association between ses and outcomes of selected complex elective surgical procedures. methods: using hospital discharge registries (icd-ix-cm codes) of milan, bologna, turin and rome we identified patients undergoing cardiovascular operations (coronary artery bypass grafting, valve replacement, carotid endarterectomy, repair of unruptured thoracic aorta aneurysm) (n = 20,194) and cancer resections (pancreatectomy, oesophagectomy, liver resection, pneumonectomy, pelvic evisceration) (n = 2,300) in four italian cities, 1997-2000. an area-based income index was calculated. post-operative mortality (in-hospital or within 30 days) was the outcome. logistic regression adjusted for gender, age, residence, comorbidities, concurrent and previous surgeries. results: high income patients were older and had fewer comorbidities. mortality varied by surgery type (cabg 4,1%, valve 7,5%, endartectomy 1,2%, aorta aneurysm 10,2%, cancer 6.7%). low income patients were more likely to die after cabg (or = 1. abstract background: an important medical problem of renal transplant patients who receive immunosuppression therapy, is the development of a malignancy during the long term follow-up. however, existing studies are not in agreement over whether patients who undergo renal transplantation have an increased risk of melanoma. objective: the aim of this study was to determine the incidence of melanoma in renal transplantation patients in the northern part of the netherlands. methods: we linked a cohort of 1125 patients who received a renal transplantation in the university medical centre groningen between 1989 and 2003 with the cancer registry of the comprehensive cancer centre north-netherlands, to identify all melanoma patients in this cohort. results: only 1 patient developed a melanoma following the renal transplantation; no significant increase in the risk of melanoma was found. conclusion: although several epidemiologic studies have shown that the risk of melanoma is increased in renal transplantation patients who receive immunosuppression therapy to prevent allograft rejection, this increased risk was not found in the present study. the lower level of immunosuppressive agents given in the netherlands might be responsible for this low incidence. abstract background: socio-economic health inequalities are usually studied for self-reported income, although the validity of self-reports is uncertain. objectives: to compare self-reports of income by respondents to health surveys with their income according to tax registries, and determine to what extent choice of income measure influences the health-income relation. methods: around 22.000 respondents from the dutch permanent survey on living conditions were linked to data from dutch tax and housing registries of 2001. both self-reported and registry-based measures of household equivalent income were calculated and divided into deciles. the association with less than good self-assessed health was studied using prevalence rates and odds ratios. results: around 18% of the respondents did not report their income. around 27% reported an income 3 deciles lower or higher than the actual income value. the relation between income and health was influenced by choice of income measure. larger health inequalities were observed with selfreports compared to registry-based measures. while a linear healthincome relation was found using self-reported income, a curvilinear relation (with the worst health in the second lowest deciles) was observed for registry-based income. conclusion: choice of the income source has a major influence on the health-income relation that is found in inequality research. abstract background: while many health problems are known to affect immigrant groups more than the native dutch population, little is known about health differences within immigrant groups. objectives: to determine the association between self assessed health and socioeconomic status (ses) among people of turkish, moroccan, surinamese and antillean origin. methods: data were obtained from a social survey held among immigrants 20-59 years in the netherlands, with almost 2000 respondents per immigrant group. ses differences in the prevalence of 'poor' self-assessed health were measured using prevalence rate ratios estimated with regression techniques. results: within each immigrant group, poor health was much more common among those with low ses. the health of women was related to their educational level, occupational position, household income, financial situation and (to a lesser extent) their parents' education. similar relationships were observed for men, except that income was the strongest predictor of poor health. the health differences were about as large as those known for the native dutch population. conclusion and discussion: migrant groups are not homogenous. also within these groups, low ses is related to poor general health. in order to identify subgroups where most health problems occur, different socioeconomic indictors should be used. abstract background: genetic damage quantification can be considered as biomarker of exposure to genotoxic agents and as early-effect biomarker regarding cancer risk. objectives: to assess genetic damage in newborns and its relationship with anthropometrical, sociodemographic variables, maternal tobacco consumption and pollution perception. design and methods: the bio-madrid study recruited 150 trios (mother/father/newborn) from 2 areas in madrid to assess the impact of pollutants in humans. parents answered a questionnaire about socio-economic characteristics, pregnancy, life-style and perception of pollution. genetic damage in newborns were measured with the micronucleus(mn) test in peripheral lymphocytes poisson regression models were fitted using mn frequency per 1000 binucleated cells as dependent variable. explanatory variables included sex, parents age, tobacco, area and reported pollution level. results: the mean frequency of mn was 3.94 per 1000 (range:2-10). no differences were found regarding area, sex and maternal tobacco consumption. mn frequency was higher in underweighted newborns and in those residing near heavy traffic roads. in recent years minimally invasive surgery procedures underwent rapid diffusion and laparoscopic cholecystectomy has been among the first to be introduced. after its advent, increasing rates of overall and laparoscopic cholecystectomy have been observed in many countries. we evaluated the effect of the introduction of laparoscopic procedure on the rates of cholecystectomy in friuli venezia giulia region, performing a retrospective study. from regional hospitals discharge data we selected all records with procedure code of laparoscopic (icd 9 cm: 5123) or open (5122) cholecystectomy and diagnosis of uncomplicated cholelithiasis (icd 9 cm: 574.0; 574.1; 574, 2) or cholecystitis (575,0; 575,1), in any field, from 1993 to 2004. in the 12 year study period, the number of overall cholecystectomies increased from 1546 to 2039 (+31,9%), mainly for the relevant increase of laparoscopic interventions from 3 procedures, (0,2% of overall cholecystectomies), to 1697 (83,2%). rates of laparoscopic cholecystectomies increased from 0,1 to 46,8 per 100 admitted patients with diagnosis of cholelithiasis or cholecystitis. the introduction of laparoscopic cholecystectomies was followed not only by a shift towards laparoscopically performed interventions but also by an increase in overall cholecystectomies in friuli venezia giulia region. abstract background: although a diminished doses scheme of 7-valent pneumococcal conjugate vaccination (pcv7) may offer protection against invasive pneumococcal disease, it might affect pneumococcal carriage and herd immunity. long term memory has to be evaluated. objective: to compare the influence of a 3 and 2-doses pcv7-vaccination scheme on pneumococcal carriage, transmission, herd immunity and anti-pneumococcal antibody levels. methods: in a prospective, randomized, controlled trial infants are randomly allocated to receive pcv7 at ages 2 and 4 months; ages 2, 4 and 11 months and the age of 24 months only. nasopharyngeal (np) swabs are regularly obtained from infants and family members. the np swabs are cultured by conventional methods and pneumococcal serotypes are determined by quellung reaction. antibody levels are obtained at 12 and 24 months from 80 infants in group i and ii and from 30 infants in group iii. one thousand infants are needed to detect a 10% difference in pneumococcal carriage (a = 0.05, ß = 0.80) between the three groups. results: so far, 852 infants have been included. preliminary results show that prior to vaccination pneumococcal carriage was 16%. conclusion: this trial will provide insight into the effects of a diminished dose scheme on herd immunity and long-term antipneumococcal antibody development. abstract background: oil-spills cause important environmental damages and acute health problems on affected populations. objectives: to assess the impact of the prestige oil-spill in the hrqol of the exposed population. design and methods: we selected 1350 residents in coastal areas heavily affected by the oil-spill and 1350 residents in unaffected inland villages through random sampling, stratified by age and sex. hrqol was measured with the sf-36 questionnaire in personal interviews. individual exposure was also explored. mean differences in sf-36 scores >3 points were considered 'clinically relevant'. odds ratios (or) summarized the association between area of residence (coast vs inland) and suboptimum hrqol (lower than percentile 25th), adjusting for possible confounders. results: neither clinically relevant nor statistically significant differences were observed in most of the sf-36 scales regarding place of residence or individual exposure. worse scores (inland = 79,2; coast = 75,9; p<0,001) abstract background: patient comorbidities are usually measured and controlled in health care outcome research. hypertension is one of the most commonly used comorbidity measures. objectives: this study aims to assess underreporting of hypertension in ami patients, and to analyze the impact of coding practices among italian regions or hospitals' type. methods: a cohort of ami hospitalisations in italy from november 2002 to october 2003 was selected. 4820 patients with a previous hospital admission reporting a diagnosis of complicated hypertension within the preceding 22 months were studied. a logistic model was constructed. both crude and adjusted probability of reporting a hypertension in ami admissions, depending from the number of diagnosis fields compiled in discharge abstracts, and presence of other diseases were estimated. results: in 57.9% of patients hypertension was not reported. probability of reporting hypertension increased with the number of compiled diagnosis fields (adjusted ors range: 1.50-2.17). there were no significant differences among italian regions, while private hospitals' reporting was less accurate. disorders of lipoid metabolism were more probably coded with hypertension (adjusted or: 4.37). conclusions: information from both ami and previous hospitali-sations would be needed to include hypertension in a comorbidity measure. abstract background: the angiotensin converting enzyme inhibitors (acei) should be considered the standard initial treatment of the systolic heart failure. this treatment is not recommended in patients with hypotension, although figures of systolic blood pressure around 85-90 mmhg during the treatment are allowed if the patient remains asymptomatic. objectives: to know the proportion of patients with systolic heart failure receiving treatment with acei, and the proportion of these patients with signs oh hypotension. design and methods: the electronic clinical records of all the patients diagnosed of systolic heart failure were reviewed. the electronic information system covers a 60% of the population of the basque country, approximately. diagnosis of heart failure was defined as the presence of any of the following cie-9 codes: 428 or 402.11 or 402.91. to evaluate the blood pressure, the last available determination was considered. results: out of 9464 patients with left heart failure, 4108 (43.4%) have been prescribed acei. among the 328 patients with blood pressure lower than 85mmhg (systolic) or than 60mmhg (diastolic), 163 (49.7%) were also receiving this treatment. conclusions: acei are clearly underprescribed in the basque country for the treatment of heart failure. attention should be given to the group at risk of hypotension. abstract background: epidemiologic studies have shown an association between c-reactive protein (crp) and cardiovascular endpoints in population samples. methods: in a longitudinal study of 1006 myocardial infarction (mi) survivors, crp was measured repeatedly (up to 8 times) within a period of 13 months. data on disease history and life style were collected at baseline. we examined the association between different variables and the level of crp using a random effects model. results: in total 5835 crp samples were collected in athens, augsburg, barcelona, helsinki, rome and stockholm. mean levels of crp were 2.6, 2.4, 3.4, 2.0, 2.5, 2.8 [mg/l] respectively. body mass index (bmi) and chronic bronchitis (ever diagnosed) had the largest effect on crp (38% (for 5 kg/m 2 ) and 32% change from the mean level, respectively, p<0.05). age classes showed a cubic function with a minimum at ages 50 to 60. glycosylated hemoglobin (hba1c) <6.5% as a measure of long-term blood glucose control and being male were found to be protective ()21% and )17% respectively, p<0.05). conclusion: it was shown that bmi and history of bronchitis are important in predicting the level of crp. other variables, like alcohol intake, play a minor role in this large sample of mi patients. abstract background: during the last decades a remarkable increase in incidence rates of malignant lymphoma was seen. although some reasons are known or suspect underlying risk factors are not well understood. objectives: we studied the influence of medical radiation (x-ray, radiotherapy and szintigraphy) on the risk of malignant lymphoma. methods: we analysed data from a population-based case-control study with 710 incident lymphoma cases in germany from 1999-2003. after informed consent cases were pair-matched with controls recruited from registration office by age, gender and study region. data was collected in a personal interview. we analysed data using conditional logistic regression. results: the linear model shows an or = 0.99/msv due to x-ray exposure and or = 0.63 (95%-ci = 0.5-0.79) comparing higher with lower exposure. radiotherapy shows an or = 0.76 (n = 31 cases). there is no association between all lymphomas and szintigraphies but in the subgroup containing multiple myeloma, cll, malt-and marginalcell lymphoma we found an or = 1.86 (95%-ci = 1.02-3.40) in the multivariate model. discussion: no excess risk was observed for x-ray examinations. ionising radiation may increase risk for specific lymphoma subgroups. however, it should be noted that numbers in the subgroups are small and that radiation dose may be somehow inaccurate as no measures were available. abstract background: varus-alignment (bow-leggedness) is assumed to correlate with knee osteoarthritis (oa), but it is unknown whether varus-alignment precedes the oa or whether varus-alignment is a result of oa. objective: to assess the relationship between varusalignment and the development, as well as progression, of knee oa. methods: 1,501 participants in the rotterdam study were selected. knee oa at baseline and at follow-up (mean follow-up 6.6 years) was defined as kellgren & lawrence (k&l) grade 3 2, and progression of oa as an increase of 3 1 k&l degree. alignment was measured by the femoro-tibial angle on baseline radiographs. multivariable logistic regression for repeated measurements was used. results: of 2,664 knees, 35.2% showed normal alignment, 64.3% varus-alignment, and 0.5% valgus-alignment. comparison of high varus-alignment versus normal, low and mediate varus-alignment together, showed a two-fold increase in the development of knee oa. (or = 1.83; 95%ci = 1.17-2.85). the risk of progression was higher in the high varus group compared to the normal, low and mediate varus group (or = 2.53; 95%ci = 1.04-6.19). stratification for overweight gave similar odds ratio's in the overweight group, but weaker odds ratio's in the non-overweight group. conclusion: a higher value of varus-alignment is associated with the onset and progression of knee oa. abstract background: echocardiographic image quality in copd patients can be hampered by hyperinflated lungs. cardiovascular magnetic resonance imaging (cmr) may overcome this problem and provides accurate and reproducible information about the heart without geometric assumptions. objective: to determine the value of easily assessable cmr parameters compared to other diagnostic tests in identifying heart failure (hf) in copd patients. design and methods: participants were recruited from a cohort of 405 copd patients = 65 years. a panel established the diagnosis of hf during consensus meetings using all diagnostic information, including echocardiography. in a nested case-control study design, 37 copd patients with hf (cases) and a random sample of 41 copd patients without hf (controls) received cmr. the diagnostic value of cmr for diagnosing hf was quantified using univariate and multivariate logistic modelling and roc-area analyses. results: four easily assessable cmr measurements had significantly more added diagnostic value beyond clinical items (roc-area 0.91) than amino-terminal pro b-type natriuretic peptide (roc-area 0.80) or electrocardiography (roc-area 0.77). a 'cmr model' without clinical items had an roc-area of 0.88. conclusion: cmr has excellent capacities to establish a diagnosis of heart failure in copd patients and could be an alternative for echocardiography in this group of patients. abstract background: the prevalence of overweight (i.e, body mass index [bmi] > = 25 kg/m2) is increasing. new approaches to address this problem are needed. objectives: 1) to assess the effectiveness of distance counseling (i.e., by phone and e-mail/internet) on body weight and bmi, in an overweight working population. 2) to assess differences in effectiveness of the two communication methods. design and methods: 1386 overweight employees (67% male; mean age 43.9±8.6 years; mean bmi 29.6±3.5 kg/m2) were randomized to a control group receiving general information on overweight and lifestyle (n = 460), a phone based intervention group (n = 462) and an internet based intervention group (n = 464). the intervention took 6 months and used a cognitive behavioral approach, addressing physical activity and diet. the primary outcome measures, body weight and bmi, were measured at baseline and at six months. statistical analyses were performed with multiple linear regression. results: the intervention groups (i.e., phone and e-mail combined) lost 1.5 kg (bmi reduced by 0.5 kg/m2) over the control group (p = 0.000). the phone group lost 0.5 kg more than the internet group (p = 0.179). abstract objective: although an inverse gradient education-mortality has been shown in the general population, little is known about this trend in groups with higher risks of death.we examine differences in mortality by education and hiv-status among injecting drug users (idus) before and after introduction of highly active antiretroviral therapy (haart) in 1997. methods: communitybased cohort study of idus recruited in 3 aids prevention centres (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) abstract background: pancreatic cancer is an aggressive cancer with low survival time, with health-related quality of life (hrqol) being of major importance. objectives: the aim of our study was to assess both generic and disease-specific hrqol in patients with pancreatic cancer. methods: patients with suspected pancreatic cancer were consecutively included at admission to the hospital. hrqol was determined with the disease-specific european organization for research and treatment of cancer (eortc) health status instrument and generic euroqol (eq-5d). results: a total of 57 patients (mean age 62 years ±11, 49% men) were admitted with suspected pancreatic cancer. of these patients, 45 (79%) had pancreatic cancer confirmed as final diagnosis. hrqol was significantly impaired in patients with pancreatic cancer for most eortc and eq-5d scales in comparison to norm populations. the ed-5d visual analogue scale (vas) and utility values were significantly correlated to the five functional scales, to the global health scale and to some but not all of the eortc symptom scales/items. conclusions: hrqol was severely impaired in patients with pancreatic cancer. there was a significant correlation between most eortc and eq-5d scales. our results may facilitate further economic evaluations and aid health policy makers in resource allocation. abstract background: organised violence has health impact both on those who experience the violence directly and indirectly. the numbers of people affected by mass violence is alarming. substantial knowledge on the long-term health impact of organized violence is of importance for public health and for epidemiology. objectives: to investigate research results of long term mental health impact of organised violence. design and methods: a search of papers for the keywords genocide, organised violence, transgenerational effects, mental health was carried out in pubmed, science citation index and psychinfo. results: the systematic review on the long-term health impact of genocide showed that exposure to organised violence has an impact on mental health. methodological strenghts and weaknesses varied between studies. the found mental health consequences were associated with the country of research and the time of study. overall data showed organised violence has transgenerational impact on mental health of individuals and societies. conclusion: longitudinal studies have to be carried out to get further insight into the long-term health effects of organised violence. discussion: research results on mental health effects of organised violence have to be analysed in the context of changing concepts of illness. overweight is increasing and associated with various health problems. there are no well-structured primary care programs for overweight available in the netherlands. therefore, we developed a 12-month multidisciplinary treatment program in a primary care setting. the aim of the present study is to determine the feasibility and efficacy of a multidisciplinary treatment program on weight loss and risk profile in an adult overweight population. hundred participants of the utrecht health project are randomised to either a dietetic group or a dietetic plus physiotherapy group. the control group consist of another 50 participants recruited from the utrecht health project and receives routine health care. body weight, waist circumference, blood pressure, serum levels, energy-intake and physical activity are measured at baseline, halfway and at the end of the treatment program. feasibility of the treatment program is assessed by response, compliance and program-associated costs and workload. efficacy is determined by analysing changes in outcome measures between groups over time using t-tests and anova repeated measurements. the treatment program is considered effective with at least a 5% difference in mean weight change over time between groups. positive evaluation of the multidisciplinary treatment program for overweight may lead to implementation in routine primary health care. abstract background: examining patient's quality of life (qol) before icu admission will permit to compare and analyze its relation with other variables. objectives: analyze qol of patients admitted to a surgical icu before admission and study its relation with baseline characteristics and outcome. design and methods: the study was observational and prospective in a surgical icu, enrolling all patients admitted between november 2004 and april 2005. baseline characteristics of patients, history of co morbidities and quality of life survey score (qolss) were recorded. assessment of the relation between each variable or outcome and the total score of qolss was performed by multiple linear regression. results: total qolss demonstrated worse qol in patients with hypertension, cerebrovascular disease, renal insufficiency, severely ill (as measured by saps and asa physical status), and in older patients. there was no relation between qol and longer icu los. conclusions: preadmission qol correlates with age, severity of illness, comorbidities and mortality rates but is an able to predict longer icu stay. discussion: qolss appears to be a good indicator of outcome and severity of illness. abstract background: transient loss of consciousness (tloc) has a cumulative lifetime incidence of 35%, and can be caused by various disorders. objectives: to assess the yield and accuracy of initial evaluation (standardized history, physical examination and ecg), performed by attending physicians in patients with tloc, using follow-up as a gold standard. design and methods: adult patients presenting with tloc to the academic medical centre between february 2000 and may 2002 were included. after initial evaluation physicians made a certain, likely or no initial diagnosis. when no diagnosis was made additional cardiological testing, expert history taking and autonomic function testing were performed. the final diagnosis, after 2 years follow-up, was determined by an expert committee. results: 503 patients were included. after initial evaluation, 24% of the patients were diagnosed with a certain and 40% with a likely cause for their episodes. overall diagnostic accuracy was 91% (95%ci 88-94%); 96% (95%ci 91-98%) for the certain diagnoses and 88% (95%ci 83-92%) for the likely diagnoses. conclusion and discussion: attending physicians make a diagnosis in 64% of patients with tloc after initial evaluation, with high accuracy. the use of abundant additional testing can be avoided in many patients. abstract background: the possibility of an influenza pandemic is one of the major public health challenges of today. risk perceptions among the general public may be important for successful public health measures to better control an outbreak situation. objectives: we investigated risk perception and efficacy beliefs related to an influenza pandemic in the general population in 8 countries in europe and asia. design and methods: telephone interviews were conducted in 2005. risk perception of an influenza pandemic was measured on a 5-point scale and outcome-and self-efficacy on a 4point scale (low-high). the differences in risk perception by country, sex and age were assessed with a general linear model including interaction effects. results: 3,403 persons were interviewed. the mean risk perception of flu was 3.14 and was significantly higher in europe (3.21) compared to asia (3.03) (p<0.001) and higher in women (3.23) than men (3.02) (p<0.001). outcome-and self-efficacy were lower in europe than asia. conclusion: in europe higher risk perceptions and lower efficacy beliefs were found as compared to asia. in developing preparedness plans for an influenza pandemic specific attention should therefore be paid to risk communication and how perceived self-efficacy can be increased. abstract background: increased survival of patients with cf has prompted interest towards their hrqol. objectives: 1.to measure hrqol and its predictors in cf patients cared for at the bambino gesuc hildren's hospital in rome; 2. to assess the psychometric properties of the italian version of the cf specific hrqol instrument (cystic fibrosis questionnaire, cfq). design and methods: crosssectional survey. all cf patients aged 7 years or more were asked to complete the cfq (age-specific format). psychological distress was assessed through standardized questionnaires in patients (achenbach and general health questionnaire, ghq) and their parents (ghq and sf-36). results: one-hundred-eighteen patients (58 males, 60 females, age range 7 to 39 years) participated in the study (response rate 97%). internal consistency of cfq was satisfactory (cronbach alpha from 0.60 to 0.88); all item-test correlation were greater than 0.40. average cfq standardized scores were very good in all domains (>70 on a 0-100 scale), except perceived burden of treatments (57) and degree of socialization (45). multiple regression analysis was performed to identify factors associated with different hrqol dimensions. conclusion: support interventions for these patients should concentrate on finding a balance between need to prevent infections and promotions of adequate, age-appropriate social interactions. abstract background: the metabolic syndrome (metsyn) -a clustering of metabolic risk factors with diabetes and cardiovascular diseases as the primary clinical outcomes -is thought to be highly prevalent with an enormous impact on public health. to date, consistent data in germany are missing. objective: the study was conducted to examine the prevalence of the metsyn (according to ncap atp iii-definition) among german patients in primary care. methods: the german-wide cross-sectional study run two weeks in october 2005 with 1503 randomly selected general practitioners included. blood glucose and serum lipids were analyzed, waist circumference and blood pressure assessed, data on smoking, dietary and exercise habits, regional and sociodemographic characteristics collected. abstract background: excessive infant crying is a common and often stress inducing problem than can ultimately result in child abuse. from previous research is known that maternal depression during pregnancy is related to excessive crying, but so far little attention is paid to paternal depression. objective: we studied whether paternal depression is independently associated to excessive infant crying. design and methods: in a prospective multiethnic population-based study we obtained depression scores of 3,451 mothers and 2,606 fathers at 20 weeks pregnancy using the brief symptom inventory, and information on crying behaviour of 2,747 infants at 2 months. we used logistic regression analyses in which we adjusted for depression of the mother, level of education, smoking and alcohol use. results: paternal depressive symptomatology was related to the widely used wessel's criteria for excessive crying (adusted odds ratio 2.34, 1.22 -4.49). conclusion: our findings indicate that paternal depressive symptomatology might be a risk factor for excessive infant crying. discussion genetic as well as other direct (e.g. interaction between father and child) or indirect (e.g. marital distress or poor circumstances) mechanisms could explain the found association. abstract background: in studying genetic background of congenital anomalies the comparison of affected cases to non-affected controls is popular method. investigation of case-parent triads uses observation of cases and their parents exclusively. methods: both casecontrol approach and log-linear case-parent triads model were implemented to spina bifida (sb) cases and their parents (61 triads) and 267 controls in analysis of impact of the c667t and a1298c mthfr polymorphisms on occurrence of sb. results: observed frequencies for 677tt genotype were 11,5% in sb children, 6,6% in mothers, 9,8% in fathers, 8,6% in controls and for 1298cc genotype were 4,9% of sb children, 4,9% of mothers, 11,5% of fathers and 7,9% of controls. both genotype frequencies in sb triads did not differ significantly from controls. case-control approach showed nonsignificant increase in risk of having sb for 677t allele carriers either in homozygous (or = 1,7) or heterozygous form (or = 1,2) and for 1298c allele carriers in heterozygous form (or = 1,3). log-linear model revealed significant relative risk of sb in children with both 677tt and ct genotype (rr = 3,77 and rr = 2,37 respectively). child's genotype at a1298c and mother's genotypes did not contribute to the risk. conclusions: caseparent triads approach adds new information regarding impact of parental imprinting on congenital anomalies. abstract background: previous studies showed an association of autonomic dysfunction with coronary heart disease (chd) and with depression as well as an association of depression with chd. however, there is limited information on autonomic dysfunction as potential mediator of the adverse effect of depression on chd. objectives: to examine the role of autonomic dysfunction as a potential mediator of the association of depression with chd. design/ methods: we used data of 1309 participants aged 45-83 years of the ongoing population-based cross-sectional carla study (54% male). time-and frequency-domain parameters of heart rate variability (hrv) as a marker of autonomic dysfunction were calculated. prevalent myocardial infarction (mi) was defined as selfreported physician-diagnosed mi or diagnostic minnesota code in the electrocardiogram. depression was defined based on the cesd-depression scale. logistic regression was used to assess associations between depression, hrv and mi. results: in ageadjusted logistic regression models, there was no statistically significant association of hrv with depression, of depression with mi, or of hrv with mi in men and women. discussion/conclusion: the present analyses do not support the hypothesis of an intermediate role of autonomic dysfunction on the causal path from depression to chd. abstract background: hypertension is an established risk factor for cardiovascular disease. however, prevalence of untreated or uncontrolled hypertension is often high (even in populations at high risk). objectives: to assess the prevalence of untreated and of uncontrolled hypertension in an elderly east german population. design and methods preliminary data of a cross-sectional, populationbased examination of 1556 men and women aged 45-83 years were analysed. systolic (sbp) and diastolic blood pressure (dbp) were measured and physician-diagnosed hypertension and use of antihypertensive drugs were recorded. prevalence of hypertension was calculated according to age and sex. results: of all participants, 78.5 % were hypertensive (81.8 % of men, 74.8 % of women). of these, 29.7 % were untreated, 43.8 % treated but uncontrolled, and 26.6 % controlled. women were more often properly treated than men. the prevalence of untreated hypertension was highest in men aged 45-55 years (60.9 %) and lowest in men and women aged > = 75 years (12.6 %). uncontrolled hypertension increases with age in both sexes. conclusion and discussion: in this elderly population, there is a high prevalence of untreated and uncontrolled hypertension. higher awareness in the population and among physicians is needed to prevent sequelae such as cardiovascular disease. abstract background: exposure to pesticides is a potential risk factor for subfertility, which can be measured by time-to-pregnancy (ttp). as female greenhouse workers constitute a major group of workers exposed to pesticides at childbearing age, a study was performed among these and a non-exposed group of female workers. objectives: to measure the effects of pesticide exposure on time-topregnancy. design and methods: data were collected through postal questionnaires with detailed questions on ttp, lifestyle factors, and work tasks (e.g. application of pesticides, re-entry activities, and work hours) during six months prior to conception of the most recent pregnancy. associations between ttp and exposure to pesticides were studied in cox's proportional hazards models among 398 female greenhouse workers and 524 referents. results: the initial fecundability ratio (fradjusted) for greenhouse workers versus referents was 1.11 (95%ci: 0.96-1.29). this fr proved to be biased by the reproductively unhealthy worker effect. restricting the analyses to fulltime workers only gave an fradjusted of 0.89 (95%ci: 0.67-1.19). among primigravidous greenhouse workers, an association was observed between prolonged ttp and gathering flowers (fr = 0.46, 95%ci: 0.18-1.19). conclusion and discussion: this study adds some evidence to the hypothesis of adverse effects of pesticide exposure on time-topregnancy, but more research is needed. abstract background: hfe-related hereditary hemochromatosis (hh) is an iron overload disease for which screening is recommended to prevent morbidity and mortality. however, discussion has risen on the clinical penetrance of the hfe-gene mutations. objective: in the present study the morbidity and mortality of families with hferelated hh is compared to a normal population. methods: c282yhomozygous probands with clinically overt hfe-related hh and their first-degree relatives filled in a questionnaire on health, diseases and mortality among relatives. laboratory results on serum iron parameters and hfe-genotype were collected. the self-reported morbidity, family mortality and laboratory results were compared with an age and gender matched subpopulation of the nijmegen biomedical study (nbs), a population-based survey conducted in the eastern part of the netherlands. results: twohundred-twenty-eight probands and 743 first-degree relatives participated in the hefas. serum iron parameters were significantly elevated in the hefas population compared to the nbs controls. also, the morbidity within hefas families was significantly increased for fatigue, hypertension, liver disease, myocardial infarction, osteoporosis and rheumatism. mortality among siblings, children and parents of hefas probands and nbs participants was similar. discussion: the substantially elevated morbidity within hefas families justifies further exploration for a family cascade screening program for hh in the netherlands. abstract objectives: to evaluate awareness levels and effectiveness of warning labels in cigarette packs, among portuguese students enrolled in the 7th to the 12th grades. design and methods: a cross sectional-study was carried out in may (2004) in a high school population (7th-12th grades) in the north of portugal (n = 1005). a confidential self-reported questionnaire was administered. warning labels effectiveness was evaluated by changes in smoking behaviour and cigarette consuption, during the period between june/2003 (before the implementation of the tobacco warnings labels in portugal) and may/2004. continuous variables were compared by the t-test for paired samples and kruskal-wallis test. crude and adjusted odds ratios and confidential intervals were calculated by logistic regression analysis. results: the majority of students (71.8%) have a high level of awareness about warning labels content. this knowledge was significantly associated with school grade and current smoking status. none of these variables was significantly associated with changes in smoking behaviour. although not reaching statistic significance, the majority of teenagers (75.4%) increased or kept their smoking pattern. awareness level was not associated with smoking prevalence or consumption decreases. conclusions: current warning labels are ineffective in changing smoking behaviour among portuguese adolescents. abstract background: injuries are an important cause of morbidity. the presence of pre-existing chronic conditions (pecs) have been shown to be associated with higher mortality. objectives: aim of this study is to evaluate the association between pecs and risk of death in elder trauma patients. methods: an injury surveillance, based on the integration between emergency, hospital, and mortality databases of lazio region, year 2000, was used. patients were the elder people visited at the emergency departments, and hospitalised. pecs were evaluated on the basis of the charlson comorbidity index (cci). to measure the effect of pecs on the probability of death, we used logistic regression. results: 8145 patients were admitted to the hospital. the 17.9% of the injured subjects were affected by one or more chronic conditions. risk of death for non urgent and urgent patients increased at increasing cci score abstract background: c-reactive protein (crp) was shown to predict prognosis in heart failure (hf). objective: to assess variability of crp over time in patients with stable hf. methods: we measured high-sensitivity crp (hscrp) 3 times (3-week intervals) in patients with stable hf. patients whose hscrp was >1 mg/dl or whose clinical status deteriorated were excluded. two consecutive hscrp measurements were available for 50 patients: 37 men, mean(sd) age 69.6(11.9) years, 82% depressed left ventricular systolic function. forty-four patients had a third measurement. using the cutoff point of 0.3mg/dl for prediction of adverse cardiac events we assessed the proportion of patients who changed risk category. results: median(p25-p75) baseline hscrp was 0.19mg/dl(0.12-0.34). hscrp varied largely particularly for higher levels. the 5th and 95th percentiles of differences between first two measurements were )0.20mg/dl and +0.55mg/dl. correlation coefficient between these measurements: 0.55, p<0.001. eleven (22%) patients changed risk category, kappa = 0.53, p<0.001. among patients whose first two measurements were concordant, 16.7% changed category in third measurement, kappa = 0.65, p<0.001. conclusion: large variability in hscrp in stable hf may decrease the validity of risk stratification based on single measurements. it remains to be demonstrated whether the pattern of change over time adds predictive value in hf patients. abstract background: instrumental variables can be used to adjust for confounding in observational studies. this method has not yet been applied with censored survival outcomes. objectives: to show how instrumental variables can be combined with survival analysis. design and methods: in a sample of 415 patients with type-1 diabetes who started renal-replacement therapy in the netherlands between 1985 and 1996, the effect of pancreas-kidney transplantation versus kidney transplantation on mortality was analyzed using region as the instrumental variable. because the hospital could not be chosen with this type of transplantation, patients can be assumed to be naturally randomized across hospitals. we calculated an adjusted difference in survival probabilities for every time point including the appropriate confidence interval (ci95%). results: the 5-year difference in survival probabilities between the two transplantation methods, adjusted for measured and unmeasured confounders, was 0.37 (ci95%: 0.18-0.57) favoring the pancreas-kidney transplantation. this is substantially larger than the intention-to-treat estimate of 0.13 (ci95%: 0.01-0.25) where policies are compared. conclusion and discussion: instrumental variables are not restricted to uncensored data, but can also be used with a censored survival outcome. hazard ratios with this method have not yet been developed. the strong assumptions of this technique apply similarly with survival outcomes. 11.2] . sir of coronary heart disease was 5.7 [95%ci: 4.8-6.7] and remained significantly increased up to 30 years of follow-up. cox regression analysis showed a 3.2-fold (95% ci, 1.6-6.4) increased risk of congestive heart failure after anthracyclines and a 5.3-fold (95% ci, 1.6-16.8) increased risk of coronary heart disease after radiotherapy to the mediastinum. conclusion: the incidence of several cardiac diseases was strongly increased after treatment for hl, even after prolonged follow-up. anthracyclines increased the risk of congestive heart failure and radiotherapy to the mediastinum increased the risk of coronary heart disease. abstract background: the concept of reproductive health is emerging as an essential need for health development. objectives: to know the opinions of parents, teachers and students about education of reproductive health issues to students of mid and high schools. design and methods: focus group discussions (fgd) as a qualitative research was chosen. a series of 24 group discussions with participation of 162 persons (64 students, 50 teachers, and 48 parents) was held. each group had included 6 to 9 persons. results: all the participants noted to a true need in education of puberty health in order to provide essentials for pre-adolescent students to adopt the psycho-and somatic changes of puberty. however, a few fathers and a group of mothers believed that education of family planning is not suitable for students. a need for education of aids and marital problems for students was the major concern in all groups. the female students emphasized a need for programming counseling in pre-marital period. conclusion: essentials in puberty health, family planning, aids and marital problems should be provided in mid-and high schools in order to narrow the knowledge gap of the students. abstract background: the association between social support and hypertension in pregnancy remains controversial. objective: the objective of this study was to investigate whether level of social support is a protective factor against preeclampsia and eclampsia. design and methods: a case-control study was carried out in a public high-risk maternity hospital in rio de janeiro, brazil. between july 2003-may 2004, all 225 cases, identified at diagnosis, and 459 controls, matched on gestational age, were included in the study. participants were interviewed about clinical history, socio-demographic and psychosocial characteristics. the principal exposure was the level of social support available during the pregnancy, using the medical outcomes study scale. adjusted odds ratios were estimated using multivariate conditional logistic regression. results: multiparous women with a higher level of social support had a lower risk of presenting with preeclampsia and eclampsia (or = 0.7), although this association was not statistically significant (95% ci 0.4-1.2). in primiparous women, a higher level of social support was seen amongst cases (or = 2.1; 95% ci 1.5-2.9). an interaction between level of social support and stressful life events was not identified. these results contribute to increased knowledge of the relationship between preeclampsia and psychosocial factors in low-income pregnant and puerperal women. abstract background: current case-definitions for cfs/me are designed for clinical-use and not appropriate for health needs assessment. a robust epidemiological case-definition is crucial in order to achieve rational allocation of resources to improve service provision for people with cfs/me. objectives: to identify the clinical features that distinguish people with cfs/me from those with other forms of chronic fatigue and to develop a reliable epidemiological case-definition. methods-primary care patient data for unexplained chronic fatigue was assessed for symptoms, exclusionary and comorbid conditions and demographic characteristics. 101 cases were assigned to disease and non-disease groups by three members of the chief medical officer's working group on cfs/me (reliability-cronbach's alpha 0.644). results: preliminary multivariate analyses were conducted and classification and regression tree analysis included a 10-fold cross-validation approach to prevent over fitting. the results suggested that there were at least four strong discriminating variables for cfs/ me with 'post-exertional malaise' being the strongest predictor. risk and classification tables showed an overall correct classification rate of 81.2%. conclusion: the analyses demonstrated that the application of the combination of the four discriminating variables (the defacto epidemiological case-definition) and predefined comorbid conditions had the ability to differentiate between cfs/me and non-cfs/me cases. abstract background: infection with high-risk human papillomavirus (hpv) is a necessary cause for cervical cancer. vaccines against the most common types (hpv16, hpv18) are being developed. relatively little is known about factors associated with hpv16 or hpv18 infection. we investigated associations between lifestyle factors and hpv16 and hpv18 infection. methods: 5031 uk women aged 20-59 years with a recent abnormal cervical smear underwent hpv testing and completed a lifestyle questionnaire. hpv16 and hpv18 status was determined using type-specific pcrs. associations between lifestyle factors and hpv status were assessed by multivariate logistic regression models. results: 16.5% (95%ci 15.4%-17.6%) of women were hpv16-positive. 8.6% (95% ci 7.9%-9.5%) were hpv18-positive. for both types, the proportion testing positive decreased with increasing age, and increased with increasing grade of cytological abnormality. after adjusting for these factors, significant associations remained between (i) hpv16 and marital, employment, and smoking status and (ii) hpv18 and marital status and contraceptive pill use. gravidity, ethnicity, barrier contraceptive use and socio-economic status were not related to either type. conclusions we identified modest associations between several lifestyle factors and hpv16 and hpv18. studies of this type help elucidate hpv natural history in different populations and will inform development of future vaccine delivery programmes. in ageing men testosterone levels decline, while cognitive function, muscle and bone mass, sexual hair growth, libido and sexual activity decline and the risk of cardiovascular diseases increase. we set up a double-blind, randomized placebo-controlled trial to investigate the effects of testosterone supplementation on functional mobility, quality of life, body composition, cognitive function, vascular function and risk factors, and bone mineral density in older hypogonadal men. we recruited 237 men with serum testosterone levels below 17 nmol/l and ages 60-80 years. they were randomized to either four capsules of 40 mg testosterone undecanoate (tu) or placebo daily for 26 weeks. primary endpoints are functional mobility and quality of life. secondary endpoints are body composition, cognitive function, aortic stiffness and cardiovascular risk factors and bone mineral density. effects on prostate, liver and hematological parameters will be studied with respect to safety. measure of effect will be the difference in change from baseline visit to final visit between tu and placebo. we will study whether the effect of tu differs across subgroups of baseline waist girth, testosterone level, age, and level of outcome under study. at baseline, mean age, bmi and testosterone levels were 67 (yrs), 27 (kg/m2) and 13.x (nmol/l), respectively. abstract at a student population, the carie's prevalence was 56,28%. objectives: to evaluate the efficiency between two types of oral health education programmes and the adherence towards tooth brushing. study design: case control study: 333 youngsters took part, 134 in the case group. an health education programme was carried out in 8 schools and included two types of strategies: a participative strategy (learning based on the colouring of the dental plaque) towards a case group; and a traditional strategy (oral expository method) towards a control group. during the outcome of the programmes, the oral health condition evaluation was done through cpo index, the adherence towards tooth brushing and the (iho's) oral hygiene index. results: in the initial dental exam the (iho) average was of 1,60. three months after the application of the oral health programme, there was a general decrease in the average of iho's to 1,30. discussion and conclusion: in the case group the decrease was higher: 0,36 to 0,20. the students submitted to a session of oral health education based on the colouring of the dental plaque showed an lower iho's average and higher knowledge. this can be due to the teaching session being more active, participative and demonstrative. abstract background: violence perpetuated by adolescents is a major problem in many societies. objectives: the aim of this study is to examine high school students' violent behaviour and to identify predictors. design and methods: a cross-sectional study was conducted in timis county, romania between may-june 2004. the sample consisted of 149 randomly selected classes, stratified proportionally according to grades 9-12, high school profile, urban and rural environment. the students completed a self administered questionnaire in their classroom. a weighting factor was applied to each student record to adjust for non-response and for the varying probabilities of selection. results: a total of 2908 students were included in the survey. during the last 12 months, 24.3% of adolescents got mixed into a physical fight outside school and 14.5% on school property. abstract background: drug use by adolescents has become an increasing public health problem in many countries. objectives: the aim of this study is to identify prevalence of drug use and to examine high school students' perceived risks of substance use. design and methods: a cross-sectional study was conducted in timis county, romania between may-june 2004. the sample consisted of 149 randomly selected classes, stratified proportionally according to grades 9-12, high school profile, urban and rural environment. the students completed a self administered questionnaire in their classroom. eighteen items regarding illicit drug use suggesting different intensity of use were listed. the response categories were 'no risk', 'slight risk', 'moderate risk', 'great risk' and 'don't know'. results: a total of 2908 students were included in the survey. the lifetime prevalence of any illicit drug was 5.3%. significant beliefs associated with drug use are: trying marijuana once or twice (p<0.001), smoking marijuana occasionally (p = 0.003), trying lsd once or twice (p = 0.035), trying cocaine once or twice (p = 0.001), trying heroine once or twice (p = 0.002). conclusion: the overall drug use prevalence is small. however, use of some drugs once or twice is not seen as a very risky behaviour. abstract background: the health ombudsman service was created in ceara´, brazil, in 1991, with the objective of receiving user opinions about public services. objectives: to describe user profiles, evaluating their satisfaction with health services and the ombudsman service itself. design and methods: a transversal and exploratory study with a random sample of 292 users who had used the service in the last three months. the data were analyzed with the epi info program. results: women were those who used the service most (74.1%). the users sought the service for complaints (64.7%), guidance (12.3%) and commendation (11.5%). users made the following complaints about health services: lack of care (57.8%), poor assistance (52.0%) lack of medication (8.6%). in relation to the ombudsman service, the following failures were mentioned: lack of autonomy (14.7%), delay in solving problems (8.0%) and few ombudsmen (6.6%). conclusion: participation of the population in use of the serviced is small. the service does not satisfy the expectations of users, it is necessary to publicize the service and try to establish an effective partnership between users and ombudsmen so that the population finds in the ombudsman service an instrument to put into effect social control and improve the quality of health services. in chile, the rates of breast cancer and diabetes have dramatically increased in the last decade. the role of insulin resistance in the development of breast cancer, however, remains unexplored. we conducted a hospital-based case-control to assess the relationship of insulin resistance (ir) and breast cancer in chilean pre and postmenopausal women. we compared 175 women, 33-86 y, with incident breast cancer diagnosed by biopsy and 209 controls with normal mammography. insulin and glucose were measured in blood and ir was calculated by homeostasis model assessment method. anthropometric measurements and socio-demographic and behavioural data were also collected. odds ratios (ors) and 95% confidence intervals (cis) were estimated by multivariate logistic regression. the risk of breast cancer increased with age. ir was significantly associated to breast cancer in postmenopausal women (or = 2.03, 95%ci = 1.11-3.74), but not in premenopausal (p>0.05). socioeconomic status and smoking appeared as important risk factors for breast cancer. obesity was not associated with breast cancer at any age (p>0.05). in these women, ir increased the risk of breast cancer only after menopause. overall, these results suggest a different risk pattern for breast cancer before and after menopause. keywords: insulin resistance; breast cancer; chile. abstract background: previous european community health indicators (echi) projects have proposed a shortlist of 82 indicators as a common conceptual structure for health information. the european community health indicators and monitoring (echim) is a 3-year project to develop and implement health indicators and to develop health monitoring. objectives: our aim is to assess the availability and comparability of the echi-shortlist indicators in european countries. methods: four widely used health indicators i) perceived general health ii-iii) prevalence of any and certain chronic diseases or conditions iv) limitations in activities of daily living (adl) were evaluated. our evaluation of available sources for these indicators is based on the european health interview & health examination surveys database (171 surveys in chile, breast cancer, obesity and sedentary behaviour rates are increasing. the role of specific nutrients and exercise in the risk of breast cancer remains unclear. the aim of the present study was to evaluate the role of fruits and vegetables intake and physical activity in the prevention of breast cancer. we undertook an age matched case-control study. cases were 170 women with breast cancer histologically confirmed and controls were 170 women with normal mammography, admitted to the same hospital. a structured questionnaire was used to obtain dietary information and measurement of physical activity was obtained from the international physical activity questionnaire. odds ratios (ors) and 95% confidence intervals (cis) were estimated by conditional logistic regression adjusted by obesity, socioeconomic status and smoking habit. a significant association was found with fruit intake (or = 0.57, 95%ci = 0.35-0.94). the consumption of vegetables (or = 0.91, 95%ci = 0.80-1.04), moderate (or = 1.00, 95%ci = 0.62-1.59) and high physical activity (or = 1.13, 95%ci = 0.51-2.50) were not observed as protective factors. in conclusion, the consumption of fruit is protective in breast cancer. these findings need to be replicated at chile to support the role of diet and physical activity in breast cancer and subsequence contribution in public health policy. keywords: diet; physical activity; breast cancer; chile. the role of trace elements in pathogenesis of liver cirrhosis and its complications is still not clearly understood. serum concentrations of zinc, copper, manganese and magnesium were determinated in 100 patients with alcoholic liver cirrhosis and 50 healthy subjects by means of plasma sequential spectrophotometer. serum levels of zinc were significantly lower (median 0.77 vs 11.0lmol/l, p = 0.001) in patients with liver cirrhosis in comparison to controls. serum levels of copper were significantly higher in patients with liver cirrhosis (23.71 vs 13.32lmol/l, p<0.001) as well as manganese (4.60 vs 0.02lmol/l, p = 0.001). concentration of magnesium was not significantly different between patients with liver cirrhosis and controls (0.94 vs 0.88 mmol/l, p = 0.060). there was no difference in trace elements concentrations between child-pugh groups. zinc level was significantly lower in patients with hepatic encephalopathy in comparison to cirrhotic patients without encephalopathy (0.60 vs 0.96lmol/l, p = 0.020). manganese was significantly higher in cirrhotic patients with ascites in comparison to those without ascites (6.05 vs 2.60lmol/l, p = 0.039). correction of trace elements concentrations might have beneficial effect on complications and maybe progression of liver cirrhosis. it would be recommendable to provide analyzis of trace elements as a routine. abstract background: respiratory tract infections (rti) are very common in childhood and knowledge of pathogenesis and risk factors is required for effective prevention. objective: to investigate the association between early atopic symptoms and occurrence of recurrent rti during first 4 years of life. design and methods: in the prospective prevention and incidence of asthma and mite allergy birth cohort study, 4146 children were followed from birth to the age of 4 years. information on atopic symptoms, potential confounders, and effect modifiers like passive smoking, daycare attendance and presence of siblings was collected at ages 3 months and 1 year by parental questionnaires. information on rti was collected at ages 1, 2, 3, and 4 years. results: children with early atopic symptoms, i.e. itchy skin rash and/or eczema or doctordiagnosed cow's milk allergy at 1 year of age had a slightly higher risk to develop recurrent rti (aor 1.20 (0.83-1.73); and 1.98 (1.06-3.70), respectively). the association between atopic symptoms and recurrent rti was stronger in children whose mother smoked during pregnancy and who had siblings (aor 4. 12 (1.47-11.49) the aim : the aim of the study was to assess the relative risk (rr) of obesity and abdominal fat distribution on the insulin resistance (ir), diabetes, hyperlipidemia and hypertension in polish population. materials and methods: 6000 subjects at age 35-75, were randomized and invited to the study. in 2838 participants anthropometric and blood pressure examination was performed. fasting lipids, fasting and after glucose load glucose and insulin were determined. ir was defined as the upper quartile of the homa-ir distribution for the normal glucose tolerant population. results: overweight and obesity was observed in 39,7% and 25,1% of subjects. visceral obesity was found in 2143 subjects (88,9%-men and 73,4%-women). rr of ir in obesity was 3,94 (95% ci:3,09-5,04), for obese subjects at age below 45 was 6,6 (95% ci:3,6-12,0). in men with visceral obesity rr of ir was the highest for men aged below 55. rr of diabetes was increasing with the increase of body weight, in obese subjects with abdominal fat distribution was 2,88 (95%ci:2,20-3,79). the same was observed for the hypertension and hyperlipidemia. conclusions: obesity and the abdominal fat distribution seems to be an important risk factor of ir, diabetes, hypertension, hiperlipidemia, especially in the younger age groups. abstract background: age as an effect modifier in cardiovascular risk remains unclear. objective: to evaluate age-related differences in the effect of risk factors for acute myocardial infarction (ami). methods: in a population-based case-control study, with data collected by trained interviewers, 696 consecutive male cases of first myocardial infarction (participation rate 98%) and 867 randomly selected male control dwellers (participation rate 70%) were compared. effect-measure modification was evaluated by the statistical significance of a product term of each independent variable with age. unconditional logistic regression was used to estimate ors in each age stratum (<46 years/>45 years). results: there was a statistically significant interaction between education (>9 vs. <5 years), sports practice, diabetes and age: the adjusted (education, ami family history, dyslipidemia, hypertension, diabetes, angina, waist circumference, sports practice, alcohol and caffeine consumption, and energy intake) ors (95%ci) were respectively 0.16 (0.07-0.33), 0.76 (0.45-1.28) and 8.35 (1.64-42.6 ) in younger, and 0.46 (0.30-0.69), 0.36 (0.24-0.52) and 1.84 (1.08-3.16) in older participants. conclusions: in males, age has a significant interaction with education, sports practice and diabetes in the occurrence of ami. the effect is evident in the magnitude but not in the direction of the association. abstract there are few studies on the role of diet in lung cancer etiology. thus, we calculated both, squamous cell and small cell carcinoma risks in relation to the frequency of consumption of vegetables, cooked meat, fish and butter in silesian male in industrial area of poland. in the case-control study, the studied population comprised 237 men with squamous cell carcinoma and 122 men with small cell carcinoma, and 649 healthy controls. multivariate logistic regression was employed to calculate lung cancer risk in the relation to simultaneous influence of dietary factors. the relative risk was adjusted for age and smoking. we observed a significant decrease in lung cancer risk related to more frequent consumption of raw vegetables, cooked meat and fish. however, stronger protective effect was reported for squamous cell carcinoma. frequent fish consumption significantly decreases the risk especially in cigarette smokers. the frequent consumption of pickles lowers squamous cell carcinoma risk in all cases but small cell carcinoma risk only in smokers. the presence of butter, cooked meat, fish and vegetables in diet significantly decreases the lung cancer risk especially in smokers. the association between diet and lung cancer risk is more pronounced for squamous cell carcinoma. abstract background: in functional disease research selection mechanisms need to be studied to assure external validity of trial results. objective: we compared demographic and disease-specific characteristics, history, co-morbidity and psychosocial factors of patients diagnosed, approached and randomised for a clinical trial analysing the efficacy of fibre therapy in irritable bowel syndrome (ibs). design and methods: in primary care 1078 patients were diagnosed with ibs by their gp in the past two years. characteristics were compared between (1) randomised patients (n = 108); (2) patients who did not give their informed consent (n = 235); (3) patients who decided not to participate (n = 270); and (4) those not responding to the mailing (n = 465). results: the groups showed no significance differences in age and gender (74% females, mean age 41 years, s.d. 12). patients consulting their gp for the trial compared to patients not attending their gp showed significant more severe ibs symptoms, more abdominal pain during the previous three months, and a longer history of ibs (p<0,001). patients randomised have more comorbidity (p = 0,001). conclusion and discussion: patients included in this ibs trial differ from no participating and excluded patients mainly in ibs symptomatology, history and comorbidity. this may affect the external validity of the trial results. abstract objectives: to evaluate smoking prevalence among teenagers and identify associated social-behavioral factors. study design and methods: a cross sectional-study was carried out in may (2004) in high school population (7th-12th grades) in the north of portugal (n = 1005). a confidential self-reported questionnaire was administered. crude and adjusted odds ratios and confidence intervals were calculated by logistic regression analysis. results: overall smoking prevalence was 19.5% (boys = 26.1%; girls = 14.6%) (or = 2.06; ci 95% = 1.50-2.83; p<0.001). smoking prevalence was significantly and positively associated with gender, smoking parents, school failure and school grade; in the group of students with smoking relatives, smoking was significantly associated with parents who smoke near the student (or = 4.32; ci 95% = 2.41-7.74; p<0.001); in the group of the secondary grade (10th-12th grades) smoking was significantly associated with belonging to 'non science-courses' (or = 1.81; ci 95% = 1.18-2.78; p = 0.007). conclusions: smoking is a growing problem among portuguese adolescents, increasing with age, prevailing among males, although major increases have been documented in the female population. parents' behaviours and habits have an important impact in their children's smoking behaviour. school failure is also an important factor associated with smoking. there is a need for further prevention programmes that should include families and consider students' social environment. abstract background: social environment of school can contribute to etiology of health behaviors. objective: to evaluate the role of school context for substance abuse in youth. design: a cross-sectional study was carried out in 2005, using self-completed classroomadministered questionnaire. subjects: from a representative sample of 2893 students, a sub-sample of 1880 students was selected (including 85/130 classes with at least 15 persons without missing data)*. methods: substance abuse was measured by: tobacco smoking at present, episodes of drunkenness and marijuana use in the lifetime. overall index was created as main independent variable, ranging 0-9 (cronbach's alpha = 0.71). class membership, type of school, gender, place of domicile, and school climate were included as contextual variables, measured on individual or group level. results: on individual level, the mean index was equal to 3.5 (sd = 2.9), and ranged from 3.1 in general comprehensive schools to 4.6 in basic vocational schools and from 1.0 to 7.4 for separated classes. about 16.3% of total variance in this index may be attributed to differences between classes. conclusion: individual differences in substance abuse in youth could be partly explained by factors at school level. * project no 2 po5d 064 27. abstract background: rates of c-section in brazil are very high, 33.8% in 2004. brazil illustrates an extreme example of medicalization of birth. c-section, as any major surgery, increases the risk of morbidity, which can persist long after discharge from hospital. objectives: to investigate how social, reproductive, prenatal care and delivery factors interact after hospital discharge, influencing post partum complications. design and methods: a cross-sectional study of 200 women gathered information through home interviews and clinical examination during post-partum. a hierarchical logistic regression model of factors associated with post-partum complications was applied. results: physical and emotional post partum complications were almost twice as high among women having c-section. most of this effect were associated with lower socioeconomic conditions which influences, were mainly explained by longer duration of delivery (even in the presence of medical indications), and less social support when returning home. conclusion: risk of c-section complications is higher among women from the lower socioeconomic strata. social inequalities mediate the association between type of delivery and postpartum complications. discussion: c-section complications should be taken into account when decisions concerning type of delivery are made. social support after birth, from the public health sector, has to be provided for women in socioeconomic deprivation. the relationship between unemployment and increased mortality was previously reported in western countries. the aim of this study was to assess the influence of the changes in unemployment rate on survival in general population in northern poland at the time of economic transition. to analyze the association between the unemployment and risk of death we collected survival data from 62736 death certificates and data on rates of unemployment from 8 regions of gdansk county from period 1991-1996. kaplan-meier method and cox proportional hazard model were used in univariate and multivariate analysis. a change of unemployment (percentage) in the year of death in the area of residence, sex and educational level (6 categories) were included into multivariate analysis. the change of unemployment rate was associated with significantly worse overall survival: hazard ratio 1.02 95% confidence interval 1.016 to 1.024. the highest risk associated with the change of unemployment in the area of residence was for death from congenital defects (hazard ratio 1.16 95% confidence interval 1.04 to 1.3) and for death from cardiovascular diseases (hazard ratio 1.036 95% confidence interval 1.032 to 1.042 abstract background: there is no evidence from randomized trials as to whether or not educational interventions improve voluntary reporting systems in terms of quantity or quality. objectives: evaluation of the effectiveness of educational outreach visits aimed at improving adverse drug reaction (adr) reporting by physicians design and methods: cluster-randomized controlled trial covering all health system physicians in northern portugal. four spatialclusters assigned to intervention group (n = 1388) received outreach visits tailored to training needs detected in previous study and 11 clusters were assigned to the control (n = 5063). the main was the total number of reported adr; the second was the number of serious, unexpected, high-causality and new-drug-related adr. a follow-up was conducted for a period of 30 months. results: the intervention increased reports as follows: total adr, 9.7-fold (p<0.0001); serious adr, 6.1-fold (p = 0.001); high-causality adr, 8.5-fold (p<0.001); unexpected adr, 32.6-fold (p<0.001); and newdrug-related adr, 8.2-fold (p = 0.002). the intervention had its maximum effect during the first four months (23.3-fold increase, p<0.001), yet the effect was nonetheless maintained over the four 4month periods post-intervention (p = 0.06). discussion and conclusion: physician training based on academic detailing visits improves reporting quality and quantity. this type of intervention could result in sizeable improvements in voluntary reporting in many countries. there were no evidence of differences in absolute indications between the years. conclusion: most of the increase in rates in the period may be attributable to relative and non-medical indications. discussion policies to promote rational use of c-sections should take into account the role played by obstetrician's convenience and the increased medicalization of birth on cesarean rates. abstract background: the changing environment has led to unhealthy dietary habits and low physical activity of children resulting in overweight/obesity and related comorbid conditions. objective: idefics is a five-year multilevel epidemiological approach proposed under the sixth eu framework to counteract the threatening epidemic of diet-and lifestyle-induced morbidity by evidence-based interventions. design and methods: a population-based cohort of 17.000 children 2 to 10 years old will be established in nine european countries to investigate the aetiology of these diseases. culturally adapted multi-component intervention strategies will be developed, implemented and evaluated prospectively. results: idefics compares regional, ethnic and sex-specific distributions of the above disorders and their key risk factors in children within europe. the impact of sensory perception, genetic polymorphisms and the role of internal/external triggers of food choice and children's consumer behaviour are elucidated. risk profile inventories for children susceptible to obesity and its co-morbid conditions are identified. based on controlled intervention studies an evidencebased set of guidelines for health promotion and disease prevention is developed. conclusions: provision of effective intervention modules, easy to implement in larger populations, may reduce future obesity related disease incidence. discussion: transfer of feasible guidelines into practice requires involvement of health professionals, stakeholders and consumers. abstract background: non-medically indicated cesarean deliveries increase morbidity and health care costs. brazil has one of the highest rates of caesarean sections in the world. variations in rates are positively associated with socioeconomic status. objectives: to investigate factors associated with cesarean sections in public and private sector wards in south brazil. design and methods: cross sectional data from post partum interviews and clinical records of 216 consecutive deliveries (112 in the main public and 104 in a private maternity) was analyzed using logistic regression. results: multiple regression showed privately insured women having much higher cesarean rates than those delivering in public sector wards (or = 9.4; ci95%: 4.5-32.6). obstetricians individual rates varied from 38%-100%. doctors working in both, public and private sectors had a higher rates of cesarean in private wards (p<0.01). wanting and having a cesarean was significantly more common among privately insured women. conclusion: women from wealthier families are at higher risk of cesarean, particularly those willing this type of delivery and whose obstetrician works in the private sector. discussion: women potentially at lower clinical risk are more like to have a caesarean. the obstetricians' role and women's preferences must be further investigated to tackle this problem. abstract background: in the netherlands, bcg-vaccination is offered to immigrant children and children of immigrant parents in order to prevent severe tuberculosis. the effectiveness of this policy has never been studied. objectives: assessing the effectiveness of the bcg-vaccination policy in the netherlands. design and methods: we used data on the size of the risk population per year (from statistics netherlands), number of children with meningitis or miliary tuberculosis in the risk population per year, and vaccination status of those cases (from the netherlands tuberculosis register) over the period 1996-2003. we estimated the vaccine efficacy and annual risk of acquiring meningitis or miliary tuberculosis by log-linear modelling and treating the vaccination coverage as missing data. results: in the period 1996-2003 13 cases of meningitis or miliary tuberculosis were registered. the risk for unvaccinated to children to acquire such a serious tuberculosis infection was 4.54 (95%ci 2.26-9.62) per 100000 per year; the reduction in risk for vaccinated children was 81% (95%ci 31-94%). conclusion and discussion: this means that, discounting future effects with 4%, a 1088 (95%ci: 560-2500) extra children should be vaccinated to prevent one extra case of meningitis or miliary tuberculosis. given that bcg-vaccination is relatively inexpensive, the current policy could even be cost-saving. abstract background: psychotic symptom experiences in the general population are frequent and often longlasting. objectives: the zurich cohort study offered the opportunity of differentiating the patterns of psychotic experiences over a span of 20 years. design and methods: the zurich study is based on a stratified community sample of 591 persons born in 1958 (women) and 1959 (men). the data were collected at six time points since 1979. we examined variables from two subscales of the scl-90-r -'paranoid ideation' and 'psychoticism' -using factor analysis, cluster analysis and polytomous logistic regression. results: two new subscales were derived representing 'thought disorders' and 'schizotypal signs'. continously high symptom load on one of these subscales (both subscales) was found in 7% (1.7%) of the population. cannabis use was the best predictor of continuously high symptom load in the 'thought disorders' subscale, whereas several variables representing adversity in childhood / youth were associated with continuously high symptom load in the 'schizotypal signs' subscale. conclusion and discussion: psychotic experiences can be divided at least in two different syndromes -thought disorders and schizotypal signs. despite similar longitudinal course patterns and also similar outcomes these syndromes rely on different risk factors, thus possibly defining separate pathways to psychosis. abstract background: the reasons for the rise in asthma and allergies remain unclear. to identify influential factors several european birth cohort studies on asthma and allergic diseases have been initiated since 1985. objective: the aim of one work package within the global allergy and asthma european network (ga2len), sponsored by the european commission, was to identify and compare european birth cohorts specifically designed to examine asthma and allergic diseases. methods: for each study, we collected detailed information (mostly by personal visits) regarding recruitment process, study setting, follow-up rates, subjective/objective outcomes and exposure parameters. results: by june 2005, we assessed 18 european birth cohort studies on asthma and allergic diseases. the largest 5 recruited over 3000 children each. most studies determined specific immunoglobulin e levels to various allergens or used the isaac questionnaire for evaluation of asthma or allergic rhinitis symptoms. however, the assessment of other objective and subjective outcomes (e.g. lung function or definitions of eczema) were rather heterogeneous across the studies. conclusions due to the unique cooperation within the ga2len project a common database was established containing study characteristics of european birth cohorts on asthma and allergic diseases. the possibility to pool data and perform meta-analyses is currently being evaluated. abstract background: birth weight is an important marker of health in infancy and health trajectories later in life. social inequality in birth weight is a key component in population health inequalities. objective: to comparatively study social inequality in birth weight in denmark, finland, norway, and sweden from 1980 to 2005. design and methods as part of the nordic collaborative project on health and social inequality in early life (norchase), register-based data covering all births in all involved countries 1980-2005 was linked with national registries on parental socioeconomic position, covering a host of different markers including income, education and occupation. also, nested cohort studies provide opportunity to test hypotheses of mediation. results: preliminary results show that the social inequality in birth weight, small for gestational age, and low birth weight has increased in denmark through out the period. also, preliminary results from finland, norway and sweden will be presented. discussion: crosscountry comparisons pose several methodological challenges. these challenges include characterizing the societal context of each country so as to correctly interpret inter-country differences in social gradients, along with dealing with differences in the data collection methods and classification schemes used by different national registries. also, strategies for influencing policy will be discussed. abstract background: modifying the availability of suicide methods is a major issue in suicide prevention. objectives: we investigated changes in the proportion of firearm suicides in western countries since the 1980's, and their relation to the change of legislation and regulatory measures. design and methods: data from previous publications, from the who mortality database, and from the international crime victims survey (icvs) were used in a multilevel analysis. results: multilevel modeling of longitudinal data confirmed the effect of the proportion of households owning firearms on firearm suicide rates. several countries stand out with an obvious decline in firearm suicides since the 1980s: norway, united kingdom, canada, australia, and new zealand. in all of these countries legislative measures have been introduced which led to a decrease in the proportion of households owning firearms. conclusion and discussion: the spread of firearms is a main determinant of the proportion of firearm suicides. legislative measures restricting the availability of firearms are a promising option in suicide prevention. abstract background: fatigue is a non-specific but frequent symptom in a number of conditions, for which correlates are unclear. objectives: to estimate socio-demographic and clinical factors determining the magnitude of fatigue. methods: as part of a follow-up evaluation of a cohort of urban portuguese adults, socio-demographic and clinical variables for 563 consecutive participants were collected through personal interview. lifetime history of chronic disease diagnosis was inquired (depression, cancer, cardiovascular, rheumatic, and respiratory conditions), anthropometry was measured, and haemoglobin determined. krupp's 9-item fatigue severity scale was applied and severe fatigue defined as mean score over 4. mean age (sd) was 62.3 (9.9) and 58.8% of participants were females. logistic regression was used to compute adjusted odds ratios, and attributable fractions were estimated using the formula ar = 1-s(?j/orj). results: adjusted for age and clinical conditions, female gender (or = 1.56, 95%ci: 1.08-2.26) and education (under 5-years schooling: or = 1.61, 95%ci: 1.10-2.34) were associated with severe fatigue. obesity (or = 1.87, 95%ci: 1.21-2.90) and diagnosed cardiovascular disease (or = 2.23, 95%ci: 1.28-3.90) also increased fatigue. attributable fractions were 21.1% for gender, 14.2% for education, 9.8% for obesity, and 10.9% for cardiovascular disease. conclusion: gender and education have large impact on severe fatigue, and, to a lesser extent, obesity and cardiovascular disease. abstract introduction: analysis of infant mortality allows identification of death contributing factors and assessment of child health care quality. objective: to study characteristics of infant and fetal mortality using data from a committee for prevention of maternal and infant mortality, in sobral, brazil. methods: all cases of infant deaths between 2002 and 2004 were analyzed. medical records were reviewed and mothers, interviewed. using a tool to identify preventable deaths (seade classification -brazil) the committee characterized causes of death. meetings with governmental groups involved in family health care took place to identify death contributing factors. results: in 2002, infant mortality decreased from 29.7 to 18.9. in the next 2 years there was an increase from 23.1 to 26.6. the increase in 2003 was due to respiratory illnesses. in 2004, was due to diarrhea. analysis of preventable deaths indicated a reduction from 32 to 20 deaths that could have been prevented by adequate gestational care, and an increase in preventable deaths by early diagnosis and treatment. conclusion: pre-natal and delivery care improved whereas care for children less than 1 yr old worsened. analysis of death causes allowed a reduction of infant mortality rate to 16.44 abstract objective: to identify dietary patterns and its association with metabolic syndrome. design and methods: we evaluated 2166 noninstitutionalised adults. diet was assessed using a semi-quantitative dietary frequency questionnaire, and dietary patterns were identified using principal components analysis followed by cluster analysis (k-means method) with bootstrapping (choosing the clusters presenting the lowest intra-cluster variance). metabolic syndrome (mets) was defined according to the ncep-atp-iii. results: the overall prevalence of metabolic syndrome was 20.6%. in the population sample 4 clusters were identified in females -1.healthy, 2.milk/soup; 3.fast food; 4.wine/low calories; and 4 in males -1.milk/carbohydrates; 2. codfish/soup; 3.fast food; 4.low calories. in males, using milk/carbohydrates as the reference and adjusting for age and education, high blood pressure (or = 1.72; 95%ci:1.04-2.85) and high triglycerides (or = 1.61;95%ci:1.00-2.60) were associated with the fast food pattern, and low calories pattern presented higher frequency of high blood pressure (or = 1.61; 95%ci:1.01-2.55). in females, after age and education adjustment, no significant association was found either with metabolic syndrome or its individual features and the dietary patterns identified. conclusion: we found no specific dietary pattern associated with an increased prevalence of metabolic syndrome. however, a fast food diet was significantly more frequent in males with dyslipidemia and high blood pressure. abstract aim: to determine the prevalence of stress urinary incontinence (sui) before, during pregnancy and following childbirth, and also to analyse the impact of a health education campaign about sui prevention, following childbirth in viana district, portugal. methods: participants (n = 336), interviewed during hospitalization, after birth and two months later at health centres, were divided into two groups: a first group of non-exposed and a second exposed to a health education campaign. this second group was encouraged to perform an exercise programme and given a 'suiprevention-treatment' brochure, approved by the regional health authority. results: sui prevalence was 5.4%(95%ci: 3.0-7.8) before pregnancy, 51.5%(95%ci: 46.1-56.9) during pregnancy and 10.2(95%ci: 6.8-13.7) four weeks after birth. less than half of the women with sui sought help from healthcare professionals. statistical significant differences were found between groups: sui knowledge level and practice of pelvic floor muscles re-education exercises were higher in the exposed group (2.6 and 5.1 times, respectively). conclusions: sui affects a great number of women but only a small percentage reveals it. this campaign improved women knowledge and modified their else behaviors. healthcare professionals must be aware of this reality, providing an early and continuous intervention that would optimise the verified benefits of this campaign. abstract background: social inequalities have been associated with poorer developmental outcomes, but little is known about the role of the area of residence. objectives: examine whether the housing infrastructure of the area modifies the effect of socio-economic conditions of the families on child development. design and methods: community-based survey of 3052 under-fives in southern brazil applied hierarchical multi-level linear regression to investigate determinants of child development, measured by a score from the denver developmental screening test. results: in multivariable models, the mean score of child development increased with maternal and paternal education and work qualification, family income and better housing and was higher when the mother was in paid work (all p<0.001). paternal education had an effect in areas of lower housing quality only; the effect of occupational status and income in these areas were twice as large as in better-provided areas (likelihood test for all interactions p<0.05). this model explained 37% of the variation in developmental score between the areas of residence. conclusion: the housing quality and sanitation of the area modified the effects of socioeconomic conditions on child development. discussion: housing and sanitation programs are potentially beneficial to decrease the negative effect of social disadvantage on child development. abstract background: it is known that both genetic and environmental factors are involved in the early development of type1 diabetes (t1d), and that incidence varies geographically. however we still need to explain why there is variation in incidence. objectives: in order to better understand the role of non-genetic factors, we decided to examine whether prevalence of newborns with high risk genotypes or islet autoantibodies varies geographically. design and methods: the analysis was performed on a cohort of 29912 newborns born to non-diabetic mothers, between september 2000 and august 2004, who were included in diabetes prediction in ska˚ne study (dipis) in sweden. neighbourhoods were defined by administrative boundaries and variation in prevalence was investigated using multi-level regression analysis. results: we observed that prevalence of newborns with islet autoantibodies differed across the 33 municipalities of ska˚ne (s = 0.16, p <0.01), with highest prevalence found in wealthy urban areas. however there was no observed difference in the prevalence of newborns with high risk genes. conclusion and discussion: newborns born with autoantibodies to islet antigens appear to cluster by region. we suggest that non-genetic factors during pregnancy may explain some of the geographical variation in the incidence of t1d. abstract background: risk assessment is a science-based discipline used for decision making and regulatory purposes, such as setting acceptable exposure limits. estimation of risks attributed to exposure to chemical substances are traditionally mainly the domain of toxicology. it is recognized, however, that human, epidemiologic data, if available, are to be preferred to data from laboratory animal experiments. objectives: how can epidemiologic data be used for (quantitative) risk assessment? results: we described a framework to conduct quantitative risk assessment based on epidemiological studies. important features of the process include a weight-of-theevidence approach, estimation of the optimal exposure-risk function by fitting a regression model to the epidemiological data, estimation of uncertainty introduced by potential biases and missing information in the epidemiological studies, and calculation of excess lifetime risk through a life table to take into account competing risks. sensitivity analyses are a useful tool to evaluate the impact of assumptions and the variability of the underlying data. conclusion and discussion: many types of epidemiologic data, ranging from published, sometimes incomplete data to detailed individual data, can be used for risk assessment. epidemiologists should better facilitate such use of their data, however. abstract background: high-virulence h. pylori (hp) strains and smoking increase the risk of gastric precancerous lesions. its association with specific types of intestinal metaplasia (im) in infected subjects may clarify gastric carcinogenesis pathways. objectives: to quantify the association between types of im and infection with highvirulence hp strains (simultaneously caga+, vacas1 and va-cam1) and current smoking. design and methods: male volunteers (n = 201) underwent gastroscopy and completed a self-administered questionnaire. participants were classified based on mucin expression patterns in biopsy specimens (antrum, body and incisura). hp vaca and caga were directly genotyped by pcr/reverse hybridization. data were analysed using multinomial logistic regression (reference: normal/superficial gastritis), models including hp virulence, smoking and age. results: high-virulence strains increased the risk of all im types (complete: or = 3.08, 95%ci:1.20-7.89; incomplete: or = 9.23, 95%ci:2.20-38.72; mixed: or = 6.27, 95%ci:2.55-15.43) but smoking was only associated with an increased risk of complete im (or = 2.99 95%ci:1.15-7.78). compared to non-smokers infected with lowvirulent strains, infection with the high-virulence hp increased the risk of im similarly for smokers (or = 11.00, 95%ci:3.59-33.71) and non-smokers (or = 9.65 95%ci:3.82-24.40). conclusion: gastric precancerous lesions, with different potential for progression, are differentially modulated by hp virulence and smoking. the risk of im associated with high-virulence hp is not further increased by smoking. abstract background: in may 1999, the portuguese government created the basic urgency units (buu). these buu must attend at least 40.000 persons, be open 24 hours per day, and be at maximum 60 minutes of distance to all the users. objectives: determine the optimal location of buu, considering the existing health centers, in the viseu district, north portugal. methods: from a matrix of distances between population and health centers an accessibility index was created (sum of distances traveled by population to reach a buu). the location-allocation models were used to create simulations based on p-median model, maximal covering location problem (mclp) and set covering location problem (sclp). the solutions were ranked by weighting the variables of accessibility (50%), number of doctors in the health centers (5%), equipments (20%), distance/time (20%) and total number of buu (5%). results: the best solution has 3 buu, 89 doctors, attends 59 000 users and the accessibility index is 8.859 km. conclusions: it was proved that it is impossible to attend all the criterion for creation of a buu. in some areas with low population density, to sum at least 40 000 persons in a buu, the travel time is necessarily more than 1 hour. background: a prospective observational study of fatigue in staff working a 10 day/5 off/8 night/5 off roster of 12 hour shifts was conducted at a fly-in/fly-out fertilizer mine in remote northern australia. objectives: to determine whether fatigue in staff increased: from the start compared to the finish of shift; with the number of consecutive shifts; and from day-compared to nightshift. methods: data of sleep diaries, the mackworth clock test and the swedish occupational fatigue inventory were obtained at the start and finish of each shift from august to november 2004. results: a total of 51 staff participated in the study. reaction times, sleepiness and lack of energy scores were highest at the finish of nights 1 to 4. the reaction times increased significantly at both the start and finish of day 8 onwards, and at the finish of night 7. reaction times and lack of motivation were highest during nightshift. conclusions: from the above results, a disturbed diurnal rhythm and decreased motivation during night-shift; and a roster of more than eight consecutive shifts can be inferred as the primary contributors to staff fatigue. discussion: the implications for changes to workplace practices and environment will be discussed. the aim of this survey was to assess the impact of a meta-analysis comparing resurfacing with nonresurfacing of the patella on the daily practice of experts. participants in this study were 87 experts which had participated in a previous survey on personal preferences regarding patella resurfacing. these experts in the field of patella resurfacing were identified by a thorough search of medline, an internet search (with googletm search engine), and personal references from the identified experts. participants of the 'knee arthroplasty trial' (kat) in the united kingdom were also included. two surveys were sent to the participants, one before and one after the publication of the meta-analysis. the response rate is 39 questionnaires or 45%. the vast majority of responders are not persuaded to change change their practice after reading the metaanalysis. this is only in part due to the fact that best evidence and practice coincide. other reasons given are methodology related, an observation which is shared by the authors of the review, which force the orthopedic community to improve its research methodology. reasons such as 'i do not believe in meta-analysis' either demands a fundamental discussion or demands the reader to take evidence based medicine more seriously. abstract background: patients with type 2 diabetes (dm2) have a 2-3 fold increased risk of cardiovascular disease. delegating routine tasks and computerized decision support systems (cdss) such as diabetes care protocol (dcp) may improve treatment of cardiovascular risk factors hba1c, blood pressure and cholesterol. dcp includes consultation-hours exclusively scheduled for dm2 patients, rigorous delegation of routine tasks in diabetes care to trained paramedics, and software to support medical management. objective: to investigate the effects of dcp, used by practice assistants, on the risk of coronary heart disease for patients with dm2. design and methods: in an open-label pragmatic trial in 72 general practices with 7893 patients, hba1, blood pressure and cholesterol were examined before and prospectively one year after implementation of dcp. the primary outcome was the change in the 10 year ukpds coronary heart disease (chd) risk estimate. results: the median 10 year ukpds chd risk estimate improved significantly from 21.7% to 19.0%. hba1 decreased from 7.2% to 6.9%, systolic blood pressure from 148.4 to 144.0 mmhg and total cholesterol from 5.1 to 4.7 mmol/l. (all p<0.01). conclusion: delegating routine task in diabetes care to trained paramedics and using cdss improves the cardiovascular risk of dm2 patients. tuberculosis in exposed immigrants by tuberculin skin test, ifn-g tests and epidemiologic abstract background: currently immigrants in western countries are only investigated for active tuberculosis (tb) by use of a chest x-ray. recent latent tuberculosis infection (ltbi) is hard to diagnose in this specific population because the only available test method, the tuberculin skin test (tst), has a low positive predictive value (ppv). recently interferon-gamma (ifn-g) tests have become available that measure cellular responses to specific m. tuberculosis antigens and might have a better ppv. objective: to determine the predictive value of tst and two different ifn-g tests combined with epidemiological characteristics for developing active tb in immigrants who are close contacts of smear positive tb patients. methods in this prospective cohort study 800 close contacts will be included. demographic characteristics and exposure data are investigated. beside their normal examination they will all have a tst. two different ifn-g tests will be done in those with a tst induration of ?5 mm. these contacts will be followed for 2 years to determine the occurrence of tb. results since april 2005, 13 municipal health services have started with the inclusion. preliminary results on the predictive value of tst, both ifn-g tests and epidemiological characteristics will be presented. abstract background: different factors contribute to the quality of ed (emergency department) care of an injured patient. objective: determine factors influencing the disagreement between er diagnoses and those assigned at hospital admission in injuried patients, and evaluate if disagreement between the diagnoses could have worsened the outcome. methods: all the er visits of the 60 emergency departments of lazio region for unintentional injuries followed by hospitalisation in 2000. concordant diagnoses were established on the basis of the barell matrix cells. logistic regression was used to assess the role of individual and er care factors on the probability of concordance. a logistic regression where death within 30 days was the outcome and concordance the determinant was uses. results: 22,892 injury er visits were considered. in 62.2% cases, the er and discharge diagnoses were concordant. higher concordance was found with increasing age and less urgent cases. factors influencing concordance were: the hour of the visit, er level, initial outcome, length of stay in hospital. patients who had non concordant diagnoses had a 30% higher probability of death. conclusions: a correct diagnosis at first contact with the emergency room is associated with lower mortality. methods: a cohort of consecutive patients treated for secondary peritonitis were sent the posttraumatic stress syndrome inventory (ptss-10) and impact of events scale-revised (ies-r) 4-10 years following their surgery for secondary peritonitis. results: from the 278 patients operated upon between 1994 and 2000, questionnaires were sent to the 131 long-term survivors of which 88% responded (n = 101). ptsd-related symptoms were found in 17% of patients by both questionnaires. patients admitted to icu (n = 39) were significantly older, with higher apache-ii scores, but reported similar ptsd symptomology scores compared to non-icu patients (n = 61). traumatic memories during icu and hospital-stay were most predictive for higher scores. adverse memories did not occur more often in the icu group than in the hospital-ward group conclusions: longterm ptsdrelated symptoms in patients with secondary peritonitis were very barthé lé my 53 c cabanas ruiz-carrillo 429 de la cruz den boon jimé nez-moleó n mü ller-nordhorn 36 national evaluation team 436 279 rich-edwards in the netherlands. design and methods we used the populationbased databases of the netherlands cancer registry, the eindhoven cancer registry (ecr) and the central bureau of statistics. patients from the ecr were followed until 1-1-2005 for vital status and relative survival was calculated. results: the number of breast cancer cases increased from 7900 in 1989 to 11.500 in 2003, an annual increase of 1.3% (p<0.001). the death rate decreased 1,4% annually (p<0.001), which resulted in 3400 deaths in 2003. the relative 5-yr survival was less than 50% for patients diagnosed in the seventies, this increased to over 80% for patient diagnosed since 2000, patients with stage i disease even have a 96% 5-yr relative survival. conclusion: the alarming increase in breast cancer incidence is accompanied with a serious improvement in survival rates. this results in a large number of women (ever) diagnosed with breast cancer, about 119,000 in 2005 of whom 80% demand some kind of medical care. abstract background: nine % of the population in the netherlands belongs to non-western ethnic minorities. perceived health is worse and health care use different from dutch natives. objectives. which factors are associated with ethnic differences in self-rated health? which factors are associated with differences in utilisation of gp care? methods: during one year all contacts with gps were registered. adult surinam, antillean, turkish, moroccan and dutch responders were included (total n: 10.252). we performed multivariate analyses of determinants of self-rated health and on the number of contacts with gps. results: self-rated health differ from native dutch: surinam/antillean (or 2.4) and turkish/moroccan patients (or 4.7/3.8) , especially in turkish/moroccan females. more turks visit the gps at least once a year (or 1.5). less surinamese (or 0.5) and antillean patients (or 0.9) visit their gps than the dutch do. people from ethnic minorities in good health visit their gps more often (3.9 -4.4 consults per year vs. 3.4). incidence rates of acute respiratory infections and chest complaints were significantly higher than in the dutch. conclusions: ethnicity is independently associated with self-rated health. higher use of gp-care by ethnic minorities in good health, points towards possible inappropriate use of resources. the future: do they fulfil it? first results of the limburg youth monitoring project abstract background: incidence of coronary heart disease (chd) and stroke can be estimated from local, population-based registers. it is unclear, to what extent local register data are applicable on a nationwide level. therefore, we compared german register data with estimates derived with who global burden of disease (gbd) method. methods: incidence of chd and stroke was computed with the gbd method using official german mortality statistics and prevalences from the german national health survey. results were compared to estimates from the kora/monica augsburg register (chd) and the erlangen stroke project in southern germany. results: gbd estimates and register data showed good agreement: chd (age group 25-74 years) 155,862 (gbd) versus 159,245 (register) and stroke (all ages) 157,104 versus 167, 892 incident cases per year. chd incidence among all age groups was estimated with the gbd method to be 250,000 per year (no register data available). chd incidence in men and stroke incidence in women were underestimated with the gbd method as compared to register data. conclusions: gbd method is a useful tool to estimate incidence of chd and stroke. the computed estimates may be seen as lower limit for incidence data. differences between gbd estimates and register data are discussed. abstract background: children with mental retardation (mr) are a vulnerable not much studied population. objectives: to investigate psychopharmacotherapy in children with mr and to examine possible factors associated with psychopharmacotherapy. methods: participants were recruited through all facilities for children with mental retardation in friesland, the netherlands, resulting in 865 participants, 4-18 years old, including all levels of mental retardation. the dbc and the pdd-mrs were used to assess general behavior problems and pervasive developmental disorders (pdd). information on medication was collected through a parent-interview. logistic regression was used to investigate the relationship between the psychotropic drug use and the factors dbc, pdd, housing, age, gender and level of mr. results: 10% of the participants used psychotropic medication. main factors associated with receiving psychopharmacotherapy were pdd (or 2.31) and dbc score (or 1.03). living away from home and mr-level also played a role whereas gender and age did not. dbc score was associated with clonidine, stimulants and anti-psychotics. pdd was the main factor associated with anti-psychotics use (or 5.7). discussion: psychopharmacotherapy is especially prevalent among children with mr and comorbid pdd and general behavior problems. although many psychotropic drugs are used off-label, specific drugs were associated with specific psychiatric or behavior problems. abstract background: increased survival in children with cancer has raised interest on the quality-of-life of long-term survivors. objective: to compare educational outcomes of adult survivors of childhood cancer and healthy controls. methods: retrospective cohort study including a sample of adult survivors (495) treated for childhood cancer in the three existing italian paediatric research hospitals. controls (501) were selected among siblings, relatives or friends of survivors. when these controls were not available, a search was carried out in the same area of residence of the survivors though random digit dialling. data collection was carried out through a telephone-administered structured questionnaire. results: significantly more survivors than controls needed school support (adjusted odds ratio -oradj-1.61, 95% ci 1.22-2.13); failed at least a grade after disease onset (oradj 1.46, 95% ci 1.09-1.97); achieved a lower educational level (oradj 1.77, 95% ci 1.27-2.45) and did not reach an educational level higher than their parents' (oradj 1.66, 95% ci 1.19-2.32). subject's age, sex, parents' education and area of residence were taken into account as possible confounders. conclusions: these findings suggest the need to provide appropriate school support to children treated for childhood cancer. abstract background: in italy supplementation with folic acid (fa) in the periconceptional period to prevent congenital malformations (cms) is quite low. the national health institute has recently launched (2005) a programme to improve awareness about the role of fa in reducing the risk of serious defects also by providing 0.4 mg fa tablets free of charge to women planning a pregnancy. objectives: we analysed cms that are or may be sensitive to fa supplementation in order to establish an adequate baseline to allow a fa impact assessment in the next years and to investigate spatial differences among cms registries, time trends and time-space interactions. design and methods data collected over 1996-2003 by the italian registries members of eurocat and icbdsr on births and induced abortions with neural tube defects, ano-rectal atresia, omphalocele, oral clefts, cardiovascular, limb reduction and urinary system defects. results: all the cms showed statistically significant differences among registries with the exception of ano-rectal atresia. the majority of cms by registry showed stable or increasing trends over time. conclusions results show the importance of fa intake during the periconceptional period. differences among registries indicate also the need of having a baseline for each registry to follow trends over time. abstract country-specific resistance proportions are more biased by variable sampling and ascertainment procedures than incidence rates. within the european antimicrobial resistance surveillance system (earss) resistance incidence rates and proportions can be calculated. in this study, the association between antimicrobial resistance incidence rates and proportions and the possible effect of differential sampling of blood cultures was investigated. in 2004, earss collected routine antimicrobial susceptibility test data from invasive s. aureus isolates, tested according to standard protocols. via a questionnaire denominator information was collected. the spearman correlation coefficient and linear regression were used for statistical analysis. this year, 735 of 1205 hospitals and 483 of 758 laboratories from 28 of 30 earss countries responded to the questionnaire. they reported of, overall, 18,729 s. aureus isolates. in the different countries, mrsa proportions ranged from <1% to 40% and incidence rates per 1,000 patient days from 0.26ae10-2 to 19.29ae10-2. overall, the proportions and rates highly correlated. blood culturing rates only influenced the relationship between mrsa resistance proportions and incidence rates for eastern european countries. in conclusion, resistance proportions seem to be very similar to resistance incidence rates, in the case of mrsa. nevertheless, this relationship appears to be dependent of some level of blood culturing. . key: cord-004584-bcw90f5b authors: nan title: abstracts: 8th ebsa european biophysics congress, august 23rd–27th 2011, budapest, hungary date: 2011-08-06 journal: eur biophys j doi: 10.1007/s00249-011-0734-z sha: doc_id: 4584 cord_uid: bcw90f5b nan o-003 structure determination of dynamic macromolecular complexes by single particle cryo-em holger stark max-planck-institute for biophysical chemistry, goettingen, germany macromolecular complexes are at the heart of central regulatory processes of the cell including translation, transcription, splicing, rna processing, silencing, cell cycle regulation and repair of genes. detailed understanding of such processes at a molecular level requires structural insights into large macromolecular assemblies consisting of many components such as proteins, rna and dna. single-particle electron cryomicroscopy is a powerful method for threedimensional structure determination of macromolecular assemblies involved in these essential cellular processes. it is very often the only available technique to determine the 3d structure because of the challenges in purification of complexes in the amounts and quality required for x-ray crystallographic studies. in recent years it was shown in a number of publications that it is possible to obtain near-atomic resolution structures of large and rigid macromolecules such as icosahedral viruses. due to a number of methodological advances there are now also great perspectives for high-resolution single particle cryo-em studies of large and dynamic macromolecules. successful high-resolution structure determination of dynamic complexes requires new biochemical purification strategies and protocols as well as state of the art electron microscopes and high-performance computing. in the future cryo-em will thus be able to provide structures at near-atomic resolution and information about the dynamic behavior of macromolecules simultaneously. detection and rapid manipulation of phosphoinositides with engineered molecular tools tamas balla section on molecular signal transduction, program for developmental neuroscience, nichd, nih, bethesda, md 20892, usa polyphosphoinositides (ppis) are ubiquitous lipid regulators of a variety of cellular processes serving as docking sites and conformational switches for a large number of signaling proteins. the localization and dynamic changes in ppis in live cells have been followed with the use of protein domain gfp chimeras. in this presentation we will show experimental systems that allow rapid manipulation of the levels of ppis in specific membrane compartments. we are also actively pursuing strategies that will allow us to map the distribution and possible functional diversity of the phosphatidylinositol (ptdins) pools within intact cells since they are the precursors of ppis. we will show our most recent progress in addressing this question: the use of a ptdins specific plc enzyme isolated from listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of ptdins in living cells. these studies reveal that a significant metabolically highly active ptdins pool exists associated with tiny mobile structures within the cytoplasm in addition to the known er and pm ptdins pools. we will show our most recent data on the consequences of ptdins depletion within the various ptdins pools on ppi production and on the morphology and functions of various organelles. the functionality of proteins is known to be intimately related to the motion of their constituents on the atomic/molecular level. the study of microscopic motion in complex matter is often reduced to the observation of some average mean square atomic displacement, a first, very partial characterization of the dynamics. the marked crossover in the temperature dependence of such quantities in hydrated proteins around 200 k, the so called ''dynamic transition'' has been originally observed a quarter of century ago. the origin, nature and the key characteristics of the atomic motions behind this remarkable evolution of the mean square displacement in proteins remained controversial over the past decades. recent analysis of mö ssbauer, dielectric relaxation and neutron scattering spectroscopic data provide unambiguous evidence that this phenomenon is caused by the temperature dependence of a relaxation process spread over several orders of magnitude in the time domain, similarly to the b relaxation process observed in glasses. the review and critical analysis of the available data highlights the inherent ambiguities of commonly used data fitting approaches. emerging evidence from model independent observations tend to exclude some of the proposed mechanisms. microbial rhodopsins: light-gated ion channels and pumps as optogenetic tools in neuro-and cell biology e. bamberg, c. bamann, r.e. dempski, k. feldbauer, s. kleinlogel, u. terpitz, p. wood department of biophysical chemistry, max-planck-institute of biophysics, frankfurt, germany microbial rhodopsins are widely used in these days as optogenetic tools in neuro and cell biology. we were able to show that rhodopsins from the unicellar alga chlamydomonas reinhardtii with the 7 transmembrane helix motif act as light-gated ion channels, which we named channelrhodopsins(chr1,2). together with the light driven clpump halorhodopsin chr2 is used for the non-invasive manipulation of excitable cells and living animals by light with high temporal resolution and more important with extremely high spatial resolution the functional and structural description of this new class of ion channels is given (electrophysiology, noise analysis,flash photolysis and 2d crystallography). new tools with increased spatial resolution and extremely enhanced light sensitivity in neurons are presented. a perspective for basic neurobiology and for medical applications is given. extracellular signals consists of the induction of specific gene expression patterns and the re-organization in space and time of stereo-specific macromolecular interactions that endow the cell with its specific morphology. we develop quantitative experimental and computational approaches to derive and conceptualize physical principles that underlie these dynamics of signal processing and cellular organization. we have an experimental emphasis on functional microscopic imaging approaches at multiple resolutions to study the localization and dynamics of protein reactions/ interactions, maintaining the inherent spatial organization of the cell. we have a strong recursion between computation of molecular dynamics in realistic cell geometries as sampled by microscopy, and experiments that reveal the dynamic properties of networks in living cells. we investigate the cellular topography of activities that transmit signals from receptors at the cell surface. here we ask, how spatial partitioning of intracellular signalling activities is achieved by the causality structure of the signalling network, and how this partitioning affects signal response. this entails the experimental elucidation of connections between reactions and the determination of enzyme kinetic parameters in living cells. o-008 molecular photovoltaics mimic photosynthesis michael grä tzel laboratory of photonics and interfaces, institute of chemical science and engineering, station 6, ecole polytechnique fé dé rale, ch-1015 lausanne, switzerland e-mail: michael.graetzel@epfl.ch the field of photovoltaic cells has been dominated so far by solid state p-n junction devices made e.g. of crystalline or amorphous silicon, profiting from the experience and material availability of the semiconductor industry. however, there is an increasing awareness of the possible advantages of devices referred to as ''bulk'' junctions due to their interconnected three-dimensional structure. their embodiment departs completely from the conventional flat p-n junction solid-state cells, replacing them by interpenetrating networks. this lecture focuses on dye sensitized mesoscopic solar cells (dscs), which have been developed in our laboratory. imitating natural photosynthesis, this cell is the only photovoltaic device that uses a molecular chromophore to generate electric charges from sunlight and that accomplishes the separation of the optical absorption from the charge separation and carrier transport processes. it does so by associating the molecular dye with a film constituted of tiny particles of the white pigment titanium dioxide. the dsc has made phenomenal progress, present conversion efficiencies being over 12 percent for single junction and 16 percent for tandem cells, rendering the dsc a credible alternative to conventional p-n junction devices. molecularly single-molecule imaging and tracking techniques that are applicable to living cells are revolutionizing our understanding of the plasma membrane dynamics, structure, and signal transduction functions. the plasma membrane is considered the quasi-2d non-ideal fluid that is associated with the actinbased membrane-skeleton meshwork, and its functions are likely made possible by the mechanisms based on such a unique dynamic structure, which i call membrane mechanisms. my group is largely responsible for advancing highspeed single molecule tracking, and based on the observations made by this approach, i propose a hierarchical architecture of three-tiered meso-scale (2-300 nm) domains as fundamental organizing principles of the plasma membrane. the three tiers i propose are the following. [tier 1] 30-200 nm compartments made by partitioning the entire plasma membrane by the membrane-associated actinbased meshwork (membrane skeleton: fences) and its associated transmembrane proteins (pickets). since the entire plasma membrane is partitioned by these structures, and the membrane skeleton provides important platforms for the molecular interactions and pools, membrane compartments are the most basic tier for the plasma membrane organization. [tier 2] meta-stable 1-5 nm raft domains that can be turned into stable *10-20-nm domains (receptor-cluster rafts), based on ligand-induced homo-dimers of glycosylphosphatidylinositol (gpi)-anchored receptors (coupling with [tier 3]) and facilitated by raft-lipid interactions. [tier 3] protein complexes of various sizes (3-10 nm) and lifetimes. i will also talk about how domains of tiers 2 and 3 are coupled to the membrane partitioning (tier 1). the concept of the three-tiered domain architecture of the plasma membrane and the cooperative interactions of different tiers provides a good perspective for understanding the mechanisms for signal transduction and many other functions of the plasma membrane. introduction: in the present study we investigate the effects of electromagnetic fields (emf) on the binding of norfloxacin (nrf) to human serum albumin (hsa) by fluorescence, three-dimensional fluorescence and uv-visible spectroscopic approaches. hsa is the most abundant protein in human blood plasma which works as a carrier that transports different materials in the body. nrf is used to treat variety of bacterial infections. it works by stopping the bacterial growth. methods: hsa, nrf and potassium phosphate buffer were purchased from sigma. fluorescence spectrofluorometer, uv-vis spectrophotometer, three-dimensional fluorescence and a home-built emf generator apparatuses were used. results: results obtained from this study indicated that nrf has a strong ability to quench hsa in 280 nm. in addition, there was a slight blue shift, which suggested that the microenvironment of protein became more hydrophobic after addition of nrf. moreover, synchronous fluorescence demonstrated that the microenvironment around tyrosine (tyr) had a trivial increase. these, and the results of hsa-nrf in the presence of emf with 1 khz, illustrates the same results inferred from quenching and blue shift. however, there was a significant decrease in k sv of nrf with hsa in presence of emf exposure. moreover, the binding parameters including the number of binding sites and the binding constant were calculated form hill equation. conclusion: it was shown that nrf could induce conformational changes in hsa both in the absence and presence of emf with no significant difference. yet, the affinity is decrease significantly in the presence of emf. the clinical implications are discussed in detail. characterization of the biochemical properties and biological function of the formin homology domains of drosophila we characterised the properties of drosophila melanogaster daam-fh2 and daam-fh1-fh2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. the results show that while the daam-fh2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the daam-fh1-fh2 fragment a profilindependent increase in the formation of actin structures is observed. the trachea specific expression of daam-fh1-fh2 also induces phenotypic effects leading to the collapse of the tracheal tube and lethality in the larval stages. in vitro, both daam fragments catalyze actin nucleation but severely decrease both the elongation and depolymerisation rate of the filaments. profilin acts as a molecular switch in daam function. daam-fh1-fh2, remaining bound to barbed ends drives processive assembly of profilin-actin, while daam-fh2 forms an abortive complex with barbed ends that does not support profilinactin assembly. both daam fragments also bind to the sides of the actin filaments and induce actin bundling. these observations show that the drosophila melanogaster daam formin represents an extreme class of barbed end regulators gated by profilin. electron spin echo studies of free chain-labelled stearic acids interacting with b-lactoglobulin rita guzzi, luigi sportelli, rosa bartucci dipartimento di fisica, università della calabria, 87036 rende (cs), italy b-lactoglobulin (blg) binds non-covalently fatty acids within its central calyx, a cavity in the barrel formed by the strands ba-bh. we present results of pulsed electron paramagnetic resonance (epr) spectroscopy on the interaction of blg with stearic acids spin-labelled at selected positions, n, along the acyl chain (n-sasl, n = 5,7,10,12,16). d 2 o-electron spin echo envelope modulation (eseem) fourier transform spectra indicate that all segments of the bound chains in the protein binding site are accessible to the solvent. the extent of water penetration decreases progressively on moving from the first segments toward the terminal methyl end of the chain. about 50% of the nitroxides in the upper part of the chain (n = 5,7) are h-bonded by a single water molecule and this fraction reduces to 30% at the chain terminus (n = 12,16). a lower fraction of the nitroxides are h-bonded by two water molecules, and it decreases from about 15% to a vanishingly small value on going down the chain. echo-detected ed-epr spectra reveal subnanosecond librational motion of small amplitude for both 5-and 16-sasl in the protein cavity. the temperature dependence of the librations is more marked for 16-sasl and it arises mainly from an increase in librational amplitude with increasing temperature. fusion peptides (fp) pertaining to the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus are essential for the fusion between viral and host cellular membranes. here we report a biophysical characterization of the interaction of two putative fps with model membranes. fluorescence and dsc experiments showed that both peptides bind stronger to anionic than to zwitterionic lipid membranes. esr spectra showed that toac-sars ifp rotational dynamics is modulated by lipid composition and ph as compared to the spectrum of this peptide in solution. however, stearic acid spin labels reported no changes on the dynamic structure of zwitterionic micelles, whereas the whole chain of anionic surfactants was perturbed by the peptides. finally, cd data revealed a predominant b-strand structure for sars fp and an a-helix for sars ifp in the presence of micelles, in contrast to their disordered structures in buffer. overall the results point out that electrostatic and hydrophobic interactions are both important to the energetic behavior of peptide membrane interaction. these findings might provide a useful rationale for the elucidation of one of the steps involved in the fusion process, and thus help understanding the more general way of action of fps at a molecular level. interaction of filamentous actin and ezrin within surface modified cylindrical nanopores daniela behn 1,2 and claudia steinem 1 1 institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße 2, 37077 gö ttingen, germany, 2 ezrin is a member of the ezrin-radixin-moesin (erm) protein family that acts as a dynamic linker between the plasma membrane and the actin cytoskeleton and is hence involved in membrane organization, determination of shape and surface structures and other cellular processes. the protein is highly enriched in microvilli of polarized epithelial cells, where it binds filamentous actin (f-actin) with its c-terminal domain, while the n-terminal domain is connected to the plasma membrane via specific binding to l-a-phosphatidylinositol-4,5-bisphosphate (pip 2 ). nanoporous anodic aluminum oxide (aao) films provide similar dimensions as microvilli and are thus a versatile template to investigate the interaction of ezrin with f-actin within spationally confined areas. owing to their optical transparency, functionalized aaos can be used to measure the binding process of ezrin to a pip 2 containing solid supported membrane by means of time resolved optical waveguide spectroscopy (ows). confocal laser scanning microscopy (clsm) will elucidate, whether f-actin binding to ezrin takes place within or atop the nanopores. furthermore, elasticity mapping of f-actin filaments by means of atomic force microscopy will allow determining binding forces and the lateral tension of the actin cytoskeleton. in vitro application of porphyrin photosensitisers on mcf7, hela and g361 tumour cell lines binder s., kolarova h., bajgar r., tomankova k., daskova a. deparment of medical biophysics, faculty of medicine of palacky university, olomouc, czech republic tumour treatment presents a challenge to all scientists and clinicians. contemporary methods like radiotherapy, chemotherapy or surgery have many undesirable side effects. photodynamic therapy (pdt) seems to be one of alternatives which can be helpful in malignant cell therapy. pdt is not only limited to cancer treatment but is also used as an alternative for cardiovascular, skin and eye disease treatment. pdt employs photosensitive agents which need to be activated by light which is not harmful to a patient. the activated photosensitive agent provokes a formation of reactive oxygen species leading to cell damage or death. the phototoxicity of the two porphyrin photosensitizer (tmpyp, zntpps 4 .12h 2 o) on the malignant cell lines (g361, hela, mcf7) irradiated with the 1 jcm -2 doses was evaluated by ros production assay, mtt assay and comet assay. our results indicate higher efficiency of tmpyp over zntpps 4 .12h 2 o. as for the photodynamic effectiveness of the used photosensitizers on chosen cell lines we found that hela cell line is the most sensitive to phototoxic damage induced by tmpyp. p-018 nmr analysis of the respiratory syncytial virus m2-1 protein structure and of its interaction with some of its targets c. sizun 1 the respiratory syncytial virus (rsv) is a major cause of acute respiratory tract infections (bronchiolitis, pneumonia) in human and a leading cause of viral death in infants and immunocompromised patients. rsv genome is formed of a single non-segmented negative strand rna which transcription and replication is ensured by a specific rna-dependent rna polymerase complex formed of the large (l) polymerase subunit and of several cofactors. this complex has no cellular counterpart and represents an ideal target for antiviral drugs. among the cofactors, m2-1 acts as an antitermination factor and increases the polymerase processivity. its central domains has been shown, in vitro, to bind the phosphoprotein p and genomic rna in a competitive manner. here we report the nmr structure of this central domain and its interaction with p and rna fragments. m2-1 shares structural similarity with vp30, a transcription factor of ebola virus. the binding surfaces for rna and p are distinct but overlapping. rna binds to a basic cluster located next to residues found to be critical for transcription both in vitro and in vivo by mutational analysis. we speculate that m2-1 might be recruited by p to the transcription complex, where interaction with rna takes place, stabilized by additional elements. force spectroscopy at the membrane-cytoskeleton interface: interactions between ezrin and filamentous actin julia a. braunger 1,2 , ingo mey 1 and claudia steinem 1 1 institute for organic and biomolecular chemistry, georg-august-university of gö ttingen, tammannstraße 2, 37077 gö ttingen, germany, 2 ggnb doctoral program: imprs ezrin, a member of the erm (ezrin/radixin/moesin) protein family, provides a regulated linkage between the plasma membrane and the actin cytoskeleton. it contributes to the organization of structurally and functionally distinct cortical domains participating in adhesion, motility and fundamental developmental processes. ezrin is negatively regulated by an intramolecular interaction of the terminal domains that masks the f-actin binding site. a known pathway for activation involves the interaction of ezrin with phosphatidylinositol 4,5bisphosphate (pip 2 ) in the membrane, followed by phosphorylation of the threonine 567 residue in the c-terminal domain. to date, it is unclear to what extent both regulatory inputs contribute to the activation. we developed an in vitro system that facilitates the specific analysis of the interaction forces between ezrin and f-actin by means of atomic force spectroscopy (afm). applying ezrin wild type and the pseudophosphorylated mutant protein ezrin t567d, respectively, permits to monitor the individual influence of phosphorylation on the f-actin-ezrin interaction. thus, a thorough characterization of the acting forces at the ezrin-actin interface will elucidate the activation mechanism of ezrin. delivery system even more efficient, we have constructed nano-carrier by coating of ldl by polyethylene glycol (peg) . the hydrophilicity of peg should reduce the interaction of ldl with other serum proteins and consequently decrease the redistribution of loaded drug from ldl to the (lipo)proteins. dynamic light scattering was used for determination of hydrodynamic radius of ldl-peg particles. cd spectroscopy measurements didn't reveal structural changes of apoliprotein b-100 (ligand for ldl receptors on cell surface), after conjugation of peg with ldl. interaction of ldl-peg complexes with hypericin (hyp) a natural photosensitizer was studied by fluorescence spectroscopy. we have demonstrated accumulation of higher number of hyp in ldl-peg than ldl particles. however, the kinetics of hyp redistribution from hyp/ldl-peg complex to free ldl have similar parameters as those for the kinetics of hyp transfer between non-modified ldl molecules. we suggest that hyp molecules are mostly localized in the vicinity of the surface of the ldl-peg particles and they are prone to redistribution to other serum proteins. grant support: lpp-0072-07, vega-0164-09. modification of the head-group of aminophospholipids by glycation and subsequent lipid oxidation affect membrane's structure causing cell death. 1 these processes are involved in the pathogenesis of aging 2 and diabetes. 3 non-enzymatic glycation forms in the first step a schiff base (sb), which rearranges to a more stable ketoamine, amadori product, which leads to the formation of a heteregenous group of compounds (ages). although several studies have been focused on identification of aminophospholipid glycation products, 4 less attention has been paid to kinetic mechanism of the reaction. for that reason, in the present work, we compare the kinetic reactivity of polar head-group of phosphatidylethanolamine (pe) and phosphatidylserine (ps), the two target phospholipids components of mammalian cell membranes. the reaction of pe and ps's head-group with glycating compounds (glucose and arabinose) was studied in physiological conditions by using nmr spectroscopy. the obtained formation rate constants for sb are lower than those determined for the sb of the peptide ac-phe-lys with the same carbohydrates. 5 it suggests that the phosphate group may delay the glycation process. moreover, the ps's head-group has a carboxilic group in the structure, which affects the stability of the sb. we developed ultrasensitive, elisa-like nanoimmuno assays suitable for proteomics/interactomics studies in low sample volumes. we exploit the approach of dna microarray technologies applied to proteomics [1] , in combination with atomic force microscopy (afm) to generate functional protein nanoarrays: semisynthetic dna-protein conjugates are immobilized by bioaffinity within a nanoarray of complementary ssdna oligomers produced by afm nanografting (ng). a nanoarray of different antibodies or synthetic molecular binders can be generated in a single operation, once the dna nanoarray is produced. moreover, ng allows adjusting the packing density of immobilized biomolecules to achieve optimum bio-recognition. afm-based immunoassays with these nanoarrays were shown to achieve detection limit of hundreds of femto molar, in few nanoliters volumes, with very high selectivity and specificity [2] . to detect the hybridization efficiency of our devices, we run a combined experimental-computational study that provides quantitative relations for recovering the surface probe density from the mechanical response (afmcompressibility measurements) of the sample. nucleoside analogues used as anticancerous drugs can be rapidly degraded within treated cells, constituting a major obstacle of their therapeutic efficiency. among the enzymes responsible for this degradation, the cytosolic 5'nucleotidase ii (cn-ii) catalyses the hydrolysis of some nucleoside monophosphates. in order to improve the efficacy of anticancerous drugs and to define the precise role of cn-ii, new original inhibitors have been developed against cn-ii. virtual screening of chemical libraries on the crystal structure has allowed us to identify very promising candidates that turned to be competitive inhibitors of cn-ii. one molecule was included in the anticancerous treatment of tumoral cell lines in order to evaluate the potential benefit and could induce in fine a sensitization of certain anticancerous drugs. we also explore other inhibitors targeting the allosteric sites of this enzyme using a strategy that takes into account the dynamics of cn-ii. the chemical structures of the newly identified allosteric inhibitors as well as the atomic interactions with enzyme residues will be presented. the final goal of this study is to find molecules that can freeze the enzyme in a conformation for which its dynamics is severely limited and therefore its function. native mass spectrometry to decipher interactions between biomolecules sarah cianferani laboratoire de spectromé trie de masse bio-organique, université de strasbourg, iphc, 25 rue becquerel 67087 strasbourg, france. cnrs, umr7178, 67037 strasbourg, france mass spectrometry is generally understood as ''molecular mass spectrometry'' with multiple applications in biology (protein identification using proteomic approaches, recombinant protein and monoclonal antibody characterization). an original and unexpected application of mass spectrometry emerged some twenty years ago: the detection and the characterization of intact biological noncovalent complexes. with recent instrumental improvements, this approach, called native ms, is now fully integrated in structural biology programs as a complementary technique to more classical biophysical approaches (nmr, crystallography, calorimetry, spr, fluorescence, etc.). native ms provides high content information for multiprotein complexes characterization, including the determination of the binding stoichiometries or oligomerization states, sitespecificities and relative affinities. recent developments of ion mobility / mass spectrometry instruments (im-ms) provide a new additional level for ms-based structural characterization of biomolecular assemblies allowing size and shape information to be obtained through collisional cross section measurements. these different aspects of native ms for structural characterization of biomolecular assemblies will be illustrated through several examples, ranging from multiprotein-complexes to protein/nucleic acid assemblies. complex coacervation is a process which may result by electrostatic interaction between charged polysaccharides. it depends essential on ph, ionic strength and biopolymers properties like ratio, concentration and charge density. in this case, the main work was to study the structural properties of a colloidal system of opposite charge -chitosan and gum-arabic by atomic force microscopy (afm). according to some of complexes show tendency to agglomerate. this depends on the molar ration of the macromolecules and their relative molecular weights. afm micrographs show, too, that some formation of irregular aggregates by both polymers were due to presence of noncharged polar monomers in chitosan molecule. at higher gum-arabic/chitosan ratios biopolymer concentrations, coacervates appear like a core-shell miccelar structure composed of hydrophobic core (charge neutralized segments) stabilized by the excess component (positive zeta potential) and non-charged segments of gum arabic. interaction of human serum albumin with rutin theoretical and experimental approaches ícaro p caruso 1 human serum albumin (hsa) is the principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. flavonoids are a large class of naturally occurring polyphenols widely distributed in plants. rutin (quercetin-3-rutinoside) is the glycoside between flavonoids quercetin and disaccharide rutinose. like other flavonoids, rutin displays anti-inflammatory and anti-oxidant properties. the interaction between hsa and rutin was investigated by fluorescence spectroscopy, ab initio and molecular modeling calculations. fluorescence titration was performed by keeping the hsa concentration (4 lm) constant and stoichiometrically varying the rutin concentration (1-4 lm) . the emission spectra were obtained in the range of 305 to 500nm, with the excitation wavelength at 295nm. the obtained fluorescence data were corrected for background fluorescence and for inner filter effects. the stern-volmer quenching constant values were 3.722 9 10 5 and 1.868 9 10 5 m -1 at 298 and 303 k, respectively. from the modified stern-volmer association constants 2.285 9 10 5 (at 298 k) and 2.081 9 10 5 m -1 (at 303 k) were calculated the thermodynamic parameters dh = 14.048 kj mol -1 , dg 298k = -30.557 kj mol -1 and dg 303 k = -30.834 kj mol -1 , and ds = 55.4 kj mol -1 k -1 . fluorescence quenching method was used also to study the binding equilibria thus determining the number of binding sites 1.085 and 1.028, and binding constant 1.094 9 10 6 m -1 and 0.255 9 10 6 m -1 at 298 and 303 k, respectively. the efficient quenching of the trp214 fluorescence by rutin indicates that the binding site for the flavonoid is situated within subdomain iia of hsa. the distance r = 2.397 nm between the donor (hsa) and the acceptor (rutin) was obtained according to fluorescence resonance energy transference (fret). wavelength shifts in synchronous fluorescence spectra showed the conformation of hsa molecules is changed in the presence of rutin. the structure of rutin utilized in molecular modeling calculation was obtained by gaussian 98 program. the optimization geometry of rutin was performed in its ground states by using ab initio dft/b3lyp functional with 6-31g(d,p) basis set used in calculations. the molecular electrostatic potential (mep) was calculated to provide the molecular charge distribution of rutin. the gap energy value between the homo and lumo of the rutin molecule was about 4.22 ev which indicates that rutin is classified as a reactive molecule. from molecular modeling calculation the interaction between hsa and rutin was investigated using the autodock program package. the three-dimensional coordinates of human serum albumin were obtained from the protein data bank (entry pdb code 1ao6) and of rutin were obtained from output optimization geometry of dft. the best energy ranked result shows that rutin is localized in the proximity of single tryptophan residue (trp214) of hsa that is in agreement with the fluorescence quenching data analysis. the effect of toxofilin on the structure of monomeric actin lívia czimbalek, veronika kollá r, roland kardos, gá bor hild university of pé cs, faculty of medicine, department of biophysics, pé cs, hungary actin is one of the main components of the intracellular cytoskeleton. it plays an essential role in the cell motility, intracellular transport processes and cytokinesis as well. toxoplasma gondii is an intracellular parasite, which can utilise the actin cytoskeleton of the host cells for their own purposes. one of the expressed proteins of t. gondii is the 27 kda-sized toxofilin. the long protein is a monomeric actinbinding protein involved in the host invasion. in our work we studied the effect of the actin-binding site of toxofilin 69-196 on the g-actin. we determined the affinity of toxofilin to the actin monomer. the flourescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of toxofilin. in the presence of toxofilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. the results of the completed experiments can help us to understand in more details what kind of cytoskeletal changes can be caused in the host cell during the invasion of the host cells by intracellular parasites. t7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. both tmpcp and bmpcp and their peptide conjugates proved to be efficient photosensitizers of virus inactivation. the binding of porphyrin to phage dna was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by dna bound porphyrin. mechanism of photoreaction (type i. versus type ii) and the correlation between dna binding, singlet oxygen production and virus inactivation capacity was also analyzed. dna binding reduced the virus inactivation due to the reduced absorbance and singlet oxygen production of bound photosensitizer, and altered mechanism of photoinactivation. as optical melting studies of t7 nucleoprotein revealed, photoreactions of porphyrin derivatives affected the structural integrity of dna and also of viral proteins, even if the porphyrin did not bind to np or was selectively bound to dna. anesthesia is a medical milestone (friedman & friedland, medicine's 10 greatest discoveries, 2000) and local anesthetics (la) are the most important compounds used to control pain in surgical procedures. however, systemic toxicity is still a limitation for la agents as well as low solubility, as for tetracaine (ttc). approaches to improve la effects include macrocyclic systems formation, such as in cyclodextrins (cd). we have studied complexes formed between ttc and b-cd or hydroxylpropyl (hp)-b-cd through nmr and other (uv-vis, fluorescence, dsc and x-ray diffraction) techniques. at ph 7.4 a 1:1 stoichiometry of complexation was detected for both complexes, with association constants of 777 m -1 and 2243 m -1 for ttc:b-cd and ttc:hp-b-cd, respectively. the nuclear overhauser nmr data disclosed trough the space proximities between hydrogens h h and h iat the aromatic ring of ttc -and hydrogens from the inner cavity of the cyclodextrins, allowing us to propose the topology of ttc:cd interaction. complex formation did not curb ttc association with model (liposomes) and biological membranes since the total analgesic effect (infraorbital nerve blockade in rats) induced by 15mm ttc increased 36% upon complexation. supported by (fapesp # 06/121-9, 03838-1) brazil. p-033 itc as a general thermodynamic and kinetic tool to study biomolecule interactions philippe dumas 1 , dominique burnouf 1 , eric ennifar 1 , sondes guedich 1 , guillaume bec 1 , guillaume hoffmann 1 isothermal titration calorimetry (itc) is a powerful technique for thermodynamic investigations that is little used to obtain kinetic information. we have shown that, in fact, the shape of the titration curves obtained after each ligand injection is strictly governed by the kinetics of interaction of the two partners. a simple analysis allowed us to explain several facts (e.g. the variation of time needed to return to equilibrium during a titration experiment). all simplifications were further released to obtain a very realistic simulation of an itc experiment. the method was first validated with the binding of the nevirapine inhibitor onto the hiv-1 reverse transcriptase by comparison with results obtained by biacore tm . importantly, for more complex systems, the new method yields results that cannot be obtained in another way. for example, with the e. coli transcription-regulator thiamine pyrophosphate riboswitch, we could resolve kinetically and thermodynamically the two important successive steps: (1) the binding of the tpp ligand and (2) the subsequent rna folding. our results show that initial tpp binding is controlled thermodynamically by tpp concentration, whereas the overall transcription regulation resulting from rna folding is kinetically controlled. gfps, due to their tendency to dimerize at high concentration. we have characterized for the first time the selfassociation properties of cfp (cyan fluorescent protein), the fluorescent protein mostly used as fret donor. we found that the fluorescence quenching observed at high expression level in the cell cytoplasm and the fluorescence depolarization measured at high concentration in vitro are insensitive to the a206k mutation, shown to dissociate other gfp dimers. both phenomena are satisfactorily accounted for by a model of non-specific homo-fret between cfp monomers due to molecular proximity. modeling the expected contributions to fluorescence depolarization of rotational diffusion, homo-fret within a hypothetical dimer and proximity homo-fret shows that cfp has a homo-affinity at least 30 times lower than gfp. this difference is due to an intrinsic mutation of cfp (n146i), originally introduced to increase its brightness and that by chance also disrupts the dimers. biomolecular recognition typically proceeds in an aqueous environment, where hydration shells are a constitutive part of the interacting species. the coupling of hydration shell structure to conformation is particularly pronounced for dna with its large surface to volume ratio. conformational substates of the phosphodiester backbone in b-dna contribute to dna flexibility and are strongly dependent on hydration. we have studied by rapid scan ftir spectroscopy the isothermal b i -b ii transition on its intrinsic time scale of seconds. correlation analysis of ir absorption changes induced by an incremental growth of the dna hydration shell identifies water populations w 1 (po 2 --bound) and w 2 (non-po 2 --bound) exhibiting weaker and stronger h-bonds, respectively, than those in bulk water. the b ii substate is stabilized by w 2 . the water h-bond imbalance of 3-4 kj mol -1 is equalized at little enthalpic cost upon formation of a contiguous water network (at 12-14 h 2 o molecules per dna phosphate) of reduced !(oh) band width. in this state, hydration water cooperatively stabilizes the b i conformer via the entropically favored replacement of w 2 -dna interactions by additional w 2 -water contacts, rather than binding to b i -specific hydration sites. such water rearrangements contribute to the recognition of dna by indolicidin, an antimicrobial 13-mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the b i conformer in a water-mediated induced fit. in combination with cd-spectral titrations, the data indicate that in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the dna hydration shell allows for entropic contributions similar to those assigned to water upon dna ligand recognition in solution. segmental-labeling expression of sh3 domains of cd2ap protein to study interaction with their ligand i.f. herranz-trillo 1 , j.l. ortega-roldan 1 , n.a.j. van transient and low affinity interactions within the cell can be enhanced by the combination of more than one domain. up to now most of the effort has been put on the study of the regulation in the affinity and specificity of the binding to isolated single domains but little is known about the effect of the presence of a second or third domain. multiple examples of proteins containing tandem domains exist in the genome like the cin85/cms family of adaptor proteins. in this family all three n-terminal sh3 domains are involved in a wide variety of different interactions, they share higher similarity among themselves than to any other sh3 domains, suggesting an overlapping specificities in binding. cd2 associated protein (cd2ap) is an adaptor protein and belongs to this family, its n-terminus consists of three sh3 domains and the interaction of each one of them with its target(-s) might be ultimately modulated by the presence of its next-door-neighbor. in this work we present the expression and purification of the tandem cd2ap-sh3a/ sh3b produced by segmental labeling techniques that allow us to express the domains with different isotopic label, improving the nmr signal and facilitating to study the interaction of the natural ligand in the presence of nextdoor-neighbor domain. there are plenty of molecules that exert their effects at the cell membrane. the evaluation of these interactions, frequently quantified by the nernst lipid/water partition constant (kp), helps to elucidate the molecular basis of these processes. we present here a recently derived and tested method to determine kp for single solute partitions using fpotential measurements. the concept was then extended to the interaction of supramolecular complexes with model membranes. a simultaneous double partition with an aqueous equilibrium is considered in this partition model. the results were validated by dynamic light scattering -dls, f-potential, fluorescence spectroscopy and laser confocal microscopy experiments. we evaluated the interaction of supramolecular complexes (peptides derived from dengue virus proteins with oligonucleotides) with luv to study our biophysical models. dengue virus (dv) infects over 50-100 million people every year and may cause viral hemorrhagic fever. no effective treatment is available and several aspects of its cellular infection mechanism remain unclear. the extension of the interactions of these complexes with biomembranes helps to elucidate some steps of dv life cycle. the aggregation of amphotericin b in the lipid membranes induced by k + and na + ions: langmuir monolayers study marta arczewska, mariusz gagoś department of biophysics, university of life sciences in lublin, poland the polyene antibiotic amphotericin b (amb) is currently the drug of choice in the treatment of fungal infections despite its undesirable side effects. according to the general conviction, the biological action of the drug is based on the formation of transmembrane channels which affect physiological ion transport, especially k + ions. this work reports the results of langmuir monolayers study of the effect of k + and na + ions on the molecular organizations of amb in the model lipid membrane. the two-component monolayers containing amb and phospholipid (dppc) have been investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of k + and na + ions. the strength of the amb-dppc interactions and the stability of the mixed monolayers were examined on the basis of surface pressure measurements, the compressional modulus and the excess free energy of mixing. the obtained results proved a high affinity of amb towards lipids in the presence of k + than na + ions. the most stable mixed monolayers were formed with the 1:1 and 2:1 stoichiometry in the presence of k + and na + ions, respectively. this research was financed by ministry of education and science of poland within the research project n n401 015035. microcalorimetric study of antibiotic amphotericin b complexes with na + , k + and cu 2+ ions arkadiusz matwijczuk, grzegorz czernel, mariusz gagoś department of biophysics, university of life sciences in lublin, poland amphotericin b (amb) as a metabolite of streptomyces nodosus is one of the main polyene antibiotics applied in the treatment of deep-seated mycotic infections. we presented microcalorimetric (dsc) study of molecular organization of amphotericin b in lipid membranes induced by na + , k + and cu 2+ ions. the analysis of dsc curves indicates the influence of na + and k + ions on the main phase transition of pure dppc lipid. for the molar fractions of 3, 5, 10, 15 mol% amb in dppc we observed the thermal shift towards higher temperatures in respect to pure lipid, both in the presence of na + and k + ions. this result may be connected with the changes in dynamic properties of the model membrane system. in case of amb-cu 2+ complexes in aqueous solution at two ph values, 2.0 and 10.6, the dsc measurements reported endothermic heat effect. this phase transition was related to the dissociation process of amb-cu 2+ complexes. the formation of amb-cu 2+ complexes are accompanied by changes of the molecular organization of amb especially disaggregation. these all observed effects might be significant from a medical point of view. this research was financed by ministry of education and science of poland within the research project n n401 015035. membrane proteins and peptides are acting in an environment rich in other proteins or peptides. aim of our study was to understand how such molecular crowding and resulting intermolecular interactions can influence the behavior of membrane proteins, using various antimicrobial peptides and membrane proteins as examples. in the case of antimicrobial peptides we have previously described a change in their alignment in the membrane at a characteristic threshold concentration. to understand whether this change is due to unspecific crowding or specific peptidepeptide interactions, we tested if this re-alignment depends on the presence of additional peptides. in most cases we found a similar re-orientation behavior irrespective of the added peptide type, indicating unspecific crowding. when pairing pgla and magainin-2, however, we observed a distinctly different sequence of pgla re-orientation in the membrane, indicating a specific interaction between these two peptides, which correlates well with their known synergistic activity. a rather different effect of crowding was observed for the larger channel protein mscl, which was found to form clusters of functionally active proteins in the membrane. we propose that this clustering is caused by lipid-mediated protein-protein interactions. water, hydrophobic interaction, and protein stability j. raul grigera and c. gaston ferrara instituto de física de líquidos y sistemas bioló gicos (iflysib), conicet-unlp, la plata, argentina although there are several forces maintaining protein structure, it is well know that hydrophobic interaction is the dominant force of protein folding. then, we can infer that any factor that alters hydrophobic interaction will affect the protein stability. we have study by computer simulation a model system consisting in solution of lenard-jones particles in water (spc/e model) at different pressures and temperatures and analyzed the solubility i.e. the aggregation properties, of such a system. from the obtained data we are able to build up the phase surface determining the critical point. the computing results where compared with experimental data of binary mix of non polar substance in water and of protein denaturation, finding high coincidence on the critical point. since the behavior of our model system can only be due to hydrophobic effects, the coincidences with the denaturation of proteins allow us to conclude that the dominant factor that determine temperature and pressure denaturation of proteins is the hydrophobic interaction. the temperature and pressures at which the denaturation, as well the disaggregation of simple non-polar particles, starts agree with what we could expect based on the cross over line of the low to high density structure water transition. the functional reconstitution of a mitochondrial membrane protein into a lipid bilayer was studied using a quartz crystal microbalance. the 6xhis-tagged protein was immobilised via specific binding to a cu 2+ terminated sensor surface, with a change in frequency indicating approximately 75% coverage of the sensor surface by the protein. a lipid bilayer was reconstituted around the protein layer, with a final change in frequency that is consistent with the remaining area being filled by lipid. incubation with a specific ligand for the protein resulted in a significant change in frequency compared to the interaction with the surface or lipid alone. the change is greater than expected for the mass of the ligand, indicating a possible conformational change of the protein, such as the opening of a channel and increased water content of the layer. electrical impedance measurements on the same system have provided additional evidence of protein-lipid bilayer formation, and it is intended that this system will be studied with neutron reflectometry to characterise potential ligand induced channel formation. valuable functional and structural information about this membrane protein was obtained by using surface sensitive techniques to study the protein in a biomimetic lipid bilayer. visualizing and quantifying hiv-host interactions with fluorescence microscopy jelle hendrix 1, *, zeger debyser 2 , johan hofkens 3 and yves engelborghs 1 1 laboratory for biomolecular dynamics, university of leuven, belgium, 2 laboratory for molecular virology and gene therapy, university of leuven, belgium, 3 laboratory for photochemistry and spectroscopy (*present address), university of leuven, belgium protein-chromatin interactions are classically studied with in vitro assays that only provide a static picture of chromatin binding. fluorescence correlation spectroscopy (fcs) is a non-invasive technique that can be used for the same purpose. being applicable inside living cells it provides dynamic real-time information on chromatin interactions. transcriptional co-activator ledgf/p75 has well characterized protein and chromatin interacting regions. we studied ledgf/p75 in vitro and inside living cells with fcs and other techniques (luminescent proximity assay, spot/half-nucleus fluorescence recovery after photobleaching, continuous photobleaching). protein-protein interactions in living cells can be monitored with fluorescence cross-correlation spectroscopy (fccs) using fluorescent proteins as genetic labels. advantages over using fö rster resonance energy transfer (fret) are the independence from intermolecular distance and knowledge of absolute protein concentrations. we characterized fccs with fluorescent proteins in vitro and then studied the intracellular complex of ledgf/p75 and the hiv-1 integrase (in) enzyme both with fret and fccs. nucleus and its compartment nucleolus are a seat of enormous biosynthetic activity in human cancer cells. nucleolar proteins, e.g. b23 or c23, play an important role in regulation of cell division and proliferation. one of the strategies how to intermit malignant cell proliferation is affecting, e.g. by drug treatment, a net of intracellular protein interactions to bring the cell on a way of apoptosis. a cytostatic agent actinomycin d initiates apoptosis in human cancer cells, as well as in normal peripheral blood lymphocytes. at the same time, translocation of b23 and c23 into nucleoplasm is observed in the treated cells. therefore interaction between nucleolar and apoptotic proteins comes into a question. co-immunoprecipitation, fluorescence microscopy and yeast two hybrid analysis are used to answer it. in co-immunoprecipitation experiments, tumor suppressor p53 showed up to be a promising candidate for the interaction. fluorescence deposits mostly constituted by variants of transthyretin (ttr), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol [1] . nowadays, the only effective therapy for ttr amyloidosis is liver transplantation. new therapeutic strategies are being developed taking advantage of our current understanding of the molecular mechanisms of amyloid formation by ttr [2] . a significant effort has been devoted to the search and rational design of compounds that might decrease ttr tetramer dissociation, for example, through ligand binding at the thyroxine binding sites of ttr [3, 4] . here, we use isothermal titration calorimetry (itc) to characterize the thermodynamic binding signature of potential ttr tetramer stabilizers, previously predicted by computerassisted methods [3] . itc allows the measurement of the magnitude of the binding affinity, but also affords the characterization of the thermodynamic binding profile of a protein-ligand interaction. high affinity/specificity ttr ligands, enthalpically and entropically optimized, may provide effective leads for the development of new and more effective drug candidates against ttr amyloidosis. we have established a set of vectors to promote easy cloning of ecfp and eyfp fusions with any protein of interest. we exploit these fluorescent fusion proteins to study protein-protein interactions by fluorescence lifetime of ecfp. the decrease of ecfp lifetime reveals fret between ecfp and eyfp and hence the interaction between proteins in question. groel-groes chaperonin complex is required for the proper folding of eschericia coli proteins. bacteriophage t4 and its distant relative coliphage rb49 encode co-chaperon proteins (respectively gp31 and coco) that can replace groes in the chaperonin complex. gp31 is also required in the folding of the major capsid protein of the phage. prd1 is a large membrane-containing bacteriophage infecting gram-negative bacteria such as e. coli and salmonella enterica. it has 15 kb long linear dsdna genome and the capsid has an icosahedral symmetry. the groel-groes chaperonin complex is needed in the assembly of prd1. we have found evidence that prd1 protein p33 can work similar way as other viral co-chaperones and substitute groes in chaperonin complex. fluorescence lifetime studies between proteins groel and p33 reveals an interaction that backs up the theory. structural modification of model membranes by fibrillar lysozyme as reaveled by fluorescence study a.p. kastorna v.n. karazin kharkiv national university, 4 svobody sq., kharkiv, 61077, ukraine recent experimental findings suggest that protein aggregation, leading to the formation and depositions of amyloids play a central role in the neurodegenerative diseases, type ii diabetes, systemic amyloidosis, etc. in the present study we focused our efforts on investigation of the influence of fibrillar lysozyme on the structural state of model lipid membranes composed of phosphatidylcholine and its mixtures with cardiolipin (10 mol %) and cholesterol (30 mol %). to achieve this purpose, two fluorescent probes with different bilayer location, 1,6-diphenyl-1,3,5-hexatriene (dph) distributing in membrane hydrocarbon core and 6-lauroyl-2-dimethylaminonaphthalene (laurdan) locating at lipid-water interface, have been employed. the changes in membrane viscosity under the influence of amyloid lysozyme were characterized by fluorescence anisotropy of dph. this fluorescence parameter was not markedly affected by fibrillar protein in all types of model membranes. the changes in emission spectra of laurdan were analysed by the generalized polarization value (gp). it was found that adding of amyloid lysozyme resulted in the increment of gp value. our data suggest that lysozyme fibrils cause reduction of bilayer polarity and increase of lipid packing density. isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions. however, reliable determination of the dissociation constant (k d ) is typically limited to the range 100 lm [ k d [ 1 nm. nevertheless, interactions characterised by a higher or lower k d can be assessed indirectly, provided that a suitable competitive ligand is available whose k d falls within the directly accessible window. unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of highand low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-and low-affinity protein-ligand interactions. interaction of myoglobin with oxidized polystyrene surfaces studied using rotating particles probe m. kemper 1,2 , d. spridon 1 , l.j. van ijzendoorn 1 , m.w.j. prins 1,3 1 eindhoven university of technology, department of applied physics, eindhoven, the netherlands, 2 dutch polymer institute, eindhoven, the netherlands, 3 philips research, eindhoven, the netherlands the interaction of proteins with polymer surfaces is of profound importance for the sensitivity of biosensors. polymer surfaces are often treated in order to tune their chemical and physical properties, for example by oxidation processes. to get a better understanding of the association of proteins to treated polymer surfaces, we use the rotating particles probe (x.j.a. janssen et al., colloids and surfaces a, vol. 373, p. 88, 2011). in this novel technique, protein coated magnetic particles are in contact with a substrate and the binding is recorded for all individual particles using a rotating magnetic field. we investigate the interaction of myoglobin coated magnetic particles to spincoated polystyrene surfaces that have been oxidized with a uv/ozone treatment. the surfaces have been characterized by xps, afm and water contact angle measurements. we will demonstrate a clear influence of polystyrene oxidation on the binding fractions of the myoglobin coated particles. we interpret the results in terms of dlvo-theory: electrostatic as well as electrodynamic properties of the surfaces will be influenced by the oxidation. interact with monomeric and/or filamentous actins. twinfilin is a 37-40 kda protein composed of two adf-homologue domains connected by a short linker. in our work we studied the effects of the mouse twinfilin-1 (twf1) on the monomeric actin. we determined the affinity of twf1 to the atp-actin monomer with fluorescence anisotropy measurement (k d = 0.015 lm). the fluorescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of twf1. in the presence of twinfilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. it was confirmed with stopped-flow experiments that the kinetics of nucleotide-exchange of actin decreased in the presence of twf1. we determined the thermodynamic properties of twf1 and investigated the effect of twinfilin on the stability of actin monomer with differential scanning calorimetry. the twf1 stabilized the stucture of the g-actin. our results can help us to understand the regulation of actin cytoskeleton in more details. magnetic np have attracted attention due to their potential of contrast enhancement of magnetic resonance imaging and targeted drug delivery, e.g. tumor magnetic hyperthermia therapy. potential nephrotoxicity of single i.v. administration of fe 3 o 4 np was studied in female wistar rats i.v. administered either placebo (10% v/v rat serum in 0.9% nacl), suspension of tio 2 np (positive control, bimodal 84/213 nm distribution), or fe 3 o 4 np (bimodal 31/122 nm distribution) in doses of 0.1, 1.0 or 10.0 mg/kg. rats were sacrificed 24h, 7-, 14-and 28-days after np injection (n=9-10/each group). administration of np did not alter kidney size significantly; renal function of np administered rats as monitored by plasma creatinine and urea concentrations, creatinine clearance and protein excretion rate did not differ significantly in either interval from rats administered placebo. one week after administration significant rise in plasma ca, its urinary and fractional excretion was observed in rats administered 10 mg fe 3 o 4 /kg. plasma mg levels rose in this group 1 and 2 weeks after administration. no significant changes in the expression of tnf-a, tgf-b, and collagen iv genes in renal cortex were revealed. no obvious nephrotoxic effects were observed in rats after a single i.v. dose of fe 3 o 4 np. study was supported by 7fp ec eu: nanotest (development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics.), grant no.: 201335. biomimetic supramolecular assemblies for studying membrane interactions in vitro and in vivo s. kolusheva, r. jelinek ben-gurion university, beer-sheva, israel we designed a novel biomimetic sensor, composed of conjugated polydiacetylene (pda) matrix embedded within lipid vesicles. the system is capable of detecting various compounds occurring within lipid membranes through rapid colorimetric as well as fluorescent transitions. the colorimetric response of the sensor is correlated to the extent of compound-membrane binding and permeation and quantified binding sensitivity to lipid composition. we describe a new disease diagnostic approach, denoted ''reactomics'', based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (pda), a chromatic polymer. we show that reactions between sera and such a lipid/pda vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. through attachment of chromatic polydiacetylene (pda) nanopatches onto the plasma membrane, real-time visualization of surface processes in living cells is possible. the ras protein is mutated in 30% of human tumors. ras acts as a switch, transmitting a growth signal in an active gtp-bound form and turning the signal off in an inactive gdp-bound form. the switch off is accomplished by gtp hydrolysis, which is catalyzed by ras and can be further accelerated by gtpase activating proteins (gaps). mutations which prevent hydrolysis cause severe diseases including cancer. we investigate the reaction of the ras gap protein-protein complex by time-resolved ftir spectroscopy. 1 detailed information on the mechanism and the thermodynamics of the reaction was revealed: 2 first, the catalytic arginine-finger of gap has to move into the gtp binding pocket, then cleavage of gtp is fast and h 2 po 4 hydrogen-bonded in an eclipsed conformation to the b-phosphate of gdp is formed. further, we performed for the first time atr-ftir spectroscopy of ras in its native environment, a lipid membrane. 3 in this setup we are able to do difference spectroscopy of the immobilized protein. interactions with other proteins can be determined in a similar way as in spr experiments but with the additional information from the infrared spectra. galectins are a family of animal lectins that specifically bind b-galactosides and have gained much attention due to their involvement in several biologic processes such as inflammation, cell adhesion and metastasis. in such processes, several issues are still not clear including the mechanisms of interaction with different carbohydrates. galectin-4 (gal-4) is a tandem-repeat type galectin that contains two carbohydrate recognition domains (crd-i and crd-ii) connected by a linking peptide. in this study, we performed spectroscopic studies of the carbohydrate-recognition domains from human gal-4. our goals are two-fold: (1) to monitor conformational changes in each domain upon its binding to specific ligands and then to correlate the observed changes with structural differences between the crds and (2) to investigate the interaction between the crds and lipid model membranes. to achieve such objectives we used a combined approach of spectroscopic techniques involving circular dichroism and electron spin resonance. overall the results obtained so far show that crd-i and crd-ii have distinct behaviors in terms of carbohydrate recognition and membrane binding. this may be due to specific differences in their structures and certainly suggests a non-equivalent role in protein function. hemoglobin influence on lipid bilayer structure as revealed by fluorescence probe study o.k. kutsenko, g.p. gorbenko, v.m. trusova v.n. karazin kharkov national university, kharkov, ukraine hemoglobin (hb) is a red blood cell protein responsible for the oxygen transport. its affinity for lipid bilayers represents interest for gaining insight into protein biological function as well as for some applied aspects such as development of blood substitutes or biosensors. hb influence on lipid bilayer structure was investigated using fluorescent probes pyrene and prodan. model membranes were prepared of phosphatidylcholine (pc) and its mixtures with phosphatidylglycerol (pg) and cholesterol (chol). hb penetration into membrane interior is followed by the increase of relative intensity of pyrene vibronic bands and decrease of prodan general polarization value suggesting an enhancement of bilayer polarity. this implies that hb incorporation into membrane interior decreases packing density of lipid molecules, promoting water penetration into membrane core. chol condensing effect on lipid bilayer prevents protein embedment into bilayer, thus decreasing membrane hydration changes as compared to pc bilayers. in the presence of anionic lipid pg hb-induced increase of bilayer polarity was found to be most pronounced, pointing to the modulatory role of membrane composition in hb bilayer-modifying propensity. we present optimized sialic acid-based mimics binding in the low nanomolar range. molecular interactions were determined with surface plasmon resonance (spr), characterizing the affinity and the kinetics of binding. furthermore, isothermal titration calorimetry (itc) was applied to dissect the standard free energy of binding (dg°) into the standard enthalpy of binding (dh°) and the standard entropy of binding (ds°). in order to pass the cell membranes, most of these medicines has to be administrated to patients as nucleoside pro-drugs and not directly as triphosphorylated forms. because of the poor phosphorylation of the nucleoside analogues used in therapy, it is important to understand and to optimize their metabolism. our aim is to understand how compounds of chirality l turn away 3-phosphoglycerate kinase (pgk) from its normal glycolytic function to be converted into the triphosphate forms. in order to elucidate pgk mechanism and substrate specificity, we have measured the kinetics of the different steps of the enzymatic pathways by rapid mixing techniques and studied the influence of the nature of the nucleotide substrate thereon. we first performed an extensive study with d-and l-adp (see poster by p. lallemand). we are now extending the studies to other nucleotide diphosphates (some of them used in therapies). changes in the nature of the nucleobase or deletion of hydroxyl group of the sugar affect the efficiency of phosphorylation by pgk, either by decreasing dramatically their affinity or by altering the phospho-transfer step itself. structural explanations are given based on docking data. probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research, drug discovery and membrane biophysics. previous studies showed that enrofloxacin metalloantibiotic has potential as an antimicrobial agent candidate, since it exhibits antimicrobial effect comparable to that of free enrofloxacin but a different translocation route. these differences in uptake mechanism can be paramount in counteracting bacterial resistance. in view of lipids role in bacterial drug uptake, the interaction of these compounds with different e. coli model membranes were studied by fluorescence spectroscopy. partition coefficients determined showed that lipid/antibiotic interactions were sensitive to liposomes composition and that the metalloantibiotic had a higher partition than free enrofloxacin. these results corroborate the different mechanism of entry proposed and can be rationalized on the basis that an electrostatic interaction between the metalloantibiotic positively charged species, present at physiological ph, and the lipids negatively charged head groups clearly promotes the lipid/antimicrobial association. oligomerization and fibril assembly of amyloid b peptide amyloid b peptide (ab) forms a large amount of extracellular deposits in the brain of alzheimer's disease (ad) patients and it is believed that this peptide is related to the pathogenesis of that disease. the most abundant monomeric form of physiological ab (*90%) is constituted by 40 amino acids and is benign, but by an unknown mechanism this endogenous material becomes aggregated and neurotoxic. increasing evidence suggests that membrane interaction plays an important role in ab neurotoxicity. in this work it will be studied the interactions of ab(1-40) with ctac (cationic), sds (anionic), pfoa (anionic with fluorine atoms) and og (nonionic) amphiphiles in monomeric and micellar forms. the results demonstrated that ab(1-40) forms fibrils with different morphologies in the presence of micelles. in addition, the presence of micelles accelerates the formation of fibrils and decreases the lifetime of oligomers. we present here the exploitation of the powerful approach of surface plasmon resonance imaging and mass spectrometry coupling for protein fishing in biological fluids such as human plasma at the same sensitivity. on one hand, multiplex format spri analysis allows direct visualization and thermodynamic analysis of molecular avidity, and is advantageously used for ligand-fishing of captured bio-molecules on multiple immobilized receptors on a spri-biochip surface. on the other hand, maldi mass spectrometry is a powerful tool for identification and characterization of molecules captured on specific surface. therefore, the combination of spri and ms into one concerted procedure, using a unique dedicated surface, is of a great interest for functional and structural analysis at low femtomole level of bound molecules. to reach these goals, particular surface engineering has been engaged to maintain a high level of antibody grafting and reduce non-specific adsorption. thus, various chemistries have been tested and validated towards biological fluids such plasma, keeping in mind the capacity of the in situ investigation by ms. finally, signal to noise ratio was magnified leading to the characterization of protein lag3, a potential marker of breast cancer, in human plasma. atenolol incorporation into pnipa nanoparticles investigated by isothermal titration calorimetry mihaela mic, ioan turcu, izabell craciunescu, rodica turcu, national institute for r&d of isotopic and molecular technologies, 400293 cluj-napoca, romania e-mail: mihaela.mic@itim-cj.ro poly(n-isopropylacrylamide) (pnipa) is a thermo-sensitive hydrogel undergoing a volume phase transition at about of 32°c close to the body temperature. this volume phase transition is envisaged as a key property for drug binding and release. the purpose of our research is the thermodynamic characterization of the binding of atenolol by pnipa polymeric nanoparticles. the thermodynamic parameters which characterize the binding process are obtained using the isothermal titration calorimetry (itc) as the main investigation technique. when polymeric nanoparticles bind drug molecules, heat is either generated or absorbed depending on the amount of bond molecules and also on the exothermic or endothermic character of the binding process. the heat measurement allows the determination of binding constants, reaction stoichiometry and the thermodynamic profile of the interaction. itc technique has been used to investigate the binding properties of nanoparticles which shrink from the swollen to the collapsed state. the capacity of such nanogels to bind atenolol molecules is directly related to relevant differences between the binding properties in the swollen and in the collapsed state respectively. aggregation study of x-(alkyldimethylamonium)alkylaldonamide bromides p. misiak 1 , b. ró _ zycka-roszak 1 , e. woźniak 1 , r. skrzela 1 , k.a. wilk 2 1 department physics and biophysics, wrocław university of environmental and life sciences, wrocław, poland, 2 department of chemistry, wrocław university of technology, wrocław, poland sugar-based surfactants are of considerable research interest because they have improved surface and performance properties, reduced environmental impact, and have potential pharmaceutical and biomedical applications. x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lenghs (n = 10, 12, 14, 16) belonging to cationic sugar-based surfactants were newly synthesised. the aggregation processes of c n gabs were studied by means of isothermal titration calorimetry (itc), electric conductance method and molecular modelling methods. the critical micelle concentrations (cmc), the degree of micelle ionization (b), the enthalpies (dh m ) and the entropies (ds m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (dg m 0 (hy)) were calculated. the obtained values were compared with those for dodecyldimethylethylammonium bromide and literature data for analogical glucocationic surfactants. the latest compounds differ from c n gab surfactants by substitution of sugar chain by gluco ring. molecular modelling methods were used to relate the molecular properties of the compounds with their experimentally studied properties in solution. this work was supported by grant n n305 361739. every year over 50 million people are infected with dengue virus (denv), transmitted by a mosquito (aedes aegypti). this enveloped virus, member of the flaviviridae family, has four distinct serotypes. it has a single stranded positive rna molecule with a single open reading frame that encodes a single poliprotein, which, after appropriate processing by viral and host proteases, gives rise to three structural proteins (c, prm and e) and seven non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5) [1] . the surface of the immature virion is composed of e and prm heterodimers that are arranged as trimers protruding from the membrane [2] . the virus is thought to enter the host cell via a receptor-mediated endocytosis, although, if any, the specific dengue receptors have not been described. once inside the cell, the acidified environment inside the endocytic vesicle triggers an irreversible trimerization of the envelope (e) protein, inducing the release of the nucleocapsid (composed of viral rna and multiple copies of c protein) to the cytoplasm, thus starting the infection process, where the poliprotein is translated and processed, originating all viral proteins. considering the structural proteins c and e, these are essential for the viral infection, specifically, protein c is thought to be involved in the viral assembly and specific encapsidation of the genome and protein e (a class ii fusion protein) plays a major role in the fusion process. as recently described by some studies [3] , protein c is composed of four a helices connected by four short loops and has a highly hydrophobic region forming a concave groove that could interact with lipid membranes and a region with an increased concentration of positive charges, possibly interacting with the viral rna. as for protein e, it is composed of three b stranded domains. it is proposed that the fusion loop is located in domain ii of this protein and the putative receptor binding sites, considered essential for the viral entry, are supposedly located in domain iii. in this work, we describe the identification of the membrane active regions of both these proteins, considering both theoretical studies, hydrophobic moments, hydrophobicity and interfaciality values as well as experimental ones, namely fluorescence spectroscopy, where a fluorescent probe is encapsulated in model membrane systems, and differential scanning calorimetry [4] . we have found one region in protein c and four regions in protein e with membranotropic activity. this is the first work describing experimentally the putative membrane interacting zones of both these proteins. this work was funded by grant bfu2008-02617-bm from ministerio de ciencia y tecnologia, spain, granted to jose villalaín. investigation of membrane-membrane interaction mediated by coiled coil lipopeptides gesa pä hler, andreas janshoff georg-august-university, tammannstrasse 6, 37077 gö ttingen, germany e-mail: gpaehle@gwdg.de specific cellular membrane interaction and fusion are crucial points in vivo which are in eukaryotic cells mediated by snare proteins. the definite mechanism behind these processes is still poorly understood, but the coiled coil formation of a snare core complex consisting of four a-helices seems to generate a fusogenic driving force. this offers the possibility to design a straightforward experimental setup to mimic the complex protein-mediated membrane-membrane interaction by using mere protein fragments or peptides attached to artificial lipid bilayers which self-assemble to a coiled coil structure. in our approach, two artificial three heptad repeat coiled coil forming peptides were synthesized and attached to maleimide functionalized membranes via an in situ-coupling reaction. thus, secondary structure changes, kinetic characteristics and binding energetics were monitored during coiled coil formation with real time ellipsometry, ir and cd spectroscopy. the lipopeptide mediated membrane-membrane interaction itself is investigated by colloidal probe spectroscopy and tirfm. these techniques and the setup of our model system allow screening the energetic and structural properties of variable coiled coil forming peptides, i.e. linker-modified or biologically inspired sequences. enzymatic reactions in nanostructured surfaces: unzipping and cutting the double helix pietro parisse 1 , matteo castronovo 2 , bojana lucic 3 , alessandro vindigni 3 , giacinto scoles 2 and loredana casalis 1 1 sincrotrone trieste s.c.p.a., trieste, italy, 2 temple university, philadelphia, usa, 3 protein-dna interactions are vital for living organisms. from viruses to plants and humans, the interactions of these two different classes of biopolymers control processes as important and diverse as the expression of genes and the replication, rearrangement, and repair of dna itself. to understand these processes at the molecular level, and to follow changes in cellular pathways due to different kinds of perturbations and/or diseases it is necessary the identification and quantification of proteins and their complex network of interactions. we have exploited the high spatial resolution given by atomic force microscopy to generate dna arrays of variable density by means of nanografting. on such nanostructures, we investigate the mechanism of different enzymatic reactions (from restriction enzymes to helicases). registering with high precision the height variation due to the action of the enzyme onto the engineered dna sequences (in the case of restriction enzymes) or taking advantage of the different mechanical properties of single and double stranded dna (in the case of helicases, where for the first time kinetic data were obtained on recq1 human helicase), we were able to monitor either the activity and/or the action mechanisms of these two important classes of enzymes. in this study an attempt has been made to investigate the structure, dynamics and stability of cyclic peptide nanotubes (cpnts) formed by the self-assembly of cyclic peptides (cps) using classical molecular dynamics (md) simulation and semiempirical quantum chemistry calculation employing pm6. the structure and energetics of monomer and various oligomeric cpnts have been investigated by considering the (cyclo-[(d-ala-l-ala) 4 ]) peptide as the model for cp. various geometrical parameters extracted from the md simulation reveal that the terminal residues are loosely hydrogen bonded to the inner subunits regardless of degree of oligomerization. the hydrogen bonds present in the inner core regions are stronger than the terminal residues. as the degree of oligomerization increases, the stability of the tube increases due to the hydrogen bonding and stacking interactions between the subunits. the results show that the binding free energy increases with the extent of oligomerization and reaches saturation beyond cpnt5. in addition, hydrophobic and electrostatic interactions play crucial roles in the formation of cpnts. analysis of both structural and energetics of formation of cpnts unveils that the selfassembly of dimer, trimer and tetramer cpnts are the essential steps in the growth of cnpts. monolayers on a langmuir trough constitute a great biomimetic model to characterize protein-protein or protein-lipid interaction, where the physical state of the interfacial layer is completely controlled. we present here three studies performed on monolayers, with a wide panel of experimental (optical, spectroscopical, rheological) techniques. i) surface properties and conformation of nephila clavipes spider recombinant silk proteins (maspi and masp2) was studied at the air-water interface: we show that the mechanism of assembly of both proteins is different, although both proteins share the same sequence pattern and a close hydrophobicity. they both exhibit a certain propensity to form b-sheets that may be important for the efficiency of the natural spinning process. ii) the dystrophin molecular organization and its anchoring in a lipidic environment depend on the rod fragment used and on the lipid nature. moreover the interaction is guided by the lateral surface pressure. this lipid packing variation is essential to understand the role of the dystrophin during compression-extension cycle of the muscle membrane. iii) we evidence that non additive behavior of mixtures of food globular proteins leads to enhanced foaming properties or to self assembled objects. nucleolar-targeting peptides (nrtps) were designed by structural dissection of crotamine, a toxin from the venom of a south-american rattlesnake. at lm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work was to pursue with the study of nrtps molecular mechanism for translocation, as well as to determine the ability of nrtp to delivery large molecules into cells. for the translocation experiments, rhodamine b-labeled nrtps were used and tested with giant multilamellar vesicles. confocal microscopy results show that there is an efficient translocation across model membranes. high levels of intracellular peptide were also seen in different cell lines and pbmc, soon after incubation with nrtp. furthermore, a conjugate of nrtp (nrtp6c) bound to b-galactosidase was prepared by chemical synthesis and tested in hela cells. this conjugate maintains enzymatic activity and is stable at 4°c for several days. the work done so far with this new family of cell-penetrating peptides revealed strong interaction and translocation with lipid model systems. moreover, successfully cellular delivery of bgalactosidase was observed and quantified. interaction of zinc phthalocyanine with ionic and non ionic surfactants: uv-vis absorption and fluorescence spectroscopy for application in photodynamic therapy m. p. romero, s. r. w. louro physic department, pontifícia universidade cató lica de rio de janeiro puc-rio, brazil among the second-generation photosensitizer (ps) developed for the treatment of neoplastic diseases by photodynamic therapy (pdt), metallo-phthalocyanines (mpc) have been proposed as an alternative to the currently used ps in clinical application. unsubstituted mpc are not soluble in physiological solvents and their in vivo administration relies upon their incorporation into carriers or their chemical conversion into water-soluble dyes by the attachment of selected substituents. in this work, uv-vis absorption and fluorescence spectroscopy were used to study the ability of different micelles for dispersing zinc phthalocyanine (znpc). the following surfactants were tested: sds, ctab, hps, tween 80, tween 20, and pluronic f127. znpc has low solubility in virtually all solvents, but dmf and dmso are observed to dissolve znpc in concentrations of the order of 0.9 and 0.2 mmol/l, respectively. stock solutions of znpc in dmf and dmso were prepared. micelles of the different surfactants containing znpc were prepared by dissolving in aqueous medium (milli-q water or phosphate buffer ph 7.4) small amounts of the stock solutions previously mixed with each surfactant. the amounts of each surfactant were calculated to give an average ratio of one znpc molecule per micelle in the final solution. the absorption and fluorescence spectra of znpc in the micellar systems were obtained, and were observed to change in time. immediately after dissolution the spectra are characteristic of monomeric znpc, suggesting formation znpccontaining nanoemulsions with the mixture of znpc-organic solvent in the hydrophobic region of the micelle. since dmso and dmf are miscible with water, the solvent diffuses out of the micelle and znpc stays inside the micelle in a monomeric or aggregated form. the different surfactants lead to different time evolution of znpc aggregation. aggregation lifetimes vary from one hour, in the case of pluronic f127, to more than twelve hours, in the case of ctab and hps. it was observed that the ionic surfactants were more efficient than non ionic ones for monomeric delivery of znpc . work partially supported by cnpq, inami and faperj. nucleobase-containing peptides are an important class of molecules comprising both artificial (synthetic nucleopeptides) and natural (peptidyl nucleosides and willardiine-containing peptides) compounds characterized in many cases by interesting biological properties. 1, 2 in this work, we report a spectroscopic study on the properties of a chiral nucleobase-bearing peptide obtained by chemical synthesis starting from commercial sources. the findings of this research strongly encourage further efforts in the field of the use of nucleopeptides as supramolecular assembling systems and open the way to novel drug delivery approaches based on nucleobase recognition. conformational plasticity. their structure depends tremendously on their local environment and confinment, and may accommodate several unrelated conformations, that are a strong challenge for the traditional characterizations of structure, supramolecular assembly and biorecognition phenomena. atomic force microscopy (afm) has been successfully exploited for both highly controllable nanolithography of biomolecules and for biorecognition studies, such as oriented prion protein -antibody interaction (sanavio et al., acs nano (2010) 4(11): 6607, bano et al. nano lett (2009) 9(7): 2614-8). here, we report different strategies for selective, oriented confinement of alphasynuclein at the nanoscale for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of biomolecular interactions in confined assemblies. a new class of cell penetrating peptides (cpps) was generated by splicing the (1-9) and (38-42) segments of crotamine, a toxin from crotalus durissus terrificus venom [1] . as they localize preferably on the nucleolus, these novel cpps were named nucleolar-targeting peptides (nrtps). the extent of nrtp partition to zwitterionic (popc; popc:cholesterol 67:33) and anionic (popg; popc:popg 70:30) lipid vesicles was studied following the intrinsic tyr or trp fluorescence of the peptides. the partition curves into popc zwitterionic vesicles were characterized by downward slopes and higher partition coefficients (k p *10 4 -10 5 ). for pure popg, an upward curve and smaller partition coefficient point out for a different type of membrane-peptide interaction. popc:popg membranes present characteristics of both types of interaction. from red edge excitation shift and quenching experiments similar conclusions were attained. leakage assays ruled out lipid vesicle disruption by crotamine or nrtps. further studies on nrtp cellular translocation mechanism and large molecule delivery are currently in progress. dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. we characterized the behaviour of dys r11-15, a five spectrin-like repeats from the central domain of human dystrophin, in the presence of liposomes and monolayers as membrane models. interaction of dys r11-15 depends on the lipid nature, anionic or zwitterionic, with suvs, and on the lipid packing when comparing luvs to suvs. lateral pressure of lipid monolayers modifies the protein organization and leads dys r11-15 to form a regular network as revealed by afm. trypsin proteolysis assays show that the protein conformation is modified following its binding to monolayer and suvs. label free quantification by nano-lc/ms-ms allowed identifying the helical amino acid sequences in repeats 12 and 13 that are involved in the interaction with anionic suvs. results indicate that dys r11-15 constitutes a part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid-packing and lipid nature. we provide here strong experimental evidence for a physiological role of the central domain of dystrophin on sarcolemma scaffolding through modulation of lipid-protein interactions. extracellular matrix proteins. overexpression of the mmps has been associated with a variety of diseases ranging from periodontal disease and arthritis to tumor invasion and metastasis. the majority of the more powerful synthetic inhibitors of mmps incorporate a hydroxamate group, but exhibit low selectivity and are toxic. in a recent modeling study, astaxanthin (ast), a carotenoid with potent antioxidant property, has been shown to be a potential inhibitor of mmp-13 function by occupying a binding site near the active center of the enzyme (bika 0 di et al. 2006). in our ongoing project, we investigate the binding of ast to the catalytic domain of mmps using biochemical and ultimately crystallization to validate the proposed action of ast. along these lines, the catalytic domain of mmp-13 (cdmmp-13) was expressed in e.coli bl21(de3) codon-plus and refolded using a novel effective refolding method. our results reveal that ast has a potent inhibitory effect on cdmmp-13 activity, however, determination of ic 50% or k i is difficult due to fast oxidation and structural instability of ast. ongoing work aims at optimizing the inhibition conditions and improving the refolding yield to allow analyzing structure and function of the ast-bound mmp-13 in more detail. hyaluronic acid (hyaluronan, ha) is a linear polysaccharide with a molar mass in the range of 10 5 to 10 7 da and is built from alternating units of glucuronic acid and n-acetylglucosamine. synthesized in the cellular plasma membrane, it is a network-forming and space-filling component in the extracellular matrix of animal tissues. here, we create hyaluronic acid films atop a porous alumina substrate, where they act as a barrier for macromolecular transport depending on their length and geometry. the geometry of the hyaluronic acid switches between a fully stretched and a mushroomlike state and is dependent on the concentration of hyaluronic acid. to bind hyaluronic acid selectively atop the nanoporous anodic aluminum oxide (aao), the aao is orthogonally functionalized by silane chemistry. by means of time resolved optical waveguide spectroscopy (ows) the transport of macromolecules, e.g. avidin, across the hyaluronic acid barriers can be recorded as a function of the pore diameter and hyaluronic acid concentration in a time resolved and label free manner. confocal laser scanning microscopy (clsm) provides an alternative method to investigate the orthogonal functionalization of the pores and to elucidate whether a molecule can cross the barrier at the pore entrance. we functionalized gold surfaces with a hydroxy-terminated self-assembled thiol monolayer exposing an adjustable fraction of biotin moieties. [1] by in-situ acetylation or fluorination, surface properties could be fine-tuned to different protein immobilization scenarios. using streptavidin as a linker protein, immobilization of human abcc3 [2] in liposomal and planar bilayer systems was possible. abcc3-containing proteoliposomes doped with a biotinylated anchor lipid were successfully tethered to our streptavidin-coated surfaces. biotinylation of the extracellular glycosylation of abcc3 allowed direct immobilization with inside-up orientation and subsequent assembly of a lipid bilayer. outside-up orientation was achieved by exploiting the c-terminal histidin tag of recombinant abcc3 for immobilization via ni 2+ and biotinnitrilotriacetate. all systems were thoroughly characterized by quartz crystal microbalance, atomic force microscopy and surface plasmon resonance techniques with respect to monitoring abcc3mediated substrate transport in real time. because of its role in the apoptotic pathway, conformational transitions of cytochrome c (cyt c) have gain on interest. in native cyt c, met 80 and his 18 residues serve as heme axial ligands. cyt c interaction with the membrane causes the disruption of the iron-met80 bond. this allows the binding of others endogenous ligands forming alternative low spin species 1,2 or induces peroxidase activity through the formation of a five coordinated high spin iron specie. acquisition of this peroxydase activity by cyt c has been shown to be a key stage before its release from the mitochondria 3 . in order to study these non native low spin species by checking the possible amino acids able to bind the human cyt c heme, different mutants have been designed and produced: h26q, h33n, and the double one h26q/h33n. sds in countries where seafood is an integrate part of the diet, fish are among the most common food allergen sources. the major fish allergen parvalbumins are abundant in the white muscle of many fish species. parvalbumin belongs to the family of ef-hand proteins and has a globular shape containing six helical parts. high pressure is known to unfold proteins. we performed high pressure ftir experiments, to explore the p-t phase diagram of cod parvalbumin, gad m 1, to test the possibility of its inactivation by high pressure treatment. the infrared spectrum of parvalbumin is characteristic for the helical conformation, in agreement with the crystal structure. a marked transition in the structure of the parvalbumin was observed with the central point of 0.5 gpa (at room temperature). the amide i position shifts to a wavenumber which is between the helical and the unfolded position. we assign this change to a native-molten globule transition. it was reversible as seen from the infrared spectra. it has been proven in the past that reflectometric interference spectroscopy (rifs) is a powerful measurement system for the detection of protein-protein interactions 1 . we present here the development of a reflectometric sensor which allows for the detection of membrane-protein interactions in the micromolar regime. in this study we employ two different instrumental assemblies. the first installation enables direct detection and quantification of the interaction of membrane proteins with solid supported lipid bilayers. in the second assembly the original instrument is combined with an upright fluorescence microscope. the advantages of this installation are the direct optical control of the experiment as well as a smaller sensing area. the set-up allows for the detection of interactions on lipid-patches of just several micrometers in diameter. the aim of this work is an experimental system that enables the measurement of transport processes through lipid membranes. we attempt to achieve this by covering a closed porous substrate with a lipid membrane. the first steps to reach this goal were done by spanning membranes over anodized aluminum oxide substrates. initiation of actin polimerization in cells requires nucleation factors. a pointed-end-binding protein of f-actin -the lei-omodin2-acts as a strong filament nucleator in muscle cells. the dynamical, structural and kinetic properties of a protein can provide important information to understand the intramolecular events underlying its function. we are interested in how does the leiomodin2 regulate the actin polimerization. our aim is to determine the dissociation constant of the actin-leiomodin2 complex, and study a possible side-binding effect of the leiomodin2. the cardiac leiomodin2 of rattus norvegicus is a 50 kda molecular weight protein, which contains a 17 kda n-terminal, a 18 kda leucin reach repeat (lrr) and a 15 kda c-terminal region. the n-term and the lrr regions are together tropomodulin homologues. we expressed the wild type the n-term+lrr the lrr+c-term and the c-term protein fragments by using a ptyb1 vector that contains an amplicillin resistance gene. the expression of the proteins was carried out with the twin-cn (ne bio-labs) kit, which is a chitin-intein self-cleavage and purification system. the nucleation activity of leiomodin and the polimerization speed of actin in the presence of tropomyosin and leiomodin were studied by using pyrene-actin polimerization assay. we measured the stoichiometric, conformational and kinetic properties of the leiomodin-actin complexes with co-sedimentation assay, fluorescence spectroscopic and rapid-kinetic methods. the results showed that the rate of actin polimerization depended on the leiomodin2 concentration. the nucleator activity of leiomo-din2 was ionic strength dependent. the data also confirmed that leiomodin2 is a side-binding and pointed-end binding protein of f-actin. the binding of leiomodin2 to the sides of the actin filaments was slower than to the pointed-end of the f-actin. the structure of f-actin was changed by the sidebound leiomodin2. these observations will contribute to the better understanding of the development and function of thin filaments in cardiac and other muscle tissues. leukemias are one of the most common malignancy worldwide. there is a substantial need for new chemotherapeutic drugs effective against these diseases. doxorubicin (dox), used for treatment of leukemias and solid tumors is poorly efficacious when administered systemically at conventional doses. therefore, in our study, to overcome these limitations, we used transferrin (trf) as a drug carrier. we compared the effect of dox and doxorubicin-transferrin conjugate (dox-trf) on human leukemic lymphoblasts (ccrf-cem). the in vitro growth-inhibition test, xtt assay, indicated that dox-trf was more cytotoxic for leukemia cells than dox alone. in our researches we also evaluated the alternations of mitochondrial transmembrane potential (dw m) , and production of reactive oxygen species (ros). we monitored the dw m using dye jc-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine) . the level of ros was studied using the fluorescent probe dcfh 2 -da (2', 7'dichlorodihydrofluorescein diacetate). the results demonstrate that dox-trf induced, decrease of mitochondrial membrane potential and significantly higher production of ros compared with dox treated cells. moreover, all these results seem to be correlated with dna fragmentation analyzed by dna ladder. the tested processes were partially inhibited by the antioxidant, n -acetylocysteine (nac). the changes induced by dox-trf conjugate and free drug were suggest the different mechanism of action of dox alone and conjugated with transferrin. time-resolved detection of protein-protein interaction masahide terazima kyoto university, kyoto, 606-8502, japan revealing molecular mechanism of a protein reaction has been a central issue in biophysics. for that purpose, a variety of time-resolved spectroscopic methods have been developed. however, most of them can monitor only dynamics associated with an optical transition and it has been very difficult to trace processes without optical transition. we used the pulsed laser induced transient grating (tg) method to study spectrally silent reactions of various proteins in time-domain. here we will show studies on pixd. pixd is a 17 kda short protein which consists of the bluf domain and additional short helices, and is involved in phototactic movement. the photochemical reaction studied by absorption spectroscopy revealed that this protein exhibits typical photochemistry of the bluf proteins. the red-shifted intermediate is generated within a 100 ps. the spectrum does not change after this initial reaction, and returns back to the dark state with a time constant of 12 s at room temperature. we studied the reaction of this protein by our method and found that the proteinprotein interaction is drastically changed during the reaction. the details and the biological meaning will be presented. human ileal bile acid-binding protein (i-babp) plays a key role in the enterohepatic circulation of bile salts. previously we have shown that the protein has two binding sites and triand dihydroxy bile salts bind with strong and moderate positive cooperativity, respectively. positive cooperativity is thought to be related to a slow conformational change in the protein. our current study is directed at the structural and dynamic aspects of molecular recognition in human i-babp using nmr spectroscopy and other biophysical techniques. as a first step in the investigation, 15 n relaxation nmr experiments have been employed to characterize the backbone motion in the apo and holo protein on a wide range of timescales. our results show a moderately decreased ps-ns flexibility in the ligated protein, with most significant ordering near the portal region. in addition, the measurements indicate a slow ls-ms fluctuation at four distinct segments in the apo protein, a motion not observed in the doubly-ligated form at room temperature. our studies support the hypothesis of an allosteric mechanism of binding cooperativity in human i-babp. to shed more light on the molecular details of the binding mechanism, a site-directed mutagenesis study is in progress. cationic porphyrin-peptide conjugates were recently shown to enhance the delivery of peptide moiety to the close vicinity of nucleic acids but their interaction with dna is not yet studied. we synthesized two cationic porphyrin-peptide conjugates: tetra-peptides were linked to the tri-cationic meso-tri(4-n-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-n-methylpyridyl)-15,20di-(4-carboxyphenyl)porphyrin. dna binding of porphyrins, and their peptide conjugates was investigated with comprehensive spectroscopic methods. evidences provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and cd signals reveal that peptide conjugates of di-and tricationic porphyrins bind to dna by two distinct binding modes which can be identified as intercalation and external binding. the peptide moiety does not oppose the interaction between the dna and cationic porphyrins. we compared the effect of complexation on structural stability of dna and nucleoprotein complex : hela nucleosomes and t7 phage. uv and cd melting studies revealed that the porphyrin binding increases the melting temperature of dna and destabilizes the dna protein interaction in the nucleosomes but not in the t7 phage. the wide nucleotide specificity of 3-phosphoglycerate kinase (pgk) allows its contribution to the effective phosphorylation (activation) of nucleotide-based pro-drugs against hiv. here the structural basis of the nucleotide-pgk interaction is characterised in comparison to other kinases, namely pyruvate kinase (pk) and creatine kinase (ck) by enzyme kinetic and structural modelling studies. the results evidenced favouring the purine vs. pyrimidine base containing nucleotides for pgk rather than for pk or ck. this is due to the exceptional ability of pgk in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate chain for catalysis. the unnatural l-configurations of the nucleotides (both purine and pyrimidine) are better accepted by pgk than either by pk or ck. further, for the l-forms the absence of the ribose oh-groups with pgk is better tolerated for the nucleotides with purine rather than pyrimidine base. on the other hand, positioning the phosphate chain of both purines and pyrimidines with l-configuration is even more important for pgk, as deduced from the kinetic studies with various nucleotide-site mutants. these characteristics of the kinase-nucleotide interactions can provide a guideline in drug-design. the role of the enzyme types atp-ases in the muscle contraction g. vincze-tiszay 1 , j. vincze 1 , e. balogh 2 1 hheif, budapest, hungary, 2 nové zá mky, slovakia the myofibrilla assuring muscle contraction gains energy to the slipping in mechanisms and the degree of efficiency of this process will decisively be determined by the velocity of recombination of the atp molecule. in this there play a particular part the na + -k + -atp-ase and mg ++ -atp-ase enzymes. chemical reactions taking place in the living organism are catalyzed by enzymes, so the recombination from adp to atp, too. this transport process can be modelled from the energetic point of view on the basis of the general transport theorem through the following formula: grad a x dx dt: from the point of view of muscle contraction it is of interest that, dependent from the type of the motions whether the length of time is very short, some seconds, or we can speak about a long lasting process. in the first case one can compare the decomposition of the atp with the avalanche effect while in the spot. its degree of efficiency is determined by the migration and linkage velocity of the ions. conclusion: the degree of efficiency of the muscle contraction is determined by the quantities of the two enzymes (na + -k + -atp-ase and mg ++ -atp-ase) as related to each other. [1] . experiments with deletion mutants have shown that the aminoterminal domain contains a beta sheet with an ordered array of acidic residues, which mediates the attachment to basic calcium phosphates [2, 3] . the inhibition is based on the formation of nanometer-sized, spherical mineral-fetuin-a colloids, denoted as calciprotein particles (cpps) [2, 4] . the initially formed cpps show hydrodynamic radii in the range of 50 nm and are only transiently stable. after a distinct lag time, they are subject to a morphological change towards larger prolate ellipsoids with hydrodynamic radii of 100-120 nm [5] . in this context, we studied the role of fetuin-a in the formation and ripening of cpps. on the one hand, dynamic light scattering (dls) was used to study the influence of temperature, fetuin-a concentration and mineral ion supersaturation on the kinetics of cpp ripening [6] . on the other hand, the protein fetuin-a was investigated by means of small angle x-ray scattering (saxs) and fluorescence correlation spectroscopy (fcs degradation of the mrna cap (mrna 5' end) by dcps (decapping scavenger) enzyme is an important process of the gene expression regulation, but little is known about its mechanism. the biological role of dcps and its potential therapeutic applications, e.g. as a novel therapeutic target for spinal muscular atrophy, make it an interesting object for biophysical investigations. the ability of dcps to act on various short capped mrnas will be presented. we have examined the substrate specificity and binding affinity of the enzyme in a quantitative manner, employing experimental physics' resources, such as atomic force microscopy (afm) and fluorescence spectroscopy for enzyme kinetics and timesynchronized-titration method (tst 2007) . in this study we extended the application of mqd-ihc to investigate potential biomarkers associated with prostate cancer (pca) invasiveness and lethal progression. objectives: to establish a mqd-ihc protocol using qd light-emitting nanoparticles 1) to detect the expression/activation of critical cell signaling proteins at the single cell level; 2) to image the plasticity and lethal progression of human pca with specific emphasis on emt and c-met signaling; and 3) to examine the utility of mqd-ihc in clinical pca specimens to determine its invasion ability and predict its metastatic capability. results: we analyzed the co-expression and activation of osteomimicry associated biomarkers: b2-microlgobulin (b2-m), phosphorylated cyclic amp responsive element binding protein (pcreb) and androgen receptor (ar) in 2,100 cells from 14 localized pca tissue areas (gleason 3 and 4) of 10 patients with known metastatic status. the overall median % triple positive for b2-m + /pcreb + /ar + cells was 51.5%. the median triple positive for the samples with metastatic potential was 61% compared with those without metastatic potential (median = 0%); p = 0.01 by a wilcoxon rank sum test. the results were confirmed in 11 pca bone metastatic specimens. we also investigated the c-met signaling in castration-resistant human pca model or crpc xenografts and the clinical pca specimens and found that the downstream signal components including pakt and mcl-1 were activated. conclusion: to validate our findings, additional clinical specimens with confirmed survival data will be analyzed and the cell-signaling-network-based mqd-ihc will be automated by vectra image analysis system in a high throughput manner with the hope to predict the lethal progression of pca prior to clinical manifestation of distant metastases. protein ligand binding is an important field of biopharmaceutical research. there are many techniques for quantitative determination of the ligand binding. the combination of isothermal titration calorimetry (itc) and thermal shift assay provides a robust estimate of the binding constant. many binding reactions are coupled to the absorption or release of protons by the protein or the ligand, conformational changes of the protein and other processes. to correlate the structural features of binding with the energetics of binding one needs to carry out a detailed thermodynamic study of the binding reaction and to determine dependencies such as ph, buffer and temperature. here we present a detailed thermodynamic description of radicicol binding to human heat shock protein hsp90 and determined proton linkage contributions to observed binding thermodynamics. we calculated the pk a of the group responsible for proton linkage, the protonation enthalpy of this group and intrinsic thermodynamic parameters for radicicol binding. the intrinsic enthalpy of radicicol binding to hsp90 is one of the largest enthalpies observed for any protein -ligand binding. the structural features responsible for such large binding enthalpy and very favorable intrinsic binding gibbs free energy are discussed. neuronal systems and modelling o-111 optogenetic electrophysiology walther akemann, amelie perron, hiroki mutoh, and thomas knö pfel laboratory for neuronal circuit dynamics, riken brain science institute, japan the combination of optical imaging methods with targeted expression of protein-based fluorescent probes enables the functional analysis of selected cell populations within intact neuronal circuitries. we previously demonstrated optogenetic monitoring of electrical activity in isolated cells, brain slices and living animals using voltage-sensitive fluorescent proteins (vsfps) generated by fusing fluorescent proteins with a membrane-integrated voltage sensor domain. however, several properties of these voltage reporters remained suboptimal, limiting the spatiotemporal resolution of vsfpbased voltage imaging. a major limitation of vsfps had been a reduced signal-to-noise ratio arising from intracellular aggregation and poor membrane targeting upon long-term expression in vivo. to address this limitation, we generated a series of enhanced genetically-encoded sensors for membrane voltage (named vsfp-butterflies) based on a novel molecular design that combines the advantageous features of vsfp2s and vsfp3s with molecular trafficking strategies. the new sensors exhibit faster response kinetics at subthreshold membrane potentials and enhanced localization to neuronal plasma membranes after long-term expression in vivo, enabling the optical recording of action potentials from individual neurons in single sweeps. vsfp-butterflies provide optical readouts of population activity such as sensoryevoked responses and neocortical slow-wave oscillations with signal amplitudes exceeding 1% dr/r 0 in anesthetized mice. vsfp-butterflies will empower optogenetic electrophysiology by enabling new type of experiments bridging cellular and systems neuroscience and illuminating the function of neural circuits across multiple scales. opsin molecules are a burgeoning new tool for temporally precise neuronal stimulation or inhibition. opsin properties are commonly characterized in cell culture or acute brain slice preparations using whole cell patch clamp techniques, where neuronal membrane voltage is fixed at the resting potential. however, in vivo, where neurons are firing action potentials, opsins are exposed to large fluctuations in membrane voltage and transmembrane ionic concentrations which can influence opsin function. in the case of implanted light delivery devices, stimulation light power varies as a function of brain tissue volume. we therefore investigated the stability of opsin properties across a variety of in vivo-like stimulation conditions. we find that off-kinetics of excitatory opsins vary significantly with holding membrane potential; channelrhodopsin (chr2) slowing with depolarisation and chief (chr1/chr2 hybrid) in contrast, accelerating. new chr2 point mutation variants demonstrate stability across all membrane potentials. we additionally explore responses to initial and subsequent light pulses and find that chief has the unique property of accelerating kinetics after the first light stimulation. inhibitory opsins vary in sensitivity to light in a manner which correlates with their off-kinetics. slower opsins, such as mac (leptosphaeria maculans), have higher sensitivity at low light power densities, saturating early relative to fast inhibitory opsins such as arch (archaeorhodopsin) and nphr (halorhodopsin). we discuss the relative merits of stability versus versatility of opsins under variable stimulation conditions. it has been previously shown that overexpression of ndm29 ncrna in a sknbe2-derived neuroblastoma (nb) cell line leads to cell differentiation, with a decrease of malignant potential. here we use the patch-recording technique to characterize the ionic channel apparatus of nb cells expressing ndm29 at its basal level (mock cells) or at 5.4 fold higher levels (s1 cells). the two cell lines shared very similar pools of functional k channels, but s1 cells displayed larger ttx-sensitive na currents and were able to generate action potentials, while mock cells were not. in addition, while mock cells barely express functional gaba receptors, in the majority of s1 rapid application of gaba elicited a current with a ec 50 =11.4 lm; this current was antagonized by bicuculline (10 lm) and potentiated by zaleplon (ec 50 = 35 nm). in mock cells, real time pcr evidenced a high level of gaba a a 3 subunit, while in s1 cells a significant expression of a 1 and a 4 was detected, whereas a 3 mrna was downregulated by 70%, confirming the development of functional gaba a receptors. in the same cell lines, the presence of specific markers and the secretion of specific cytokines confirmed that ndm29 expression leads to a differentiation process toward a neuron-like, rather than glial-like, phenotype. was planned therefore to reconstitute a model of brain tumors in rats by orthotopic implantation of xenogenic transformed human cells. iron is important element used for chemical reaction catalysis and physiological cell functions. the reason of iron deposition is still unknown. under conditions prevailing in human brain it is expected the formation of an amorphous or minute crystalline phase. we used light, scanning (sem) and transmission electron microscopy (tem), energy-dispersive microanalysis, electron diffraction and electron paramagnetic resonance (epr) for investigation of iron deposits in globus pallidus of human brain. sem revealed iron rich particles with na, si, p, s, cl, ca and cu around glial cells. tem revealed bumpy, solid particles of platy and sometimes rounded shape with the size of 2 lm to 6 lm. these ones were identified as hematite. epr measurements showed the presence of fe(iii) and cu(ii), but little amount of fe(ii) can not be excluded. we consider low-temperature process of hematite formation in human globus pallidus in aqueous environment influenced by organic and inorganic factors. chemical processes leading to nanoparticles formation can be associated with neurodegenerations such as alzheimer or parkinson disease. over the past 50 years our understanding of the basic biophysical mechanisms governing the spatio-temporal dynamics of neuronal membrane potentials and synaptic efficacies has significantly expanded and improved. much research has focussed on how ionic currents contribute to the generation and propagation of action potentials, how subthreshold signals propagate along dendritic trees, how the active properties of dendrites shape the integration of incoming signals in a neuron, and how pre-and postsynaptic activities â and potential heterosynaptic effects a determine the way synaptic efficacies change on the short-and long-term. yet, despite these advances, there have been no systematic efforts to relate the basic dynamical repertoire of neurons to the computational challenges neural circuits face, and in particular to explain systematically how the biophysical properties of neurons are adapted to process information efficiently under the constraints of noise and uncertainty in the nervous system. as an initial step in this direction, i will show how various biophysical properties of neurons, in particular short-term synaptic plasticity and dendritic non-linearities, can be seen as adaptations to resolve an important bottleneck in neuronal information processing: the loss of information entailed by the conversion of analogue membrane potentials to digital spike trains. the optogenetic toolbox has greatly expanded since the first demonstration of genetically-targeted optical manipulation of neural activity. in addition to the cation channel channelrhodopsin-2 (chr2), the panel of excitatory opsins now includes an array of chr2 variants with mutations in critical residues, in addition to other, related cation channels, and channel hybrids. the inhibitory opsin panel has similarly expanded beyond the first-described halorhodopsin (nphr), a chloride pump, to include trafficking-enhanced versions of nphr as well as the proton pumps mac and arch. while the expansion of available opsins offers researchers an increasingly powerful and diverse selection of tools, it has also made it increasingly difficult to select the optimal tool for a given experiment. one cannot extract a comparison of opsins from the current literature, since studies differ across multiple variables known to contribute to opsin performance (e.g. expression method, light power density, stimulation protocols, etc.). here, we provide the first empirical comparison of both excitatory and inhibitory opsins under standardized conditions. furthermore, we identify the set of parameters that describe the properties of an opsin in a way that is maximally relevant for biological application. o-120 subcellular compartment-specific distribution of voltage-gated ion channels zoltan nusser institute of experimental medicine, hungarian academy of sciences, budapest, hungary voltage-gated na + (nav) channels are essential for generating the output signal of nerve cells, the action potential (ap). in most nerve cells, aps are initiated in the axon initial segment (ais). in vitro electrophysiological and imaging studies have demonstrated that dendritic nav channels support active backpropagation of aps into the dendrites, but the subunit composition of these channels remained elusive. here, i will present evidence for the somato-dendritic location of nav channels in hippocampal pyramidal cells (pcs). using a highly sensitive electron microscopic immunogold localization technique, we revealed the presence of the nav1.6 subunit in pc proximal and distal dendrites, where their density is 40-fold lower than that found in aiss. a gradual decrease in nav1.6 density along the proximo-distal axis of the dendritic tree was also detected. we have also investigated the subcellular distribution of kv4.2 voltage-gated k + channel subunit and found a somato-dendritic localization. in contrast to that of nav1.6 channels, the density of kv4.2 first increases then decreases as a function of distance from the somata of pcs. such subcellular compartment-specific distribution of voltage-gated ion channels increases the computational power of nerve cells. keywords: memory, extra cellular matrix, random walk we expose first a biological model of memory based on one hand of the mechanical oscillations of axons during action potential and on the other hand on the changes in the extra cellular matrix composition when a mechanical strain is applied on it. due to these changes, the stiffness of the extra cellular matrix along the most excited neurons will increase close to these neurons due to the growth of astrocytes around them and to the elastoplastic behavior of collagen. this will create preferential paths linked to a memory effect. in a second part, we expose a physical model based on random walk of the action potential on the array composed of dendrites and axons. this last model shows that repetition of the same event leads to long time memory of this event and that paradoxical sleep leads to the linking of different events put into memory. myelinated nerve fibres were studied with fluorescent microscopy and laser interference microscopy. ca 2+ redistribution during prolonged stimulation, changes in morphology and rearrangement of cytoplasmic structures were compared in normal conditions and after membrane modification by lysolecithin and methyl-b-cyclodextrin. lysolecithin is a detergent known to provoke demyelination, and methyl-b-cyclodextrin extracts cholesterol from membranes. cholesterol extraction could lead to disruption of membrane caveolae-like microdomains or ''rafts'' and solubilisation of different proteins connected to them. our data suggest that methyl-b-cyclodextrin and lysolecithin lead to different changes in morphology and distribution of cytoplasmic structures. the effect was different for different regions of the nerve (node of ranvier, paranodal and internodal regions). the agents also altered the kinetics of ca 2+ response to stimulation in myelinated fibres. extracellular carbonic anhydrase contributes to the regulation of ca2+ homeostasis and salivation in submandibular salivary gland nataliya fedirko, olga kopach, nana, voitenko lviv national university, human and animal physiology, lviv, ukraine the maintenance of ph in the oral cavity is important for the oral heath since even a minor drop in ph can result in dental caries and damage to the teeth. submandibular salivary gland (smg) is main source of fluid and electrolytes enriched saliva therefore its core for oral ph homeostasis. smg secretion is activated by acetylcholine (ach) in [ca 2+ ] idependend manner and accompanied with oral ph acidic shifts. ph shifts could be due to changes in buffering capacity that is regulated by carbonic anhydrase (ca). despite the expression of different subtypes of ca in smg the role of ca in the regulation of smg function is unclear yet. we found that ca inhibition by benzolamide (bz) decreased of fluid secretion in vivo extracellular na 2+ concentration in situ. the latter confirm the ability of ca to modify both primarily and final saliva secretion. we also found correlation between the secretion and ca 2+ -homeostasis since bz-induced decrease of: in striated muscle ca 2+ release from the sarcoplasmic reticulum (sr) occurs when ryanodine receptors (ryr-s) open either spontaneously or upon the stimulation from dihydropyridine receptors that are located in the adjacent transverse-tubular membrane and change their conformation when the cell is depolarized. recent observations demonstrated that muscles from animal models of ptdinsp phosphatase deficiency suffer from altered ca 2+ homeostasis and excitation-contraction coupling, raising the possibility that ptdinsp-s could modulate voltage-activated sr ca 2+ release in mammalian muscle. the openings of a single or a cluster of ryr-s can be detected as ca 2+ release events on images recorded from fibres loaded with fluorescent ca 2+ indicators. to elucidate the effects of ptdinsp-s on ca 2+ release events, images were recorded from skeletal muscle fibers enzymatically isolated from the m. flexor digitorum breavis of mice utilizing a super-fast scanning technique. a wavelet-based detection method was used to automatically identify the events on the images. three different ptdinsp-s (ptdins3p, ptdins5p, and ptdins(3,5)p) were tested. all these ptdinsp compounds decreased the frequency of spontaneous ca 2+ release events. supported by the hungarian national science fund (otka 75604), té t. calcium sparks elicited by 1 mmol/l caffeine and by a depolarization to -60 mv were recorded at high time resolution on both x-y (30 frames/s) and line-scan images (65 lines/ ms) on intact skeletal muscle fibers of the frog. while a typical spark appeared in one frame only, 17.3 and 26.0% of spark positions overlapped on consecutive frames following caffeine treatment or depolarization, respectively. while both caffeine and depolarization increased the frequency of sparks, as estimated from x-y images, the morphology of sparks was different under the two conditions. both the amplitude (in df/f 0 ; 0.49 ± 0.025 vs. 0.29 ± 0.001; n = 22426 vs. 23714; mean ± sem, p \ 0.05) and the full width at half maximum (in lm; parallel with fiber axis: 2.33 ± 0.002 vs. 2.21 ± 0.005; perpendicular to fiber axis: 2.07 ± 0.003 vs. 1.88 ± 0.004) of sparks was significantly greater after caffeine treatment than on depolarized cells. these observations were confirmed on sparks identified in line-scan images. in addition, x-t images were used to analyze the time course of these events. calcium sparks had significantly slower rising phase under both conditions as compared to the control. on the other hand, while the rate of rise of signal mass was decreased after depolarization, it increased in the presence of caffeine. prolonged depolarisation of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. skeletal muscle excitation-coupled calcium entry relies on the interaction between the 1,4 dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. in this study we exploited tirf microscopy to monitor with high spatial resolution excitationcoupled calcium entry (ecce) in primary coltures of human skeletal muscle cells harbouring mutation in the ryr1 gene linked to malignant hyperthermia and central core disease. we found that excitation-coupled calcium entry is strongly enhanced in cells from patients with central core disease compared to individuals with malignant hyperthermia and controls. in addition, excitation-coupled calcium entry induces generation of reactive nitrogen species and causes the nuclear translocation of nfatc1. the activation of nfatc1 dependent genes is consistent with an increase of the il6 secretion from primary coltures human myotubes from ccd patients and with fibre type 1 predominance of skeletal muscle of ccd patients. membrane lipids, microdomains & signalling p-132 ftir and calorimetric investigation of the effects of trehalose and multivalent cations on lipid structure sawsan abu sharkh, jana oertel, and karim fahmy division of biophysics, institute of radiochemistry, helmholtz-zentrum dresden-rossendorf, germany e-mail: s.sharkh@hzdr.de the structure of membrane lipids is of fundamental importance for the integrity of cell and organelle membranes in living organisms. membrane lipids are typically hydrated and their headgroup charges counter-balanced by solvated ions. consequently, water loss can induce severe structural changes in lipid packing (lyotropic transitions) and can lead to the damage of lipid membranes even after rehydration. this can be one out of several factors that affect the viability of organisms undergoing desiccation. many organisms, however, are resistant to even extreme water loss. some of them synthesize trehalose which has been shown to be associated with survival of desiccation in phylogenetically diverse organisms (yeast, nematodes, brine shrimp, insect larvae, resurrection plants, and others). here we have studied hydration sensitive transitions in model lipids to determine the effect of trehalose and electrostatics on lipid order. hydration pulse-induced time-resolved fourier-transform infrared (ftir) difference spectroscopy was used to address hydration-dependent lipid structure as a function of trehalose. in combination with differential scanning calorimetry and studies of langmuir-blodget films we arrive at a structural and energetically consistent picture of how trehalose can affects lipidic phase behaviour and support a native lipid structure under water loss. experiments were performed on model lipids with different headgroups and native lipids from desiccation-tolerant organisms. controlled self-assembly and membrane organization of lipophilic nucleosides and nucleic acids: perspectives for applications martin loew 1 , paula pescador 3 , matthias schade 1 , julian appelfeller 1 , jü rgen liebscher 2 , oliver seitz 2 , daniel huster 3 , andreas herrmann 1 , and anna arbuzova 1 1 humboldt universitä t zu berlin, institute of biology/ biophysics, berlin, germany, 2 humboldt universitä t zu berlin, institute of chemistry, berlin, germany, 3 universitä t leipzig, department of medical physics and biophysics, leipzig, germany lipophilic conjugates of nucleosides and nucleic acids such as dna, rna, and peptide nucleic acid (pna) -combining assembly properties of amphiphiles and specific molecular recognition properties of nucleic acids -allow numerous applications in medicine and biotechnology. we recently observed self-assembly of microtubes, stable cylindrical structures with outer diameters of 300 nm and 2-3 lm and a length of 20-40 lm, from a cholesterol-modified nucleoside and phospholipids. morphology and properties of these microtubes and functionalization with lipophilic dna will be characterized. we also observed that lipophilic nucleic acids, pna and dna differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes as visualized by hybridization with respective complementary fluorescently-labeled dna strands. upon heating, domains vanished and both lipophilic nucleic acid structures intermixed with each other. reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. by linking specific functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces. conformational dependent trafficking of p-glycoprotein with rafts zsuzsanna gutayné tó th, orsolya bá rsony, katalin goda, gá bor szabó and zsolt bacsó university of debrecen, mhsc, department of biophysics and cell biology, debrecen, hungary p-glycoprotein (pgp), an abc-transporter playing a prominent role in multidrug resistance, demonstrate conformationdependent endocytosis on the surface of 3t3-mdr1 cells. these cell surface transporters have a uic2 conformationsensitive-antibody-recognizable subpopulation, which is about one-third of the rest persisting long on the cell surface and perform fast internalization via rafts. we have identified that the rapid internalization is followed by quick exocytosis, in which the other subpopulation is not or only slightly involved. the exocytosis presents a cholesterol depletion dependent intensification, in contrast to the internalization, which is inhibited by cyclodextrin treatment. this continuous recycling examined by total internal reflection (tirf) microscopy increases the amount of the raft associated subpopulation of pgps in the plasma membrane, and it might have a role in restoring the cholesterol content of the membrane after cholesterol depletion. our presentation will summarize related endocytotic, exocytotic and recycling processes and that how does our data fit into our current notions regarding to the cholesterol and sphingomyelin trafficking. membrane nanodomains based on phase-segregating lipid mixtures have been emerged as a key organizing principle of the plasma membrane. they have been shown to play important roles in signal transduction and membrane trafficking. we have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-nta) head groups for selective targeting of histagged proteins into liquid ordered or liquid disordered phases. in giant unilamellar vesicles strong partitioning of tris-nta lipids into different lipid phases was observed. for a saturated tris-nta lipid, at least 10-fold preference for the liquid ordered phase was found. in contrast, an unsaturated nta lipid shows a comparable preference for the liquid disordered phase. partitioning into submicroscopic membrane domains formed in solid supported membranes was confirmed by superresolution imaging. single molecule tracking of his-tagged proteins tethered to solid supported membranes revealed clear differences in the diffusion behavior of the different nta-lipids. by using bsa as a carrier, multivalent nta lipids were efficiently incorporated into the plasma membrane of live cells. based on this approach, we established versatile methods for probing and manipulating the spatiotemporal dynamics of membrane nano domains in live cells. il-9 is a multifunctional cytokine with pleiotropic effects on t cells. the il-9r consists of the cytokine-specific a-subunit and the c c -chain shared with other cytokines, including il-2 and -15, important regulators of t cells. we have previously shown the preassembly of the heterotrimeric il-2 and il-15r, as well as their participation in common superclusters with mhc glycoproteins in lipid rafts of human t lymphoma cells. integrity of lipid rafts was shown to be important in il-2 signaling. we could hypothesize that other members of the c c cytokine receptor family, such as the il-9r complex, may also fulfill their tasks in a similar environment, maybe in the same superclusters. co-localization of il-9r with lipid rafts as well as with the il-2r/mhc superclusters was determined by clsm. molecular scale interactions of il-9ra with il-2r and mhc molecules were determined by microscopic and flow cytometric fret experiments. the role of lipid rafts in il-9r signaling was assessed by following the effect of cholesterol depletion on il-9 induced stat phosphorylation. our results suggest the possibility that preassembly of the receptor complexes in common membrane microdomains with mhc glycoproteins may be a general property of c c cytokines in t cells. to unravel the molecular processes leading to fas clustering in lipid rafts, a 21-mer peptide corresponding to the single transmembrane domain of the death receptor was reconstituted into model membranes that display liquid-disordered/ liquid-ordered phase coexistence, i.e. mimicking cells plasma membranes. using the intrinsic fluorescence of the peptide two tryptophans residues (trp 176 and trp 189 ), fas membrane lateral organization, conformation and dynamics was studied by steady-state and time-resolved fluorescence techniques. our results show that the fas has preferential localization to liquid disordered membrane regions, and that it undergoes a conformational change from a bilayer inserted state in liquid-disordered membranes to an interfacial state in liquidordered membranes. this is a result of the strong hydrophobic mismatch between the (hydrophobic) peptide length and the hydrophobic thickness of liquid-ordered membranes. in addition, we show that ceramide, a sphingolipid intimately involved in fas oligomerization and apoptosis triggering, does not affect fas membrane organization. overall, our results highlight ceramide role as an enhancer of fas oligomerization, and unravel the protective function of fas transmembrane domain against non-ligand induced fas apoptosis. organization and dynamics of membrane-bound bovine a-lactalbumin: a fluorescence approach arunima chaudhuri and amitabha chattopadhyay centre for cellular and molecular biology, hyderabad 500 007, india e-mail: amit@ccmb.res.in many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. interestingly, apo-bovine alactalbumin (bla), a soluble protein, specifically interacts with negatively charged membranes and the membranebound protein exhibits a molten globule conformation. we have used the wavelength-selective fluorescence approach to monitor the molten globule conformation of bla upon binding to negatively charged membranes as compared to zwitterionic membranes. tryptophans in bla exhibit differential red edge excitation shift (rees) upon binding to negatively charged and zwitterionic membranes, implying differential rates of solvent relaxation around the tryptophan residues. our results utilizing fluorescence anisotropy, lifetime and depth analysis by the parallax approach of the tryptophans further support the differential organization and dynamics of the membrane-bound bla forms. in addition, dipole potential measurements and dye leakage assays are being used in our ongoing experiments to explore the mechanism of bla binding to membranes. these results assume significance in the light of antimicrobial and tumoricidal functions of a-lactalbumin. role of long-range effective protein-protein forces in the formation and stability of membrane protein nano-domains nicolas destainvill laboratoire de physique thé orique, université paul sabatier toulouse 3 -cnrs, toulouse, france we discuss a realistic scenario, accounting for the existence of sub-micro-metric protein domains in plasma membranes. we propose that proteins embedded in bio-membranes can spontaneously self-organize into stable small clusters, due to the competition between short-range attractive and intermediate-range repulsive forces between proteins, specific to these systems. in addition, membrane domains are supposedly specialized, in order to perform a determined biological task, in the sense that they gather one or a few protein species out of the hundreds of different ones that a cell membrane may contain. by analyzing the balance between mixing entropy and protein affinities, we propose that protein sorting in distinct domains, leading to domain specialization, can be explained without appealing to preexisting lipidic micro-phase separations, as in the lipid raft scenario. we show that the proposed scenario is compatible with known physical interactions between membrane proteins, even if thousands of different species coexist. lipid rafts are cholesterol and sphingolipid-enriched functional microdomains present in biomembranes. rafts have been operationally defined as membrane fractions that are detergent insoluble at low temperature. here we have characterized drms from erythrocytes treated with the nonionic detergents brij58 and brij98, at 4°c and 37°c, and compared them to drms obtained with triton x-100 (tx100). we have also investigated the effect of cholesterol depletion in drms formation. brij drms were enriched in cholesterol as well as tx100 drms. hptlc analysis showed a very similar distribution of phosphatidylcholine-pc, phosphatidylethanolamine-pe and sphingomyelin-sm in brij drms to that found in ghost membranes. sm-enriched drms were obtained only with tx100 while pe content was decreased in tx100 drms, in comparison to brij drms. immunoblot essays revealed that rafts markers (flotillin-2 and stomatin) were present in all drms. contrary to tx100 drms, analysis of electron paramagnetic resonance spectra (with 5-doxyl stearate spin label) revealed that brij drms are not in the liquid-ordered state, evincing the differential extraction of membrane lipids promoted by these detergents. supported by fapesp/cnpq (brazil). several biological membrane mimics have been built to investigate the topology of molecules in membranes. among them ''bicelles'', i.e., mixtures of long-chain and short-chain saturated phospholipids hydrated up to 98%, became very popular because they orient spontaneously in magnetic fields. disk-shaped systems of 40-80 nm diameter and 4-5 nm thickness have been measured by electron microscopy and solid state nmr and can be oriented by magnetic fields with the disc-plane normal perpendicular to the field. we have been developing recently lipids that contain in one of their chains a biphenyl group (tbbpc) affording an orientation parallel to the magnetic field, in the absence of lanthanides. a large number of hydrophobic molecules including membrane proteins have been successfully embedded and static nmr afforded finding the orientation of protein helices in membranes; mas nmr provided the 3d structure of peptides in bicelles. biphenyl bicelles keep their macroscopic orientation for days outside the field, thus leading to combined nmr and x-rays experiments. tbbpc allows also construction of lm vesicles showing a remarkable oblate deformation in magnetic fields (anisotropy of 3-10) and opens the way to applications for structural biology or drug delivery under mri. imaging membrane heterogeneities and domains by super-resolution sted nanoscopy christian eggeling, veronika mueller, alf honigmann, stefan w. hell department of nanobiophotonics, max planck institute for biophysical chemistry, am fassberg 11, 37077 gö ttingen, germany cholesterol-assisted lipid and protein interactions such as the integration into lipid nanodomains are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of [200nm of a conventional far-field optical microscope. we report the detection of the membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (sted) far-field nanoscopy. by combining a (tunable) resolution of down to 30 nm with tools such as fluorescence correlation spectroscopy (fcs), we obtain new details of molecular membrane dynamics. sphingolipids or other proteins are transiently (* 10 ms) trapped on the nanoscale in cholesterol-mediated molecular complexes, while others diffuse freely or show a kind of hopping diffusion. the results are compared to sted experiments on model membranes, which highlight potential influences of the fluorescent tag. the novel observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity. ca2+ controlled all-or-none like recruitment of synaptotagmin-1 c2ab to membranes sune m. christensen, nicky ehrlich, dimitrios stamou bio-nanotechnology laboratory, department of neuroscience and pharmacology, nano-science center, lundbeck foundation center biomembranes in nanomedicine, university of copenhagen, 2100 copenhagen, denmark e-mail: ehrlich@nano.ku.dk & stamou@nano.ku.dk synaptotagmin-1 (syt) is the major ca2+ sensor that triggers the fast, synchronous fusion of synaptic vesicle with the presynaptic membrane upon ca2+-mediated membrane recruitment of the cytosolic c2ab domain. the ca2+-dependent recruitment of syts c2ab domain to membranes has so far been investigated by ensemble assays. here we revisited binding of wild type c2ab and different c2ab mutants of syt to lipid membranes using a recently developed single vesicle assay. the hallmark of the single vesicle approach is that it provides unique information on heterogeneous properties that would otherwise be hidden due to ensemble averaging. we found that c2ab does not bind to all vesicles in a homogenous manner, but in an all-or-none like fashion to a fraction of the vesicles. the fraction of vesicles with bound c2ab is regulated by the amount of negatively charged lipids in the membrane as well as by [ca2+] . this ca2+ controlled all-ornone like recruitment of syt to membranes provides a possible explanation for the strongly heterogeneous behavior of the in vitro model system for neuronal membrane fusion. furthermore, heterogeneity in release probability among synaptic vesicles is a critical property in determining the output of a neuronal signaling event. new insights in the transport mechanism of ciprofloxacin revealed by fluorescence spectroscopy mariana ferreira, sílvia c. lopes, paula gameiro requimte, faculdade de ciê ncias da universidade do porto, porto, portugal keywords: fluoroquinolones; liposomes; proteoliposomes; ompf; ciprofloxacin; fluorescence; anisotropy. fluoroquinolones are antibiotics that have a large spectrum of action against gram negative and some gram positive bacteria. the interaction between these species and liposomes has been cited as a reference in the understanding of their diffusion through phospholipid bilayer and can be quantified by the determination of partition coefficients between a hydrophobic phase (liposomes) and an aqueous solution. it is also known that some porins of the bacterial membranes are involved in transport mechanism of many fluoroquinolones. ompf, a well characterized membrane protein characteristic of the outer membrane of gram negative bacteria assumes the conformation of homo-trimer, whose monomers have two tryptophan residues (one located at the interface of monomers and the other at the interface lipid/protein). thus, we proceeded to study the interaction of ciprofloxacin, a second generation fluoroquinolone, with unilamellar liposomal vesicles and ompf proteoliposomes of pope/popg, pope/popg/cardiolipin and e. coli total. partition coefficients (kp's) and the association with ompf proteoliposomes were determined by steady state fluorescence spectroscopy under physiological conditions (t=37°c; ph 7.4). the membrane mimetic systems used were characterized by dls and fluorescence anisotropy. motivation is whether there exist differences in the patternforming capabilities of two adhesion molecules of different roles: cd44, mediating ,,dynamic'' adhesion in cell rolling and icam-1, mediating ,,static'' adhesion during the formation of immune-synapse. homo-and hetero-associations of cd44, icam-1 and the mhci is investigated on the nm-and lmdistance levels on ls174t colon carcinoma cells in two different conditions of lymphocyte homing: (1) with ifnc and tnfa, both lymphokines up-regulating the expression level of mhci and icam-1 and down-regulating that of cd44. (2) crosslinking of cd44 and icam-1 representing receptor engagement. the observations are explained by assuming the existence of a kinase cascade-level crosstalk between the cd44 and icam-1 molecules which manifests in characteristic complementary changes in the properties of cell surface receptor patterns. for the characterisation of cluster morphology new colocalization approaches were developed: (i) ,,number of first neighbours'' distribution curves, (ii) ,,acceptor photobleaching fret-fluorescence intensity fluctuation product'' correlation diagrams, and (iii) ,,random gradient-kernel smoothing assisted decay'' of pearson-correlations, and (iv) k-function formalism. analyzing janus kinases in living cells with single-color fcs using confocal and tir-illumination thomas weidemann 1 , hetvi gandhi 1 , remigiusz worch 1,3 , robert weinmeister 1 , christian bö kel 2 , and petra schwille 1 1 biophysics research group, technische universitä t dresden, germany, 2 crtd, center for regenerative therapies dresden, technische universitä t dresden, germany, 3 institute of physics, polish academy of science, warsaw, poland cytokine receptors of the hematopoietic superfamily transduce their signal through non-covalently bound janus kinases. there are only 4 such kinases in humans (jak1, 2, 3 and tyk2), which associate to 46 different cytokine receptor chains. here we study the dynamics of gfp-tagged jak1 and jak3 in epithelial cells with fluorescence correlation spectroscopy (fcs). jak1 and jak3 behave differently in various aspects: in the absence of receptors, jak1 still binds the membrane, whereas jak3 diffuses homogeneously in the cytoplasm. we used fcs under total internal reflection illumination (tir-fcs) and determined the membrane binding affinity of jak1 to be 60±36 nm. the association of jak3 with the common gamma chain (c c ) is very tight as shown by fluorescence recovery after photobleaching (frap). molecular brightness analysis of single-point fcs shows that jak1 diffuses as a monomer in the rather small cytoplasmic pool, whereas jak3 diffuses as dimers, which undergo a defined oligomerization. the degree of oligomerization decays at higher concentrations, indicating that some unknown, saturable scaffold is involved. characterizing the binding and mobility schemes of the janus kinases may be important to further elucidate their specific and redundant effects in signal transduction. plasma membrane (pm)-enriched fraction obtained through subcellular fractioning protocols are commonly used in studies investigating the ability of a compound to bind to a receptor. however, the presence of mitochondria membranes (mi) in the pm-enriched fraction may compromise several experimental results because mi may also contain the interest binding proteins. aiming to analyze the subcellular fractioning quality of a standard sucrose density based protocol, we investigated (a) the na + k + -atpase (pm marker) and succinate dehydrogenase -sd (mi marker) activities; (b) the immunocontent of the adenine nucleotide translocator (ant -mi membrane marker) in both pm-and mi-enriched fractions. since several binding protocols may require long incubation period, we verified the quality of both fractions after 24 hours of incubation in adequate buffer. our results show that pmand mi-enriched fractions exhibit contamination with mi or pm, respectively. we did not observe any effect of incubation on na + k + -atpase activity and ant content in both fractions. surprisingly, sd activity was preserved in the pm-but not in mi-enriched fraction after incubation. these data suggest the need of more careful use of pm-enriched fraction preparation in studies involving pm proteins characterization. human neutrophil peptide 1 (hnp1) is a human cationic defensin that present microbial activity against various bacteria (both gram-positive and negative), fungi and viruses. hnp1 is stored in the cytoplasmic azurophilic granules of neutrophils and epithelial cells. in order to elucidate the mode of action of this antimicrobial peptide (amp), studies based on its lipid selectivity were carried out. large unilamellar vesicles (luv) with different lipid compositions were used as biomembranes model systems (mammal, fungal and bacterial models). changes on the intrinsic fluorescence of the tryptophan residues present in hnp1 upon membrane binding/insertion were followed, showing that hnp1 have quite distinct preferences for mammalian and fungal membrane model systems. hnp1 showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a high partition for ergosterol-containing membranes (as fungal membranes) and low interaction with cholesterolcontaining membranes (as in mammalian cells). these results reveal that lipid selectivity is the first step after interaction with the membrane. further insights on the hnp1 membrane interaction process were given by fluorescence quenching measurements using acrylamide, 5-doxylstearic acid (5ns) or (16ns). nanoparticles (np) are currently used in many industrial or research applications (paints, cosmetics, drug delivery materials…). recent papers demonstrate clearly their activity with biological membranes (nanoscale holes, membrane thinning, disruption). different studies of the np-membrane interaction suggest that parameters are particularly important, such as the np size, their surface properties or their aggregation state. composition of biological membranes being particularly complex, supported lipid bilayers (slb) composed of a restricted number of lipids are usually used as simplified membrane model. moreover, these two-dimensional systems are convenient for surface analysis techniques, such as atomic force microscopy (afm), giving information on the morphology of the slb and its mechanical properties. in this work, we study the behaviour of slbs made of lipids representative of the membrane fluid phase (popc) or of the raft phase (sphingomyelin). these slbs are deposited on planar surfaces (mica or glass) previously recovered with silica beads (10 or 100 nm in diameter) in order to mimick the np-membrane interaction. we will present in this work our first results obtained by afm and fluorescence microscopy. it is well known that the eukaryotic nuclei are the sphere of lipids active metabolism. the investigations demonstrated the existence of numerous enzymes in nuclei which modulate the changes of nuclear lipids during different cellular processes. although the nuclear membrane is accepted as the main place of the lipids localization, nearly 10% of nuclear lipids are discovered in chromatin fraction. the ability of chromatin phospolipids to regulate dna replication and transcription was already demonstrated. the chromatin phospolipids seems to play an important role in cell proliferation and differentiation as well as in apoptosis. it seems also possible that chromatin phospholipids may be participated in realization of cisplatin antitumor effects. the 24-hour in vivo effect of cisplatin on rat liver chromatin phospholipids was investigated. the phospholipids of rat liver chromatin were fractionated by microtlc technique. the quantitative estimation of fractionated phospholipids was carried out by computer program fugifilm science lab. 2001 image gauge v 4.0. the alteration of total phospholipids content as well as the quantitative changes among the individual phospholipids fractions in rat liver chromatin after in vivo action of cisplatin was established. the total content of chromatin phospholipids was significantly decreased after the cisplatin action. four from five individual phospholipids fractions were markedly changed after the drug action. two cholin-content phospholipids, particularly phosphatidylcholine and sphingomyelin exhibit diversity in sensitivity to this drug: the increase of sphingomyelin content accompanied by quantitative decrease of phosphatidylcholine. the quantity of cardiolipin was markedly increased while the amount of phosphatidylinositol was decreased after the cisplatin treatment. the phosphatidylethanolamin content remained unchanged after the drug action. it seems that high sensitivity of chromatin phospholipids exhibited to cisplatin action may play an important role in antitumor effects of this drug. membrane lipids and drug resistance in staphylococci r. d. harvey institute of pharmaceutical science, king's college london, 150 stanford street, london se1 9nh, uk staphylococci express numerous resistance mechanisms against common antimicrobials, including peptide components of the innate immune system which have been trumpeted as being likely candidates to replace our increasingly ineffective antibiotics. the membrane phospholipid lysylphosphatidylglycerol (l-pg) appears to play a key role in staphylococcal drug resistance, since its absence in mutant bacteria renders them susceptible to a range of cationic antimicrobials. the current assumption about the role l-pg plays in drug resistance is that of facilitating charge neutralisation of the plasma membrane, leading to loss of affinity towards cationic moieties. we have investigated this phenomenon using a range of model membrane systems composed of both synthetic lipids and reconstituted natural lipid extracts, using such techniques as stopped-flow fluorescence, circular dichroism and neutron scattering. our conclusions indicate that the initial assumptions about the role of l-pg in drug resistance are over-simplistic and certainly do not tell the whole story of the physical and biological properties of this fascinating moderator or membrane behaviour. our findings show that l-pg does not inhibit antimicrobial drug action by charge dampening, hinting at a different protective mechanism. modulation of a-toxin binding by membrane composition m. schwiering, a. brack, c. beck, h. decker, n. hellmann institute for molecular biophysics, university of mainz, mainz, germany although the alpha-toxin from s. aureus was the first poreforming toxin identified, its mode of interaction with membranes is still not fully understood. the toxin forms heptameric pores on cellular and artificial membranes. the present hypothesis is that the initial binding to the membrane occurs with low affinity, and that an efficient oligomerisation, relying on clusters of binding sites, is the reason for the overall high affinity of the binding process. in order to separate the effects of increasing concentration of binding sites from this topological effect, we investigated the oligomer formation based on pyrene-fluorescence for a series of lipid compositions, where the fraction of toxin binding lipids (egg phospatidylcholine (epc) or egg sphingomyelin (esm)) was varied while their concentration remained constant. the results indicate that an increased local density of toxin binding sites occurring due to phase separation facilitates oligomer formation. furthermore, the change in local environment (number of neighboring cholesterol molecules) upon domain formation also enhances oligomer formation.we thank the dfg (sfb 490) for financial support, s. bhakdi and a. valeva for production of the toxin and helpful discussions. we explored quercetin effects on lipid bilayers containing cholesterol using a spectrofluorimetric approach. we used the fluorescent probe laurdan which is able to detect changes in membrane phase properties. when incorporated in lipid bilayers, laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. this behavior is seen in recorded spectra as a shift of maximum emission fluorescence from 440 nm at temperatures below lipids phase transition to 490 nm at temperatures above lipids phase transition values. emission spectra of laurdan were analyzed as a sum of two gaussian bands, centered on the two emission wavelengths allowing a good evaluation of the relative presence of each population. our results show that both laurdan emission states are present with different shares in a wide temperature range for dmpc liposomes with cholesterol. quercetin leads to a decrease in the phase transition temperature of liposomes, acting in the same time as a quencher on laurdan fluorescence. this paper is supported by the sectorial operational programme human resources development, financed from the caveolins are essential membrane proteins found in caveolae. the caveolin scaffolding domain of caveolin-1 includes a short sequence containing a crac motif (v 94 tkywfyr 101 ) at its c-terminal end. to investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav-1(v94-l102) nonapeptide encompassing this motif and including the first residue of cav-1 hydrophobic domain (l102), in dodecylphosphocholine (dpc) micelles and in dmpc/dhpc bicelles, as membrane mimics. nmr data revealed that this peptide folded as an amphipathic helix located in the polar head group region. the two tyrosine sidechains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face (le lan c. et al., 2010, eur. biophys. j., 39, 307-325). to investigate the interactions between the crac motif and the lipids, we performed molecular dynamics simulations in two different environment: a dpc micelle and a popc bilayer. the results obtained are in good agreement with nmr data and the comparison between both systems provided insight into the orientation of the crac motif at the membrane interface and into its interactions with lipids. this work was partially supported by the strategic grant posdru/21/1. our study suggests that conformation and positioning of hydroxyl groups significantly affects thermotropic properties of sphingolipids and sterol interaction. the polymorphism of a new series of bolaamphiphile molecules based on n-(12-betainylamino-dodecane)-octyl b-d-glucofuranosiduronamide chloride is investigated. the length of the main bridging chain is varied in order to modify the hydrophilic/lipophilic balance. the other chemical modification was to introduce a diacetylenic unit in the middle of the bridging chain to study the influence of the pp stacking on the supramolecular organisation of these molecules. dry bolaamphiphiles self-organize in supramolecular structures such as lamellar crystalline structure, lamellar fluid structure and lamellar gel structure. the thermal dependence of these structures, as well as the phase transition is followed by smallangle and wide-angle x-ray scattering. once the thermal cycle is accomplished, the system remains in the kinetically stabilized undercooled high-temperature phase at temperature of 20°c. subsequently, the time dependence of the relaxation to the thermodynamically stable phase is followed and very slow relaxation on the order of hours or days is observed. the study of polymorphism and the stability of various phases of this new series of bolaamphiphiles is interesting for potential application in health, cosmetics or food industry, is undertaken in this work. alkylphospholipids have shown promising results in several clinical studies and among them perifosine (opp) is promising for breast cancer therapy. antitumor effect was much better in estrogen receptor negative (er-) than in estrogen receptor positive (er+) tumors in vivo. it is believed that apl do not target dna, but they insert in the plasma membrane and ultimately lead to cell death. liposomes made of opp and different amount of cholesterol (ch) showed diminished hemolytic activity as compared to micellar opp, but in most cases cytotoxic activity was lower. in order to find optimal liposomal composition and to understand better the difference in the response of er+ and er-cells the interaction opp liposomes with er+ and er-cells was studied. for liposomes with high amount of ch both cell types showed slow release of the liposome entrapped spin probe into the cytoplasm. liposmes with low amount of ch interact better with cells but the release is faster for er-as for er+ cells at 37°c. experiments with nitroxide-labeled opp (sl-opp) liposomes suggest that the exchange of sl-opp between liposomes and cellular membranes is fast. however, translocation of sl-opp across the plasma membrane is slow, but seems to be faster for opp resistent, er+ cells as for er-cells at 37°c. estimation of a membrane pore size based on the law of conservation of mass krystian kubica 1 , artur wrona 1 and olga hrydziuszko 2 1 institute of biomedical engineering and instrumentation, wroclaw university of technology, wroclaw, poland, 2 centre for systems biology, university of birmingham, birmingham, uk the size of biomembrane pores determines which solutes or active compounds may enter the cell. here, using a mathematical model of a lipid bilayer and the law of conservation of mass, we calculate the radius of a membrane pore created by rearranging the lipid molecules (the pore wall was formed out of the lipid heads taken from the membrane regions situated directly above and below the pore, prior its formation). assuming a constant number of lipid molecules per bilayer (with or without the pore) and based on the literature data (60% decrease in the area per chain for a fluid-to-gel transition and a matching change of one chain volume not exceeding 4%) we have shown that the pore radius can measure up to 4.7nm (for a 7nm thick lipid bilayer) without the lipid molecules undergoing a phase transition. a further assumption of area per chain being modified as a consequence of the lipids conformational changes has resulted in an increase of the calculated radius up to 7.1nm. finally, a comparison of the pore volume with the corresponding volume of the lipid bilayer has led to a conclusion that for the system under consideration the membrane pore can only be created with the lipids undergoing fluidto-gel conformational changes. the key signaling pathway involves tyrosine phosphorylation of signal transducers and activators of transcription (stat1 and stat2) by receptor-associated janus kinases. we aim to unveil of the very early events of signal activation including ligand-induced receptor assembly and the recruitment of the cytoplasmic effector proteins stat1 and stat2 in living cells. to this end, we have explored the spatiotemporal dynamics of stat recruitment at the membrane on a single molecule level. highly transient interaction of stats to membrane-proximal sites was detected by tirf-microscopy, allowing for localizing and tracking individual molecules beyond the diffraction limit. thus, we obtained a pattern of the spatio-temporal recruitment of stat molecules to the plasma membrane revealing distinct submicroscopic structures and hotspots of stat interaction with overlapping recruitment sites for stat1 and stat2. strikingly, these stat binding sites were independent on receptor localization and expression level. simultaneous superresolution imaging of the cytoskeleton revealed the organization of stat recruitment sites within the cortical actin skeleton. characterization of molecular dynamics on living cell membranes at the nanoscale level is fundamental to unravel the mechanisms of membrane organization andcompartmentalization. we have recently demonstr ated the feasibility of fluorescence correlation spectroscopy (fcs) based on the nanometric illumination of near-field scanning optical microscopy (nsom) probes on intact living cells [1] . nsom-fcs was applied to study the diffusion of fluorescent lipid analogs on living cho cells. the experiments allowed to reveal details of the diffusion hidden by larger illumination areas and associated with nanoscale membrane compartmentalization. the technique also offers the unique advantages of straightforward implementation of multiple color excitation, opening the possibility to study nanoscale molecular cross-correlation. furthermore, the nsom probe evanescent axial illumination allows to extend diffusion study to the membrane-proximal cytosolic region. as such, nsom-fcs represents a novel powerful tool to characterize the details of many biological processes in which molecular diffusion plays a relevant role. the growing interest in supported lipid bilayers (slbs) on conductive substrates, such as gold, is due to the possibility of designing lipid-based biosensor interfaces with electrochemical transduction. due to the hydrophobicity of gold it is still a challenge to deposit planar and continuous bilayers without previous surface modification. most studies on gold concern single-phase slbs without cholesterol or gangliosides, two vital components of biomembranes. in this work the experimental conditions suitable for the formation of complex slbs with phase-separation directly on gold are exploited. the mixtures dopc/dppc/cholesterol (4:4:2) with 0 or 10 mol % of ganglioside gm1, which should yield lipid raft-like domains according to reported phase diagrams, were studied. slb with lipid rafts were successfully formed onto bare au (111), although surface modification with 11-mercapto-undecanoic acid sam stabilized the slbs due to its charge and hydrophilicity. the different experimental conditions tested had an impact on nano/microdomains organization observed by atomic force microscopy in buffer solution. surface characterization through the combined use of ellipsometry, cyclic voltammetry and afm allowed to optimize the conditions for the formation of more planar and compact slbs. it is widely accepted that the conversion of the soluble, nontoxic amyloid b-protein (ab) monomer to aggregated toxic ab rich in b-sheet structures is central to the development of alzheimer's disease. however, the mechanism of the abnormal aggregation of ab in vivo is not well understood. we have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by ab, the toxicity and physicochemical properties of which are different from those of ab amyloids formed in solution [1, 2] . in this presentation, we report a detailed mechanism by which ab-(1-40) fibrillizes in raft-like lipid bilayers composed of gm1/cholesterol/sphingomyelin. at lower concentrations, ab formed an a helix-rich structure, which was cooperatively converted to a b sheet-rich structure above a threshold concentration. the structure was further changed to a seed-prone b structure at higher concentrations. the seed recruited ab in solution to form amyloid fibrils. [ hepatitis c virus (hcv) has a great impact on public health, affecting more than 170 million people worldwide since it is the cause of liver-related diseases such as chronic hepatitis, cirrhosis and hepatocarcinoma. hcv entry into the host cell is achieved by the fusion of viral and cellular membranes, replicates its genome in a membrane associated replication complex, and morphogenesis has been suggested to take place in the endoplasmic reticulum (er) or modified er membranes. the variability of the hcv proteins gives the virus the ability to escape the host immune surveillance system and notably hampers the development of an efficient vaccine. hcv has a single-stranded genome which encode a polyprotein, cleaved by a combination of cellular and viral proteases to produce the mature structural proteins (core, e1, e2, and p7) and non-structural ones (ns2, ns3, ns4a, ns4b, ns5a and ns5b), the latter associated with the membrane originated from the er in the emerging virus. the ns4b protein, a fundamental player in the hcv replicative process and the least characterized hcv protein, is a highly hydrophobic protein associated with er membranes. it has recently been shown that the c-terminal is palmitoylated and the n-terminal region has potent polymerization activity. the expression of ns4b induces the formation of the so called membranous web, which has been postulated to be the hcv rna replication complex. thus, a function of ns4b might be to induce a specific membrane alteration that serves as a scaffold for the formation of the hcv replication complex and therefore has critical role in the hcv cycle. due to the high hydrophobic nature of ns4b, a detailed structure determination of this protein is very difficult. the ns4b protein is an integral membrane protein with four or more transmembrane domains. the c-terminal region of ns4b is constituted by two a helices, h 1 (approximately from amino acid 1912 to 1924) and h 2 (approximately from amino acid 1940 to 1960), which have been studied as potential targets for inhibiting hcv replication. previous studies from our group, based on the study of the effect of ns4b peptide libraries on model membrane integrity, have allowed us to propose the location of different segments in this protein that would be implicated in lipid-protein interaction. additionally, the h 1 region could be an essential constituent in the interaction between protein and membrane. in this study we show that peptides derived from the c-terminal domain of ns4b protein of hcv are able to interact with high affinity to biomembranes, significantly destabilizing them and affecting their biophysical properties. there were also differences in the interaction of the peptide depending on the lipid composition of the membranes studied. we have also applied fluorescence spectroscopy, infrared spectroscopy and differential scanning calorimetry which have given as a detailed biophysical study of the interaction of the peptide with model biomembranes. this work was supported by grant bfu2008-02617-bmc (ministerio de ciencia y tecnología, spain) to j.v. a semi-quantitative theory describing the adhesion kinetics between soft objects as living cells or vesicles was developed. the nucleation-like mechanism has been described in the framework of a non-equilibrium fokker-planck approach accounting for the adhesion patch growth and dissolution (a. raudino, m. pannuzzo, j. chem. phys. 132, 045103 (2010)). a well known puzzling effect is the dramatic enhancement of the adhesion/fusion rate of lipid membranes by water-soluble polymers that do not specifically interact with the membrane surface. we extend the previous approach by molecular dynamics simulations in the framework of a coarse-grained picture of the system (lipid+polymer+ions embedded in an explicit water medium) in order to test and support our previous analytical results. simulations show that the osmotic pressure due to the polymer exclusion from the inter-membrane spacing is partially balanced by an electrostatic pressure. however, we also evidenced an interesting coupling between osmotic forces and electrostatic effects. indeed, when charged membranes are considered, polymers of low dielectric permittivity are partially excluded from the inter-membrane space because of the increased local salt concentration. the increased salt concentration means also a larger density of divalent ions which form a bridge at the contact region (stronger adhesion). the overall effect is a smaller membrane repulsion. this effect disappears when neutral membranes are considered. the model could explain the fusion kinetics between lipid vesicles, provided the short-range adhesion transition is the rate-limiting step to the whole fusion process. the role of ceramide acyl chain length and unsaturation on membrane structure sandra n. ceramide fatty acid composition selectively regulates distinct cell processes by a yet unknown mechanism. however, evidence suggests that biophysical processes are important in the activation of signalling pathways. indeed, ceramide strongly affects membrane order, induces gel/fluid phase separation and forms highly-ordered gel domains. the impact of ceramide n-acyl chain in the biophysical properties of a fluid membrane was studied in popc membranes mixed with distinct ceramides. our results show that: i) saturated ceramide has a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (c24:1 ceramide) or no (c18:1 ceramide) ability to form gel domains at physiological temperature, ii) differences between distinct saturated species are smaller and are related mainly to domain morphology, and iii) very long chain ceramide induces the formation of tubular structures probably associated with interdigitation. these results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology. extra high content of cholesterol (chol) in fiber-cell membranes in the eye lens leads to chol saturation and formation of cholesterol bilayer domains (cbds). it is hypothesized that high enrichment in cholesterol helps to maintain lens transparency and protect against cataractogenesis. in model studies, the cbd is formed in a phospholipid bilayer when cholesterol content exceeding the cholesterol solubility threshold, thus, the cbd is surrounded by phospholipid bilayer saturated with cholesterol. in the present study, we carried out molecular dynamics (md) simulation of two bilayers: a palmitoyloleoylphosphatidylcholine (popc) bilayer (reference) and a 1:1 popc-chol bilayer, to investigate the smoothing effect of the saturating amount of cholesterol on the bilayer. to our knowledge, this effect has not been studied so far so this study is certainly providing new results. our results indicate that saturation with cholesterol significantly narrows the distribution of vertical positions of the center-of-mass of the popc molecules and the popc atoms in the bilayer and smoothes the bilayer surface. we hypothesize that this smoothing effect decreases light-scattering and helps to maintain lens transparency. the phospholipid content of staphylococcus aureus membranes displays a high degree of variability (1-3). the major phospholipids found in s. aureus are phosphatidylglycerol (pg), cardiolipin (cl) and lysylphosphatidylglycerol (lpg), (1) the concentrations of which are environment dependent and see fluctuations on exposure to high concentrations of positively charged moieties (4) . up regulation of lpg has a suspected role in neutralisation of the plasma membrane in response to cationic threats. studies have been conducted to probe biomimetic models of this theory however our focus is to look at atomic details of membrane extracts in the presence of magaininf5w. s. aureus 476 lipid extracts from cells grown at ph 5.5 and 7.0, studies by neutron diffraction with and without peptide at two contrasts. d-spacings were assessed by vogt area fitting and bragg's law. bilayer separation at low ph was *1-2 å less than ph 7.0. with peptide, bilayer separations of ph 5.5 and 7.0 extracts were reduced by *2 å and *4 å , respectively. reduced pg content of low ph extracts is suggested to reduce d-spacing, however presence of peptide further reduces this, possibly by an anion neutralisation effect. abnormal d-spacing on increased humidity may be due to the breakdown of lpg. activation of neutrophils releasing hocl and apoptosis of vein endothelial cells are the events documented to occur in the course of atherosclerosis. as lipid chlorohydrins, which are the key products of the reaction between hocl and unsaturated fatty acid residues, were found in atherosclerotic plaques, we decided to check their biological activity in the context of their ability to act as the mediators of hoclinduced oxidative stress and apoptosis in the culture of immortalized human umbilical vein endothelial cells (hu-vec-st). the concentration of reactive oxygen species was found to be elevated after 1 h cell incubation with phospahtidylcholine chlorohydrins. this effect was at least partially caused by the leakage of superoxide anion from mitochondria and followed by depletion of gsh and total thiols. the significant decrease of antioxidant capacity of cell extracts was also observed. the intracellular red-ox imbalance was accompanied by the increase of the ratio between phosphorylated and dephosphorylated forms of p38 map kinase. after longer incubation a significant number of apoptotic cells appeared. summing up, phosphatidylcholine chlorohydrins may be regarded as signaling molecules, able to initiate signalling pathways by induction of oxidative stress. giant unilamellar vesicles (guvs) are a valuable tool in the study of lateral distribution of biological membrane components. guv dimensions are comparable to typical cell plasma membranes and lipid phase separation can be observed through fluorescence microscopy. guv studies frequently require immobilization of the vesicles, and several methods are available for that effect. one of the most common methodologies for vesicle immobilization is the use of avidin/streptavidin coated surfaces and biotin labeled lipids at very low concentration in the vesicles. here, we analyze the effect of using this methodology on lipid domain distribution for different lipid compositions. we show that as a result of non-homogeneous distribution of biotin labeled lipids between liquid disordered, liquid ordered and gel phases, distribution of lipid domains inside guvs can be dramatically affected. monitoring membrane permeability: development of a sicm approach christoph saßen 1,2 and claudia steinem 1 1 institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße 2, 37077 gö ttingen, germany, 2 ggnb doctoral program: imprs, physics of biological and complex systems scanning ion conductance microscopy (sicm) utilises a nanopipette containing an electrode as a probe for surface investigations with resolutions of 1/3 of the inner pipette diameter. experiments are conducted under physiological conditions, in situ and without mechanical contact of probe and sample. hence, sicm serves as a well-suited technique for the investigation of soft objects such as cells or artificial lipid membranes. using pore-suspending membranes (psm) as a model system, interactions of melittin as an example for cell penetrating peptides (cpps) and lipid membranes are investigated by means of sicm. formation of a range of solvent free psm from lipid vesicles has been achieved as confirmed by means of fluorescence microscopy and sicm. application of melittin results in rupturing of the lipid bilayer. putative insights gained from this assay are critical concentrations of membrane permeabilising ccps and answers to mechanistic questions, e.g. whether ccps translocate only or form pores within the lipid bilayer. positioning of the z-ring in escherichia coli prior to cell division is regulated by intracellular pole-to-pole oscillation and membrane binding of min proteins, allowing assembly of ftsz filaments only at the center plane of the cell. in order to investigate the influence of membrane geometry on the dynamic behavior of membrane binding of min proteins, we combined concepts of synthetic biology and microfabrication technology. glass slides were patterned by a gold coating with microscopic windows of different geometries, and supported lipid bilayers (slb) were formed on these microstructures. on slbs, min-proteins organize into parallel waves. confinement of the artificial membranes determined the direction of propagation.min-waves could be guided along curved membrane stripes, in circles and even along slalom-geometries. in elongated membrane structures, the protein waves always propagate along the longest axis. coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and viscosity of the aqueous media above the bilayer. this indicates the existence of an inhomogeneous and dynamic protein gradient above the membrane. minimal systems for membrane associated cellular processes petra schwille bio technology center biotec, technical university of dresden, germany the strive for identifying minimal biological systems, particularly of subcellular structures or modules, has in the past years been very successful, and crucial in vitro experiments with reduced complexity can nowadays be performed, e.g., on reconstituted cytoskeleton and membrane systems. in this overview talk, i will first discuss the virtues of minimal membrane systems, such as guvs and supported membranes, in quantitatively understand protein-lipid interactions, in particular lipid domain formation and its relevance on protein function. membrane transformations, such as vesicle fusion and fission, but also vesicle splitting, can be reconstituted in these simple subsystems, due to the inherent physical properties of selfassembled lipids, and it is compelling question how simple a protein machinery may be that is still able to regulate these transformations. as an exciting example of the power of minimal systems, i show how the interplay between a membrane and only two antagonistic proteins from the bacterial cell division machinery can result in emergence of protein self-organization and pattern formation, and discuss the possibility of reconstituting a minimal divisome. quantitative microscopic analysis reveals cell confluence regulated divergence of pdgfr-initiated signaling pathways with logically streamlined cellular outputs á rpá d szö } or, lá szló ujalky-nagy, já nos szö ll} osi, gyö rgy vereb university of debrecen, department of biophysics and cell biology, debrecen, hungary platelet derived growth factor receptors (pdgfr) play an important role in proliferation and survival of tumor cells. pdgf-bb stimulation caused a redistribution of pdgf receptors towards gm1 rich domains, which was more prominent in confluent monolayers. pdgf-bb stimulation significantly increased relative receptor phosphorylation of the ras / mapk pathway specific tyr716 residues and the pi3-kinase / akt pathway specific tyr751 residues in nonconfluent cultures. tyr771 residues that serve as adaptors for ras-gap which inactivates the mapk pathway and tyr1021 residues feeding into the plc-gamma / camk-pkc pathway were the docking sites significantly hyperphosphorylation following ligand stimulation in confluent cells. we found that p-akt facilitated cell survival and pmapk dependent proliferation is more activated in dispersed cells, while phospholipase c-gamma mediated calcium release and pkc-dependent rhoa activation are the prominent output features pdgf stimulus achieves in confluent cultures. these observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs, namely, cell division and survival in sparse cultures and inhibition of proliferation joined with promotion of migration in confluent monolayers that appear contact inhibited. a thermodynamic approach to phase coexistence in ternary cholesterol-phospholipid mixtures jean wolff, carlos m. marques and fabrice thalmann institut charles sadron, université de strasbourg, cnrs upr, 22, 23 rue du loess, strasbourg cedex, f-67037, france e-mail: thalmann@ics-cnrs.unistra.fr we present a simple and predictive model for describing the phase stability of ternary cholesterol-phospholipid mixtures. assuming that competition between the liquid and gel organizations of the phospholipids is the main driving force behind lipid segregation, we derive a phenomenological gibbs free-energy of mixing, based on the calorimetric properties of the lipids main transition. gibbs phase diagrams are numerically obtained that reproduces the most important experimental features of dppc-dopc-chol membranes, such as regions of triple coexistence and liquid orderedliquid disordered segregation. based on this approach, we present a scenario for the evolution of the phase diagram with temperature. results for other phospholipid species, such as popc or psm will also be presented. interleukin-2 and -15 receptors play a central role in the activation, survival and death of t lymphocytes. they form supramolecular clusters with mhc i and ii glycoproteins in t cells. in damaged or inflamed tissues the extracellular k+ concentration increases, which can depolarize the membrane. the common signaling beta and gamma chains of il-2/15r are phosphorylated upon cytokine binding and get a permanent dipole moment, thus their conformation, interactions, mobility and activity may be sensitive to the membrane potential. we induced depolarization on ft 7.10 t lymphoma cells by increasing the ec. k+ level or by blocking kv 1.3 voltage gated k+ channels with margatoxin. fcs measuremens showed that the lateral mobility of fab-labeled il-2/ 15r and mhc i and ii decreased upon depolarization, while that of gpi-linked cd48 did not change. fret efficiency measured between some elements of the il-receptor/mhc cluster increased, which may reflect an increase of cluster size. il-2-induced receptor activity, as monitored by measuring stat5-phosphorylation, increased upon depolarization, whereas il-15 induced phosphorylation did not change. our results may reveal a novel regulatory mechanism of receptor function by the membrane potential. cytokines play an important role in t cell activation and immunological memory, whereas mhcs are known for the role in antigen presentation. we applied rnai to silence the expression of mhc i in order to study its possible role in receptor assembly and function. fret data indicated that the association of il-2r and il-15r with mhc i as well as between il-2r and il-15r weakened. fcs indicated an increase of receptor mobility also suggesting the partial disassembly of the clusters. mhc i gene silencing lead to a remarkable increase of il-2/il-15 induced phosphorylation of stat5 transcription factors. in search for the molecular background of this inhibition of signaling by mhc i we checked il-2 binding and the formation of the receptor complex (il-2r alpha -il-2r beta association), but we did not find a difference as compared to the control. our results suggest that mhc i plays an organizing role in maintaining supramolecular receptor clusters and inhibits il-2r signaling, revealing a nonclassic new function of mhc i beyond its classical role in antigen presentation. interleukin-4 (il-4) is an important cytokine involved in adaptive immunity. il-4 binds with high affinity the singlepass transmembrane receptor il-4ra. the occupied complex, il-4/il-4ra then engages either il-2rc or il-13ra1, to form an activated type i or ii receptor, respectively. this formation of heterodimers is believed to trigger cross-activation of intracellular janus kinases. here we follow a fluorescently labeled ligand through various stages of receptor activation in hek293t: using fluorescence correlation spectroscopy (fcs), we see that the receptor chains diffuse as monomers within the plasma membrane. using dual-color fccs provides direct evidence for ligand induced co-diffusion of occupied il-4ra and il-13ra1. in contrast, type i complexes containing il-2rc could not be observed. however, ectopic expression of gfp-tagged il-2rc/jak3 induced stable fluorescent speckles in or close to the plasma membrane. we identified these structures as early sorting endosomes by colocalization of surface markers like eea1 and rab gtpases. the il-4ra chain is continuously trafficking into these compartments. these observations suggest that the formation of a type i il-4r heterodimer may require internalization and that early endosomes serve as a platform for il-4 signaling. among the membrane associated proteins, the ras family, which is lipid-anchored g protein, plays a key role in a large range of physiological processes and, more importantly, is deregulated in a large variety of cancer. in this context, plasma membrane heterogeneity appears as a central concept since it ultimately tunes the specification and regulation of ras-dependent signaling processes. therefore, to investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by fluorescence correlation spectroscopy at different spatial scales (spot variable fcs, svfcs) (1). we have shown in a variety of cell types that lipidbased nanodomains are instrumental for cell membrane compartmentalization. we have also observed that thesenanodomains are critically involved in the activation of signaling pathways and are essential for physiological responses (2-3). more recently, weextend the application of svfcs to characterizethe dynamics of k-rasproteinat the plasma membrane. as major result, we demonstrated that the rate of k-ras association/dissociation from the membrane is fast but vary as a functional of the activation state of the molecule as well as of specific intracellular protein interactions. we have so demonstrated that an helical lid sub-domain in the sbd is essential for monomeric as binding, but not for the anti-aggregation activity of the chaperone, suggesting that hsp70 is able to interact with pre-fibrillar oligomeric species formed during as aggregation and that, then, the mechanism of binding for these species is different from that of the monomeric protein. aggregation of the acylphosphatase from sulfolobus solfataricus (sso acp) into amyloid-like protofibrils is induced by the establishment of an intermolecular interaction between a 11-residue unfolded segment at the n-terminus and the globular unit of another molecule. we have used data from hydrogen/deuterium exchange experiments, intermolecular paramagnetic relaxation enhancements and isothermal titration calorimetry measurements on an aggregation-resistant sso acp variant lacking the 11-residue n-terminus to characterize the initial steps of the aggregation reaction. under solution conditions that favour aggregation of the wild-type protein, the truncated protein was found to interact with a peptide corresponding to the n-terminal residues of the full length protein. this interaction involves the fourth strand of the main b-sheet structure of the protein and the loop following this region and induces a slight decrease in protein flexibility. we suggest that the amyloidogenic state populated by sso acp prior to aggregation does not present local unfolding but is characterized by increased dynamics throughout the sequence that allow the protein to establish new interactions, leading to the aggregation reaction. amyloid-like aggregates alter the membrane mobility of gm1 gangliosides martino calamai and francesco pavone university of florence, lens -european laboratory for non-linear spectroscopy, sesto fiorentino, florence, italy neuronal dysfunction in neurodegenerative pathologies such as alzheimer's disease is currently attributed to the interaction of amyloid aggregates with the plasma membrane. amongst the variety of toxic mechanisms proposed, one involves the binding of amyloid species to gm1 gangliosides. gm1 takes part into the formation of membrane rafts, and exerts antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. in this study, we investigated the effects of amyloid-like aggregates formed by the highly amyloidogenic structural motif of the yeast prion sup35 (sup35nm) on the mobility of gm1 on the plasmamembrane of living cells. preformed sup35nm aggregates were incubated with cells and gm1 molecules were subsequently labeled with biotinylated ctx-b and streptavidin quantum dots (qds). single qds bound to gm1 were then tracked. the mobility of gm1 was found to decrease dramatically in the presence of sup35nm aggregates, switching from brownian to mainly confined motion. the considerable interference of amyloid-like aggregates with the lateral diffusion of gm1 might imply a consequent loss of function of gm1, thus contributing to explain the toxic mechanism ascribed to this particular interaction. insights into the early stages of fibrillogenesis of insulin using mass spectrometry harriet l. insulin is a vital hormone in metabolic processes as it regulates the glucose levels in the body. insulin is stored in the b cells of the pancreas as a hexamer, however its biologically active form is the monomer. the formation of fibrillar aggregates of insulin rarely occurs in the body; however localised amyloidosis at the site of injection for diabetes patients and aggregation of pharmaceutical insulin stocks present problems. in the current study oligomers formed early in the process of fibril assembly in vitro are observed by mass spectrometry (ms). ms is the only technique which allows early species to be characterised as it can identify different oligomeric orders by mass to charge ratio and show protein abundance and aggregation propensity. on mobility ms is used to examine rotationally averaged collision cross sections of oligomers in the aggregating solution. a wide array of oligomers is observed and the stability of specific species is remarked. the presence of multiple conformations for the highly charged oligomers is particularly noted and their assignment confirmed using fourier transform ion cyclotron resonance ms and collision induced dissociation. molecular modelling has been used to further explore the conformational space the oligomers inhabit. amyloid fibrils consisting of different proteins have been recognized as an accompanying feature of several neurodegenerative diseases. many proteins without known connection to any diseases have been found to form amyloid fibrils in vitro, leading to suggestion that the ability to form fibrils is the inherent property of polypeptide chain. the observed common character of protein amyloid formation enables to seek further clues of fibrillation mechanism by studying generalized sequenceless polypeptide models, e.g. polylysine. we have studied conformational transitions of polylysine, with different chain length at various ph, ionic strength and temperature by means of novel approachviscometric method. this polypeptide undergoes alfa-helix to beta-sheet transition and forms amyloid fibrils in special conditions. temperature induced a-helix to b-sheet transitions occurs at ph interval form 10 to 11.8 and with increasing chain length is slightly shifted to the lower ph. we have found narrow ph interval, in which the thermal transition is fully reversible, suggesting the high sensitivity of polypeptide conformation on subtle changes in charge on its side chains. this work was supported within the projects vega 0079,0038 and 0155, cex sas nanofluid and by esf project no. 26220120033. understanding the mechanisms of the conversion from the native state of a protein to the amyloidal state represents a fundamental step in improving the purification, storage and delivery of protein-based drugs and it is also of great relevance for developing strategies to prevent in vivo protein aggregation. amyloid fibrils have a structural arrangement of cross b-sheet but they can also experience different packing into three dimensional superstructures, i.e. polymorphism. it is well known that, among others, both the geometric confinement of the molecules and shear forces can affect the final morphology of the aggregates. importantly, due to the complexity and crowding of the cellular region, such parameters also play a crucial role in in vivo processes. we present an experimental approach to study in vitro amyloid aggregation in a controlled and uniform shear force field and within microscale environments. in particular we focus on the effect of these two parameters on the formation of spherical aggregates, known as spherulites. using micro channels of different cross-sections from 5 to1000 lm x 12 lm and flow rates in the range of hundreds of ll/min, the number and diameter of spherulites within the channels have been characterized using crossed polarizers optical microscopy. inhibition of insulin amyloid fibrillization by albumin magnetic fluid k. insulin amyloid aggregation causes serious problems for patient with insulin dependent diabetes undergoing long-term treatment by injection, in production and storage of this drug and in application of insulin pumps. recent studies indicate that protein amyloid aggregation causes the cell impairment and death; however, the prevention of amyloid aggregation is beneficial. we have investigated ability of albumin magnetic fluid (amf) to inhibit insulin amyloid aggregation by spectroscopic and microscopic techniques. albumin magnetic fluid consists of magnetic fe 3 o 4 nanoparticles sterically stabilized by sodium oleate and functionalized with bovine serum albumin (bsa) at various weight ratios bsa/fe 3 o 4 . we have found the positive correlation between inhibiting activity of afm and nanoparticle diameter and zeta potential. the ability of amf to inhibit formation of amyloid fibrils exhibits concentration dependence with ic 50 values comparable to insulin concentration. the observed features make amf of potential interest as agents effective in the solving of problems associated with insulin amyloid aggregation. (this work was supported within the projects vega 0079, 0155 and 0077, cex sas nanofluid, apvv-0171-10, sk-ro-0012-10 and esf project 26220220005). amyloid formation of peptides causes diseases like alzheimer's and parkinson's disease. however, the conditions for the onset of the neurotoxic beta-sheet formation are poorly understood. we focus on aggregation triggers and their interplay: interactions with hydrophobic-hydrophilic interfaces, orientation of peptides in 2d, metal ion complexation and lipid layers. the tailor-made model peptides exhibit defined secondary structure propensities and metal ion binding sites. the interactions of the peptide with the air-water interface and with metal ions are studied using surface sensitive methods connected to film balance measurements. x-ray diffraction, x-ray reflection, infrared reflection-absorption spectroscopy and total reflection x-ray fluorescence were applied to reveal the layer structure, peptide conformations and metal ion binding at the interface. we found that amyloid formation in 2d is dominated by the hydrophobic-hydrophilic interface and not comparable to the bulk behaviour. the interface can enhance or inhibit betasheet formation. the effect of metal ion complexation depends on the arrangement of the binding sites in the peptide and the preferred metal complexation geometry. the two triggers interface and metal ion complexation, can oppose each other. effect of apoe isoform and lipidation status on proteolytic clearance of the amyloid-beta peptide hubin, e. alzheimer's disease (ad) is the most common type of dementia in the elderly. the most important genetic risk factor identified for ad is the isoform, e2, e3 or e4, of apolipoprotein e (apoe), a lipid-carrying protein. one hallmark of ad is the accumulation of amyloid-beta peptide (ab) in the brain which is thought to result from an imbalance between the production of ab and its clearance. previous studies report an important role for apoe in ab degradation. we sought to determine the effect of apoe isoform and lipidation status on the degradation of soluble ab by proteinases such as insulin-degrading enzyme and neprilysin. in this study an in vitro ab clearance assay based on the competition between ab and a fluorogenic peptide substrate is developed to quantify ab degradation. to elucidate the proteolytic clearance mechanism, the fragments resulting from cleavage are identified by mass spectrometry and further analyzed to identify the interacting stretch of the ab sequence with the different apoe isoforms. the results suggest that apoe influences the rate of ab degradation. the aggregation of proteins into fibrillar nanostructures is a general form of behaviour encountered for a range of different polypeptide chains. the formation of these structures is associated with pathological processes in the context of alzheimer's and parkinson's diseases but is also involved in biologically beneficial roles which include functional coatings and catalytical scaffolds. this talk focuses on recent work directed at understanding the kinetics of this process through the development and application of experimental biophysical methods and their combination with kinetic theories for linear growth phenomena. lbs contains not only as, but also other proteins including 14-3-3 proteins. 14-3-3 proteins exist mainly as a dimer and its exact functions are remain unclear. however, recent work has shown that the association of 14-3-3(eta) with as in lbs. herein we show how 14-3-3(eta) can modulate as in vitro aggregation behavior, by rerouting it toward the formation of stable non-fibrillar aggregates. we also show that the resulting populations of fibrillar and pre-fibrillar aggregates exhibit a modified toxicity in vivo with respect to the unperturbed aggregates. interestingly, 14-3-3(eta) does not show any binding affinity for monomeric as, nor for the mature fibrillar aggregates. we provide evidence that it acts on the oligomeric species which form during the amyloidogenesis process of aggregation. since 14-3-3(eta) can influence the toxicity of amyloidogenesis without perturbing the functional as monomers, we are convinced that once fully understood, its mode of action could represent a promising model to mimic with synthetic drugs and peptides. what makes an amyloid toxic: morphology, structure or interaction with membranes? more than 40 human diseases are related to amyloids. in order to understand why some amyloids may become toxic to their host and some others are not, we first developed a genetical approach in the yeast saccharomyces cerevisiae. we have chosen the amyloid/prion protein het-s prion forming domain (218-289) from the fungi podospora anserina, which is not toxic in yeast. some toxic amyloids mutants were generated by random mutagenesis. in vitro the most toxic mutant called ''m8'' displays very peculiar nanofibers, which polymerized mainly in amyloid antiparallel b-sheets whereas the non-toxic wt exhibits a parallel polymerization. we further established the dynamic of assembling of the m8 toxic amyloid, in comparison to the wt non-toxic amyloid, and showed the presence of specific oligomeric intermediates also organized in antiparallel b-sheet structures. a more global structure/toxicity study on more than 40 mutants clearly identified an antiparallel b-sheet signature for all the toxic mutants. therefore size, intermediates and antiparallel structures may account for amyloid toxicity in yeast but we still wonder what their cellular targets are. recently, we established the first evidences that toxic mutants may specifically bind in vitro to lipids, particularly negatively charged. interconnected mechanisms in abeta (1-40) we present an experimental study on the fibril formation of ab(1-40) peptide at ph 7.4. the kinetics of this process is characterized by the occurrence of multiple transient species that give rise to final aggregates whose morphology and molecular structure are strongly affected by the growth conditions. to observe in details the aggregation pathway as a function of solution conditions, we have used different experimental techniques as light scattering, thioflavin t fluorescence, circular dichroism and two-photon fluorescence microscopy. this approach gives information on the time evolution of conformational changes at molecular level, on the aggregates/fibrils growth and on their morphologies. the selected experimental conditions allowed us to highlight the existence of at least three different aggregation mechanisms acting in competition. a first assembly stage, which implies conformational conversion of native peptides, leads to the formation of small ordered oligomers representing an activated conformation to proceed towards fibril growth. this process constitutes the rate limiting step for two distinct fibril nucleation mechanisms that probably implicate spatially heterogeneous mechanisms. the formation of amyloid fibrils of amylin 10-29 was studied by means of molecular dynamics (md) and energy partition on three peptide ß-sheet stack systems with the same amino acid composition: wild type amylin 10-29 (amyl 10-29), reverse amylin 10-29 (rev-amyl 10-29) and scrambled amylin version scr-amyl 10-29. the results show that for amylin10-29 peptides, amino acid composition determines the propensity of a peptide to form amyloid fibrils independent of their sequence. the sequence of amino acids defines the shape and the strength of amyloid protofibril, which conforms with the atom force microscope (afm) data [1] . md show that the 6x6revamyl-10-29 stack has looser selfassembly than the 6x6amyl-10-29 stack, which conforms with the results of fourier transform infrared spectroscopy (ftir) measurements for the peptides studied [1] . the results of md show that 6x6amyl-10-29 could have a turn, which consists with ftir data [1] . data on ab aggregation kinetics have been characterized by a large spread between experiments on identical samples that contain so many molecules that stochastic behaviour is difficult to explain unless caused by uncontrolled amounts of impurities or interfaces. we have therefore spent considerable effort to eliminate sources of inhomogeneity and reached a level of reproducibility between identical samples and between experiments on separate occasions that we can now collect data that can lead to mechanistic insights into the aggregation process per se, and into the mechanism of action of inhibitors. data on ab42 aggregation will be shown that give insight into the influence of physical parameters like peptide concentration, shear and ionic strength, as well as the effect of inhibitory proteins, model membranes and the effects of sequence variations. monte carlo simulations of amyloid formation from model peptides corroborate the finding from experiments and underscore that the very high level of predictability and reproducibility comes from multiple parallel processes. negatively-charged membranes were reported to catalyze ''amyloid-like'' fiber formation by non-amyloidogenic proteins [1] . our study aims to elucidate the factors that govern the formation of these amyloid-like fibers. lysozyme was selected as a model of non-amyloidogenic protein and was fluorescently-labeled with alexa fluor 488 (a488-lz). first, a488-lz partition towards phosphatidylserine-containing liposomes was characterized quantitatively using fluorescence correlation spectroscopy (fcs), in order to calculate the protein coverage of liposomes. secondly, the interaction between a488-lz and negatively-charged lipid membranes was studied using both steady-state and time-resolved fluorescence techniques. this interaction was found to switch from a peripheral binding to the anionic headgroups, at high lipid/ protein molar ratio (l/p), to a partial insertion of protein into the hydrophobic core of the membrane, at low l/p. finally, the lipidprotein supramolecular complexes formed at low l/p were characterized by fluorescence lifetime imaging microscopy (flim). the mean lifetime of a488-lz in these supramolecular structures is much lower compared to the values obtained for the free and bound a488-lz at high l/p. the fiber characterization will be complemented by fcs studies. [ the conversion of normal prp c to its pathological isoform prp sc is a key event in prion diseases and is proposed to occur at the cell surface or more probably in acidic late endosomes. a convergence of evidence strongly suggests that the early events leading to the structural conversion of the prp seem to be in relation with more or less stable soluble oligomers, which could mediate neurotoxicity. as commonly shared by other amyloidogenic proteins, membrane-bound monomers undergo a series of lipid-dependent conformational changes, leading to the formation of oligomers of varying toxicity rich in b-sheet structures (annular pores, amyloid fibrils). here, we have used a combination of biophysical techniques (dynamic and static light scattering, fluorescence techniques, and quartz crystral microbalance) to elucidate the interaction of native monomeric prp and that of purified b-rich oligomeric prp on model lipid membranes. under well established conditions, three b-sheet-rich oligomers were generated from the partial unfolding of the monomer in solution, which were found to form in parallel. from single mutation and/or truncation of the full length prp, the polymerization pathway is strongly affected, revealing the high conformational diversity of prp. in our previous work, we identify the minimal region of the prp protein leading to the same polymerization pattern of the full length prp. soluble 12-subunits and 36-subunits oligomers were obtained depending on the single mutation or truncation and purified. we compare their structural properties (ftir, cd) when associated with anionic lipid bilayers and study their propensities to permeabilize the membrane. fluorescence kinetics suggest different mechanisms of membrane perturbation for the monomer and the prp oligomers. deciphering this complex network of lipid interactions and conformational diversity of the prp protein will help for understanding of how amyloidogenic proteins induce neurotoxicity. the traditional view of the lipid bilayer described as a ''sea'' of lipids where proteins may float freely, is going to be inadequate to describe the increasingly large number of complex phenomena which are known to take place in biological membranes. membrane-assisted protein-protein interactions, formation of lipid clusters, protein-induced variation of the membrane shape, abnormal membrane permeabilities and conformational transitions of membrane-embedded proteins are only a few examples of the variegated ensemble of events whose tightly regulated cross-talk is essential for cell structure and function. experimental work on the above mentionated problems is very difficult and some time not accessible, especially when the studied systems have a fast dynamics. due to the large size of the systems usually involved in this multifaceted framework, a detailed molecular description of these phenomena is beyond the possibilities of conventional amyloid aggregation, a generic behavior of proteins, is related to incurable human pathologies -amyloid-related diseases, associated with formation of amyloid deposits in the body. all types of amyloid aggregates possess rich bsheet structural motif. the recent data confirm the toxic effect of aggregates on the cells, however, it was found that reduction of amyloid aggregates plays important role in prevention as well as therapy of amyloidosis. we have investigated effect of phytoalexin derivatives on amyloid fibrillization of two proteins, human insulin and chicken egg lysozyme, by tht and ans fluorescence assays. we have found that amyloid aggregation of both studied proteins was significantly inhibited by phytoalexin derivates cyclobrassinin and benzocamalexin. for most effective phytoalexins the estimated ic 50 values were at low micromolar concentration. the observed inhibiting activity was confirmed by transmission electron microscopy. our data suggest the potential therapeutic use of the most effective phytoalexins in the reduction of amyloid aggregation. (this work was supported within the projects vega 0079, 0155, cex sas nanofluid, apvv-0171-10, sk-ro-0012-10 and esf project 26220220005). the amyloid pore hypothesis suggests that interactions of oligomeric alpha-synuclein (as) with membranes play an important role in parkinson's disease. oligomers are thought to permeabilize membranes and interfere with ca 2+ pathways. permeabilization by as requires the presence of negatively charged phospholipids. whether as can bind and permeabilize membranes with physiologically relevant lipid compositions has not been extensively explored. here we report on the binding of as to giant unilamellar vesicles (guvs) with physiologically relevant lipid compositions. comparing different protocols of oligomer preparation, leakage assays on both large unilamellar vesicles (calcein release) and guvs (hpts efflux assay) show that as is not able to permeabilize these membranes. the presence of cholesterol has a stabilizing effect on these membrane systems. in agreement with these findings, we do not observe concentration dependent as toxicity using in vivo mts assays. however, in the calcein release assay, different as preparations show differences in kinetics and as concentrations that cause 100% leakage. these results motivate us to critically reassess the amyloid pore hypothesis, and suggest that membrane permeabilization may be attributable only to a very specific as species. alpha-synuclein oligomers impair membrane integrity-a mechanistic view martin t. stö ckl, mireille m. a. e. claessens, vinod subramaniam nanobiophysics, mesa+ institute for nanotechnology, university of twente, enschede, the netherlands one of the most prevalent neurodegenerative diseases is parkinson's disease (pd), which is accompanied with the loss of dopaminergic neurons. although the mechanisms leading to the death of these cells are still unclear, the protein alpha-synuclein (as) is one of the pivotal factors. previous studies indicate that especially oligomeric forms of as show a detrimental effect on membrane integrity. as an intact membrane is crucial to many cellular processes, the impairment of the membrane integrity is a likely pathway for neuronal death. we use different phospholipid bilayer model systems to investigate the mechanisms underlying this process. atomic force microscopy in combination with suspended asymmetric phospholipid bilayers, which closely mimic the plasma membrane, allows the identification of the binding sites, the measurement of penetration depths of the as oligomers into the phospholipid bilayer, and the detection of membrane thinning or creation of membrane defects. using an approach based on phospholipid vesicles we were able to observe for the first time that as oligomers cause an enhanced lipid-flip flop. suggesting that the loss of lipid asymmetry is a novel mechanism which may contribute to or trigger neuronal death in pd. amyloid protein-membrane interactions and structuretoxicity relationship study h.p. ta (toxic) has a much higher and more specific effect on negatively charged phospholipids (dops, dopi and dopg) than in the case of wt (non-toxic). therefore the insertion of protein into phospholipid monolayers, which occurred similarly for both wt and m8, is not a key factor for these effects (h.p. ta et al. langmuir, in press). we are now using unilamellar vesicles as a membrane model to investigate the amyloid protein (toxic and nontoxic) -phospholipid interactions. results confirmed the high specific and strong effects of m8 on negatively-charged membrane. in this project, we study the chemical, physical and biological properties of fibrillar networks. the formation and the network mechanics are investigated by combining droplet-based microfluidics with optical microscopy and small angle x-ray scattering (saxs). the chosen system, fibrin network formation, plays an important role in blood coagulation processes. crosslinking of fibrinogen induced by an enzymatic reaction with thrombin leads to 3d fibrin network formation. the fibrillar networks are formed within picoliter droplets of aqueous solutions in an continuous oil phase. droplets containing fibrinogen and thrombin can be produced of different sizes and stored for fibrin network formation. the formation of the fibrillar networks is imaged by fluorescence microscopy. to analyze the elastic properties of the networks, the droplets flow through a microchannel device of alternating width in order to squeeze and stretch the networks. additionally, saxs experiments will give structural information about the molecular dimensions of the networks. the amyloid beta peptide (ab), implicated in alzheimer's disease (ad), is released from the amyloid precursor protein (app) by secretase-induced cleavage. this process results in the release of a range of ab peptides varying in length. the brains of ad patients often contain longer ab peptides while the total concentration of ab is unaffected. longer peptides are more hydrophobic having far-reaching consequences for their toxicity and aggregation. as ab is necessary for normal neuronal function, research activities into ad therapeutic development currently explore the possibilities of modulating c-secretase activity to produce short ab peptides. whether such an approach effectively ameliorates the toxic effect of ab has not been explored yet. to answer this question, we studied the impact of heterogeneity in ab pools in an in vitro biophysical and in cellulo context using microelectrode array to assay the synaptic activity of primary neurons. we show that various lengths of the ab peptide and mixtures thereof aggregate with distinct kinetics and notoriously affect synaptotoxic and cytotoxic response. we also show that small amounts of less abundant peptides ab38 and ab43 induce aggregation and toxicity of ab40 while the behavior of ab42 is unaffected. one of the most important irreversible oxidative modifications of proteins is carbonylation, a process of introducing the carbonyl group in reaction with reactive oxygen species. importantly, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. however, it is still largely unclear how carbonylation affects protein structure, dynamics and aggregability on the atomic level. here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. we perform an exhaustive set of molecular dynamics simulations of native villin headpiece together with every single combination containing carbonylated versions of its seven lysine, arginine and proline residues, the quantitatively most important carbonylable amino acids. surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. on the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, chargeneutral mutations in their place, and, by comparison with experimental results, demonstrate that this by itself significantly increases intrinsic aggregation propensity of both structured, native proteins and their unfolded states. finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics and aggregability using site-directed mutagenesis. septins are an evolutionarily conserved family of gtp-binding proteins involved in important cellular processes, such as cytokinesis and exocytosis, and have been implicated in neurological diseases, such as alzheimer's and parkinson's diseases. the focus of this study was two septins of schistosoma mansoni, (the causative agent of schistosomiasis in south america) named smsept5 and smsept10, which were produced in a recombinant system. our objective was to verify if these septins from a simpler organism display similar characteristics to human septins. analysis of protein structure by circular dichroism showed that both recombinant smseptins produced were folded. the gtpase activity assay showed that smsept5 was able to hydrolyze gtp, whereas smsept10 was not. aggregation studies for amyloid fibril detection by right angle light scattering and thioflavin t fluorescence assay were performed. both proteins showed a temperature dependent increase in light scattering and fluorescence emission of tht probe. this indicated that s. mansoni septins tend to aggregate into amyloid-like fibers in high temperatures, with thresholds of 30°c for smsept5 and 37°c for smsept10. these results are in accordance to that previously reported for human septins. in our work we investigated the response to standard chemotherapy of blood lymphocytes of patients suffering with melanoma. dna single and double strand breaks were determined using comet assay; intracellular levels of marker proteins were detected using immunocytochemistry. ultimately this set of parameters allows to characterize two mechanisms of dna repair (base excision repair, ber and mismatch repair, mmr) which together with apoptosis proneness underlie response of tumor cells to chemotherapy. cell death caused by o 6 mg adducts is promoted by mmr system by inducing unrepaired double strand breaks in dna. there was a linear correlation between the level of dsdna breaks in lymphocytes after 1-st cycle of chemotherapy and mmr efficiency in them. the level of double strand breaks in dna after 1-st cycle of chemotherapy is predictive of clinical outcome. otherwise damage at the level of ssdna (ap-sites and single strand breaks) and ber mechanism associated with it couldn't be a good prognostic factor of chemotherapy. high level of double strand breaks in dna in blood lymphocytes of melanoma patients 48 hours after 1-st cycle of chemotherapy appears to be a marker of a good prognosis. self-assembly and stability of g-quadruplex: counterions, pressure and temperature effects e. baldassarri jr., p. mariani, f. spinozzi, m. g. ortore saifet dept. & cnism, marche polytechnic university, ancona, italy the important role of g-quadruplex in biological systems is based on two main features: composition and stability of telomeres, and activity of telomerase. the g-quadruplex structures are formed by supramolecular organization of basic units called g-quartets that are planar rings constituted by four guanosines linked by hoogsten hydrogen bonds. gquadruplex requires the presence of monovalent cations playing a key role in stabilizing these structures, since they give rise to coordination bonds needed for the stacking of more tetrads. we performed x-ray diffraction experiments at different pressures (ranging from 1 to 2000 bar), and small angle x-ray scattering (saxs) changing the temperature (between 20-60°c retinoic acid receptor (rar) is a member of the nuclear receptor superfamily. this ligand-inducible transcription factor binds to dna as a heterodimer with the retinoid x receptor (rxr) in the nucleus. the nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. we studied the diffusion of egfp-rar by fluorescence correlation spectroscopy (fcs) in order to uncover the molecular interactions determining receptor mobility. in the absence of ligand we identified two distinct species with different mobilities. the fast component has a diffusion coefficient of d 1 = 1.8-6 lm 2 /s corresponding to small oligomeric forms, whereas the slow component with d 2 = 0.06-0.12 lm 2 /s corresponds to interactions of rar with the chromatin or other large structures. the rar ligand binding domain fragment also has a slow component probably as a result of indirect dna-binding via rxr, with lower affinity than the intact rar:rxr complex. importantly, rar-agonist treatment shifts the equilibrium towards the slow population of the wild type receptor, but without significantly changing the mobility of either the fast or the slow population. by using a series of mutant forms of the receptor with altered dna-or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of rar during ligand activation. formation of inactive nuclear with high level of dna compaction in sperm cells is accompanied by a substitution of linker histones h1 by a number of other proteins. among them sperm-specific histones (ssh), which are characterized by elongated arginine-rich polypeptide chain compared to the somatic h1. the secondary and tertiary structure of the ssh and their interactions with dna were studied using spectroscopic and thermodynamic approaches. the histones were isolated from sperm of marine invertebrates and rat thymus. all studied ssh demonstrate no considerable compaction of dna in solutions of low ionic strength. however, at physiological conditions, ssh h1 from s.intermedius and a.japonica compact dna more intensively than other ssh. the somatic h1 from rat thymus revealed a minimal ability to compact the dna. we suggest that the ssh h1 are able to interact with dna not only in the major groove but also in the minor groove of the double helix inducing considerable structural changes in dna and facilitating the formation of the supercompact sperm chromatin. the authors are grateful for the financial support from the russian foundation for basic research (grants § 10-04-00092 and 09-08-01119) and from administration of saint-petersburg. ionizing radiation causes modification and destruction of nitrogenous bases in dna molecule. there are also local breakages of hydrogen bonds both in the lesion sites mentioned above and in other sites of the macromolecule. to reveal the amount of some of these damages we applied cd and uv absorption spectroscopy. radiation-induced changes in dna structure influence its uv absorption spectrum in different ways: partial denaturation causes hyperchromic effect, while destruction of the bases results in hypochromic shift. at the same time both of them result in the same changes in dna cd spectra: the decrease in intensity. we attempted to segregate the described damages in dna structure and studied the influence of dna ionic surroundings on the radiation effect. it is shown that the radiation efficiency of base destruction and partial denaturation increases with decreasing concentration of nacl in irradiated solution. udu (ugly duckling) has been first identified from a zebrafish mutant and shown to play an essential role during erythroid development; however, its roles in other cellular processes remain largely unexplored. facs analysis showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased dna damage in udu mutants. we further showed that the extensive p53-dependent apoptosis found in udu mutants is a consequence of activation in the atm-chk2 pathway. udu appears not to be required for dna repair, because both wild-type and udu embryos similarly respond to and recover from uv treatment. yeast two-hybrid and coimmunoprecipitation data demonstrated that pah-l repeats and sant-l domain of udu interacts with mcm3 and mcm4. furthermore, udu was colocalized with brdu and heterochromatin during dna replication, suggesting a role in maintaining genome integrity. recently, we started to work on the second zebrafish homolog, udu2, and its mammalian counterpart, gon4l. preliminary data showed that udu2 and gon4l mrna injection can rescue zebrafish udu mutant phenotypes. furthermore, pah-l and sant-l domains of udu2 and gon4l can bind to mcm3 and mcm4 and they are localized in the nucleus. these data suggest that udu2 and gon4l are functionally equivalent to zebrafish udu. their molecular mechanism leading to udu phenotypes is currently under investigation. chromatin condensation: general polyelectrolyte association and histone-tail specific folding nikolay korolev, nikolay berezhnoy, abdollah allahverdi, renliang yang, chuan-fa liu, james p. tam, lars nordenskiö ld school of biological sciences, nanyang technological university, 60 nanyang drive, 637551, singapore the major component of chromatin, dna, is a densely charged polyanion. electrostatic interactions between dna and dnapackaging proteins contribute decisively to formation of its elementary unit, the nucleosome, and are also important in chromatin folding into higher-order structures. we investigate condensation of dna and chromatin and find that electrostatics and polyelectrolyte character of dna play dominant role in both dna and chromatin condensation. by comprehensive experimental studies and using novel oligocationic ligands, we suggest simple universal equation describing dna condensation as a function of oligocation, dna and monovalent salt concentrations and including the ligand-dna binding constant. we found that similar dependence was also observed in condensation of the nucleosome arrays. next, we studied how general electrostatic and specific structural alterations caused by lysine acetylations in the histone tails influence formation of 30-nm chromatin fibre and intermolecular nucleosome array association. for the first time, a structural model is suggested which explains critical dependence of chromatin fibre folding on acetylation of the single lysine at position 16 of the histone h4. exceptional importance of the h4 lys 16 acetylation in general and gene specific transcriptional activation has been known for many years but no structural basis for this effect has yet been proposed. detection of specific dna sequences is central to modern molecular diagnostic. ultrasensitive raman measurements of nucleic acids are possible through exploiting the effect of surface-enhanced raman scattering (sers). in this work, the sers spectra of genomic dnas from leaves of different apple trees grown in the field, have been analyzed [1] . a detailed comparative analysis of sers signatures of genomic dnas is given. sers wavenumbers (cm -1 ) are reported here for all types of vibrations of plant genomic dnas, including bands assigned to localized vibrations of the purine and pyrimidine residues, localized vibrations of the deoxyribosephosphate moiety, etc. proposed band assignments are given. a strong dependence of the sers spectra on dna concentration and on time have been observed. in biochemical fields, nucleic acids might be used to explore the interaction between dna and small molecules, which is important in connection with probing the accurate local structure of dna and with understanding the natural dnamediated biological mechanisms [2] . the ph-dependent structure of dna studied by fourier transform infrared spectroscopy the region of the infrared spectrum studied covered the wave number range from 4000 cm -1 to 700 cm -1 . ir spectra show that in ph 8.0-9.0 interval carbonyl (c=o) band at 1689 -1 cm (assigned to guanine) is reduced in intensity and slightly shifted to lower frequencies. at the ph 10.0 significantly decreases band intensity at 1712 cm -1 due to unbounded c2=o of thymine and shifts to lower frequencies, indicating at the transition of this group in bounded form, supposedly by means of excess polarized hydroxyl ions. together this, in basic region a new intense absorption band has been observed in 1500-1300cm -1 frequency interval, corresponding to o-h group in-plane bending vibration (1410-1310cm -1 ). as for acidic conditions, it was observed that under the extreme ph (*2.8) value carbonyl absorption region shifts to higher frequencies and absorption intensity significantly increased, indicated at releasing of c=o groups from h-bonding between base pairs. moreover, bands intensity at 1418cm -1 and 1022cm -1 corresponding to out-of-plan deformation of nh 2 groups increased due to rupture of connections between the dna strands. during the last decade it was found that in many cases specific structural organization of multi-molecular protein and dna-protein complexes determines their functioning in living cells. although these functioning structures are usually unique, it is often possible to identify their common structural elements. one of the interesting examples of such universal elements are hmgb domains: structurally conservative functional domains of non-histone proteins hmgb1/2 also identified in many nuclear proteins. using afm, thermodynamic approaches, circular dichroism and molecular absorption spectroscopy in far-uv and mid-ir regions we have studied structural organization of the complexes between dna and different proteins, including hmgb1, hmgb-domain recombinant proteins and linker histone h1. we have demonstrated, that interaction with dna leads to increasing both a-helicity of the proteins and thermal stability of dna. also, this interaction may result in formation of highly ordered supramolecular complexes facilitated by hmgb-domains. the c-terminal sequence of hmgb1/2 regulates affinity of the proteins to dna and can be ''inactivated'' by interaction with histone h1. based on the data obtained a model of the interaction of multy-domain hmgb-proteins with dna is suggested. darmstadt, germany, 4 lmu biozentrum; munich, germany *these authors contributed equally to this work chromatin in living cells displays considerable mobility on a local scale. this movement is consistent with a constrained diffusion model, in that individual loci execute multiple, random jumps. to investigate the connection between local chromatin diffusion (lcd) and the changes in nuclear organization, we established a stable hela cell line expressing gfp-pcna. this protein, a core component of the replication machinery, serves as a cell-cycle marker and allows us to visualize sites of ongoing dna synthesis within the nucleus. to monitor lcd, we labeled discrete genomic loci through incorporation of cy3-dutp. this experimental system, in conjunction with particle tracking analysis, has enabled us to quantitatively measure chromatin mobility throughout the cell cycle. our results demonstrate that lcd is significantly decreased in s-phase. to explore the connection between dna replication and reduced chromatin movement, we undertook a more detailed examination of lcd in s-phase nuclei, correlating chromatin mobility with sites of replication. our results demonstrate that labeled chromatin in close proximity to gfp-pcna foci exhibit significantly decreased mobility. we therefore conclude that presence of active replication forks constrains the movement of adjacent chromatin. single-molecule studies of dna replication antoine m. van oijen zernike institute for advanced materials, groningen university, nijenborgh 4. 9747 ag groningen, the netherlands e-mail: a.m.van.oijen@rug.nl advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. in a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. we are applying a single-molecule approach to study dna replication. i will present recent results of single-molecule studies of replication in bacterial and eukaryotic systems. by combining the stretching of individual dna molecules with the fluorescence observation of individual proteins, we visualize the dynamic interaction of replication factors with the fork. in the bacteriophage t7 replication system, we show that dna polymerases dynamically associate with and dissociate from the fork during replication. further, i will present new data from single-molecule replication studies in x. laevis oocyte extracts. we have developed a novel imaging scheme that permits single-molecule fluorescence experiments at concentrations of labeled protein that were hitherto inaccessible. using this method, we visualize, in real time, origin firing and fork movement. in force-extension diagrams of reference models of naked dna (freely jointed chain, wormlike chain) as well as extensionrotation diagrams of naked dna have been successfully recovered. of note, plectonemic structures are most efficiently simulated thanks to ode's collision detection code. new insights into nucleosome and chromatin fiber structure and dynamics will be presented. the study of the pkm.101 plasmid effect on the repair of dna j. vincze 1 , i, francia 2 , g. vincze-tiszay 1 1 hheif, budapest, hungary, 2 univ. debrecen, debrecen, hungary in our experiments was studied the effect of pkm.101 plasmid on repair of single strand breaks in dna induced by 60cogamma irradiation in e.coli k 12 ab 1157 (wild type) and its different rec mutant cells. the pkm.101 resistant-factor in case of the control decreases the sensitivity of radiation, which as we suppose, is reached by the help of dna conformation change. it can be supposed from the well known effect of radiation biology that by the effect of pkm.101, the ratio of dna radiation sensitive volumes by appearing its new conformation decreases. the pkm.101 r-factor in rec mutants in case of gamma irradiation shows effects in two ways. one is the ''chemical'' connection between the r-factor and dna, though the other relate to positive and negative ''induced'' radiation resistance from the local type effect of the connection of an r-factor and a rec mutant, and the two radiation resistant effects are added algebraically. as a result from the view of biology we have to categorize the radiation resistance and the connected repair processes as two different classes according to the change either in the chemical or in the induced radiation resistant effect. recent studies have indicated that two trimethylated peptides (k6, k7), derived from the parental hybrid peptide ca(1-7)m(2-9), strongly interact with a bacterial membrane model (mixture of zwitterionic and negatively charged lipids), but not with a membrane model of mammalian erythrocytes (zwitterionic lipids) [1] . a reduction of the cytotoxicity effect and an improvement of the therapeutic index have also been reported for the derivatives when compared with the parental ca(1-7)m(2-9) [2] . in this work, with the aim of providing insight on the interaction phenomena of the indicated peptides with zwitterionic and negatively charged membrane models, a systematic molecular dynamics study was carried out. full hydrated bilayers of dmpc:dmpg (3:1) and pope:popg(3:1) were studied in the presence of each peptide, and results analyzed in terms of peptide structure and membrane composition. lipid-water and lipid-lipid interactions at the membrane/water interface play important role in maintaining the bilayer structure, however, this region is not easily available for experimental studies. we performed molecular dynamics simulations of two bilayers composed of two different types of lipids: (1) dioleoylphosphatidylcholine (dopc); (2) galactolipid monogalactosyldiacylglycerol (mgdg). to investigate the properties of the membrane/water interface region, we performed analysis of lipid-lipid interactions: direct, via charge pairs (dopc) and hydrogen bonds (mgdg) as well as indirect, via water bridges. we also examined water-lipid interactions. existence of well-defined entities (lipids) linked by different types of interactions (hydrogen bonds, charge pairs, water bridges) makes the analysis of the membrane/ water interface region a suitable for a graph theoretical description. we applied a network analysis approach for comparative analysis of simulated systems. we note a marked difference between the organization as well as the dynamics of the interfacial region of the two bilayers. l-nucleoside analogues form an important class of antiviral and anticancer drug candidates. to be pharmacologically active, they need to be phosphorylated in multiple steps by cellular kinases. human phosphoglycerate kinase (hpgk) was shown to exhibit low specificity for nucleotide diphosphate analogues and its catalytic efficiency in phosphorylation was also affected. to elucidate the effect of ligand chirality on dynamics and catalytic efficiency, molecular dynamics simulations were performed on four different nucleotides (d-/l-adp and d-/l-cdp) in complex with hpgk and 1,3-bisphospho-d-glycerate (bpg). the simulation results confirm high affinity for the natural substrate (d-adp), while l-adp shows only moderate affinity for hpgk. the observed short residence time of both cdp enantiomers at the active site suggests very weak binding affinity which may result in poor catalytic efficiency shown for hpgk with d-/l-cdp. analysis of the simulations unravels important dynamic conditions for efficient phosphorylation replacing the single requirement of a tight binding. using the van der waals density functional based on the semilocal exchange functional pw86 together with a longrange component of the correlation energy [1] implemented in the siesta program code, we have calculated the band structure of the double stranded dna. the unit cell was built by taking together 10 gc (or at) homogenous base pairs and we have considered the translational symmetry as the periodic boundary condition. the results obtained are compared with the oligomer calculations taking up to seven base pairs. the band structure obtained with this van der waals density functional is also compared with results obtained with other exchange-correlation functionals as well as with band structure obtained by the hartree-fock crystal-orbital method taking into account the helical symmetry of the double stranded dna. the role of different parts of dna (base pairs, sugar-phosphate backbone, na ions) is also presented. transmembrane (tm) proteins comprise some 15% to 30% of the proteome but owing to technical difficulties, relatively few of these structures have been determined experimentally. computational modeling techniques can be used to provide the essential structural data needed to shed light on structurefunction relationships in tm proteins. low-resolution electrondensity maps, obtained from cryo-electron microscopy (cryo-em) or preliminary x-ray diffraction studies, can be used to restrict the search in conformational space. at the right resolution, the locations of tm helices can be roughly determined even when the amino acids are not visible. when these data are combined with physicochemical characteristics of amino acids (such as their hydrophobicity) and with evolutionary conservation analysis of the protein family, the location of the amino acids can be modeled. the modelstructure may provide molecular interpretations of the effects of mutations. moreover, it can be used to suggest molecular mechanisms and to design new mutations to examine them. the overall approach will be demonstrated using two human proteins: copper transporter 1 (ctr1), which is the main copper transporter in the human cell, and the 18 kda translocator protein (tspo) of the outer mitochondria. modelstructures of these proteins and their functional implications in health and disease will be discussed. calcium channels play a crucial role in many physiological functions and their selectivity mechanism is still an unresolved question and a subject of debate. a physical model of selective ''ion binding'' in the l-type calcium channel is constructed, and consequences of the model are compared with experimental data. this reduced model treats only ions and the carboxylate oxygens of the eeee locus explicitly and restricts interactions to hard-core repulsion and ion-ion and ion-dielectric electrostatic forces. according to the charge/space competition mechanism, the charge of structural ions attracts cations into the filter, while excluded volume effects are trying to keep them out. this is a competition between energy and entropy, where the balance of these terms minimizes free energy and determines selectivity. experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed. the model pore rejects alkali metal ions in the presence of biological concentrations of ca2+ and predicts the blockade of alkali metal ion currents by micromolar ca2+. conductance patterns observed in varied mixtures containing na+ and li+, or ba2+ and ca2+, are predicted. ca2+ is substantially more potent in blocking na+ current than ba2+. in apparent contrast to experiments sing buffered ca2+ solutions, the predicted potency of ca2+ in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with ca2+ for the pore. these experiments depend on the problematic estimation of ca2+ activity in solutions buffered for ca2+ and ph in a varying background of ulk salt. equilibrium binding affinity (expressed as the occupancy of the selectivity filter by various ions) is computed by equilibrium grand canonical monte carlo (gcmc) simulations. the conductivity of the channel is estimated from the equilibrium concentration profiles using the integrated nernst-planck equation. our simulations show that the selectivity of l-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. the reduced system selects for the cation that delivers the largest charge in the smallest ion volume. we have also performed dynamic monte carlo (dmc) simulations for a model ca channel to simulate current directly and present our results for the dynamical selectivity (expressed as the flux carried by various ions). we show that the binding affinity of ca2+ versus na+ is always larger than the dynamical selectivity because ca2+ ions are tightly bound to the binding site of the selectivity filter of the channel and, at the same time, their mobility and drift velocity is smaller in this region. carotenoids are used in light-harvesting complexes with the twofold aim to extend the spectral range of the antenna and to avoid radiation damages. the effect of the polarity and conformation of the environment is supposed to be responsible for the tuning of the electronic, optical and vibrational properties of peridinin carotenoid both in solution and in protein matrix. we investigate the effect of vibrational properties of peridinin in different solvents by means of vibrational spectroscopies and qm/mm molecular dynamics simulations 1 . the shift of vibrational fingerprints in the 1500-2000 cm -1 frequency region, due to the solvent polarity and proticity, is studied in three cases: cyclohexane (apolar/aprotic), deuterated acetonitrile (polar/aprotic) and methanol (polar/ protic). the frequencies and vibrational modes of the carbonyl, the allene, and the polyene chain were identified using effective normal mode analysis 2 and compared with the present and previous experimental data 3 . on the basis of our calculations and experiments in different solvents, we propose a classification of the four peridinins of the high-salt pcp form. the controlled self-assembly of functional molecular species on well defined surfaces is a promising approach toward the design of nanoscale architectures. by using this methodology, regular low-dimensional systems such as supramolecular clusters, chains, or nanoporous arrays can be fabricated. small biological molecules such as amino acids represent an important class of building blocks that are of interest for molecular architectonic on surfaces because they inherently qualify for molecular recognition and selfassembly [1] . the interaction between amino acids and solid surfaces is decisive for the development of bioanalytical devices or biocompatible materials as well as for a fundamental understanding of protein-surface bonding. we investigate the adsorbtion mechanism of the cysteine on au(111) surfaces by means of the dft [2] . our main concern is to describe the molecule-metal bonding mechanism. therefore we present a complex study, including the full determination of the density of states for the free and adsorbed molecule, the determination of molecule-surface bonding energy. the method of crystal orbital overlap populations is used in order to determine the contribution of specific atomic orbitals to the molecule-metal bond. it is now widely accepted that myoglobin (mb) is not simply an o 2 storage/delivery system but, depending on oxygen concentration, it exerts other fundamental physiological roles. recent studies revealed a widespread expression and, in particular, an over-expression in response to hypoxia, in various non-muscle tissues, including tumor cells. in human five different mb isoforms are present. the two most expressed ([90%) differ only at the 54 th position, k54 (mb-i) and e54 (mb-ii) respectively. since high-altitude natives from tibet are characterized by a higher mb concentration and locomotion efficiency, together with the observation that the mb overexpression is totally attributable to mb-ii, the idea that the latter might be one of the responses to high-altitude evolutionary adaptation, i.e. hypoxic environment, started to emerge. however, this is not yet supported by any structure/function investigation. we performed hundred nanoseconds md simulations on human mbs to investigate the structure and dynamics of both protein and surface water. important differences have been protein kinases play key roles in cell signaling and constitute crucial therapeutic targets. in normal cell, upon substrate binding, tyrosine kinase receptor kit undergoes extensive structural rearrangement leading to receptor dimerization and activation. this process is initiated by the departure of the juxta membrane region (jmr) from the active site, allowing the activation loop (aloop) deployment. the deregulation of kit activity is associated with various forms of cancer provoked by abnormalities in signal transduction pathways. mutations v560g (jmr) and d816h/v (a-loop) have been reported as oncogenic and/or drug-resistant. to contribute further in the understanding of kit activation/ deactivation mechanisms, we applied a multi-approach protocol combining molecular dynamics (md), normal modes analysis (nma) and pocket detection. disturbing structural effects, both local (a-loop) and long-range (jmr), were evidenced for kit d816h/v in the inactive state. nma showed that jmr is able to depart its position more easily in the mutants than in the wild type. pockets analysis revealed that this detachment is sufficient to open an access to the atp binding site. our results provided a plausible conception of mutant dimerization and a way to explore putative allosteric binding sites. transmembrane association of leukocyte integrin heterodimer might be mediated by a polar interaction choon-peng chng 1 and suet-mien tan 2 1 biophysics group, a*star institute of high performance computing, and, 2 school of biological sciences, nanyang technological university, republic of singapore the lateral association of transmembrane (tm) helices is important to the folding of membrane proteins as well as a means for signaling across the cell membrane. for integrin, a hetero-dimeric protein important for cell adhesion and migration, the association of its a-and b-subunits' tm helices plays a key role in mediating bi-directional mechanical signaling across the membrane. we found evidence from experiment and simulation for a polar interaction (hydrogenbond) across leukocyte integrin alb2 tm that is absent in the better-studied platelet integrin aiibb3 [1] . our coarse-grained molecular dynamics simulations of tm helix-helix selfassembly showed more native-like packing achieved by alb2 within the simulation timescale as compared to its 'lossof-function' b2t686g mutant or aiibb3 [2] . association free energy profiles also showed a deeper minimum at a smaller helix-helix separation for alb2, suggestive of tighter packing. the likely conservation of this polar interaction across the b2 integrin family further reinforces its importance to the proper functioning of leukocyte integrins. active extrusion of drugs through efflux pumps constitutes one of the main mechanisms of multidrug resistance in cells. in recent years, large efforts have been devoted to the biochemical and structural characterization of rnd efflux pumps in gram-negative bacteria, in particular the acrb/a-tolc system of e.coli. specific attention has been addressed to the active part of the efflux system, constituted by the acrb unit. despite the presence of several data, crucial questions concerning its functioning are still open. the understanding of the structure-dynamics-function relationship of mexb, the analogous transporter in p. aeruginosa, encounters even more difficulties, because of the lack of structural data of the transporter in complex with substrates. to shade some light on the activity of mexb, we performed computational studies on mexb interacting with two compounds, meropenem and imipenem, the first known to be a good substrate, and the second a modest one. several techniques were used in the present work, ranging from flexible docking [1] to standard and targeted molecular dynamics (md) simulations. starting from the published crystal structure [2] we identified the most probable poses of the two compounds in both the original experimental and in the md-equilibrated structures. we used information from acrb binding pocket in order to find relevant binding sites of the two compounds in the analogous binding pocket of mexb. meropenem frequently lies with appropriate orientation in a pocket similar to the one identified for doxorubicin in acrb [3] , while the occurrence of imipenem poses in the same pocket is very scarce. additionally, when present in the pocket, imipenem is located in a position that renders very unlikely its extrusion toward the oprm docking domain during the simulation of the functional peristalsis. the analysis of the trajectories has provided a complete inventory of the transporter and antibiotic hot spots, which is key information in terms of screening and design of antibiotics and inhibitors. clathrins are three-legged proteins with the intriguing ability to self-assemble into a wide variety of polyhedral cages. the nucleation and growth of a clathrin lattice against the cytosolic face of a cell membrane enables the endocytosis of membrane proteins and various external molecules, by wrapping the membrane around the cargo to produce a coated transport vesicle within the cell. clathrins can also self-assemble, in slightly acidic solutions devoid of auxiliary proteins, into empty cages. our simulations of this process, using a highly coarsegrained model, indicate that the key to self-assembly is neither calthrin's characteristic puckered triskelion shape, nor the alignment of four legs along all cage edges, but an asymmetric distribution of interaction sites around the leg's circumference. based on the critical assembly concentration, the binding strength in these cages is estimated at 25 to 40 k b t per clathrin. the simulations also answer the long-standing conundrum of how flat patches of purely hexagonal clathrin lattices, which in cryo electron microscopy are frequently seen to decorate cell membranes, can produce highly curved cages containing twelve pentagonal faces interdispersed between hexagonal faces. we present experimental evidence supporting this pathway. in eukaryotic cells, the exchange of macromolecules between the cytoplasm and the nucleus is mediated by specialized transport factors. by binding to these transporters, cargo molecules, which are otherwise excluded from entering the nucleus, can traverse the nuclear pore complex efficiently. most of the proteins mediating nuclear import and export exhibit a characteristic _-solenoid fold, which provides them with an unusual intrinsic flexibility. crm1 is an essential nuclear export receptor, which recognizes a very broad range of export cargoes. crm1-dependent nuclear export is ran-gtpase-driven, and recognition of rangtp and cargo is highly cooperative. however, recent crystal structures show that the binding sites for export cargos and rangtp are located at distant parts of crm1 [1] [2] [3] . we have used a combined approach of all-atom molecular dynamics simulations and small-angle x-ray scattering to study rangtp and cargo binding to crm1. we have found that the allosteric effect in crm1-dependent nuclear export arises from a combination of subtle structural rearrangements and changes in the dynamic properties of crm1. light-induced phototactic responds in green algae chlamydomonas reinhardtii are mediated via microbial-type rhodopsins, termed channelrhodopsin-1 (chr1) and channelrhodopsin-2 (chr2)1, which carry the chromophore retinal covalently linked to lysin via a schiff base and were shown to be directly light-gated ion-channels2. the n-terminal putative seven-transmembrane region of chr2 was shown to be responsible for the generation of photocurrents and exhibits sequence similarity to the well understood proton pump bacteriorhodopsin (br) and the sensory rhodopsin anabaena sensory rhodopsin (asr)3. as for the majority of membrane proteins, there is no 3d-structural data for chr2 available yet. here we present homology models of chr2 using two high-resolution x-ray template structures of br (1qhj4) respectively asr (1xio5) in order to get structural and functional insights into chr2. with both homology models we performed molecular dynamics (md) simulations in a native membrane/solvent environment using gromacs 4.0.36. comparison of energetic and structural results revealed obvious advantages of the br-based homology model of chr2. here we show that the br-based homology model is a reliable model of chr2 exhibiting structural features already found experimen-tally7. our br-based homology model of chr2 allows predictions of putative crucial residues within chr2. so we proposed several mutations within the chr2 sequence which are already accomplished. electrophysiologic and spectroscopic studies of these mutations are underway in order to confirm the functional relevance of these residues and to contribute to an optimized usage of chr2 as a powerful tool in optogenetics. ( neuroglobin is a recently discovered globin protein predominantly expressed in brain. its biological function is still elusive. despite the fact that neuroglobin shares very little sequence homology to the well-known globins as mioglobin and hemoglobin, they all have a characteristic globin fold with heme molecule bound to the distal pocket. the structural investigations and co binding kinetics revealed existence of cavities and tunnels within the protein matrix, where small ligands can be stored even for hundreds of microseconds [1] . in human neuroglobin there is one internal disulfide bond possible which existence is proved to have significant effect on ligand affinity [2] . in this study effects of temperature, ph, distal histidine mutation and presence of disulfide bond on co rebinding to neuroglobin are investigated by flash photolysis experiments. in parallel, the molecular dynamics simulations are performed in corresponding conditions in order to investigate structural change of neuroglobin and especially changes in distribution of internal tunnels and cavities able to bind diatomic ligands in response to different physical conditions listed above. the thrombospondin family, being extracelluar proteins, is known to be implicated in various physiological processes such as wound healing, inflammation, angiogenesis and neoplasia. the signature domain of thrombospondins shows high sequence identities and thus allows us to transfer results obtained, studying this complex calcium reach part of the proteins, from one member of the family to the other. the domain is known to play a key role in hereditary diseases such as psach or med. in this part of thrombospondins lies a binding site to integrins, important for cell attachment. it is further known that the lectin like globe binds to cd-47, a feature known to be important in cancer research. as the theoretical unit we are trying to resolve these problems by numerical means and are constantly challenged by the size, where thrombospondin can be a huge trimeric protein as one strand can measure 430 kda, and the large variety of subdomains found in this proteins. we are thus facing a multiscale problem which can range from solving, by means of quantum mechanics a specific ion binding site, to large scale abstraction by continuum mechanics. in our talk we will show you our newest results that we obtained by simulating calcium rich c-terminal domain which is known to be conserved across the entire family, and give you an outlook into the future of our research. the process of swift heavy ions energy deposition while penetrating a solid or scattering on its surface can result in a strong and nonequilibrium excitation of matter. an extremely localized character of this excitation, meanwhile, can make possible both selective changes in chemistry of matter 1 and its surface nanomodifications 2 . since possible applications have been found in bio-and it-technologies (cancer curing and nanostructuring respectively) in the last decade, the heavy ion bombardment technique has attracted a lot of scientific interest 3, 4 . the processes of fast energy deposition into the solid and its further dissipation, however, are essentially perturbed with highly excited and nonequilibrium state of both lattice and electron systems. at such conditions therefore, the precision in treatment of processes of electron thermalization, fast electron heat conduction, and phase transformation of the overheated solid becomes crucial. having several physical models to handle the mentioned processes, it is nevertheless difficult to describe all of them within a scale of a single computational approach. our work is aimed on elaboration of the atomistic-continuum model 5 of heavy ion bombardment of solids. in particular, the model will be applied to study the formation of nanohillocks in the experiments on swift heavy ion xe + scattering on srtio 3 surface 2 . [3] g. aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). the presence of the human aquaporin 5 (hsaqp5) in cells proximal to airinteracting surfaces (eyes, lacrimal glands, salivary glands, lungs, stomach etc.) suggest its potentially important role in ''wetting'' these surfaces. the high-resolution x-ray structure of the hsaqp5 tetramer (pdb code 3d9s) exhibits two important features: (i) lack of the four fold symmetry, common in most of the aquaporins, and (ii) occlusion of the central pore by a phosphatidylserine lipid tail. in this study we investigate the importance of these two features on the transport properties of the human aqp5 by means of molecular dynamics simulations. we found that the asymmetry in the tetramer leads to a distribution of monomeric channel structures characterized by different free energy landscapes felt by the water molecules passing through the channel. furthermore, the structures' distribution is influenced both by the presence/absence of the lipid tail in the central pore, and by the lipid composition of the bilayer that solvates the hsaqp5 tetramer. elucidating the modular structure of the protein g c2 fragment and human igg fc domain binding site using computer simulations hiqmet kamberaj faculty of technical sciences, international balkan university, skopje, r. of macedonia protein-protein recognition plays an important role in most biological processes. although the structures of many protein-protein complexes have been solved in molecular detail, general rules describing affinity and selectivity of proteinprotein interactions break down when applied to a larger set of protein-protein complexes with extremely diverse nature of the interfaces. in this work, we will analyze the non-linear clustering of the residues at the interface between proteins. the boundaries between clusters are defined by clustering the mutual information of the protein-protein interface. we will show that the mutations in one module do not affect residues located in a neighboring module by studying the structural and energetic consequences of the mutation. to the contrary, within their module, we will show that the mutations cause complex energetic and structural consequences. in this study, this is shown on the interaction between protein g c2 fragment and human igg fc domain by combining molecular dynamics simulations and mutual information theory, and computational alanine scanning technique. the modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. the results test our understanding of the dominant contributions to the free energy of protein-protein interactions, can guide experiments aimed at the design of protein interaction inhibitors, and provide a stepping-stone to important applications such as interface redesign. membrane proteins can form large multimeric assemblies in native membranes that are implicated in a wide range of biological processes, from signal transduction to organelle structure. hydrophobic mismatch and membrane curvature are involved in long range forces largely contributing to such segregation. however, the existing assembly specificity is thought to be coded in the atomic details of protein surface and topology. these are best described in high resolution structures and atomistic molecular dynamics simulations. in order to explore more systematically such forces and energetics arising at intermediate time scales and resolution, we use coarse grained molecular dynamics simulations applied to 20 membrane systems spanning over 5 to 15us. as a first glimpse we study how proteins induce lipid perturbations using a previously developed conformational entropy estimator. we show that in the model membrane where hydrophobic mismatch is present, lipid perturbations extend up to *40a from the protein surface. however, significant variations in perturbation profiles are seen. parameters such as protein shape, surface topology, and amino acid physicochemical properties are studied to discover the parameters governing such perturbations. crossing energy barriers with self-guided langevin dynamics gerhard kö nig, xiongwu wu, bernard brooks national institutes of health, national heart, lung and blood institute, laboratory of computational biology, rockville, md, usa even with modern computer power, the applicability of molecular dynamics simulations is restricted by their ability to sample the conformational space of biomolecules. often high energy barriers cause normal molecular dynamics simulations to stay trapped in local energy minima, leading to biased results. to address this problem, self-guided langevin dynamics (sgld) were developed. it enhances conformational transitions by accelerating the slow systematic motions in the system. this is achieved by calculating the the local average of velocities and adding a guiding force along the direction of systematic motions. thus, the efficiency of sgld is governed by three factors: a.) the friction constant involved in the langevin dynamics b.) the local averaging time and c.) the guiding factor that determines the guiding force. however, the guiding force also causes deviations from the original ensemble that have to be corrected by reweighting the data, thus decreasing the efficiency. here, we explore the three-dimensional parameter space of sgld for several benchmark systems with particularly rough energy surfaces. based on our data, we supply guidelines for the optimal selection of sgld parameters, to allow the extension of our method to other biological problems of interest. propagation of d816v/h mutation effects across kit receptor e. laine, i. c. de beauchê ne, c. auclair and l. tchertanov lbpa, cnrs -ens de cachan, france receptor tyrosine kinases (rtks) regulate critical biological processes. constitutive activation of rtks provokes cancers, inflammatory diseases and neuronal disorders. biological data evidenced that oncogenic mutations of the rtk kit, located either in the juxtamembrane region (jmr) or in the activation loop (a-loop) -as is the case of d816v/h, displace the equilibrium of conformational states toward the active form. we present a multi-approach study that combines molecular dynamics (md), normal modes (nm) and pocket detection to characterize and compare the impact of d816v/h on the structure, dynamics and thermodynamics of kit. we have evidenced a local structural destabilization of a-loop induced by the mutation and a long-range effect on the structure and position of jmr. we have further correlated these observations with experimental data and decipher some details about the activation mechanisms of the mutants, involving leverage of the jmr negative regulation and release of an access to the catalytic site. through the identification of ''local dynamic domains'' and the recording of interactions within the protein, we propose a model of the mutational effects propagation, which highlights the importance of both structural distortion and local conformational fluctuations. investigation of biological active azolidinones and related heterocycles refer to one of the most successful scientific projects of dh lnmu. it is based on three strategic vectors: organic synthesis, pharmacological research, rational design of ''drug-like'' molecules (including in silico approaches). while applying the research strategy we succeeded in gaining a number of interesting results that make possible to extend the field, especially in the scope of ''drug-like'' molecules design, notably it has focused on the search of new anticancer agents. anticancer activity screening was carried out for more than 1000 compounds (us nci protocol (dtp) based on obtained directed library over 5000 new compounds, among them 167 compounds showed high activity level. for the purpose of optimization and rational design directions of highly active molecules with optimal ''drug-like'' characteristics and discovering of possible mechanism of action sar, compare analysis, molecular docking and qsa(p)r were carried out. obtained results allowed to form main directions of possible anticancer activity mechanisms, which probable are apoptosis-related. nowadays the investigation of cellular and molecular aspects of anticancer effects is in progress. regulation of (bio)chemical reactions by mechanical force has been proposed to be fundamental to cellular functions [1, 2, 3] . atomic force microscopy experiments have identified the effect of mechanical force on the reactivity of thiol/disulfide exchange, a biologically important reaction [4] . in order to understand the influence of the force at an atomistic level, we have performed hybrid quantum mechanicsmolecular mechanics (qm/mm) transition path sampling simulation of the reaction under external forces. the results of the simulations supported the experimental findings and demonstrated that the location of the transition state on the free energy surface was shifted due to force [5] . in contrast to our findings, however, a recent experimental study suggests only a weak coupling between the mechanical force and the reaction rate [6] . in this study, the reactants were covalently linked to a stilbene molecule. in this system a force can be applied by photo-isomerization from the relaxed trans to the strained cis configuration. a drawback of this system is that one cannot easily determine the forces that acting on the reaction coordinate. therefore, we have developed a force distribution analysis method for quantum mechanical molecular dynamics simulations. the results of the analysis show how isomerization of stilbene alters the forces acting on the reacting atoms. the force distribution is essential for understanding how chemistry is controlled by external forces. [ conformational space modelling (csm) is a promising method for membrane protein structure determination. it is based on the concept of the side-chain conformational space (sccs), which is formed by the allowed side-chain conformations of a given residue. each sccs can be calculated from a 3d structure or measured via epr-sdsl experiments. for structure determination a set of singly spinlabelled mutants is needed. the final structure is obtained by altering an initial (possibly random) 3d structure until the best fit between the calculated and measured sccs for the whole set is found. such optimization is computationally intensive; therefore csm includes several empirical approximations. one of them describes the effect of the lipid tails on the sccs. the implementation is not trivial as lipids diffuse in the plane of the membrane and the lipid tails behave differently at different membrane depths. to unravel this relationship adaptive biasing force md simulations were used. an alanine peptide helix was made in silico, spin-labelled at the middle and inserted perpendicularly into a dmpc membrane. the free energy of the spin-label orientation at various membrane depths was calculated. a 3d free energy surface describing the membrane ''depth'' effect was obtained. it is known that b-cyclodextrins (bcds) are able to modify the cholesterol content of cell or model membranes. however the molecular mechanism of this process is still not resolved. using molecular dynamics simulations, we have been able to study the bcd-mediated cholesterol extraction from cholesterol monolayers and lipid-cholesterol monolayer mixtures. we have investigated many conditions that would affect this process (e.g. lipid-cholesterol ratio, lipid chain unsaturation level) our results can be summarized as follow: i) dimerization of bcds, ii) binding of the dimers to the membrane surface assuming either a tilted (parallel to the membrane normal) or untilted (90°respect to the normal of the membrane) configuration, iii) the latter configuration is suitable to extract cholesterol at a reasonable computational time scale (100-200 ns), however, this process may be affected by unfavorable energy barriers (from 10 to 80 kj mol -1 ), iv) desorption of the complex brings cholesterol in solution, v) the bcd-cholesterol complex is able to transfer cholesterol to other membranes. with a clearer understanding of the basic molecular mechanism of the bcd mediated process of cholesterol extraction, we can begin to rationalize the design of more efficient bcds in numerous applications. the mechanism of the complex formation of biopolymers with ligands including the solvent molecules is an actual problem of modern biophysical and biological science. polypeptides form a secondary structure and mimic the motifs of the protein architecture. the study of complexation between polypeptides and solvent molecules leads to deeper understanding of the basic interaction of proteins with environment at atomic level. besides polypeptides are promising for the development of applications which encompass some of the following desirable features: anti-fouling, biocompatibility, biodegradability, specific biomolecular sensitivity. on this account polypeptides have a great significance for a variety modern applications ranging from the nanoscale medicine devises up to food technology and others. we compare the results of calculations of complexes between helical polypeptides (polyglutamic acid in neutral form and poly-c-benzyl-l-glutamate) and water molecules at dft pbe level and the results of ftir-spectroscopic study of the film of wetted polypeptides. vibrational spectroscopy is one of the most useful experimental tools to study non-covalent bonded complexes, and calculated spectra in comparison with experimental data are reliable test for reality of simulated complexes. platelet aggregation at the site of vascular injury is vital to prevent bleeding. excessive platelet function, however, may lead to thrombus formation after surgery. therefore, an accurate measure and control of platelet aggregation is important. in vitro platelet aggregometry monitors aggregate formation in platelet rich plasma triggered by agonists such as adp, epinephrine or collagen. the fraction of aggregated platelets is plotted versus time, and platelet function is assessed by analyzing the plot's morphology. we propose new measures of platelet function based on a compartmental kinetic model of platelet aggregation induced by adp. our model includes three compartments: single, aggregated and deaggregated platelets. it is simpler than earlier models and agrees with experimental data. the model parameters were determined by non-linear least squares fitting of published data. we associated the kinetic parameters with the activity of the adp receptors p2y1 and p2y12. to this end, we studied published data obtained in the presence and in the absence of specific antagonists of these receptors. comparison of kinetic parameters of healthy subjects with those of patients with myeloproliferative disorder (mpd) shows that the function of p2y12 is significantly reduced in mpd. coarse-grained modeling of drug-membrane interactions manuel n. melo, durba sengupta, siewert-jan marrink groningen biomolecular sciences and biotechnology institute, university of groningen, groningen, the netherlands the martini coarse-grained (cg) forcefield was used to simulate the actions of the antimicrobial peptide alamethicin and of the anti-tumoral drug doxorubicin. both drugs were shown to interact strongly with a fluid phospholipid bilayer, and aggregate there, in agreement with experimental results. because doxorubicin may establish intermolecular h-bonding, and thus lower its dipole moment, the cg representation of a doxorubicin dimer was adjusted. this less polar dimer was then tested for translocation and/or pore formation. contrary to results of atomistic simulations, alamethicin aggregates did not spontaneously open pores. they did so, however, when the size of the water beads was decreased. several small independent pores could then be observed. the magnitude of the permeability of these pores is analyzed and compared to experimental values. the occurrence of multiple small pores could indicate that the different conductance levels experimentally observed for alamethicin might simply result from the association of different numbers of these small pores. polarization refers to the asymmetric changes in cellular organization in response to external or internal signals. neuronal polarization begins with the growth of a single neurite shortly after cell division, followed by the growth of a second neurite at the opposite pole. this early bipolar shape is critical for brain function, as it defines axis of migration and consequently proper three dimensional organization and nerve circuitry. however, it is not known if a direct relationship exists between the formation of the second, opposite, neurite and the mechanisms involved in the formation of the first. we tackled this issue through mathematical modeling, based on membrane traffic (exocytosis-endocytosis), and lateral diffusion. with this approach, we demonstrated that a single pole of molecular asymmetry is sufficient to induce a second one at the opposite side, upon induction of growth from the first pole. our work gives mathematical proof that the occurrence of a single asymmetry in a round cell is sufficient to warrant morphological bipolarism. trypsin is one of the best characterized serine proteases. enzyme acylation process is required for substrate degradation. there is a lot of information about how this process undergoes. however, in order to obtain a more detailed description of the catalytic triad mechanism, a qm/mm approach was used. we used the hybrid qm/mm potential implemented in amber11 package. in the qm calculations a dft hamiltonian was used. we develop an approach based on the adaptively biased md in order to obtain the free energy surface of the conformational space defined by the reaction coordinates. this approach presents some characteristics of steered md and umbrella sampling procedures. our results offer information about the lowest energy trajectory, the barrier profile of the reaction, and the geometry of the transition state. this method also provides a further insight into the role of specific residues in the reaction. substituting asp102, member of the catalytic triad, for ala we were able to detect an increase of the barrier profile. this was due to the loss of the interaction of carbonyl group of asp102 with nd of his57, which make ne of this residue a worst proton acceptor. this results show our approach as a valuable method in the study of enzymatic mechanisms. the intracellular media comprise a great variety of macromolecular species that are not individually concentrated, but being preset in the same compartment they exclude each other's volume and produce crowding. crowding has a profound impact on protein structure and determines conformational transitions and macromolecular association. we investigated macromolecular association on a 50% w/w bovine serum albumin (bsa) solution by time-domain terahertz (thz) spectroscopy and molecular modeling. molecular crowding was simulated by including two bsa molecules in the same water box. we generated *300 such dimeric models, computed their thz spectra by normal modes analysis and compared them with the experimental data. the best bsa dimer model was selected based on the agreement with the experiment in the lowest frequencies domain of up to 1 thz. symmetry constraints improve accuracy of ion channels models. application to two-pore-domain channels adina l. ion channels are important drug targets. structural information required for structure-based drug design is often filled by homology models (hm). making accurate hm is challenging because few templates are available and these often have substantial structural differences. besides, in molecular dynamics (md) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate hm refinement using md. we evaluate the ability of symmetry-constrained md simulations to improve hm accuracy, using an approach conceptually similar to casp competition: build hm of channels with known structure and evaluate the efficiency of various symmetry-constrained md methods in improving hms accuracy (measured as deviation from x-ray structure). results indicate that unrestrained md does not improve accuracy, instantaneous symmetrization improves accuracy but not stability during subsequent md, while gradually imposing symmetry constraints improves both accuracy (by 5-50%) and stability. moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting hm accuracy. we further used this method to refine hm of trek channels. we also propose a gating mechanism for mechanosensitive channels that was further experimentally confirmed. nucleotide modifications and trna anticodon-mrna codon interactions on the ribosome olof allné r and lennart nilsson karolinska institutet, stockholm, sweden molecular dynamics simulations of the trna anticodon and mrna codon have been used to study the effect of the common trna modifications cmo 5 u34 and m 6 a37. in trna val these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. previous data suggest entropic effects are mainly responsible for the extended reading capabilities but detailed mechanisms have remained unknown. the aim of this paper is to elucidate the details of these mechanisms on an atomic level and quantify their effects. we have applied: extensive free energy perturbation coupled with umbrella sampling, entropic calculations of trna (free and bound to the ribosome) and thorough structural analysis of the ribosomal decoding center. human neuroserpin (hns) is a serine protease inhibitor (serpin) of a tissue-type plasminogen activator (tpa). the conformational flexibility and the metastable state of this proteins underlies to misfolding and to dysfunctional mutations causing a class of rare genetic diseases which share the same molecular basis. the conformational transition of the native form, triggered upon the cleavage at reactive center loop (rlc), releases a complex of the cleaved form bound to the inactivated target protease. without rlc cleavage a stable inactive latent form can be obtained by intra/inter molecular loop insertion leading to polymerization. this work concerns the study of the three above mentioned forms of hns by md simulations to investigate the relation between their conformational stability and. the starting native and cleaved configurations are based on the x-ray structure, while the latent form is here modelled. the results of the simulation reveal a striking conformational stability along with the intrinsic flexibility of selected regions of the fold.the analysis of the essential collective modes of the native hns shows that the initial opening of the b-sheet a coincides with several changes in the local pattern of salt bridges and of hydrogen bonds. regulation of ubiquitin-conjugating enzymes: a common mechanism based on a pattern of hydrophobic and acidic residues enzyme temperature adaptation generally involves a modulation of intramolecular interactions, affecting proteins dynamics, stability and activity [1] [2] . in this contribution, we discuss studies of different classes of extremophilic enzymes, focusing on cold-adapted variants, as well as their mesophilic-like mutants, performed by all-atom molecular dynamics simulations with particular attention to structural communication among residues within the threedimensional architecture [3] [4] . common adaptation strategies turned out to be based on improved local flexibility in the proximity of the functional sites, decrease in interconnected electrostatic interactions, and modulation of correlated motions and networks of communicating residues. specific differences related to the diverse protein folds can also be detected. bneurexins and neuroligins are cell adhesion molecules and play important role in synapse junction formation, maturation and signal transduction between neurons. mutations in genes coding these proteins occurs in patients with cognitive diseases like autism disorders, asperger syndrome and mental retardation [1] . it has been found that the complex bneurexin-neuroligin has also an important role in angiogenesis [2] . herein we will present molecular foundations of bneurexin-neuroligin interactions obtained from all-atom molecular dynamics simulations of bneurexin, neuroligin and their complex (3b3q) [3] . 50 ns md trajectories (charmm force field) were analyzed and roles of ca2+ and n-actetyl-d-glucosamine posttranslational modifications in intermolecular interactions were scrutinized. advances in hardware and software have enabled increasingly long atomistic molecular dynamics simulations of biomolecules, allowing the exploration of processes occurring on timescales of hundreds of microseconds to a few milliseconds. increasing the length of simulations beyond the microsecond time scale has exposed a number of limitations in the accuracy of commonly employed force fields. such limitations become more severe as the size of the systems investigated and the length of the simulations increase. here i will describe the force field problems that we have encountered in our studies, how we identified and addressed them, and what we have learned in the process about the biophysics of the systems we are investigating. while the quest for a ''perfect'' force field is not over (and may never be), our work has greatly improved the accuracy and range of applicability of simple physics-based force fields, to the point that reliable predictions can now be obtained from millisecond-timescale simulations of biomolecules. local anesthetics (la) are pain-relief drugs, widely used in medicine and dentistry. the relatively short duration of analgesia still restricts their clinical use for the treatment of chronic pain. nowadays, intensive research is focused on anesthetics entrapped into liposomes to enhance their activity and pharmacokinetic properties [1] . in this work, we investigated the encapsulation of prilocaine (plc), an aminoamide local anesthetic, into a small unilamellar liposome. on the line of our previous work [2] , we have carried out molecular dynamics (md) simulations using a coarse grain model up the microsecond time scale. in this way, we compare the effects of the concentration of la at fisiological ph. we were able to capture important features of the plc-vesicle interactions. the behavior of plcs at fisiological ph is essentially a combination of high and low ph: we found that all neutral plc molecules rapidly diffuse into the hydrophobic region of the vesicle adopting an asymmetric bimodal density distribution. protonated plc molecules (pplc) initially placed in water were instead only found on the external monolayer, with a high rate of exchange with the water phase and no access to the inner part of the liposome in a concentration dependent way. we focus on applications of molecular and mesoscale simulation methodologies to the cellular transport process of endocytosis, i.e., active transport mechanisms characterized by vesicle nucleation and budding of the cell membrane orchestrated by protein-interaction networks, and functionalized carrier adhesion to cell surfaces. we discuss theoretical and computational methodologies for quantitatively describing how cell-membrane topologies are actively mediated and manipulated by intracellular protein assemblies. we also discuss methods for computing absolute binding free energies for carrier adhesion. we present rigorous validation of our models by comparing to diverse range of experiments. the importance of delta-opioid receptors as target of a large number of drugs is well recognized, but the molecular details of interaction and action of the compounds are largely unknown. in an effort to shade some light on this important issue we performed an extensive computational study on the interaction of two compounds, clozapine and desmetilclozapine, with a delta-opioid receptor. according to experiments, the lacking of a single methyl group in desmetilclozapine with respect of clozapine makes the former more active than the latter, providing a system well suited for a comparative study. we investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by metadynamics, an algorithm that allows the simulation of events that are otherwise out of range for standard molecular dynamics simulations. our results point out that the action of the compound is related to the spatial distribution of the affinity sites it visits during its permanency. desmetilclozapine has a larger distribution of residues, which is interacting with, than clozapine. however, large conformational changes of the receptor were not observed in the presence of both compounds. thus, a more dynamical picture of ligand-receptor affinity is proposed on the basis of the results obtained, involving the competition among different stable states as well as the interaction with the solvents. such information might be useful to provide hints and insights that can be exploited in more structure-and-dynamics-oriented drug design. the coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. in particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. we study such phenomena using a method or investigating local flexibility on the residue scale, which combines a reduced protein representation with brownian dynamics simulations. we performed calculations on the enzyme guanylate kinase in order to study its mechanical response when submitted to anisotropic deformations. the resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinities that were observed experimentally. further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechanhiv-1 protease autocatalyses its own release from gag and gagpol precursor polyproteins into mature functional proteins. as it is functional in the dimeric form, whilst initially, only a single monomer is embedded within each gagpol chain, the question arises as to what cut's the cutter. two individual monomers in different gagpol chains are known to come together to form an embedded-dimer precursor protease. mature-like protease activity is concomitant with n-terminal intramolecular cleavage of this transient embedded-dimer precursor, but how this crucial maturation-initiating step is physically achieved, has remained unknown. here, we show via 400 all-atom explicit solvent molecular dynamics simulation runs of 400 ns each that the n-terminal of an immature-like protease, with the n-terminal initially unbound as in the gagpol polyprotein, can self-associate to the active site and therefore be cleaved under conditions of thermodynamic equilibrium, identifying possible binding pathways at atomic resolution, in agreement with previous indirect experimental evidence [1] . the binding pathway predominantly makes use of the open conformation of the beta-hairpin flaps characterised by us previously [2] , and the n-terminal binds across the entire active site in good agreement with crystal structures of a cleavage-site peptidebound protease. the n-terminus serves two roles, firstly in the maturation of the protease itself by self-associating to the protein and then as a stabilizing component of the dimer interface in a mature protease. targeting the prior mechanism could be the focus of a novel therapeutic strategy involving immature protease inhibition. [ knotted proteins are the object of an increasing number of experimental and theoretical studies, because of their ability to fold reversibly in the same topologically entangled conformation. the topological constraint significantly affects their folding landscape, thus providing new insight and challenges into the funnel folding theory [1] . recently developed experimental methods to trap and detect knots have suggested that denaturated ensembles of the knotted proteins may be knotted [2] . we present numerical simulations of the early stage of folding of the knotted proteins belonging to protein families mtase (methyltransferase) and sotcase (succinyl-ornithine transcarbamylase), and of their unknotted homologues [3] . our results show that native interactions are not sufficient to generate the knot in the denaturated configurations. however, when non-native interactions are included we observe formation of knots only in the chains whose native state is knotted. in addition, we find that the knots are formed through a universal mechanism. such a knot formation mechanism correctly predicts the fraction of the knotted proteins found in nature and can be used to make qualitative prediction on their folding times. shape and motility and also for numerous signaling processes. adhesion is based on non-covalent interactions between transmembrane proteins and the extracellular matrix. cells are able to create two-dimensional assemblies of integrins, so called focal adhesions, which they use to stick to the substrate and collect information about the environmental properties. the goal of this work is a deeper understanding of the formation and the stability of these adhesion clusters. bond cluster formation and disintegration is dynamically modeled with the aid of monte carlo simulations. in the model, a membrane is attached to a flat surface via a variable number of adhesion bonds. the spacial configuration of these adhesion points subjected to an inhomogeneous stress field maps into a distribution of local membrane/ surface distances. we introduce a model which explicitly accounts for the membrane elasticity and demonstrate that such models are able to explain the spontaneous formation of adhesion bond clusters. structure based models are successful at conjugating the essence of the energy landscape theory of protein folding [1] with an easy and efficient implementation. recently their realm expanded beyond single protein structure, been used profitably to widely study large conformational transitions [2] [3] . still, when dealing with conformational transition between two well-defined structures an unbiased and realistic description of the local backbone and sidechain interactions is necessary. the proposed model merges a precise description of these interactions with a structure-based long range potential that takes into account different conformers. we present the results of the activation of the catalytic domain of human csrc tyrosine kinase for which we reconstructed the transition free energy and the description of the activation loop flexibility. the excellent model performances in terms of speed and the satisfactory accuracy of the description of the system and its flexibility are promising for a more systematic study of the tyrosine kinase family activation mechanisms. [ we introduce a previously undescribed technique for modelling the kinetics of stochastic chemical systems. we apply richardson extrapolation, a sequence acceleration method for ordinary differential equations, to a fixed-step tau-leaping algorithm, to produce an extrapolated tau-leaping method which has weak order of accuracy two. we prove this mathematically for the case of linear propensity functions. we use four numerical examples, two linear and two nonlinear, to show the higher accuracy of our technique in practice. we illustrate this by using plots of absolute error for a fixed-step tau-leap and the extrapolated tau-leap. in all cases, the errors for our method are lower than for a fixedstep tau-leap; in most cases they are second order of accuracy. the major tripartite efflux pump acrab-tolc is responsible for the intrinsic and acquired multidrug resistance in escherichia coli. at heart of the extrusion machinery there is the homotrimeric transporter acrb, which is in charge of the selective binding of structurally and chemically different substrates and energy transduction. the effects of conformational changes, which have been proposed as the key features of the extrusion of drugs, are investigated at molecular level using different computational methods like targeted molecular dynamics. simulations, including almost half a million atoms, have been used to assess several hypotheses concerning the structure-dynamics-function relationship of the acrb protein. the results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. in addition, we provide evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. using these atomistic simulations the role of specific amino acids during the transitions can be identified, providing an interpretation of sitedirected mutagenesis experiments. additionally, we discuss a possible role of water molecules in the extrusion process. virus inhibitory peptide (virip), a 20 amino acid peptide, binds to the fusion peptide (fp) of human immunodeficiency virus type 1 (hiv-1) gp41 and blocks viral entry. molecular dynamics (md) and molecular mechanics/poisson-boltzmann surface area (mm/pbsa) free energy calculations were executed to explore the binding interaction between several virip derivatives and gp41 fp. a promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations and the use of multiple internal dielectric constants for the mm/pbsa calculations depending on the amino acids sequence. bases on these results, a virtual screening experiment was carried out to design enhanced virip analogues. a selection of peptides was tested for inhibitory activity and several improved virip derivatives were identified. these results demonstrate that computational modelling strategies using an improved mm/pbsa methodology can be used for the simulation of peptide complexes. as such, we were able to obtain enhanced hiv-1 entry inhibitor peptides via an mm/pbsa based virtual screening approach. an essential step during the hiv life cycle is the integration of the viral cdna into the human genome. hiv-1 integrase mediates integration in a tight complex with the cellular cofactor: ledgf/p75 [1] . disruption of the interaction interferes with hiv replication and therefore provides an interesting new drug target for antiretroviral therapy [2, 3] . here we present the structure based discovery and optimization of a series of small molecule inhibitors that bind to hiv-1 integrase and block the interaction with ledgf/p75. the work flow was set up according to a funnel principle in which a series of virtual screening tools were applied in such way to discard at each step molecules unlikely to be active against the desired target (including 2d filtering, pharmacophore modelling and molecular docking) the activity and selectivity of the selected molecules were confirmed in an alpha screen based assay, that measure protein-protein interaction in vitro, and furthermore by in vivo experiments. active compounds proceeded towards crystallographic soaking into the receptor protein crystals. these crystal structures not only validated the binding mode and activity of the hit compounds, but furthermore were used as a platform for structure based drug design which resulted in the rational discovery of new hit compounds and optimized lead compounds. in vitro and in vivo experiments validated the mechanism of action of these compounds and show that they are a novel class of antiretroviral compounds with in vivo inhibitory activity by targeting the interaction between ledgf/p75 and hiv-1 integrase. cross resistance profiling indicate that these compounds are active against current resistant viral strains. [4] currently the most potent inhibitors show an in vivo ic 50 of 55nm. these compounds are promising for future pharmaceutical optimizations to be used in the clinic as new antiretroviral agents. crystallography was used to validate the binding mode of the discovered inhibitors. insights in the interaction of the ligand-protein complex allowed for rational design of optimized inhibitors. ligand-induced structural changes are small, thermal fluctuations can play a dominant role in determining allosteric signalling. in thermodynamic terms, the entropy change for subsequent binding is influenced by global vibrational modes being either damped or activated by an initial binding event. one advantage of such a mechanism is the possibility for long range allosteric signalling. here, changes to slow internal motion can be harnessed to provide signalling across long distances. this paper considers homotropic allostery in homodimeric proteins, and presents results from a theoretical approach designed to understand the mechanisms responsible for both cooperativity and anticooperativity. theoretical results are presented for the binding of camp to the catabolite activator protein (cap) [1] , where it is shown that coupling strength within a dimer is of key importance in determining the nature of the allosteric response. results from theory are presented along side both atomistic simulations and simple coarse-grained models, designed to show how fluctuations can play a key role in allosteric signalling in homodimeric proteins. [ reversibly switchable fluorescent proteins (rsfps) can be switched between a fluorescent (on) and a nonfluorescent (off) state which is accompanied by a cis-trans isomerization of the chromophore on the molecular level 1,2. this unique property has already provided new aspects to various microscopy techniques, such as high resolution microscopy, fcs or monochromatic multicolor microscopy 3-5. despite of their established potential, rsfps still have a major drawback: the wavelength for fluorescence excitation is always one of the two switching wavelengths. the imaging process thus inevitably results in the switching of a small fraction of the rsfps, which might hinder or complicate some experiments. we developed a new reversibly switchable fluorescent protein which eliminates the problem of the coupling between switching and fluorescence excitation. this fluorescent protein follows an unusual and currently unknown mechanism of switching between a fluorescent and a nonfluorescent state. it is brightly fluorescent and exhibits an excellent signal to noise ratio. in parallel studies [2] , qd-based ligands (egf, mabs) were targeted to egfr in gliomas. cell-cultures, animal models and ex vivo biopsies of human high-grade as well as low-grade gliomas showed high probe specificity.. the aim is to define more precisely the tumor boundaries at the time of resection. we used the programmable array microscope designed for sensitive, high-speed optical sectioning, particularly of living cells. the pam is based on structured illumination and conjugate detection using a digital micromirror device (dmd) [3] located in a primary image plane. the unique feature is the rapid, (re)programmable adjustment of local excitation intensity, dynamic, on-the-fly optimization is thus achieved, e.g. multipoint frap [4] , light exposure minimization and object tracking [5] , or super-resolution strategies. the features and operation of the 3rd generation pam will be presented. contraction of muscle cells, motility of microorganisms, neuronal activity, and other fast cellular processes require microscopic imaging of a three-dimensional (3d) volume with a video-rate scanning. we present 3d video-rate investigations of structural dynamics in biological samples with the multicontrast third-and second-harmonic generation as well as fluorescence microscope. the multidepth scanning is achieved by two combined laser beams with staggered femtosecond pulses. each of the beams is equipped with a pair of deformable mirrors for dynamic wavefront manipulation enabling multidepth refocusing with simultaneous corrections for optical aberrations. combined, more than 250 frames per second lateral scanning with fast refocusing enables the 3d video-rate imaging of dynamically moving structures. in addition, combination of two laser beams is accomplished at two perpendicular polarizations enabling live imaging of sample anisotropy, which is important for structural studies particularly with the second harmonic generation microscopy. investigations of beating chick embryo hearts with the 3d video-rate scanning microscope revealed multidirectional cardiomyocyte contraction dynamics in myocardial tissue. intricate synchronization of contractions between different layers of myocytes in the tissue will be presented. the video-rate 3d microscopy opens new possibilities of imaging fast biological processes in living organisms. confocal fluorescence microscopy is an invaluable tool to study biological systems at cellular level also thanks to the synthesis of always new specific fluorescent probes, e.g. multiprobe labelling enables complex system characterization. however, only the recent employment of narrowband tunable filters overcomes the problems due to the use of the broadband ones. the possibility of acquiring the emission spectra in a spatially resolved way extends simple image intensity studies into characterization of complex probeenvironment relationship through the sensitivity of fluorescence spectra to the local molecular environment differences. consequently, fluorescence microspectroscopy (fms) is able to provide the spectral information in a well defined spatial region allowing the researcher to simultaneously obtain spatial and spectroscopic information. our instrument has been specially built to study live cells and their interaction with nanomaterials, drug carriers and modified cell environment. other main characteristics are reducing the bleaching effect and employing a white light source that does not limit the use to specific probes. graphical tools, such as colour coded images, have also been introduced to provide explicit and straightforward visual information. high speed fpga based multi-tau correlation for singlephoton avalanche diode arrays jan buchholz 1,2 , jan krieger we demonstrate the use of fret-imaging (forster resonance energy transfer) as an assay to directly monitor the dynamics of cross-bridge conformational changes in single fibres of skeletal muscle. we measured nm-distances of several fret pairs located at strategic positions to sense myosin head conformational changes: we focused our attention on the essential light chain, elc (specifically labelling a modified elc and exchanging it with the natural elc of the fibre) and we investigated its interaction with the sh1 helix, with the nucleotide binding pocket and with actin. we characterized fret in single rigor muscle fibres, determining distances in agreement with those from the crystallographic data. the results demonstrate the viability of the approach in sensing different fret efficiencies over a few nanometres, an essential requirement to follow the expected small fret variations in contracting muscle fibres. we are now performing dynamic experiments on rigor and active fibres by applying small stretch/release cycles to alter the interaction distances (estimated time resolution of nearly 20ms/frame). in this configuration, it will be possible to measure functional changes, shedding light on the myosin head dynamics during contraction. focal stimulation of cultured neurons is crucial since it mimics physiological molecules release. indeed, the nervous system finely tunes the activity of each synapse by regulating the secretion of molecules spatially and temporally. currently used techniques have some drawbacks such as a poor spatial resolution or a low flexibility. we propose a novel approach based on optical tweezers (ot) [1] to overcome these limitations. ot allow an ease manipulation with sub-micrometric precision of silica beads, which can be functionalized with any protein. for a proof-of-principle study we coated 1,5lm large beads with brain-derived neurotrophic factor (bdnf) or bovine serum album (bsa) as control. we showed that a single bead was able to activate the bdnf receptor trkb, inducing its phosphorylation. moreover bdnf beads but not control beads were able to induce c-fos translocation into the nucleus [2] , indicating that the whole pathway was activated. finally, we positioned vectors in proximity to the growth cones of cultured hippocampal neurons [3] . control beads didn't affect the normal development of these structures while bdnf beads significantly did. these findings support the use of the ot technology for long-term, localized stimulation of specific subcellular neuronal compartments. a key role of its photoactivity, due to the singlet oxygen production, which has a very short lifetime (ns-ls, depending of hyp environment). hyp sub-cellular localization depends on its concentration in the medium, incubation time and used delivery system. variations in activity of protein kinase c, (anti-apoptotic pkca and pro-apoptotic pkcd) in correlation with the activity of bcl-2 protein, cytochrome c release from mitochondria and decreasing of mitochondrial membrane potential after photodynamic action were monitored. the study was performed for two different delivery modes of hyp to u-87mg glioma cells-hyp alone (membrane diffusion) vs. hyp loaded in low density lipoprotein (ldl) (endocytosis). confocal fluorescence microscopy, flow-cytometry and specific fluorescence labeling were used as main experimental techniques. our results show that hyp photoaction strongly affects apoptotic response of the cells and that the dynamics of this action significantly depends on used delivery system. correlation analysis between the monitored parameters (see above) determined for both delivery system is presented and critically discussed. surface contamination by bacteria is a natural and spontaneous process occurring in any natural environment on biotic (mucosa, tissues…) and abiotic surfaces (medical equipments, food surfaces…). whatever the bacterial nature (gram-positive or -negative), the environmental fluid (air, water, blood…) and the receptor surface (implants, medical equipments, food surfaces…), the surface contamination initiated by the first adherent bacteria can evolve to a three dimensional structure named biofilm (cohesive bacteria assembly ensured by an autoproduced extracellular organic matrix). the mechanisms by which these biofilms offer protective environment to viral particles or hypertolerance to antimicrobial action are not yet elucidated. to reach a better understanding of biofilm reactivity, we reported for the first time successful applications of correlative time-resolved optical microscopy approach by time-lapse (tl), frap, fcs, flim, for real-time analysis of molecular mobility and reaction inside biofilms. by means of non-biological or biological (virus, biocides and antibiotics) reactive compounds, significant advances to understand the roles of the extracellular matrix and bacteria physiological properties were obtained, an important step to improve pathogenic biofilm inactivation. here we present a feasibility study to develop two-photon microscopy (2pm) into a standard diagnostic tool for noninvasive, skin cancer diagnosis. the goal is defining experimental parameters to maximize image quality of optical biopsies in human skin, while avoiding tissue damage. possible diagnostic indicators will be compared for healthy tissue, benign, and malignant melanocytic lesions. we report on preliminary results of a study on 2pm imaging of ''ex-vivo'' biopsy samples, were autofluorescence intensity and contrast between lesion and surrounding tissues were optimised varying excitation wavelength, detection band, and dispersion pre-compensation. moreover, we determined modulation functions for laser power and detector gain to compensate losses in deep tissue imaging. as the main process of photo-damage, thermo-mechanical modifications were quantified and damage threshold powers were determined. in order to image structural changes in ordered tissue like collagen fibres, second-harmonic generation signals were recorded and optimised. in-vivo two-photon imaging of the honeybee antennal we adapted a two-photon microscope for in-vivo imaging of the honeybee olfactory system, focusing on its primary centres, the antennal lobes. the setup allowed obtaining both 3d-tomographic measurements of the antennal lobe morphology and time-resolved in-vivo calcium imaging of its neuronal activity. the morphological data were used to precisely measure the glomerular volume in both sides of the brain, investigating the question of volumetric lateralization. functional calcium imaging allowed to record the characteristic glomerular response maps to external odour stimuli applied to the bees' antennae. compared to previous neural imaging experiments in the honeybee, this work enhanced spatial and temporal resolution, penetration depth, and it minimized photo-damage. final goal of this study is the extension of the existing functional atlases of the antennal lobe to 3d and into the temporal dimension by investigating the time-resolved activity pattern. the use of voltage sensitive fluorescent dyes (vsd) for noninvasive measurement of the action potential (ap) in blood perfused heart have been hindered by low interrogation depth, high absorption and auto-fluorescence of cardiac tissue. these limitations are diminished using new near infrared (nir) vsd (di-4-anbdqbs). here we validated toxicity and photo-toxicity of these dyes in guinea pig and human cardiac muscle slabs. application of nir vsd showed no effect on cardiac muscle contraction force or relaxation. optical action potentials closely tracked kinetics of microelectrode recorded aps in both field and electrode stimulated preparations. for phototoxicity assessment dye (50 lm) preloaded cardiac slabs were exposed to prolonged laser radiation of various power. microelectrode ap recordings show that exposure to prolonged laser radiation (10min.; 2mw/mm 2 ) of dye loaded tissue had no statistically significant effect on apd50 or conduction velocity, thus indicating no or weak photo-toxicity on the nir vsd. in contrast, exposure to 5 min. laser radiation of phototoxic dyes (mitotracker deep-red) preloaded tissue caused significant reduction in apd50 (by 13%) and conduction velocity (30%). thus, due to the low photo-toxicity, nir vsd are well suited for in vivo cardiac imaging. streptomycesetes are filamentous gram-positive soil bacteria well known for their complex morphological development and secondary metabolite production. during their life cycle spores germinate to form a network of hyphae, which later develops into aerial mycelium when cross-walls are generated and spores are formed. we have examined and compared the last stage of the differentiation process in a wild-type s. coelicolor (m145) and its dcabb mutant lacking a calmodulin-like calcium binding protein. the strains were grown on four kinds of media: smms, smms with 10 % saccharose, r5 and r5 with reduced calcium in order to study the effect of environment and osmotic stress on the sporulation of the two strains to assess the function of cabb protein. from the cultures pictures were taken at 48 hours and after 7 days using phase contrast, atomic force and confocal laser scanning microscope and the sizes of spores were measured. our results showed that the dcabb mutant made smaller spores, its differentiation and stress response were slower. we could conclude from it that the aberrant protein slows the metabolism, the signal transduction and has effects on sporulation, septation and air-mycelium formation. based on it we can tell that the cabb has a significant role in normal development. the mobility and reaction parameters of molecules inside living cells can be conveniently measured using fluorescent probes. typically fluorescence correlation spectroscopy (fcs) based on confocal microscopy is used for such measurements. this implies high time-resolution but only for a single spot at a time. in order to achieve a high time-resolution at multiple spots, we built a single plane illumination microscope (spim) equipped with high-speed image acquisition devices and a high-na detection optics. this allows us to do parallel fcs measurements in a thin plane (width * 2-3 lm) of the sample. our setup is equipped with a fast emccd camera (full frame time resolution 2000ls) and a 32932 pixel array of spads. the spad array has a full frame time resolution of 3-10ls, which is even fast enough to resolve the typical motion time-scale of small molecules (like egfp) inside living cells. the performance of the system is characterized by diffusion measurements of water-soluble fluorescent beads, as well as fcs measurements in living cells. our data acquisition system uses programmable hardware for some tasks and is fast enough to allow real-time correlation of 1024 pixels, as well as saving the complete dataset for later evaluation. electron cryo-microscopy (cryo-em) covers a larger size range than any other technique in structural biology, from atomic resolution structures of membrane proteins, to large noncrystalline single molecules, entire organelles or even cells. electron crystallography of two-dimensional (2d) crystals makes it possible to examine membrane proteins in the quasi-native environment of a lipid bilayer at high to moderately high resolution (1.8-8å ). recently, we have used electron crystallography to investigate functionally important conformational changes in membrane transport proteins such as the sodium/proton antiporters nhaa and nhap, or the structure of channelrhodopsin. ''single particle'' cryo-em is well suited to study the structure of large macromolecular assemblies in the 3.3 to 20å resolution range. a recent example is our 19å map of a mitochondrial respiratory chain supercomplex consisting of one copy of complex 1, two copies of complex iii, and one of complex iv. the fit of the x-ray structures to our map indicates short pathways for efficient electron shuttling between complex i and iii by ubiquinol, and between complex iii and iv by cytochrome c. electron cryo-tomography can visualize large protein complexes in their cellular context at 30-50å resolution, and thus bridges the gap between protein crystallography and light microscopy. cryo-et is particularly suitable for studying biological membranes and large membrane protein complexes in situ. we found that long rows of atp synthase dimers along ridges of inner membrane cristae are an ubiquitous feature of mitochondria from all 6 species we investigated (2 mammals, 3 fungi, 1 plant). the proton pumps of the respiratory chain appear to be confined to the flat membrane regions on either side of the ridges. this highly conserved pattern suggests a fundamental role of the mitochondrial cristae as proton traps for efficient atp synthesis. single-particle analysis: advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. chief among these fluorophores are the on/off photoswitchable and green-to-red photoconvertible fluorescent proteins. irisfp was recently reported as the first fluorescent protein to combine irreversible photoconversion from a green-emitting to a red-emitting state with reversible on/off photoswitching in both the green and red states. the introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging, but the properties of irisfp are far from being optimal from a spectroscopic point of view and its tetrameric organization complicates its use as a fusion tag. we have demonstrated how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins by developing and characterizing a new set of such enhanced optical highlighters derived from meo-sfp and dendra2. one of these, which we called nijifp, was identified as a promising new multi-photoactivatable fluorescent protein with optical properties that make it ideal for advanced fluorescence-based imaging applications. introducing to medicine and biology concept of optical markers in tremendous way has changed the recent status of these two important disciplines. this was mainly due to strong development in imaging techniques which recently allow us to investigate both static as well dynamic properties of living cells, their components and their interactions with external factors. recently used molecular markers including organic dyes, fluorescent proteins or chelates containing lanthanide ions have several significant limitations. one of the alternatives for molecular markers are inorganic quantum dots (ie. cdse, cds) which are recently commonly used in many academic works. however, even if they are much better from physicochemical point of view, from the application point of view at this moment they are rather useless mainly because of their high risk of toxicity. one of the solution combining advantages of both concepts is to make nontoxic inorganic nanocrystals doped by lanthanide ions. in this work, we will present optical results obtained for nayf 4 nanocrystals doped by different lanthanide ions. the aim of this work was to design and to synthesize these markers and to understand physical processes responsible for their emission and to optimize these processes to the physical limits. intravital microscopy has fostered a full blown of publication regarding the behavior of cells in different tissues and physiological conditions. however, a few papers describe how the motility parameters can be used to understand whether an interaction is occurring and, on balance, the distinction between interacting and non interacting cells is performed visually on the image time stack. here we describe a multi-parameter approach that allows to discern among different cell behaviors on an objective ground and we demonstrate its effectiveness valuating the mutual fate of natural killer (nk) and dendritic (dc) cells at the draining lymph-nodes in inflammatory and stationary conditions. the method is time saving and allows a wide scale characterization of the lymphocyte tracks and to build up statistics of the cell-cell interaction duration. this has allowed the development of a numerical model of the nk-dc interaction, based on a molecular-stochastic dynamic approach, whose output can be directly compared to the data. hemozoin is formed during malaria infection of red blood cells, the malaria parasite cleaves hemoglobin, leaving free heme which is then toxic to the parasite. the free heme is then bio-crystalized to form hemozoin which allows the parasite to remain viable. the hemozoin is released during the breakdown of the red blood cells, is small and can be difficult to resolve spatially. since it contains an abundance of heme protein, which has a strong absorbance at 532 nm, it can be readily detected and tracked by using resonant raman scattering spectroscopy. here we use slit-scanning confocal raman imaging to detect the hemozoin and resolve it against the background molecules. inside a red blood cell, hemoglobin is the strongest background signal since it also contains large amounts of heme. nevertheless, the discrimination is possible, and the time-resolved observation of hemozoin is an important tool to understand effects of malaria since the hemozoin can trigger the immune response and cause inflammation in tissue. muscle performance at the molecular level is determined by the elementary displacement (working stroke) produced by the motor protein myosin ii, and its dependence on load. we developed a laser trap assay (the optical leash) capable of applying controlled loads to a single myosin head before the working stroke is initiated and probing actin-myosin interaction on the microsecond time scale. we found that the working stroke size depends both on load and on the detachment pathway followed by myosin. in a first pathway, myosin detaches very rapidly from actin (\1ms) without producing any movement. in a second pathway, myosin steps and remains bound to actin for a time inversely proportional to atp concentration; the working stroke remains constant (*5 nm) as the load is increased, until it suddenly vanishes as the isometric force is reached (5.7±0.6pn). a third dissociation pathway becomes more populated as the force is increased, due to premature unbinding of myosin from actin, resulting in a working stroke that decreases with load. taken together, these results give a new insight into the molecular mechanism of load dependence of the myosin working stroke, which is a primary determinant of skeletal muscle performance and efficiency. previously we have deleted either or both of these terminal helices genetically. surprisingly, all mutants rotated in the correct direction, showing that the shaft portion is dispensable. here we inquire if the rest of the c rotor, the globular protrusion that accounts for *70 % of the c residues, is also dispensable. keeping the n-and c-terminal helices that constitute the shaft, we have replaced the middle *200 residues with a short helix-turn-helix motif borrowed from a different protein. the protrusion-less mutant thus made retained a high atpase activity and rotated fast in the correct direction. this may not be unexpected because, in crystal structures, most of the removed residues do not contact with the a 3 b 3 ring. combined with the previous results, however, the present results indicate that none of the c residues are needed for rotation. the rotary mechanism of a molecular engine, the vacuolar proton-atpase, working in a biomembrane csilla ferencz 1 , pá l petrovszki 1 , zoltá n kó ta 1 the rotary mechanism of vacuolar proton-atpase (v-atpase) couples atp hydrolysis and trans-membrane proton translocation. we tested the effect of oscillating electric (ac) field on v-atpase activity in yeast vacuoles. the ac technique has several advantages over direct observations: it can be applied on native membranes, there are no labels and attachments involved, and the target protein is in its natural environment. this was/is the first of its kind of experiment on v-atpase, and we got strikingly different results from previous studies on other proteins: both low and high frequency ac field reduces atpase activity in a wide frequency range. a sharp resonance is seen at 88.3 hz, where the atpase activity reaches or exceeds the control (no ac) level. we think that the resonance happens at that of the 60 degrees rotor steps, meaning that the rotation rate of the rotor is around 15 hz, under the given conditions. synchronisation of individual atpases by slow or matching, but not fast ac is likely via a hold-and-release mechanism. we can explain the above observations by assuming that the ac field interacts with the proton movements, and if we consider the estimated geometry of the hydrophilic proton channels and the proton binding cites on the rotor. [2] . the ttss constitutes a continuous protein transport channel of constant length through the bacterial envelope [3] . the needle of the type three secretion system is made of a single small protein (protomer). we analyzed the assembly and the structure of the ttss needle using different biophysical methods including fourier transform-infrared spectroscopy, nmr spectroscopy and x-ray crystallography. we show that the ttss needle protomer refolds spontaneously to extend the needle from the distal end. the protomer refolds from a-helix into b-strand conformation to form the ttss needle [4] . regulated secretion of virulence factors requires the presence of additional protein at the ttss needle tip. x-ray crystal structure analysis of the tip complex revealed major conformational changes in both the needle and the tip proteins during assembly of the s. typhimurium ttss. our structural analysis provides the first detailed insight into both the open state of the ttss needle tip and the conformational changes occurring at the pathogen-host interface [5] . [ the membrane-bound component f o of the atp synthase works as a rotary motor and plays the central role of driving the f 1 component to transform chemi-osmotic energy into atp synthesis. we have conducted molecular dynamics simulations of the membrane-bound f o sector with explicit lipid bilayer, in which the particular interest was to observe the onset of helix motion in the c ring upon the change of the protonation state of asp61 of the c 1 subunit, which is the essential element of the boyer's binding-change mechanism. to investigate the influence of transmembrane potential and ph gradient, i.e., the proton motive force, on the structure and dynamics of the a-c complex, different electric fields have been applied along the membrane normal. correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. the deuterium order parameter (s cd ) profile calculated by averaging all the lipids in the f o -bound bilayer was not very different from that of the pure bilayer system, which agrees with recent 2 h solid-state nmr experiments. however, by delineating the lipid properties according to their vicinity to f o , we found that the s cd profiles of different lipid shells are prominently different. lipids close to f o formed a more ordered structure. similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. the lipids in the proximity of f o exhibited very significantly reduced diffusional motion. the numerical value of s cd was anti-correlated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. our findings will not only help for elucidating the dynamics of f o depending on the protonation states and electric fields, but may also shed some light to the interactions between the motor f o and its surrounding lipids under physiological condition, which could help to rationalize its extraordinary energy conversion efficiency. this work has been published 1 in march 2010, and it was selected as one of the two featured articles of that issue. the research project is directed toward the construction of a synthetic bio-inorganic machine that consists in a single actin filament that interacts with a linear array of myosin ii motors regularly disposed on a nano-structured device. the motor array is intended to simulate the unique properties of the ensemble of motor proteins in the half-sarcomere of the muscle by providing the condition for developing steady force and shortening by cyclic interactions with the actin filament. the mechanical outputs in the range 0.5-200 pn force and 1-10,000 nm shortening will be measured and controlled the bacterial flagellar motor is a membrane-embedded molecular machine that rotates filaments, providing a propulsive force for bacteria to swim. the molecular mechanism of torque (turning force) generation is being investigated through the study of the properties and three-dimensional structure of the motor's stator unit. we are taking both topdown and bottom-up approaches, combining data from the molecular genetics studies, cross-linking, x-ray protein crystallography and molecular dynamics simulations. we have recently determined the first crystal structure of the protein domain that anchors the proton-motive-force-generating mechanism of the flagellar motor to the cell wall, and formulated a model of how the stator attaches to peptidoglycan. the work presented at the meeting will inform the audience on our latest work that establishes the relationship between the structure, dynamics and function of a key component of the bacterial flagellar motor, the motility protein b (motb). this work will be put in the perspective of the mechanism of rotation, stator assembly, anchoring to peptidoglycan and interaction with the rotor, and discussed in the light of the elementary events composing the cycle of electrochemical-to-mechanical energy conversion that drives flagellar rotation. members of the conserved kinesin-5 family fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plusend directed motors. the mechanisms that regulate kinesin-5 function are still not well understood. we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin-5 cin8 using single-molecule motility assays and single-molecule fluorescence microscopy and found that cin8 motility is exceptional in the kinesin-5 family. in vitro, individual cin8 motors could be switched by ionic conditions from rapid (up to 50 lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert of 99 amino acids in loop 8 of cin8 induced bias towards minus-end motility and strongly affected the directional switching of cin8 both in vivo and in vitro. we further found that deletion of the functionally overlapping kinesin-5 kip1 and of the spindle-organizing protein ase1 affected cin8 velocity and processivity, but directionality was not affected. the entirely unexpected finding of switching of cin8 directionality in vivo and in vitro demonstrates that the ''gear box'' of kinesins is much more complex and versatile than thought. many biological motor molecules move within cells using stepsizes predictable from their structures. myosin-vi, however, has much larger and more broadly-distributed stepsizes than those predicted from its short lever arms. we explain the discrepancy by monitoring qdots and gold nano-particles attached to the myosin vi motor domains using high-sensitivity nano-imaging. the large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). these results suggest there exist two tilt-angles during myosin-vi stepping, which correspond to the pre-and post-powerstrokes states and regulate the leading head. the large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with adp. switching between these two mechanisms in a strain sensitive, adpdependent manner allows myosin-vi to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring. http://www.fbs.osaka-u.ac.jp/labs/yanagida/, http:// www.qbic.riken.jp/. ferritin deposits iron in oxyhydroxide iron core surrounded by protein shell. the iron core structure may vary in different ferritins in both normal and pathological cases. to study iron core variations the mö ssbauer spectroscopy with a high velocity resolution was applied for comparative analysis of normal and leukemia chicken liver and spleen tissues, human liver ferritin and commercial pharmaceutical products imferon, maltoferò and ferrum lek as ferritin models. mö ssbauer spectra of these samples measured with a high velocity resolution at room temperature were fitted using two models: homogenous iron core (one quadrupole doublet) and heterogeneous iron core (several quadrupole doublets). the results of both fits demonstrated small variations of mö ssbauer hyperfine parameters related to structural variations of the iron cores. these small structural variations may be a result of different degree of crystallinity, the way of iron package, nonequivalent iron position, etc. obtained small differences for normal and leukemia tissues may be useful for distinguishing ferritins from normal and pathological cases. this work was supported in part by the russian foundation for basic research (grant # 09-02-00055-a). invasion of epithelial cells by salmonella enterica is mediated by bacterial ''effector'' proteins that are delivered into the host cell by a type iii secretion system (ttss). the collaborative action of these translocated effectors modulates a variety of cellular processes leading to bacterial uptake into mammalian cells. type iii effectors require the presence in the bacterial cytosol of specific tts chaperones. effectors are known to interact with their chaperone via a chaperone binding domain (cbd) situated at their n-terminus. this work focus on sopb, an effector with phosphoinositide phosphatase activity and particularly its interaction with the specific chaperone sige by biochemical, biophysical and structural approaches. we have co-expressed sopb with its specific chaperone sige and purified the complex, determined the limits of the cbd and purified the sopb cbd /sige complex. the structure of sige has been solved previously but no crystals could be obtained for structure determination of both complexes. we used saxs experiment combined with biophysical approach to analyse the interaction between sopb and its chaperone as well as the quaternary structure on the complex that will be described in this presentation. guanylate monophosphate kinase (gmpk) is a cytosolic enzyme involved in nucleotide metabolic pathways. one of the physiological roles of gmpks is the reversible phosphoryl group transfer from atp to gmp (its specific ligand), yielding adp and gdp. the gmpk from haemophilus influenzae is a small protein, with a number of 208 amino acids in the primary structure. in order to determine the secondary structure changes of this enzyme, as well as some physical characteristics of its complexes with gmp and atp ligands, circular dichroism (cd) and atr -ftir studies were performed. the enzyme and its ligands were dissolved in tris -hcl buffer, at ph 7.4 and 25°c. from the cd spectra the content of the secondary structure elements of gmpk and gmpk/gmp, gmpk/atp (with and without mg 2+ ) were determined. the major secondary structure elements of gmpk from haemophilus influenzae were a-helix (* 37 %) and b-sheet (* 36 %). atr -ftir experiments show that the amide i and amide ii bands of the gmpk are typical for a protein with great a-helix content. from the second derivative spectra, the content of the secondary structure elements were estimated. these data were in agreement with those obtained by cd. assembly of the mature human immunodeficiency virus type 1 capsid involves the oligomerization of the capsid protein, ca. the c-terminal domain of ca, ctd, participates both in the formation of ca hexamers, and in the joining of hexamers through homodimerization. intact ca and the isolated ctd are able to homodimerize in solution with similar affinity (dissociation constant in the order of 10 lm); ctd homodimerization involves mainly an a-helical region. in this work, we show that peptides derived from the dimerization helix (which keep residues energetically important for dimerization and with higher helical propensities than the wild-type sequence) are able to self-associate with affinities similar to that of the whole ctd. moreover, the peptides have a higher helicity than that of the wild-type sequence, although is not as high as the theoretically predicted. interesting enough, the peptides bind to ctd, but binding in all peptides, but one, does not occur at the dimerization interface of ctd (helix 9). rather, binding occurs at the last helical region of ctd, even for the wild-type peptide, as shown by hsqc-nmr. as a consequence, all peptides, but one, are unable to inhibit capsid assembly of the whole ca in vitro. the peptide whose binding occurs at the ctd dimerization helix has an val?arg mutation in position 191, which has been involved in dimer-dimer contacts. these findings suggest that event keeping the energetically important residues to attain ctd dimerization within a more largely populated helical structure is not enough to hamper dimerization of ctd. putp is an integral membrane protein located in the cytoplasmic membrane of escherichia coli, being responsible for the coupled transport of na + and proline. it belongs to the family of sodium solute symporters (sss). structural data for putp is not available, but secondary structure predictions together with biochemical and biophysical analyses suggest a 13 transmembrane motif. from a recent homology model based on the x-ray structure of the related na + /galactose symporter vsglt, previously published electron paramagnetic resonance (epr) studies, and recent crystallographic and epr studies on the cognate bacterial homolog of a neurotransmitter:na + symporter, leut, it has been proposed that helices viii and ix as well as the interconnecting ''loop 9'' region determine the accessibility of the periplasmic cavities which bind sodium and proline. we performed site-directed spin labeling of ''loop 9'' in combination with epr spectroscopy to investigate the structural features of this region and possible conformational changes induced by sodium and proline. analyses of spin label mobility and polarity as well as accessibility to paramagnetic quenchers allow us to refine this region in the present homology model. furthermore, our data suggest conformational changes in this region upon substrate binding including an overall motion of a helical segment. fatty acid-binding proteins (fabp) are a family of low molecular weight proteins that share structural homology and the ability to bind fatty acids. the common structural feature is a b-barrel of 10 antiparallel b-strands forming a large inner cavity that accommodates nonpolar ligands, capped by a portal region, comprising two short a-helices. b-fabp exhibits high affinity for the docosahexaenoic (dha) and oleic acid (oa). it is also postulated that b-fabp may interact with nuclear receptors from ppar family. in the present work, we used molecular biology and spectroscopic techniques to correlate structure, dynamics and function. site-directed mutagenesis was used to produce 5 mutants of b-fabp with a nitroxide spin probe (mtsl) selectively attached to residues located at the portal region. esr spectra of the labeled b-fabp mutants were sensitive to the location of the mutation and were able to monitor interactions in three cases: (1) shsp are ubiquitous proteins involved in cellular resistance to various stress (oxidative, heat, osmotic…). they are able to prevent aggregation of non-native proteins through the ability of forming large soluble complexes and preventing their nonspecific and insoluble aggregation. in consequence to this molecular chaperone function, they can regulate many processes (resistance to chemotherapy, modulation of the cellular adhesion and invasion, inflammatory response in skin), and the modulation of their expression has been found to be a molecular marker in cancers, spermatogenesis, or cartilage degeneration. furthermore, they are involved in several pathologies: myopathies, neuropathies, cancers, cataracts. among the 10 human members (hspb1-10), this study focused on hspb1 (hsp27 involved in some cancers), hspb4 (lens specific), hspb5 (lens, muscle, heart, lung) and hspb5-r120g (responsible for a desmin-related myopathy and a cataract). as shsp form large, soluble (but polydisperse in mammals) hetero-oligomers, molecular biology, biochemistry, biophysics and bioinformatics were successfully combined to compare the functional/dysfunctional assemblies in order to understand the critical parameters between shsp members depending upon their tissue and cellular localization. ionizing radiation is a type of radiation that contains enough energy to displace electrons and break chemical bonds. this can promote the removal of at least one electron from an atom or molecule, creating radical species, namely, reactive oxygen species (ros) [1, 2] . these are often associated with damages at cellular level, such as, dna mutations, cell cycle modifications and, in animal cells, cancer. to overcome this problem, organisms developed different protection/repair mechanisms that enable them to survive to these threats. dna glycosylases are enzymes that are part of base excision repair (ber) system, mainly responsible for dna repair. they can recognize a dna lesion and, in some cases, are able to remove the mutated base. here we propose to study one of those enzymes, endonuclease iii, which contains a [4fe-4s] cluster [3, 4] . samples were exposed to different doses of uv-c radiation and the effects were studied by electrophoretic and spectroscopic methods. [ na,k-atpase is an integral protein present in the plasma membrane of animal cells, and consists of two main subunits: the a and b. cholesterol is an essential constituent of the animal membrane cells. in order to study the interaction between na,k-atpase and cholesterol, we have used the dsc technique, and a proteoliposome system composed by the enzyme and dppc:dppe, with different percentage in mol of cholesterol. the heat capacity of purified na,k-atpase profile exhibits three transitions with 31, 189 and 60 kcal/mol at 55, 62 and 69°c. multiple components in the unfolding transition could be attributed either to different steps in the pathway or to independent unfolding of different domains. this denaturation of na,k-atpase is an irreversible process. for the proteoliposome, we also observed three peaks, with 180, 217 and 41 kcal/mol and 54, 64 and 72°c. this increase in dh indicates that the lipids stabilize the protein. when cholesterol was used (from 10 to 40 mol %), the first transition was shifted to a lower temperature value around 35°c. these results confirm that cholesterol has an influence on the packing and fluidity of lipid bilayer and changes in lipid microenvironment alter the thermostability as well as the activity of na,k-atpase. financial support: fapesp. we have undertaken to study the structure and function of peroxisomal multifunctional enzyme type 2 (mfe-2) from different organisms. mfe-2 is a key enzyme in long-and branched-chain fatty acids breakdown in peroxisomes. it contains two enzymes in the same polypeptide and also consists of differing amount of domains depending on the species. crystal structure and enzyme kinetics of drosophila melanogaster mfe-2 has revealed the domain assembly and raised a question about existence of a substrate channeling mechanism. small-angle x-ray scattering studies have further resolved the assembly of domains in the human mfe-2. mutations in the mfe-2-coding gene in humans may cause d-bifunctional protein deficiency -a metabolic disease characterized by accumulation of fatty acyl-coa intermediates due to inactive or residually active mfe-2 protein. we have also studied the structure, stability and dynamics of such mutant proteins both experimentally and in silico. the latest results on all these studies will be presented. ftsz is a protein that plays a key role in bacterial division, forming a protein ring directly related to the constriction of the membrane. this process has been observed to occur without the help of molecular motors. nonetheless, the details of the self-assembly and subsequent force generation of the septal ring are still obscure. afm observations allows the study of the behaviour of ftsz solutions on a substrate with unprecedented resolution, permitting the identification of individual protein filaments. the different resulting structures can be compared to monte carlo models for a 2d lattice accounting for the essential interactions between monomers. these include a strong longitudinal bond that allows a limited flexibility (i.e., curvature of the filaments) and a weaker lateral interaction. the work we present follows this approach, focussing on the latest experiments with ftsz mutants. by using these mutants, it is possible to choose the specific region of the monomer that will anchor to the substrate, thus generating new structures that provide an insight into monomer-monomer interactions. in this way, we explore the anisotropy of the lateral bond in ftsz, a factor that has not been taken into account before but may prove to be of importance in fstz behaviour in vivo. modeling protein structures and their complexes with limited experimental data dominik gront university of warsaw, pasteura 1, 02-093 warsaw, poland conventional methods for protein structure determination require collecting huge amounts of high-quality experimental data. in many cases the data (possibly fragmentary and/or ambiguous) on itself cannot discriminate between alternative conformations and a unique structure cannot be determined. small angle xray scattering is an example of such a ''weak'' experiment. the spectrum encodes only several independent degrees of freedom that provide a global description of a molecular geometry in a very synthetic way. in this contribution we utilized both local information obtained from nmr measurements and global description of a macromolecule as given by saxs profile combined with a knowledge-based bimolecular force field to determine tertiary and quaternary structure of model protein systems. saxs curve as well as various kinds of local nmr data such as isotropic chemical shifts and their tensors, j-couplings, rdc, backbone noe and redor from nmr in solid phase are parsed with the ''experimental'' module of bioshell toolkit and utilized by rosetta modeling suite to generate plausible conformations. obtained results show the new protocol is capable to deliver very accurate models. noenet-use of noe networks for nmr resonance assignment of proteins with known 3d structure dirk stratmann, carine van heijenoort and eric guittet structural genomics programs today yield an increasing number of protein structures, obtained by x-ray diffraction, whose functions remain to be elucidated. nmr plays here a crucial role through its ability to readily identify binding sites in their complexes or to map dynamic features on the structure. an important nmr limiting step is the often fastidious assignment of the nmr spectra. for proteins whose 3d structures are already known, the matching of experimental and back-calculated data allows a straightforward assignment of the nmr spectra. we developed noenet, a structure-based assignment approach. it is based on a complete search algorithm, robust against assignment errors, even for sparse input data. it allows functional studies, like modeling of protein-complexes or protein dynamics studies for proteins as large as 28 kd. almost any type of additional restraints can be used as filters to speed up the procedure or restrict the assignment ensemble. noenet being mainly based on nmr data (noes) orthogonal to those used in triple resonance experiments (jcouplings), its combination even with a low number of ambiguous j-coupling based sequential connectivities yields a high precision assignment ensemble. we observed, that t. thermophilus isopropylmalate dehydrogenase (ipmdh) has higher rigidity and lower enzyme activity at room temperature than its mesophilic counterpart (e. coli), while the enzymes have nearly identical flexibilities under their respective physiological working conditions, suggesting that evolutionary adaptation tends to maintain optimum activity by adjusting a ''corresponding state'' regarding conformational flexibility. in order to reveal the nature of the conformational flexibility change related to enzymatic activity, we designed a series of mutations involving non conserved prolines specific to thermophilic ipmdh. proline to glycine mutations substantially increased conformational flexibility and decreased conformational stability. the mutant enzyme variants did not show enhanced catalytic activity, but the non arrhenius temperature dependence of enzyme activity of the wild type was abolished. this phenomenon underlines the fact that the delicate balance between flexibility, stability and activity which is required for the environmental adaptation of enzymes can be easily disrupted with mutations even distant from the active site, providing further evidence that optimization of proper functional motions are also a selective force in the evolution of enzymes. the kinetoplastids trypanosoma brucei, t. cruzi and leishmania major are responsible for causing great morbidity and mortality in developing countries. the all a-helical dimeric dutpases from these organisms represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared to the trimeric human enzyme. to aid in the development of dutpase inhibitors we have been structurally characterizing the enzymes from these species. here we present the structure of the t. brucei enzyme in open and closed conformations, completing the view of the enzymes from the kinetoplastids. furthermore, we sought to probe the reaction mechanism for this family of enzymes as a mechanism has been proposed based on previous structural work but has not received any further verification. the proposed scheme is similar to that of the trimeric enzyme but differs in detail. using tryptophan fluorescence quenching in the presence of the transition state mimic alf 3 we have been able to identify which species is the likely transition state in the reaction. the crystal structure of t. brucei in complex with this transition state analogue confirms the nature of the nucleophilic attack clearly showing how it differs from trimeric enzymes. the structure of factor h-c3d complex explains regulation of immune complement alternative pathway circular dichroism (cd) spectroscopy is a widely used technique for studying the secondary structure (ss) of proteins. numerous algorithms have been developed for the estimation of the ss composition from the cd spectra. although, these methods give more or less accurate estimation for proteins rich in a-helical structure, they often fail to provide acceptable results on mixed or b-rich proteins. the problem arises from the diversity of b-structures, which is thought to be an intrinsic limitation of the technique. the worst predictions are provided for proteins of unusual b-structures and for amyloid fibrils. our aim was to develop a new algorithm for the more accurate estimation of ss contents for a broader range of protein folds with special interest to amyloid fibrils. using synchrotron radiation cd (srcd), we were able to collect high quality spectra of amyloid fibrils with good s/n ratios down to 175nm. the novel reference dataset with spectra that significantly differ from present reference sets, extends the information content for ss determination. our algorithm takes into account the diverse twist of the b-sheets that has a great influence on the spectral features. for the first time, we can reliably distinguish parallel and antiparallel b-structure using cd spectroscopy. monitoring the assembly of the membrane protein insertase alexej kedrov 1 , marko sustarsic 2 , arnold j.m. driessen 1 1 groningen biomolecular sciences and bioengineering institute, university of groningen, the netherlands, 2 university of oxford, uk molecular forces that govern membrane protein integration and folding remain a major question in current molecular biology and biophysics. each nascent polypeptide chain should acquire its unique three-dimensional folded state within a complex environment formed by the anisotropic lipid membrane and the membrane-water interface. secyeg translocase and members of a recently described yidc/oxa1/alb3 chaperone family are recognized as primary players in the membrane protein genesis. these proteins, so called insertases, serve as membraneembedded molecular pores where the newly synthesized protein is loaded prior its release into the bilayer. here we apply fluorescence correlation spectroscopy to monitor the assembly of the insertase:ribosome:nascent polypeptide chain complexes in solution and reconstituted into nanodiscs and model membranes. results provide insights on molecular mechanisms and dynamics of the insertase functioning. conformational changes during gtpase activity induced self-assembly of human guanylate binding protein 1 revealed by epr spectroscopy correct assembly and regulation of multi-component molecular machines is essential for many biological activities. the type iii secretion system (t3ss) is a complex molecular machine that is a key virulence determinant for important gram-negative pathogens including shigella, yersinia and salmonella species [1] [2] . the t3ss consists of multiple copies of *25 different proteins (totalling *7mda), spans both bacterial membranes and drives insertion of a contiguous pore into the host-cell membrane. virulence factors are secreted via this apparatus directly into the host cell. in all t3ss various levels of regulation occur with switching between secretion off and on states overlaid on control of which substrates are secreted. genes involved in a variety of these switches have been identified but the molecular mechanisms underlying these functions is poorly understood. we are studying the t3ss of shigella flexneri, the causative agent of dysentery and will present the structure of the so-called ''gatekeeper protein'' mxia. the diacylglycerolacyltransferase1 (dgat1) is an integral protein from the reticulum endoplasmic membrane that plays an essential role in triacylglyceride synthesis. in cattle, this enzyme is associated to the fat content regulation on milk and meat. in this study, synthetic peptides corresponding to both dgat1 binding sites (sit1 and sit2) were designed, purified and employed to investigate the enzyme interaction with substrates and membrane models. different binding specificities in the interaction with phospholipid vesicles and micelles were noted. sit1 showed to bind more strongly in nonpolar membrane models, while sit2 was electrostatically attracted to negative phospholipid surfaces. the binding of both peptides was followed by significant conformational changes (like unordered to helix transition) in circular dichroism spectra and a 20nm blue shift in fluorescence emission. the binding of sit1 and sit2 peptides to negative liposome gave dissociation constants (k d ) of 170 and 0.44lm, respectively, and a leakage action 24-fold higher to sit2. the difference in specificity is related to the features of the putative substrates (acyl-coas and diacylglycerol) and can be attributed to the distinct role of each dgat1 binding site during lipid synthesis. supported by fapesp. ftsz, the bacterial homologue of tubulin, assembles into polymers in the bacterial division ring. the interfaces between monomers include a gtp molecule, although the relationship between polymerization and gtpase activity is still controversial. a set of short ftsz polymers were modelled and the formation of active gtpase structures was monitored using molecular dynamics. only the interfaces nearest the polymer ends exhibited geometry adequate for gtp hydrolysis. conversion of a mid-polymer interface to a close-to-end interface resulted in its spontaneous rearrangement from an inactive to an active conformation. fluorescent proteins (fps) have become extremely valuable tools in the life sciences. due to the latest advances in the light microscopy, there is a steady need for fps with improved spectral properties. mirisfp is a monomeric fp that can be switched reversibly between a bright green fluorescent and a dark state by illumination with light of specific wavelengths [1] . structurally, this photo-switching is based on a cis-trans isomerization of the chromophore. upon illumination with violet light, mirisfp can be irreversibly photoconverted from the green-emitting to a red-emitting form. the red form can again be switched reversibly between a fluorescent and a dark state. to elucidate the mechanistic details of the photoinduced reactions, we have generated mirisgfp1. this variant can still undergo reversible photoswitching, but lacks the ability to photoconvert to the red state so that the photoinduced transitions of the green form can be studied without 'artifacts' due to green-to-red photoconversion. using uv/visible spectroscopy, we have characterized the on-and off-switching processes in great detail. several light-activated reaction pathways have been identified. they are highly intertwined so that the net effect achieved with light of a particular wavelength depends on the relative probabilities to photoinduce the various processes. phosducin (pd) is a g t bc-binding protein that is highly expressed in photoreceptors. pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. dephosphorylated pd binds g t protein bc-heterodimer with high affinity and inhibits its interaction with g t a or other effectors, whereas phosphorylated pd does not. therefore pd down-regulates the light response in photoreceptors. phosphorylation of pd at s54 and s73 leads to the binding of the 14-3-3 protein. the 14-3-3 proteins function as scaffolds modulating the activity of their binding partners and their role in pd regulation is still unclear. the 14-3-3 protein binding may serve to sequester phosphorylated pd from g t bc or to decrease the rate of pd dephosphorylation and degradation. we performed several biophysical studies of the 14-3-3:pd complex. analytical ultracentrifugation was used to determine the complex stoichiometry and dissociation constant. conformational changes of pd induced both by the phosphorylation itself and by 14-3-3 binding were studied using the time-resolved fluorescence spectroscopy techniques. mö ssbauer spectroscopy with a high velocity resolution was used for comparative study of various oxyhemoglobins for analysis of the heme iron electronic structure and protein structure-function relationship. samples of pig, rabbit and normal human oxyhemoglobins and oxyhemoglobins from patients with chronic myeloleukemia and multiple myeloma were measured using mö ssbauer spectrometric system with a high velocity resolution at 90 k. mö ssbauer spectra were fitted using two models: one quadrupole doublet (model of equivalent iron electronic structure in a-and b-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in a-and b-subunits of hemoglobins). in both models small variations of mö ssbauer hyperfine parameters (quadrupole splitting and isomer shift) were observed for normal human, rabbit and pig oxyhemoglobins and related to different heme iron stereochemistry and oxygen affinity. small variations of mö ssbauer hyperfine parameters for oxyhemoglobins from patients were related to possible variations in the heme iron stereochemistry and function. the different types of silk produced by orb-weaving spiders display various mechanical properties to fulfill diverse functions. for example, the dragline silk produced by the major ampulate glands exhibits high toughness that comes from a good trade-off between stiffness and extensibility. on the other hand, flagelliform silk of the capture spirals of the web is highly elastic due to the presence of proline and glycine residues. these properties are completely dictated by the structural organization of the fiber (crystallinity, degree of molecular orientation, secondary structure, microstructure), which in turn results from the protein primary structure and the mechanism of spinning. although the spinning process of dragline silk begins to be understood, the molecular events occurring in the secretory glands and leading to the formation of other silk fibers are unknown, mainly due to a lack of information regarding their initial and final structures. taking advantage of the efficiency of raman spectromicroscopy to investigate micrometer-sized biological samples, we have determined the conformation of proteins in the complete set of glands of the orb-weaving spider nephila clavipes as well as in the fibers that are spun from these glands. domain of rgs3 at 2.3å resolution was solved. the stoichiometry of 14-3-3 f /rgs3 protein complex was elucidated using the analytical ultracentrifugation. to map the interaction between 14-3-3f and rgs3 protein we performed a wide range of biophysical measurements: h/d exchange and cross link experiments coupled to mass spectrometry, fret (fö rster resonance energy transfer) time-resolved fluorescence experiments, time-resolved tryptophan fluorescence spectroscopy and saxs (small angle x-ray scattering) measurements. based on all these results we build 3d model of 14-3-3 f /rgs3 protein complex. our model revealed new details on architecture of complex formed by 14-3-3 proteins. to date all known structure of 14-3-3 proteins complexes suggests that the ligand is docked in the central channel of 14-3-3 protein. our results indicate that the rgs domain of rgs3 protein is located outside the central channel of 14-3-3f protein interacting with less-conserved residues of 14-3-3f. the receptor for advanced glycation end-products (rage) is a multiligand cell surface receptor involved in various human diseases. the major alternative splice product of rage comprises its extracellular region that occurs as a soluble protein (srage). although the structures of srage domains were available, their assembly into the functional full length protein remained unknown. here we employed synchrotron small-angle x-ray scattering to characterize the solution structure of human srage. the protein revealed concentration-dependent oligomerization behaviour, which was also mediated by the presence of ca 2+ ions. rigid body models of monomeric and dimeric srage were obtained from the scattering data recorded at different solvent conditions. the monomer displays a j-like shape while the dimer is formed through the association of the two n-terminal domains and has an elongated structure. the results provided insight into the assembly of i) the heterodimer srage:rage, which is responsible for blockage of the receptor signalling, and ii) rage homodimer, which is necessary for signal transduction, paving the way for the design of therapeutical strategies for a large number of different pathologies. clpb is a hexameric aaa? atpase that extracts unfolded polypeptides from aggregates by threading them through its central pore. the contribution of coiled-coil m domains is fundamental for the functional mechanism of this chaperone, and its location within the protein structure in previous structural models is contradictory. we present cryo-electron microscopy structural analysis of clpb from e. coli in several nucleotide states. the study reveals a novel architecture for clpb and shows that m domains form an internal scaffold located in the central chamber of clpb hexamers. this inner structure transmits local signals due to atp binding and hydrolysis by aaa? domains. surprisingly, coiled-coil m domains are seen to bend significantly around a hinge region that separates two structural motifs. our results present a new framework to understand clpb-mediated protein disaggregation. streptomyces clavuligerus isoenzymes involved in clavulanic acid biosynthesis: a structural approach clavulanic acid (ca) is a potent b-lactamase inhibitor produced by streptomyces clavuligerus. n 2 -(2-carboxyethyl) arginine synthase (ceas) and proclavaminate amidino hydrolase (pah) catalyze the initial steps in the biosynthesis of ca. recently ceas1and pah1genes (paralogous of ceas and pah) were related to the ca biosynthesis but their products have not been studied yet. here we present the initial structural analysis of ceas1 and pah1 using biophysical techniques. pah1 and ceas1 genes were isolated from the genomic dna of s. clavuligerus and overexpressed in e. coli. the recombinant proteins were purified by affinity chromatography and analyzed by size exclusion chromatography, non-denaturing page, dynamic light scattering, far-uv circular dichroism (cd) and fluorescence spectroscopy. our results showed that pah1 and ceas1 were obtained as hexamer and dimer respectively. both proteins showed an a/b folding, being stable up to 35°c. above this temperature protein unfolding was observed but the complete unfolding was not observed, even at 100°c. moreover ceas1 and pah1 showed to be stable over a wide ph range (ph 5.5 -9.5). we are currently working on improving ceas1 crystals which are a promising step towards the elucidation of the ceas1 structure. supported by fapesp. • synchrotron radiation circular dichroism • mass spectrometry following vuv photoionisation • fluorescence imaging with lifetime and spectral measurements here we will present the srcd experiment. high photon flux of 10 10 photons / sec, improved detector performances as well as user-orientated software developments have proven to be the garants for successful data collections,which considerably increased the information content obtained. the exploration into the charge transfer region of the peptide bonds is adding specifically new insights. low sample volumes of as little as 2ll per spectra as well as convenient sample chamber handling allow for economic and efficient data collections. typical spectra acquisition from 280 to 170nm, last for 9 min for three scans with a 1nm step size. prior to high resolution based techniques, srcd spectrawill answer questions about folding states of macromolecules including dna, rna and sugar macromolecules as well as their complexes with proteins and specially membrane proteins. sporulation in bacillus subtilis begins with an asymmetric cell division producing a smaller cell called the forespore, which initially lies side-by-side with the larger mother cell. in a phagocytosis-like event, the mother cell engulfs the forespore so that the latter is internalised as a cell-within-a-cell. engulfment involves the migration of the mother cell membrane around the forespore until the leading edges of this engulfing membrane meet and fuse. this releases the forespore, now surrounded by a double membrane, into the mother cell cytoplasm. membrane migration during engulfment is facilitated by the interacting proteins spoiiq and spoiiiah that are membrane-associated and expressed in the forespore and the mother cell respectively 1 . they interact in the intercellular space and function initially as a molecular zipper and later they participate in a more elaborate complex in which spoiiq and spoiiiah are integral components of an intercellular channel. this channel is a topic of much current interest, having initially been proposed as a conduit for the passage from the mother cell to the forespore of a specific, but putative, regulator of the rna polymerase sigma factor, r g 2 and later as a gap junction-like feeding tube 3 through which the mother cell supplies molecules for the biosynthetic needs of the forespore. here we present data on the structure and interactions of spoiiq and spoiiiah gleaned from biophysical methods and protein crystallography. these data lead to a plausible model for the intercellular channel. the glycine receptor (glyr) is a chloride permeable ligand gated ion channel and that can mediate synaptic inhibition. due to a possible involvement in the pathophysiology of temporal lobe epilepsy, the different properties of glyrs containing alpha3l and k subunit isoforms are currently investigated. previous characterizations of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution as observed by diffraction-limited fluorescence microscopy. we studied these isoforms, when separately expressed in transfected hek 293t cells, by using single molecule tracking (smt) in living cells and direct stochastic optical reconstruction microscopy (dstorm) on fixated cells. for both techniques the glyrs are stained using a primary antibody directly labeled with alexa fluor dyes. the dstorm experiments support the observation that alpha3l glyr are clustered, while the alpha3k glyrs are more uniformly spread. the analysis of the short range diffusion coefficients obtained by smt reveals the presence of heterogeneous motion for both isoforms. the k-isoform has a higher fraction of fast diffusion. in contrast, the l-isoform is more associated with slow diffusion and appears to undergo hindered diffusion. since nanoparticles are suitable for tumor therapy due to their passive targeting to cancer cells by enhanced permeability and retention effect [1] , it is important to understand mechanisms of their delivery into the living cancer cells. in this respect we have developed a modular spectral imaging system based on a white light spinning disk confocal fluorescence microscope and a narrow tunable emission filter. firstly, interaction of polymer nanoparticles and cells labeled with spectrally overlapping probes was examined. the use of fluorescence microspectroscopy (fms) allowed co-localization, which showed that the size of polymer nanoparticles strongly influences their transfer across the cell plasma membrane. next, the delivery of liposomes (composed of cancerostatic alkylphospholipid (opp) and cholesterol) labeled with environment-sensitive fluorescent probe was monitored. we were able to detect a very small shift in emission spectra of cholesterol-poor opp liposomes inside and outside the cells, which would not be possible without the use of fms. this shift implies that the delivery of these liposomes into cancer cells is based on fusion with the cell membrane [2] . [ high-resolution optical imaging techniques make now accessible the detection of nanofeatures in bio-and soft-matter by non-ionizing visible radiation. however, high-resolution imaging is critically dependent by the fluorescent probes used for reporting on the nano-environment. on account of our long-standing interest in the development of fluorescent probes, we set out to design and engineer new fluorescent systems for nanoscale imaging and sensing of biological specimens and soft-matter. these fluorophores report on fast subtle changes of their nanoscale environment at excited state and are meant to fulfill these requirements: a) optical responses (intensity, wavelength-shift, lifetime, anisotropy) predictably related to the environmental polarity, viscosity, macromolecular structure, b) high brightness allowing for single-molecule detection, c) easily conjugable to biomolecules or macromolecules of interest. notably, we aim at conjugating these properties with the capability of nanoscopy imaging based on stimulated emission depletion or stochastic reconstruction optical microscopy. in this lecture the main features and applications of the engineered probes will be reviewed and future developments in this exciting field will be discussed. foamy virus (fv) is an atypical retrovirus which shares similarities with hiv and hepatitis b viruses. despite numerous biochemical studies, its entry pathway remains unclear, namely membrane fusion or endocytosis. to tackle this issue, dual color fluorescent viruses were engineered with a gfp labeled capsid and a mcherry labeled envelope. using high resolution 3d imaging and 3d single virus tracing, we followed the entry of the fluorescent viruses in living cells with a precision of 30nm in the plane and 40 nm along the optical axis. to distinguish between the two possible pathways, we developed a novel colocalization analysis method for determining the moment along every single trace where the colors separate, i.e. the fusion event. the combination of this dynamical colocalization information with the instantaneous velocity of the particle and its position within the reconstructed 3d cell shape allows us to determine whether the separation of capsid and envelope happens at the cell membrane or from endosomes. we then compared two types of fv and demonstrated, consistently with previous ph-dependency studies, that the prototype fv can enter the cell by endocytosis and membrane fusion, whereas the simian fv was only observed to fuse after endocytosis. phosphatidylinositol 4,5-bisphosphate (pi(4,5)p 2 ) is a minor component of the plasma membrane known to a critical agent in the regulation of synaptic transmission. clustering of pi(4,5)p 2 in synaptic active zones is important for synaptic transmission. however, pi(4,5)p 2 does not spontaneously segregate in fluid lipid membranes and another mechanism must be responsible for the lateral segregation of this lipid in active zones. clustering of pi(4,5)p 2 is expected to be associated with lipid-protein interactions and possibly partition towards lipid rafts in the plasma membrane. here we analyze the influence of protein palmitoylation on the formation of pi(4,5)p 2 clusters and on synaptic protein-pi(4,5)p 2 interaction by means of fö rster resonance energy transfer measurements by fluorescence lifetime imaging (fret-flim) and fret confocal microscopy. . during sporulation an entire chromosome is transferred into the forespore. this process starts by the formation of an asymmetrically located division septum that leads to the formation of two unequally sized compartments: a large mothercell and a smaller forespore. the septum traps about 30% of the chromosome to be transferred into the forespore. the remaining (*3mbp) are then translocated from the mother cell into the forespore by an active mechanism involving the spo-iiie dna translocase. the mechanisms of translocation, particularly the control of the directionality, still remains unknown and various models have been proposed so far. since each model predicts very different distribution of spoiiie proteins at the sporulation septum, we used palm microscopy (photoactivated localization microscopy) to investigate proteins localization in live-sporulating bacteria. using this technique, we showed that spoiiie proteins are forming a single tight focus at the septum with a characteristic size of around 30nm. more surprising, the focus is usually localized in the mother cell compartment and the mean distance between the spoiiie focus and the septum is 35nm. our data suggest that during the translocation process, spoiiie proteins are only forming stable complexes on the mother cell side, allowing then for a control of the chromosome translocation from the mother cell to the forespore. morphogenetic gradients determine cell identity in a concentration-dependent manner and do so in a way that is both incredibly precise and remarkably robust. in order to understand how they achieve this feat, one needs to establish the sequence of molecular mechanisms starting with morphogen gradient formation and leading to the expression of downstream target genes. in fruit flies, the transcription factor bicoid (bcd) is a crucial morphogen that forms an exponential concentration gradient along the embryo ap axis and turns on cascades of target genes in distinct anterior domains. we measured bcd-egfp mobility in live d. melanogaster embryos using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. we found that bcd-egfp molecules had a diffusion coefficient on the order of * 7 lm 2 /s during nuclear cycles 12-14, both in the cytoplasm and in nuclei. this value is large enough to explain the stable establishment of the bcd gradient simply by diffusion. on the other hand, in the context of the extremely precise orchestration of the transcription of the hunchback bcd target gene, it is too slow to explain how a precise reading of bicoid concentration could be achieved at each interphase without the existence of a memorization process. single molecule studies of key processes during the initiation of innate and adaptive immune response the two pillars of the vertebrate immune system are the innate and adaptive immune response, which confer resistance to pathogens and play a role in numerous diseases. here we exploit single molecule fluorescence imaging on live cells to study the key molecular processes that underpin these responses. the first project looks at the changes in the organisation of toll-like receptor 4 (tlr4) on the cell surface of macrophages upon activation via lipopolysaccharide (lps), as it is currently not known whether a higher level of tlr4 organisation is required for the signalling process. macrophages natively express tlr4 at a low level which allows for oligomerisation to be analysed in live cells by dynamic single molecule colocalisation (dysco) using data obtained by totalinternal reflection fluorescence (tirf) microscopy. the experiments of the second project aim at determining the critical initial events in t-cell triggering by labelling key proteins like the tcr receptor and cd45 on the surface of live t-cells and following how their spatial distribution changes following the binding of the t-cell to a surface. this enables us to distinguish between the different models of t-cell triggering which are based on aggregation, segregation or a conformational change of the tcr. the study of cells using scanning force microscopy the motility of unicellular parasites in mammalians seems very interesting, yet very complex. in a world, were inertia cannot be used for propulsion, in a world at low reynolds numbers, most of our everyday strategies of self-propulsion do not work. one class of parasites that know their way around, the flagellate trypansome, manage not only to survive in the blood stream, which is a lot faster than its own propulsion velocity and where the trypanosome is constantly attacked by its host's immune response, but also to penetrate the bloodbrain-barrier, which actually should be to tight to enter. even though trypanosomes are known for more than 100 years, their motility behaviour is not completely elucidated yet. now, using high-speed darkfield-microscopy in combination with optical tweezers in microfluidic devices and analyzing the recorded data, new light has been shed on the motility of these parasites. astonishing results show that trypanosomes are very well adapted to their hosts environment, they even can abuse red blood cells for their self-propulsion and use the bloodstream itself to drag antibodies bound to their surface to their cell mouth, where the antibodies are endocytosed and digested. the first part of the presentation will discuss nanoparticle (quantum dot, qd) biosensors and nanoactuators that exploit novel and unusual fret phenomena in the induction/detection of protein aggregation [1] , reversible on-off qd photoswitching [2] , and ph sensing [3] . the second part of the presentation will feature the application of an integrated chemical biological fret approach for the in situ (in/on living cells) detection of conformational changes in the ectodomain of a receptor tyrosine kinase (the receptor for the growth factor egf) induced by ligand binding [4] . the measurements were conducted with a two-photon scanning microscope equipped with tcspc detection; novel methods for lifetime analysis ad interpretation were employed to confirm the concerted domain rearrangements predicted from x-ray crystallography. the study of protein-protein interactions in vivo is often hindered by the limited acquisition speed of typical instrumentation used, for instance, for lifetime imaging microscopy. anisotropy polarization is altered by the occurrence of foerster resonance energy transfer (fret) and anisotropy imaging was shown to be comparatively fast and simple to implement. here, we present the adaptation of a spinning disc confocal microscope for fluorescence anisotropy imaging that allowed to achieve in vivo imaging at high spatial and temporal resolution. we demonstrate the capabilities of this system and in-house developed analysis software by imaging living caenorhabditis elegans expressing constitutive dimeric and monomeric proteins that are tagged with gfp. measuring intracellular viscosity: from molecular rotors to photodynamic therapy of cancer marina k. kuimova department of chemistry, imperial college london, exhibition road, sw7 2az, uk viscosity is one of the main factors which influence diffusion in condensed media and is crucial for cell function. however, mapping viscosity on a single-cell scale is a challenge. we have imaged viscosity inside live cells using fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent [1] [2] [3] . this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to 1009 higher than that of water. we demonstrated that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt) [2] . we also demonstrated that the ability of a fluorophore to induce apoptosis in cells during pdt [4] , or to act as a benign molecular rotor, measuring viscosity, can be controlled by carefully selecting the excitation wavelength in viscous medium [5] . in the field of biophysics and nanomedicine, the cellular reaction and the kinetics of gene expression after transfection of live cells with plasmid dna or gene-silencing sirna is of great interest. in a previous study on the transfection kinetics of non-viral gene-transfer [1] we realised that the development of single-cell arrays would be a great step towards easy-to-analyse, high-throughput transfection studies. the regular arrangement of single cells would overcome the limitations in image-analysis that arise from whole populations of cells. in addition to that, the analysis of expression kinetics at the single-cell can help to identify the cell-to-cell variability within a cell population. in order to develop suitable single-cell arrays, we are currently adjusting the different parameters of such a microenvironment (e.g. size, shape, surface-functionalisation) in order to end up with a defined surrounding for single-cell transfection studies. in addition to that, we try to find the optimal uptake pathway for each of the different applications. the neurodegenerative disorder alzheimer's disease (ad) causes cognitive impairment such as loss of episodic memory with ultimately fatal consequences. accumulation and aggregation of two proteins in the brain -amyloid beta and tau -is a characteristic feature. these soluble proteins aggregate during the course of the disease and assemble into amyloid-like filaments. recently it was found that the toxicity of soluble amyloid beta oligomers must also be taken into account for the pathogenesis of cognitive failure in ad. if oligomers are the predominant toxic species it would be pertinent to determine how they disrupt and impair neuronal function. the prion protein (prp) receptor has been proposed to mediate amyloid beta binding to neuronal cells. we have characterised the interaction of the amyloid beta and the prp receptor expressed on hippocampal and neuroblastoma cells at the single-molecule level. we do not detect any colocalisation of either the 40 or 42 amino acids variants with the prp receptor. bacterial biofilms are of the utmost importance in the study of environmental bioremediation and the design of materials for medical applications. the understanding of the mechanisms that govern cell adhesion must be analysed from the physics point of view in order to obtain quantitative descriptors. the genus rhodococcus is widely spread in natural environments. the species are metabolically diverse and thus they can degrade a wide range of pollutants. due to their high hydrophobicity, these cells are very resistant to harsh conditions, are able to degrade hydrophobic substances (e.g. oil) and attach to high-contact angle surfaces. the hydrophobicity of several strains of rhodococcus is measured and mapped using chemical force microscopy (cfm) in the present study. cfm relies on the functionalisation of scanning force microscopy (sfm) tips using hydrophobic or hydrophilic groups. in cfm, the microscope is operated in the forcevolume mode, which combines adhesion data with topographic images. the careful control of the tip chemistry permits the study of interactions between the functional groups on the tip and the bacterial surface, thus allowing the assessment of hydrophobicity. in order to perform a cfm study, the cells need to be firmly anchored to a substrate under physiological conditions (i.e. under a nutrient media or a saline buffer). to this end, several adhesive surfaces have been tested in order to find the one that gives the best results. optical microscopy is arguably the most important technique for the study of living systems because it allows 3d imaging of cells and tissues under physiological conditions under minimally invasive conditions. conventional far-field microscopy is diffraction-limited; only structures larger than *200 nm can be resolved, which is insufficient for many applications. recently, techniques featuring image resolutions down to *20 nm have been introduced such as localization microscopy (palm, storm) and reversible saturable optical fluorescent transition microscopy (resolft, sted). these methods are well suited for live-cell imaging and narrow the resolution gap between light and electron microscopy significantly. we have used palm imaging to study the formation and disassembly of focal adhesions of live hela cells in a high resolution pulse-chase experiment using monomeric irisfp [1] . mirisfp is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green-to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. in our experiments a subpopulation of mirisfp molecules is photoconverted to the red form by irradiating a specified region of the cell with a pulse of violet light. migration of tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by palm imaging, now using the photoswitching capability of the red species. real-time image reconstruction developed in our lab [2] allowed instant control imaging parameters. live cell imaging of cancer cells is often used for in-vitro studies in connection with photodynamic diagnostic and therapy (pdd and pdt). especially in presence of a photosensitizer, this live cell imaging can only be performed over relatively short duration (at most 1 hour). this restriction comes from the light-induced cell damages (photodamages) that result from rapid fluorescence photobleaching of photosensitizer. while these studies reveal exciting results, it takes several hours to discover the detailed effects of the photosensitizer on cell damage. up to our knowledge, however, there is no general guideline for modification of excitation light dose to achieve that. in this paper, the relation between excitation light doses, photobleaching of photosensitizer (pvp-hyperycin) and cell vitality are investigated using human lung epithelial carcinoma cells (a549). the strategy of this paper is to reduce the excitation light dose by using a low-power pulsed blue led such that the structures are visible in time-lapse images. fluorescence signals and image quality are improved by labelling the cells with an additional non-toxic marker called carboxyfluorescein-diacetate-succinimidyl-ester (cfse). in total we collected 2700 time-lapse images (time intervals 2 min) of dual-marked a549 cells under three different light intensities (1.59, 6.34 and 14.27 mw/cm 2 ) and a variety of pulse lengths (0.127, 1.29, 13, 54.5 and 131 ms) over five hours. we have found that there is a nonlinear relationship between the amount of excitation light dose and cell vitality. cells are healthy, i.e. they commence and complete mitosis, when exposed to low light intensities and brief pulses of light. light intensities higher than 6.34 mw/cm 2 together with pulse durations longer than 13 ms often cause cell vesiculation, blebbing and apoptosis. in all other cases, however, we found no cell death. in the future, this striking nonlinearity will be studied in more detail. progressive advances in scanning ion conductance microscopy (sicm) [1] enabled us to convert ordinary scanning probe microscope (spm) in to versatile multifunctional technique. as an imaging tool, ion conductance microscopy is capable to deliver highest possible topographical resolution on living cell membranes among any other microscopy techniques [2] . also, it can visualize surfaces complexity of those makes them impossible to image by other spms [3] . ion conductance microscopy combined with a battery of powerful methods such as fluorescence resonance energy transfer (fret) [4] , patch-clamp, force mapping, localized drug delivery, nano-deposition and nano-sensing is unique among current imaging techniques. the rich combination of ion conductance imaging with other imaging techniques such as laser confocal and electrochemical [5] will facilitate the study of living cells and tissues at nanoscale. ) and coleoptiles of wheat (triticum aestivum l.) seedlings, which were growing in light and dark conditions, were used to determine fluorescence of whole cells. fluorescence emission spectrum was monitored by fluorescent microscopy using the spectrometer usb 4000. fluorescence intensity f490, f680, f710 and f740 was determined and data was statistically analyzed in annova. we observed that bgf, rf and frf intensity increased in the first leaves with the age of the seedlings. in the coleoptiles was observed great bgf intensity increase with the age of the seedlings. in the coleoptiles decreased rf intensity of the 144 and 196 hours old seedlings, and bgf intensity decreased of 196 hours old seedlings. it was found that emission spectrum and fluorescence intensity changes are induced by the lack of light and salt (nacl) stress. analysis of fluorescence spectrum can quickly and accurately indicate the outset of light and salt stress in plants. there are analogical changes in fluorescence emission spectrum of plant cells in senescence and stress conditions. it was assumed that environmental stress and senescence have common mechanisms in plants. this changes can be monitored by fluorescent microscopy. triple-colour super-resolution imaging in living cells markus staufenbiel, stephan wilmes, domenik lisse, friedrich roder, oliver beutel, christian richter and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastraße 11, 49076, germany, markus.staufenbiel@biologie.uniosnabrü ck.de super-resolution fluorescence imaging techniques based on single molecule localisation has opened tremendous insight into the sub-micrometre organisation of the cell. live cell imaging techniques such as fluorescence photoactivation localization microscopy (fpalm) are currently limited to dual-colour detection due to the restricted availability of red-fluorescent photoswitchable proteins. we employed photoswitching of the oxazine dye atto655 under reducing conditions for super-resolution imaging in the cytoplasm of living cells. for efficient and specific covalent labelling of target proteins, we have made use of the halotag system. atto655 was coupled to the halotag ligand (htl) and fast reaction of htl-atto655 with the halotag enyzme was confirmed in vitro by solid phase binding assays. efficient labelling of the membrane cytoskeleton using lifeact fused to the halotag was observed and super-resolution imaging was readily achieved. based on this approach, we managed to follow the nanoscale dynamics of the actin cytoskeleton as well as clathrincoated pits using clathrin light chain fused to the halotag. we combined this technique with fpalm for triplecolour super-resolution imaging of the spatial distribution of membrane receptors in context of the membrane skeleton. the erbb family of receptor tyrosine kinases consists of four transmembrane proteins that transduce signals across the membrane to control cell fate. growth factor binding results in homo-and hetero-interactions between these receptors at the membrane. erbb receptors are implicated in many cancers, making them a target for therapeutic drugs. to date, studies of erbb interactions have been limited to individual family members or specific pairs, giving an incomplete picture of the highly complex behaviour controlling positive and negative feedback loops and signalling outcomes. to investigate erbb receptor interactions, we have developed tirf-based single molecule fluorescence microscopes capable of simultaneously imaging three, and soon five, fluorescence probes in live cells. we have also developed a catalogue of extrinsic fluorescent probes for 1:1 labelling of both endogenous and transfected erbb family members in mammalian cells, plus a bayesian approach to the analysis of single molecule data. this allows us to track active and inactive erbb family members at the basal surface of a model breast cancer cell line that expresses physiological levels of all four receptors. we present here initial characterisation of the entire erbb family together in the cell membrane. the human genome contains more than 800 g proteincoupled receptors (gpcrs); overall, 3-4% of the mammalian genome encodes these molecules. processes controlled by gpcrs include neurotransmission, cellular metabolism, secretion, and immune responses. however it is the stoichiometry of these receptors that is the most controversial. the starting point for understanding gpcr function was the idea that these receptors are monomeric. on the other hand a lot of recent studies favour the concept that gpcr form dimers and are not capable of signalling as independent monomers. recent single molecule studies try to solve this dilemma by suggesting that gpcrs form transient dimers with a lifetime of *100 ms. however questions remain about the physiological relevance of the preparations necessary for these studies, since they have not been performed on endogenous receptors. here, we directly image individual endogenous receptors using an equimolar mixture of two colour fluorescent fab fragments. we can then determine the receptors stoichiometry by quantifying its dynamic single molecule colocalisation (dy-sco) recorded by total-internal reflection fluorescence (tirf) microscopy. we have recently investigated the domain dynamics of pgk (1) . structural analysis by small angle neutron scattering revealed that the structure of the holoprotein in solution is more compact as compared to the crystal structure, but would not allow the functionally important phosphoryl transfer between the substrates, if the protein would be static. brownian large scale domain fluctuations on a timescale of 50 ns was revealed by neutron spin echo spectroscopy. the observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable function. [ many physiological and pathological processes involve insertion and translocation of soluble proteins into and across biological membranes. however, the molecular mechanisms of protein membrane insertion and translocation remain poorly understood. here, we describe the ph-dependent membrane insertion of the diphtheria toxin t domain in lipid bilayers by specular neutron reflectometry and solid-state nmr spectroscopy. we gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. at ph 6, the native tertiary structure of the t domain unfolds, allowing its binding to the membrane. the membrane-bound state is characterized by a localization of the c-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. in contrast, the amphiphilic n-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. at ph 4, repulsive interactions vanish; the n-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. the c-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. these helices do not adopt a transmembrane orientation. interestingly, the t domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. we propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane. the limited stability in vitro of mps motivates the search of new surfactants (1) (2) (3) (4) . fss with a polymeric hydrophilic head proved to be mild towards mps (1) .new fss were designed with chemically defined polar heads for structural applications. lac-derivative was efficient in keeping several mps water soluble and active but formed elongated rods (2) . the glu-family was synthesized, characterized in by sans and auc and for its biochemical interest. the formation of rods is related to the low volumetric ratio between the polar head and hydrophobic tail. the surfactant bearing two glucose moieties is the most promising one, leading to both homogeneous and stable complexes for both br and the b 6 f. it was also shown be of particular interest for the structural investigation of membrane proteins using sans (3). by combining elastic and quasi-elastic neutron scattering data, and by applying theory originally developed to investigate dynamics in glassy polymers, we have shown that in lyophilised apoferritin above t * 100 k the dynamic response observed in the pico-to nano-second time regime is driven by ch 3 dynamics alone, where the methyl species exhibit a distribution of activation energies. our results suggest that over the temporal and spatial range studied the main apoferritin peptide chain remains rigid. interestingly, similar results are reported for other smaller, more flexible lyophilised bio-materials. we believe this work elucidates fundamental aspects of the dynamic landscape in apoferritin which will aid development of complex molecular dynamic model simulations of super-molecules. a detailed appreciation of the relationships between dynamics and biological function will require analysis based on such models that realize the full complexity of macromolecular material. biological systems must often be stored for extended periods of time. this is done by lyophilisation in the presence of lyoprotectants, such as sugars, which results in stable products at ambient conditions. [1] in an effort to understand the mechanism of preservation and stabilization, the interactions between sugars and liposome vesicles, which serve as a simple membrane model, have been studied extensively. amongst the common sugars, trehalose has superior preservative effects [1] and accumulates to high concentrations in many anhydrobiotic organisms. despite many experimental and numerical studies three mechanisms are proposed: vitrification [2] , preferential exclusion [3] and water replacement [4] . to gain more insight into the stabilization mechanism we have recently investigated the effect of trehalose on the bending elasticity of fully hydrated unilamellar vesicles of 1,2-dipalmitoyl-phosphatidylcholine (dppc) in d 2 o at temperatures below and above the lipid melting transition (tm) using neutron spin-echo. the data was analyzed using the zilman-granek theory. at all temperatures measured, trehalose stiffens the bilayer suggesting strong interactions between trehalose and the lipid. trehalose appears to broaden the melting transition but does not change the tm. this agrees with observations using differential scanning calorimetry. influence of macromolecular crowding on protein stability sté phane longeville, clé mence le coeur laboratoire lé on brillouin, gif-sur-yvette, france cell interior is a complex environment filled with a variety of different objects with respect to shape and size. macromolecules are present at a total concentration up to several hundred grams per litre and the overall occupied volume fraction can reach ö & 0.3-0.4. under crowding environment protein-protein interaction play a fundamental role. the crowding environment can affect some physical, chemical, and biological properties of biological macromolecules [1, 2] . traditionally, protein folding is studied in vitro at very low concentration of proteins. under such conditions, small globular single chain proteins can unfold and refold quite rapidly depending mainly to the nature of the solvent. such processes have been very intensively studied, since folding of proteins into their native structure is the mechanism, which transforms polypeptide into its biologically active structure. protein misfolding is involved in a very high number of diseases [4] (e.g. alzheimer, parkinson, and kreuzfeld-jacob diseases, type ii diabetes, …). theoretically, the problem was studied by the introduction of the concept of excluded volume [5] . in recent papers [6, 7] , minton uses statistical thermodynamic models to address the question. he predicted that inert cosolutes stabilize the native state of proteins against unfolding state mainly by destabilizing the unfolded state and that the dimension of the unfolded state decreases with increasing the concentration of cosolute in a measurable way. small angle neutron scattering (sans) is a technique of choice for such study because, by using appropriate mixtures of light and heavy water, it is possible to match the scattering length density of the solvent to the one of the cosolute and thus to measure the conformation of a molecule at low concentration in a presence of a high concentration of another one. we will present a complete experimental study of the mechanism that leads to protein stabilization by macromolecular crowding [7, 8] . coupled dynamics of protein and hydration water studied by inelastic neutron scattering and molecular dynamics simulation hiroshi nakagawa 1,2 , mikio kataoka 1,3 1 japan atomic energy agency, tokai, japan, 2 juelich centre for neutron science, forschungszentrum juelich gmbh, 3 nara institute of science and technology, ikoma, japan proteins work in an aqueous environment at ambient temperature. it is widely accepted that the proteins are flexible and mobile. the flexibility and mobility, that is, protein dynamics are essential for protein functions. neutron incoherent scattering is one of the most powerful techniques to observe protein dynamics quantitatively. here i will talk about dynamics of protein and its hydration water. the structure of a soluble protein thermally fluctuates in the solvated environment of a living cell. understanding the effects of hydration water on protein dynamics is essential to determine the molecular basis of life. however, the precise relationship between hydration water and protein dynamics is still unknown because hydration water is ubiquitously configured on the protein surface. we found that hydration level dependence of the onset of the protein dynamical transition is correlated with the hydration water network. hydration water dynamics change above the threshold hydration level, and water dynamics control protein dynamics. these findings lead to the conclusion that the hydration water network formation is an essential property that activates the anharmonic motions of a protein, which are responsible for protein function. thermal motions and stability of hemoglobin of three endotherms (platypus -ornithorhynchus anatinus, domestic chicken -gallus gallus domesticus and human -homo sapiens) and an ectotherm (salt water crocodile -crocodylus porosus) were investigated using neutron scattering and circular dichroism. the results revealed a direct correlation between the dynamic parameters, melting -, and body temperatures. on one hand, a certain flexibility of the protein is mandatory for biological function and activity. on the other hand, intramolecular forces must be strong enough to stabilize the structure of the protein and to prevent unfolding. our study presents experimental evidence which support the hypothesis that the specific amino acid composition of hb has a significant influence on thermal fluctuations of the protein. the amino acid sequence of hb seems to have evolved to permit an optimal flexibility of the protein at body temperature. macromolecular resilience was found to increase with body and melting temperatures, thus regulating hb dynamics. where k is the trap spring constant, a is the subdiffusion exponent and e a is the mittag-leffler function. the parameters obtained by fitting this equation to the experimental msds are summarized in table 1 . at short lag times we have not found any difference between the two cell types, contrarily to the previous results obtained by afm 2 . for both cell lines the subdiffusion exponent, a was found close to , the value predicted by the theory of semiflexible polymers. but the crossover frequency x 3 , was found smaller for the cancerous cells for all datasets. it corresponds to passage to the confined regime at longer times. we attribute it to the bigger impact of molecular motors. we study the spatiotemporal evolution of fibrous protein networks present in the intracellular and extracellular matrices. here, we focus on the in vitro actin network dynamics and evolution. in order to study the hierarchical self-assembly of the network formation in a confined environment and provide external stimuli affecting the system minimally, we introduce a new microfluidic design. the microfluidic setup consists of a controlling channel to which microchambers of different shapes and sizes are connected through narrow channels. this design results in having mainly convective transport in the controlling channel and diffusive transport into the microchamber. rhodamine labeled actin monomers diffuse into the chamber. after polymerization is induced, they form a confined entangled network. cross-linking proteins can then be added to increase the network complexity. moreover, we can generate gradients of reactants across the microchambers and vasodilator-stimulated phosphoprotein (vasp) is a crucial regulator of actin dynamics. it is important in cellular processes such as axon guidance and migration, promoting assembly of filopodia and lamellipodia. vasp's multiple domain structure increases the range of interactions it has with actin monomers, filaments, and other proteins and it displays multiple binding modes both in vitro and in vivo, including barbed end elongation and filament bundling. however, it is not fully understood how vasp affects the structural and mechanical properties of actin networks. we characterize vasp-mediated bundling of actin networks in a simplified in vitro system using confocal microscopy and quantify mechanical properties with rheology measurements. we show that the network properties differ from other actin bundling proteins and reflect vasp's multiple domain structure, displaying a complex bundling phase space that depends upon solution conditions. we observe the formation of large bundle aggregates accompanied by a reduction in network elasticity at high protein ratios. in addition, we change vasp's actin binding mode and eliminate bundling by introducing free actin monomers. finally, we show preliminary results from a biomimetic system that extends the range of actin-vasp interaction. cell migration or proliferation? the go or grow hypothesis in cancer cell cultures tamá s garay, é va juhá sz, jó zsef tímá r, balá zs heged} us 2 nd department of pathology, semmelweis university, budapest, hungary background: cancer related death is constantly growing in the past decades. the mortality of solid tumors is mostly due to the metastatic potential of tumor cells which requires a fine adjustment between cell migration and cell proliferation. as the metabolic processes in the cell provide a limited amount of available energy (i.e. atp) the various biological processes like cell motility or dna synthesis compete for the atp available. the go or grow hypothesis postulates that tumor cells show either high migration or proliferation potential. in our study we investigate on a large series of tumor cell lines whether this assumption stands for malignant cells. materials and methods: twenty tumor cell lines derived from malignant mesothelioma (mesodermal origin) and malignant melanoma (neuroectodermal origin) were subjected to three-days-long time-lapse videomicroscopic recordings. cell motility and proliferation were characterized by the probability of cell division within 24 hours and the 24-hour migration distance of the cells. results: we found a wide range in both the cell migratory activity and the proliferation capacity in our series. the 24-hour migration distance ranged from 40 to 300 micron and from 10 to 130 micron in mesothelioma and melanoma cells, respectively. the lowest 24-hour cell division probability was found to be 0.21 in both the melanoma and mesothelioma series while the highest proliferation activity reached 1.1 and 1.4 in melanoma and mesothelioma, respectively. interestingly, in the melanoma cell lines we found a significant positive correlation (r=0,6909; p=0,0186) between cell proliferation and cell migration. in contrast our mesothelioma cell lines displayed no correlation between these two cellular processes. conclusions: in summary our findings demonstrate that the investigated tumor cells do not defer cell proliferation for cell migration. important to note the tumor cells derived from various organ systems may differ in terms of regulation of cell migration and cell proliferation. furthermore our observation is in line with the general observation of pathologists that the highly proliferative tumors often display significant invasion of the surrounding normal tissue. many cell types are sensitive to mechanical signals. one striking example is the modulation of cell proliferation, morphology, motility, and protein expression in response to substrate stiffness. changing the elastic moduli of substrates alters the formation of focal adhesions, the formation of actin filament bundles, and the stability of intermediate filaments. the range of stiffness over which different primary cell types respond can vary over a wide range and generally reflects the elastic modulus of the tissue from which these cells were isolated. mechanosensing also depends on the type of adhesion receptor by which the cell binds, and therefore on the molecular composition of the specific extracellular matrix. the viscoelastic properties of different extracellular matrices and cytoskeletal elements also influence the response of cells to mechanical signals, and the unusual non-linear elasticity of many biopolymer gels, characterized by strain-stiffening leads to novel mechanisms by which cells alter their stiffness by engagement of molecular motors that produce internal stresses. the molecular mechanisms by which cells detect substrate stiffness are largely uncharacterized, but simultaneous control of substrate stiffness and adhesive patterns suggests that stiffness sensing occurs on a length scale much larger than single molecular linkages and that the time needed for mechanosensing is on the order of a few seconds. to explore potential role of cytoskeletal component in cardiomyocyte for adaptation to extreme conditions was carried out the comparative study of expression of cytoskeletal sarcomeric protein titin in myocardium of ground squirrels during hibernation and gerbils after spaceflight. we have revealed a two-fold increase in content of long n2ba titin isoform as compared to short n2b titin isoform in different heart chambers of hibernating ground squirrels. the prevalence of the long titin isoform is known to determine the larger extensibility of heart muscle that promotes, according to frank-starling law, the increase in force of heart contractility for pumping higher viscous blood during torpor and adapting the myocardium to greater mechanical loads during awakening. moreover, titin mrna level showed seasonal downregulation in which all hibernating stages differed significantly from summer active level. it is possible that the decline of mrna and protein synthesis during hibernation may be regarded as the accommodation for minimization of energetic expenditures. we have not revealed differences in titin mrna levels between control gerbils and gerbils after spaceflight. but we have also observed the two-fold growth in the amount of n2ba titin isoform in left ventricle of gerbils after spaceflight that is likely to be directed to the restoration of the reduced heart contractility at zero-gravity. these results suggest that the increase of the content of the long n2ba titin isoform may serve as universal adaptive mechanism for regulating of heart function in response to the extreme conditions. nuclear migration is a general term for a non-random movement of the nucleus toward specific sites in the cell. this phenomenon has been described throughout the eukaryotes from yeast to mammals. the process is however still poorly understood in mammalian cells. by using microcontact printing we are able to regulate the geometry and spreading of cultured cells. adhesive micropatterns of fibronectin provide an attachment surface for the cells whereas the passivation of the surface by pll-peg prevents protein, thus cell adhesion. live cell imaging by time-lapse microscopy has shown that under these conditions cells gain a bipolar shape, and more interestingly, the nuclei of the cells showed auto-reversed motion. our research tries to understand the molecular cues and mechanisms behind the observed cellular and nuclear movement. we have already shown that the cytoskeleton plays an important role in this phenomena but the exact players and the detailed mechanism remain to be clarified. in order to identify the most important components and their relationship have drug treatments and sirna experiments have been applied. although our research focuses mainly on the motility of the nucleus, it may also help to get a better understanding of the general theme of cell migration. cell motility involves a number of strategies that cells use to move in their environments in order to seek nutrients, escape danger and fulfil morphogenetic roles. when these processes become uncontrolled, pathological behaviours, like cancer or metastasis of cancerous cells, can occur. here we present a new method for the contextual quantification of cellular motility, membrane fluidity and intracellular redox state, by using the ratiometric, redox-sensitive protein rxyfp and the ratiometric fluidity-sensitive probe laurdan. we provide evidence that dynamic redox and fluidity changes are correlated with signaling processes involved in cellular motility. these findings may pave the way to novel approaches for the pharmacological control of cell invasiveness and metastasis. manipulation of cellular mechanics anna pietuch, andreas janshoff georg-august university, tammanstraße 6, 37077 gö ttingen, germany, e-mail: anna.pietuch@chemie.unigoettingen.de rheological properties of cells determined by the underlying cytoskeleton (cortex) are key features in cellular processes like cell migration, cell division, and cell morphology. today it is possible to investigate local cellular elastic properties under almost physiological conditions using the afm. by performing force indentation curves on local areas on a cell surface the use of contact mechanic models provides the young's modulus, comprising information about the elastic properties of cells. the administration of cytoskeleton modifying substances into the cell is achieved by microinjection. we are also investigating morphological changes and rearrangements of the cytoskeleton in time resolved impedance measurements. electric cell-substrate impedance sensing is a label-free and minimal invasive technique which allows monitoring morphological changes of cells in real time. readout of the impedance is sensitive to changes in cell-substrate contacts as well as density of cell-cell contacts yielding important information about the integrity of the cell layer and changes in the properties of the cell membrane. we are studying the cellular response to modification of the cytoskeleton e.g. by introducing proteins which affect directly the organization of the actin structure like ezrin. mechanical characterization of actin gels by a magnetic colloids technique thomas pujol, olivia du roure, julien heuvingh pmmh, espci-cnrs-umpc-p7. paris, france the actin polymer is central in cell biology: it is a major component of cytoskeleton and it plays a fundamental role in motility, division, mechanotransduction…. its polymerization just beneath the cell membrane generates forces responsible for cell movement. actin filaments form a network whose architecture is defined by the nature of the binding proteins and depends on the location in the cell. for example, in the structure which leads cell migration, the lamellipodium, the gel is branched due to its interaction with arp2/3 protein complex. determining the mechanical properties of such actin network is a crucial interest to understand how forces are generated and transmitted in living cells. we grow a branched actin network from the surface of colloids using the arp2/3 machinery. the particles are super paramagnetic and they attract each other via dipole-dipole interaction to form chain. by increasing the magnetic field we apply an increasing force to the gel and we optically measure the resulting deformation. from those measurements we deduce a young modulus for a large amount of data. we are characterizing different networks by varying the concentration of the capping and branching proteins and we show how mechanics can be regulated by the different proteins. microtubules (mts) are central to the organization of the eukaryotic intracellular space and are involved in the control of cell morphology. in fission yeasts cells mts transport polarity factors to poles where growth is located, thus ensuring the establishment and maintenance of the characteristic spherocylindrical shape. for this purpose, mt polymerization dynamics is tightly regulated. using automated image analysis software, we investigated the spatial dependence of mt dynamics in interphase fission yeast cells. we evidenced that compressive forces generated by mts growing against the cell pole locally reduce mt growth velocities and enhance catastrophe frequencies. in addition, our systematic and quantitative analysis (in combination with genetic modifications) provides a tool to study the role of ?tips (plus-end tracking proteins) such as mal3 and tip1 in the spatial regulation of mt dynamics. we further use this system to decipher how the linear transport by mt interferes with the feedback circuitry that assures the correct spatial distribution of tea1, the main polarity factor in fission yeast cells. the dynamics of the cytoskeleton are largely driven by cytoskeletal motor proteins. complex cellular functions, such as mitotsis, need a high degree of control of these motors. the versatility and sophistication of biological nanomachines still challenges our understanding. kinesin-5 motors fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plus-end directed motors. here we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin-5 cin8 using single-molecule motility assays and single-molecule fluorescence microscopy. in vivo, the majority of cin8 motors moved slowly towards mt plus-ends, but we also observed occasional minus-end directed motility episodes. in vitro, individual cin8 motors could be switched by ionic conditions from rapid (up to 50 lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert in loop 8 of cin8 induced bias towards minus-end motility and strongly affected the directional switching of cin8 both in vivo and in vitro. the entirely unexpected in vivo and in vitro switching of cin8 directionality and speed demonstrate that kinesins are much more complex than thought. these results will force us to rethink molecular models of motor function and will move the regulation of motors into the limelight as pivotal for understanding cytoskeleton-based machineries. morphological and dynamical changes during tgf-b induced epithelial-to-mesenchymal transition david schneider 1 , marco tarantola 2 , and andreas janshoff 1 1 institute of physical chemistry, georg-august-university, gö ttingen, germany, 2 max planck institute for dynamics and self-organization, laboratory for fluid dynamics, pattern formation and nanobiocomplexity (lfpn), goettingen, germany the epithelial-to-mesenchymal transition (emt) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. besides its importance for numerous developmental processes like embryogenesis, emt has also been held responsible for the development and progression of tumors and formation of metastases. the influence of the cytokine transforming growth factor 1 (tgf-b1) induced emt on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines nmumg, a549 and mda-mb231 was investigated by time-resolved impedance analysis and atomic force microscopy (afm) performing force-indentation measurements. the three cell lines display important differences in cellular morphology mirrored in changes of their elastic response (young modulus), as well as their dynamics upon tgf-b1 treatment. impedance based measurements of micromotility reveal a complex dynamic response to tgf-b1 exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. additionally, the investigation of cellular elasticity via afm depicts the different cytoskeletal alterations depending on the metastatic potential of the used cell type. physics of cellular mechanosensitivity studied with biomimetic actin-filled liposomes bjö rn stuhrmann, feng-ching tsai, guido mul, gijsje koenderink fom institute amolf, amsterdam, the netherlands biological cells actively probe the mechanical properties of their tissue environment by exerting contractile forces on it, and use this information to decide whether to grow, migrate, or proliferate. the physical basis for cell contractility is the actin cytoskeleton, which transmits motor generated stresses to mechanosensitive adhesions sites that anchor the cell to the tissue. the origins of mechanosensing are far from understood due to the complex interplay of mechanical effects and biochemical signaling that occurs far from equilibrium. we use a quantitative biophysical approach based on biomimetic constructs to elucidate physical principles that underlie active mechanosensing in biological cells. we have built realistic in vitro models of contractile cells by encapsulating cross-linked actin networks together with myosin motors in cell-sized membranous containers (liposomes). our method has several advantages over prior methods, including high liposome yield, compatibility with physiological buffers, and chemical control over protein/lipid coupling. i will show contour fluctuation spectra of constructs and first data on mechanical response obtained by laser tweezers microrheology. our work will yield novel insights into stress generation and stiffness sensing of cells. setting up a system to reconstitute cytoskeleton-based protein delivery and patterning in vitro nú ria taberner, liedewij laan, marileen dogterom fom institute amolf, amsterdam, the netherlands keywords: microtubules, fission yeast, cell polarity, protein patterning, plus end binding proteins. many different cell types, from mobile fibroblasts [1] to fission yeast cells [2] , display non-homogenous protein patterns on their cell cortex. in fission yeast the cell-end marker protein tea1 that among others is responsible for recruiting the actin dependent cell-growth machinery, is specifically located at the growing cell ends. tea1 travels at the tips of growing microtubules and is delivered to the cell ends [2] . we aim to in vitro reconstitute a minimal microtubule plus-end tracking system that leads to cortical protein patterning in functionalized microfabricated chambers. our model will allow us to perturb microtubule-based transport and diffusion independently and evaluate the resulting protein patterns. [ the tropomyosins (tm) are dimeric actin-binding proteins that form longitudinal polymers along the actin filament groove. there is a great variety of isoforms, but the division of labour between the individual tms and their significance is poorly understood. as in most cell types, also in the neurons several isoforms are present, whose spatio-temporal localisation is differentially regulated. the neuron-specific brain 3 isoform (tmbr-3) can be found in the axon of the mature cells. we aimed to clone, express and charaterise this protein in terms of its effects on the kinetic parameters of the actin filament. using a pet28a construct we purified native, tag-free protein, and examined if it influences the rate of actin polymerisation or the stability of the filaments in the presence of either gelsolin or latrunculin-a, two depolymerising agents. in cosedimentation experiments the affinity of tmbr-3 to actin was* 3lm, about six times that of skeletal muscle tropomyosin. the net rate of actin polymerisation was reduced by 17% in the presence of tmbr-3. the depolymerisation induced by gelsolin or latrunculin-a was inhibited in a concentration-dependent manner. tmbr-3 seems to stabilise actin filaments against disassembly without significant effect on the net polymerisation. cell mobility and metastatic spreading: a study on human neoplastic cells using optical tweezers f. tavano the primary causes of death in cancer patients are local invasion and metastasis but their mechanisms are not yet completely understood. metastatization is accompanied by alterations of the cytoskeleton and membrane structure leading to changes in their biomechanical properties [1] . in this study we analyzed by means of optical tweezers the mechanical properties of two different breast adenocarcinoma cell lines corresponding to different metastatic potential. ot were used to grab the plasma membrane by a 1,5 um silica bead and form a plasma membrane tether. we measured the force exerted by the cell membrane on the bead and drew the force-elongation curves. fitting data in the kelvin body model [2] we found out the values for the viscoelastic parameters influencing the pulling of the membrane tethers. the first cell line analyzed, mcf-7, associated to a low metastatic potential showed tether stiffness of 153 pn/um in average. the second cell line, mda-mb 231, poorly differentiated with a high metastatic potential had a tether stiffness of 36pn/um in average, that is a four times lower value. these results seems to confirm the hypotesis that metastasis prone cells are softer than less aggressive cancer cells, and support the use of ot for these measurements for its sub-pn force resolution and because cells are manipulated without damage. [ tubulin polymerization promoting protein (tppp/p25) is a brain-specific protein that primary targets the microtubule network modulating its dynamics and stability. tppp/p25 is a disordered protein with extended unstructured segments localized at the n-and c-terminals straddling a flexible region. tppp/p25 is primarily expressed in oligodendrocytes where its multifunctional features such as tubulin polymerization promoting and microtubule bundling activities are crucial for the development of the projections in the course of oligodendrocyte differentiation enabling the ensheathment of axons with a myelin sheath that is indispensable for the normal function of the central nervous system. microtubule network, a major constituent of the cytoskeleton, displays multiple physiological and pathological functions in eukaryotic cells. the distinct functions of the microtubular structures are attained by static and dynamic associations of macromolecules and ligands as well as by post-translational modifications. tppp/p25 is actively involved in the regulation of microtubule dynamics not exclusively by its bundling activity, but also by its tubulin acetylation-promoting activity. atypical histone deacetylases, such as nad-dependent sirt2 and histone deacetylase-6, function outside of the nucleus and control the acetylation level of cytosolic proteins, such as tubulin. acetylation-driven regulation of the microtubule network during cellular differentiation is an ambiguous issue. tppp/p25 has been recently identified as an interacting partner and inhibitor of these deacetylases and their interaction decreased the growth velocity of the microtubule plus ends and the motility of the cells. we have established cell models for the quantification of the acetylation degree of microtubule network in correlation with its dynamics and stability as well as in relation to aggresome formation, that mimics the pathological inclusion formation. the intracellular level of tppp/p25 is controlled at posttranscription level by microrna and at protein level by the proteosome machinery. under pathological circumstances this disordered protein displays additional moonlighting function that is independent of its association with microtubule system or deacetylases; it enters aberrant proteinprotein interaction with a-synuclein forming toxic aggregates within the neuronal and glial cells leading to the formation of inclusions characteristic for parkinson's disease and multiple system atrophy, respectively. the cell membrane separates the intracellular from the extracellular environment while intimately interacting with the cytoskeleton in numerous cellular functions, including cell division and motility. cell shape changes are for a large part mediated by the contractile actomyosin network forming the cortex underneath the cell membrane. to uncover molecular mechanisms of cell shape control based on actin-membrane interactions, we built a novel biomimetic model system: a cell-sized liposome encapsulating an actively contracting actin-myosin network. our fabrication method is inspired by a recent report of liposome preparation by swelling of lipid layers in agarose hydrogel films. 1 we extensively characterize important liposomal properties, finding diameters between 7 and 20 lm, unilamellarity, and excellent and uniform encapsulation efficiency. we further demonstrate chemical control of actin network anchoring to the membrane. the resulting liposomes allow quantitative tests of physical models of cell shape generation and mechanics. in the cohesive structure of the cytoskeleton functionally distinct actin arrays orchestrate fundamental cell functions in a spatiotemporally controlled manner. emerging evidences emphasize that protein isoforms are essential for the functional polymorphism of the actin cytoskeleton. the generation of diverse actin networks is catalyzed by different nucleation factors, like formins and arp2/3 complex. these actin arrays also exhibit qualitative and quantitative differences in the associated tropomyosin (tm) isoforms. how the molecular composition and the function of actin networks are coupled is not completely understood. we investigated the effects of different tm isoforms (skeletal muscle, cytoskeletal 5nm1 and br3) on the activity of mdia1 formin and arp2/3 complex using fluorescence spectroscopic approaches. the results show that the studied tm isoforms have different effects on the mdia1-, and arp2/3 complexmediated actin assembly. the activity of the arp2/3 complex is inhibited by sktm and tm5nm1, while tmbr3 does not have any effect. all three tm isoforms inhibited the activity of mdia1. these results contribute to the understanding of the mechanisms by which tropomyosin isoforms regulate the functional diversity of the actin cytoskeleton. chronic thromboembolic pulmonary hypertension (cteph) is a dual pulmonary vascular disorder, which combines major vascular remodelling with small-vessel arteriopathy. the presence of fibroblasts in the clot, occluding the pulmonary arteries, and its composition create a microenvironment with increased collagen level, which might affect the local endothelial function. in this study, human pulmonary artery endothelial cells (hpaec) were exposed to collagen type i to address the effect of the thrombotic microenvironment on the vessel wall forming cells. the hpaecs, cultured under standard conditions were treated with 1, 10 and 100lg/ml of collagen type i for 6h and 24h. the changes in the endothelial cell barrier function were investigated by performing permeability and migration test as well as ve-cadherin staining. collagen type i treatment led to a decrease in ve-cadherin signal in hpaec. the loosening of cell-cell contacts could be proven with a significant increase in permeability after 6h of collagen treatment with different concentrations. besides the loosening of the cell-cell contacts, the hpaec migration was also dose dependently retarded by collagen application over time. our data show that collagen-rich microenvironment leads to a disruption of the junctional proteins in hpaecs, indicating an environmental induced possible alteration in the function of endothelial cells in the clots of cteph patients. the implementation of miniaturisation and high throughput screening has quickened the pace of protein structure determination. however, for most proteins the process still requires milligram quantities of protein with purity [ 95 %. these amounts are required as a result of unavoidable losses during purification and for the extensive screening of crystallisation space. for integral membrane proteins (imps), one of the initial steps in the structure determination procedure is still a major bottleneck -the over-expression of the target protein in the milligram quantity range. with a view of developing guidelines for over-expression of human imps, a systematic approach using the three most common laboratory expression systems (e. coli, s. cerevisiae, sf9 insect cells) was implemented. initial expression levels were determined by either partial purification using ni 2? -nta (e. coli), green fluorescence protein (gfp) fluorescence using a c-terminal gfp tagged protein (s. cerevisiae) or flag tagged partial purification (sf9 cells). the results show that e. coli is suitable for the over-expression of human imps in the required quantity range however protein size and complexity is an important factor. the yeast system is fast and affordable but, for the group of human imps tested, the expression levels were borderline. finally for the insect cell system, the timelines are slower and it is in comparison costly to run, however, it can produce relatively large quantities of human imps. the cu?-atpase copb of enterococcus hirae is a bacterial p-type atpase involved in resistance to high levels of environmental copper by expelling excess copper. the membrane protein copb was purified from an over-expressing strain and solubilized in dodecyl-maltoside. by uv circular dichroism the secondary structure is predicted to contain 40-50 % a-helices and 10-15% b-sheets in agreement with estimates based on homology with the ca atpase serca1. we present cd-spectroscopic data on thermal unfolding of the protein to address the influence of the binding of the atp analogs atpcs and the fluorescent analog mant-atp on the protein stability. such analogs are used to mimic functional states of the atpase but undergo different interactions with the binding site that are not well characterized. we propose a competition-based assay for nucleotide binding using cdspectroscopy to deduce the occupancy of the nucleotidebinding site by non-fluorescent nucleotides. alternatively, the change of intrinsic fluorescence of mant-atp upon binding to the atpase is exploited in these assays. finally, we show how the simultaneous measurement of protein cd and nucleotide fluorescence in thermal denaturation experiments may help to determine the stability of several functional conformational states of copb. showing the steady-state distribution of electric potential, ionic concentrations are obtained efficiently. channel current, a summation of drift and diffusive currents, can be further computed from the flux of ionic concentrations. the influence of finite size effect will be also addressed. effect of cholesterol and cytoskeleton on k v 10.1 membrane distribution jimé nez-garduño am 1,2 , pardo la 2 , ortega a 1 , stü hmer w 2 1 unam, mexico-city, mexico, 2 mpi-em, gö ttingen, germany the potassium channel k v 10.1 is expressed nearly exclusively in the central nervous system. besides its function as an ion channel, k v 10.1 has also been associated with non-canonical signaling functions. various membrane proteins associated with cholesterol-sphingolipids enriched microdomains are involved in signaling pathways. in this work we studied the membrane distribution of k v 10.1 in highly purified brain-tissue plasma membranes as a function of cholesterol content versus cytoskeletal proteins. the results show that one fraction of kv10.1 associates to cholesterol-rich domains or detergent resistant membranes (drm) and another fraction to non-drm domains. the kv10.1 fraction inserted in drm is dependent on cholesterol as well as on cytoskeleton proteins. depletion of cholesterol leads to a doubling of k v 10.1 current density. we suggest that k v 10.1 coexists in two different populations: one where the transmembrane domain fits cholesterol enriched membranes and another able to fit into a less packed lipid bilayer. the importance of this distribution on signaling processes needs to be further investigated. we use the reduced model of an axis-symmetric water-filled channel whose protein wall has a single charged site. the channel length, radius and fixed charge are selected to match experimental data for gramicidin a. the ion current, occupancy and escape rate are simulated by the 1d self-consistent bd technique with account taken of the electrostatic ion-ion interaction. the bath with non-zero ion concentration on one side of the channel is modelled via the smoluchowski arrival rate. it is shown that: a) the occupancy saturates with michaelis-menten kinetics. b) the escape rate starts from the kramers value at small concentrations and then increases with concentration due to the electrostatic amplification of charge fluctuations. the resulting dynamics of the current can be described by modified reaction rate theory accounting for ionic escape over the fluctuating barrier [1] . many membrane-protein functions are amenable to biophysical and biochemical investigation only after the protein of interest has been reconstituted from a detergent-solubilised state into artificial lipid bilayers. unfortunately, functional reconstitution has remained one of the main bottlenecks in the handling of numerous membrane proteins. in particular, gauging the success of reconstitution experiments has thus far been limited to trial-and-error approaches. to address this problem, we have established high-sensitivity isothermal titration calorimetry (itc) as a powerful method for monitoring the reconstitution of membrane proteins into liposomes. itc has previously been employed for characterising liposome solubilisation and reconstitution in the absence of protein. here we show that itc is also excellently suited for tracking the complex process of membrane-protein reconstitution in a non-invasive and fully automated manner. the approach is exemplified for the prokaryotic potassium channel kcsa, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. electrophysiological experiments performed in planar lipid membranes confirmed that kcsa regained its functional activity upon itc-guided reconstitution. gating currents of low-voltage-activated t-type calcium channels family má ria karmažínová , ľ ubica lacinová institute of molecular physiology and genetics, sav, bratislava, slovak republic t-type calcium channels are distinguished by relatively low voltage threshold for an activation and steep voltage dependence of activation and inactivation kinetics just above the activation threshold. kinetics and voltage dependence of macroscopic inward calcium current through ca v 3 channels was described in a detail. in contrast, very little information is available on gating current of these channels. therefore we compared gating currents measured from all three ca v 3.1, ca v 3.2 and ca v 3.3 channels. voltage dependencies of charge movement differ dramatically from those for macroscopic current. first, their slope factors are several-fold bigger that slope factors of macroscopic current activation. second, activation mid-point for ca v 3.3 channels on-gating is shifted to more positive membrane potentials by about 20 mv compare to ca v 3.1 and ca v 3.2 channels, whose activation mid-points are similar. the same is truth for off-gating voltage dependences. kinetics of both onand off-gating is remarkably faster for ca v 3.1 and ca v 3.2 channels compare to ca v 3.3 channels. further, more charge is moved per unit of macroscopic current amplitude in ca v 3.3 channels compare to ca v 3.1 and ca v 3.2 channels. supported by apvv-0212-10 and vega 2/0195/10. the local anaesthetic lidocaine (lid) is generally believed to reach its binding site in the intracellular vestibule of the voltage-gated sodium channel via the cell membrane. qx222 (qx) is a permanently charged, quaternary amine analogue of lid, that can access this binding site via a hydrophilic route across the channel protein. the mutation i1575e of the adult rat muscle-type sodium channel (rna v 1.4) opens such a hydrophilic pathway. when bound to the internal vestibule, lid stabilizes both fast and slow inactivated states. we wondered whether qx, once bound to the internal vestibule, exerts a similar modulatory action on inactivated states as lid. the construct i1575e was expressed in tsa201 cells and studied by means of the patch-clamp technique. when applied from the extracellular side 500 lm qx stabilized the slow but not the fast inactivated state in i1575e. when applied internally, qx entered the channel, but stabilization of inactivated states could not be observed. these results suggest that binding site for use-dependent block is in the inner vestibule of the channel, fast inactivation is modulated only by the hydrophobic form of lid, and the binding site for modulation of slow inactivation by qx is only accessible from the extracellular side of the channel. we observed that lipophilicity (quantified by the logarithm of the calculated water-octanol partition coefficient, logp) is important in determining both kr and ki, but had a greater effect on ki. distribution coefficients (logd) discriminated better between kr and ki than partition coefficients (logp). the ratio of positively charged/neutral forms (quantified by the acidic dissociation constant, pka) was a significant determinant of resting affinity: predominantly charged compounds tended to be more potent against resting channels, while neutral compounds tended to be more state-dependent. aromaticity was more important for inactivated state affinity. the acidification of intracellular compartments is critical for a wide range of cellular processes. a recent candidate for ph regulation within early endosomes and tgn is the highly conserved intracellular na ? /h ? exchanger 7 isoform (nhe7), whose mutation leads to neurological syndromes in human patients. however, due to its intracellular localization, nhe7 biochemical features are still poorly characterized and its biological function remains elusive. we have developed somatic cell genetic techniques that enable the selection of variant cells able to resist h ? killing through plasma membrane expression of h ? extruders. this enabled us to obtain stable cell lines with forced plasma membrane expression of nhe7. we used them to measure its functional and pharmacological parameters with high accuracy, using fast transport kinetics. to summarize, this exchanger displays unique features within the nhe family, especially with respect to its affinity for its substrates, lithium, sodium and protons and for its guanidine-derived inhibitors. taken together with our results on the subcellular localization of the native nhe7, these unique biochemical features provide new insights on the biological function and pathological implications of this intracellular na ? /h ? exchanger. analysis of the collective behaviour of ion channels j. miśkiewicz, z. trela, s. przestalski wrocław university of environmental and life sciences, physics and biophysics department, ul. norwida 25, 50-375 wrocław, poland a novel approach to the analysis of the ion current recordings is proposed. the main goal of the standard patch clamp technique is to measure single channel activity (however the whole cell configuration is also used in various researches). in the presented study the ion channels time series recordings were several (up to four) ion channels were present are analysed and the collective behaviour of ion channels is investigated. the time ion current time series are converted into dwelltime series and the channel activity is analysed. the hypothesis of collective ion channels behaviour is verified and the influence of organolead compounds (met3pbcl) on collective ion channel activity is measured. the analysis is performed on the sv cation channels of vacuolar membrane of beta vulgaris. the aim of our computed study was to examine the possible binding site of primaquine (pq) using a combined homology protein modeling, automated docking and experimental approach. the target models of wild-type and mutant-types of the voltage-dependent sodium channel in rat skeletal muscle (rna v 1.4) were based on previous work by tikhonov and zhorov. docking was carried out on the p-loop into the structure model of rna v 1.4 channel, in the open state configuration, to identify those amino acidic residues important for primaquine binding. the threedimensional models of the p-loop segment of wild types and mutant types (w402. w756c, w1239c and w1531a at the outer tryptophan-rich lip, as well as d400c, e755c, k1237c and a1529c of the deka motif) helped us to identify residues playing a key role in aminoquinoline binding. in good agreement with experimental results, a 1000-fold inhibition loss was observed, tryptophan 756 is crucial for the reversible blocking effects of pq. as a result, w756c abolished the blocking effect of primaquine in voltage-clamp assays. hydrogen bond formation accelerates channel opening of the bacterial mechanosensitive channel mscl yasuyuki sawada and masahiro sokabe department of physiology, nagoya university graduate school of medicine, nagoya, japan the bacterial mechanosensitive channel mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer (tm1, tm2) a-helices, and its 3d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. to address this question, we performed molecular dynamics (md) simulations for the opening of mscl embedded in the lipid bilayer. in the closed state of mscl, neighboring tm1 inner helices are crossed each other near the cytoplasmic side and leu19 and val23 in the constricted part form a stable hydrophobic environment called gate. upon membrane stretch, phe78 in tm2 outer helices was dragged by lipids, leading to an opening of mscl. thus phe78 was concluded to be the major tension sensor. during opening, tm1inner helices were also dragged and tilted, accompanied by the outward sliding of the crossings. this led to a slight expansion of the gate associated with an exposure of oxygen atoms of the backbone to the inner surface of the gate. this allows water penetration in the gate and formation of hydrogen bonds between water and the exposed oxygen, which in turn weakened the hydrophobic interaction at the crossings, causing a further opening of the gate and water permeation. mitochondrial bk ca , mitobk ca has been proposed to be cardioprotective and formed by proteins of *50 to *125 kda. thus, we investigated the molecular characteristics of this channel in isolated mitochondria from murine heart. labeling of adult mouse cardiomyocytes with plasmalemma bk ca antibodies, mitotracker, and wheat germ agglutinin yielded remarkable mitochondrial but not plasma membrane localization. nanoscale fluorescence microscopy (stimulated emission depletion) revealed 7 to 15 of *40-50 nm bk ca clusters per mitochondria. further, western blot analysis of purified mitochondria showed the presence of a full length *125 kda protein. systematic rt-pcr exon scanning of isolated cardiomyocyte mrnas were consistent with a full length *125 kda alpha-subunit protein and revealed the expression of three splice inserts. insertless-bk ca robustly localized to the plasma membrane of cho cells but when a c-terminal splice insert was present bk ca was readily targeted to the mitochondria (protein proximity index was *1.0 indicating 100% colocalization). hence, cardiac mitobk ca is composed by full-length bk ca protein but with splice inserts which may facilitate its targeting to mitochondria. supported by nih and aha. patch-clamp technique was used to examine effect of trimethyl-lead and -tin on the sv channel activity in the red beet (beta vulgaris l.) taproot vacuoles. it was found that the addition of both investigated compounds to the bath solution inhibit, in a concentration-dependent manner, sv currents. when single channel properties were analyzed, only little channel activity can be recorded in the presence of 100 lm of organometal. compounds investigated decreased significantly (by about one order of magnitude) the open probability of single channels. the recordings of single channel activity obtained in the presence and in the absence of organometal showed that compounds only slightly (by ca. 10%) decreased the unitary conductance of single channels. it was also found that organometal diminished significantly the number of sv channel openings, whereas it did not change the opening times of the channels. taken together, these results suggest that organometal binding site is located outside the channel's selectivity filter and that the inhibitory effect of both compounds investigated on sv channel activity probably results from organometal-induced disorder in compatibility between membrane lipids and membrane proteins. the research was financed by polish ministry of science and higher education by grant no. nn305 336434. electrophysiological investigation of the hvdac1 ion channel in pore-spanning membranes conrad weichbrodt, claudia steinem georg-august-universitä t gö ttingen, iobc, tammannstr. 2, d-37077 gö ttingen, germany, e-mail: cweichb@gwdg.de the human voltage-dependent anion channel 1 (hvdac1) plays an important role in cell life and apoptosis since it is the main porin of the outer mitochondrial membrane. as hvdac1 is believed to play a pivotal role in apoptosis-related diseases such as stroke, alzheimer, parkinson and cancer, the alterations of its electrophysiological properties under different conditions are of great value. to perform different investigations, refolded hvdac1 is reconstituted in artificial membranes which typically consist of dphpc with a cholesterol fraction of 10-30%. they are prepared via the mü ller-rudin-technique on a functionalized porous alumina substrate containing pores with a diameter of 60 nm. the quality of these so-called nano-black-lipid-membranes (nano-blms) is verified via electrochemical impedance spectroscopy (eis), hvdac1 is reconstituted and single channel recordings are made. membranes are also prepared by spreading proteoliposomes on hydrophobized porous silicon nitride with pores of 1 lm diameter. information about altered gating-characteristics and related conductivities is gained by application of holding potentials up to ±100 mv and evaluation of the resulting currents. the hvdac1 was a kind gift of prof. c. griesinger, mpibpc, gö ttingen. pharmacological inhibition of cardiac herg k ? channels is associated with increased risk of arrhythmias. many drugs bind directly to the channel, thereby blocking ion conduction. ala-scanning mutagenesis identified residues important for drug block. two aromatic residues y652 and f656 were found to be crucial for block of most compounds. surprisingly, some cavity blocking drugs are only weakly affected by mutation y652a. in this study we provide a structural interpretation for this observation. md simulations on the y652a mutant suggest side chain rearrangements of f656 located one helical turn below y652. loss of p-p stacking induces reorientation of f656 from a cavity facing to a cavity lining conformation, thereby substantially altering the shape of the binding site. docking studies reveal that due to their rigid shape and compactness y652 insensitive drugs can still favorably interact with the reoriented f656 aryl groups, while molecules with more extended geometries cannot. the ankyrin transient receptor potential channel trpa1 is a transmembrane protein that plays a key role in the transduction of noxious chemical and thermal stimuli in nociceptors. in addition to chemical activation, trpa1 can be activated by highly depolarizing voltages but the molecular basis of this regulation is unclear. the transmembrane part of the tetrameric trpa1 is structurally related to the voltagegated k ? channels in which the conserved charged residues within the fourth transmembrane region (s4) constitute part of a voltage sensor. compared to these channels, the voltage-dependence of trpa1 is very weak (apparent number of gating charges * 0.4 versus 12 in k ? channels) and its putative voltage-sensing domain most likely lies outside the s4 because trpa1 completely lacks positively charged residues in this region. in the present study we used homology modelling and molecular dynamics to create models of the transmembrane part and the proximal cytoplasmic c terminus of trpa1. in combination with electrophysiological data obtained from whole cell patchclamp measurements we were able to point out several positively charged residues which mutation strongly alter the voltage sensitivity of trpa1 channel. these may be candidates for as yet unrecognized voltage sensor. photosynthesis p-511 action of double stress on photosystem 2 aliyeva samira a., gasanov ralphreed a. institute of botany, azerbaijan national academy of sciences, baku, azerbaijan the simultaneous effect of photoinhibitory illumination and toxic action of heavy metals ions (cd 2? and co 2? ) on activity of ps2 in vitro measuring by millisecond delayed fluorescence (ms-df) of chlorophyll a was studied. during action on chloroplasts only of cd 2? (10 -3 m) the fast component of ms-df, which originates via radiative recombination of reaction center with the camn 4 -cluster or y z on donor side of ps2, is inhibited stronger than at action of only co 2? . the steadystate level at cd 2? treatment is remain stable, while at co 2? action it is increased. simultaneous action of cd 2? and photoinhibitory illumination (4000 lmol photons m -2 s -1 ) have shown that fast component of ms-df was inhibited faster with time than in case of action of co 2? and excess light. result indicates that damage sites of action cd 2? and co 2? are donor and acceptor side of ps2, accordingly. we assume that binding site of cd 2? is y z or camn 4 -cluster, one of the recombination partners with p 680 ? on the donor side of ps2. thereby, action of cd ions on donor side of ps2 leads mainly to development of mechanism of donor-side photoinhibition. field instrument for determination of the photosynthetic capacity of intact photosynthetic bacteria e. asztalos 1 , z. gingl 2 and p. maró ti 1 1 department of medical physics and informatics, university of szeged, hungary, 2 department of experimental physics, university of szeged, hungary a combined pump and probe fluorometer and spectrophotometer with high power laser diodes has been constructed to measure fast induction and relaxation of the bacteriochlorophyll fluorescence yield and light-induced absorption changes in intact cells of photosynthetic bacteria. the construction is the upgraded version of our previous set up 1 with better time resolution (5 ls). the compact design of the mechanics, optics, electronics and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems. the versatility and excellent performance of the apparatus will be demonstrated on different fields: 1) organization and redox state of the photosynthetic apparatus of the whole cells under different growth conditions deduced from fluorescence characteristics including the lag phase, the amplitude and the rise time of the variable fluorescence, 2) electron transfer in the reaction center, cytochrome bc 1 complex and in between obtained from relaxation of the fluorescence and 3) re-reduction kinetics of the oxidized primary donor of the reaction center and energetization and relaxation of the intracytoplasmic membrane tracked by absorption changes at 798 and 525 nm, respectively. previous work has established that the iron stress induced protein a (isia) synthesized by cyanobacteria under stress conditions, has at least two functions: light harvesting [1] and photoprotection [2] . under prolonged iron starvation isia becomes the main chlorophyll-binding protein in the cell and occurs without a photosystem association. these isia aggregates have a strong ability to dissipate light energy and there is evidence of carotenoid participation in the quenching mechanism via downhill energy transfer from chlorophyll to the s1 state of a carotenoid [3] . in the present work we have measured the temperature dependence of the fluorescence of carotenoid depleted mutants (echinenone and/or zeaxanthin) and isia monomers in order to investigate the role of carotenoid and aggregation in the quenching process. pigment analysis confirms the absence of the carotenoid mutated in its biosynthesis but shows that it is mainly replaced. the monomers are lacking two carotenoids, echinenone and one of the two ß-carotenes found previously in isia aggregates. temperature dependent fluorescence shows that quenching properties are affected in the monomers and the mutants lacking zeaxanthin. soon exhausted oil resources and global climate change have stimulated research aiming at production of alternative fuels, ideally driven by solar energy. production of solar fuels needs to involve the splitting of water into protons, energized electrons and dioxygen. in photosynthetic organisms, solar-energy conversion and catalysis of water splitting (or water oxidation) proceed in an impressive cofactor-protein complex denoted as photosystem ii (psii). the heart of biological water-oxidation is a protein-bound manganese-calcium complex working at technically unmatched efficiency. in an attempt to learn from nature, the natural paragon is intensely studied using advanced biophysical methods. structural studies by x-ray spectroscopy with synchrotron radiation play a prominent role in this endeavor. time-resolved methods provide insights in the formation of intermediate states of the reaction cycle. an overview is presented focusing on (i) the efficiency of solar energy usage in psii, (ii) the interrelation between electron transfer and proton relocations, and (iii) the mechanism of water oxidation. as an outlook, new results on water oxidation by biomimetic manganese and cobalt oxides, which may become a key element in future solar-fuel systems, are presented. the peridinin-chlorophyll-protein (pcp) is a light-harvesting complex (lhc) that works as antenna in the photosynthetic process of dinoflagellates. the protein contains both chlorophylls and carotenoids molecules, the latter being responsible to extend the spectral range of captured light to regions where chlorophylls are transparent. pcp crystal structures [1] reveal that each chlorophyll is surrounded by 3 or 4 molecules of the carotenoid peridinin, located in non-equivalent positions. the different protein environment of the sites might be responsible of a spectral shift of the pigments with the functional role to extend the absorption spectra of the complex and enhance its light harvesting capabilities. high resolution x-ray diffraction data on a reconstructed pcp, the refolded peridinin-chlorophyll a-protein (rfpcp) [2] , and on the less common high salt-pcp (hspcp) opened the way to the mechanistic understanding of peridinin spectral tuning, peridinin chlorophyll energy transfer and photoprotective mechanism [3] . the two pcp forms differ in various features: spectral properties, molar mass, amino acid sequence and, above all, pigment stoichiometry, the peridinin:chlorophyll ratio being 4:1 for the rfpcp and 3:1 for the hspcp [4] . in the present work we perform classical molecular dynamics simulations of the rfpcp and the hspcp in explicit water solution. we analyse the structure and dynamics of the proteins and of their pigments to characterize the different peridinin sites in both pcp forms in terms of quantities that can affect the chromophore spectra, such as distorsion, fluctuations and nature of the protein environment. the comparison between the data suggests correspondences between the pigments of the two forms. quantum and mixed quantum/ classical molecular dynamics simulations are also under progress to investigate the effect of the protein environment on the electronic and optical properties of the pcp pigments. peculiar applications, like in optoelectronics, biosensors, photovoltaics. among the existing carrier matrices conductive metal oxides (e.g. indium tin oxide, ito), carbon nanotubes, graphenes, silicon (si) are the most frequently used materials because of their unique characteristics such as good conductivity, good optical properties and excellent adhesion to substrates. in our work we combined purified photosynthetic reaction center protein (rc) and porous silicon (psi) investigating the morphology and optoelectronic properties of the bio-nanocomposite material. ftir spectroscopy, scanning electron microscopy and energydispersive x-ray spectroscopy indicated the binding of the protein to the psi. specular reflectance spectra showed a red shift in the characteristic interference peak of the psi microcavity which was saturated at higher concentration of the protein. the binding was more effective if the functionalization was done by the si-specific oligopeptide compared to the classical covalent binding via aminopropyl-triethoxysilane (aptes). excitation by single saturation flashes indicated that the rc still exhibited photochemical turnover after the binding. the role of reactive oxygen species (ros) in plant stress, both as damaging agent and as potential signal molecule is often assessed in experiments using photosensitized elicitor dyes. for these studies, it is essential to know how efficiently these chemicals generate ros, whether they are specific ros sources, as well as their cellular localization and additional side effects. the present study addresses these issues using a variety of dyes known and traditionally applied as singlet oxygen ( 1 o 2 ) sources. rose bengal (rb), methylene violet (mvi), methylene blue (mb), neutral red (nr) and indigo carmine (ic) were studied as putative ros sources in tobacco leaves. ros products of photosensitized dyes were measured in vitro, using spin trapping epr spectroscopy. dye concentrations and irradiation concentration leading to equal absorbed excitation quanta were determined spectrophotometrically. in vivo studies were carried out using tobacco leaves infiltrated with water solutions of the putative 1 o 2 sources. cellular localizations were identified on the basis of the dyes' fluorescence. rb, nr and mvi reached into mesophyll cells and were used to study the effects of these dyes on photosynthesis. photochemical yields and quenching processes were compared before and after photosensitization of the elicitor dyes inside the leaf samples. chlorophyll-chlorophyll charge transfer quenching is the main mechanism of non-photochemical quenching in higher plants alfred r. holzwarth max-planck-institut fü r bioanorganischechemie, stiftstraße 34-36, d-45470 mü lheim a.d.ruhr, germany non-photochemical quenching (npq) in plants protects against photochemical destruction of the photosynthetic apparatus under excess light conditions.while one location of the npq process has been shown to be centered on the major light harvesting complex ii (lhcii) (q1 type or qequenching), an additional quenching center responsible for qi type (identical to q2 center) quenchinghas been suggested to be located on the minor light-harvesting complexes upon accumulation ofzeaxanthin (zx), in particularon cp24 and cp29 we have performed femtosecond transient absorption and time-resolved fluorescence measurements of npq quenching in intact leaves of higher plants, on isolated light harvesting complexes in the minor (non-aggregated)light harvesting complex cp29 reconstituted with violaxanthin (vx) or zx, and in the isolated major lhc ii complex in the aggregated state. in all of these situations we find the formation of chl-chl charge transfer (ct) states to be the dominant quenching mechanism. the yield of formation of carotenoid cation states and/or carotenoid s 1 state is either extremely low or absent, thus excluding their involvement in npq quenching as a major quenching mechanism. single-molecule spectroscopy (sms) is a powerful technique that allows investigation of fluorescence properties from single fluorescing systems. this technique enabled us to investigate the dynamics of the fluorescence intensity and spectral profiles of single, isolated light harvesting complex (lhc) on timescales of milliseconds to seconds, during continuous laser illumination. we were able to observe how each complex can rapidly switch between different emission states [1, 2] and to characterise the intensity and the spectral dynamics of major and minor antenna complexes from plants, in two different environments, mimicking the light harvesting and the light dissipating state, respectively. the results will be discussed with respect to the current models for nonphotochemical quenching (npq) mechanisms [3, 4, 5] , a vital photoprotection mechanism during which the lhcs of plants switch between a state of very efficient light utilisation and one in which excess absorbed excitation energy is harmlessly dissipated as heat. phaeodactylum tricornutum is one of the most utilized model organisms in diatom photosynthesis research mainly due to availability of its genome sequence (1). it's photosynthetic antennae are the fucoxanthin chlorophyll a/c binding proteins (fcps) which share a high degree of homology with lhcs of higher plants and green algae (2) . for detailed investigation of the antenna system of p.tricornutum, a transgenic strain expressing recombinant his-tagged fcpa protein was created which simplified the purification of a specific stable trimeric fcp complex consisting of fcpa and fcpe proteins. excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated (3). we investigated in detail the existence of specific antenna system for psi and psii in p.tricornutum as in case of higher plants. our studies indicated that at least the main light harvesting proteins fcpa and fcpe are most probably shared as a common antenna by both psi and psii. harvesting complex ii (lhciib) from spinach or in native thylakoid membranes by picosecond time-resolved fluorescence. the domain size was estimated by monitoring the efficiency of added exogeneous singlet excitation quenchers -phenyl-p-benzoquinone (ppq) and dinitrobenzene (dnb). the fluorescence decay kinetics of the systems under study were registered without quenchers and with quenchers added in a range of concentrations. stern-volmer constants, k 0 sv and k sv -for aggregates (membranes) and detergentsolubilized complexes, respectively, were determined from the concentration dependence of the ratio of the mean fluorescence lifetimes without/with quencher (s 0 , s). the ratio k 0 k sv • s 0 /s 0 0 was suggested as a measure of the functional domain size. values in the range of 15-30 were found for lhcii macroaggregates and 12-24 for native thylakoid membranes, corresponding to domain sizes of 500-1000 chlorophylls. although substantial, the determined functional domain size is still orders of magnitude smaller than the number of physically connected pigment-protein complexes; thus our results imply that the physical size of an antenna system beyond these numbers has little or no effect on improving the light-harvesting efficiency. the interaction between photosynthetic reaction center proteins (rcs) purified from purple bacterium rhodobacter sphaeroides r-26 and functionalized and non-functionalized (single (swnt) and multiple (mwnt) walled) carbon nanotubes (cnt-s) has been investigated. both structural (afm, tem and sem microscopy) and functional (flash photolysis and conductivity) techniques showed that rcs can be bound effectively to different cnts. both physical sorption and binding through -nh 2 or -cooh groups gave similar results. however, it appeared that by physical sorption some sections of the cnts were covered by multiple layers of rcs. after the binding the rcs kept their photochemical activity for a long time (at least for three months, even in dried form) and there is a redox interaction between the cnt and rcs. the attachment of rc to cnts results in an accumulation of positive and negative charges followed by slow reorganization of the protein structure after excitation. in the absence of cnt the secondary quinone activity decays quickly as a function of time after drying the rc onto a glass surface. the special electronic properties of the swnt/protein complexes open the possibility for several applications, e.g. in microelectronics, analytics or energy conversion and storage. the decay of the high fluorescence state generated by actinic illumination of different durations was measured in whole cells of various strains and mutants of photosynthetic purple bacteria. although similar method is used in higher plants, its application in photosynthetic bacteria is novel and highly challanging. the available data are restricted and only the re-oxidation of the reduced primary quinone (q a -? q a ) is usually blamed for the decay kinetics. here, we will analyse the complexity of the kinetics over a very broad time range (from 5 ls to 5 s) and show that the dark relaxation of the bacteriochlorophyll fluorescence reflects the overlap of several processes attributed to the intra-and interproteinous electron transfer processes of the reaction center (rc) and cytochrome bc 1 complex of the bacterium. in the shorter (\ 1 ms) time scale, the dominating effect is the re-reduction of the oxidized primary donor (p ? ? p) that is followed by the re-oxidation of the acceptor complex of the rc by the cytochrome bc 1 complex. as the life times and amplitudes of the components depend on the physiological state of the photosynthetic apparatus, the relaxation of the fluorescence can be used to monitor the photosynthetic capacity of the photosynthetic bacteria in vivo. circular dichroism (cd) spectroscopy is an indispensable tool to probe molecular architecture. at the molecular level chirality results in intrinsic cd, pigment-pigment interactions in protein complexes give rise to excitonic cd, whereas ''psitype'' cd originates from large densely packed chiral aggregates. it has been well established that the anisotropic cd (acd), measured on samples with defined orientation, carries specific information on the architecture of molecules. however, acd can easily be distorted by linear dichroism of the sample or instrumental imperfections -which might be the reason why it is rarely studied in photosynthesis research. here we present acd spectra of isolated intact and washed, unstacked thylakoid membranes, photosystem ii membranes (bby), and tightly-stacked lamellar macroaggregates of the main light-harvesting complex ii (lhcii). we show that the acd spectra of face-and edge-aligned stacked thylakoid membranes and lhcii lamellae exhibit profound differences in their psi-type cd bands. marked differences are also seen in the excitonic cd of bby and washed thylakoid membranes. thus acd provides an additional dimension to the light induced conformation changes of quinone depleted photosynthetic reaction centers (rcs) purified from the carotenoid-less rhodobacter sphaeroides r-26 were investigated by transient absorption (ta) and grating (tg) methods. surprisingly, the decay of the ta signal measured at 860 nm was divided into a 15 ns and a 40 ls components. the latter coincides with the life time of the tg signal, which was assigned earlier [nagy et al. (2008) febs lett. 582, 3657-3662] to spectrally silent conformational changes. the nature of the 40 ls phase was investigated further. although, the probability of chlorophyll triplet formation under our measuring conditions was small, possible contribution of the triplet states was also studied. the presence of carotenoid in the wild type rcs eliminated the 40 ls component indicating the role of carotenoid in the energy transfer within the rcs. there was no significant effect of the molecular oxygen on the ta. this fact may be explained if the chlorophyll triplets inside the protein have reduced accessibility to molecular oxygen. a differential effect of osmotic potential and viscosity on conformation changes accompanying the primary charge separation was measured by the effect of ficoll, glucose and glycerol as compared the ta to the tg signals. variable chlorophyll fluorescence: in part a yield change due to light-induced conformational change gert schansker 1 , szilvia z. tó th 1 , lá szló ková cs 1 , alfred r. holzwarth 2 and gy} oz} o garab 1 1 institute of plant biology, biological research center, hungarian academy of sciences, szeged, hungary, 2 max-planck-institut fü r bioanorganische chemie, mü lheim an der ruhr, germany on a dark-to-light transition the chlorophyll fluorescence rises from a minimum intensity (f 0 ) to a maximum intensity (f m ). conventionally, this rise is interpreted to arise from the reduction of the primary quinone acceptor, q a , of photosystem ii -although this cannot explain all presently available observations. in untreated leaves, at room temperature, the fluorescence rise follows the reduction of the electron transport chain (etc). once induced, *30-40 % of the variable fluorescence intensity relaxes within 100 ms in darkness and can be re-induced within 3 ms as long as the etc remains reduced. analyzing the fluorescence relaxation kinetics, ?/-dcmu, *30% of the amplitude cannot be explained by q a -re-oxidation. special properties of this phase determined on dcmu-inhibited samples: at cryogenic temperatures (below -40°c), where the q a -/s 2 recombination is blocked, it still relaxes and it exhibits a strong temperature dependence with an apparent e a & 12 kj/mol, whereas the reduction of q a is nearly temperature insensitive. a fluorescence yield change, driven by light-induced conformational change in the reaction center complex, can explain all these observations. tuning function in bacterial light-harvesting complexes katia duquesne 1 , edward o'reilly 2 , cecile blanchard 1 , alexandra olaya-castro 2 and james n. sturgis 1 1 lism, cnrs and aix-marseille university, marseille, france, 2 department of physics, university college, london, uk purple photosynthetic bacteria are able to synthesize a variety of different light-harvesting complexes, sometimes referred to as lh2, lh3 and lh4. here we have investigated the structural origins of these different forms and the manner in which the sequence tunes the absorption spectrum of the light-harvesting system. we then consider the functional consequences of this tuning for the organization of the light harvesting system and on the ecology of the organisms. specifically by spectroscopic techniques, in particular circular dichroism and resonance raman spectroscopy, we have been able to obtain information on the organization of the bacteriochlorophyll binding sites in the unusual lh4 of roseobacter denitrificans. this provides a picture of how different peripheral light-harvesting complexes are able to modulate the absorption spectrum. the structure and organization of this complex is the put in the context of the the recently published variability of the light-harvesting complexes. in particular the observation of their ability to form mixed complexes containing different polypeptides. we examine quantitatively the possible reasons for maintaining such variability by considering the transport properties of membranes containing either pure or mixed complexes and show that mixed complexes can permit light-harvesting to continue during adaptation. we then consider the different constraints that may be behind this type of adaptation in different bacteria and the conditions under which different types of antenna system might be optimal finally we integrate this into the evolutionary context of adaptation to variable light intensity and the ecological niches where such organisms are found. interaction between photosynthetic reaction centers and ito t. szabo 1 , g. bencsik 2 , g. kozak 1 , cs. visy 2 , z. gingl 3 , k. hernadi 4 , k. nagy 5 , gy. varo 5 and l. nagy 1 1 departments of medical physics and informatics, 2 physical chemistry and materials science, 3 technical informatics and of, 4 applied and environmental chemistry, university of szeged, hungary, 5 institute of biophysics, has biological research center, szeged, hungary photosynthetic reaction center proteins (rc) purified from rhodobacter sphaeroides purple bacterium were deposited on the surface of indium-tin-oxide (ito), a transparent conductive oxide, and the photochemical/-physical properties of the composite was investigated. the kinetics of the light induced absorption change indicated that the rc was still active in the composite and there was an interaction between the protein cofactors and the ito. the electrochromic response of the bacteriopheophytine absorption at 771 nm showed an increased electric field perturbation around this chromophore on the surface of ito compared to the one measured in solution. this absorption change is associated with the chargecompensating relaxation events inside the protein. similar life time, but smaller magnitude of this absorption change was measured on the surface of borosilicate glass. the light induced change in the conductivity of the composite as a function of the concentration showed the typical sigmoid saturation characteristics unlike if the chlorophyll was layered on the ito which compound is photochemically inactive. in this later case the light induced change in the conductivity was oppositely proportional to the chlorophyll concentration due to the thermal dissipation of the excitation energy. the supramolecular organization of photosystem ii in vivo studied by circular dichroism spectroscopy tü nde tó th 1,2 , herbert van amerongen 2,3 , gy} oz} o garab 1 , lá szló ková cs 1 1 institute of plant biology, biological research center, hungarian academy of sciences, hungary, 2 wageningen university, laboratory of biophysics, wageningen, the netherlands, 3 microspectroscopy centre, wageningen university, wageningen, the netherlands the light reactions of photosynthesis in higher plants take place in granal chloroplast thylakoid membranes, which contain chirally organized macrodomains composed of photosystem ii (psii) supercomplexes associated with light harvesting antenna complexes (lhciis). the physiological relevance of this hierarchic organization, which often manifest itself in semicrystalline assemblies, has not been elucidated but the diversity of the supramolecular structures and their reorganizations under different conditions indicates its regulatory role. the present work focuses on the structural and functional roles of different components of lhcii-psii supercomplexes. we used various growth conditions, influencing the protein composition, and different arabidopsis mutants (kocp24, kocp26, kopsbw, kopsbx, dgd1), with altered organization of the membranes, and measured their circular dichroism (cd) spectra as well as their chlorophyll fluorescence kinetics to characterize the chiral macro-organization of the chromophores and the functional parameters of the membranes, respectively. we show that the formation of chiral macrodomains require the presence of supercomplexes. our data also reveal specific functions of some of the protein or lipid compounds in the light adaptation processes of plants. excitation energy transfer and non-photochemical quenching in photosynthesis rienk van grondelle department of physics, vu university, de boelelaan 1081, 1081hv, amsterdam, the netherlands the success of photosynthesis relies on two ultrafast processes: excitation energy transfer in the light-harvesting antenna followed by charge separation in the reaction center. lhcii, the peripheral light-harvesting complex of photosystem ii, plays a major role. at the same time, the same light-harvesting system can be 'switched' into a quenching state, which effectively protects the reaction center of photosystem ii from over-excitation and photodamage. in this talk i will demonstrate how lhcii collects and transfers excitation energy. using single molecule spectroscopy we have discovered how lhcii can switch between this light-harvesting state, a quenched state and a red-shifted state. we show that the switching properties between the light-harvesting state and the quenched state depend strongly on the environmental conditions, where the quenched state is favoured under 'npq-like' conditions. it is argued that this is the mechanism of non-photochemical quenching in plants. photobiology in the soil: arrested chlorophyll biosynthesis in pea epicotyl sections beá ta vitá nyi, katalin solymosi, annamá ria kó sa, bé la bö ddi eö tvö s university, institute of biology, department of plant anatomy, pá zmá ny p. s. 1/c, h-1117 budapest, hungary the key regulatory step of chlorophyll (chl) biosynthesis is the nadph:protochlorophyllide oxidoreductase (por) catalyzed reduction of protochlorophyllide (pchlide)which is light activated in angiosperms. this process is usually described on artificially dark-grown plants. in this work, we studied epicotyl segments developed under the soil surface, which were dissected from pea plants grown under natural light conditions. using 77 k fluorescence spectroscopy, pigment analyses, electron microscopy and fluorescence microscopy, we found that upper segments showed transitional developmental stages, i.e. chl appeared besides pchl(ide) and etio-chloroplasts were typical. in regions under 5 cm depth, however, the characteristics of the segments were similar to those of plants germinated artificially in complete darkness, i.e. only pchl(ide) and etioplasts were present. the results of this work prove that these latter symptoms may occur in shaded tissues of fully developed, photosynthetically active plants grown under natural conditions. in this overview talk it will be shown how atomistic computations can complement experimental measurements in our quest to understand biological electron and proton transfer reactions. at first the molecular simulation methods for calculation of important electron transfer parameter such as reorganization free energy, electronic coupling matrix elements and reduction potentials will be explained. then three applications will be discussed where such computations help interpret experimental data on a molecular level. the first example concerns electron tunneling between heme a and heme a3 in the proton pump cytochrome c oxidase. this reaction is very fast, occurring on the nano-second time scale, and it is unclear if this is due to an unusually low reorganization free energy or due to high electronic coupling. carrying out large-scale all-atom molecular dynamics simulation of oxidase embedded in a membrane, we do not find evidence for unusually small values of reorganization energy as proposed previously, implying that the nanosecond tunneling rate between heme a and a3 is supported by very efficient electronic coupling. the second example is on electron transport in a deca-heme 'wire'-like protein, used by certain anaerobic bacteria to transport electrons from the inside of the cell to extracellular substrates. the crystal structure of such a protein has been solved recently for the first time. however, it is unclear if and in which direction the wire structure supports electron transport. here we present results of heme reduction potential calculations that help us reveal the possible electron flow in this protein. in a third example we explain how quantum mechanical/molecular mechanical methods (qm/mm) recently helped us understand why the catalase from h. pylori is prone to undergo an undesired protein radical migration reaction during catalysis. proton pumping activity of purple and brown membranes regenerated with retinal analogues k. bryl 1 , k.yoshihara 2 1 university of warmia and mazury, department of physics and biophysics, olsztyn, poland, 2 suntory institute for bioorganic research, wakayamadai, osaka 618, japan the retinal protein bacteriorhodopsin (br) acts as a light-driven proton pump in the purple membrane (pm) of halobacterium salinarium (h.s.). the aim of these studies was to clarify whether the specific crystalline structure of protein and protein-substrate interactions are significant for h ? transfer into the aqueous bulk phase. two membrane systems were prepared: purple membranes (br arranged in a two-dimensional hexagonal lattice) and brown membranes (br not arranged in a crystalline lattice) were regenerated with 14-fuororetinals. light-induced proton release and reuptake as well as surface potential changes inherent in the regenerated systems reaction cycles were measured. signals of optical ph indicators residing in the aqueous bulk phase were compared with signals of ph indicator covalently linked to the extracellular surface of proteins and with surface potential changes detected by the potentiometric probe. the energies of activation of proton transfer have been calculated. experimental results and thermodynamic parameters (energies of activation) suggest the different mechanism of proton transfer into the aqueous bulk phase in these two systems. the implications for models of localized-delocalized energy coupling by proton gradients will be discussed. iron regulation is a vital process in organisms and in most of them it is accomplished through the metal solubilisation and storage by ferroxidase enzymes of the ferritin family, which have the ability of sequester, oxidize and mineralize ferrous ions using oxygen or hydrogen peroxide as substrate. dnabinding proteins from starved cells (dps) belong to this ferritin's family. dps belongs to the sub-type designated as miniferritins and, besides iron storage and release capability, is responsible for hydrogen peroxide resistance showing the ability to form stable complexes with dna. the preferable cosubstrate of this enzyme is h 2 o 2 although the reaction can occur in the presence of oxygen with lower rate [1, 2] . in this work, the electrochemical behaviour of the recombinant dps from pseudomonas nautica, was assessed as a function of metal content in anaerobic environment with h 2 o 2 as co-substrate. the obtained electrochemical results together with spectroscopic studies allowed inferring some new hypothesis on the dps iron uptake mechanism. for myoglobin electrostatically immobilized on au-deposited mixed self-assembled monolayers (sams) of the composition: -s-(ch 2 ) 11 -cooh/-s-(ch 2 ) 11 -oh. our approach allows for a soft switching of the haem group charge state and accurate probing of the accompanying reorganizational dynamics of conformational (quasi-diffusional) and quantum (e.g. protonrelated) modes. the electron transfer rate constants were determined with h 2 o or d 2 o as solvent, under the variable temperature (288-328 k) or pressure (5-150 mpa) conditions, revealing the overall reorganization free energy of 0.5±0.1 ev, activation volume of -3.1±0.1 cm 3 mol -1 and inverse solvent kinetic isotope effect of 0.7±0.1 (25°c). on the grounds of an extended charge-transfer theory, we propose specific protoncoupled et mechanism additionally coupled to the slow conformational dynamics of the protein matrix accompanied by translocation(s) of haem-adjacent water molecule(s). proton gradients across pore spanning membranes: towards on-chip energy conversion daniel frese, claudia steinem institute for organic and biomolecular chemistry, georg-august-university goettingen, germany in cell organelles, chemiosmotic potentials resulting from proton gradients across membranes are widely used to fix chemical energy in forms of atp. the high efficiency of this protein-mediated energy conversion raises interest for artificial proton gradient setups. to investigate proton transport across artificial membranes, we prepared pore spanning membranes (psms) on porous silicon substrates via painting technique. this allowed us to trap aqueous content of well-defined composition and volume inside the substrates microcavities. nigericin, a peptide that acts as an h ? -k ? -antiporter and bacteriorhodopsin, a transmembrane protein which is well known to be a light driven proton pump, were reconstituted into the pre-formed psms to achieve proton transport from one aqueous compartment to the other. changes of proton concentrations inside the pores were monitored by means of confocal laser scanning microscopy (clsm). therefore, pores were filled with pyranine, a ph-sensitive fluorescence dye and variations in intensity were measured to analyze proton translocation. we were able to show that both, nigericin and bacteriorhodopsin are capable of building up a proton gradient across psms and plan to co-reconstitute atp synthases for on-chip energy conversion by formation of atp. application of the gibbs free energy profiles to sequential biochemical reactions pé ter farkas, tamá s kiss and eugene hamori department of biological physics, eö tvö s ló rá nd tudomá ny egyetem, budapest, hungary, and department of biochemistry, tulane university, new orleans, la., usa the full understanding of the energetic details of complex metabolic reaction sequences requires a step-by-step analysis of the gibbs free energy (g) changes of the ''parasystem'' (i.e., a collection of atoms comprising all the molecules participating in a given reaction) as it gradually changes from its initialreactants state to its final-products state along the reaction pathway. knowing the respective equilibrium constants of each of the participating reaction steps and also the actual in vivo concentrations of the metabolites involved, a free-energy profile can be constructed that will reveal important information about the progress of the reaction as driven by thermodynamic forces. this approach will be illustrated on some biochemical reactions including the glycolytic/gluconeogenetic pathways. furthermore, the often misleading text-book representation of enzymatic catalysis will be reexamined and explained in thermodynamic terms using the free-energy profiles of both the non-catalyzed and the enzyme-catalyzed reactions. redox-active proteins can be diversely functionalized at metaldeposited self-assembled monolayers (sams) of a widely variable composition and thickness. the voltammetric methodology in combination with the advanced data processing procedures allow for comprehensive kinetic data within the congruent series of nano-devices and the subsequent calculation of the key physical parameters, such as the medium reorganization energy of et, the donor-acceptor electronic coupling, effective relaxation time (related to the protein's and environment's fluctuational dynamics), etc. in our studies the ''model'' redox protein, cytochrome c (cytc), was either freely diffusing to sam terminal groups (mode 1), or attached to sams through the electrostatic interaction (mode 2), or specific ''wiring'' (mode 3). another redox-active protein, azurin, was confined at terminal sam groups through the hydrophobic interaction (mode 4). diverse experimental strategies including the variation of sam thickness, solution viscosity temperature and hydrostatic pressure, allowed for a severe demonstration of the full adiabatic and nonadiabatic control (thinner and thicker sams, respectively), and the intermediary regime, in a nice agreement with the major theoretical predictions. proton transfers in a light-driven proton pump j.k. lanyi dept. physiology & biophysics, university of california, irvine, usa illumination of bacteriorhodopsin causes isomerization of alltrans retinal to 13-cis,15-anti and a cyclic reaction ensues, in which the protein and the chromophore undergo conformational changes with an overall ten millisecond turn-over time and a proton is transported from one membrane side to the other. with crystal structures of six trapped intermediate states and plausible structural models for the remaining two intermediates, structures are now available for the initial bacteriorhodopsin state and all intermediates. they reveal the molecular events that underlie the light-induced transport: protonation of the retinal schiff base by asp85, proton release to the extracellular membrane surface, a switch event that allows reprotonation of the schiff base from the cytoplasmic side, side-chain and main-chain motions initiated in the cytoplasmic region, formation of a single-file chain of hydrogen-bonded water molecules that conducts the proton of asp96 to the schiff base, and reprotonation of asp96 from the cytoplasmic surface. the observed changes can be summarized as a detailed atomic-level movie in which gradual relaxation of the distorted retinal causes a cascade of displacements of water and protein atoms results in vectorial proton transfers to and from the schiff base. electron transfer (et) processes are fundamental in photosynthesis, respiration and enzyme catalysis. the relative importance of superexchange and sequential mechanisms in biological et is still a matter of debate. the identification of any ''stepping stones'' necessary for electron hopping is a key point in the understanding of long range et. hence, the study of a single event in the sequence of reactions occurring in these phenomena is a fundamental but formidable task. muon spin relaxation (lsr) has been shown to be sensitive to charge transport on a molecular lengthscale. the muon is a very sensitive probe of electron transport, as any changes to the electronic density sampled by the muon can change its spin polarization, which can easily be measured. in this context, a very useful tool is the detection of the so-called avoided level crossing (alc) resonances [1] . the enhancement in the loss of polarization of the muon's spin on these resonances dramatically increases sensitivity. we show that a laser pump -lsr probe technique can measure et processes at particular, and most importantly known, sites within the amino acids chain, and therefore track the time evolution of the electron over the molecule. keywords: photosynthesis, reaction center, electron transfer, proton transfer, fourier transform infrared, l210dn, isotopic labeling, band assignment, histidine, mechanism in photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake. in the photosynthetic reaction center (rc) of rhodobacter sphaeroides, fast formation of the charge separated state p ? q a is followed by a slower electron transfer from the primary quinone q a to the secondary quinone q b and the uptake of a proton from the cytoplasm to q b . previous fourier transform infrared (ftir) measurements on rc suggested an intermediate x in the q a -q b to q a q b transition. mutation of the amino acid aspl210 to asn (l210dn mutant) slows down proton uptake and oxidation of q a -. using time-resolved ftir spectroscopy we characterized this rc mutant and proposed specific ir bands that belong to the intermediate x. to study the role of the iron-histidine complex located between q a and q b , we performed fast-scan ftir experiments on the l210dn mutant marked with isotopically labelled histidine. we assigned ir bands of the intermediate x between 1120 cm -1 and 1080 cm -1 to histidine vibrations. these bands show the protonation of a histidine, most likely hisl190, during the q a -q b to q a q b transition. based on these results we propose a new mechanism of the coupling of electron and proton transfer in photosynthesis. complex i of respiratory chains is an energy transducing enzyme present in most bacteria and in all mitochondria. it is the least understood complex of the aerobic respiratory chain, even though the crystallographic a-helical structures of bacterial and mitochondrial complexes have been recently determined [1] [2] . this complex catalyses the oxidation of nadh and the reduction of quinone, coupled to cation translocation across the membrane. rhodothermus marinus complex i, our main model system, is a nadh:menaquinone oxidoreductase and has been extensively characterized. we have made an exhaustive study in order to identify all the subunits present in the complex [3] . the nature of the coupling charge of r. marinus complex i was investigated using inside-out membrane vesicles, which were active with respect to nadh oxidation and capable of creating and maintain an nadh-driven membrane potential (dw) positive inside. it was observed that this bacterial complex i is able of h ? and na ? transport, although to opposite directions. the coupling ion of the system was shown to be the h ? being transported to the periplasm, contributing in this way to the establishment of the electrochemical potential difference, while na ? is translocated to the cytoplasm [4] . the sodium ion extrusion from the membrane vesicles was due to the activity of complex i, since it was sensitive to its inhibitor rotenone, and it was still observed when the complex i segment of the respiratory chain was isolated by the simultaneous presence of cyanide and external quinones. additional studies have shown that although neither the catalytic reaction nor the establishment of the dph requires the presence of na ? , the presence of this ion increased the proton transport. combining all these results, a model for the coupling mechanism of complex i was proposed, suggesting the presence of two different energy coupling sites, one that works only as a proton pump (na ? independent), and the other functioning as a na ? /h ? antiporter (na ? dependent) [4] . this model was reinforced by further studies performed in the presence of the na ? /h ? antiporter inhibitor, 5-(n-ethyl-n-isopropyl)-amiloride (eipa) [5] . a deeper inside into the coupling mechanism of this enzyme was provided by studying the influence of sodium ions on energy transduction by complexes i from escherichia coli and paracoccus denitrificans. it was observed that the na ? / h ? antiporter activity is not exclusive of r. marinus complex i, since the e. coli enzyme is also capable of such a transport, but is not a general property given that the p. denitrificans enzyme does not performed sodium translocation [6] . due to the fact that r. marinus and e. coli enzymes reduce menaquinone while p. denitrificans complex i reduce ubiquinone, it is suggested that the na ? /h ? antiporter activity may be correlated with the type of quinone used as substrate. under anaerobic conditions some bacteria can use nitrate instead of oxygen in a process called denitrification. during denitrification, the reduction of no to n 2 o is catalyzed by a membrane-bound enzyme nitric oxide reductase (nor). this enzyme is an important step in the evolution of a respiratory system: nor belongs to the superfamily of o 2 -reducing heme-copper oxidases and is assumed to be the evolutionary ancestor of cytochrome c oxidase. the understanding of nor functioning was limited by the lack of structural information, but recently the first structures (cnor type from ps. aerug. and qnor type from g. stearoth.) were solved [1] [2] . we will present results of the first computational studies of nor (both cnor and qnor types) [2] [3] . the studies include: (i) large-scale all-atom md simulations of proteins in their natural environment (i.e. embedded in membrane and solvent), which were performed to describe water dynamics inside the protein and to identify potential proton transfer pathways, and (ii) free-energy calculations by the empirical valence bond (evb) method [4] for the explicit proton translocations along the pathways established by md. among important findings are new proton pathways, which were not predicted from the x-ray structure and could be identified only by means of computer simulations. simulations also reveal that, despite a high structural similarity between cnor and qnor, these enzymes utilize strikingly different proton uptake mechanisms. our results provide insights into the functional conversion between no and o 2 reductases, and into evolution proton transfer mechanisms and respiratory enzymes in general. the genome of the bacterium geobacter sulfurreducens (gs) encodes for 111 c-type cytochromes (1). genetic studies using cytochrome deficient gs strains and proteomic studies identified cytochromes that were produced under specific growth conditions (2) (3) (4) . a putative outer-membrane cytochrome, omcf, is crucial for fe 3? and u 6? reduction and also for microbial electricity production (4). omcf is a monoheme c-type cytochrome with sequence similarity to soluble cytochromes c 6 of photosynthetic algae and cyanobacteria (4). the structure of oxidized omcf was determined (5) being the first example of a cytochrome c 6 -like structure from a nonphotosynthetic organism. the structural features of omcf hinted a different function compared to cytochromes c 6 from photosynthetic organisms, being an excellent example of how structurally related proteins are specifically design by nature to perform different physiological functions. in order to elucidate omcf structural-functional mechanism, isotopic labeled protein ( 15 n and 13 c) was produced and its structure in the reduced form determined by nmr. single point-mutations at key residues were produced by site-directed mutagenesis and their impact on the structural and functional properties of omcf will be presented. in the early 90s, the search for the source of nitrogen monoxide (no) production in mammals led to the discovery of three major isoforms of no-synthases (nos): the neuronal nos (nnos), the inducible nos (inos), and the endothelial nos (enos) ( (1)). 20 years later, based on genomic analysis, numerous nos-like proteins have been identified in the genome other organism and in particular of several bacteria (bacillus anthrax, staphylococcus aureus… (2)). in spite of superimposable 3d structures and the ability to catalyse no production, all these enzymes are carrying different (if not opposite) physiological activities including cgmp signalling, cytotoxic activities, anti-oxidant defence, metabolism… moreover, noss become increasingly associated to oxidative-stress related pathologies ranging from neurodegenerative disorders, cardiovascular and inflammatory diseases, diabetes, cancers (3)…. this apparent paradox seems related to the belief that the strong similarity of sequence and structure of no-synthases must lead to a unique and identical functioning (no production) for all isoforms. this is blatant for bacterial nos-like proteins that are lacking the essential components required for no biosynthesis but remain considered as genuine no synthases. this approach might remain an obstacle to the understanding of nos actual biological role and could prevent the design of efficient nos-targeted therapeutic strategies. to elucidate this ''nos paradox'' our group has initiated a multidisciplinary approach that aims to relate the wide diversity of nos biological activities to variations in the catalytic mechanism of noss, to modifications of their regulation patterns and to adaptations to their physiological environment. in this context we have been investigating the mechanism of bacillus subtilis nos-like proteins with a special focus on the features that are specific to nos mechanism: i) electron and proton transfers and the role of the substrate and the pterin cofactor ii) oxygen activation and the role of the proximal ligand iii) the very molecular mechanism and the variations in the nature of reaction intermediates. for that matter we have been using a combination of radiolytical techniques (cryoreduction with 60 co y-irradiation, pulse-radiolysis with the elyse electron accelerator), stateof-the-art spectroscopies (epr, atr-ftir and resonance raman, and picoseconds uv-visible absorption spectrosocopies), organic synthesis (synthesized substrates and cofactors analogues) biochemistry and molecular biology (site-directed mutagenesis). we will present our results on the coupling of electron and proton transfers, on the tuning of the proximal ''push effect'' and we will discuss the conditions that favour for each nos isoform no production versus other reactive nitrogen and oxygen species. photosynthetic iron oxidation (pio) is an ancient form of photosynthesis with relevant consequences in the shaping of the planet. this form of metabolism may have been involved in the deposition of geological structures known as banded iron formations, which hold key information regarding the co-evolution of photosynthesis and earth. rhodopseudomonas palustris tie-1 and rhodobacter ferroxidans sw2 both use ferrous iron as an electron donor to support photosynthetic growth (i.e. photoferrotrophy). the sw2 foxeyz operon can stimulate light-dependent iron oxidation by other bacteria. it codes a two-heme cytochrome, a pyrroloquinoline quinone protein and an inner membrane transporter, respectively. in tie-1 the pioabc operon is required for photoferrotrophy. it codes for a ten-heme cytochrome, an outer membrane beta-barrel and a high potential iron-sulfur protein (hipip) respectively. here we present functional and structural characterization of proteins involved in pio. this molecular characterization is essential for understanding this mode of bioenergetic metabolism, and may one day aid the development of biotechnological applications like microbial fuel cells and bioremediation. alongside classical, cytochrome respiratory pathway, phycomyces blakesleeanus possess alternative, cyanideresistant respiration (crr) facilitated by alternative oxidase (aox). in order to study role of oxygen in regulation of crr, the effects of cyanide on respiration of 24h old mycelia in aerated (control), hypoxic and anoxic conditions were measured. mycelium was incubated in these conditions for 1.5h, 3h and 5h. after 1.5h, aox activity was increased only in specimens incubated in anoxic conditions (13.6%). after 3h, increase in aox activity was significant in both hypoxic and anoxic specimens (18.9% and 18.8%, respectively), with even greater increase after 5h, 20.7% for hypoxic and 23.3% for specimens in anoxic conditions. mycelia treated for 5h was then oxygenated for 10 minutes. this induced decrease in aox activity of 17% in anoxic and even 23.9% in hypoxic mycelia. aox is recognized as one of the mechanisms for maintaining low levels of reduced ubiquione which can function in conditions in which cytochrome chain is disabled, such as anoxia. this is in concordance with results obtained on p. blakesleeanus, where aox levels rise in hypoxic and anoxic conditions and decrease close to control level shortly after introduction of oxygen into the system. influence of escherichia coli f 0 f 1 -atpase on hydrogenase activity during glycerol fermentation k. trchounian 1,2 , g. sawers 2 , a. trchounian 1 1 department of biophysics, yerevan state university, 0025 yerevan, armenia, 2 institute for microbiology, martin-luther university halle-wittenberg, 06120 haale, germany e. coli encodes four hydrogenases (hyd); only three of these, hyd-1, hyd-2 and hyd-3 have been well characterized. hyd-2 has been shown recently to reversibly evolve hydrogen during glycerol fermentation at ph 7.5 [1] . proton reduction was inhibited by n,n'dicyclohexylcarbodiimide suggesting a link with the proton-translocating f 0 f 1 -atpase. indeed, at ph 7.5 in an e. coli mutant (dk8) lacking f 0 f 1 overall hyd-activity was reduced to approximately 50% of the wild type activity; hyd-2, but not hyd-1, was detected in an in-gel activity assay. f 0 f 1 is therefore suggested to be required for hyd-1 activity. at ph 6.5 in glycerol medium hyd-activity in dk8 was *10% of wild type activity and hyd-1 and hyd-2 exhibited only weak activity. this indicated a significant f 0 f 1 contribution towards hyd-activity as ph decreased. furthermore, at ph 5.5 hydactivity was negligible and only a very weak activity band corresponding to hyd-2 could be observed. these results suggest that f 0 f 1 -atpase is essential for hydrogenase activity during glycerol fermentation at ph 5.5. taken together, the results suggest an interdependence between hyd-1, hyd-2 and f 0 f 1 -atpase activity. [ ion channels and transporters control many facets of cancer cell biology 1 and blocking the activity of these impairs tumor cell growth in vitro and in vivo. this new paradigm has opened new opportunities for pharmaceutical research in oncology 1,2 . we have contributed to this field showing that k v 11.1 (herg1) channels are aberrantly expressed in several human cancers where they control different aspects of the neoplastic cell biology such as proliferation and apoptosis, invasiveness and angiogenesis, the latter through the regulation of vegf secretion (reviewed in 1 ). the herg1dependent effects were shown in vitro and, more recently, in vivo. in preclinical models of both leukemia 1 and colorectal cancer 3 , herg1 overexpression confers a higher malignancy to neoplastic cells. moreover, herg1 blockers have therapeutic potential, since preclinical tests showed that treatment with specific herg1 blockers overcame chemoresistance in acute leukemias 4 as well as reduced gi cancer growth, angiogenesis and metastatic spread 3 . the overall message emerging from our data is that the herg1 protein represents a novel biomarker and drug target in oncology. up to now, herg1 was considered an ''antitarget'' due to the cardiac side effects that many (but not all) herg1 blockers produce and that result in lengthening the electrocardiographic qt interval. we report here recent studies on known and newly developed herg1 blockers that exhibit no cardiotoxicity and are more specific for the herg1 channels expressed in cancer cells. we reported previously that the increase of the cholesterol content of the cell membrane (in vitro) modified the biophysical parameters of the gating of kvl.3 k ? ion channels in human t-lymphocytes. in our present study we aimed to determine the effect of hypercholesterolemia on the biophysical parameters of kv1.3gating and the proliferation of t cells. t-lymphocytes were isolated from the peripheral blood of patients with cholesterol level considered normal (\5.2 mmol/l,control group) and patients with hypercholesterinaemia (hc). whole-cell k? currents were measured in patchclamped t cells and the kinetic (activation and inactivation kinetics) and equilibrium parameters (voltage-dependence of steady-state activation) of kv1.3 gating were determined. lymphocyte proliferation was measured using cfse staining with and without anti-cd3 and anti-cd28 stimulation. our results indicate that the biophysical parameters of kv1.3 gating are similar in the control group and in the 'hc' samples. the cfse-based assay showed that hypercholesterolemic t cells had higher spontenaous activation rate compared to control group. however, t cells from high cholesterol level patients challenged by anti-cd3 and anti-cd28 exhibited lower proliferation rate than control cells. generalized epilepsy with febrile seizures plus (gefs?, omim 604233) is a childhood genetic epilepsy syndrome correlated to mutations in the ancillary b-subunit of neuronal voltage-gated sodium channels (nachs). b1-subunit is non-covalently associated with nach a-subunits, serving as modulator of channel activity, regulator of channel cell surface expression and as cell adhesion molecule. the first and best characterized gefs? mutation is the c121w. this mutation changed a conserved cysteine residue into a tryptophan, disrupting a putative disulphide bridge that should normally maintain an extracellular immunoglobulinlike fold. in this study, we investigated the presence of this putative disulphide bond using 2-d-diagonal-sds-page, where the proteins were separated in the first dimension in absence of a reduction agent and in presence of it in the second dimension. this method allows to visualize the protein above the diagonal experimentally confirming that the disulphide bond is intramolecular. duchenne muscular dystrophy (dmd) is associated with severe cardiac complications. recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. it is, however, unknown if ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiomyopathy. here, we studied na and ca channel impairments in dystrophic neonatal cardiomyocytes, derived from dmd mouse models, prior to cardiomyopathy development. dystrophin-deficiency reduced na current density. in addition, extra utrophin-deficiency altered na channel gating. moreover, also ca channel inactivation was reduced, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. to assess developmental changes, we also studied na channel impairments in dystrophic adult cardiomyocytes, and found a stronger na current reduction than in neonatal ones. the described na channel impairments slowed the action potential upstroke in adult cardiomyocytes, and only in dystrophic adult mice, the qrs interval of the ecg was prolonged. ion channel impairments precede pathology development in the dystrophic heart, and may be considered cardiomyopathy triggers. supported by austrian fwf (p19352). it has been over 15 years since sensory neuron-specific sodium channel na v 1.8 was identified. since then na v 1.8 has been shown to play a crucial role in pain pathways, and it became a prominent drug target for novel pain killers. in contrast to myelinated neurons, mechanisms that target voltagegated sodium channels to the unmyelinated c-fibre axons are largely unknown. we investigated the localisation of na v 1.8 in unmyelinated primary sensory neurons. na v 1.8 was found to be clustered in lipid rafts on unmyelinated axons. when the lipid rafts are disrupted, remarkable reduction in both the conduction velocity and the number of cells responsive to mechanical stimuli to the unmyelinated axons were seen. using a compartment culture system, we also found that disruption of rafts in the middle region of the sensory axons caused a significant reduction in the responsiveness of the neurons to chemical stimuli to nerve endings. this is due to the failure of action potential propagation through the axons. these data suggest that clustering of na v 1.8 in lipid rafts of unmyelinated fibres is a key factor for the functional properties of the channel, which may due to a change in the voltage threshold. disruption of the na v 1.8 cluster and modifying lipid rafts in primary sensory neurons may be a useful new approach to control the excitability of nociceptive neurons. ion currents in are crucially important for activation of t-lymphocytes. our aim was to investigate how the blockage of various ion channels in isolation or in combination affects mitogen-dependent activation and proliferation of t-cells. we activated human peripheral blood lymphocytes using monoclonal antibodies against the tcr-cd3 complex and cd28. we applied specific channel blockers inhibiting the major ion channels of the t-cell: either the kv1.3 (tea or anuroctoxin), ikca1 (tram-34), or the crac channel (2-apb) alone or in combination. five days after the stimulus we measured the change in cell size and cellular granulation with flow cytometry along with the proportion of dividing cells, using cfse (carboxyfluorescein succinimidyl ester) dilution assay. our measurements indicated that the ion channel blockers suppressed the proportion of dividing cells in a dosedependent manner. increasing the strength of the stimulation reduced the potency of the blockers to inhibit cell proliferation and eventually the blockers were ineffective in decreasing lymphocyte proliferation. we experienced the greatest amount of inhibition using the combination of the blockers, which indicates synergy in the regulation pathway of the various ion channels. recently, sodium dependent phosphate transporter napi2b was revealed as potential marker for breast, thyroid and ovarian cancer. in vivo, napi2b is involved in maintenance of phosphate homeostasis and mutations or aberrant expressions of its gene (slc34a2) are associated with several diseases including cancer. however, data about napi2b mrna expression in different types of cancer and correspondent normal tissues are controversial and restricted. we investigated slc34a2 gene expression level in normal ovarian tissues and different histomorphological types of ovarian tumors using real-time pcr analysis. it was found that slc34a2 gene was highly expressed in well-differentiated endometrioid and papillary serous tumors, but was not expressed in low-differentiated tumors, benign tumors and in most normal tissues. mrna expression of slc34a2 in serouse and endometrioid ovarian tumors accurately correlated with protein expression that was detected in these tumor samples by western blot analysis and immunohistochemistry in our previous investigation. upregulation of slc34a2 gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. in the present contribution a procedure for molecular motion characterization based on the evaluation of the mean square displacement (msd), through the self-distribution function (sdf), is presented. it is shown how msd, which represents an important observable for the characterization of dynamical properties, can be decomposed into different partial contributions associated to system dynamical processes within a specific spatial scale. it is shown how the sdf procedure allows us to evaluate both total msd and partial msds through total and partial sdfs. as a result, total msd is the weighed sum of partial msds in which the weights are obtained by the fitting procedure of measured elastic incoherent neutron scattering (eins) intensity. we apply sdf procedure to data collected, by in13, in10 and in4 spectrometers (institute laue langevin), on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry and hydrated (h 2 o and d 2 o) lysozyme with and without disaccharides. the nature of the dynamical transition is highlighted and it is shown that it occurs when the system relaxation time becomes shorter than the instrumental energy time. finally, the bioprotectants effect on protein dynamics and the amplitude of vibrations in lysozyme are presented. we evaluated q for genotoxicity in mcf-7 breast cancer cells in the presence or absence of doxorubicin (dox), docetaxel (dtx) and paclitaxel (ptx), anticancer drugs commonly used in chemotherapy of different solid tumors. damage to dna was determinated by comet assay. the cells, after treatment with investigated compounds, were washed up and cultured in fresh medium for 0, 24, 48, 72 and 96 hours. we have found that q by itself caused significant dna damage. moreover flavonol enhanced genotoxic effect of anticancer drugs. the highest amount of dna in the comet tail was observed 48 h after treatment with combination of q with dox. similar changes were found in cells incubated with combination of q with taxanes -ptx or dtx. however, damage to dna in this case was considerably lower than damage caused by combination of q with dtx. our results confirmed anticancer and genotoxic activity of quercetin, which makes it a promising candidate for a potential use as a modulator of cytotoxicity and anticancer activity of anthracycline and taxane chemotherapeutics. although the health effects of low-frequency and intensity electromagnetic fields (lfi-emfs) are controversial, increasing evidence suggests that lfi-emfs are capable for initiating various healing processes. many (bio)physical ideas were suggested to explain the influence of lfi-emfs in living systems but the main effect of lfi-emfs on cell functions remains vague. however, some effects of lfi-emfs may be explained by redox and membrane processes. during diseases, cells not only demonstrate altered biochemical processes but also produce altered non-linear bioelectromagnetic complex patterns. thus, it is reasonable to use non-linear bioelectric and bioelectromagnetic signals from cells of the body for potential therapeutic applications that may be more effective than the artificial lfi-emfs signals. our novel emost (electromagnetic-own-signal-treatment) method is based on the utilization of the non-linear, bioelectric and bioelectromagnetic signals of the patients without any electromagnetic wave modulation and inversion of recorded output signals of subjects. here, we report our some restorative results after emost application. we also suggest that the possible effects of the emost may be achieved via redox-related processes. background or leak potassium conductances are a major determinant of resting potential and input resistance, two key components of cell excitability. these currents are not passive but finely tuned and adapted to cell specific functions. k 2p channels producing these currents are tightly regulated by a variety of chemical and physical stimuli including temperature, membrane stretch, free fatty acids, ph, ca 2? , neurotransmitters and hormones as well as protein partners. these different stimuli converge on gating mechanisms that show remarkable conservation between intracellular k2p channels (twik1 channels) and k2p channels located at the plasma membrane (trek1/2 channels). living at the edge: volume 2 the conduction system of interfacial forces into the alveolar type ii cell in previous investigations, using new microscopic approaches, we found that the presence of an a li leads to a paradoxical situation: it is a potential threat that may cause cell injury, but also a important stimulus: at ii cells respond promptly, and show sustained ca 2?signals that activate exocytosis. exocytosed surfactant, in turn, clearly prolonged the time to irreversible cell damage, and may be an adaptive and evolutionary defense mechanism against the harmful nature of surface forces. recently we published that at ii cells are sensing the a li but how this stimulus is conducted and converted into the cell, is still obscure. currently we are searching for potential calcium sources and it seems that the cells signal ca 2? by extracellular ca 2? entry probably through mechanosensitive channels. specialdesigned gene chips allowed a whole genome profiling of a li exposed single cells. these cells react with rapid changes on the transcriptional level: cellular pathways that are involved include e.g. defense response and lipid metabolism, and we identified genes associated with several lung diseases and injuries. we summarize, interface forces are strong, they are acting on the cells and triggering cellular events that are closely related with classical concepts in mechanotransduction, it is very plausible that those forces play a crucial role in the lung surfactant homeostasis. calorimetric method and instrumentation were worked out and applied for investigation aqueous solutions of proteins. thermal effects were analyzed by own construction heat-fluxtype dsc cell designed for temperature range from the boiling point of water down to 120 k. the achieved sensitivity of heat flow rate (hf) of the instrument is better than 50%w tested using 1 ll of 150 mm aqueous solution of nacl. from the integral value of hf -the total enthalpy change dh total , the enthalpies of transitions were separated from the heat capacities. using the method, several types of proteins (bsa, erd10, ubq, a-, b-casein, and wt, a53t, a-synuclein mutants) were investigated in that temperature range. the results are shown in detail as an illustrative example. potential applications are outlined, which include (i) the distinction between the solvent accessible surfaces of globular and intrinsically disordered proteins, (ii) the distinction between protein mutants, and (iii) the identification of monomer and polymer protein states. this method provides a possibility to study the polymerization process (amyloid formation) and to investigate in-situ the reason and circumstances of that. morphometry due to self gravity in living organism is an integral part in understanding self gravitation bio. computational studies of biological system, especially in ab-initio embryo as self gravitating mass, floating over amniotic fluid, as if maintaining anti -gravitation (extrinsic) mechanism, would be an interesting one to explore biomechanics of intrinsic gravity. since the work would be of exploratory nature, both from the point of view of identifying appropriate stage of development of embryological morulla and available computational logics, it was contemplated to initiate functional study with a few reported ultrasound evidential works on small animals like mice, grasshopper including compact human embryo as mathematical structure that may allow the formal definition of concepts such as convergence, mapping and continuity. as many of the finite-dimensional function analysis in topological vector spaces are available, we initiated our simulation and studies on concentrating locally compact banach spaces. 3 institute for x-ray physics, university of gö ttingen, germany we report on hard x-ray phase contrast imaging of black lipid membranes (blms), which are freely suspended over a micro machined aperture in an aqueous solution. this new way of membrane structure analysis allows investigating bio molecular and organic substances in aqueous environments by parallel and divergent beam propagation imaging, using partially coherent multi-kev x-ray radiation. the width of the thinning film is significantly smaller than the detector pixel size, but can be resolved from quantitative analysis of the intensity fringes in the fresnel diffraction regime down to its native thickness of about 5nm. to our knowledge this is the first time that such small features of a very weak phase object have been visualized by direct x-ray imaging techniques. we have put forward a simplified but extendable model, which enables the theoretical description of image formation and characterization of membrane thickness and its decrease during the thinning process from a bulk to a bimolecular film. on the basis of recent experiments, future investigations will be performed to study the interactions of membranes, as they are for example known from synaptic fusion, with high spatial resolution. shown that the acid incorporates mainly in the exterior part of the erythrocyte membrane, inducing creation of echinocytes. this suggests that it interacts predominantly with the outer part of the lipid layer of erythrocytes and liposomes. it was also shown that the cga decreases the packing order of the hydrophobic part of the membranes, without changing the anisotropic fluorescence of the hydrophobic part. one of the unique features of single molecule absorption/ emission is their anisotropy due to the well-defined transition dipoles for both processes allowing the determination of the molecule's 3d orientation. therefore, several techniques have been proposed in order to determine the full 3d orientation of dipole emitters on a single molecule level. we recently demonstrated a technique that combines emission distribution and polarization detection [1, 2, 3] . as the method is an intensity distribution technique and based on single photon detection in principle, one can extend the 3d orientation determination to fluorescence correlation spectroscopy (fcs) as well as dynamical anisotropy measurements. this allows for the determination of the dynamics in 3d orientation of single molecules down to a nanosecond timescales. the 3d orientation is particularly interesting in non-isotropic environments. a lipid membrane is such a non-isotropic environment of enormous importance in biological systems. we therefore use giant unilamellar vesicle (guv) labeled with dyes like dio as a model system. due to the defined curvature of such vesicles all possible dipole orientations can be achieved. this allows us to show the capabilities of our method on different timescales and to quantify the error in determination of 3d orientation dynamics in lipid membranes. [ the aim of the studies was to determine the changes that occur in a biological membrane and the model lipid membrane as a result of interaction with strawberry leaf extract. numerous studies conducted all over the world have documented a beneficial effect of polyphenolic compounds on the human organism. however, the mechanism of the interaction on the molecular and cell level is not yet known. in the work presented, the effect of strawberry leaf extract on the erythrocyte and black lipid membranes has been investigated. the applied methods -spectroscopic, fluorimetric and electric -allowed to determine the hemolytic and antioxidant activity, and the packing order in the erythrocyte membrane as well as the electric capacity of blms. the results obtained indicate that the extract is efficient in protecting membrane lipids against oxidation, does not induce hemolysis, increases osmotic resistance and decreases packing order in the hydrophilic region of the erythrocyte membrane. moreover, it increases stability and life-time of flat lipid membranes, without altering their specific capacity. supported lipid bilayers are an abundant research platform for understanding the behavior of cell membranes as they allow for additional mechanical stability and enable characterization techniques not reachable otherwise. however, in computer simulations these systems have been studied only rarely up to now. we present systematic studies on different length scales of the changes that a support inflicts on a phospholipid bilayer using molecular modeling. we characterize the density and pressure profiles as well as the density imbalance induced by the support. it turns out that the changes in pressure profile are strong enough that protein function should be impacted leading to a previously neglected mechanism of transmembrane protein malfunction in supported bilayers. we determine the diffusion coefficients and characterize the influence of corrugation of the support. we also measure the free energy of transfer of phospholipids between leaflets using the coarsegrained martini model. it turns out that there is at equilibrium about a 2-3% higher density in the proximal leaflet. these results are in agreement with data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. we are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. we also study the influence of surface roughness and curvature on the behavior. simulations in atomistic detail are performed for selected systems in order to confirm the findings. [ the inverse bar (i-bar) domain is part of the superfamily of the membrane-deforming protein is bin-amphiphysin-rvs (bar) proteins which induce either positive or negative membrane curvature both in vitro and in cells. generation of membrane curvature by these membrane deforming proteins often works together with actin dynamics. i-bar shares its function between actin bundling and membrane binding but it is still obscured what molecular mechanisms are responsible for these functions. the aim of our project is to investigate the detailed membrane binding properties of the i-bar of irsp53 and its relations to the actin cytoskeleton. in vitro fret experiments and fluorescence quenching studies were carried out between the i-bar and liposomes made up from different lipid constructs. we have found that the i-bar has preference to bind to the negatively charged lipids however it can also bind to the uncharged lipids. the fluorescence quenching studies reflected that the accessibility of the i-bar surface was higher toward the negatively charged lipids than for the uncharged ones. tns fluorescence assay reflected that the i-bar domain binds to the surface of the micells rather than penetrating into its core. lipid bilayers present a well-known order-disorder chain transition at ambient temperatures. this transition may become anomalous if the lipid head-group presents ionic dissociation at low ionic strength, as detected by several experimental techniques: between the gel and the liquid phases an intermediate phase appears as a shoulder in the specific heat, a deep in turbulence or a maximum in conductivity. we propose a statistical model which allows ionic dissociation of the polar group on the membrane surface and thus introduces competition between the hydrophobic interaction of hydrocarbonic chains, which favours the gel phase, at low temperatures, and the electrostatic interaction of charged head-groups, which favours the fluid phase, at higher temperatures. the model presents an intermediate fluid phase with higher dissociation and charge ordering on the membrane surface, beyond a sharp gel-fluid transition. the model presents increasing temperature of the main transition by addition of salt, as well as the shrinking of the anomalous region as chain length increases. model thermodynamic behavior is compared to results for pgs, phospholipids with a glycerol head-group. the well-programmed membrane fusion systems, operating in a weakly acidic environment, have attracted attention in the fields of biochemistry, biophysics, and pharmacy because these acidic conditions are generally observed in endosomal membranes or tumor tissues. we have reported a selective liposomal membrane fusion system toward a sugar-like cyclic cis-diol structure on the target liposome. this system consists of a lapidated phenyl boronic acid derivative as membrane -bound fusogen and phosphatidylinositol as target. here we report the preparation of a boronic acid / ph-responsive polypeptide conjugate as a novel membrane fusion device and the development of a target selective liposomal membrane fusion system with endosomal ph-responsiveness. during the course of lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays to characterize membrane fusion behavior, we clearly observed a liposomal membrane fusion phenomenon when the ph of the experimental system was changed from 7.4 (physiological) to 5.0 (endosomal). our highly effective methods, which include a target selective liposomal membrane fusion, can be useful in the area of nanomedicine such as hybridoma technology and liposomebased drug or gene delivery. complete and reversible chemical denaturation of an a-helical membrane protein jana broecker, sebastian fiedler, sandro keller molecular biophysics, university of kaiserslautern, erwin-schrö dinger-str. 13, 67663 kaiserslautern, germany the question of how an unordered polypeptide chain assumes its native, biologically active conformation is one of the greatest challenges in molecular biophysics and cell biology. this is particularly true for membrane proteins. chemical denaturants such as urea have been used successfully for in vitro un-and refolding studies of soluble proteins and b-barrel membrane proteins. in stark contrast with these two protein classes, in vitro unfolding of a-helical membrane proteins by urea is often irreversible, and alternative denaturation assays using the harsh detergent sodium dodecyl sulphate suffer from a lack of a common reference state. here we present the complete and reversible chemical denaturation of the bacterial a-helical membrane protein mistic out of different micellar environments by urea. we applied multidimensional spectroscopy and techniques typically used in b-barrel membrane protein unfolding. mistic unfolds reversibly following a two-state equilibrium that exhibits the same unfolded reference state. this allows for a direct comparison of the folding energetics in different membrane-mimetic systems and contributes to our understanding of how a-helical membrane proteins fold as compared with both b-barrel membrane proteins and water-soluble proteins. in recent years, buckwheat has been of great interest in the world markets of healthy food, due to its high energy value, the content of unsaturated fatty acids, mineral constituents and vitamins. its seeds contain flavonoids which are natural, efficient antioxidants. the aim of the present studies was to investigate the effect of buckwheat extracts on the properties of biological membrane, which is the main site of the interaction between the substances buckwheat contains and the organism. the research was conducted on red blood cells and their isolated membranes, using the spectrophotometric, microscopic and fluorimetric methods. from the results obtained it follows that the compounds contained in buckwheat extracts increase the osmotic resistance of erythrocytes, making them less sensitive to the medium's osmotic pressure, induce changes in cell shape, producing increased number of echinocytes, and decrease the packing order of the polar heads of membrane lipids. it can thus be inferred that the compounds contained in the extracts penetrate the hydrophobic region of the erythrocyte membrane and alter its properties. .due to its small size, symmetric structure, amphipathicity, proteolytic stability and testable mode of activity, the gs backbone is a convenient model system to examine the structure-activity relationship of individual amino acid substitutions. we have previously reported the structure analysis of two gs analogues in which either the val or leu residues on the hydrophobic face of the molecule were substituted by the aliphatic 19 f-labeled amino acid 4f-phg. using 19 f-ssnmr in oriented lipid bilayers, we observed a re-alignment of the peptide that is compatible with the formation of a putative pore [top.curr.chem. 273:139]. here, we present novel analogs of gs with different 19 f-prolines in the b-turn region, and with cf 3 -bpg in place of leu. based on these 19 f-ssnmr results and supported by cd, dsc and activity tests, we could demonstrate that all analogues are structurally intact and antimicrobially active. we observe, however, differences in the re-alignment propensity when comparing these gs analogues in dlpc and dmpc bilayers. these differences can be rationalized in terms of molecular shape being changed upon incorporation of unnatural amino acids at various sites of the molecule. the beta-propiolactone (bpl) is an inactivating reagent commonly used to produce viral vaccine preparations (whole virions or split-virions). although bpl has been reported to inactivate nucleic acids, its mechanism of action on proteins and the outcome on viral infection remains ill-defined. in this work, h3n2/victoria/210/2009 influenza virus strain has been submitted to various bpl inactivation conditions (from 2lm to 1 mm). cell infection ability was progressively reduced and entirely abolished at 1 mm bpl. to clarify the bpl effect, we focused on membrane fusion infection steps using kinetic fluorescence molecule leakage from liposome and lipid fret assays combined with cryo electron microscopy. membrane fusion measured at ph 5 on gm3 liposomes was reduced in a dose-dependent manner. interestingly the fusion activity was partially restored using the proton-ionophore monensin as confirmed by cryoem images. in addition, a decrease of molecule leakage irrespective to bpl concentration was measured suggesting that the hemagglutinin affinity for gm3 was slightly modified even at low bpl concentration. altogether these results strongly suggest that bpl treatment impairs m2 protein activity likely by preventing proton transport and bring new light on the mechanism of action of bpl. cellular membranes have a heterogeneous lipid composition, potentially forming nano-domains or membrane rafts, believed to be platforms of altered fluidity involved in protein sorting and trafficking 1 . an alternative mechanism, potentially leading to protein sorting, has recently been proposed, suggesting that the curvature of membranes can also actively regulate protein localization 2 . recently we showed that a variety of protein anchoring motifs are membrane-curvature sensors and thus up concentrate in regions of high membrane curvature 3 . furthermore the curvature sensing ability of the anchoring motifs persisted independently of their structural characteristics. this leads us to speculate that curvature sensing might be an inherent property of any curved membrane and as a consequence, the lipid composition of the bilayer could potentially regulate this recruitment by membrane curvature. thus there might be an intimate, yet unrecognized, link between the way raft-like membrane domains and membrane-curvature promotes the localization of membrane-anchored proteins. we examined how changing the lipid composition of liposomes influenced the recruitment by membrane curvature of a model amphiphilic protein-anchoring motif. employing our single liposome curvature assay, we tested lipid mixtures with different ratios of dopc, sphingomyelin and cholesterol, giving rise to liposome populations of different phase-states. we found an amplified recruitment by membrane curvature for all raft-like l o phase-state mixtures when compared to the l d phase-state counterparts. based on these findings we suggest a synergetic effect when combining a raft-like lipid phase-state and high membrane curvature, resulting in a highly potent mechanism for selective localization of membrane-anchored proteins. keywords: non-lamellar lipid structure, phase transition, minerval, 2-hydroxylated fatty acid. minerval (2-hydroxyoleic acid), a potent antitumoral drug, is known to modulate the lipid membrane structure by decreasing the lamellar-to-non-lamellar phase transition temperature (t h ). a series of 2-hydroxy fatty acid derivatives, varying in acyl chain length and degree of unsaturation, have been analyzed in terms of their ability to stabilize the inverted hexagonal (h ii ) phase in palmitoyl-oleoyl-phosphatidylethanolamine membranes. differential scanning calorimetry and 31 p-nuclear magnetic resonance showed that mono-and polyunsaturated, but not saturated, 2-hydroxylated fatty acid molecules were able to decrease the t h . lipid vesicles mimicking the lipid composition of a cell membrane were solubilized at 4°c in the presence of triton x-100. the results demonstrated that the amount of detergent-resistant membranes, which are related to liquid ordered (lo) structures, decreased in the presence of 2-hydroxylated fatty acids. the so-called lipid membrane therapy focuses on the reversion of cell disfunction through the modulation of the membrane structure, thus altering the activity of membrane-associated proteins. the ability to modify the biophysical properties of a lipid membrane makes the studied 2-hydroxylated fatty acid molecules be prospective candidates for the use in the lipid membrane therapy. ]. however, a significant downward divergence occurs above 4 mol % of probe content, which might indicate deviations to ideal mixing in fluid phase. results for b-py-c 10 -hpc, in mixtures of popc with 10 and 20 mol % pops were indistinguishable from those obtained with pure popc vesicles; however excimer formation in pure pops bilayers appears to be appreciably higher. we also compared the excimer formation findings with quenching of the same probes by low concentrations of doxyl quencher groups labeled acyl phospholipid chain at the same depth of the pyrenyl group. the results are also scrutinized by the same two-dimensional kinetic formalism and good correlation was also found. derek marsh max-planck-institut fü r biophysikalische chemie, 37070 gö ttingen, germany the amassing of comprehensive data on the lipid composition of biological membranes by lipidomics initiatives provides a potent challenge to the membrane biophysicist interested in lipid structure. this resolves itself essentially into two aspects. the first systematises the dependence of membrane biophysical parameters on lipid molecular structure. lipid volumes, membrane dimensions, chain-melting temperatures and enthalpies, nonlamellar phase formation and structure, critical micelle concentrations and thermodynamics of membrane formation, membrane-membrane interactions and lipid transfer are amongst the properties of central biophysical interest. the relevant structural parameters are lipid chain length, degree of unsaturation, chain branching and headgroup configuration. the second, more complex and less well developed, aspect concerns the lipidlipid interactions that determine the membrane properties of lipid mixtures. in part, these can be obtained from binary phase diagrams, and the more limited number of ternary phase diagrams -notably with cholesterol -that are available. extrapolation to higher order mixtures lies in the future. i shall attempt to summarise some of the progress in these directions. the immediate aim is a second edition of my handbook of lipid bilayers, which, in addition to a vastly expanded database, will include interpretative features and will be available in the early part of next year. lateral diffusion dynamics in phosphatidylcholine/cholesterol bilayers has been mostly accessed by means of epr, nmr and fcs spectroscopic techniques. reliable stady-state florescence quenching analysis of diffusion-controlled processes has been ampered by the lack of a self-consistent kinetic formalism for the two-dimensional (2d) counterpart of the classical stern-volmer analysis for three-dimensional (3d) solvents. we studied the excimer formation of phospholipid-labeled pyrenyl probes (proportion of 4 mol %) in mixed popc/cholestrol mlv liposomes by combined steady-state and timeresolved fluorescence the findings are in very good agreement with the theoretical predictions of the kinetic formalism specific for fluorescence quenching processes occurring in the small hydrophobic head group, the closely packed acyl chains, and the capability of interfacial hydrogen bonding have been suggested to govern the characteristic membrane behavior of long chain saturated ceramides: the self-segregation, and the formation of hexagonal phases and highly ordered gel phases. while it has been shown that structural alterations of the ceramide acyl chains induce position dependent effects on their behavior, we wanted to study the effect of interfacial properties, including hydrogen bonding, on ceramide membrane properties. the h-bond donor functions of 2nh and 3oh in the sphingosine backbone of palmitoylceramide were disrupted either separately or simultaneously by replacing the hydrogen with a methyl-group. when the lateral phase behavior of mixed bilayers containing cholesterol/sphingomyelin-rich domains was studied in the presence of the ceramide analogs, the 3o-methylated ceramide appeared to form a thermally stable, sterol-excluding gel phase with sphingomyelin, whereas the 2o-methylated ceramide failed in both thermal stabilization and sterol displacement. the doubly methylated analog was the poorest ceramide mimic. together with the possible steric effects induced by the methylations, the lack of 2nh h-bond donor function impaired ceramide membrane behavior to a greater degree than the lack of 3oh h-bond donor function. sugar-based surfactants are made from renewable resources using the ''green chemistry'' methods, are easily biodegradable and used in washing agents, cosmetics, and drug carriers. besides, there are attempts to use them as nonviral vectors in gene therapy. we studied the influence of new x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lengths (n = 10, 12, 14, 16) on the thermotropic phase behavior of dppc, and dppc/chol bilayers by means of differential scanning calorimetry. the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). we analyzed the changes in the temperatures, enthalpies and shapes of the main phase transitions as a function of concentration. molecular modeling methods were also used. cytotoxicicity of the c n gabs was determined in the cell line l929 and a549. for cytotoxicity test, the cells were seeded in 96-well plates 1 ml of 2á10 6 cells/ml in the culture medium eagle'a or dulbecco with 2% calf serum, penicillin and streptomycin was deposited into each plates. the cells were treated with various doses of surfactants and incubated. the minimal concentration which was toxic to approximately 50% of cells was taken as tccd 50 . this work was supported by grant n n305 361739. membrane fusion is ubiquitous in life requiring remodeling of two phospholipid bilayers. as supported by many experimental results and theoretical analyses, merging of membranes seems to proceed via similar sequential intermediates. contacting membranes form a stalk between the proximal leaflets which expand radially into a hemifusion diaphragm (hd) and subsequently open to a fusion pore. direct experimental verification of the hd is difficult due to its transient nature. using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (guvs) containing fluorescent membrane protein anchors and fluorescent lipid analogues in the presence of divalent cations. time resolved imaging revealed that fusion was preceded by displacement of peptides and lipid analogues from the guv-guv contact region being of several lm in size. a detailed analysis showed that this structure is consistent with the formation of an hd. a quantitative model of the hemifusion equilibrium and kinetics of the growing hd was developed. bilayer tension could be shown to drive hd expansion and interleaflet tension was found to act as a counterforce, because the outer leaflets are compressed upon hd growth. the model and its predictions fit nicely with observations above. concentration effects of trehalose in the equivalent polarity of fluid popc bilayers c. nobre 2 , d. arrais 1 , j. martins 1,2 1 ibb -cbme, faro, portugal, 2 dcbb -fct, universidade do algarve, faro, portugal trehalose is an important disaccharide, formed by two units of glucose linked by a a-1,1 glicosidic bond. it is capable of replacing water molecules in the hydration shell of the phospholipid headgroups, in cases of extreme dehydration, by establishing hydrogen bonds with their -co and -po groups, preserving this way the membrane structure. the polarity gradient is a significant feature of lipid bilayers and is influenced by the amounts of water within this medium. it is therefore important to understand the effects of different concentrations of trehalose in simple model membranes. using the pyrene empirical polarity scale, we monitorized changes in the polarity values when varying trehalose concentration in the bounding aqueous phase. for lower concentrations (until 0.25 m), we observed a decrease in polarity, comparing with popc bilayers in pure water. for higher trehalose concentrations (above 0.5 m), the polarity values are indistinguishable from those popc in water. using the freeze and thaw technique we obtained the same results, except for the lower trehalose concentrations. general anesthetics are indispensible tools of daily surgery. yet, their molecular mode of action remains elusive. while one school favors specific (direct) interactions with proteins of the central nervous system, another school adheres to a nonspecific modulation of biophysical membrane properties. one of the strongest arguments against lipid theories is the absence of stereo-specific effects in model membranes, as opposed to their detection by electrophysiological measurements on ionchannels. we have combined x-ray scattering and molecular dynamics simulations on palmitoyl-oleoyl-phosphatidylcholine bilayers with fluorescence microscopy on live cells to study the effects of the stereoisomers of ketamine on membrane properties. we find significant effects of both enantiomers on the distribution of lateral pressures at clinically relevant concentrations, being more pronounced for s-(?)-ketamine. we further calculated the effect of the lateral pressure profile changes on the opening probability of an ion-channel using crystallographic information. the observed channel inhibition compares remarkably well with clinically observed effects of the enantiomers. we thus provide first evidence for a stereo-specific, but indirect effect of general anesthetics on ion-channels. dependence of gramicidin a channel lifetime on membrane structure obtained from x-ray scattering measurements horia i. petrache department of physics, indiana university purdue university indianapolis, in 46202, usa the activity of ion channels, in particular the lifetime of their conducting (open) state depends on the physical properties of lipid bilayers [1, 2] which in turn depend on lipid headgroup and acyl chain composition. in order to investigate this dependence, we have performed measurements of gramicidin a (ga) channel lifetimes in three different lipid series. in each series, the lipid headgroups were phosphatidylcholine (pc), phosphatidylethanolamine (pe), and phosphatidylserine (ps), while the acyl chains consisted of symmetric monounsaturated di(18:1), mixed (16:0)(18:1), and methylated di(16:0-4me). in order to minimize the effect of headgroup electrostatics, measurements where performed in 1m kcl salts. we show how ga lifetimes depend on headgroup and acyl chain composition and on structural parameters determined by x-ray scattering. for the lipids considered, ga lifetimes cover a range from 0.7 seconds in the dope lipid to 18 seconds in dphps. in this range, we find a gaussian dependence of ga lifetime on bilayer thickness, consistent with hydrophobic matching models. we discuss different aspects of channel-lipid interactions and to what extent measurements of ga lifetime in binary mixtures are consistent with measurements in pure lipid systems. [ the aim of the studies was to determine the effect of chlorogenic acid (cga), which is the main constituent of plant extracts, on properties of the model membranes. its effect was studied on temperature of the main phase transition of various lipids with and without presence of cholesterol, using the differential scanning calorimetry (dsc) method and the fluorimetric method. in particular, the degree of packing order of the hydrophilic phase of liposomes was determined using the laurdan and prodan probes, and fluorescence anisotropy of the hydrophobic phase with the probes dph and tma-dph. it had also been studied the effect of chlorogenic acid on the structure and capacity of black lipid membranes (blms), formed of egg lecithin and lipids extracted from erythrocytes. the results obtained indicate that cga lowers the main phase transition temperature slightly, without changing the fluorescence anisotropy in the hydrophobic part of the bilyer, and causes a decease in the packing order of the hydrophilic phase. by monitoring the capacity during blm formation we have found that the presence of chlorogenic acid accelerates the process of lipid self-organization into a bilayer, and increases stability and life of the blms. however, the was no effect of cga on specific capacity of the membranes, and thus on thickness of the liposome membrane hydrophobic layer. this work was sponsored by the ministry of science and education, scientific project no. n n312 422340 and n n304 173840. many examples have recently been found where biological processes in the lipid bilayer are affected by the changes in the physicochemical properties of the membrane, e.g. the local curvature, the membrane tension and certainly the membrane structure. it has been shown that the activity of polyene antibiotics is strongly correlated to the phase diagram in a membrane composed of a mixture of popc and ergosterol or cholesterol (j membrane biol, 237:41-49, (2010)). it is known that polyene action is quite sensitive to the type of sterol in the membrane, which enables its medical use, mainly as antifungals. it has been proposed that this selectivity of the drug to fungi is related to structure modulation by the sterols (see for example, biophys. j., 85, 2323, (2003)) and therefore the correlation found could be due to structural differences between popc/ergosterol and popc/ cholesterol along the corresponding phase diagrams. to investigate this, molecular dynamics simulations of the above mixtures along their phase diagrams were performed. it was found that there are indeed marked differences in structure along the phase diagrams, but for the sterol-sterol distribution function. an analysis of the behavior of this observable and the implications on polyene action is discussed. acyl transfer from lipids without enzyme catalysis: a new paradigm for membrane protein ageing? john sanderson, catherine pridmore, jackie mosely, paul yeo durham university, department of chemistry, durham, uk membrane proteins are recycled in cellulo with half-lives ranging from minutes to days. in other systems, such as enveloped viruses, proteins may equally remain membranebound for periods of days. it is therefore of interest to examine the behaviour of proteins in model membranes over extended periods in order to determine the long-term stability of the mixed systems, both in kinetic terms (attainment of equilibrium states) and chemical terms (reactivity). the reactivity of proteins towards membranes has been examined using the peptide melittin as a model for membrane proteins. acyl transfer from phospholipids to the peptide was found to occur over a period of several days, in the absence of any enzyme catalysis. transfer was detectable after 2 days and reached 50% conversion in 8 days. using tandem mass spectrometry approaches, the sites of melittin modification were localised. these sites included the side chain of lysine, opening the possibility that this residue may be modified in any membrane protein where this residue has an appropriate disposition. these observations challenge preconceptions concerning the membrane as an inert medium and highlight potential new mechanisms for membrane protein ageing. interaction of poly(l-arginine) with negatively charged bilayers studied by ft-ir spectroscopy christian schwieger, alfred blume martin-luther-university, halle-wittenberg, institute of chemistry, van-dankelman-platz 3, 06108 halle / saale, germany e-mail: christian.schwieger@chemie.uni-halle.de oligoarginine residues attached to macromolecules are known to facilitate the transport through lipid membranes. since the mechanism of this transport is still unclear, the effect is often called ''arginine magic''. we studied the interaction of poly(larginine) (pla) of different molecular weight with negatively charged lipid bilayers. we have shown by calorimetric and monolayer techniques that the interaction is due to a combination of electrostatic and hydrophobic forces. now we present an ft-ir spectroscopic study to reveal the effect of pla binding on membrane organisation and peptide conformation. we will show that pla binding reduces the lipid miscibility of negatively charged (pg or pa) and zwitterionic (pc) lipids within the bilayer. from the shift of the c=o stretching vibration we deduce that arginine side chains penetrate into the hydrophobic/ hydrophilic interface and replace hydration water molecules. the binding reduces the rotational freedom of the lipid molecules, as could be shown by an analysis of the ch 2 -streching vibrations. pla binds in a b-sheet conformation to pg or pa gel phase membranes whereas its structure in bulk is random coil. the shift of the guanidyl vibration frequencies shows that also hydrogen bonds contribute to the pla -lipid interactions. neutron scattering studies of model membrane as a function of hydration and temperature federica sebastiani 1,2 , alessandra filabozzi 1 and giovanna fragneto 2 1 dipartimento di fisica, università degli studi di roma ''tor vergata'', roma, italy, 2 institut laue-langevin, grenoble, france cell membranes carry out highly specialised functions in living materials. the composition of bacterial membranes is essential to understand the mechanism of action of antimicrobial peptides. in order to understand the role of the various components contributing to the overall behaviour, we have reproduced the membrane of bacillus subtilis and carried out neutron diffraction studies on d16 (small momentum-transfer diffractometer) and d17 (reflectometer used as a diffractometer), at ill. an ordered and homogeneous sample has been obtained by using the widely studied dmpc. the measured d-spacing of dmpc as a function of the relative humidity (rh) is related to the physical and chemical conditions affecting the sample. consequently the reliability of the humidity chamber, which has been previously upgraded, has been stated. moreover, the most suitable preparation technique has been set up. in order to investigate the component roles within bacillus subtilis membrane, three samples of phospholipids were prepared (with pope, popg and cardiolipin). neutron diffraction measurements, performed at controlled rh and temperature, suggested the presence of interesting phase transitions or coexistence of phases. the rupture of membrane vesicles near solid surfaces annamá ria taká ts-nyeste, imre deré nyi department of biological physics, eö tvö s university, h-1117 budapest pazmany p. stny. 1/a, hungary the behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. in spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion, and finally spread along the surface forming a supported lipid bilayer. in our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion front. all these processes are supposed to follow an overdamped dynamics and coupled to each other through membrane tension. our numerical simulations reveal that the rupture process consists of three well distinguishable phases: a fast initial volume loss; followed by a slow volume loss; ending with a final burst and surface spreading. the second phase can be skipped if either the first phase advances far enough or the third phase sets in early enough. the smaller the vesicle, the further the first phase can advance. the third phase can start earlier if either the surface is smooth enough, or the adhesion energy is large enough, or the line tension is small enough. when the second phase is not skipped the time needed for the rupture process can take very long with a large variance. in the realistic range of the material properties (line tension, bending rigidity) the process is qualitatively always the same, so the most decisive parameter remains the size of the vesicle: the smaller the vesicle the faster and easier it ruptures. (3)). we chose four plant-derived polyphenols (flavonoids and stilbenes) of documented biological activity to study their influence on lipid domain number, area, shape, and borderlength. we found that resveratrol elevated the number of domains per vesicle, decreased their area and markedly increased the total length of domain border without affecting domains' circular shape. surprisingly, no such effect was observed for piceatannol differing from resveratrol by one hydroxyl group only. neither genistein nor 8-prenylnaringenin changed the morphology of lipid domains significantly. the possible mechanism of resveratrol-induced effect on lipid domains' morphology could be its selective accumulation in the interfacial regions between liquid ordered and liquid disordered domains. putative cholesterol recognition amino acid consensus (crac) motif in hiv coreceptors cxcr4 and ccr5 mikhail a. zhukovsky, albrecht ott biological experimental physics department, saarland university, 66041 saarbruecken, germany we identified a cholesterol recognition amino acid consensus (crac) motif in transmembrane domain 5 (tmd5) of two g protein-coupled receptors (gpcrs), human chemokine receptors cxcr4 and ccr5, coreceptors of human immunodeficiency virus (hiv). we suggest that residues belonging to this crac motif are involved in cholesterol binding to cxcr4 and ccr5 that is responsible for cholesterol requirement for cxcr4 and ccr5 conformation and function and for the role that cell cholesterol plays in the cell entry of cxcr4-using and ccr5-using hiv strains. putative crac sequences involve residues v 214 /l 216 -y 219 -k 225 in cxcr4 and l 208 /v 209 /v 211 -y 214 -k 219 in ccr5. in cxcr4, crac motif is highly conserved across chordata species, whereas in ccr5, crac motif is less conserved. t curve describe quantitatively the interfacial landscape around the protein molecules and can be used for the distinction between the globular and idp states. the behavior of the t 1 and t 2 data showed that there are two reorientation types present for every protein solutions below 0°c, irrespective for the nature of the protein or the solvent composition. local field fluctuation and the bpp models were applied, which failed for the buffered protein solutions and for the idps dissolved in water. a main cause of the failure is the changing h in the analyzed temperature range. this case is valid for the solutions of idps and for buffered solutions of both protein types. another cause can be the active relaxation channels other than dipolar when ions of quadrupolar nuclei are present. ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. this trait is exhibited by rtx (repeat in toxin) motifs found in more than 250 virulence factors secreted by gram-negative pathogenic bacteria. we investigated several cyaa rtx polypeptides of different lengths ranging from 111 to 706 residues. we showed that the rtx proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they adopt premolten globule conformations and exhibit a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the high mean net charge. calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the rtx proteins. we propose that the intrinsically disordered character of the rtx proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. these results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing rtx motifs, could be finely regulated by the calcium gradient across bacterial cell wall. occupational exposure to heavy metals has been recognized to be a risk factor for parkinson's disease via metaltriggered deposition of alpha-synuclein (as) 1,2 . in the present work, al 3? induced conformational change and instant oligomerization of as have been studied using fret and fcs as main techniques. donor and acceptor were labeled in the c-terminal at positions a107c and a140c. the average lifetime of donor in the presence of acceptor increases with the increase of al 3? concentration, indicating as adopts a more extended conformation upon al 3? binding. the intrinsic tyr fluorescence rises sharply within the mixing dead time, reflecting an enhanced hydrophobicity of the tyr environment and a fast conformational change of as. al 3? also induces an immediate oligomerization of as as monitored by fcs. the diffusion coefficient of as changes from 85 ± 5 lm 2 /s as monomer state to 23 ± 5 lm 2 /s as oligomer state. the oligomerization is supposed to be induced by the ligand bridging of trivalent al ions. nearly 2% of human genes encode protein-kinases (pk), enzymes involved in cellular signaling and several other vital biochemical functions, which transfer phosphate groups from atp to specific target molecules, modifying their activity. [1] deregulated pk have been linked to numerous diseases including cancer and diabetes, making them attractive targets for drug design. [2] conformational transitions play a central role in regulating the phosphorylation activity. pk adopt an on state that is maximally active and one or more inactive states that shows minimal activity. [3] the similarity of the relatively rigid and largely conserved atp binding site makes the design of selective inhibitors binding to the active state very difficult. indeed some of the best cancer therapies available are based on inhibitors, as imatinib, that bind to inactive states peculiar to a small subset of pk (abl, c-kit and pdgfr in the case on imatinib). thus, understanding the atomic details of the active to inactive transitions in kinases has a great importance. here we study a particular active-toinactive transition of c-src, a fundamental proto-oncogene involved in cancer and metastasis, by using multi-microsecond long fully solvated molecular dynamics simulations, metadynamics and ptmetad calculations [4, 5] . the results, validated by mutagenesis, x-ray crystallography and binding kinetics, are suggestive of a functional role for the conformational transition. moreover, we were able to single out the most important residues affecting the conformational transition and to show that even a very conservative amino-acid substitution can have a dramatic effect on the conformational free energy landscape. the time-scales of protein folding events range over many orders of magnitude. in order to understand the complex folding mechanisms, peptides with well-defined secondary structure are often used as model systems as they may be regarded as smallest folding units of proteins. the formation of secondary structure elements occur on the nanosecond to low microsecond time scale. thus, stopped-flow techniques are too slow whereas pulsed laser techniques are capable to trigger folding processes in nanoseconds and to analyze faster folding events. we study ns-to-ls peptide dynamics by temperature-jump infrared spectroscopy. after initiation of a nanosecond temperature jump, the spectral response is monitored at single wavelengths in the amide i region reflecting the dynamics of the peptide backbone. relaxation rates are obtained. the helix-to-coil relaxation of polyglutamic acid is a multi-step process and requires more complex models than two-state kinetics. however, there are kinetic steps that are well described by single-exponential behavior and a two-state model. we demonstrate how equilibrium and time-resolved infrared spectroscopic data can be combined to deduce folding rates. unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the thermodynamics and kinetics of protein folding and stability. however, a major limitation of this approach lies in the large uncertainty inherent in the extrapolation of the free energy of unfolding in the absence of denaturant from free energy values measured at finite denaturant concentrations. here we show that this limitation can be overcome by combining multiple spectroscopic signals-including fluorescence, circular dichroism, and absorbance-recorded in a quasi-simultaneous and fully automated way at different wavelengths. we have optimised the number of wavelength values used, the integration time per data point, the increment in the denaturant concentration, and the weighting scheme applied for global data fitting. compared with the traditional approach based on the use of a single or a few wavelengths, we could thus improve the precision of the free energy value by an order of magnitude. we exemplify and validate this novel approach using representative, well-studied globular proteins and explain how it can be exploited to quantify subtle changes in membrane-protein stability which have thus far remained elusive. the rates of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. it is often found experimentally that a linear fit to the reciprocal of the reaction rate as a function of the viscosity of the external medium has a non-zero 1 , the physical basis of pressure unfolding is still largely unknown. we report here a specific study of cavities contributions to the volume difference between unfolded and folded states (dv u ), using four single point mutants of staphylococcus nuclease (snase). each mutation is localised in a strategic position on the protein structure and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the snase 3d structure. measuring hsqcs peaks intensities up to 2500 bar monitored the equilibrium high pressure unfolding and leads us to precise estimations of dv u for more than two-thirds of the 143 residues of each mutant. so-fast hmqc experiments 2 were also performed to measure folding and unfolding rates from 200 bar pressure jumps. high-pressure fluorescence experiments were done on six additional alanine mutants to complement the nmr study, allowing a more complete exploration of the local pressure sensitivity along the protein 3d structure. all these highly reliable measurements shed light on the real signification of the thermodynamic parameter dvu, and bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. determination of contributing factors to the volume change magnitude between unfolded and folded states (dv u ) is a longstanding question in the high-pressure field. we provide here new experimental and computational data using two wellcharacterized model proteins: notch ankyrin repeat domain (nank) and staphylococcal nuclease (snase). the repetitive nature of the nank protein was used to study influence of the protein size on dv u in a systematic way with a set of deletion mutants. high-pressure fluorescence data provided new evidences that neither peptide bonds hydration nor side chains differential hydration could be considered as major contributor to the measured dv u value. additional molecular dynamics (md) simulations rather suggested that the heterogeneous distribution of void volume in the folded states structures could explain the dv u variations among the nank deletion mutants. the specific issue of the void volume contribution to dv u values was studied using 10 cavity mutants in snase, allowing a large structural mapping of the alanine mutations on this globular protein. combination of x-ray crystallography, highpressure fluorescence, high-pressure nmr and md simulations provided a first clear determination of the void volume contribution to the dv u values. these results also bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. we expressed an ig domain (i27) and a 170-residue-long fragment of the pevk domain in order to investigate the effect of temperature and pressure on their conformation. ftir spectroscopy is a useful method for investigating the secondary structure of proteins. we analyzed the amide i band to obtain information on protein structure. fluorescence labeling was also used in some experiments. to generate high pressures, a diamond anvil cell was employed. the ftir and fluorescence spectra of the protein fragments were recorded across the pressure and temperature ranges of 0-1 gpa and 0-100°c, respectively. moderate changes were observed in the conformation of the pevk fragments in the explored range of the t-p plane, suggesting that the domain is a highly flexible, random-coil across the entire studied t-p range. by contrast, the i27 domain showed quite stable secondary structure. intrinsically disordered proteins participate in important regulatory functions in the cell, including regulation of transcription, translation, the cell cycle, and numerous signal transduction events. disordered proteins often undergo coupled folding and binding transitions upon interaction with their cellular targets. the lack of stable globular structure can confer numerous functional advantages, including, paradoxically, both binding promiscuity and high specificity in target interactions. nmr is unique in being able to provide detailed insights into the intrinsic conformational preferences and dynamics of unfolded and partly folded proteins, and into the mechanism of coupled folding and binding. the function of intrinsically disordered protein domains in transcriptional regulation and signaling will be described, with particular reference to the general transcriptional coactivators cbp and p300, the tumor suppressor p53, and the adenovirus e1a oncoprotein. the globular domains of cbp/p300 are targets for coupled folding and binding of disordered transactivation motifs of numerous transcription factors and viral oncogenes, which compete for binding to limiting amounts of cbp/p300. many intrinsically disordered proteins contain multipartite interaction motifs that perform an essential function in the integration of complex signaling networks. the role of multipartite binding motifs and post translational modifications in regulation of p53-mediated signaling pathways will be discussed. the early vascular network is one of the simplest functioning organs in the embryo. its formation involves only one cell type and it can be readily observed and manipulated in avian embryos or in vitro explants. the early vascular network of warm-blooded vertebrates self-organizes by the collective motility of cell streams, or multicellular ''sprouts''. the elongation of these future vascular network segments depends on a continuous supply of cells, moving along the sprout towards its tip. to understand the observed self-organization process, we investigate computational models containing interactions between adherent, polarized and self-propelled cells. by comparing the simulations with data from in vivo or simplistic in vitro experiments, we explore the role of active migration, leader cells, invasion of the ecm, and cell guidance by micromechanical properties of adjacent cell surfaces. boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron-10. the biophysics of bnct is very complicated; primarily due to the complexity of element composition of the brain. moreover; numerous components contributes to the over all radiation dose both to normal brain and to tumor. simple algebraic summation cannot be applied to these dose components, since each component should at first be weighed by its relative biological effectiveness (rbe) value. unfortunately, there is no worldwide agreement on these rbe values. thermal neutrons were formerly employed for bnct, but they failed to prove therapeutic efficacy. later on; epithermal neutrons were suggested proposing that they would be enough thermalized while transporting in the brain tissues. however; debate aroused regarding the optimum source neutrons energy for treating brain tumors located at different depths in brain. insufficient knowledge regarding the rbe values of different bnct dose components was a major obstacle. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding condition on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating 65 different source neutron energies, between 0.01 ev and 13.2 mev. mcnp4b monte_carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about 30 kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. simulation of mutation induction by inhaled radon progenies in the bronchial epithelium balá zs g. madas, á rpá d farkas and imre balá shá zy hungarian academy of sciences kfki atomic energy research institute, konkoly-thege mikló s ú t 29-33., budapest, h-1121, hungary radon is considered as the second most important cause of lung cancer after smoking. to understand the mechanisms leading from radon exposure to cancer formation is of crucial importance. this study focuses on the description of mutation induction by radon progenies in the bronchial epithelium. computational fluid and particle dynamics approach was applied to determine the radio-aerosol deposition distribution in the central airways. a numerical replica of a small fragment of the bronchial epithelium was prepared based on experimental data. microdosimetric computations were performed to quantify the cellular radiation burdens at the very site of deposition accumulation. a mutagenesis model was applied supposing that radiation induces dna damages and enhances the cell turnover rate. the results show that both considered mutagenic effects of densely ionising radiation contribute significantly to mutation induction and mutation rate depends non-linearly on exposure rate. furthermore, simulations suggest that the local maintenance capacity of the bronchial epithelium can be exhausted by chronic exposure to radon progenies with activity concentration characteristic of some uranium mines. the present work demonstrates possible applications of numerical modelling in radon related carcinogenesis studies. the neural crest is a group of cells found in all vertebrate embryos. it forms in the neural folds at the border of the neural plate and gives rise to a huge variety of cells, tissues and organs. one of the astonishing characteristic of neural crest cells is that they are able to migrate very long distances in the embryo. the neural crest has been called the ''explorer of the embryo'' as it is one of the embryonic cell types that migrate most during development, eventually colonizing almost every tissue. in this talk i will discuss our recent finding about neural crest migration. we have shown that neural crest cells, classically described as mesenchymal cells, migrate in large clusters cytokinesis relies on tight regulation of the mechanical properties of the cell cortex, a thin acto-myosin network lying under the plasma membrane. although most studies of cytokinetic mechanics focus on force generation at the equatorial acto-myosin ring, a contractile cortex remains at the poles of dividing cells throughout cytokinesis. whether polar forces influence cytokinetic cell shape is poorly understood. combining cell biology and biophysics, we demonstrate that the polar cortex makes cytokinesis inherently unstable and that any imbalance in contractile forces between the poles compromises furrow positioning. we show that limited asymmetric polar contractions occur during normal cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations and division failure. a theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. we further propose that blebs, membrane protrusions that commonly form at the poles of dividing cells, stabilise the position of the cleavage furrow by acting as valves releasing cortical contractility. taken together, our findings show that the physical properties of the entire cell are integrated into a finetuned mechanical system ensuring successful cytokinesis. collective motion of individual cells marks the onset of the transition to multicellularity in many microorganisms. this transition is often mediated by intercellular communication signals between cells. here, we show, in contrast, that the transition from single cell to collective motion in an ensemble of gliding bacterial cells can be understood as a dynamical selfassembly process of self-propelled rods. experiments were carried out with a mutant of the bacterium myxococcus xanthus moving by means of the a-motility system only and without undergoing reversals. the collective motion phase is confined to a monolayer and is characterized by the organization of cells into larger moving clusters. a transition to collective motion is detected in experiments by image analysis, that reveals a qualitative change of the cluster-size distribution at a critical cell packing fraction around 17%. this transition is characterized by a scale-free power-law cluster size distribution with an exponent 0.88. we provide a theoretical model for cluster formation of self-propelled rods that reproduces the experimental findings for the cluster size distribution. our findings suggest that the interplay of selfpropulsion of bacteria and volume exclusion effects of the rodshaped cell bodies is sufficient to explain the onset of collective motion and the related changes in the cluster statistics. despite much speculation on the existence of structurally distinct oligomeric species associated with the conversion of certain monomeric proteins into amyloid fibrils, it has not previously been possible to observe them directly or to relate them to any key mechanistic steps involved in the interconversion process. we have developed a novel application of singlemolecule intermolecular fret to investigate in unprecedented detail the aggregation and disaggregation of alpha-synuclein, the protein whose pathogenic deposition as intracellular lewy bodies is a characteristic feature of parkinson's disease. our study reveals that a range of oligomers of different size and structure are formed, even at physiologically relevant concentrations. interestingly, the resistance to degradation of the aggregated state of alpha-synuclein, which is a wellwe focused on the structure-dynamics interplay and showed how the fractal-like properties of proteins lead to such anomalous dynamics. we used diffusion, a method sensitive to the structural features of the protein fold and them alone, in order to probe protein structure. conducting a large scale study of diffusion on over 500 pdb structures we found it to be anomalous, an indication of a fractal-like structure. taking advantage of known and newly derived relations between vibrational dynamics and diffusion, we demonstrated the equivalence of our findings to the existence of structurally originated anomalies in the vibrational dynamics of proteins. more specifically, the time dependent vibrational mean square displacement (msd) of an amino acid is predicted to be subdiffusive. the thermal variance in the instantaneous distance between amino acids is shown to grow as a power law of the equilibrium distance. the autocorrelation function in time of the instantaneous distance between amino acids is shown to decay anomalously. our analysis offers a practical tool that may aid in the identification of amino acid pairs involved in large conformational changes. more recently, we studied the effect of the hydrodynamic interaction between amino acids using a zimm-type model. we computed the time-dependent msd of an amino acid and the time-dependent autocorrelation function of the distance between two amino acids, and showed that these dynamic quantities evolve anomalously, similar to the rouse-type behavior, yet with modified dynamic exponents. we also studied the dynamic structure factor s(k,t) of proteins at large wavenumbers k, kr g [ [1, with r g the gyration radius, that are sensitive to the protein internal dynamics. we showed that the decay of s(k,t) is dominated by the spatially averaged msd of an amino acid. as a result, s(k,t) effectively decays as a stretched exponential. we compared our theory with recent neutron spin-echo studies of myoglobin and hemoglobin for the rouse and zimm models of hydrodynamic friction. in addition, i will mention two other projects currently underway: (i) a new elastic network model that accounts for the tensorial aspects of protein elasticity and is a combination of stretch-compress springs and bond-bending energies. (ii) the unfolding of a protein under the exertion of a large pulling force. allosteric regulation of enzymatic activity is crucial for controlling a multitude of fundamental cellular processes. yet the molecular level details underlying regulation often remain poorly understood. here we employed single molecule activity studies to dissect the mechanistic origin of enzymatic activity regulation. as a model system we employed a lipase and measured its activity as a function of accessibility to surface tethered liposomes (1), which are known regulators of its activity. our results surprisingly revealed that the lipase oscillates between 2 states of different activity. we accurately quantified for the first time both the interconversion rates between activity states and the inherent activity of these states. based on these we calculated the energetic landscape of the entire reaction pathway and identified that regulatory interactions redistributed the probability to reside on preexisting enzymatic activity states but did not alter the activity of these states. our findings provide the missing link between conformational and activity substates supporting and represent the first direct validation of the textbook hypothesis of conformational selection for regulation of enzymatic activity to identify the potential targets of cgmp in arabidopsis plants we adopted a proteomic approach to isolate possible cgmp-binding proteins. purification of soluble cgmp-binding proteins was performed using cgmp-agarose-based affinity chromatography procedure. next eluted proteins were analyzed by sds-page which revealed ten bands. we focused the subsequent analysis on low-molecular peptides of 15, 16 and18 kda which were bound cgmp more intensively. after 2d-ief-page of the proteins isolated by cgmp-agaroseaffinity chromatography eight most abundant protein spots in the low-molecular area were visualized. these spots of interest were excised from the gel and in gel digested by trypsin. then tryptic peptides were analyzed by maldi-tof mass spectrometry and identified as isoforms of nucleoside diphosphate kinase (ndpk) from arabidopsis. thus, our data suggest that ndpk is a potential target of cgmp signaling in arabidopsis. dual-color fluorescence-burst analysis (dcfba) was applied to measure the quaternary structure and high affinity binding of the bacterial motor protein seca to the protein-conducting channel secyeg reconstituted into lipid vesicles. dcfba is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. seca binds to secyeg as a dimer with a nucleotideand preprotein-dependent dissociation constant. one of the seca protomers binds secyeg in a salt-resistant manner, while binding of the second protomer is salt-sensitive. since protein translocation is salt-sensitive we conclude that the dimeric state of seca is required for protein translocation. a structural model for the dimeric assembly of seca while bound to secyeg is proposed based on the crystal structures of the thermatoga maritima seca-secyeg and the escherichia coli seca dimer. • dcfba is a flurorescence based single molecule technique that allows assessment of the stoichiometry of ligands bound to membrane receptors • dimeric seca binds asymmetrically to the protein-conducting membrane channel secyeg • monomeric seca binds secyeg but dimeric seca is required for protein translocation • protein translocation depends on receptor cycling of the dimeric seca if the dna charge is sufficiently neutralized by counter-ions, electrostatic interactions between helical charge patterns can cause attraction [1] . helix specific interactions also cause tilt, in one direction, between two dna fragments [1] . in braids and supercoils, this impetus to tilt breaks positive-negative supercoil symmetry. we show that these effects may cause spontaneous braiding of two molecules, lowering the dna pairing energy [2] . the pairing is more energetically favourable for homologues (same base pair text) than for nonhomologous pairs. this might explain pairing between only homologues observed in nacl solution [3] . also, we construct a simple model for a closed loop supercoil, including chiral electrostatic interactions. there are very interesting effects, for sufficient charge neutralization and groove localization of counter-ions. i.) positive super-coils are more energetically favourable than negative ones. ii.) a transition between loosely and tightly wound supercoils as one moves from negative to positive values of the supercoiling density. iii.) in positive super-coils the chiral interaction underwinds dna. [ von willebrand factor (vwf) is a large multimeric protein that is crucial for the force sensing cascade triggering primary hemostasis. it mediates binding of activated thrombocytes to injured epithelial tissue and serves as a transporter for coagulation factor viii. while it was shown that the hemostatic activity of vwf is affected by shear stress [1] , the exact impact that shear forces have on the inflammatory cascade remains unclear. it is assumed that hydrodynamic forces lead to partial unfolding of vwf, which in consequence exposes more binding sites. in order to observe shear-induced changes of the protein's functionality, we measure conformational changes of vwf under flow with fluorescence correlation spectroscopy (fcs). we aim to measure the degree of uncoiling of vwf multimers under various buffer conditions, e.g. in the presence of colloids, vesicles or platelets. as only large multimers show significant hemostatic activity we intend to monitor the molecular weight distribution of vwf. shifts in this distribution indicate various pathological conditions making our multimer analysis a fast diagnostical tool for vwf-related diseases. this will serve as a basis for studies of vwf binding to collagen, fviii, gpib, vesicles and membranecoated nanoparticles under shear flow. [ data on mechanical properties of medically important proteins located in neural junctions are very limited. contactins (cntn) and paranodin proteins, located in extracellular part of ranvier nodes, are important for proper brain wiring. here we study a new series of fniii modules from human cntn-1 and -4 using a single molecule afm force spectroscopy and advanced, all-atom steered molecular dynamics (smd) computer simulations. mutations in cntns are responsible for numerous brain disorders including autism or pathological development of odor maps. perhaps mechanical properties of individual fniii mutated protein modules are compromised, thus we address this problem. a comparison of our afm force spectra with those of reference proteins will be presented [1] [2] , and the molecular level interpretation fniii nanomechanics, based on our smd data will be given. we believe that these data should help to understand a role of cntn in regulation of sodium ion channels in both normal and autistic subjects. supported in part by polish ministry of education and science, grant no. n202 262038, the computational center task in gdansk and license for accelrys software. recent achievements in rational dna-motors engineering demonstrate the possibility to design nano-motors and nanorobots capable of performing externally controlled or programmed tasks. a major obstacle in developing such a complex molecular machine is the difficulty in characterizing the intermediates, the final products and their activity. typically, non in-situ gel and afm and in-situ bulk fluorescence methods are used. i will present two dna-motors recently developed and studied using in-situ single-molecule fluorescence resonance energy transfer (smfret), alternating laser excitation (alex) and total internal reflection fluorescence spectroscopy (tirf), and will demonstrate that these methods can improve the way we design, construct, measure and understand highly complex dna-based machines. a motor made of bipedal dna-walker, which walks on a dna track embedded on a dna-origami, capable of long walking distance and maintaining structural stability, will be presented. the motor is non-autonomous; it receives ss-dna fuel/anti-fuel commands from outside (as in shin & pierce, jacs, 2004). the motors assembly stages and singlemotor's walking steps are monitored using smfret. the second motor is based on published bipedal autonomous dna-motor (seeman, science 2009). it is characterized by coordinated activity between the different motor domains leading to processive, linear and synchronized movement along a directionally polar track. to prove that the motor indeed walks, the authors chemically froze the motor at each step and use a complicated radioactive gel assay. i will demonstrate that using single-molecule approach, we are able to directly and in-situ measure single-motor's movements in few simple experimental steps, and measure its structural dynamics and kinetics. translation by a single eukaryotic ribosome using single molecule total internal reflection fluorescence microscopy, we observed translation of a short messenger rna (mrna) strand by single eukaryotic ribosomes. the ribosome-mrnas complexes are fixed to a microscope coverslip through the mrna, and mrnas are located through fluorescently labelled oligonucleotides hybridized to it downstream start codon. because of the ribosome helicase activity, the double strand formed by the oligonucleotide and the mrna is opened while the ribosome translates this region of the mrna. thus, the loss of the fluorescence signal allows us to measure the distribution of translation speed of single ribosomes. careful attention was given to photobleaching for the data analysis. this experiment opens the door to the study of eukaryotic translation at the single molecule level. erythrocyte hyperaggregation, a cardiovascular risk factor, has been associated to high plasma concentrations of fibrinogen. using atomic force microscopy (afm)-based force spectroscopy measurements, we have recently identified the erythrocyte membrane receptor for fibrinogen, an integrin with a a3 or a3-like subunit [1] . after this, we extended the study to the influence of erythrocyte aging on fibrinogen binding [2] . force spectroscopy measurements showed that upon erythrocyte aging, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence (from 18.6% to 4.6%) but not its force. this observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen blood content, which disturbs the blood flow. our data also show that sialic acid residues on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating the binding to its receptor. antimicrobial peptides are usually polycationic and amphiphilic with high affinity for bacterial membranes. in order to characterize their therapeutic potential it is crucial to disclose which properties of the peptide/lipids are important for target selectivity, and to examine the peptide structure and its association with lipid bilayers. in this work, first experiments have been carried out on a promising peptide called sb056, which might represent the basis for developing a novel class of antibiotics. with the goal of enhancing the activity of a new semi-synthetic sequence, two identical peptides (wkkirvrlsa) were assembled via a lysine-linker, carrying also an octanoyl-lipid anchor. a highly active compound was obtained, but its structure and mode-of-action remain unexplored. this dendrimeric peptide and its linear deca-peptide counterpart are being studied in parallel to highlight the relevant properties and differences between dendrimeric structure and the sequence. monolayer intercalation is investigated with microtensiometry, fluorescence spectroscopy is applied to study thermodynamics and kinetics of the binding process. circular dichroism, nmr and md simulations are employed with the aim of elucidating the 3d structure in the membrane-bound state. the capability of proteins to build structures via self-organization is fascinating biophysicists since decades. with the advent of single-molecule methods, namely fluorescence correlation spectroscopy (fcs) and fluorescence resonance energy transfer (fret), the process of complex formation is becoming accessible to direct observation. coronaviruses (cov) are enveloped positive-stranded rna viruses. for sars-cov, it was shown that coronaviruses encode a rna-dependent rna-polymerase (rdrp) build from non-structural protein 7 (nsp7) and non-structural protein 8 (nsp8). this hexadecameric nsp7-nsp8 complex is a hollow, cylinder-like structure assembled from eight copies of nsp8 and held together by eight nsp7 molecules [1, 2] . we are aiming at understanding the assembly process and conformational changes of the complex for the related feline coronavirus. first results implicate that nsp8 alone forms a dimer, where interchain fret is more efficient than intrachain fret. for the complex the results indicate that nsp7-nsp8 form a heterodimer which is different from sars-cov. our experiments highlight the potential of single-molecule fret for the study of protein complex formation. diffracted x-ray tracking (dxt) has been considered as a powerful technique for detecting subtle dynamic motion of the target protein at single molecular level. in dxt, the dynamics of a single protein can be monitored through trajectory of the laue spot from the nanocrystal which was labeled on the objective protein. in this study, dxt was applied to the group ii chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an atp dependent manner. a mutant group ii chaperonin from thermococcus strain ks-1 with a cys residue at the tip of the helical protrusion was immobilized on the gold substrate surface and was labeled with a gold nanocrystal. we monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the torsional motion of the chaperonin in the presence of atp condition was 10 times larger than that in the absence of atp condition. and uv-light triggered dxt study using caged atp revealed that the chaperonin twisted counterclockwisely (from the top view of chaperonin) when the chaperonin closed its chamber, and the angular velocity from open to closed state was 10 % faster than that from closed to open state. peptides or proteins may convert (under some conditions) from their soluble forms into highly ordered fibrillar aggregates. in vivo such transitions can lead to neurodegenerative disorders such as alzheimer's disease. alzheimer's disease is characterised by the extracellular deposition of abeta peptide in amyloid plaques, and the intracellular formation of neurofibrillary tangle (nft) deposits within neurons, the latter correlating well with disease severity. the major constituent of nft deposits are paired helical filaments (phf) composed of a microtubule-associated protein known as tau. studying the process by which tau forms these large aggregates may be an essential step in understanding the molecular basis of alzheimer's disease and other tauopathies. we have applied a two-colour single molecule fluorescence technique, and single molecule intermolecular fret measurements to study the soluble oligomers of tau which are formed during the aggregation and disaggregation of phf's. the neuronal protein alpha-synuclein is considered to play a critical role in the onset and progression of parkinson's disease. fibrillar aggregates of alpha-synuclein are the main constituents of the lewy bodies that are found in the brains of parkinson patients. however, there is growing evidence suggesting that oligomeric aggregates are significantly more toxic to cells than fibrillar aggregates. very little is known about the structure and composition of these oligomeric aggregates. we present results using single-molecule photobleaching approaches to determine the number of monomeric subunits constituting the oligomers. our results show that the oligomers have a narrow size distribution, consisting of *13-20 monomers per oligomer. fluorescence correlation spectroscopy data confirm the narrow size distribution and additionally indicate a very loose packing of the oligomers. in combination with bulk fluorescence spectroscopy results of tryptophan containing mutants of alpha-synuclein, we present a structural model for the alpha-synuclein oligomer. gold colloids are widely used for in vitro and in vivo imaging. compared to the traditional optical tags sers-coded nanoparticles show a narrow emission bandwidth with structured spectra typical of the molecule used, a wider excitation bandwidth, higher emission intensity, a better photo-stability, and a lower toxicity. this is why in cancer therapy, besides being considered good tools for the delivery of anti-tumor drugs, aunp can be also good optical tags for the analyses of both np localization by laser scanning microscopy and the process of drug release inside the cells by raman. in our work we used 10 nm diameter aunp loaded with rhodamine 6g, a molecule with a high raman and fluorescence efficiency, and with a chemical structure similar to doxorubicin, the antitumoral drug used in our system. the data showed that aunp are internalized by cells and sers can be performed. 10 nm and 60 nm diameter aunp loaded with doxorubicin were incubated at different time points with a549 cell line (human adenocarcinomic alveolar basal epithelial cells). only 60 nm aunp showed intense raman emission typical of the doxorubicin phonon transitions. in recent years biomedical applications of diamond nanoparticles have become of significant interest, which rises questions of their biocompatibility and mechanisms of interactions with cells. the aim of this study was to compare the effect of nonmodified diamond nanoparticles (dnps) and dnps modified by the fenton reaction on human endothelial cells. dnps (\10 nm particle size, sigma) were modified by the fenton reaction introducing surface -oh groups. immortalized human endothelial cells (huvec-st) were incubated with 2-100 lg/ml dnps in the optimem medium. diamond nanoparticles modified by the fenton reaction had smaller hydrodynamic diameter estimated by dynamic light scattering and the surface potential (zeta potential) measured using laser-doppler electrophoresis. they were more cytotoxic as evaluated by the mtt reduction assay. dnps augmented generation of reactive oxygen species in the cells, estimated by oxidation of 2',7'-dichlorofluorescin, the effect being higher for the fenton-modified dnps after 48-h incubation. cellular production of nitric oxide, estimated with daf-fm, was also affected by dnps; after 72h, fentonmodified oh, in contrast to non-modified diamond, decreased no production. diamond nanoparticles affected also the cellular level of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione s-transferase). we aim at investigating how photoreactions of proteins can be controlled by means of intense thz radiation tuned in resonance to specific vibrational modes, much in analogy to coherent control experiments conducted by fs nir laser pulses [1] . for this we will combine a time-resolved ir difference spectroscopic setup with uniquely intense, tunable narrow bandwidth thz radiation (3 -280 lm) at the ps beamline of the thz free electron laser felbe. these experiments will be performed on bacteriorhodopsin (br) which is the sole protein of the purple membrane of the archaebacterium halobacterium salinarum [2] . upon illumination, the chromophore retinal isomerizes around the c 13 -c 14 double bond [3] and br pumps a proton from the cytoplasmic to the extracellular side. this proton gradient is used by the bacterium to drive photosynthetic atp production under low oxygen tension [4] . in our experiment, the photoreaction is initiated by a visible laser pulse as in standard experiments, but then the sample will be irradiated by a thz pulse from the free electron laser tuned into resonance with low-energy vibrational modes which is supposed to influence the photoreaction [1] . such vibrational control will be monitored by time-resolved ftir spectroscopy using the step-scan technique [5] . liposomes are increasingly studied as nanoscale drug delivery systems and biomembrane models. however the exact structure dynamics and mechanical behavior of liposomes is little known. atomic force microscopy (afm) is a powerful tool to characterize nanoscale morphology and enables the mechanical manipulation of submicron-sized vesicles. a drawback of afm, however, is that liposomes may flatten and rupture on substrates to form patches or supported planar bilayers (spb). our aim was to obtain better understanding of factors affecting liposomes on substrates and find experimental conditions at which liposomes preserve their structural integrity. in the presence of divalent cations dppc liposomes formed spb on mica. vesicles sedimented subsequently preserved their integrity and showed stronger attachment to spb. in addition to cross-bridging lipid head groups, divalent cations influence the surface charge of liposomes, thereby modulating liposome-substrate and liposome-liposome interfacial interactions. preserved vesicles stabilized by divalent cations may provide a unique experimental system for studying membrane-protein interactions. the influence of e.g. ph and ionic strength on various chromatographic bead -biomass combinations. to analyze the force curves, possible elastic contributions, e.g. from deforming cellular membranes, have to be decoupled from the interaction forces. then, bead-biomass interactions will be modeled using (extended) dlvo theory and resulting data can also be compared to real-life eba processes. the project aims for a better understanding of the interaction forces in chromatography and might help to improve the process quality of eba. long-term non-invasive in vivo monitoring of the survival, migration, homing and fate of transplanted cells is of key importance for the success of cell therapy and regenerative medicine. tools for in vivo magnetic resonance (mr) imaging of labeled cells are therefore being developed. we have prepared superparamagnetic iron oxide nanoparticles by the coprecipitation of fe(ii) and fe(iii) salts and oxidation. to stabilize the particles and to facilitate their internalization by the cells, the nanoparticles were coated with several novel low-and highmolecular weight compounds including d-mannose, poly(llysin), poly(n,n-dimethylacrylamide) and dopamine-hyaluronate conjugate. the surface-modified magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. the particles were then investigated in stem cell experiments in terms of real time cell proliferation analysis, viability, labeling efficiency and differentiation. the iron oxide concentration of the labeled cells was assessed using mr relaxometry. the advantages/disadvantages of particular iron oxide coatings will be discussed and the optimal coating suggested. excellent contrast was achieved by labeling the cells with dopamine-hyaluronate-coated nanoparticles. support of the as cr (no. kan401220801) is acknowledged. in the last decades the interest towards the fabrication of innovative bio-sensors with improved sensitivity and reliability for medical-diagnostics applications has been constantly risen. among the different techniques, microfluidic systems are playing a major role. in order to detect extremely low concentrations of biomolecules (pm and fm), attention should be placed on the controlled, selective functionalization of micro-and nano-channels. in this work we propose a new approach to functionalize gold patches inside fluidic channels. we start from self-assembled monolayers (sams) of thiolated molecules on a gold electrode deposited inside the channel. then, by using an electrochemical approach [1, 2] we remove molecules from the sam at selected locations, by applying a negative voltage to the electrode. the newly exposed gold surface can be re-functionalized by using a thiolated biomolecule (i.e an antibody) capable to bind specific proteins flowing inside the channel. the cycle can be applied to other electrodes in the microfluidic system, creating a multiplexing device which, as we will show, can differentially measure ionic current flows in different channels. optically actuated micromanipulation and micro-probing of biological samples are increasingly important methods in the today's laboratory. microbeads as probes are the most commonly used tools in this field, although the only manipulative motion they allow is translation. we present polymerized 3d microstructures which can also be used for optical micromanipulation with more degree of freedom than microbeads. the two-photon polymerization (tpp), based on focused fs laser beam into appropriate photopolymers is a powerful method to build structures of arbitrary complexity with submicrometer resolution. the presented tools have the advantage of being capable of twisting and rotational manipulative motion, and also that the position of biological manipulation and optical trapping is spatially separated. different manipulative interfaces, the positioning stability and surface activation of the manipulators will be discussed. the potential application of multiplexed quantum dot labeling, mqdl, in clinical detection, prognosis and monitoring therapeutic response has attracted high interests from bioengineers, pathologists and cancer biologists. mqdl is superior comparing with conventional organic dye staining in its narrow emission bandwidths, wide signal dynamic ranges, high detection sensitivity, and low noise to signal ratios. however, the majority of the mqdl application has been limited to identification of specific cell type or cancer subtype and the improvement in labeling methodology. in this study, we focused on simultaneous detection and analysis of 5 proteins in the c-met activation pathway, i.e. rankl, vegf, nrpln-1, p-c-met and mcl-1, which are known to be associated with human prostate cancer progression and metastasis. two experimental systems were analyzed: 1) fixed xenograft tissues from an established ltl313 castration resistance human prostate cancer or crpc model; and 2) clinical prostate tissue specimens from localized cancer and bone metastasis. in the presentation we will report our experience in 1) the mqdl protocol optimization for the sequential reactions of individual primary antibody, the biotinylated secondary antibody and streptavidin-coated qd conjugate with nuclear dapi staining; and 2) the multiplexed image catching, image unmixing, and subsequent per cell base quantification. for future multi-specimen analyses and validation, we will introduce a high throughput vectra image analysis system. carbon nanotubes (cnts) 1 are already quite popular among many scientific and technological disciplines 2 . in recent years they have been targeted for biotechnological and medical applications. in this work we have investigated the nanostructural self assemblies of biological lipid molecules in presence of cnts. advantage of using highly aligned cnts 3 for this purpose being the possibility of studying the interactions of lipid molecules on the macromolecular surface as well as in the confinement of aligned cnts. we have observed various lyotropic nanostructures that are found for corresponding lipids in the bulk under dry and hydrated conditions. 4 nanostructural studies were mainly performed using small and wide angle x-ray scattering techniques. this work is crucial for designing the nano-micro-fluidic architectures and supported model membranes where both -functionalization of cnts and nanostructural assembling of lipids, could be employed simultaneously. alternative of the commonly used measuring tools in protein research and medical diagnostics. thazolidone derivatives are novel synthetic compounds possessing various biological activities. we selected three such compounds les-3120, 3166, 3372 which passed national cancer institute in vitro tests. annexin v/pi and dapi staining, dna electrophoresis in agarose gel, and western-blot analysis using specific antibodies against 30 cellular proteins involved in apoptosis were applied to study molecular mechanisms of tumor cell death induced by these compounds. it was found that molecular targets of thiazolidones in target cells strongly depend on structure of their side groups: les-3120 containing isatine fragment, activated caspase-8 involved in receptor-mediated apoptosis, while les-3166 possessing benzthiazol residue, induced mitochondrial apoptosis mediated by caspase-9, and les-3372 which has a unique chlorine atom in side chain, also led to mitochondrial apoptosis mediated by aif (apoptosis-inducing factor). to increase anticancer potential of these molecules, in silico study was performed and most active groups of les-3120 and les-3372 were combined into one molecule. in vitro studies showed that such hybrid molecule called les-3661 possessed 10 times higher anticancer potential (ic 50 =1 lm) comparing with initial compounds. we report on the synthesis and characterization of vaterite microcontainers for controlled drug release. moreover, we present experiments on possible release strategies of encapsulated substances via recrystallization, ph controlled, or by desorption methods. vaterite spherical particles were fabricated with controllable average sizes from 400±12nm till 10±1hm. we considered two ways of functionalization of the containers: encapsulation of the substances during the vaterite synthesis or their adsorption onto the prepared particles. as model experiments, vaterite containers, encapsulating rhodamine 6g, were imaged by two-photon microscopy, showing dye release into the aqueous medium due to recrystallization to calcite within 3 days. differently, in ethanol only small amounts of the encapsulated markers were diffusion released after one week. the release mechanisms can be further controlled by covering the microcontainers with additional polymer layers to increase diffusion and recrystallization time. a change of the ph from neutral to acid conditions leads to the destruction of the vaterite matrix followed by a quick release of the encapsulated materials. these flexible control mechanisms make this system an interesting candidate for pharmaceutical applications. magnetic nanoparticles (np) in combination with therapeutic molecules represent one of most promising methods for targeted drug delivery. one of major current limitations of magnetic drug targeting is to achieve efficient concentration of magnetic carrier-drug complexes at the targeted sites due to poor mobility of nanoparticles in tissue structures. interstitial delivery is hindered by microscopic extracellular matrix, which represents a major barrier for nanoparticles motilities. in order to achieve efficient magnetic drug targeting it is crucial to know particle mobility in a given in vivo environment as well as to apply magnetic field having appropriate field gradient which drags magnetic nps. we used gel magnetophoresis in order to measure motilities of different magnetic nps (co-ferrite, c-fe 2 o 3 ) in agarose gel. numerical modeling using fem method was used to determine appropriate settings of magnets, which generate sufficient magnetic field gradient. further, we used the numerical modeling to evaluate the magnetic force on the nps for different geometries. we obtained that one of crucial factors which determines final mobility in tissue is formation of larger aggregates of nanoparticles under physiological conditions and interaction of nanoparticles with surrounding matrix. defining the forces required to gate mechanosensitive channels in mammalian sensory neurons kate poole and gary lewin department of neuroscience, max delbrueck center for molecular medicine, robert-roessle str 10, 13125, berlin-buch, germany our sense of touch and mechanical pain is based on mechano-electrical transduction (met) at the terminal endings of subsets of dorsal root ganglion (drg) neurons innervating the skin. to quantify the stimulus strengths required to gate mechanosensitive channels in these subsets of neurons, we developed an approach using microstructured surfaces. the drg neurons are grown on laminin-coated pdms pillar arrays, mechanical stimuli are applied by deflecting individual pili and the deflection is monitored using light microscopy. as the pili behave as light-guides, the center of each pilus can be determined from a fit of the intensity values, allowing detection of movements of a few nanometers. the response to such stimuli is monitored using whole-cell patch-clamp. pili deflections of 10nm can gate the rapidly adapting-current in mechanoreceptor cells, while deflections above 150nm are required for gating of slowly adapting-currents in nociceptors. smaller stimuli are required to generate currents via pili deflection (10nm) vs neurite indentation (70-100nm), suggesting that gating occurs at the cell-substrate interface. we have also characterized the met currents present in n2a cells which we show are modulated by the substrate to which the cells are attached. enhanced stimulation of toll-like receptor 9 via immunostimulatory nanoparticles jan rother, anna pietuch, andreas janshoff georg-august university gö ttingen, institute of physical chemistry, tammanstr. 6, d-37077 gö ttingen, germany e-mail: jrother@gwdg.de among the toll-like receptor family (tlrs), the tlr9 has been subject of intensive research because of its predominant localization in the lysosomes of immune cells and its ligand rendering it a potential candidate for immunotherapy of autoimmune diseases and cancer. additionally, a use as an adjuvant in vaccination is aimed using synthetic cpg-oligodeoxynucleotides (cpg-odn's). albeit, immunostimulatory cpg-odns already showed promising results in animal experiments and clinical trials, several groups found that tlr9 is also expressed by tumor cells. first experiments show that activation of tlr9 displayed by cancerous cells leads to a decreased apoptosis rate and proliferation posing unpredictable threat to tumor patients exposed to cpg-odns. therefore, detailed knowledge about the impact of cpg-odns on cancer cells is inevitable for a save use in pharmaceutics. herein, we describe a sophisticated way to address tlr9 in cancer cells using cpg-odn functionalized ''superparamagnetic'' mno-and c-fe 2 o 3 -nanoparticles (nps) to stimulate tlr9 in a549 cells. analysis of impedimetric measurements revealed a cytotoxic effect of the mno-nps. cells treated with immunostimulatory fe 2 o 3 -nps showed an increased micromotility as well as a higher long-term correleation of the impedance signal. biomedical diagnostics like high-sensitivity, single-molecule study, easy sample preparation. furthermore, sers allows to conduct non-invasive studies of conformations of the molecules without destruction of living cells, i.e. in vivo [1] . this work presents a sers study of cytosolic hemoglobin (hb c ) using silver nanoparticles (agnps). the hb c was isolated from cytoplasm of red blood cells taken from rat erythrocytes and diluted. agnps were prepared by developing leopold and lendl method [2] . three types of colloids were prepared at various temperatures (25, 40 and 60°c). the resulted agnps were characterized by uv-vis-, ftirspectroscopy, dls and tem. reduction of ag ions leads to the formation of predominantly spherical agnps but also silver nanorods, faceted and aggregated agnps in small quantities with a surface plasmon resonance band in the range of 413 -445 nm. for agnps synthesized at 25°c, for example, a bimodal size distribution was observed (about 7 and 45 nm medium sizes, respectively). sers measurements were optimized for each type of agnps. it was demonstrated that agnps gave strong raman enhancement from hb c and types of sers spectra differ from each other. nano-zno is characterized by unique properties, low toxicity and high biocompatibility instead of a lot of others nanomaterials. for this fact nanoparticles of zno have great potential for applications in biosystems, for example biolabeling, biosensoring, delivery systems and others, which can be used in genetics, pathology, criminology, safety of food and many other industries. for these bioapplications are necessary surface modifications, which can made to the nanostructures to better suit their integration with biological systems, leading to such interesting properties as enhanced aqueous solubility, bio-recognition or applicability for biological systems. for synthesis of zno nanoparticles in aqueous solution we used 11-mercapto-undecanoic acid (mua) as stabilizing agent. the coating of nanoparticles with mua could allow their solubility in the water and the binding through carboxyl groups present in its structure. we defined the optimal ph for mua modificated nano-zno solubility and their ability interaction with positive charges. we studied the optical properties of pure and surface modificated nanoparticles and their conjugates with cytochrome c and also the effect of ph on the interaction between nano-mua and horse cytochrome c. the permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. an adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. as the binding of antibiotic molecules in the channels of interest is significantly weaker than that of preferentially diffusing nutrients in substrate-specific pores, the resolution of conductance measurements has to be significantly increased to be able to resolve the events in all cases. due to the limited time resolution, fast permeation events are not visible. here we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. molecular details of solute permeation can be revealed by combining electrophysiology and all atom computer modeling. [ novel functionalized nanocomposites (nc) were designed and synthesized on the basis of polymeric surface-active oligoelectrolytes. the developed technology permits controlling: 1) quality and quantity of structural blocks of nc, and size unimodality; 2) branching at specific sites in polymer chain of nc; 3) providing nc with reactive chemical groups; 4) covalent conjugation of specific bio-targeting molecules. provided bioactive elements were: a) specific anticancer drugs, antibiotics, alkaloids; b) dna and sirna; c) immunoglobulins and lectins; d) lipids and amino acids; e) polyethylene glycol. fluorescent, luminescent, super-paramagnetic, or x-ray detectable compounds were also incorporated in nc to make them detectable and measurable. biocompatible nc possessing low toxicity towards mammalian cells in vitro and in vivo (mice) were created. they were effective in delivery of: 1) drugs (doxorubicine and antibiotics) for chemotherapy in vitro and in vivo; 2) dna for transfection of mammalian, yeast and bacterial cells; 3) protein antigens for animal immunization and specific lectins for targeting apoptotic cells. these and other approaches in application of developed nc and nanobiotechnologies are considered. this work was supported by stcu grants #1930, #4140, #4953. in the anti-cancer drug delivery domain, nanotechnologies are a promising tool, providing a good tissue distribution and a low toxicity. drug delivery vehicles relying on solid nanoparticles have been proposed, among which diamond nanoparticle (size\20 nm) is a very promising candidate [1] . we have investigated the delivery of sirna by nanodiamonds (nd) into cells in culture, in the context of the treatment of a rare child bone cancer (ewing sarcoma), by such a gene therapy. sirna was bound to nds after nds coating by cationic polymers, so that the interaction is strong enough to pass the cell membrane without loss of the drug and does not prevent its subsequent release. the cellular studies showed a specific inhibition of the gene expression at the mrna and protein level by the nd vectorized sirna. we also uses the fluorescence of color center created in the nanodiamonds [2] to monitor the release of fluorescently-labeled sirna in the intracellular medium. this technique brings a quantitative insight in the efficiency of sirna to stop cell proliferation. considering the success of the cell model we recently started the drug delivery in tumor xenografted on nude mice. silica nanoparticles are stable aqueous suspension of condensed siloxane nanocomposites, having an average diameter between 10 and 100 nm. particles containing organic functional groups on their surface are called organically modified silica nanoparticles (ormosil). due to the various chemical and physical properties of the surface groups, ormosil nanoparticles may have an enormous variety of biological applications, such as in vivo bioimaging, non-viral gene delivery or targeted drug delivery. our aim was to synthesize both void and fluorescent dye doped amino functionalized ormosil nanoparticles through the microemulsion method and use them for gene delivery. the obtained nanoparticles have been characterized by transmission electron microscopy and dynamic light scattering. furthermore, the nanoparticles have been investigated to exploit their transfection efficiency and the possible toxicity caused by surfactants used in the synthesis. the transfection efficiency was tested on various cell cultures. our further aim is the in vivo transfection of salivary glands using ormosil nanoparticles. our work has shown that the nanomedicine approach, with nanoparticles acting as a dna-delivery tool is a promising direction for targeted gene therapy. in vivo amperoetric cells for detection of fast diffusing, physiologically important small molecules lívia nagy, bernadett bareith, tü nde angyal, erika pinté r, gé za nagy university of pé cs, pé cs, hungary h 2 s is a naturally occurring gas that is toxic in high concentration. it exists also in different tissues of living animals sometimes in concentrations as high as 20 lm. it is generally accepted, that h 2 s has important roles in modulating different, physiologically important biochemical processes similarly to other, fast diffusing molecules like no, co and h 2 o 2 . for investigation of the physiological effects of these species their local concentration in the studied biological media is important to know. this means methods needed for measuring the instantaneous concentration with high spatial resolution in living tissues without major invasion. electrometric micro, and ultramicro sensors are often gain application in experimental life sciences for measurement of local ion concentration or following neurotransmitter species in vivo measurements. in our work efforts are being carried out to improve the applicability of selective electrometric sensors in life science experiments. as a result of these work an improved h 2 s measuring cell and improved electrode and method was developed for measurement of electroactive small molecules like no or h 2 o 2 . in the poster to be presented the structure, the working principles and the performances of the different sensors mentioned will be described. bacteriorhodopsin (br) is the only protein in the purple membrane of the halophilic organism halobacterium salinarium. it is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of the covently bound retinal chromophore. its stability, as well as its photoactivity in dried films, has made br an attractive material for biomolecular devices. such studies, however, have used br within the membrane, on relatively large surfaces. here, conducting-probe atomic force microscopy (c-afm) analysis was performed after isolating the protein from its native membrane environment while keeping its basic trimeric structure, and demonstrated that the molecular conductance of br can be reversibly photoswitched with predictable wavelength sensitivity. intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine mutant located on the intracellular side of the protein which, combined with a 75% delipidation, generated protein trimers homogenously orientated on the surface. c-afm proximal probe analysis showed a reproducible 3 fold drop of br mean resistance over *5 cycles of interspersed illuminations at the same gold-br-gold junction when k[495 nm, while no shift was observed with other wavelengths. capture of circulating tumor cells with a highly efficient nanostructured silicon substrates with integrated chaotic micromixers shutao wang 1 ). this core technology shows significantly improved sensitivity in detecting rare ctcs from whole blood, thus provides an alternative for monitoring cancer progression. by assembling a capture-agent-coated nanostructured substrate with a microfluidic chaotic mixer, this integrated microchip can be applied to isolate ctcs from whole blood with superb efficiency. ultimately, the application of this approach will open up opportunities for early detection of cancer metastasis and for isolation of rare populations of cells that cannot feasibly be done using existing technologies. this technology helped to find a needle in a haystack and will open up the opportunity for single cell genomic and epigenetic sequencing and gene expression profiling. results from further development of this technology will assist the physicians in follow-up patients and testing vigorously the concept of personalized oncology with individualized therapy. this novel technology has recently been reviewed and highlighted by nature medicine the growing crisis in organ transplantation and the aging population have driven a search for new and alternative therapies by using advanced bioengineering methods. the formation of organized and functional tissues is a very complex task: the cellular environment requires suitable physiological conditions that, presently, can be achieved and maintained by using properly-designed bioreactors reproducing all specific functions and bioactive factors that assure viability/regeneration of cells cultured in an appropriate scaffold. the creation of biomimetic environment requires the use of biomaterials such as membranes with specific physico-chemical, morphological and transport properties on the basis of the targeted tissue or organ. tailor-made membranes (organic, functionalized with specific biomolecules, in hollow-fiber configuration), designed and operated according to well-defined engineering criteria are able to sustain specific biotransformations, to provide adequate transport of oxygen, nutrients and catabolites throughout the cellular compartment, and to supply appropriate biomechanical stimuli of the developing tissue. in this talk the author will show the development of membrane engineered constructs focusing on liver and neuronal systems. the role of membrane surface and transport properties in providing instructive signals to the cells for the guiding of proliferation and differentiation will be discussed. membrane bioreactors, which through the fluid dynamics modulation may simulate the in vivo complex physiological environment ensuring an adequate mass transfer of nutrients and metabolites and the molecular and mechanical regulatory signals, will be presented. here we present a novel but simple system for cell-based assays enabling simultaneous testing of multiple samples on a same tissue without cross-contamination between neighbouring assays, as well as sequenced or repeated assays at the same tissue location. the principle of this method lies in the spatially-controlled diffusion of test compounds through a porous matrix to the target cells. a simple microfabrication technology was used to define areas where diffusion processes are allowed or inhibited. we performed proof-of-principle experiments on madin-darby canine kidney (mdck) epithelial cells using hoechst nuclear staining and calcein-am cell viability assay. fluorescent staining superimposed properly on membrane pattern with a dose-dependent response, indicating that both compounds specifically and selectively diffused to the target cells. mdck cells similarly treated with cytochalasin b showed their actin network rapidly altered, thus demonstrating the suitability of this system for drug screening applications. such a well-less cell-based screening system enabling multiple compounds testing on a same tissue and requiring very small volumes of test samples appears interesting for studying potential combined effects of different biochemicals applied separately or sequentially. it is generally believed that all-optical data processing is the most promising direction to achieve serious improvements both in capacity and speed of internet data traffic. one of the bottlenecks of the state-of-the-art photonic integration technology is to find the proper nonlinear optical (nlo) materials that are supposed to serve as cladding media in waveguidebased integrated optical circuits performing light-controlled active functions. recently, the unique chromoprotein bacteriorhodopsin (br) has been proposed to be used as an active, programmable nlo material in all-optical integrated circuits. in integrated optical applications of br, its light-induced refractive index change is utilized. in this paper we exploit the refractive index changes of a dried br film accompanying the ultrafast transitions to intermediates i and k, which allows even sub-ps switching, leading beyond tbit/s communication rate. in the experiments direct pulses of a femtosecond laser system at 800 nm were used along with synchronized ultrafast laser pulses at 530 nm. we believe that the results may be the basis for the future realization of a protein-based integrated optical device, and represent the first steps to a conceptual paradigm change in optical communication technologies. last years, such autoantibodies attract an increasing attention of researchers as potential cancer biomarkers. since the sera of cancer patients typically contain a unique set of antibodies that reflect the tumor-associated antigens expressed in a particular malignant tissue, diagnosing and predicting the outcome of disease such as breast cancer based on serum autoantibody profiling is an attractive concept. to create a representative panel of antigens for detecting of breast cancer autoantibody profile we selected 18 breast cancer associated antigens. these antigens were identified by screening of tumor cdna libraries with autologous sera using serex (serological investigation of recombinantly expressed clones) approach. all antigens were cloned, expressed, purified in bacteria and tested with sera of breast cancer patients and healthy donors in large-scale allogenic screening using elisa. the utility of selected tumor associated antigens for detecting of autoantibody profile in different types of breast cancer was evaluated. (controlled by architectural software) is carried out according to a design template, consistent with the geometry and composition of the desired organ module. structure formation occurs by the post-printing fusion of the discrete bio-ink units. when the bio-ink units contain more than one cell type, fusion is accompanied by sorting of the cells into the physiologically relevant pattern. thus structure formation takes place through self-assembly processes akin to those utilized in early embryonic morphogenesis. we demonstrate the technology by detailing the construction of vascular and nerve grafts. spherical and cylindrical bio-ink units have been employed to build fully biological linear and branching vascular tubular conduits and multiluminal nerve grafts. upon perfusion in a bioreactor the constructs achieved desirable biomechanical and biochemical properties that allowed implantation into animal models. our results show that the printing of conveniently prepared cellular units is feasible and may represent a promising tissue and organ engineering technology. femtosecond lasers have become important tools for noncontact microprocessing of biological specimens. due to the short pulse length and intensity-dependent nature of the multiphoton ionization process, fs-laser pulses affect only a small volume of a treated cell, providing a high degree of spatial localization. we employed fs-laser to address topical bioengineering and biomedical problems such as cell fusion and embryo biopsy respectively. a tightly focused laser beam (cr:f seed oscillator and a regenerative amplifier, 620nm, 100 fs, 10 hz) was used for a fusion of blastomeres of two-cell mouse embryos and for a polar body (pb) biopsy. in order to fuse blastomeres the contact border of cells was perforated by a single laser pulse. the fusion process usually completed within *60 min. in order to perform a noncontact laser based pb biopsy we initially drilled an opening in the zona pellucida with a set of laser pulses, and then extracted the pb out of zygote by means of optical tweezer (cw laser, 1064 nm). the energy of laser pulses was thoroughly optimized to prevent cell damage and increase the fusion and biopsy rates. the proposed techniques demonstrate high efficiency and selectivity and show a great potential for using fs lasers as a microsurgical tool. new insights into mechanisms of electric field mediated gene delivery maš a kanduš er and mojca pavlin university of ljubljana, faculty of electrical engineering, si-1000 ljubljana, slovenia gene electrotransfer is widely used for transfer of genetic material in biological cells by local application of electric pulses and is currently the most promising non-viral delivery method for gene therapy for a series of diseases as well as for dna vaccination. current description of the process defines several steps: electropermeabilization, dna-membrane interaction, translocation, trafficking to nucleus and into nucleus. but the mechanisms of electrotransfer are still not fully understood. we present results of the systematic in vitro analysis using pegfp of all steps involved in electrotransfection from electropermeabilization, analysis of different pulsing protocols, theoretical analysis of plasmid mobility to visualization of the processes of dna-membrane interaction. we demonstrate that in order to translate in vitro results to tissue level sub-optimal plasmid concentrations have to be used. furthermore, sofar the method of dna entry into cytoplasm was only speculated. our results suggest that it is crucial that first, membrane is electropermeabilized, then sufficient electrophoretic force is crucial for insertion of dna into destabilized lipid bilayer followed by dna translocation into cytoplasm via a slow process. efficiency of electrotransfer depends also on the stage of of cell culture -cells in dividing phase are easer to electrotransfect. gentamicin interaction with b16f10 cell membrane studied by dielectrophoresis dielectrophoresis (dep) is the translational motion of polarizable particles due to an electric field gradient. positive-dep and negative-dep correspond to particle movement forward or backward the region of high field intensity, respectively. our study reveals some of the cell membrane modifications induced by gentamicin (gt), as they are reflected in the crossover frequency f co of b16f10 murine cells incubated with gt for different concentrations and durations. f co is the ac frequency when cells turn from positive-dep to negative-dep. gentamicin is a positively charged aminoglycosidic antibiotic, with concentration-dependent killing action; it is widely used because of its low cost and reliable bactericidal activity. gt drawbacks consist in high toxicity for renal and hearing cells; the molecular mechanisms of this toxicity are still unclear. for low external medium conductivities (& 0.0012 s/m), f co of control and gt-cells was found to range from 3 to 10khz. f co shifts to higher frequencies with the increase of gt concentration and incubation time. cells dielectrophoretic behavior is discussed using the cell singleshell based model. extracellular matrix (ecm) is a major obstacle for succesful delivery of genes. chitosan is a versatile and biocompatible polysaccharide derived from chitin and is a promising gene carrier. chitosan-dna interactions, and hence dna polyplexation and release can be controlled through chitosan de-acetylation degree, molecular weight and functionalization of chitosan cationic groups. grafting of poly(ethylene glycol) peg to gene delivery vectors increases circulation time of gene delivery systems in blood vessels and reduces polyplexes charge. diffusion and unpacking of pegylated and non-pegylated chitosan-dna polyplexes through articial ecms based on collagen and collagen-hyaluronic acid (ha) gels were compared using fluorescence correlation microscopy, confocal microscopy and colocalization analysis. non-pegylated polyplexes were immobilized in the gels whereas pegylated polyplexes were diffusing. the smaller charge of pegylated polyplexes seems to decrease interactions between polyplexes and ecm components. furthermore, ha might also screen collagen fibers-pegylated polyplexes interactions. pegylated polyplexes also showed a higher degree of unpacking in gels, probably due to a looser compaction of dna by pegylated chitosan compared to non-pegylated chitosan. fabrication of vesicles, a close membrane made of an amphiphile bilayer, has great potentiality for encapsulation and controlled release in chemical, food or biomedical industries but also from a more fundamental point of view for the design of biomimetic objects. methods based on lipid film hydratation 1 , inverse emulsion techniques 2 and more recently microfluidic techniques such as double emulsion 3 or jetting 4 method are limited either by a low yield, a low reproducibility, a poor control on the size, or by the presence of remaining solvent or defects. we propose a fast and robust method 5 easy to implement: continuous droplet interface crossing encapsulation (cdice), that allows the production of defect-free vesicles at high-yield with a control in size and content. the vesicles have controlled bilayer composition with a polydispersity in size lower than 11%. we have shown that solutions as diverse as actin, cells, micrometric colloids, protein and high ionic strength solutions can easily be encapsulated using this process. by adjusting the parameters of our set-up, we are able to produce vesicles in the range 4-100 lm in diameter, stable for weeks. we believe this method open new perspectives for the design of biomimetic systems and even artificial tissues. under appropriate conditions. the extremely variable d3 domain of flagellin subunits, comprising residues 190-283, protrudes at the outer surface of flagellar filaments. the d3 domain has no significant role in the construction of the filament structure. thus, replacement of d3 may offer a promising approach for insertion of heterologous proteins or domains without disturbing the self-assembly of flagellin subunits. our work aims at the construction of flagellinbased fusion proteins which preserve the polymerization ability of flagellin and maintain the functional properties of the fusion partner as well. in this work a fusion construct of flagellin and the superfolder mutant of green fluorescent protein (gfp) was created. the obtained gfp variant was highly fluorescent and capable of forming filamentous assemblies. our results imply that other proteins (enzymes, binding domains etc.) can also be endowed by polymerization ability in a similar way. this approach opens up the way for construction of multifunctional filamentous nanostructures. generation 5 polyamidoamine (pamam) dendrimer has been shown to be highly efficient nonviral carriers in gene delivery. however, their toxicity limits their applications. in this study, to improve their characteristics as gene delivery carriers, g5 pamam dendrimer was modified with anti-tag72 nanobody through hetrobifunctional peg, then complexed with t-bid coding pdna, yielding pamam-peg-anti-tag72 nanobody/pdna nanoparticles (nps). nuclear magnetic resonance (nmr) spectroscopy, zeta sizing and gel retardation assay results provided evidence that the nanovector was successfully constructed. the transfection efficiency of vector/pdna complexes were evaluated in vitro. real time pcr results also demonstrated that anti-tag72 nanobody modified nps are more efficient in t-bid killer gene expressing in colon cancer cell line than the unmodified nps. in conclusion, pamam-peg-anti-tag72 nanobody showed great potential to be applied in designing tumour-targeting gene delivery system. 3 dept of chemistry, faculty of science, national university of singapore, singapore, 4 dept of biochemistry, yong loo lin school of medicine, national university of singapore, singapore, 5 division of bioengineering, faculty of engineering, national university of singapore, singapore macromolecular crowding (mmc) is a biophysical tool which has been used extensively to enhance chemical reactions and biological processes by means of the excluded volume effect (eve). the in vivo stem cell microenvironment contains macromolecules which are crucial for stem cell selfrenewal and cell fate determination. in order to mimic this physiological microenvironment, crowders are included in cell culture medium. we have observed that the ex vivo differentiation of human mesenchymal stem cells (hmscs) into the adipogenic lineage is significantly amplified when a crowder mixture comprising ficoll 70 and ficoll 400 is added to the culture medium. stem cell differentiation is modulated by soluble chemical substances as well as interactions between cells and the extracellular matrix (ecm), and both these external influences may be affected by mmc. measurements we have performed by fluorescence correlation spectroscopy (fcs) show that ficoll additives cause anomalous subdiffusion within a crowder concentration range of 0 to 300 mg/ml. the diffusion of fluorophorelabelled molecules in artificial lipid bilayers and membranes of living cells is not changed by crowders, suggesting that these crowders do not directly alter membrane properties and cell surface signalling. however, we have data to suggest that crowders increase actin polymerization reaction rates in vitro. we have also observed that crowders are taken up by stem cells and that they localize to specific compartments. based upon our observations, we hypothesize that crowders can influence stem cell differentiation by influencing molecular kinetics. lignocellulose-based composites are becoming extremely important and perspective sustainable and renewable natural materials. fibre modification enhancing their existing properties can be obtained to broaden the application areas. in response to shortcomings of traditional chemical and physical methods, enzymes and chemo-enzymatic methods have emerged as eco-friendly catalysts working under mild conditions and enable tailoring of the material surface properties by substrate specificity and regional selectivity. recently, binding of different functional molecules to lignin-rich fibres by using an oxidative enzyme (e.g. laccase) has been reported leading to their functionalisation through free radical reactions. by the application of electron paramagnetic resonance spectroscopy (epr) laccase action was inspected. consumption of substrates was investigated and their polymerization traced. stable radical intermediates were detected with epr when substrate molecules were in contact with active enzymes. secondly, oxidation of mediators like nitroxides was determined via epr spectroscopy of stable water-soluble nitroxide radicals. finally, the generation of short-lived radicals as well as their reduction was measured via epr spin trapping using dmpo as sensitive water soluble spin trap. mammalian ovary hormone stimulation (ohs) is known to be an inalienable stage of reproductive biotechnology as well as human infertility treatment. the basic aim of the ohs is to receive a stock of valuable oocytes and early embryos for subsequent utilization in the reproductive technology, experimental work et al. however, it is known that ohs itself affects the character of ovulation and oocyte quality, which in its turn affects the development of embryos and even has distant consequences. the wideness of cell parameters and appropriate methods for investigation of gamete/embryo quality are very important. the aim of this study is determination of specific electric conductivity of mouse oocytes and early embryos which have been received after ohs in comparison with the ones that have been received in natural animal sex cycle. using techniques of electroporation the dependence of specific electric conductivity of mouse oocytes, zygotes, 2-cell and 8-cell embryos on the external electric field intensity has been studied. it is shown that the whole pool of oocytes that were obtained in the result of ohs consists of two groups of oocytes that don't differ from each other morphologically, but differ by their electric parameters and resistance to electric breakdown. at the zygote stage, dividing of embryos into two groups is preserved, but is less expressed. at the stage of 2-cell and 8-cell dividing of embryos into two groups on their electric conductivity disappeared but certain scattering of the parameters due to individual embryo peculiarities is observed. the obtained data show that ohs may lead to latent changes of oocyte state that in their turn affect embryo quality. many microbes synthesize and accumulate granules of polyhydroxyalkanoates (pha, biodegradable storage materials alternative to traditional plastics), which help them survive under stresses. in particular, the plant-growth-promoting rhizobacterium azospirillum brasilense, that is under investigation worldwide owing to its agricultural and biotechnological significance, can produce poly-3hydroxybutyrate (phb) [1] . in our work, phb synthesis in a. brasilense cells was studied under various stresses using diffuse reflectance ftir spectroscopy. phb in cells was determined from the band intensity ratio of the polyester m(c=o) at *1740 cm -1 to that of cell proteins (amide ii band at *1550 cm -1 ), showing a. brasilense to be able to produce phb up to over 60% of cells' dry weight. stresses induced phb accumulation, enhancing ir absorption in phb specific regions. analysis of a few structure-sensitive phb vibration bands revealed changes in the degree of intracellular phb crystallinity (related to its enzymatic digestion rate) at different stages of bacterial growth, reflecting a novel trait of the bacterial adaptability to an enhancing stress, which is of great importance to agricultural biotechnology. the aim of this work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. the d3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. d3 is not essential for filament formation. the concept in this project is to replace the d3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. to test the feasibility of this approach, xylanase a (xyna) from b. subtilis was chosen as a model enzyme for insertion. with the help of genetic engineering, a fusion construct was created in which the d3 domain was replaced by xyna. the flic(xyna) chimera exhibited catalytic activity as well as polymerization ability. these results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created ( table 1) . the support of the hungarian national office for research and technology and the hungarian scientific research fund (otka) (grants ck77819, nk77978, nanoflag) is acknowledged. cluster phases of membrane proteins an alternative scenario for the formation of specialized protein nano-domains (cluster phases) in biomembranes dna fragmentation induced in human fibroblasts by accelerated 56 fe ions of differing energies swift heavy ion irradiation of srtio 3 under grazing incidence'' proc. natl. acad. sci. usa 105 forespore engulfment mediated by a ratchet-like mechanism a channel connecting the mother cell and forespore during bacterial endospore formation a feeding tube model for activation of a cell-specific transcription factor during sporulation in bacillus subtilis the scanning ion-conductance microscope imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy nanoscale live-cell imaging using hopping probe ion conductance microscopy beta2-adrenergic receptor redistribution in heart failure changes camp compartmentation simultaneous noncontact topography and electrochemical imaging by secm/sicm featuring ion current feedback regulation timasheff in protein-solvent interactions the roles of water in foods on motor and electrical oscillations in urinary tract: computer evaluation daniele martin 1,2 , viktor foltin 1,2 , erich gornik 2 , rumen stainov 3,4 , tanya zlateva 4 nü rnberg. icsd e.v. postfach (pob) 340316, d-80100 mü nchen method: parameters: motor patterns (guinea-pig) -frequency/f, amplitudes/a (% init. length, isot. & intracell. rec.) of spontaneous phasic/spc & tonic/stc contractions, also electrical spikes/s, bursts/b, burst plateaus/bp (neu et al. biophys.j. 125/6a/jan2008 stretch (3-80mn), k-/ca-influence induced specific changes in motor/electrical parameters. special computer programme reflects exactly biophysical parameters. conclusion: acc. to earlier/recent results mechano-sensitive ca ?? -activated k ? -channels participate in electrical oscillations of detrusor/ureteral myocytes. further experiments/evaluations incl effect of hydrophobic mismatch on the light-induced structural changes in bacterial reaction centers s. s. deshmukh, h. akhavein, and lá szló ká lmá n department of physics mechanism of proton transfer in nitric oxide reductase: computational study andrei pisliakov riken advanced science institute, wako-shi proteins 74, 266. acknowledgments this work was supported by project grant ptdc/qui/70182/ 2006 (cas) and doctoral grants sfrh th anniversary conference aicr this work was supported by the italian association for cancer research (airc), the istituto toscano tumori and the associazione noi per voi differential hydration, void volume: which factor provides the main contribution to dv u ? inserm umr 1054 fr; roumestand@cbs.cnrs.fr introduction: globalization needs new organizational models also for biophysics. reports on necessity of int. institutes for biophysics (iib) c/o int. universities (proposed by british nobel laureate b.russell) are given conception: proposals for ebsa-discussion: 1. enlargement of executive committee by a. honorary & presidents (permanent 1-3: moral support & 1-3 fixed term), b. interdisciplinary commission: scientists from biology, medicine, physics, etc. (feps/iups, iuphar, iupab, etc). 2. implication of interdisciplinary topics to esba/iupab congressprogrammes, 3. also for biophysical journals. 4. organization of common interdisciplinary sessions not only to biophysical, but also to other congresses. 5. co-operation between esba/iupab with int. interdisciplinary organisations (waas, icsd/ias, eur. academies) for creation of iib by network of national ones: successive common personnel, possibility for whole life work, etc. conclusion: realization of proposals 1.-5. could increase scientific/political authority of ebsa/iupab, leading to model for renewal of scientific organizations collective migration of neural crest cells: a balance between repulsion and attraction roberto mayor university college london goodilin 2,3 , olga v single-molecule cut-and-paste surface assembly (smcp) has been also used to build up a biotin scaffold that streptavidin utilizing specific molecular interactions, for example between dna-binding proteins and dna or antibodies and antigens, this technique is capable of providing a scaffold for the controlled self-assembly of functional complexes. furthermore, this allows for the introduction of smcp into protein science. we aim to employ dna-binding zinc-finger variants and gfp-binding nanobodies as shuttle-tags fused to the proteins of interest. thus a fully expressible system that can be used for the step-wise assembly of individual building blocks to form single-molecule cut-and-paste surface assembly optically monitoring the mechanical assembly of single molecules nanoparticle self-assembly on a dna-scaffold written by single-molecule cut-and-paste torsional motion analysis of group ii chaperonin using diffracted x-ray tracking nanomechanical manipulation of mason-pfizer monkey retroviral rna fragment with optical tweezers melinda simon 1 , zsolt má rtonfalvi 1 , pasquale bianco 1 , beá ta vé rtessy 2 , mikló s kellermayer 1 micro-viscosimeter generated and manipulated by light andrá s buzá s 1 , lá szló oroszi 1 , ló rá nd kelemen 1 , pá l ormos 1 temesvá ri krt proc. natl. acad. sci. usa 105 neural signal recordings with a novel multisite silicon probe gergely má rton 1 , anita pongrá cz 1 , lá szló grand 2,3 , é va vá zsonyi 1 pé ter pá zmá ny catholic university, faculty of information technology, h-1083, 50/a prá ter st multiscale pattern fabrication for life-science applications francesco valle 1 , beatrice chelli 1 , michele bianchi 1 , eva bystrenova 1 , marianna barbalinardo 3 , arian shehu 1 , tobias cramer 1 , mauro murgia 1 , giulia foschi 1 miroslava kuricova 1 , jana tulinska 1 , aurelia liskova 1 , eva neubauerova 1 , maria dusinska 2,1, ladislava wsolova acceleration neuronal precursors differentiation induced by substrate nanotopography gianluca grenci 1 , jelena ban 2 , elisabetta ruaro 4 , massimo tormen 1 , marco lazzarino 1,3 and vincent torre 2 light-induced structural changes are reported near the primary electron donor of bacterial reaction centers (brc) dispersed in detergent micelles and in liposomes from lipids with different fatty acid chain lengths. in this study we present evidence for the correlation between the light-induced increase of the local dielectric constant, determined by the analysis of the electrochromic absorption changes, and the lifetime of the charge-separated state at physiologically relevant temperatures. the increase of the local dielectric constant induced a significant decrease of the oxidation potential of the primary electron donor and a slow proton release, which appears to be the rate limiting step in the overall process. systematic selection of the head group charges of detergents and lipids, as well as the thickness of the fatty acid chains of the liposome forming lipids can increase the lifetime of the charge-separated state by up to 5 orders of magnitude. such extensions of the lifetime of the charge-separated state were reported earlier only at cryogenic temperatures and can provide new opportunities to utilize the brc in energy storage. ontogenesis of photosynthetic bacteria tracked by absorption and fluorescence kinetics m. kis, e. asztalos, p. maró ti department of medical physics and informatics, university of szeged, hungarythe development of photosynthetic membrane of rhodobacter sphaeroides was studied by absorption spectroscopy and fast induction of bacteriochlorophyll fluorescence in different phases of the growth, under various growing conditions (oxygen content, light intensity etc.) and in synchronous cell population. the results are: 1) the newly synthesized components of the membranes were imbedded immediately into the proteinous scaffold independently on the age of the cell (no ,,transient'' membranes were observed). 2) under aerobic conditions, the pigments were bleached and under anaerobic conditions the pigment systems showed greening. the relative variable fluorescence (f v /f max ) had small age-dependent (but not cellcycle-related) changes. the fluorescence induction kinetics was sensitive marker of the aerobiosis: the f v /f max ratio dropped from 0.7 to 0.4 and the photochemical rate constant from 5á10 4 s -1 to 3á10 4 s -1 with an apparent halftime of about 4-5 hours after change from anaerobic to aerobic atmosphere.3) the electrogenic signal (absorption change at 525 nm) reflected the energetization of the membrane which showed cell-cycle dependent changes. that included periodic production and arrangement of protein-lipid components of the membrane synchronized to the cell division. interfacial water in b-casein molecular surfaces: wide-line nmr, relaxation and dsc characterization t. verebé lyi 1 , m. bokor 1 , p. kamasa 1 , p. tompa 2 , k. tompa 1 1 research institute for solid state physics and optics, hungarian academy of sciences, pob. 49, 1525 budapest,hungary, 2 institute of enzymology, biological research center, hungarian academy of sciences, pob. 7, 1518 budapest, hungarywide-line proton nmr fid, echoes, spin-lattice and spin-spin relaxation times were measured at 82.55 mhz frequency in the -70°c to ?40°c temperature range, in lyophilized bcasein and aqueous and buffered solutions, and dsc method were also applied. the motivation for the selection of b-casein is the uncertainty of structural order/disorder. naturally, the nmr and thermal characteristics were also evaluated. the melting of hydration water could be detected well below 0°c and the quantity of mobile water molecules (hydration) was measured. the hydration vs. melting temperature curve has informed us on the bonding character between the protein surfaces and water molecules. the generally used local field fluctuation model and the bpp theory were applied in the interpretation, and the limits of the models were concluded. the torsional properties of dna play an important role in cellular processes such as transcription, replication, and repair. to access these properties, a number of single-molecule techniques such as magnetic tweezers have been developed to apply torque to dna and coil it. i will briefly refer to investigations of dna-protein interactions using these techniques, and describe what has been learnt. i will then focus on the development of novel magnetic techniques that go beyond standard magnetic tweezers, such as the magnetic torque tweezers 1 and the freely-orbiting magnetic tweezers 2 . these approaches allow one to quantify conjugate variables such as twist and torque. for example, the magnetic torque tweezers rely on high-resolution tracking of the position and rotation angle of magnetic particles in a low stiffness angular clamp. we demonstrate the experimental implementation of this technique and the resolution of the angular tracking. subsequently, we employ this technique to measure the torsional stiffness c of both dsdna molecules and reca heteroduplex filaments. lastly, i will describe novel applications of the optical torque wrench 3, 4 . the optical torque wrench is a laser trapping technique developed at cornell capable of applying and directly measuring torque on microscopic birefringent particles via spin momentum transfer. we have focused on the angular dynamics of the trapped birefringent particle 4 , demonstrating its excitability in the vicinity of a critical point. this links the optical torque wrench to non-linear dynamical systems such as neuronal and cardiovascular tissues, non-linear optics and chemical reactions, which all display an excitable binary ('all-or-none') response to input perturbations. based on this dynamical feature, we devise a conceptually novel sensing technique capable of detecting single perturbation events with high signal-to-noise ratio and continuously adjustable sensitivity.for the first time we report a comparative approach based on surface enhanced raman spectroscopy (sers) and raman spectroscopy to study different types of haemoglobin molecules in living erythrocytes. in erythrocytes there are two fractions of haemoglobin: cytosolic (hb c ) and membranebound (hb m ). the concentration of hb m is less then 0,5% and therefore it is impossible to study hb m with traditional optical techniques. modifications of cellular membrane can affect conformation of hb m . therefore, it can be used as a sensitive marker of pathologies. firstly, we investigated enhancement of sers signal of hb m depending on ag nanoparticles' size. we found that the intensity of sers spectra of hb m and enhancement factor increase with the decrease in ag nanoparticles' size. secondly, we investigated the dependence of haemoporphyrin conformation in both hb c and hb m ion ph values. we observed different sensitivity of hb c and hb m to the ph and found that conformational movements of haemoporphyrin (vibrations of pyrrol rings and side radicals) in hb m are sterically hampered comparably with hb c . our observation is an evidence of a benefit of application of surface enhanced raman spectroscopy to investigate properties of the hb m in erythrocytes and provide new information about conformational changes and functional properties of hb m . rna nanotechnology is an emerging field with high potential for nanomedicine applications. however, the prediction of rna three-dimensional nanostructure assembly is still a challenging task that requires a thorough understanding the rules that govern molecular folding on a rough energy landscape. in this work, we present a comprehensive analysis of the free energy landscape of the human mitochondrial trna lys , which possesses two different folded states in addition to the unfolded one. we have quantitatively analyzed the degree of rna tertiary structure stabilization, firstly, for different types of cations, 1 and, secondly, for several naturally-occurring nucleotide modifications in the structural core of the trna lys . 2,3 thus, notable variations in the rna binding specificity was observed for the divalent ions of mg 2? , ca 2? and mn 2? , that can be attributed to their sizes and coordination properties to specific ligands. furthermore, we observed that the presence of m 2 g10 modification together with the principal stabilizing m 1 a9 modification facilitates the rna folding into the biologically functional cloverleaf shape to a larger extent than the sum of individual contributions of these modifications. in order to elucidate the mechanism of the recognition, we used diffracted x-ray tracking method (dxt) that monitors real-time movements of individual proteins in solution at the single-molecule level. we found that peptides move distinctly from i-a k , and the rotational motions of peptides correlate with the type b t cell activation. in the case of diabetogenic i-a g7 , immediately after peptide exchange, all the peptides moves magnificently but the motion ceased in a week, then new ordered motion appears; the rotational motion of peptides correlate to t cell activation, which is analogous to the peptide in i-a k . the rotational motion of peptides may create transient conformation of peptide/mhc that recognized by a population of t cells. dxt measurement of peptide/mhc complex well correlated to other biological phenomenon too. our finding is the first observation that fluctuations at the level of brownian motion affect to the functions of proteins.mason-pfizer monkey virus (mpmv) is an excellent model for the analysis of retrovirus assembly and maturation. however, neither the structure of the viral rna, nor its modulation by capsid-protein binding are exactly known. to explore the structure of the mpmv genome, here we manipulated individual molecules of its packaging signal sequence with optical tweezers. the 207-base-long segment of mpmv rna corresponding to the packaging signal, extended on each side with 1200-base-long indifferent gene segments for use as molecular handles, was cloned into a pet22b vector. rna was synthesized in an in vitro transcription system. rna/ dna handles were obtained by hybridization in a pcr with complementary dna initiated with primers labeled with either digoxigenin or biotin. the complex was manipulated in repetitive stretch and relaxation cycles across a force range of 0-70 pn. during stretch, transition occurred which increased the rna chain length and likely corresponds to unfolding. the length gain associated with the unfolding steps distributed across three main peaks at *13, 20, 32 nm, corresponding to *35, 57, 85 bases, respectively. often reverse transitions were observed during mechanical relaxation, indicating that refolding against force proceeds in a quasi-equilibrium process. structural investigation of gpcr transmembrane signaling by use of nanobodies jan steyaert 1,2 1 structural biology brussels, vrije universiteit brussel, pleinlaan 2, 1050 brussel, belgium, 2 department of structural biology, vib, pleinlaan 2, 1050 brussel, belgiumin 1993, scientists at the vrije universiteit brussel discovered the occurence of bona fide antibodies devoid of light chains in camelidae. the small and rigid recombinant antigen binding fragments (15kd) of these heavy chain only antibodies -known as vhhs or nanobodies -proved to be unique research tools in structural biology. by rigidifying flexible regions and obscuring aggregative surfaces, nanobody complexes warrant conformationally uniform samples that are key to protein structure determination by x-ray crystallography:• nanobodies bind cryptic epitopes and lock proteins in unique native conformations • nbs increase the stability of soluble proteins and solubilized membrane proteins • nbs reduce the conformational complexity of soluble proteins and solubilized membrane proteins • nbs increase the polar surface enabling the growth of diffracting crystals • nbs allow to affinity-trap active protein i will focus my talk on the use of nbs for the structural investigation of gpcr transmembrane signaling to illustrate the power of the nanobody platform for generating diffracting quality crystals of the most challenging targets including gpcrs and their complexes with downstream signaling partners. dynamics of the type i interferon receptor assembly in the plasma membrane stephan wilmes, sara lö chte, oliver beutel, changjiang you, christian paolo richter and jacob piehler university of osnabrü ck, division of biophysics, barbarastrasse 11, 49076 osnabrü ck, germanytype i interferons (ifn) are key cytokines in the innate immune response and play a critical role in host defense. all ifns bind to a shared cell surface receptor comprised of two subunits, ifnar1 and ifnar2. detailed structure-function analysis of ifns has established that the ifn-receptor interaction dynamics plays a critical role for signalling specificities.here we have explored the dynamics of receptor diffusion and ifn assembly in living cells. by using highly specific orthogonal posttranslational labelling approaches combined with tirf-microscopy we probed the spatio-temporal dynamics of receptor diffusion and interaction in the plasma membrane of live cells on the single molecule level. for this purpose, we employed posttranslational labelling with photostable organic fluorophores. this allowed us to map diffusion and lateral distribution of ifnar1 and ifnar2 with very high spatial and temporal resolution by using single particle tracking (spt) and single molecule localization imaging. observed events of ''transient confinement'' and co-localization with the membrane-proximal actin-meshwork suggest partitioning of ifnar1/2 in specialized microcompartments. this will be investigated in terms influence on receptor assembly and recruitment of cytoplasmic effector proteins. cytoplasmic dynein moves through uncoordinated action of the aaa1 ring domains ahmet yildiz department of physics, and department of molecular and cell biology, university of california, berkeley, ca 94720 usa cytoplasmic dynein is a homodimeric aaa? motor that moves processively toward the microtubule minus end. the mechanism by which the two catalytic head domains interact and move relative to each other remains unresolved. by tracking the positions of both heads at nanometer resolution, we found that the heads remain widely separated and move independently along the microtubule, a mechanism different from that of kinesin and myosin. the direction and size of steps vary as a function of interhead separation. dynein processivity is maintained with only one active head, which drags its inactive partner head forward. these results challenge established views of motor processivity and show that dynein is a unique motor that moves without strictly coordinating the mechanochemical cycles of its two heads.o-715 self-controlled monofunctionalization of quantum dots and their applicaitons in studying protein-protein interaction in live cells changjiang you, stephan wilmes, sara loechte, oliver beutel, domenik lisse, christian paolo richter, and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastrasse 11, 49076 osnabrü ck, germanyindividual proteins labeled with semiconductor nanocrystals (quantum dots, qd) greatly facilitate studying protein-protein interactions with ultrahigh spatial and temporal resolution. multiplex single molecule tracking and imaging require monovalent quantum dots (mvqd) capable of orthogonally labeling proteins with high yield. for this purpose, we prepared monovalance qd-trisnta by a chemical conjugation method. our results indicated that monovalent qd-trisnta was obtained in high yield by restricting the coupling by means of electrostatic repulsion. monovalent functionalization of the qd-trisnta was confirmed by assays in vitro and in vivo. two-color qd tracking of interferon receptors ifnar1 and ifnar2 based on mvqd-trisnta were realized on live cell [1] .to broaden the multiplex toolbox of mvqds, we extended the electrostatic-repulsion induced self-control concept for mono-functionalizing quantum dots with different affinity moieties. as a first instance, we used negatively-charged biotin peptide to produce qd with biotin mono-functionalization. we confirmed our approach was a general method to rend qd monovalent by single molecule assays based on stepwise photobleaching. these mvqds facilitate obtaining spatiotemporal information of ifnars' organization in live cells. by orthogonal labeling u5a cells stably expressing ifnar2 at low level with biotin mvqd and mvqd-trisnta-ifn, we verified colocalization and colocomotion of individual ifn and ifnar2 at minute scale. combined with super-resolution imaging of ifnars' cytosolic effecter stat2, we observed the dynamic coming-and-going contact between the microcompartments of ifnar2 and stat2.a micron sized viscometer was fabricated using the couette type geometry that is capable of measuring the complex viscosity of fluids. the viscometer was produced by two photon polymerization of su8 photopolymer using a femtosecond laser system, a high na objective and a piezo translator stage. the viscometer was manipulated by holographic optical tweezers and operated in the 0.005-1 hz frequency range. video analysis algorithm was used to evaluate our measurements. we tested the viscometer with water-glycerol solutions. one of the main reasons for lack of reliability in protein analysis for disease diagnostics or monitoring is a lack of test sensitivity. this is because, for many tests, to be reliable, they need to be performed on a homogeneous, and therefore very small, sample. current in-vitro techniques fail in accurately identifying small differences in protein content, function and interactions starting from samples constituted of few or even single cells. a nanotechnology approach may overcome the current limits in low abundance protein detection. we aim at designing a microwell device for the trapping (in native environment) and the parallel characterization of rare cells (e.g. adult stem cells). such versatile device, based on soft and nanolithography, will promote cell adhesion and viability on differently functionalized bio-compatible materials, allowing for the morphological characterization of the cells, at a single cell level. in parallel, by facing our microwell device with a protein nanoarray, produced via atomic force microscopy nanolithography, we can run proteomic studies at a single/few cells level. moreover, we could foresee the possibility to deliver different stimuli to each cell, correlating the changes in chemistry/ morphology with the protein profile at a single cell level. using an electrophysiological assay the activity of nhaa was tested in a wide ph range from ph 5.0 to 9.5. forward and reverse transport directions were investigated at zero membrane potential using preparations with inside out and right side out oriented transporters with na ? or h ? gradients as the driving force. under symmetrical ph conditions with a na ? gradient for activation, both the wt and the ph-shifted g338s variant exhibit highly symmetrical transport activity with bell shaped ph dependencies, but the optimal ph was shifted 1.8 ph units to the acidic range in the variant. in both strains the ph dependence was associated with a systematic increase of the k m for na ? at acidic ph. under symmetrical na ? concentration with a ph gradient for nhaa activation an unexpected novel characteristic of the antiporter was revealed; rather than being down regulated it remained active even at ph as low as 5. these data allowed to advance a transport mechanism based on competing na ? and h ? binding to a common transport site and to develop a kinetic model quantitatively explaining the experimental results. in support of these results both alkaline ph and na ? induce the conformational change of nhaa associated with nhaa cation translocation as demonstrated here by trypsin digestion. furthermore, na ? translocation was found to be associated with the displacement of a negative charge. in conclusion, the electrophysiological assay allowed to reveal the mechanism of nhaa antiport and sheds new light on the concept of nhaa ph regulation. swimming motility is widespread among bacteria. however, in confined or structured habitats bacteria often come in contact with solid surfaces which has an effect on the swimming characteristics. we used microfabrication technology to quantitatively study the interaction of swimming cells with solid boundaries. we tracked bacteria near surfaces with various engineered topologies, including flat and curved shapes. we were able to study several surface related phenomena such as hydrodynamic trapping and correlated motion. we think that our results may help to understand how physical effects play a role in surface related biological processes involving bacteria such as biofilm formation. cell labeling efficiency of oppositely charged magnetic iron oxide nanoparticles-a comparative study raimo hartmann 1 , christoph schweiger 2 , feng zhang 1 , wolfgang. j. parak 1 , thomas kissel 2,# , pilar rivera_gil 1,# 1 biophotonics, institute of physics, philipps university of marburg, 2 pharmaceutical technology, institute of pharmacy, philipps university of marburg e-mail: kissel@staff.uni-marburg.de; pilar.riveragil@physik.uni-marburg.dethe interaction of nanomaterials with cells is a key factor when considering their translocation into clinical applications. especially an effective accumulation of nanoparticles inside certain tissues is beneficial for a great number of applications. predominantly size, shape and surface charge of nanoparticles influence their cellular internalization and distribution. to investigate this, two series of maghemite (c-fe 2 o 3 ) nanoparticles were synthesized either via aqueous coprecipitation or via thermal decomposition of organometallic precursor molecules. size and the spherical shape of both nanoparticle types were kept constant whereas the charge was changed by modifying the surface of the nanoparticles with polymers of opposite charge, in detail poly(ethylene imine) (pei) and a polymaleic anhydride derivative (pma). the positively and negatively charged c-fe 2 o 3 nanoparticles were characterized with respect to size, zeta potential, colloidal stability and magnetic properties. furthermore, the uptake rate and localization of both formulations into a549 carcinoma cells after fluorescent labeling of the carriers as well as the resulting alteration in mr-relaxation times were evaluated. membrane proteins are the target of more than 50% of all drugs and are encoded by about 30% of the human genome. electrophysiological techniques, like patch-clamp, unravelled many functional aspects of membrane proteins but usually suffer from poor structural sensitivity. we have developed surface enhanced infrared difference absorption spectroscopy (seidas) 1,2 to probe potential-induced structural changes of a protein on the level of a monolayer. a novel concept is introduced to incorporate membrane proteins into solid supported lipid bilayers in an orientated manner via the affinity of the his-tag to the ni-nta terminated gold surface 3 . full functionality of surface-tethered cytochrome c oxidase is demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c. general applicability of the methodological approach is shown by tethering photosystem ii to the gold surface 4 . in conjunction with hydrogenase, the basis is set towards a biomimetic system for h 2 -production. recently, we succeeded to record ir difference spectra of a monolayer of sensory rhodopsin ii under voltage-clamp conditions 5 . this approach opens an avenue towards mechanistic studies of voltage-gated ion channels with unprecedented structural and temporal sensitivity. initial vibrational studies on the novel light-gated channelrhodopsin-2 will be presented 6 . probing biomass-chromatographic bead interactions by afm force spectroscopy gesa helms, marcelo ferná ndez-lahore, rami reddy vennapusa, and jü rgen fritz school of engineering and science, jacobs university bremen, 28759 bremen, germany e-mail: g.helms@jacobs-university.dein expanded bed adsorption (eba), bioproducts are purified from an unclarified fermentation broth by their adsorption on chromatographic beads in a fluidized bed. the unspecific deposition of biomass onto the adsorbent matrix can severely affect the process performance, leading to a poor system hydrodynamics which then decreases the success of this unit operation. to quantify the bead-biomass interactions different chromatographic beads are attached to afm cantilevers, and force spectroscopy experiments are performed with these colloidal probes on model surfaces and cells in solution. the experiments are conducted under varying conditions to study uncovering physiological processes at the cellular level is essential in order to study complex brain mechanisms. using multisite signal recording techniques in the extracellular space, functional connectivity between different brain areas can be revealed. a novel microfabrication process flow, based on the combination of wet chemical etching methods was developed, which yields highly reproducible and mechanically robust silicon-based multielectrode devices. the fabricated shaft of the probe is 280 lm wide, 80 lm thick, has rounded edges and ends in a yacht-bow like, sharp tip. its unique shape provides decreased invasivity. the sensor contains 24 platinum recording sites at precisely defined locations. murine in vivo experiments showed that the probes could easily penetrate the meninges. high quality signals, providing local field potential, multi-and single unit activities, were recorded. the interfaces between the tissue and the platinum contacts were further improved by electrochemical etching and carbon nanotube coating of the metal sites. the integrated optical mach-zehnder interferometer is a highly sensitive device, considered a powerful lab-on-a-chip tool for specific detection of various chemical and biochemical reactions. despite its advantages, there is no commercially available biosensor based on this technique. the main reason is the inherent instability of the device due to slight changes of environmental parameters. in this paper we offer a solution to this problem that enables the optimal adjustment of the working point of the sensor prior to the measurement. the key feature is a control unit made of a thin film of the lightsensitive chromoprotein bacteriorhodopsin deposited on the reference arm of the interferometer. after showing the transfer characteristics of such a device, we demonstrate its applicability to sensing of specific protein-protein interactions. we expect our method to become a rapid and cost-efficientthe combination of unconventional fabrication technology and biomaterials allows both to realize state-of-the-art devices with highly controlled lateral features and performances and to study the main properties of the biomolecules themselves by operating at a scale level comparable with the one crucial for their activity. soft lithography and microfluidic devices offer a tool-box both to study biomolecules under highly confined environments [1] and to fabricate in an easy way topographic features with locally controlled mechanical and chemical surface properties, thus leading to a finer control of the interplay of mechanics and chemistry. i will present an application of this technology to the control of cell fate that is becoming a key issue in regenerative medicine in the perspective of generating novel artificial tissues. patterns of extracellular matrix (ecm) proteins have been fabricated, by a modified lithographically controlled wetting (lcw), on the highly antifouling surface of teflon-af to guide the adhesion, growth and differentiation of neural cells (shsy5y, 1321n1, ne-4c) achieving an extremely accurate guidance [2] . local surface topography is also known to influence the cell fate [3] , thus, integrating this parameter in the substrate fabrication could increase the complexity of the signals supplied to the cells. in this perspective we have developed a novel fabrication technique, named lithographically controlled etching (lce), allowing, in one step, to engrave and to functionalize the substrate surface over different lengthscales and with different functionalities. i will conclude showing how we have been developing ultrathin film organic field effect transistors (ofets) as label-free biological transducers and sensors of biological systems. ofets are low dimensional devices where ordered conjugated molecules act as charge transport material. unconventional patterning techniques and microfluidics have been adapted to proteins and nucleic acids to dose the molecules on the ofet channel with a high control of the concentration. in another set of experiments, we have also been addressing the signalling from neural cells and networks grown on pentacene ultra-thin film transistosr [3, 4] .advances in nanotechnology are beginning to exert a significant impact in medicine. increasing use of nanomaterials in treatment of diseases has raised concerns about their potential risks to human health. in our study, the effect of poly(lactic-co-glycolic acid) (plga) and titanium dioxide (tio 2 ) nanoparticles (nps) on function of b-and t-lymphocytes was investigated in vitro. human blood cultures were treated with plga and tio 2 nps in concentrations: 0.12; 3 and 75 lg/cm 2 for 72h. lymphocyte transformation assay was used to assess the effect of nps on lymphocyte function. lymphocytes were stimulated with mitogens: concanavalin a, phytohaemmagglutinin (t-cell response) and pokeweed mitogen (b-cell response). our findings indicate immunomodulatory effect of plga nps. proliferative response of t-and b-lymphocytes exposed in vitro to the highest dose of plga for 72h was suppressed significantly (p.01, p.05). on the other hand, we observed stimulative effect of exposure to middle dose of plga nps on b-lymphocyte proliferation (p.05). no alteration was found in lymphocyte proliferation treated in vitro with tio 2 nps for 72h. in conclusion, proliferation of lymphocytes in vitro might be one of the relevant tests for evaluation of nps immunotoxicity.embryonic stem (es) cell differentiation in specific cell lineage is still a major challenge in regenerative medicine. differentiation is usually achieved by using biochemical factors (bf) which concentration and sides effects are not completely understood. therefore, we produced patterns in polydimethylsiloxane (pdms) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. we analyzed the effect of different nanostructures on differentiation of es-derived neuronal precursors into neuronal lineage without adding biochemical factors. neuronal precursors adhere on pdms more effectively than on glass coverslips but the elastomeric material itself doesn't enhance neuronal differentiation. nano-pillars increase both precursors differentiation and survival with respect to grooves. we demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. on higher pillar neuronal differentiation reaches *80% 96 hours after plating and the largest differentiation enhancement of pillars over flat pdms was observed during the first 6 hours of culture. we conclude that pdms nanopillars accelerate and increase neuronal differentiation. key: cord-006229-7yoilsho authors: nan title: abstracts of the 82(nd) annual meeting of the german society for experimental and clinical pharmacology and toxicology (dgpt) and the 18(th) annual meeting of the network clinical pharmacology germany (vklipha) in cooperation with the arbeitsgemeinschaft für angewandte humanpharmakologie e.v. (agah) date: 2016-02-06 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-016-1213-y sha: doc_id: 6229 cord_uid: 7yoilsho nan in vitro systems and mechanistic investigations i the demand of alternative test systems which closely mirror the in vivo situation is one of the main challenges in modern toxicity testing. the major goal is the development of in vitro systems that partly display the complexity of an organism and thus may mimick in vivo conditions. despite great efforts in the past no adequate in vitro systems are available yet. on the other hand, cell cultures from almost every organ are easily accessible and therefore may help to roughly assess the toxic potential of substances at target structures. nonetheless, the complex interactions which take place in vivo cannot be addressed in single cell cultures. in the liver, hepatocytes comprise 80% of the total liver volume while non-parenchymal cells -endothelial cells, stellate cells and kupffer cells (that is, liver resident macrophages) -contribute only 6.5% of the volume, but 40% of the total cell number (kmiec 2001) . it has been increasingly recognized that in the liver neighboring non-parenchymal cells release molecules which contribute to the inflammatory damage and even aggravate it (adams et al. 2010) . in our project a human in vitro co-culture system was established by combining a hepatic and a monocytic cell line, the latter of which can be differentiated to a macrophage-like phenotype. in this system the hepatotoxicty of substances has been analyzed, and the results were compared to single cultures and to published data from in vivo studies. using ketoconazole, an antifungal, as a known hepatotoxic substance, inflammatory markers were studied and proved to be significantly upregulated only in coculture. conversely, cultures of hepatic cells only did not display this increase in inflammatory markers. at the same time, a negative substance, caffeine, failed to show any hepatotoxic potential in the co-culture system. our results demonstrate that this novel in vitro co-culture model represents a promising tool to evaluate the hepatotoxic potential of substances. in drug research, it might help to reduce animal testing as drugs with a high dili potential can be dropped early in the development phase. question: raman spectroscopy (rs) is a highly sensitive analytical method for markerfree and non-invasive identification and characterization of cells. here, we present rs as a novel tool for gentle yet precise cell analysis in three independent experiments, focusing on monitoring cellular reactions upon treatment. we could provide evidence that rs is a suitable tool to monitor cell differentiation, analyze cell modification and study cell apoptosis after drug application. methods: in a first experiment, mesenchymal stem cells (mscs) were treated with erythropoetin (epo) for certain time points and subsequently fixed with paraformaldehyde (pfa) for raman analysis. in addition, skbr3 breast cancer cells were exposed to the anti-cancer drug herceptin (20μg/ml). cells were then fixed in pfa for rs. in a last experiment, molm-13 cells were separately cultivated in microwells and treated with thymidine for different time points prior to raman analysis. results: raman spectroscopy was able to monitor differentiation of epo treated mscs and found that around 35% of treated cells showed fibroblast like raman profiles. in case of herceptin treated skbr3 cells, rs found internal changes of the cell´s metabolism as reaction on drug application. analyzing the most prominent differences in raman spectra revealed discrimination of cells to be mainly due to changes in amid i, lipid and protein content. in the last experiment, rs was able to follow apoptosis of molm-13 cells after thymidine application and discriminate early from late apoptotic states. discussion: rs is a photonic marker for gentle yet highly specific cell analysis, which allows monitoring of single cell reaction after drug treatment. thereby, rs provides information about changes within the entire metabolome on a single cell level. raman spectra are as characteristic as a "fingerprint". rs works label-free and non-invasive and thus does not impare cell viability. this allows to gain new insights in pharmacological development and toxicological survey. acknowledgement: this project received funding from the eu 7th health program grant agreement no 279288. deutsches zentrum für herzinsuffizienz, würzburg, germany extracellular signal-regulated kinases 1 and 2 (erk1/2) are essential for the regulation of cell growth and cell survival and their kinase activity is up-regulated for example in different types of cancers and pathological cardiac hypertrophy. while inhibition of erk1/2 activity by kinase inhibitors prevents tumor growth, it can also lead to exacerbated cardiomyocyte death and impaired heart function. interestingly, we have previously identified an erk autophosphorylation at threonine 188 as a prerequisite for nuclear erk1/2 signaling and erk-mediated cardiac hypertrophy. here, we investigated an alternative strategy to interfere with erk1/2 signaling: since activation of erk1/2 triggers erk dimerization, a prerequisite for erk t188autophosphorylation, we chose the erk dimer interface as a possible target to selectively interfere with erk t188 -phosphorylation. first, we investigated the impact of monomeric erk2 on cardiac function. to address this issue, we generated mice with cardiac overexpression of monomeric erk2 ∆174-177 and performed transverse aortic constriction (tac) to induce cardiac hypertrophy. compared to wild-type mice, erk2 ∆174-177 overexpression attenuated tac-induced cardiac hypertrophy, interstitial fibrosis and mrna expression levels of collagen and brain natriuretic peptide (bnp), while cardiomyocyte survival and cardiac function were largely preserved. because of the positive effects of monomeric erk ∆174-177 in the heart, we designed a peptide to interfere with endogenous erk dimerization. cross-linking and coimmunoprecipitation experiments showed that the peptide binds to erk2 and prevents its dimerization. moreover, the peptide effectively inhibited erk t188 -phosphorylation and nuclear translocation of yfp-tagged wild-type erk2 after phenylephrine stimulation. further, adenoviral or adeno-associated virus serotype 9 (aav9)-induced overexpression of the peptide in neonatal rat cardiomyocytes (nrcm) or mouse hearts resulted in a significantly reduced hypertrophic response to phenylephrine or tac, background: g i -proteins have been proposed to be cardioprotective. it's matter of debate whether this depends on the particular g i isoform and/or on the particular conditions (e.g. cardiac "stress") only. in our study we investigated effects of a gα i2knockout on cardiac function and survival in a murine heart-failure model of cardiac β 1adrenoceptor overexpression. methods and results: β 1 -adrenoceptor overexpressing mice lacking gα i2 (β 1 -tg/gα i2 -/-) were compared to wild-type (c57bl/6) mice and littermates either overexpressing cardiac β 1 -adrenoceptors (β 1 -tg) or lacking gα i2 (gα i2 -/-). at 300 days of age mortality of mice only lacking gα i2 was higher compared to wild-type or β 1 -tg, but similar to β 1tg/gα i2 -/mice. beyond 300 days mortality of β 1 -tg/gα i2 -/mice was enhanced compared to all other genotypes (mean survival time: 363±21 days). echocardiography revealed similar cardiac function of wild-type, β 1 -tg and gα i2 -/mice, but significant impairment for β 1 -tg/gα i2 -/mice (e.g. ejection fraction 14±2% versus 40±4% in wild-type mice). significantly increased ventricle-to body-weight ratio (0.71±0.06% versus 0.48±0.02% in wild types), left-ventricular size (length 0.82±0.04 cm versus 0.66±0.03 cm in wild types) and anp and bnp expression (mrna: 2819% and 495% of wild type, respectively) clearly indicated hypertrophy. gα i3 was significantly upregulated in gα i2 -knockouts (protein compared to wild-type mice: 340±90% in gα i2 -/and 394±80% in β 1 -tg/gα i2 -/-, respectively). radioligand binding experiments confirmed cardiac overexpression of β 1adrenoceptors in β 1 -tg mice. of note, overexpression levels differed depending on the particular wild-type background. on an fvb/n background we found the overexpression level to be more than 2-fold higher (b max : 1425 ± 68 fmol/mg) than on the otherwise used c57bl/6 background. accordingly, fvb/n-based β 1 -tg mice showed a significantly impaired cardiac function at an age of 300d while c57bl/6-based β 1 -tg mice did not. conclusions: gα i2 -deficiency combined with cardiac β 1 -adrenoceptor overexpression strongly impaired survival and cardiac function. on a c57bl/6 background β 1adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction at day 300 while there was overt cardiomyopathy in mice additionally lacking gα i2 . we propose an enhanced effect of increased β 1 -adrenergic drive by lack of protection via gα i2 . the observed gα i3 upregulation was not sufficient to compensate for gα i2 deficiency suggesting an isoform-specific and/or a concentration-dependent mechanism. the role of gα i3 is currently addressed in a subsequent study using β 1 -tg and gα i3deficient mice. heart failure is accompanied by morphological and functional alterations (e.g. hypertrophy, decreased contractility) which are summarized by the term "cardiac remodeling". while the β-adrenergic signaling pathway is essential for short-term modulation of cardiac performance, its chronic stimulation by elevated plasma catecholamines and the subsequent activation of camp-dependent signal transduction pathways is regarded as a fundamental factor in the pathogenesis of cardiac remodeling. however, the mechanisms mediating the transition of physiological conditions of short-term to detrimental remodeling under long-term β-adrenergic stimulation are not understood in detail so far. in this context, icer, an isoform of the camp dependent transcription factor crem (camp responsive element modulator), acts as an early response gene strongly induced by beta-adrenergic stimulation via camp responsive elements (cre) in its promoter. contrary to its cre-mediated induction, icer is a strong inhibitor of cre-mediated transcription by itself. here we study the role of icer induction in the catecholamine-induced cardiac remodeling in a time dependent manner by the use of icer deficient mice (iko) and wild type (wt) controls, which were treated with isoproterenol (iso; 10 mg/kg per d) for 6 and 24 hours and 7 days. overall 7 days of iso stimulation resulted in an elevation of cardiomyocyte length in iko (in µm; 7d iso 156±1) vs. wt cardiomyocytes (7d iso 143±2). at this time point a 29 % decrease of cardiac output and a 16 % decrease of the maximal rate of rise of left ventricular pressure (dp/dt max ) in iko vs. wt animals was detectable. the maximum increase of icer mrna in wt cardiomyocytes already occurred after 6 h (75-fold), and declined after 24 h (29-fold) to 2.5 fold increase after 7 days, while icer mrna was not detectable in iko mice. this raised the hypothesis, that the early induction of icer modulates transcriptional processes after beta-adrenergicstimulation, involved in cardiac remodeling of the heart. profiling of mrna expression levels between iko vs. wt cardiomyocytes at the different time points revealed: 55 regulated genes (up-regulated: 45 %) in untreated; 103 altered genes (up 35 %) after 6h; 1437 changed genes (up 97%) after 24 h and 131 altered genes (up 19%) after 7 days of iso treatment. in summary, the absence of icer induction in myocytes resulted in an increase of cardiomyocytes length and a decrease of heart performance after 7 days of betaadrenergic stimulation. this is preceded by upregulated mrna levels of several hundred genes at 24 h, which is going along with the induction of transcriptional inhibitor icer after a few hours of beta-adrenergic stimulation. this suggested a protective role of icer by inhibiting the progression of cardiac remodeling after betaadrenergic stimulation in an early responsive manner. (supported by the dfg) the performance of the adult heart is tightly regulated by g protein-coupled receptors. adrenergic and angiotensin receptors efficiently control heart rate and contractility. muscarinic receptors, on the other hand serve as master regulators of the conduction system, which is often lost upon myocardial infarction. this function of muscarinic receptors has been well described in the adult or late embryonic heart. here we provide evidence that muscarinic receptors are crucial to constrain pacemaker cell identity. we applied subtype-specific inhibitors of muscarinic receptors to zebrafish embryos of different stages. we observed that both, early cardiac function as well as specification are specifically regulated by muscarinic m3 receptors, while m2 receptors appear to exert a heart-specific function only at later stages. continuous m3 blockage renders zebrafish with greatly altered cardiac morphology, particularly of the conduction system. furthermore, embryos with m3 inhibition display impaired ventricular function most likely due to an av-block and substantial arrhythmia in the atrium. importantly, to observe these phenotypes it was sufficient to block m3 receptors during stages of cardiac differentiation, which is long before a heart tube has formed. we corroborated our findings regarding these morphological changes using marker gene analysis. furthermore, we obtained evidence for m3 receptors preventing a transcriptional program towards the induction of pacemaker cells at the expense of av canal cells. importantly, this is not only true during heart development. a pacemaker program is also induced in adult hearts upon m3 inhibition. taken together, we postulate that muscarinic m3 receptors confine a pacemaker lineage during early steps of heart development as well as in the adult heart. our data suggests m3 receptors as potential new therapeutic targets for the regeneration of hearts with an injured sinoatrial node. systemic inhibition of mir-21 has proved effective against fibrosis of the myocardium and in other organs. mir-21 has been reported to exert detrimental effects in cardiac fibroblasts and protective roles in cardiac myocytes and other myocardial cell types. a better definition of the cell types that contribute to the beneficial effects of inhibiting mir-21 in vivo may aid the development of strategies with enhanced therapeutic efficacy. thus far, no approach to selectively manipulate micrornas in the non-myocyte population of cardiac cells in vivo has been available. in this study, we developed an icre-encoding mml virus for application in mir-21 fl/fl mice. delivery of this vector to neonates achieved targeted genetic ablation of mir-21 in non-myocyte cardiac cells. immunohistochemistry and flow cytometry confirmed that mmlv was highly selective and effective for cardiac fibroblasts and endothelial cells. in parallel, an aav9-icre vector allowed for specific and almost complete deletion of mir-21 in cardiac myocytes. when tested in a model for chronic left ventricular pressure overload, mmlv-icremediated deletion of mir-21 in cardiac fibroblasts and endothelial cells significantly reduced cardiac fibrosis and hypertrophy and improved cardiac function. the benefit of this cell-type-specific inhibition exceeded that observed upon global genetic deletion of the mir-21 gene in mice. aav9-mediated deletion of mir-21, albeit lowering cardiac hypertrophy, had no effect on fibrosis or cardiac function. taken together, neonatal delivery of engineered icre-encoding viruses enabled for the first time a differential gene targeting in non-myocyte and myocyte cells in myocardium. non-myocyte deletion of mir-21 demonstrated that mir-21 exerts its cardiac profibrotic activity directly in cardiac fibroblasts and in endothelial cells. this novel finding should encourage tailoring of antimir-21 therapy towards cellular tropism. chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. as current anti-inflammatory pharmacotherapy is not always satisfying, there is a great demand for the discovery of new drug leads as well as novel drug targets. the synthetic carbazole alkaloid derivative c81 acts as a multikinase inhibitor. results of a thermal shift assay revealed that c81 shows by far the highest binding affinity to the bmp-2-inducible kinase (bmp2k/bike). bmp2k represents an as yet largely uncharacterized protein, which is not regulated by bmp-2 in endothelial cells. therefore, we aimed to analyze (i) the pharmacological potential of c81 and (ii) the role of bmp2k in angiogenic and inflammatory processes in the vascular endothelium. initial experiments show that only high concentrations of c81 affected the viability of human umbilical vein endothelial cells (huvecs). both c81 and the knock-down of bm2k (rnai) reduced the migratory capacity of a human microvascular endothelial cell line (hmec-1). also the proliferation of hmec-1 was reduced by c81 treatment (ic 50 : 7 µm). a tube formation assay on matrigel demonstrated that c81 significantly impaired the formation of capillary-like structures in a dose-dependent manner. interestingly, the analysis (western blot) of signaling molecules in huvecs that play a crucial role in cell proliferation (e.g. erk, akt) revealed that these pathways are not influenced, neither by c81 treatment nor by bmp2k gene silencing. in regard to inflammatory processes, c81 treatment or bmp2k silencing of huvecs decreased the adhesion of thp-1 cells, a monocytic cell line, onto the activated endothelial cells. as the interaction of leukocytes is mainly mediated by cell adhesion molecules (cams), the effect of c81 or bmp2k silencing on their expression was analyzed (flow cytometry, qpcr). while the expression of cams was strongly decreased after c81 treatment, the knock-down of bmp2k did not markedly affect their expression. furthermore, both approaches did not lead to the reduction of tnf-induced iκbα degradation (western blot) or p65 translocation into the nucleus (microscopy). our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole alkaloid derivative c81 in vitro. the precise role of bmp2k in angiogenic and inflammatory endothelial processes as well as the involved pathways during bmp2k silencing and c81 treatment will be further elucidated. moreover, since the inhibition of bmp2k seems not to be responsible for all actions of c81, we will investigate the role of other kinases affected by the compound in these processes. the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). cxcr4 seems to be part of the lipopolysaccharide sensing complex, suggesting that an intervention with cxcr4 agonists or antagonists could result in reduced tlr4 signaling. however, the role of cxcr4 and the influence of different cxcr4 ligands in acute as well as chronic inflammatory diseases are still contradictious. therefore, we aimed to characterize the systemic effects of cxcr4 activation in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by applying a sublethal lps dose (5 mg/body weight) in mice. the plasma stable cxcl12 analog ctce-0214d was synthesized and administered subcutaneously shortly before lps treatment to ensure a delayed release and thereby a prolonged effect of the drug. 24 hours following lps administration, mice were sacrificed and blood was obtained for tnf alpha, ifn gamma and blood glucose evaluation. additionally, histopathological changes and oxidative stress in the liver and spleen were assessed and liver biotransformation capacity was determined. finally, cxcr4, cxcl12 and tlr4 expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for oxidative stress and apoptosis were evaluated by means of immunohistochemistry. ctce-0214d improved the health status and distinctly reduced the lps mediated effects on tnf alpha, ifn gamma and blood glucose levels by approximately 35%, 50% or 70%, respectively. it attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. ctce-0214d diminished the lps induced expression of cxcr4, cxcl12, tlr4, nf-κb, cleaved caspase-3 and gp91 phox, whereas heme oxygenase 1 expression and activity were induced above average. furthermore, tunel staining revealed anti-apoptotic effects of ctce-0214d in all organs. the cxcr4 is undoubtedly involved in inflammation. its activation was accompanied by anti-inflammatory, anti-oxidative and cytoprotective effects as ctce-0214d attenuated tlr4 signaling, induced heme oxygenase 1 activity and mitigated apoptosis. thus, the administration of cxcl12 analogs seems to be a promising treatment option to control acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals. the neurodegenerative disease friedreich ataxia (frda) is caused by a gaa triplet repeat expansion in the first intron of the frataxin gene, which results in a reduction of the corresponding mitochondrial protein. despite several cellular and animal models the exact function of frataxin is still a matter of debate, but the role of frataxin in iron sulfur cluster biosynthesis is generally accepted. however, we still don't know which primary metabolic events are caused by a frataxin deficit and until now, there is no therapeutic option available. we developed a new cellular model for frda by using the cre/loxp recombination system in mouse embryonic fibroblasts (mef). c57bl/6j mouse strains with a loxp flanked exon 4 of the frataxin gene and an tamoxifen-inducible cre-recombinase (creer t2 ) were crossed and several mef cell lines isolated. after selection by genotype and growth manner the fx-mef 2-1 (fxn -/-) and fx-mef 2-8 (fxn +/-) cell lines were finally choosed. the generation of the homozygous or heterozygous frataxin knockout was successfully tested on rna and protein level. long maintenance of the frataxin depleted fibroblasts revealed a strong growth inhibition consistent to earlier observations in other cell systems. therefore, we established a pattern of treatment over 12 days, with medium and substance changes at day 4 and 8, which allows us to get a fully functional knockout and overcome the growth inhibition problem. endpoint measurements of known metabolic phenomena from mammalian and non-mammalian models were studied at day 12 of our novel cell system. the induced total disruption of frataxin leads to a clearly reduced aconitase activity, cell division and oxygen consumption as well as an increase in ros production. in the heterozygous knockout with residual frataxin activity no such changes were observed. in addition, our pattern of treatment enables us to monitor the full and partial frataxin knockout in the course of time, to detect early and late metabolic events after frataxin disruption. therefore we analysed the mentioned parameters (with additional atp and iron content) in parallel at day 3, 5, 7 and 10 and could identify an initial event followed by secondary consequences and parameters, which seem to play only a minor role in the frda pathogenesis. on the contrary, a partial deficit of frataxin didn't result in any differences over time and suggests that there are only cellular alterations below a critical threshold. in conclusion, our new established mammalian cellular frda model mimics typical metabolic consequences of the human disease and seems to be qualified for frda research. the model shows for the first time six different metabolic events in the course of time in parallel and reveals insights into primary and secondary events of frda pathogenesis. these observations can be used to better understand the function of frataxin and can help to develop new therapeutic strategies to address the consequences of frataxin deficiency. moreover, the transfer of this cell model into 96well plates offers the possibility for a high-throughput screening of potential therapeutic substances. the disease diphtheria is caused by the diphtheria toxin (dt) which belongs to the group of single-chain ab-type bacterial protein toxins. receptor-binding of the b-domain on the target cell surface is followed by receptor-mediated endocytosis and internalization into early endosomal vesicles. endosomal acidification triggers membrane insertion and pore formation of the transmembrane (t) domain together with translocation of the (partially) unfolded catalytic (c) domain into the cytosol. herein, dta catalyzes adp-ribosylation of elongation factor 2 which leads to disruption of protein synthesis and finally causes cell death [1] . in hela cells, these events are related to cell-rounding functioning as a specific endpoint to monitor the uptake of dta into the cytosol of the host cell. as for other adp-ribosylating toxins such as c. botulinum c2 toxin, c. perfringens iota toxin and c. difficile cdt, also in case of native dt we demonstrated the involvement of several host cell factors during the translocation step of the catalytic domain across the endosomal membrane [2, 3] . in detail, we confirmed the involvement of the host cell chaperone hsp90 and the thioredoxin reductase (trxr), the latter presumably responsible for the reduction of the interchain disulfide bond between the dta and dtb moieties [4, 5, 6] . furthermore, we identified another group of protein folding helpers, the family of peptidyl-prolyl cis/trans isomerases (ppiases) including cyclophilin a (cypa), cyp40 and fk506-binding protein (fkbp)51 as required cytosolic factors for dta translocation. to characterize the role of the protein folding helpers in more detail, we investigated their interaction with purified dta in vitro by performing dot blot analysis with immobilized recombinant host cell factors, co-precipitation of cellular factors with dta from hela lysate and isothermal titration calorimetry with purified proteins therewith determining the thermodynamic parameters of the individual binding events. thereby, we detected binding of dta to hsp90, cypa, cyp40, fkbp51 and fkbp52. the data increase the knowledge of the molecular mechanisms underlying dt uptake and especially dta translocation which can be medically used to develop novel therapeutic strategies against the disease diphtheria. [1] murphy (2011) toxins 3, 294-308. [2] barth (2011) naunyn-schmied arch pharmacol 383, 237-245. [3] kaiser et al. (2012) cell. microbiol. 14, 1193-1205. [4] dmochewitz et al. (2011) cell. microbiol. 13 , 359-373. [5] ratts et al. (2003) j. cell biol. 160, 1139-1150. [6] schnell et al. (2015) novel afflictions as for example clostridium (c.) difficile associated diseases (cdad) caused by clostridium difficile are on the increase and challenging to treat. cdad most frequent occur in hospitalized patients after prolonged treatment with antibiotics. cdad includes among others diarrhea and the severe form of pseudomembranous colitis. not only the treatment of the infection, but also the treatment of the toxins has a high clinical significance. c. difficile secretes the exotoxins a (tcda) and b (tcdb), which glycosylate and thereby inactivate rho-gtpases in mammalian cells. tcda and tcdb are considered as the causative agents of cdad. in the last few years, more and more hypervirulent strains of c. difficile were described. in these hypervirulent strains, the adp-ribosyltransferase cdt was found as a third toxin in addition to tcda and tcdb. given the lack of agents effective against antibiotic-resistant bacterial strains and bacterial exotoxins, the development of novel pharmacological strategies is needed. the antimicrobial activity of naturally occurring substances is already known for a long time. one important mechanism of the innate immune system is the production of natural peptides showing antibiotic features. in recent years, it was shown that human antimicrobial peptides as important part of the native innate immune system plays a crucial role not only in inactivation of bacteria but also in inhibition of bacterial toxins (1). prompted by these result, we found that only human α-defensin-1 (hnp-1) but not human β-defensin-1 (hbd-1), both important effectors of the innate immune system, protected cultured epithelial cells from intoxication with tcda and cdt when applied prior to the toxins to the cells. moreover, α-defensin-1 prevented also the cytotoxic effects of all three c. difficile toxins tcda, tcdb and cdt combined in the medium. the combined investigation of all three toxins might be even more suitable to mimic the situation after an infection with hypervirulent c. difficile. the inhibition of the toxins was monitored by cell rounding caused by each of the toxins. currently, the molecular mechanisms underlying the inhibitory effects are still unknown and will be investigated in different cell lines. in conclusion, our results demonstrate that hnp-1 causes a loss of cytotoxicity of the c. difficile toxins and may act as novel drugs to cure c. difficile infections that contribute to cdad. neurodegenerative diseases like parkinson´s disease (pd) are accompanied by altered gene expression levels in the brain. recent studies support a role of regulatory noncoding rnas, such as micrornas (mirnas), which silence a specific set of mrnas at the post-transcriptional level. upon their aberrant expression, they are likely involved in the pathophysiology of specific neuronal loss. manipulation of neuronal gene expression is pivotal for understanding the function of proteins and the development of new therapeutic strategies. rna interference (rnai) strategies can be employed through the administration of small interfering rnas (sirna), which mediate the specific knockdown of a selected target gene. however, the main challenge is the delivery of these rnas into the neurons of interest. in this pilot study, we present a method for delivering sirnas in polymeric nanoparticles based on low molecular weight polyethylenimines (peis). their intracerebroventricular (icv) injection leads to in vivo silencing of neuronal gene expression in the brain of mice overexpressing α-synuclein (thy1-asyn mice). in a first step, pei-complexed sirna tagged with afluorescencedye were injected to track the localization and distribution after icv administration. five days later, fluorescent cells were visible throughout the brain, with the highest fluorescence intensity around the ventricles. fluorescence was also observed in large cells of the lumbar spinal cord. moreover, preliminaryresultsdemonstrate a 42.6% knockdown (p<0.05 student's t-test, n=6) of human α-synuclein (snca) in thetargetstructurestriatum upon a single icv injection of pei-complexed specific sirna compared to the control injection group (n=9). hence, our first results support the usability and efficacy of pei nanoparticle-mediated delivery of short rnas, namely sirnas, for rapidly and efficiently reducing the expression of a neuronal target gene of interest in the brain in vivo. this may allow the development of gene therapy strategies for the treatment of neurodegenerative diseases. is a propenylic alkenylbenzene found in several plants, e.g. acorus calamus. bacontaining plant materials are used to flavor foods, and are active ingredients in traditional plant medicines. thus, human exposure results primarily from the intake of bitters and teas, as well as from calamus-containing medicines and plant food supplements. although many (positive) pharmacological properties/effects of asarone isomers are described in the literature, ba was found to be carcinogenic in rodents (liver, duodenum) when given daily or in a single dosage. early experiments indicated that ba is not activated via hydroxylation and sulfonation as it is the case for known hepatocarcinogenic allylic alkenylbenzenes such as estragole or methyleugenol. because the mechanism of metabolic activation of ba in not known, we investigated the metabolism of ba in liver microsomes and human cytochrome p450 (cyp) enzymes, the mutagenicity of ba and its metabolites in the ames fluctuation assay and the dna adduct formation in primary rat hepatocytes. we found that side-chain epoxidation (leading to diols and a ketone) was by far the most dominating metabolic route of ba in liver microsomes and human cyp enzymes. ba was mutagenic in the ames test (+s9 mix), as was the synthesized ba-epoxide (-s9 mix). furthermore, we were able to synthesize and characterize a ba epoxide-derived dna adduct with deoxyguanosine. this dna adduct was formed in a concentration-dependent manner in rat hepatocytes incubated with ba. our results strongly indicate that ba is genotoxic with the side-chain epoxide being its ultimate carcinogen. morbid obesity is an independent risk factor for cardiovascular disease, type 2 diabetes mellitus and certain types of cancer. bariatric surgery -with the roux-en-y gastric bypass (rygb) being the gold standard -has become the therapeutic option of choice as a sustained weight loss and improvement of associated morbidity is achieved in the majority of patients. there is, however, a lack of evidence focusing on bariatric surgery induced sustained weight loss and its possible impact on cancer risk. we investigated the association between obesity, oxidative stress and genomic damage after roux-en-y gastric bypass surgery (rygb) or caloric restriction induced weight loss in the obese zucker rat. obese male zucker fa/fa rats were divided into three groups: sham surgery (sham), rygb and caloric restriction (cr) and were compared with lean controls (lean; zucker fa/+ rats). shams showed impaired glucose tolerance and elevated plasma insulin levels, which were less severe in rygb and cr. oxidative stress was elevated in kidney, liver and colon tissue of sham and reduced again after weight loss induced by either rygb or bwm. urine-derived oxidization products of lipids, dna and rna increased in shams and decreased after weight loss (rygb and cr). dna double strand breaks were more frequent in shams than in the weight loss groups or lean. dna damage in zucker fa/fa rats correlated with their basal plasma insulin values. obese rats showed elevated oxidative stress and genomic damage in comparison to lean rats. after body weight loss, achieved by either rygb or caloric restriction alone, oxidative stress level and genomic damage were decreased. this may indicate a reduction of the elevated cancer risk in obesity. ) mice were treated with the nocrelated compound azoxymethane (aom) followed by the administration of dextran sodium sulfate to trigger crc. tumors were quantified by non-invasive mini-endoscopy, which revealed a non-linear increase in crc formation in wt and aag -/mice. in contrast, a linear dose-dependent increase in tumor frequency was observed in mgmt -/and mgmt -/-/aag -/mice. the data was corroborated by hockey stick modeling, which yielded similar carcinogenic threshold doses for wt and aag -/mice. o 6 -meg levels and depletion of mgmt activity correlated well with the observed dose-response in crc formation. aom dose-dependently induced double strand breaks (dsbs) in colon crypts including in lgr5-positive colon stem cells, which coincided with atr-chk1-p53 signaling. intriguingly, mgmt-deficient mice displayed significantly enhanced levels of γ-h2ax, suggesting the usefulness of γ-h2ax as an early genotoxicity marker in the colorectum. this study demonstrates for the first time a non-linear dose-response for alkylation-induced carcinogenesis and reveals dna repair by mgmt, but not aag, as a key node in determining a carcinogenic threshold at low alkylation dose levels [2] . obesity is characterized as a status where the excessive accumulation of fat in adipocytes leads to local inflammation and hypoxia; both contributing to severe obesity associated co-morbidities such as cardiovascular disease and type 2 diabetes mellitus. local inflammation is mediated by macrophages, stromal vascular cells, preadipocytes, and adipocytes as well as by a number of proinflammatory cytokines and chemokines (1). in particular, the chemokines monocyte chemoattractant protein (mcp-1, ccl2), interleukin-8 (il-8/cxcl8) and stromal cell-derived factor 1 (sdf-1a/cxcl12) secreted by stromal vascular cells, preadipocytes, and adipocytes exert paracrine effects by recruiting neutrophils, monocytes/macrophages, and t-and b-cells. interestingly, deficiency in cxcl14 was shown to attenuate obesity in mice (2) . the cc chemokine ccl2 is known to stimulate ccr2 receptors, and the cxc chemokines cxcl8 and cxcl12 to activate cxcr1 and cxcr2 or cxcr4 and cxcr7, respectively. the receptor(s) activated by cxcl14 is currently unknown. a role of cxcl14 in modulating cxcr4 signaling has been proposed. we initiated our studies to determine the presence and functional significance of chemotaxin receptors in human adipocytes and their precursor cells. to this end, the mrna expression pattern of cc chemokine receptors, cxc chemokine receptors formylated peptide receptor fpr1 and the related receptor fprl1, and cc and cxc chemokines was analyzed during in vitro adipose differentiation of human simpson-golabi-behmel-syndrome (sgbs) preadipocytes, and under conditions mimicking an inflammatory response. in particular, we focused on the expression pattern of human ccr2 receptors, since previous reports indicated a role in adipogenic differentiation. however, our comprehensive analysis using different sources of adipocytes and their precursors indicated that ccr2 receptors were absent (3) . yet, the analysis revealed appreciable levels of mrna encoding ccl2, cxcl12, and cxcl14, and ccr1, cxcr2, cxcr7, fpr1, and fprl1, and cxcr4. of interest, cxcr4-and cxcr7-mrna were found to be up-regulated under the proinflammatory conditions. to analyze the responses of adipocytes and their precursors to chemokine receptor agonists, we used chemokine-mcherry fusion proteins purified from baculovirus-infected insect cells, e.g ccl2, cxcl12, cxcl14. while sgbspreadipocytes and adipocytes did not accumulate ccl2-mcherry upon stimulation, they showed a small accumulation of cxcl12-mcherry, and a strong accumulation of cxcl14-mcherry in the endosomal compartment. similar results were obtained in murine 3t3l-1 preadipocytes. using mass spectrometry analysis, we set out to identify the cxcl14-binding putative receptor protein(s) in murine 3t3l-1 preadipocytes. (1) makki, k. et al., (2013) in personalized medicine tumors are screened for several mutations in oncogenes or tumor suppressors. however, the cellular protein content not exclusively depends on the dna. we identified new rac variants generated on the mrna level in androgenindependent prostate cancer cells. all variants represent active forms of the gtpase. they are capable to suppress rhoa-induced apoptosis and additionally, mediate the synthesis of genes which are under the control of the androgen receptor. importantly, expression of the rac variants is sufficient to support tumor growth in mice. we prove the existence of the variants and verify their clinical appearance and relevance in tissue samples of a prostate cancer patient. dna analysis, however, revealed the wildtype sequence of rac. therefore, routine analysis of patient tumor tissue would miss the detection of active rac which precludes the success of therapy. the existence of active rac variants in prostate cancer tissue that promote resistance towards androgen deprivation suggest rac inhibition as an effective add on therapeutic strategy against prostate cancer. the bacterial effector protein exotoxin y (exoy) of pseudomonas aeruginosa is delivered into host cells via the bacterial type iii secretion system. once arrived in the host cell nucleotidyl cyclase activity of exoy is activated by a yet unknown cofactor and thus has a profound effect on concentrations of cyclic nucleotides: in addition to production of cyclic amp (camp) and cyclic gmp (cgmp) there is a massive synthesis of cyclic 3', and to some extent of the corresponding cytidylyl analogue ccmp 1,2 . currently, the role of cump and ccmp during the pathogenesis of p. aeruginosa infection remains unknown 3 . one of our hypotheses is that these cyclic nucleotides fulfil a role as first messengers, e.g. in the communication between individual bacteria or bacterial populations during establishment of acute or chronic infections. to test this hypothesis, the intra-and extrabacterial concentrations of cyclic nucleotides were measured via hplc-ms/ms at different time-points in liquid cultures of p. aeruginosa, either in a complete (lb medium) or a starving medium (vogel-bonner medium). additionally, we tested if supplementation of the media with extrinsic cump or ccmp had an effect on these measured concentrations. the influence of extrabacterial cyclic nucleotides on the bacterial metabolism and homeostasis was evaluated with a microarray of bacterial total cdna extracted at different time points of p. aeruginosa liquid culture with or without extrinsic cump/ ccmp. furthermore we investigated a potential function of the cyclic nucleotides in biofilm formation. cyclic ump and cyclic cmp have differential roles in bacterial metabolism and communication. for example, whereas ccmp is synthesized by p. aeruginosa when the bacteria are in a nutrient-rich environment, we could not detect bacterial cump under any tested circumstance. in our biofilm formation assays, only ccmp had a biofilmpromoting effect, but only in very high concentrations. the currently ongoing analysis of gene expression data in the presence or absence of cump may reveal a role of this cyclic nucleotide as first messenger, too. in further studies we will elucidate the signal transduction processes underlying the observed cump / ccmp effects, for example by identifying cump and ccmp binding proteins and their coupling mechanisms to intracellular signalling cascades. synapses are complex computational platforms that transmit information encoded in action potentials but also transform their functionality through synaptic plasticity. g protein-coupled receptors (gpcrs) play a major role in modulating the strength of the synapses via the second messenger camp 1 . however the spatio-temporal dynamics of the mode of action of camp underlying synaptic plasticity are still controversial. the role of this study was to investigate the dynamics of camp signaling at the drosophila neuromuscular junction, where octopamine binding to its receptors has been shown to cause camp-dependent synaptic plasticity 2 . for this purpose, we generated a transgenic drosophila expressing the camp sensor epac1-camps 3 in motor neuron. this allowed us to directly follow the octopamine-induced camp signals in real time by fluorescence resonance energy transfer (fret) in different compartments of the motor neuron (i.e. cell body, axon, boutons). we found that octopamine induces a steep camp gradient from the synaptic bouton (high camp) to the cell body (low camp), which was due by higher pde activity in the cell body. high octopamine concentrations evoked a response also in the soma. notably, these signals were independent and isolated form each other. moreover, application of octopamine by iontophoresis to single synaptic boutons induced bouton-confined camp signals. these data reveal that a motor neuron can posses multiple and largely independent camp signaling compartments, and provide new basis to explain how camp could control neurotransmission at a level of a single synapse. 1 kandel, e.r., dudai, y. & mayford, m.r. the molecular and systems biology of memory. cell 157, 163-186 (2014) . 2 koon, a.c., et al., autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. nat neurosci. 14 (2): p. 190-9 (2010) . 3 nikolaev vo, bünemann m, hein l, hannawacker a, lohse mj novel single chain camp sensors for receptor-induced signal propagation. j. biol. chem.279, 37215-37218 (2004) cardiovascular pharmacology 039 hyaluronic acid deposition determines engineered heart muscle characteristics and can be pharmacologically targeted to enhance function s. schlick background: engineered human myocardium (ehm) can be generated from psc derived cardiomyocytes (cms) and primary fibroblasts suspended in a collagen i hydrogel (70%:30%:0.4 mg/ml). ehm development encompasses an early consolidation phase followed by functional maturation. the presence of fibroblasts is essential for consolidation into a force-generating ehm. here we assessed the hypothesis that fibroblasts of different origin support ehm formation differentially as a function of hyaluronic acid deposition. methods and results: oscillatory rheology (1% strain, 1 hz) on cell-free and cell containing collagen i hydrogels directly after casting revealed enhanced consolidation in the presence of human foreskin fibroblasts (ffbs) compared to primary adult cardiac fibroblasts (cfbs) -change in storage modulus over time (pa/min): collagen 0.03; collagen + cms 0.03; collagen + cms + cfbs 0.09, collagen + cms + ffbs 0.2. we next generated ehm with cms and ffbs or cfbs. after 4 weeks of culture under serum-free conditions, we assessed ehm function by contraction measurements. ffb-ehms developed a significantly (p<0.01) higher force of contraction (foc) per cross sectional area (csa) than cfb-ehms (maximal foc/ csa are in mn/mm 2 : 1.8±0.1, n=29 vs. 0.3±0.1, n=20). cross sectional area (csa) of tissues was greatly increased (p<0.01) in cfb-ehms (csa in mm 2 : 1.6±0.1, n=20 vs. 0.6±0.03, n=30) and nonmyocyte content was higher in cfb-ehms (5.6±0.7, n=9 vs. 3.1±0.4, n=15; x10 5 cells/ml). histological analysis revealed that cardiomyocytes were only poorly matured in cfb-ehms compared to ffb-ehms. extending ehm functional data, principal component analysis of rnaseq data revealed distinct expression patterns for ffbs and cfbs, in which hyaluronic acid synthase 2 (has2) enzyme was significantly (p<0.01) upregulated. based on these findings, we pharmacologically intervened with has2 mediated hyaluronic acid (ha) deposition by treating cfb-ehms with hyaluronidase during all 4 weeks of culture. interestingly, ecm manipulation with low concentrations of enzyme significantly (p<0.01) reduced csa (csa in mm 2 : control 1.8±0.4, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.9±0.2, n=4) with a concurrent, statistically significant (p<0.01), increase in contractile function and improved cardiomyocyte morphology on a histological level (maximal foc/csa in mn/mm 2 : control 0.2±0.05, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.4±0.08, n=4). summary and conclusions: our data suggest that ehm consolidation is influenced differentially by fibroblasts of different tissue origin with hff-ehm being functionally superior to cfb-ehm. cfb-ehm could be rescued by hyaluronidase leading to reduced ha deposition. the latter demonstrates that extracellular matrix composition is centrally involved in ehm development. angiogenesis is the process of formation of new blood vessels from the pre-existing ones. vascular endothelial growth factor (vegf) is the most studied regulator of this process. by binding to its type 2 receptor (vegfr2), it has been shown to activate a variety of different signaling-pathways leading to enhanced angiogenesis. camp, on the other hand, is a versatile second messenger which regulates various endothelial functions including barrier function. it directly activates protein kinase a (pka) or the exchange protein directly activated by camp (epac) which is a guanine exchange factor (gef) for the small monomeric gtpase rap. as human umbilical vein endothelial cells (huvec) express both camp effectors (epac1 and pka), we investigated the role of camp-signaling using a spheroid based sprouting assay as an in vitro model for angiogenesis. interestingly, the activation of β-adrenergic receptors with 5 µm of isoproterenol significantly increased the cumulative sprout length. similarly, the selective activation of epac with 30 µm of the camp analog 8-pcpt-2'-o-camp (007) significantly increased the basal and the vegf-induced cumulative sprout length. in accordance, sirna-mediated depletion of epac1 in huvec decreased the basal and vegf-induced sprouting. surprisingly, 10 µm of forskolin increased basal and vegf-induced cumulative sprout length stronger than 007, indicating an additional role of pka. in accordance, 1 µm of myristoylated pki, a membrane-permeable specific pka inhibitor, significantly attenuated the forskolin-induced increase in sprouting. in all conditions tested, 50 ng/ml of vegf always showed an additive effect to the same extent on cumulative sprout length. therefore, our data indicate that the vegf-pathway is acting independently of the camp-pathway in the regulation of the sprouting angiogenesis. the β-adrenergic receptor-mediated activation of camp signaling in huvec induces angiogenic sprouting by activation of epac1 and pka. introduction: hypertension is one major risk factor for the development of chronic heart and kidney disease. mineralocorticoid receptor (mr) antagonists are a cornerstone in the therapy of heart failure and there is first evidence for a beneficial effect on the kidney as well. inflammation plays an important role in hypertensive organ injury. thus, this study was designed to evaluate and directly compare the effect of mr deletion in endothelial cells on blood pressure and cardiac vs. renal injury in a mouse model of deoxycorticosterone acetate-induced hypertension. methods and results: mice lacking the mineralocorticoid receptor in endothelial cells (mr cdh5cre ) were created using the cre/loxp system. mr cdh5cre and cre-negative littermates (mr wildtype ) underwent unilateral nephrectomy and received 1 % nacl with drinking water for 6 weeks. the mineralocorticoid deoxycorticosterone acetate (doca, 2.5 mg/d) was delivered by subcutaneous pellets. untreated mice served as controls (ctrl). ambulatory blood pressure was determined by implantable telemetry in awake mice. doca/salt treatment increased mean blood pressure in mr wildtype (141.7 ± 8.0 vs. ctrl 97.8 ± 3.2 mmhg, p<0.001) and mr cdh5cre (150.0 ± 3.0 vs. ctrl 104.1 ± 4.7 mmhg, p<0.001) without differences between genotypes. cardiac hypertrophy after doca/salt treatment was ameliorated in mr cdh5cre mice (ventricle weight 162.4 ± 4.2 mg vs. mr wildtype 189.0 ± 10.9 mg, p<0.05). doca/salt significantly increased cardiac fibrosis and the expression of fibrotic marker genes in mr wildtype but not in mr cdh5cre mice. this was accompanied by an increased expression of the vascular cellular adhesion molecule (vcam1) in mr wildtype cardiac endothelial cells. renal function was not altered by mr deletion in endothelial cells at baseline. doca/salt treatment lead to marked interstitial fibrosis in the kidneys of mr wildtype (sirius red fibrosis score: 2.4 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) and mr cdh5cre (2.3 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) mice. mrna expression of the fibrosis marker gene col3a1 (mr wildtype 4.3 ± 0.9-fold; mr cdh5cre 3.9 ± 1.1-fold vs. ctrl) was similarly increased. periodic acid-schiff staining revealed glomerular injury in both genotypes. this was associated with a marked rise in urinary albumin / creatinine ratio (mr wildtype 20.4 ± 5.7fold; mr cdh5cre 34.3 ± 12.5-fold vs. ctrl). in the kidney vcam1 mrna expression and the number of macrophages was increased by doca/salt treatment independently from endothelial mr deletion. conclusion: in conclusion, mr deletion from endothelial cells ameliorated doca/saltinduced cardiac but not renal inflammation and remodeling independently from blood pressure. these findings suggest different mechanisms for the beneficial effect of mr antagonists in hypertensive heart vs. kidney disease. platelets are relevant cells implicated in morbidity and mortality provoked by cardiovascular thrombosis. even with the actual antiplatelet therapy there is still a substantial incidence of arterial thrombosis. therefore, a better understanding of the mechanisms involved in platelet activation and aggregation is required to develop improved antiplatelet therapies. the increase in the intracellular ca 2+ concentration due to ca 2+ entry from the extracellular space is critical for platelet activation and aggregation. ca 2+ entry follows activation of plasma membrane receptors including gqcoupled receptors for adp, thromboxane a 2 (txa 2 ) or thrombin, as well as the collagen receptor glycoprotein vi (gpvi). the cellular signalling pathways downstream these receptors involve activation of phospholipase c and second messengers that are known to mediate activation of trpc channels. trpc proteins form receptor-operated cation channels, but their regulation and permeability differ depending on the cell type. it has been proposed that trpc proteins might contribute to platelet function as constituents of agonist-activated ca 2+ entry channels; however, the experimental approaches used so far and the lack of specific agonists or antagonists have not allowed to determine the individual contribution of trpc proteins for agonist-induced ca 2+ entry in platelets, aggregation and thrombosis formation. we detected the expression of trpc3 and trpc6 in human and mouse platelets. we identified that those proteins together are essential components of a system of coincidence detection in cellular ca 2+ signalling. this coincidence detection triggered by simultaneous stimulation of both thrombin and collagen receptors is required for the phosphatidylserine exposure in human and murine platelets, indicating the role of trpc3/c6 proteins for procoagulant activity. in addition, we detected the expression of s12 trpc1 transcripts in mouse platelets. therefore, we tested trpc1/c3/c6-deficient mice in an in vivo model of arterial thrombosis where they showed reduced thrombus formation. regardless of the protective effect of trpc1/c3/c6 inactivation observed in the thrombosis model, no differences were detected in tail bleeding. to evaluate the relevance of these trpc proteins in platelet aggregation we measured in vitro platelet aggregation in platelets from trpc1/c3/c6-deficient mice and we observed that the aggregation was reduced after adp (3µm) or txa 2 -analogue (1µm) stimulation, but not after collagen stimulation (10µg/ml). we are currently analyzing the in vitro aggregation and the agonist-evoked ca 2+ response in platelets from different trpc-compound and single deficient mouse lines to understand the mechanisms behind this phenotype with regard to its complementarity to actual antiplatelet therapy. background: thrombin signaling initiates inflammatory events directly and through activation of platelets. endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. the small leucine-rich proteoglycan biglycan (bgn) is such an endogenous thrombin inhibitor that acts through activation of heparin cofactor ii (hcii). here, the effect of genetic deletion of bgn on thrombin activity, inflammation and atherosclerosis was addressed. methods and results: bgn concentrations were elevated in the plasma of patients with acute coronary syndrome. in apoe -/mice, bgn was detected in the plasma as well as in the glykokalyx of capillaries. additionally, bgn expression occurred in the subendothelial matrix of arterioles as well as in atherosclerotic plaques. in line with a role of bgn in balancing thrombin activity, apoe -/-/bgn -/0 mice exhibited higher activity of circulating thrombin and increased numbers of activated platelets than did apoe -/mice. furthermore, higher concentrations of circulating cytokines in apoe -/-/bgn -/0 mice suggested a pro-inflammatory phenotype. likewise, immunohistochemistry and facs analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions of these mice. in addition, apoe -/-/bgn -/0 mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. of note, apoe -/-/bgn -/0 mice showed progressive dilatation of the aortic arch corresponding to a decrease in collagen fibril density suggestive of an outward remodelling in the absence of bgn. no differences were evident with respect to lipid content of the aortic root plaques or circulating plasma lipids. treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in apoe -/-/bgn -/0 mice. conclusions: the present results strongly suggest a protective role of bgn during the progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation. the exposure to environmental or human-made xenobiotics including drugs induces the hepato-intestinal transcription of metabolizing enzymes and transporters. the time-span of induction is thought not to exceed xenobiotic exposure, in order to minimize disturbances of endobiotic metabolism. in contrast, we find cross-generational transmission of the induction of the phase i enzyme cyp2b10 (1200-fold in females, 700-fold in males) in 6-day old offspring of adult female mice exposed one week prior mating to tcpobop (3 mg/kg i.p.) , the model ligand of the xenosensing nuclear receptor car. such cross-generational effects of xenobiotics are of great clinical interest as they could have profound consequences on the health status of the offspring, including interferences with drug therapies. the multigenerational transmission of tcpobop-driven induction could be mediated by pre-uterine/pre-conceptional epigenetic changes of oocytes. alternatively, they could be brought about by direct intrauterine/post-conceptional contact with tcpobop released from long-term depots. to discriminate between these mechanisms we conducted embryo transfer experiments. both donor mothers and foster mothers were injected with tcpobop (3 mg/kg) prior to mating. the analysis of hepatic cyp2b10 expression in 6-day-old offspring is clearly consistent with a post-conceptional onset of tcpobop effects. thus, offspring of solvent-injected donor mothers transferred to tcpobopexposed foster mothers display a 700-fold induction while offspring from the reciprocal experiment show no changes. cesarean sections on day e18.5 followed by crossfostering proved transmission to be mediated predominantly via lactation (f1 hepatic cyp2b10 induction 3000-fold) and only to a minor part via intra-uterine exposure (300fold). this mechanism is consistent with the absence of induction transmission via the male germline. to analyze if tcpobop leads to functional consequences in drug metabolism of f0 and f1 generation, we conducted in vivo zoxazolamine paralysis assays taken as a functional test for cyp2b10 catalytic activity. in both tcpobop-pretreated f0 and in their f1 descendants, the induction reduced the duration of paralysis evoked by zoxazolamine by >50%. the characterization of cross-generational tcpobop-mediated effects on other processes controlled by car such as energy and bone metabolism is in progress. first tests indicate a transmission of anabolic effects on bone, as evidenced by the induction of serum osteocalcin expression by 45% in 12-weeks-old offspring. in summary, the car-mediated cyp2b10 induction by tcpobop is transmitted to the offspring mainly via lactation, resulting in lasting phenotypic consequences in drug and bone metabolism. the effects of similarly lipophilic drugs and anthropogenic environmental pollutants are currently being investigated. such compounds could affect offspring despite discontinuation of intake or exposure well ahead of pregnancy. age-related cognitive decline can eventually lead to dementia, the most common mental illness in elderly people and an immense challenge for patients, their families and caregivers. cholinesterase inhibitors constitute the most commonly used antidementia prescription medication. the standardized ginkgo biloba leaf extract egb 761 ® is approved for treating age-associated cognitive impairment and has been shown to improve the quality of life in patients suffering from mild dementia. a clinical trial with 96 alzheimer´s disease patients indicated that the combined treatment with donepezil and egb 761 ® had less side effects than donepezil alone (yancheva et al., 2009) . in an animal model of cognitive aging, we compared the effect of combined treatment with egb 761 ® or donepezil monotherapy and vehicle. we compared the effect of chronic treatment (15 days of pretreatment) with donepezil (1,5 mg/kg p. o.), egb 761 ® (100 mg/kg p. o.), or the combination of the two drugs, or vehicle in 18 -20 month old male ofa rats. learning and memory performance were assessed by morris water maze testing, motor behavior in an open field paradigm. in addition to chronic treatment, the substances were administered orally 30 minutes before testing. compared to the first day and to the control group, only the combination group showed a significant reduction in latency to reach the hidden platform on the second day of testing. moreover, from the second day of testing onwards, the donepezil, the egb 761 ® and the combination group required less time to reach the hidden platform compared to the first day. the control group did not reach the same latency reduction until day three. there were no effects on motor behavior. these results suggest a superiority of the combined treatment of donepezil with egb 761 ® compared to monotherapy. literature: yancheva, s., ihl, r., nikolova, g., panayotov, p., schlaefke, s., & hoerr, r. (2009) . ginkgo biloba extract egb 761(r), donepezil or both combined in the treatment of alzheimer's disease with neuropsychiatric features: a randomised, double-blind, exploratory trial. aging ment health, 13 (2) , [183] [184] [185] [186] [187] [188] [189] [190] institut für vegetative physiologie und pathophysiologie, göttingen, germany values are well below the plasma concentrations (3-28 µm; just et al. expert opin emerging drugs 20: 161-164, 2015) observed in patients treated with dantrolene. although not yet proven directly, oat3 may be involved in renal secretion of dantrolene and 5-oh dantrolene by mediating the first step, i.e. the uptake across the basolateral membrane of proximal tubule cells. the second step, the release of these compounds into the urine across the luminal membrane, is possibly mediated by mrp4. since oat3 was also detected in the cytoplasmic membrane of skeletal muscle cells (takeda et al. europ. j. pharmacol. 483: 133-138, 2004) , dantrolene may reach its target, the intracellular ryanodine receptor, ryr1, by influx through oat3, where it inhibits calcium efflux by ryr1 thereby preventing severe muscle contraction and malignant hyperthermia. this assumption, however, awaits a direct demonstration of dantrolene transport by oat3. in addition, we identified, besides dantrolene and 5-oh dantrolene, several further fdaapproved drugs such as tyrphostin ag 1478, ceefourin 1, glafenine, nalidixic acid, and prazosine, as inhibitors of es uptake by oat3. controls 1 with other defects and controls 2 with genetic disorders. all drugs used in the first trimester were identified from the database, and were cross-referenced against previously compiled lists of drugs with reactive intermediates and drugs with faa. drugs with reactive intermediates, with systemic absorption and with a daily dose ≥50mg were considered os-inducing drugs. when there was an association between os-inducing drugs and a group of birth defects, we further investigated two different faa exposure categories: concurrent exposure to both os-inducing drugs and faa drugs (os+/faa+) and exposure to os-inducing drugs only (os+/faa-). when the number of subjects allowed (at least five cases/controls were exposed), we examined the role of folic. odds ratios (ors) with 95% confidence intervals were adjusted for maternal smoking and alcohol use in the first trimester in controls 1 and additionally adjusted for maternal age in controls 2. results: a total of nine groups of birth defects were investigated. only nervous system defects were associated with os-inducing drugs. exposure rates were 65/464 (14.0%) for cases, 512/6033 (8.5%) for controls 1 and 130/1564 (8.3%) for controls 2 and adjusted ors (95%cis) were 1.71 (1.29-2.26) and 1.77 (1.27-2.46), respectively. this association was unchanged when we examined os+/faa+ and os+/faa-separately. the os+/faa+ category, however, had slightly higher or values than the os+/faa-(2.41 vs. 1.61 for controls 1, and 2.55 vs. 1.67 for controls 2). because of the low number of exposed subjects, we could only examine folic in relation to os+/faa-. using os-/faa-/folic+ as reference, we found the highest risk with os+/faa-/folicand a lesser magnitude with os+/faa-/folic+ (ors being 2 and 1.6 times respectively for both controls). conclusion: our study suggests an increased risk of having a child with nervous system defects in mothers who were exposed to os-inducing drugs during pregnancy, and a potential risk reduction with folic. background: inhibition of rho-gtpases with statins as well as specific inhibition of the small gtpase rac1 protects non-transformed cells from topoisomerase ii-(top2)-poisoninduced cleavable complex formation and thereof derived dna double-strand breaks. this effect rests at least partially on rac1-mediated regulation of topoisomerase ii activity. however, the link between rac1 and top2-poisoning is only poorly understood. furthermore, it is unclear whether mitochondrial or nuclear type ii topoisomerases are the most relevant target for top2-poison-induced cytotoxicity. here, we investigated the relevance of rac1-regulated actin cytoskeleton integrity as well as mitochondrial integrity in top2-poison-induced dna damage responses as well as cytotoxicity under situation of rac1 inhibition. methods: since endothelial cells are the first barrier for any kind of systemically administered chemicals and cardiomyocytes are particular sensitive to anthracyclines, endothelial cells (h5v) as well as cardiomyocytes (h9c2) were chosen as in vitro model systems for top2-poisoning. the cells were pre-treated with rac1 inhibitors, statins or actin cytoskeleton disruptors and were subsequently treated with the topoisomerase ii poisons doxorubicin or etoposide. to compare the levels of induced dna damage, γh2ax foci quantifications as well as the comet assay were employed. actin disruption was visualized by phalloidin-fitc staining. to be able to detect relevant changes in mitochondrial mass or integrity, high doses of top2-poisons had to be used in both cell lines. changes in mitochondrial homeostasis as well as integrity were detected by the jc1-assay, mitotracker assay as well as atp-assay. additionally, pcr-and gelelectrophoresis-based methods were used for detecting mitochondrial dna damages. selected components of the dna damage response machinery as well as factors of mitochondrial homeostasis were detected by western blot. results: disruption of the integrity of the actin cytoskeleton attenuated the dna damage response to a similar extent as seen by rac1 inhibition, pointing to a role of actin filaments in the dna damage response after genotoxic insults. the actin cytoskeleton seems to participate in genotoxin-induced dna damage, -repair or in the dna damage response as reflected by reduced numbers of nuclear h2ax-foci as well as the comet assay after treatment with doxorubicin. this was not related to nuclear import or export of doxorubicin. disturbance of mitochondrial homeostasis or integrity was only detectable at high doses of topoisomerase ii poisons. this was largely unaffected by pre-treatment with statins or rac1-inhibitor. top2-poison-induced raise in mitochondrial mass was slightly enhanced by the rac1-inhibitor and statins. interestingly, inhibition of rac1 counteracted doxorubicin-induced phosphorylation of the amp-kinase in endothelial cells but not in cardiomyocytes. conclusion: mitochondrial toxicity seems to play only a minor role in top2-poisoninduced cytotoxicity in h9c2 and h5v cells. the data point to a role of rac1-regulated filamentous (nuclear?) actin in the dna repair and/or dna damage response after treatment with top2-poisons. poly(adp-ribose) polymerase 1 (parp1) and the recq helicase werner syndrome protein (wrn) are important caretakers of the genome. they physically interact with each other and are both localized in the nucleus and in particular in the nucleoli. both participate in various overlapping mechanisms of dna metabolism, in particular genotoxic stress response and dna repair [1] . previously, we and others have shown in biochemical studies that enzymatic functions of wrn are regulated by parp1 as well as by non-covalent poly(adp-ribose)-wrn interaction [2] [3] [4] . furthermore, pharmacological parp inhibition as well as a genetic parp1 ablation in hela cells alters the recruitment kinetics of wrn to sites of laser-induced dna damage [5] . here we report a novel role for parp1 and poly(adp-ribosyl)ation in the regulation of wrn's subnuclear spatial distribution upon induction of oxidative stress. we could verify previous reports that wrn is transiently released from nucleoli upon induction of oxidative stress, camptothecin (cpt) treatment, and laser-induced dna damage in a time-dependent manner. while, cpt-induced translocation appears to be a parpindependent process, our results reveal that upon h 2 o 2 -induced oxidative stress, parp1 is essential for the translocation of wrn from the nucleoli to the nucleoplasm. parp1 activity only partially contributes to wrn release from nucleoli, underlining the importance of a direct wrn-parp1 interaction for subnuclear wrn redistribution. furthermore, we identified a novel par-binding motif within the wrn sequence that is located in its rqc domain, which also harbors the binding site for parp1 and is necessary for wrn's nucleolar localization under non-stress conditions. currently, we are testing corresponding wrn mutants to analyze if this region is responsible for the parp1-dependent release of wrn from nucleoli to sites of dna damage. in conclusion, we provide novel insight into the role of parp1 in wrn's spatio-temporal regulation in the nucleus during the oxidative stress response. host-cell reactivation (hcr) is an assay used to determine dna repair capacity of cells. in its canonical layout, the test utilised a virus or a plasmid with a marker gene, inactivated by uv-damage [1, 2] . among the infected or transfected host cells types, only those with functional dna repair pathway would re-activate the damaged dna, thus providing a rationale for identification of dna repair genes in the mutant screens. an obvious advantage of hcr is that repair can be measured in cells that have not been exposed to a damaging agent. however, because of multiple variables of the damage generation, transfection and interpretation of results, the assay has been hard to harmonise and develop into a widely accepted quantitative dna repair assay. over the last years, my team has developed and validated several major improvements of the mammalian hcr assay. exploiting sequence-specific nicking endonucleases and customised design of the reporter vectors, we proposed an innovative and very efficient technique for incorporation of synthetic oligonucleotides, containing single structurally defined dna base and backbone modifications, into desired gene elements [3] . this ad hoc approach allows examination of the repair in a stand-specific manner and at single nucleotide resolution. we efficiently applied hcr in its new layout for measurement of the nucleotide excision repair of various dna adducts. moreover, we demonstrated that the enhanced hcr assay can differentiate between the transcription-coupled (tc-ner) and global genome (gg-ner) subpathways of ner [4] . we further obtained new significant insights into the lesion-specific mechanisms of base excision repair of several endogenously occurring aberrant dna bases [5 -7] and plan to adapt the assay to the detection of mismatch repair and translesion dna synthesis. in addition to the applications in the dna repair field, the enhanced hcr assay provides a tool for investigation of the dynamics and transcriptional impact of the regulatory dna bases 5methylcytosine and 5-hydroxymethylcytosine as well as their derivatives (5formylcytosine and 5-carboxycytosine a coordinated and faithful dna damage response is of central importance for maintaining genomic integrity and cell survival. transcriptional activation of dna repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress and protection against genotoxin-mediated cell death. here we show that exposure to a low dose of benzo(a)pyrene 9, , the active metabolite of benzo(a)pyrene (b[a]p), which represents the most important carcinogen formed by incomplete combustion during food preparation and smoking, causes upregulation of several dna repair genes. combined induction of the nucleotide excision repair (ner) genes ddb2, xpc, xpf and xpg enhanced repair activity and protected cells against a subsequent bpde exposure. furthermore induction of the translesion polymerase polh was also involved in protection against bpde-induced apoptosis, however led to an enhanced mutation frequency in the surviving cells. activation of these dna repair pathways was also observed upon exposure to b[a]p and in vivo in buccal cells of male individuals upon smoking, indicating that this mechanism may be involved in the formation of smoking-related cancers. altogether, we could show that low-dose bpde exposure activates a complex network of transcriptional alterations, leading to protection against cell death, at the cost of increased mutation frequency, highlighting the danger of occasional smoking. poly(adp-ribosyl)ation (parylation) is an essential posttranslational modification with the biopolymer poly(adp-ribose) (par). the reaction is catalyzed by poly(adp-ribose) polymerases (parps) and plays key roles in cellular physiology and stress response by regulating physico-chemical properties of target proteins. of the 17 members of the human parp gene family, at least four have been shown to exhibit par-forming capacity. upon dna damage parp1 is catalytically activated and is thought to contribute to the bulk of the cellular par formation. parp inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality (mangerich and bürkle 2011, mangerich and bürkle 2015) . here we generated a genetic knock out of parp1 in one of the most widely used human cell systems, i.e. hela parp1 ko cells, via talen-mediated gene targeting and characterized these cells with regards to parylation metabolism and genotoxic stress response. furthermore, by reconstituting hela parp1 ko cells with a series of artificial and natural parp1 variants, we analyzed structure-function relationships of parp1 in a cellular environment without interfering with endogenously expressed wt-parp1. we confirmed that the parp1e988k mutant exhibits mono-adp-ribosylation activity and extended previous reports by demonstrating that the parp1l713f mutant is constitutively active in a cellular environment, leading to high cellular par levels even in unchallenged cells. additionally, both mutants exhibited significantly altered recruitment and dissociation kinetics at sites of laser-induced dna-damage, which can partially be attributed to non-covalent parp1-par interaction via at least one specific par binding motif located in zinc finger 2 of parp1. expression of both artificial mutants led to distinct cellular consequences, caused by the altered cellular biochemistry. while the expression of parp1l713f itself triggered apoptosis, parp1e988k expression led to a strong g2-arrest during cell cycle and sensitized cells to camptothecin treatment. interestingly, pharmacological parp inhibition with abt888 mitigated effects of the e988k mutant, suggesting distinct functions of mono-adp-ribosylation. finally, by reconstituting parp1 ko cells with a natural cancer-associated parp1 snp variant (v762a), as well as a newly identified parp1 mutant present in a patient of pediatric colorectal carcinoma (f304l-v762a), we demonstrate, that these variants exhibit altered biochemical and cellular properties, potentially supporting carcinogenesis. together, this study establishes a novel model to study parp1-dependent parylation during genotoxic stress response and reveals new insight into the structure-function relationships of artificial as well as natural parp1 variants in a cellular environment, with implications for parp research in general. mangerich, a. and a. bürkle (2011) . "how to kill tumor cells with inhibitors of poly(adpribosyl)ation." int j cancer128 (2): 251-265. mangerich, a. and a. bürkle (2015) . multitasking roles for poly (adp-ribosyl) ation in aging and longevity. parp inhibitors for cancer therapy, springer: 125-179. leukemic cells frequently overexpress the transcription factor wilms tumor 1 (wt1) and the persistence of wt1 expression after chemotherapy indicates remaining leukemic stem cells. hydroxyurea induces replicative stress by its ability to inhibit ribonucleotide reductase, an enzyme that catalyzes the synthesis of dntps from ntps. we demonstrate that the expression levels of wt1 determines the extent of dna damage and apoptosis in a panel of leukemic cells treated with hydroxyurea. accordingly, inhibiting apoptosis through chemical inhibition of caspases or by overexpression of mitochondrial anti-apoptotic bcl proteins prevents the hydroxyurea-induced depletion of wt1 and cell death. in addition, we show that an rna interference-mediated elimination of wt1 sensitizes leukemic cells to the pro-apoptotic and dna damaging effects of hydroxyurea. furthermore, such a loss of wt1 suppresses hydroxyurea-induced erythroid differentiation. pharmacological approaches that diminish wt1 also sensitize cells to hydroxyurea. these include the tyrosine kinase inhibitor (tki) imatinib or epigenetic modifiers belonging to the histone deacetylase inhibitor (hdaci) group. thus, an inhibition of wt1 is therapeutically exploitable for a targeting approach against leukemic cells undergoing replicative stress. our novel findings reveal that wt1 is a novel biological target of hydroxyurea and they suggest that wt1 has a previously unrecognized ability to prevent dna damage when replication forks halt and eventually collapse. fret (fluorescence resonance energy transfer)-based cell assays were developed to directly monitor receptor activation and receptor-stimulated camp response. mutant ß 1 ar were generated by insertion of cyan and yellow fluorescent proteins (cfp and yfp) into the third intracellular loop and the c-terminus, respectively (bornholz et al., cardiovasc res 97:472, 2013) and stably transfected to hek 293 cells (hekß 1 -fret). to monitor the camp response the epac1-based fret sensor of camp, was stably transfected alone (hekwt-e1) and together with a moderate level of native ß 1 ar to hek293 cells (hekß 1 -e1; nikolaev et al. jacc 50: 423, 2007) . fret-activity was measured with recently developed fluorescence detectors (12 channels) equipped with fast semiconductor technology, avoiding any movable optical and mechanical parts, using 438 nm for excitation and 483/540 nm for the emmission ratio. cells were cultivated in 96-format 12-well strips, incubated in physiological hepes-buffered salt solution and treated with ßar agonists of different selectivity and affinity to determine their ßar-subtype preference. catecholamines tested in hekß 1 -fret cells exhibited ec 50 -values (-log, m) which matched k d -values (-log, m) known from native heart receptor membranes (isoprenaline, iso: 6.9±0.1, adrenaline 5.7±0.1, noradrenaline 6.2±0.1). ßar-expression levels were controlled by radioligand binding with [ 3 h]-(-)-cgp12,177 resulting in different densities of ~4x10 6 and ~1.4x10 4 receptors/cell in hekß 1 -fret and hekß 1 -e1, respectively, whereas in hekwt-e1 cells only ~1000 ß 2 ar were found. surprisingly, the low level of ßar in hekwt-e1 cells allowed the measurement of the action of ß 2 -sympathomimetics (ß 2 sym), e.g. fenoterol, thereby amplifying receptor binding (pk d~6 .7) to an effective regulation of fret activity in the presence of 0.06 mm ibmx (pec 50~8 .3±0.1), nearly matching the ~100-fold amplification of iso (pk d~6 .9; pec 50~8 .9). in order to determine ß 1 ar-mediated side effects of ß 2 sym, hekß 1 -e1 cells characterized by a 14-fold higher level of ß 1 ar over ß 2 ar were assayed for fret activity. fenoterol maximally inhibited fret activity with a pec 50~8 .4 whereas the high affinity ß 2 sym salmeterol acted a partial agonist (~75% of iso maximum, pec 50~8 .6), both compounds being rather insensitive against the highly effective ß 1 ar-blockade with 1 µm cgp 20,712 a. for that reason hekß 1 -fret cells were used characterizing fenoterol as partial agonist (~25%) whereas salmeterol activated less than 10% of maximum receptor activation by iso. thus, it has to be concluded that the low level of effectively coupled wt-ß 2 ar present in hekß 1 -e1 cells precludes the exact determination of ß 1 ar-mediated side effects of ß 2 sym, and that cfp/yfp labelled receptors have to be used for the determination of the subtype specific intrinsic activity of an agonist. they account for about one third of all drug targets. their regulation from desensitization to internalization and alternative signal transduction is largely dependent on phosphorylation of intracellular serine and threonine residues of the activated receptor. even though the β 1 -adrenoceptor is of tremendous importance in a number of diseases its phosphorylation remains poorly understood. we addressed this question in a qualitative and quantitative way. by using radioactive phosphorylation assays and mass spectrometry, we were able to elucidate the phosphorylation pattern of the human β 1 -adrenoceptor in vitro. we identified ten previously unknown phosphorylation sites in the third intracellular loop and the receptor's c-terminus. labeling hek293 cells with stable heavy isotopes (silac) lead to the discovery of a stimulation-dependent regulation of several of these phosphorylation sites. furthermore, mutagenesis studies in stably transfected hek293 cells revealed the impact of phosphorylation for arrestin binding and internalization of the receptor. fluorescence resonance energy transfer experiments with β 1 -adrenoceptor variants carrying point mutations of putative phosphorylation sites identified two c-terminal phosphosites that determine arrestin recruitment. our current goal is to further investigate the functional implications of these newly identified phosphorylation sites on downstream signal transduction, with an emphasis on the map kinase pathway. a moderate increase in arrestin affinity to the β 2 -adrenergic receptor is sufficient to induce arrestin internalization. the homologous desensitization of g-protein-coupled receptors is a two-step process. initially, g-protein-coupled receptor kinases phosphorylate agonist-occupied receptors which are subsequently bound by arrestins. in many cases, the resulting receptorarrestin complex is then internalized via clathrin-coated pits. dependent on the identity of the receptor and the ligand, the complex between receptor and arrestin may exist only in the proximity of the plasma membrane or internalize into the cell interior. we constructed mutants of the β 2 -adrenergic receptor carrying three additional serine residues in various positions at the c-terminal tail. one of these mutants which carried the serine residues in close proximity to the endogenous grk phosphorylation sites (β 2 ar-sss) showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. the affinity of arrestin-3 to the receptor was measured by fluorescence resonance energy transfer (fret) between the receptor and arrestin-3 and by two-color fluorescence recovery after photobleaching (frap). in the fret assay, arrestin-3 dissociation from the β 2 ar-sss receptor upon agonist washout was prolonged approximately two-fold compared to the wild-type receptor. frap was performed with an n-terminally tagged receptor immobilized with an antibody against the n-terminal tag either in solution or on a micropatterned surface. in these assays, the recovery of arrestin-3 into the bleached region was prolonged between two-and fourfold for the β 2 ar-sss receptor compared to the wild-type. even though this two-to fourfold increase in affinity seemed rather modest, it resulted in the trafficking of receptorarrestin complexes to the early endosome whereas the wild-type receptor interacted only transiently with arrestin at the plasma membrane. furthermore, the increased affinity of arrestin led to more efficient internalization of the β 2 ar-sss compared to the wild-type receptor. however, recycling to the plasma membrane after agonist washout was very similar for both receptors. we conclude that even a modest change in affinity between a g-protein-coupled receptor and arrestin can lead to substantial alterations in arrestin trafficking which in turn may have effects on cellular signaling. despite recent structural research allow for better understanding of gpcr structure, the crucial aspects of the selectivity mechanism of receptor -g protein subtype coupling remain unresolved. based on the hypothesis that the affinity of the ternary complex (agonist/gpcr/g-protein) in the nucleotide-free state determines the selectivity of gpcr-g protein coupling, we set out to measure gpcr-g protein interaction in membranes of single cells. in order to quantify the affinity of gα-subunit towards gpcrs in single cell, we determined the lifetime of the receptor-g protein complex in living cells upon agonist withdrawal under conditions of gtp-depletion. therefore, we utilized förster resonance energy transfer (fret) based assays to study interactions between fluorescent muscarinic receptors and heterotrimeric g proteins in single permeabilized hek293t cells transfected with the appropriate cdnas. here we focused on muscarinic m1-, m2-, and m3-receptors and characterized the kinetics of agonist-induced binding of go/i -and gq/11-proteins to muscarinic receptors and their subsequent dissociation in the absence of nucleotides. as a measure of affinity we calculated the rate constant of g protein dissociation from the receptor after agonist withdrawal. the dissociation kinetics of go protein from m3-and m1-achrs was found to be 10-fold faster in comparison to gq. similarly, we observed a 15-fold right shift of the concentration-response curves of go proteins binding to m3-achr in comparison to gq. in order to ensure, that the affinity of the ternary complex correlates with the efficiency of g protein activation, we performed experiments on the g protein activity in intact cells expressing non-fluorescent m3-achr by using a fret-based assay. our results showed that gq activation required 10-fold lower agonist concentration compared to go activation, suggesting that indeed the stability of the ternary complex in the absence of nucleotides determines the selectivity of gpcr-g protein coupling. we further explored the subtype selectivity of m2-achr for gi family members by comparison of dissociation kinetics of gi1-, gi2, gi3-, and go-proteins from m2-achr under nucleotide depleted conditions. k off of gi1 and gi3 were found to be two-fold higher in comparison to gi2 and go proteins, indicating the higher affinity of the latter ones to m2-achr. our fret-based assay to study receptor-g-protein interactions in membranes of single cells has been proven to be a fast and reliable method to quantify the affinity of the ternary complex. the g protein subtype dependent differences in the affinity towards activated receptors correlate with the g protein coupling efficiency of this receptor. despite their tremendous pharmacological relevance and potential for the development of new drugs, our understanding of g protein-coupled receptor (gpcr) architecture and signaling mechanisms are still limited. major reasons for this are the low abundance and poor biophysical properties of gpcrs, which makes them one of the most challenging class of proteins for structural and biophysical studies. among the superfamily of gpcrs, the class b receptors comprising 15 receptors are structurally least understood because to date it has not been possible to obtain a crystal structure of this receptor class. to overcome these limitations, we have developed a method for improving functional expression and simultaneous thermo-stabilization of gpcrs by directed evolution which is based on expression of receptors in saccharomyces cerevisiae and subsequent selection of highly expressing variants by flow cytometry with fluorescent ligands. by this strategy, key residues within a receptor sequence can be rapidly identified that are responsible for improved biophysical properties without greatly affecting the pharmacological features of the receptor. we have now applied this method to the human parathyroid hormone 1 receptor, a member of the class b of gpcrs which is a major regulator of calcium homeostasis in the body and a key target for the treatment of osteoporosis. from two rounds of directed evolution in yeast we obtained several mutants of parathyroid hormone 1 receptor that exhibit strongly improved expression levels and that remain stable after solubilization in detergents. these receptor variants are ideal candidates for subsequent structural and biophysical analysis. opioid drugs exert nearly all of their clinically relevant actions through stimulation of mors (μ-opioid receptors). the molecular biology of endogenous opioid peptides and their cognate receptors has been studied extensively in vitro. for mor, signaling efficiency is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. morphine induces a selective phosphorylation of serine 375 that is predominantly catalyzed by g protein-coupled receptor kinase 5. as a consequence, the selective morphine-induced s375 phosphorylation does not lead to a robust beta-arrestin mobilization and receptor internalization. by contrast, high-efficacy opioid agonists such as fentanyl or etonitazene not only induce phosphorylation of s375 but also drive higher order phosphorylation on the flanking residues threonine 370, threonine 376, and threonine 379 in a hierarchical phosphorylation cascade that specifically requires grk2 and grk3 isoforms. as a consequence, multisite phosphorylation induced by potent agonist promotes both betaarrestin mobilization and a robust receptor internalization. however, little is known about agonist-selective phosphorylation patterns in vivo after acute and chronic drug administration. to learn more about mor regulation in vivo we have generated a new μopioid receptor knock in mouse with an n-terminal ha-tag. using these mice, we were able to study in vivo phosphorylation of an endogenous g protein-coupled receptor using both mass spectrometry and phosphosite-specific antibodies. we were also able to address the question which of the many putative mor splice variants detected on the mrna level are indeed expressed as functional receptors in mouse brain. ion channels 061 hcn4 in thalamic relay neurons is necessary for oscillatory activity in the thalamocortical system institut für physiologie i, westfälische wilhelms-universität, münster, germany hcn channels underlie the i h current and are involved, among other functions, in the genesis of epilepsy. the significance of hcn1 and hcn2 isoforms for brain function and epilepsy has been demonstrated, however the role of hcn4, the third major neuronal hcn subunit, is not known. here we show an unexpected role of hcn4 in controlling oscillations in the thalamocortical network. hcn4 is predominantly expressed in several thalamic relay nuclei, but not in the thalamic reticular nucleus and the cerebral cortex. hcn4-deficient thalamocortical relay neurons showed a massive reduction of i h and strongly reduced intrinsic burst firing. evoked thalamic oscillations in a slice preparation were completely abolished. in vivo, brain-specific hcn4 null mutants were protected against induced spike-and-wave discharges (swd), the hallmark of absence seizures. our findings indicate that hcn4 is necessary for rhythmic intrathalamic oscillations and that the channels constitutes an important component of swd generation. ludwig-maximilians-universität, walther-straub-institut für pharmakologie und toxikologie, münchen, germany trpc4 and 5 channels are members of the classical transient receptor potential (trpc) family whose activation mechanism downstream of phospholipase c (plc) largely remained elusive until now. while trpc3/6/7 channels are directly activated by diacylglycerol (dag), trpc4 and 5 channels are commonly regarded as daginsensitive. in contrast to trpc3/6/7 channels, they contain a c-terminal pdz-binding motif allowing for binding of na + /h + exchanger regulatory factor (nherf) 1 and 2. interestingly, performing electrophysiological measurements, co-immunoprecipitations and intermolecular dynamic fret experiments, we found that dissociation of nherf proteins from the c-terminus of trpc5 confers dag-sensitivity on trpc5 channels. trpc5 channels were dag-sensitive under the following experimental conditions: inhibition of protein kinase c, amino acid exchange in the c-terminal pdz-binding motif, pip 2 depletion with and without involvement of plc, over-expression of g-protein coupled receptors, down-regulation of endogenous nherf1 and 2 proteins and overexpression of a nherf1 mutant incapable of trpc5 binding. these findings strongly argue for nherf proteins as molecular determinants for channel activation. interestingly, pip 2 depletion itself caused slight trpc5 current increases while during pip 2 depletion, the membrane permeable dag analogue oag evoked even higher trpc5 currents suggesting that pip 2 depletion induces an active and dag-sensitive channel conformation. receptor mediated pip 2 depletion also resulted in dissociation of nherf1 and 2 from the c-terminus of trpc5 thereby eliciting a dag-sensitive trpc5 channel conformation. thus, our findings suggest that dag-sensitivity of trpc5 is the result of an activation cascade starting with pip 2 depletion and subsequent dynamic dissociation of nherf1 and 2 from the c-terminus of trpc5. altogether, dagsensitivity is a unifying functional hallmark of all trpc channels. the melastatin-related transient receptor potential channel trpm3 is a heat-activated nonselective cation channel expressed in sensory neurons of dorsal root ganglia. since trpm3-deficient mice show impaired inflammatory thermal hyperalgesia, the pharmacological inhibition of trpm3 may exert antinociceptive properties. fluorometric ca 2+ assays and a compound library containing approved drugs were used to identify trpm3 inhibitors and to characterize their potency and selectivity. biophysical properties of the block were assessed using electrophysiological patch-clamp methods. microfluorometry in fura-2-loaded single cells was applied to monitor [ca 2+ ] i signals in isolated dorsal root ganglion (drg) neurons. analgesic effects were assessed applying pregnenolone sulfate (pregs)-induced chemical pain and heat stimuli at mice. in the screening approach using stably transfected hek trpm3 cells we identified the nonsteroidal anti-inflammatory drug (nsaid) diclofenac, the tetracyclic antidepressant maprotiline and the anticonvulsant primidone as highly efficient trpm3 inhibitors. the compounds exhibited half-maximal inhibitory concentrations of 0.6-6 µm. the selectivity profiles of maprotiline and primidone for trpm3 were promising with no inhibitory effects on trpm2, trpm8, trpa1, trpv1, trpc5, trpc6 and p2x7 receptor channels. primidone inhibited pregs-induced [ca 2+ ] i signals in rat drg neurones, indicating a block of native trpm3 channels. consistently, primidone attenuated nocifensive responses of mice to paw-injected pregs. furthermore, intraplantar primidone reduced nociception in healthy and hyperalgesic cfa-inflamed paws in the hot plate test. the finding that an approved drug can inhibit trpm3 at concentrations that may be therapeutically relevant and thereby can act as an analgesic, provides a method to study trpm3-related effects by acutely challenging the channel´s function. pharmacological interference with trpm3 applying an approved drug or optimised successor compounds may pave the way to better understanding of physiological functions of trpm3 in humans and may represent a novel concept for analgesic treatment. excitotoxicity, calcium deregulation, mitochondrial dysfunction and neuroinflammation contribute to progressive cell death in many neurodegenerative diseases. therefore, proteins that prevent deregulation of these pathways are considered as drug targets. potential therapeutic approaches may benefit from modulation of small-conductance calcium-activated potassium (sk) channels, since recent data supports the hypothesis that sk channel activity promotes neuronal survival against cellular stress via a dual mechanism of action: i) by controlling neuronal excitability and ii) by preventing mitochondrial dysfunction and inflammation. our previous studies showed that activation of sk channels in neurons exerted protective effects through inhibition of nmdarmediated excitotoxicity. further, we revealed recently that in a model of glutamate oxytosis, activation of sk channels attenuated mitochondrial fission, prevented the release of pro-apoptotic mitochondrial proteins, and reduced cell death. however, little is known about the function of sk channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. in this study, we addressed the question whether sk channel activation affected primary mouse microglia activation upon lps and α-synuclein challenge. we found that activation of sk channels significantly reduced activation of microglia in a concentration-dependent manner, as detected by real-time xcelligence cell impedance measurements. further data on cytokine (tnf-alpha and il-6) analysis revealed that activation of sk channels attenuated α-synuclein-induced cytokine release. inhibition of glycolysis prevented microglial activation and cytokine release. although sk channel activation slightly reduced atp levels, it attenuated α-synuclein-induced no release. furthermore, glycolytic products and ampk signaling were evaluated. overall, our findings show that activation of sk channels attenuates microglial cell activation. thus, sk channels are promising therapeutic targets for neurodegenerative disorders, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease. mutant of the residue pair e103/k308 can be crosslinked efficiently in both states, the closed-and open state of the p2x2r. interestingly, oxidative crosslinking of cysteine substitution mutants of each individual residue pair significantly reduced the atpinduced current amplitudes. charge reversal or swapping mutagenesis and cysteine modification by charged mts-reagents indicated the electrostatic nature of the pairwise interactions in these four residue pairs. furthermore, preliminary data from triple, tetra and penta mutant cycle analysis indicated energetic coupling between the residue pairs e84/r290, e103/k308, e167/r290 and e167/k308 and thus indicates the cooperative interaction in a larger salt bridge network. together with the markedly reduced current amplitudes following disulfide crosslinking, our data suggest that the salt bridge network serves to stabilize the closed-state conformation of the p2x2r. the comparison of the closed-state and open-state model of the rat p2x2r showed that atp promotes a marked rearrangement of the side chains of the residues r290 and k308 to enable the strong ionic coordination of the γ-phosphate oxygen of atp. in summary, our data are in line with the concept that the electrostatic interaction of r290 and k308 with atp competitively releases e84, e103 and e167 from their strong electrostatic coupling and thus initiates a destabilization of the closed-state, which favors channel opening. fig. 1 in a similarity search using sequence motifs conserved amongst various members of the trp protein family we identified three non-annotated putative membrane proteins that we initially termed tmem1, tmem2 and tmem4. expression analysis using the nanostring ncounter system, northern blotting and rt-pcr showed that murine tmem2 is expressed in various tissues including heart, brain, lung, endothelium, colon, cardiac myocytes, cardiac fibroblasts, embryonic fibroblasts, mast cells and pancreatic acinar cells. hydropathy analysis predicts that tmem2 proteins exhibit 6 to 10 plasma-membrane spanning domains, but fluorescently labeled tmem2 fusion constructs expressed in mouse embryonic fibroblasts revealed a vesicular subcellular localization pattern. in contrast to the prediction by the psort ii algorithm, tmem2-eyfp could not be identified in the plasma membrane of fibroblasts, cardiac myocytes, mast cells or pancreatic acinar cells but showed a significant colocalization with markers and fusion constructs specific for acidic compartments including lysosomes. in tmem2 -/mice, a marked elevation of amylase and lipase plasma levels was observed. we found that constitutive but not stimulated amylase secretion from tmem2-deficient acinar cells is elevated indicating a cell autonomous defect. calcium (ca ++ ) is an important signaling molecule regulating stimulated as well as constitutive secretion from pancreatic acinar cells. microfluorimetric measurements using fura-2 or indo-1 indicate higher resting ca ++ concentrations in tmem2-/-pancreatic acinar cells correlating with elevated basal enzyme secretion. in tmem2-yfp-knock-add-on mice we identified tmem2 in organelles of the apical acinar cell pole and a partial colocalisation with lamp2 proteins. furthermore, largely increased elevations in cytoplasmic ca ++ concentration were observed upon osmotic lysis of lysosomes triggered by gly-phe β-naphthylamide (gpn) or by nh 4 cl application. the role of tmem2 for ca ++ release was evaluated by stimulation with low concentration of cholecystokinin 2pm) in the absence of extracellular ca ++ using both microfluorimetric recording of cytosolic ca ++ transients as well as electrophysiological recordings of ca ++ -activated chloride currents. these measurements revealed a higher frequency of intracellular ca ++ oscillations and a larger area under the curve of ca ++ activated chloride currents upon cck-8 stimulation indicating that tmem2 inactivation leads to an enhancement of the globalization of cck-8 evoked ca2+ release from intracellular organelles. taken together, our study identifies tmem2 as a novel regulator of ca ++ release from intracellular organelles including endo-lysosomes and as a critical determinant of constitutive protein secretion in pancreatic acinar cells. while generally highlighting toxicology as a translational science that requires academic anchoring, gundert-remy and co-workers [1] have called for efforts to improve the relevance of in vitro methodologies in predicting in vivo effects. against this background, the german society of toxicology working group on alternative approaches to animal testing proposes specific quality criteria (qc) for in vitro methods and for research work using in vitro methods. these qc may serve to evaluate in vitro methods that are developed or applied in-house or that are described in work plans, peer reviewed articles, etc. for the time being, the qc focus on in vitro cell or tissue culture methods that address human health endpoints in the context of substance-related regulatory toxicity testing. nevertheless, these qc are also generally applicable to in vitro research conducted for other toxicological purposes. relevant work from, e.g., the organisation for the economic co-operation and development has been taken into account in specifying the qc that cover the following aspects:  the 3rs impact of an in vitro method in replacing, reducing (and refining) a specific animal test for a specific toxicological endpoint. this aspect also includes scientific hurdles that, in the past, had impeded the successful development of in vitro methods for the given toxicological endpoint.  scientific relevance and reliability, i.e. which fundamental requirements should an in vitro method meet to ensure that its results are relevant and reliable.  practicability and applicability, i.e. what is the expected expenditure for the in vitro method, and have relevant authorities and industrial sectors been involved in the development of the in vitro method. qc related to the scientific relevance of research work using a specific in vitro method provide a tool to justify, e.g., the suitability of the selected test system and in vitro endpoint(s) for the given purpose; the selection of test substances, positive and negative controls; the setting and control of test concentrations; and the definition of acceptance criteria to determine the relevance of test results. the proposed qc may serve as a framework to assess the relevance of in vitro methods and in vitro research work. thereby, they aim at improving in vitro predictivity of in vivo toxicological effects, which in return contributes to reducing and replacing the need for animal testing. [1] gundert-remy, u. et al. (2015) . toxicology: a discipline in need of academic anchoring -the point of view of the german society of toxicology. arch toxicol 89: 1881-93. during the past decades, considerable progress has been made in implementing 3r approaches in routine safety assessment. in spite of these achievements, animal tests still need to be conducted if legal requirements prescribe in vivo tests or if no reliable, accepted alternative method exists. efforts to foster 3r approaches and make them 'ready for use' focus on three levels: development and validation of scientific methods/strategies, regulatory acceptance of acknowledged approaches and global harmonization of standards. strong cooperation between toxicological experts from scientific bodies, national and international authorities and industry is needed to advance on all three levels for the benefit of animal welfare. 3r approaches that have already been implemented in routine safety assessment of consumer goods do not focus solely on replacement of animal testing by use of accepted alternative methods. in cases where reliable alternative approaches are not yet available, reduction in animal numbers and refinement of testing procedures can be achieved on a case-by-case basis. a tailor-made, tiered testing strategy is usually pursued that involves knowledge on specific characteristics of the test item and makes use of all available data, including details on exposure and results obtained with structural homologues. hurdles to apply alternative approaches can even occur for established methods. as legislations give different priority to alternative approaches, it remains challenging to fulfil conflicting legal requirements in different regions of the world, or even to address horizontal legislations of the same region. furthermore, successfully validated and legally implemented alternative approaches might not always provide the safety assessor with meaningful test results. with gaining experience, limitations of test systems can become evident that affect for example the applicability domain of the method, as has been the case both for some in vitro and in vivo methods. in these cases, the new information needs to be shared not only among safety assessors, but also with method developers and regulators to facilitate refinement of scientific approaches and/or amendments of regulations. leibniz-institut für arbeitsforschung (ifado), vistox, dortmund, germany two-photon microscopy facilitates imaging of biological processes in vivo. establishing this recent technique in mouse liver allowed us to record in a real-time the sequence of events during acetaminophen (apap) induced-liver damage. although apap is intensively studied and described in vivo imaging revealed so far unknown scenarios of cell death. the hepatocytes close to the central vein of a liver lobule went within hours into cell death as commonly described due to the toxic metabolite napqi. surprisingly, we observed a distinct way of cell killing at the outer border of the dead cell area which is accompanied by bile acid decompartmentalization. there, within an hour after apap administration dilatation of bile canaliculi was observed. subsequently, bile acids containing invaginations arouse from the apical side of a hepatocyte into the cytosol. these invaginations ballooned until the bile leaked into the hepatocyte volume and subsequently the plasma membrane of the affected hepatocytes lost its integrity leading to cell death. this mechanism emerged in an environment for hepatocytes where moderate napqi levels meet intracellular high bile salt concentrations of the midzonal region. in conclusion, establishing in vivo imaging in mouse liver enabled us to identify new cellular mechanisms which cannot be discovered by conventional methods. universitätsklinikum düsseldorf, institut für toxikologie, düsseldorf, germany introduction: lung inflammation and fibrosis are considered as major toxicities after thoracic cancer radiotherapy. up to now effective pharmacological interventions for normal tissue protection are largely missing. hmg-coa reductase inhibitors (statins), which are used in the clinic for lipid-lowering purpose, are reported to have multiple inhibitory effects on genotoxic stress responses. for this reason we aim to investigate the usefulness of statins to protect normal lung cells in vitro and lung tissue in vivo from damage provoked by ionizing radiation (ir). methods: according to clinically relevant anticancer radiation regimens, we used fractionated irradiation schemes (4 x 4 gy) for both in vitro as well as in vivo experiments. we analyzed the effect of lovastatin on ir-induced dna damage formation and repair, dna damage response (ddr) and cell death in non-proliferating human lung fibroblasts, epithelial as well as endothelial cells. furthermore, we established an irradiation device that is useful to selectively irradiate the right lung of mice and investigated the influence of lovastatin on lung damage following fractionated and selective irradiation of the lung in vivo (balb/c mice). results: compared to lung fibroblasts and epithelial cells, endothelial cells exhibited the highest radiosensitivity and underwent ir-induced apoptosis which was partly prevented by lovastatin. by contrast fibroblasts and epithelial cells did not undergo apoptosis upon irradiation. lovastatin did not affect initial dna damage formation in any of these cells. in all three lung cell types lovastatin enhanced the repair of dna double-strand breaks as analyzed 24 h after the last irradiation by γh2ax nuclear foci formation. depending on the cell type lovastatin affected various components of the ddr machinery in vitro. in vivo, lovastatin prevented ir-mediated increase in breathing frequency as determined two and four weeks after fractionated irradiation. moreover, statin treatment attenuated the level of residual dna damage and ir-induced apoptosis as analyzed four weeks after irradiation. these results were mimicked when eht1864, a small molecule inhibitor of the small rho-gtpase rac1, was applied in vivo, pointing to an involvement of rac1 in statin-mediated radioprotective effects. conclusion: bearing in mind that statins are well tolerated in humans, we suggest the application of statins as a promising pharmacological strategy for the prevention of irradiation-induced damage of the lung. targeted genome engineering by crispr/cas9 is an evolving tool for generating specific knockout cell lines. co-expression of crispr/cas9 allows for efficient dna cleavage and introduction of so called indel mutations (insertion/deletion point mutations) that lead to either misfolded non-functional proteins or complete knockout. we exploited this tool to generate a bid (bh3-interacting domain death agonist) knockout cell line in neuronal ht-22 cells. bid has been shown to be involved in regulated cell death pathways like oxytosis where its activation mediates mitochondrial demise, subsequent release of apoptosis inducing factor (aif) and cell death. in the cell death model of oxytosis the cystine/glutamate antiporter (x c -) is inhibited by high extracellular glutamate concentrations. following events such as increasing lipid peroxidation and ros production resemble major characteristics of another emerging cell death pathway, called ferroptosis. in this study we generated a bid crispr/cas9-knockout cell line to elucidate the role of bid as a potential link of oxytosis and ferroptosis in the ht-22 cell line. in order to investigate the potential mechanistic overlap at the level of mitochondrial death pathways, we induced oxytosis with glutamate or ferroptosis with erastin in wild-type cells and analyzed the respective effects of the well-established inhibitors ferrostatin-1 and the bid inhibitor bi-6c9 on cell death and mitochondrial paradigms. these results were then compared to the effects of glutamate or erastin in crispr/cas9-bid-knockout cells. bi-6c9 inhibited glutamate-induced morphological changes of ht-22 cells and also prevented cell death as assessed using the mtt assay and annexin v/pi staining. similar results were observed with ferrostatin-1 in the model of erastin-induced ferroptosis. subsequent facs analysis of lipid peroxidation by bodipy staining demonstrated that bi-6c9 abolishes lipid peroxide formation in the erastin model and ferrostatin-1 in the model of oxytosis. facs analysis was further employed for the detection of mitochondrial ros formation. mitosox staining revealed a significantly decreased production of mitochondrial ros by bi-6c9 and ferrostatin-1 in the respective model systems. investigating the crispr/cas9-bid-knockout ht-22 cell line revealed that bid knockout prevented cell death, lipid peroxidation and mitochondrial toxicity in both model systems of cell death, oxytosis and ferroptosis. in conclusion, the present study exposes bid as a pivotal molecular link between the previously separated cell death pathways oxytosis and ferroptosis at the level of mitochondria. parkinson's disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. however, the molecular mechanisms underlying alphasynuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. the goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. therefore, we have generated neuron specific, adeno associated virus type 2 (aav2) expressing cytosolic as well as mitochondrial targeted alpha synuclein and egfp expressing viruses used as respective controls. overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular atp, enhanced mitochondrial ros production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. in addition, real-time analysis of mitochondrial bioenergetics using the seahorse bioscience system following aav infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. in addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. in addition, we found that inhibition of caspases by the broad spectrum caspase inhibitor qvd significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. interestingly, inhibition of caspases preserved neuronal network integrity, atp levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity at the level of mitochondria. thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in parkinson's syndromes that are associated with alpha synuclein toxicity. degradation of and adverse effects caused by tattoo and permanent make-up pigments upon sunlight exposure and laser removal have been occasionally reported in the last decades. until now, only the ban of certain azo-pigments has been addressed in the national legislation. the regulation was based on a number of studies showing the cleavage of azo-bonds by ultra violet light and laser-irradiation leading to the formation of carcinogenic aromatic amines. as a result, especially german tattoo ink manufactures switched to the use of more light-fast polycyclic pigments assuming these would be safer for this kind of application when compared to azo-pigments. to assess the potential risks of polycyclic pigments in terms of decomposition in the skin, we compared the photochemical cleavage of the widely used azo-pigment orange 13 and the polycyclic pigment copper phthalocyanine blue. main decomposition products are qualitatively and quantitatively analyzed after q-switched laser irradiation of 1 mg/ml aqueous suspensions and tattooed pig skin. irradiated specimen were extracted with ethyl acetate and analyzed with gas chromatography coupled to mass spectrometric detection (gc/ms) using liquid injection and head-space sampling techniques. we were able to confirm the cleavage of pigment orange 13 at the azo-and other weak bonds in our experimental set-up (fig.1a) . amongst other substances, the carcinogens aniline (max. conc. 1.01 ± 0.12 µg/ml) and 3,3-dichlorobenzidine (max. conc. 0.88 ± 0.18 µg/ml) are formed. despite the lack of such weak bonds, the highly stable porphyrin-like structure of copper phthalocyanine blue is as well decomposed upon laser-irradiation (fig. 1b) . here, 1,2-benzenedicarbonitrile (max. conc. 1.11 ± 0.12 µg/ml) were found as the main decomposition product in all experimental setups. concentrations of cleavage products were generally higher in aqueous suspensions compared to pig skin extracts with both pigments. additionally, the highly toxic gas hydrogen cyanide (max. conc. 35.8 ± 4.32 µg/ml) and the human carcinogen benzene (max. conc. 0.19 ± 0.06 µg/ml) were formed from both pigments, dependent on the laser wavelengths used. cyanide levels of ≥50 µg/ml evolving upon ruby laser irradiation of >1.5 mg/ml aqueous suspensions of phthalocyanine blue were proven to significantly reduce cell viability in human skin cells in vitro. reference 1 schreiver, i., hutzler, c., laux, p., berlien, h. p. & luch, a. formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. sci rep 5, 12915 (2015) . understanding the interactions between nanoscaled objects and living cells is of great importance for risk assessment, due to rising application of nanomaterials in foodrelated products. several studies show that silver nanoparticles can reach the intestinal epithelia in nanoform in a human in vitro digestion model. nevertheless, only sparse data concerning the direct quantification of cellular uptake of silver nanoparticles are available. therefore, this study was focused on a systematical quantitative comparison of the cellular uptake of differently coated silver nanoparticles of comparable size. intracellular uptake was determined quantitatively via a transwell tm -system with subsequent elemental analysis (aas) and ion beam microscopy (ibm). silver nanoparticles were coated with poly (acrylic acid) and polyvinylpyrrolidone and characterized extensively by tem, dls, saxs, zetasizer and nanosight. agpure tm as a widely used reference nanoparticle coated with tween 20 and tagat to v was also used for comparison. different intestinal cell models were applied to get closer to the complex in vivo situation: beside the widely used caco-2 model we also investigated particle uptake in a model which considered the enterocyte-covering mucus layer, as well as in a model specialized on particle uptake, the so-called m-cell model. our findings suggest that silver uptake is clearly a particle-and not an ion-related effect. the internalization of silver nanoparticles was enhanced in uptake-specialized m-cells, although no enhanced transport through the cells was observable. furthermore, the mucus did not providing a substantial additional barrier for nanoparticle internalization. rutherford backscattering spectrometry (rbs) via ibm allowed distinguishing between adsorbed an internalized material and the results were in accordance with the transwell tm -data. additionally, ibm investigations via particle-induced x-ray emission (pixe) showed intracellular association of silver with sulfur. the quantification of silver nanoparticle internalization revealed a clear particle-specific and a coating-related uptake. furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation. analyzing iron oxide nanoparticles for drug delivery -innovative investigation tools for nanotoxicology nanoparticles offer promising new possibilities for medical applications including therapy and diagnosis of various diseases. especially nanoparticle systems with magnetic cores provide a broad application spectrum as contrast agents, magnetic transporters, or heat carriers in hyperthermia treatment. for bench to bedside translation of superparamagnetic iron oxide nanoparticles (spions) for medical applications, safety issues have to be clarified. for that, reliable standards must be established on the basis of comprehensively validated physicochemical and biological characterization methods. spions consisting of maghemite and magnetite are usually of brown or black color. due to these special properties, spions and other metal oxide nanoparticles are prone to interfere with classical toxicological assays relying on optical detection of colorimetric, fluorescence or luminescence signals. particularly, nanoparticle concentration and cellular uptake are further influencing factors. consequently, for reliable analysis of nanoparticle mediated effects, alternative robust and interference-free readouts have to be established. based on long lasting experience working with spions, we suggest a combination of complementary methods to analyse nanoparticle-mediated effects: multiparameter analyses in flow cytometry deliver statistically relevant data and link uptake of nanoparticles (side scatter increase) with cellular effects in a high-content style. combination of noninvasive, label-free impedance measurements (xcelligence system) with real-time (fluorescence) microscopy enables us to monitor cellular proliferation and morphology over several days without interference by nanoparticles. additional experiments in multicellular tumor spheroids provide information about tissue infiltration and thus, more closely resemble the in vivo situation. using those complementary methods, several drug-loaded spion systems dedicated for medical applications have been successfully characterized previously. in sum, nanotoxicology is a complex and interdisciplinary challenge, where physicochemical parameters, as well as in vitro and in vivo behavior of nanoparticles have to be considered. to address these basic requirements, we are working on a stringent standardized road of characterization for iron oxide nanoparticles synthesized for medical applications. reference: lyer s, tietze r, unterweger h, zaloga j, singh r, matuszak j, poettler m, friedrich rp, duerr s, cicha i, janko c, alexiou c. nanomedical innovation: the seon concept for an improved cancer therapy with magnetic nanoparticles. nanomedicine (lond). 2015; 10 (21) acrylamide (aa) is an α,β-unsaturated compound, which is categorized as probably carcinogenic to humans [1, 2] . aa is known to arise in foods by heat treatment in the course of the maillard reaction between reducing sugars and amino acids at processing temperatures > 120 °c [3] . dietary aa exposure has mainly been estimated on the basis of dietary recall, assessing consumption of foods with known aa contents. the use of human biomarkers of aa exposure, primarily haemoglobin adducts of aa and its genotoxic metabolite, glycidamide ( ga) in red blood cells, as well as mercapturic acids excreted in the urine, is a promising alternative. such biomarkers are to be validated by exact measurement of aa uptake in duplicates of food as consumed (duplicate diet studies) [4] . we here present results of a nine-day human intervention study with 14 healthy male volunteers. aa contents were determined in duplicates of servings as consumed and kinetics of aa-associated mercapturic acids (aama and gama) monitored in total urine [5] . the study design included washout periods with an aa-minimized diet (21 -41 ng /kg bw), a low aa intake day (0.6 -0.8 µg /kg bw) as well as a high aa intake day (1.3 -1.8 µg /kg bw). after a three-day washout period an aama baseline level of 93 ± 31 nmol/d was determined. low aa intake led to an aama excretion within 24 h of 225 ± 37 nmol/d, high intake to 404 ± 78 nmol/d corresponding to an aama excretion rate of about 30% of the ingested aa dose within 24 h, whereas aama output within 72 h corresponded to 58% of the respective aa intake, the aama baseline after 3 days washout corresponds to a net exposure level of 0.2 -0.3 μg aa/kg bw/d. whether this represents a true baseline level is to be clarified in a follow-up study. in summary, this study provides important quantitative information on kinetics of urinary short-term exposure biomarkers validated by analytically verified dietary aa intake at present day food contamination levels. [1] deutsche forschungsgemeinschaft (dfg), mak-und bat-werte-liste 2013 , 2013 doi: 10.1002 iarc, iarc monographs on the evaluations of carcinogenic risks to humans 1994, 60. [3] tareke et al., j. agric. food chem. 2002, 50, 4998-5006. [4] efsa panel on contaminants in the food chain (contam), efsa journal 2015; 13(6):4104 [321 pp.] . [5] ruenz et al., arch. toxicol. 2015 doi: 10.1007 /s00204-015-1494 heinrich-heine-universität, institut für toxikologie, düsseldorf, germany objective: flavonoids are known to modulate distinct signaling pathways thereby causing different physiological effects. effects of the flavonoids baicalein and myricetin as well as several methylated derivatives were analyzed in the nematode caenorhabditis elegans and in in hct116 colon carcinoma cells and to get insights in molecular mechanisms modulated by these compounds. methods: radical-scavenging activity (teac, dcf), stress resistance (sytox, sodium arsenite), modulation of signaling pathways (nrf2/skn-1, daf16), life span. results: baicalein enhances the resistance of c. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode (median life span: + 57%). using rna interference we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on skn-1 (homolog to mammalian nrf2), but not daf-16 (homolog to mammalian foxo), another pivotal transcription factor. negletein was the only methylated derivative which was able to enhance the life span of the nematode. in hct116 cells, baicalein activates nrf2; the methylated derivatives oroxylin a and negletein showed a comparable redox-active potential in these cells, but only negletein was able to activate nrf2. the dietary flavonoid myricetin as well as the methylated derivatives laricitrin, syringetin and myricetintrimethylether strongly enhance life span of c. elegans, decreased oxidative stress (dcf) and accumulation of lipofuscin. in contrast to myricetin, the methylated compounds strongly enhanced the resistance against thermal stress. furthermore, treatment with the derivatives induced a much stronger nuclear localization of the daf-16 transcription factor. conclusion: baicalein increases stress-resistance and life span in c. elegans via skn-1 but not daf-16. experiments with methylated baicalein derivatives suggest that the redox-active potential has a minor impact on the nrf2/skn-1 activation since only distinct derivates activate this pathway. in case of myricetin, the methylation increases the stress resistance of the flavonoid. methylation seems to enhance the biofunctionality of the flavonoids. our results may be useful to understand molecular mechanisms of flavonoids and methylated derivatives used as food supplements or pharmacological extracts. the loss of progesterone during menopause is linked to common sleep complaints of the affected women. consequently, a previous study of our laboratory demonstrated sleep promoting effects of oral progesterone replacement in postmenopausal women [1] . the oral administration of progesterone, however, is compromised by individual differences in bioavailability and metabolism of the steroid. we therefore investigated the sleep-eeg effects after intranasal application of progesterone in 12 healthy postmenopausal women (50-70 yrs).in a randomized doubleblind protocol each subject received four treatments, 2 doses of intranasal progesterone (4.5mg mpp22; 9.0 mg mpp22), 10mg of zolpidem and placebo. the 4 conditions consisted of 2 experimental nights (adaptation + examination) separated by at least one week. during each examination sleep eeg was recorded from 23:00 to 07:00. simultaneously blood was collected every 20 min between 22:00 and 07:00 by long catheter for later analysis of the hormones growth hormone (gh), cortisol, melatonin and progesterone. conventional sleep-eeg was statistically evaluated by multivariate analyses of variances (manovas) with repeated measures designs after removal of two outliers, which showed a low sleep efficiency index (sei) after 4.5 and 9.0mg mpp22. univariate f-tests in the manovas pointed to the following results (significant p-values at α=0.05). sei was higher after zolpidem than after the other three treatments. after 9.0mg mpp22 sei was elevated significantly in comparison to placebo. subjects spent more time in nonrem sleep and less time in intermittent wakefulness after 9.0mg mpp22 and after zolpidem than after placebo. total sleep time was elevated and wake after sleep onset (waso) was reduced after 9.0mg mpp22 and after zolpidem. after all active treatments with mpp22 and zolpidem the time spent in sleep stage 2 was higher than after placebo. the amount of slow-wave sleep was higher after zolpidem than after placebo. in addition, the higher dose of mpp22 resulted in an increase of spindle and β frequencies combined with a decrease of δ oscillations during nonrem sleep. in comparison, administration of zolpidem resulted in strong increase of δ, spindle and high β frequencies as well as strong decrease in θ and α frequencies. nocturnal progesterone levels increased after 9.0 mg mpp22. no other changes of hormone secretion were found. our study show sleep promoting effects of 9.0 mg mpp. as expected the sleep promoting effect of zolpidem was confirmed. the spectral signature of intranasal progesterone partly resembled the well-known sleep-eeg alterations induced by gaba active compounds. progestereone levels were elevated after 9.0 mg mpp22. no other endocrine effects were observed. introduction: anticholinergic drugs or drugs with anticholinergic side effects are commonly used for the treatment of various diseases in the elderly population. elderly patients are particularly vulnerable to anticholinergic-related cognitive effects. moreover, there is a relationship between anticholinergic exposure and cognitive impairment. however, there is currently a lack of data on the anticholinergic burden in geriatric patients in germany. it was therefore the aim of this study to evaluate the anticholinergic burden in a large representative cohort of geriatric patients. materials and methods: in this retrospective cohort study, (co)-prescriptions of anticholinergic drugs as well as anti-dementia drugs were evaluated using the discharge medication of geriatric patients between january 2013 and june 2015 from the geriatrics in bavaria-database (gib-dat). anticholinergic drugs were classified according to the anticholinergic cognitive burden (acb) scale in three groups (definite anticholinergics with a score of 2 or 3 and possible anticholinergics with a score of 1). the acb scale was modified by omitting trospium and by adding the three drugs biperiden, metixen and maprotilin, which are used in germany, with a score of 3. a patient's individual score of 3 or higher is considered to be clinically relevant. in total, 130,186 geriatric patients (median age 82 years, 66.3% female, median no. of drugs 9) were evaluated. of these, 41,456 (31.8%) patients took at least one drug with anticholinergic properties. two or more anticholinergic drugs were coprescribed in 10,941 (26.4% of the patients taking anticholinergic drugs) patients. 11,241 (27.1% of the patients taking anticholinergic drugs) patients had a score of 3 or higher. the most common anticholinergic drug combinations involving two definite anticholinergic drugs were amantadine/quetiapine (58), amitriptyline/quetiapine (41) and amitriptyline/carbamazepine (35). 2,885 (7.0%) patients received anticholinergic drugs in combination with anti-dementia drugs. conclusions: one third of patients in a large geriatric population were prescribed at least one anticholinergic drug. one quarter received a co-prescription of anticholinergic drugs. caution is advised prescribing anticholinergic drugs to elderly patients especially with dementia. the antiglaucoma agents brimonidine and timolol are novel substrates of the organic cation transporters oct2 and mate1 expressed in human eye c. neul purpose: glaucoma is a leading cause of visual loss in the world population. lowering intraocular pressure by topical administration of antiglaucoma agents is still the mainstay for glaucoma treatment. 1,2 although many effective drugs exist, the major challenge is their efficient intraocular delivery, which is estimated to amount to 3 the involvement of membrane drug transporters in the intraocular delivery of the widely prescribed antiglaucoma prostanoid latanoprost has been described. 4 however, it is currently unknown whether the cationic drugs brimonidine and timolol, which are also commonly used antiglaucoma agents, are similarly transported by drug transporters and whether these transporters are expressed in human eye. brimonidine is an α 2 -adrenergic agonist, which inhibits the activity of the adenylate cyclase subsequently leading to a reduced production of aqueous humor. timolol is a β-adrenergic receptor antagonist, which blocks β-receptors on the ciliary epithelium also resulting in a reduced aqueous humor production. the aim of the present study was to determine whether brimonidine and timolol are substrates of the organic cation drug transporters oct1 (encoded by slc22a1), oct2 (slc22a2), oct3 (slc22a3) and mate1 (slc47a1). a further aim was to investigate whether these transporters are localized in different human eye substructures. experimental design: transport of brimonidine and timolol was studied using the mammalian cell line hek293 stably expressing the organic cation transporters oct1, oct2, oct3 or mate1. 5 intracellular accumulation of brimonidine and timolol was analyzed by mass spectrometry. immunohistochemistry and immunofluorescence experiments were performed to study the localization of these transporters in different substructures from glaucomatous and non-glaucomatous human eyes. results: uptake experiments revealed that brimonidine is transported by oct2 and mate1 in a time-and concentration-dependent manner, but not by oct1 or oct3. timolol is only transported by mate1, but not by the octs. as shown by immunolocalization studies, the oct2 and mate1 transporter proteins were expressed in all anterior eye substructures of non-glaucomatous and glaucomatous eyes, i.e. the cornea, the conjunctiva and the ciliary body. conclusion: our data demonstrate that oct2 and mate1 may play a role in the ocular disposition of the antiglaucoma drugs brimonidine and timolol and may contribute to interindividual variability of drug concentrations and effects. references: 1. zhang et al., nat rev drug discov. 2012 jun 15; 11(7) :541-59. 2. lavik et al., eye (lond). 2011 may; 25(5):578-86. 3. gaudana et al., pharm res. 2009 may; 26(5):1197 -216. 4. kraft et al., invest ophthalmol vis sci. 2010 51(5):2504 -11. 5. nies et al., plos one. 2011 6(7) :e22163. supported by the robert bosch foundation, stuttgart, germany. immature platelet count or immature platelet fraction as optimal predictor of antiplatelet response to thienopyridine therapy c. stratz 1 , t. nuehrenberg background: previous data suggest that reticulated platelets impact significantly on antiplatelet response to thienopyridines. it is unknown which of the parameters describing reticulated platelets is the optimal predictor of antiplatelet response to thienopyridine therapy. methods: this study is a prespecified subanalysis of the excelsiorload trial that randomized elective patients undergoing coronary stenting to loading with clopidogrel 600mg, prasugrel 30mg or prasugrel 60mg (n=300). adp-induced platelet reactivity was assessed by impedance aggregometry before loading (=intrinsic platelet reactivity) and on day 1 after loading. multiple parameters of reticulated platelets were assessed by an automated whole blood flow cytometer: immature platelet fraction (ipf, proportion of reticulated platelet of the whole platelet pool), highly immature platelet fraction (hipf), absolute immature platelet count (ipc). results: each parameter of reticulated platelets correlated significantly with adpinduced platelet reactivity: ipf (r s =0.18; p=0.002), hipf (r s =0.18; p=0.002), ipc r s =0.26; p<0.001). in a multivariable model including all three parameters, only ipc remained as significant predictor of platelet reactivity (p<0.001). after adjustment to known predictors of on-clopidogrel platelet reactivity including cytochrome p450 2c19 polymorphisms (*2 and *17), age, body mass index, diabetes, smoking and intrinsic platelet reactivity, ipc s21 was the strongest predictor of on-treatment platelet reactivity (partial η 2 =0.045; p<0.001) followed by intrinsic platelet reactivity (partial η 2 =0.034; p < 0.002). these findings prevailed when analyzing subgroups of patients on clopidogrel or on prasugrel. conclusion: immature platelet count is the strongest platelet count derived predictor of antiplatelet response to thienopyridine treatment. given its easy availability together with its even stronger association with on-treatment platelet reactivity when compared to known predictors including the cyp 2c19*2 polymorphism, immature platelet count might become the preferable predictor of antiplatelet response to thienopyridine treatment. cutaneous squamous cell carcinoma (cscc) is the second most common human cancer with continuously rising incidences worldwide. primarily caused by cumulative uvb exposure, cscc accounts for considerable costs for health care systems and poses a deadly risk especially to organ transplant recipients [1] . current chemotherapy needs to be improved, because even the topical treatment for cscc's carcinoma in situ bears limited efficacy and painful adverse effects [2] . however, animal-based approaches in preclinical development contribute to the frequent failure of investigational new drugs in clinical trials [3] . herein, we characterized a human cell-based cscc model, normal reconstructed human skin (rhs) served as control. whereas rhs exhibited low proliferation, the co-culture with cscc increased ki-67 index 23-fold in the cscc model (p£0.001). while the presence of claudin-4 and occludin were distinctly reduced, zonula occludens protein-1 was more wide-spread, and claudin-1 was heterogeneously distributed within the cscc model compared with rhs. this is in accordance to the in vivo situation [4] and likely contributes to the impaired barrier function of the cscc model, as demonstrated for 2.6-fold increased caffeine permeation. finally, the ingenol mebutate effects in the cscc model and rhs closely mimic the anti-tumor effect and the adverse reactions in patients [2] , both linked to the drug's inherent cytotoxicity. in conclusion, the thoroughly characterization of disease models fosters both advanced preclinical drug development and improved cscc treatment. funded by the german government, the berlin-brandenburg research platform bb3r with integrated graduate education was launched in 2014. the aim of this research platform, along with the associated graduate school, is to close substantial knowledge gaps in the fields of the 3rs and to find alternatives to animal experimentation within the next years. a panel of 3r experts has been set up to provide advice and assistance and to raise awareness in society for 3r-related issues. research in bb3r investigates physiological functions on different levels to establish alternative methods for preclinical drug development and basic research. the principal investigators aim at facilitating research collaborations and sustainable research activities in the region berlin-brandenburg and abroad. an integrated bb3r graduate program has been developed to offer structured training to graduate students in a specific, mandatory course program on 3r including modules on ethics and legislation. currently, 14 phd students are qualified for management positions in professional areas related to the 3rs, and three junior research groups are now ready to expand their regional research activities nationwide. furthermore, the concept of a novel lecture series for master students and undergraduates has been designed and awarded the animal welfare research award for berlin-brandenburg in teaching and education. the state government of berlin supports the research platform bb3r and will be funding an additional professorship at the fu berlin to further promote research on alternative testing. finally, co-operations with national and international partners are being built to facilitate the project-based exchange of scientists and joint research. currently, the identification and evaluation of skin sensitizers is mainly restricted to animal testing using the guinea pig maximization test, buehler test or the murine local lymph node assay. recently, an adverse outcome pathway of skin sensitization has been released by the oecd, identifying the key events leading to allergic contact dermatitis. in vitro tests address these key events and two assays are now regulatory adopted (oecd 442c and 442d). the use of the current in chemico and in vitro models is, however, limited since they do not reflect dermal penetration, complete biotransformation and cell cross-talk in an organotypic environment. in this study, we aimed to overcome these limitations by establishing reconstructed skin tissues containing langerhans cells (lcs). in vitro generated immature monocyte-derived (molcs) or mutz-3-derived cells (mutz-lcs) cultivated with keratinocytes on a dermal compartment with fibroblasts form a stratified epidermis after 14 days as indicated by the expression of epidermal differentiation markers. molcs or mutz-lcs were mainly localized in suprabasal layers of the epidermis and distributed homogeneously in accordance with native human skin. topical application of the extreme contact sensitizer 2,4-dinitrochlorobenzene (dncb) induced il-6 and il-8 secretion in skin models with lc-like cells, whereas no change was observed in control rhs lacking immune cells. increased gene expression of cd83 and pd-l1 in the dermal compartment indicated lc maturation. we confirmed the enhanced mobility from epidermal to dermal compartments for mutz-lcs and molcs in the presence of dncb. in summary, we successfully integrated immature and functional lc-like cells into reconstructed human skin. this fosters the development of animal-free test systems for advanced and potentially individualized hazard assessment of skin sensitization. computational methods for prediction of in vitro activity of new chemical structures. background: with a constant increase in the number of new chemicals synthesized every year, it becomes highly important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. in recent years, in silico prediction methods received great attention as alternatives to animal experiments for evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine (3rs). various computational approaches have been proposed for prediction of toxicity of chemicals ranging from quantitative structure activity relationship modeling to molecular similarity based methods and machine-learning methods. within the "toxicology in the 21st century" screening initiative, a crowdsourced platform was established for development and validation of computational models to predict the interference of chemical compounds in nuclear and stress receptor pathways based on a training set containing more than 10,000 compounds tested in high-throughput screening assays. methods: here we present the results of various molecular similarity-based and machine-learning-based methods over an independent evaluation set containing 647 compounds. further, we compare the performance of these methods when applied individually and together. in retrospect we also discuss the reasons behind the superior performance of an ensemble approach, that combines a molecular similarity approach and a naïve bayes machine learning algorithm in achieving best prediction rates in comparison with other individual methods, explaining their intrinsic limitations. results and conclusions: our results suggest that, although prediction methods were optimized individually for each modeled target, an ensemble of similarity and machinelearning approaches provides promising performance indicating its broad applicability in toxicity prediction. charité -universitätsmedizin berlin, structural bioinformatics group, berlin, germany a multitude of drug candidates (approx. 20 %) fail in the late drug development due to toxicity and adverse effects [1] . immunotoxicity can be divided into four types of immune-mediated adverse effects: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. current safety evaluations of drug candidates with respect to immunotoxicity are comprised of in vivo and in vitro assays. here, we present an in silico approach for predicting immunotoxic substances based on immune suppressive and not toxic compounds using the laplacian-modified naϊve baysian model as a supervised machine learning method. therefore, we examined the relations between about 51,000 compounds and 115 cancer cell lines from the national cancer institute's (nci) nci-60 growth inhibition data [2] with focus on five immune cell lines (rpmi-8226, ccrf-cem, . different fingerprints encoding the chemical structures have been evaluated for their predictive power (e. g. extended-connectivity fingerprints, substructure fingerprints) and showed good prediction rates in a retrospective analysis. acting in phase ii metabolism, sulfotransferases (sult) transform endo-and exogenous molecules such as drugs into more hydrophilic entities that are easily excreted from the human body [1] . although serving detoxification, sult-mediated transformation of molecules has also been associated with the formation of chemically reactive metabolites that could promote adverse reactions [2] . the development of a computerbased model that allows prediction of molecules susceptible to metabolism would improve drug development and drug safety [3] , and encourage the reduction of in vivo testing according to the principles of the 3rs (replacement, reduction and refinement). in our study, we developed an in silico model to predict human sult subtype 1e1 activity acting in phase ii metabolism. structure-based molecular modelling of sult activity is challenging due to the broad and overlapping substrate spectrum of sult subtypes. this low substrate specificity can be attributed to the high degree of conformational flexibility of the enzyme, particularly in the active site. therefore, molecular dynamics simulations were performed to address enzyme flexibility and the broad substrate spectrum of sult ( figure 1 ). based on a collection of selected enzyme conformations from molecular dynamics simulations and a dataset of active sult1e1 ligands, ensemble docking was utilized to generate ligand-protein complexes that served as a basis for 3d pharmacophore development. eight specific 3d pharmacophores were created that allow identification of sult1e1 substrates and inhibitors. for further refinement of the computer-based prediction, machine learning models and post-filtering steps were generated that allow classification of predicted hits. the final prediction model was used to screen the drugbank (a database comprising over 6,500 experimental and approved drugs). a major part of the predicted hits could be confirmed from literature. from the remaining hits, a representative selection of molecules was experimentally tested for sult1e1 inhibition or biotransformation. experimental results were in agreement with our computer-based models and revealed previously-unknown biotransformation by or inhibition of sult1e1 for compounds listed in the drugbank. introduction: vascularization of the dermal equivalent of full-thickness skin constructs by endothelial cells is highly desirable, because such constructs closely mimic the architecture of real skin. unfortunately, the realization of a capillary network in skin constructs is still difficult. in our study of full-thickness skin constructs, following the methodologies of küchler et al. (2011) , there were alterations in the epidermal differentiation after endothelialization of the dermal equivalent. the aim of this study was to characterize these changes on a morphological level. material and methods: non-endothelialized constructs (keratinocytes, fibroblasts) were prepared according to küchler et al. (2011) . to obtain endothelialized constructs, the dermal equivalent of the non-endothelialized constructs was enriched with endothelial cells. after two weeks of in vitro culture, the skin constructs were processed for quantitative as well as qualitative assessment by light and electron microscopy. results: both types of skin construct developed all strata, i.e., stratum basale, spinosum, granulosum, corneum of a stratified soft-cornified epidermis, although the two constructs displayed differences in every stratum: significantly more mitoses occurred in the epithelial germ layers of the endothelialized constructs (p=0.013). in addition, significantly more keratohyalin granules were counted within their stratum granulosum (p=0.010). 50% of the shapes of the spinous and the granulosum cells were irregular and these cells were separated by wide intercellular spaces. the typical epidermal lamellar bodies appeared in the endothelialized constructs more often than in the nonendothelialized ones. at the stratum granulosum -stratum corneum interface, no cohesion between the strata was present. background and novelty: during the last decade organ-on-a-chip technologies received increasing attention in the scientific community. the idea of combining different tissue types on a physiological-like system creates completely new options on how substances can be characterized without the use of animal experiments. animal models were used for the investigation of skin sensitization as a standard method until animal testing for cosmetic substances was banned in the e.u. in 2013. by combining skin models with an immunological counterpart, new data will be presented to see if the multi-organ-chip add value to the current need of alternative methods regarding skin sensitization and immunotoxicity testing. experimental approach: the multi-organ-chip platform is designed to combine different human cell and tissue types like 3d-spheroids, barrier models or biopsies in one microfluidic system. a peristaltic on-chip micropump enables circulation of medium, allowing for a constant perfusion between the compartments. first experiments were performed using a dendritic-cell-only approach in the 2-organ-chip. in subsequent cocultivation experiments ex vivo human epidermis and dendritic cells were cultivated each in one culture compartment connected by the microfluidic channels. for analysis we measured the typical activation marker cd86 and the vitality of the dendritic cells by flow cytometry. functionally different sensitizers were selected to investigate their effects in our model. finally a more complex 3d matrices including different immunological cell types to emulate in vivo-like reactions like in the human lymph node were cultivated in the 2-organ-chip. results and discussion: our data show a strong influence of pump pressure and pumping frequency on the activation of dendritic cells. hence, we established an adequate set up by cultivating the dendritic cells in cell culture inserts, preventing cell activation due to shear stress. compared to existing sensitization assays, the main advantage of the perfused 2-organ-chip sensitization assay is the presence of an epidermis equivalent, partially integrating important parameters such as metabolism and skin barrier function. we compared our data with reference cd86-values from the pbmdc (peripheral blood monocyte derived dendritic cells) skin sensitization assay. for identical substances, we observed differences in dendritic cell activation between the pbmdc assay and the 2-organ-chip perfused assay. here we present the first-time cultivation of primary derived immune cells cultivated on our microfluidic system which is a promising enhancement to integrate immunological reactions on further multi-organ combinations. due to growing social and political pressure, the interest in alternatives to animal testing has constantly increased during the past 10 years stimulating the development and validation of new in vitro test systems including reconstructed skin models. additional to toxicological studies and permeability assays, skin models are of high interest for fundamental research to elucidate basic physiological and pathophysiological processes in human skin [1, 2] . as for today, most of the in vitro skin models are grown from primary keratinocytes and fibroblasts that were either isolated from excised human skin or from juvenile foreskin following circumcision. in this project, we aimed for the generation of in vitro skin models using hair folliclederived cells. therefore, different methods to optimize cell isolation and expansion of outer root sheath (ors) cells from human hair follicles were systematically investigated. the best procedure for isolation of ors cells was the direct cell outgrowth on a cell culture insert which was co-cultured with a feeder layer of postmitotic human dermal fibroblasts. following outgrowth, the cells were either further cultivated with feeder cells in specific serum-enriched cell culture medium to obtain hair follicle-derived keratinocytes or using the same culture medium without feeder cells to obtain fibroblasts. afterwards, the generation of hair follicle-derived fibroblasts and keratinocytes was verified via the fibroblast-specific markers vimentin and desmin and the keratinocyte marker cytokeratin (ck) 14 clearly showing that vimentin and desmin are expressed in hair follicle-derived fibroblasts and in dermal fibroblasts. as expected, these cells were negative for ck14, which was abundantly expressed in hair folliclederived keratinocytes. moreover, the expression of collagen type i, iv, tgf-beta, alpha sma and il-1 alpha in hair follicle-derived fibroblasts and dermal fibroblasts showed no significant differences. ultimately, hair follicle-derived keratinocytes and fibroblasts were used to grow full-thickness skin models which were subsequently characterized with regard to epidermal differentiation, skin permeability and skin surface ph. again, no significant differences compared with skin models grown from skin-derived cells were detected showing the potential of using hair follicle-derived cells for generating in vitro skin models. [1] vávrová, k., henkes, d., strüver, k., sochorová, m., školová, b. et al. filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3d skin construct. the journal of investigative dermatology. 134, 746-753 (2014 bundesinstitut für risikobewertung, experimentelle toxikologie und zebet, berlin, germany background: the eu directive 2010/63 has been drawn up with the aim of ultimately replacing animal testing. wherever animal experimentation is necessary, the 3-rprinciple of russel and burch (replace, reduce, refine) has to be observed. the primary goal of the 3-r-principle is to replace animal testing with alternative methods. if no alternative method can be applied, the total number of animals is supposed be reduced. consequently, some animals are used multiple times in the course of an experiment. for example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. however, the directive claims that "the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal". objective: we are looking into whether multiple exposures to anesthesia cause more stress than a single exposure. methods: the most common mouse strain c57/bl6 j and anesthetics isoflurane and the combination of ketamine/xylazine are used. with regard to recent studies, the animals are anesthetized six times for 45 minutes over a period of three weeks. all parameters observed are compared between controls, animals with a single and repeated anesthesia. the interval between the administration of the anesthesias is three to four days. when the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. the grimace scale is scored 30 and 150 minutes after anesthesia. besides pain, the grimace scale can also assess anxiety, stress and malaise. the display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. furthermore, activity, food and water intake are monitored for 24 hours. a behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. motor coordination and balance are assessed by the balance beam and rota rod. the open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress. results: the first results of our study will be presented at the 82 nd meeting of dgpt. conclusion: we are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. bb3r is funded by bmbf. in 1959 the 3r-principle was defined by the british scientists william russel and rex burch in their book 'the principles of human experimental techniques'. the 3r refer to replace, reduce, and refine. they set the goals to use alternative non-animal methods (replace), to reduce the number of laboratory animals (reduce) and to minimize the distress of laboratory animals and to refine their welfare (refine). the implementation of the 3r-concept is the overall goal of scientific animal welfare. article 4 of the 'directive 2010/63/eu on the protection of animals used for scientific purposes' state that the research on refinement is as important as on replacement and reduction [1] . according to the current statistics on laboratory animals, the mouse is the most commonly used animal in experiments with approximately 70 % [2] [3] . effective pain treatment is crucial not only for ethical and legal considerations but also to achieve highquality results [4] . pain in mice is only obvious if it is severe or acute pain. however, it is difficult to identify slight or moderate pain. the determination of pain levels and effective dosages of analgesics is therefore challenging. the most commonly used analgesics for postsurgical pain treatment in mice are opioids. however, the recommendations for their use show vast dosage ranges. for example, the recommended dose of buprenorphine ranges from 0.05 to 2.5 mg/kg per bodyweight [5] [6] [7] depending on the pain model used. additionally, putative pharmacogenetic strain differences have to be considered. for example, the analgesic treatment with morphine shows mouse strain specific differences in pain sensitivity [8] . the goal of the project is the refinement of the recommendations for effective dosage of opioid analgesics in laboratory animals for mouse inbred strains by incorporating strain specific differences. regarding the phenotype, we want to identify a putative inbred strain dependent pain threshold and measure the drug level in plasma and brain. additionally the metabolic capacity and mrna expression level will be investigated. genotype identification is based on a data bank analysis used for correlation with phenotypical parameters. [1] rl 2010/63/eu. [2] bmel (2014) . anzahl der für versuche und andere wissenschaftliche zwecke verwendeten wirbeltiere. [3] eu commission (2013) . seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the european union. [4] carbone l (2011) pain in laboratory animals: the ethical and regulatory imperatives. plos one 6, e21578. [5] gv-solas, a.f. a.d. (2013) . empfehlung zur schmerztherapie bei versuchstieren. [6] carpenter, j.w., t.y. mashima, and d.j. rupiper (2001) . exotic animal formulary, 2nd edition, w.b. saunders co., phila. [7] flecknell, p. (1996) . laboratory animal anaesthesia, 2nd edition, academic press, san diego, ca. introduction: göttingen minipigs™ are frequently used large animal models for orofacial research, for example dental implant surgery. requests from experimental surgeons for detailed anatomical information can not be answered because there is no existing data, especially not age-related. because of unavailable data and the false choice of animal age, surgical interventions fail or lead to enormous post-operative suffering. therefore the aim of this study is the acquisition of detailed anatomical data of the mandibula and other organs and structures without sacrificing pigs for this reason. animals, materials and methods: ct scans of a 64-slice scanner were collected from 18 female minipigs, consisting of 6 animals aged 12 months (group 1, n=6) and 12 animals (group 2; n=12) examined at the age of 17 and 21 months. these minipigs were involved in experiments, approved by the regional office for health and social affairs, berlin. image analysis was performed using vitrea advanced® (vital images). more than 50 parameters concerning teeth, the mandibular body, frame and canal, coronoid process and mandibular condyle were defined and measured. for now, we focused on the development of the mandibular canal and the distance between the dorsal borders of the mandibular canal to the alveolar ridge at the most posterior mental foramen, parameters immensely important for planning interventions when testing new dental implants. results: the measurements by computed tomography showed variations of several parameters between left and right ramus mandibulae and within the different age groups. the volume of the canalis mandibulae increases in time. animals of the same age show significant differences in volume, with a range of up to 65% where the largest volume was 13,4 ml and the lowest 4,7 ml. the distance between the dorsal borders of the mandibular canal to the alveolar ridge decreases between 12 and 17 months of age. comparing 17 and 21 months old minipigs, no significant difference in distance could be observed. from the age of 17 months the position of the mandibular canal in relation to the alveolar ridge remains constant. conclusion: the decrease of the distance between the mandibular canal and the alveolar ridge between 12 and 17 months of age indicates ongoing anatomical changes of this parameter until the age of 17 months. therefore animals should be older than 17 months if included in long-term studies after orofacial experiments, like implant surgery of the mandibula. because of the described individual differences, the authors strongly suggest to support the planning of orofacial interventions by ct imaging or other radiographic techniques. background: laboratory housing conditions are standardized to a high level. under these conditions, the occurrence of stereotypic behaviour (sb) can be observed. stereotypies are commonly known as deviations from normal behaviour that are repetitive, invariant and without any obvious function or aim for the animal [1]. worldwide it is estimated that over 85 million animals perform sb, with the highest prevalence in laboratory animals and the agricultural sector. fvb/n is a typical inbred mouse-strain that shows different types of stereotyped movements. it is known that environmental enrichment decreases the incidence of sb, yet they still occur [2] . since animal's behaviour highly influences its metabolism and immune system, differences in handling, caring and keeping can lead to varying results, even with an identical experimental setup [3] . aim: of the study aim of the study is to observe different life stages of fvb/n-mice and immediately detect the development of sb. observations are linked to various behavioural tests and the characterisation of the metabolic and immunological phenotype. the results will lead to a better understanding of the mechanisms driving the development of sb and clarify its implication to animal welfare or to what extent the performance of stereotypies even reflect emotional suffering. the strain-related behaviour and sb are recorded with computer-assisted programmes. observational periods already begin with the parental generation. as possible indicators for later developing sb, data on reproductive success and maternal care are collected, such as different behavioural tests. the animals are characterized by a specific protocol for detecting the metabolic and immunological phenotype. finally, histological and molecular biological analyses follow. outlook: it is of paramount importance to take good care for the welfare of laboratory animals (3r-refinement). though, the knowledge about the ethological particularities of animals and the motivational base of animals performing sb are not enough to generally avoid stereotypies. therefore the meaning according to the character of sb has to be analysed more intensively to understand the needs of laboratory animals and evolve recommendations for optimizing the breeding and keeping such as for the assessment of possible distress in animals performing sb. objective: thermoregulation is a vital function in both humans and animals with the serotonin (5-ht) system, in particular the 5-ht 1a receptor, playing a major role. activating 5-ht 1a receptors by the 5-ht 1a receptor agonist 8-hydroxy-2-(dipropylamino)tetralin (8-oh-dpat) leads to reduced body temperature. while there is consensus that hypothermia is induced by the stimulation of postsynaptic 5-ht 1a receptors in rats and humans, the regulatory mechanisms in mice are less clear. in our group, within phenotyping a transgenic mouse line permanently overexpressing the 5-ht 1a receptor in serotonergic projection areas, bert et al. (2008, pmid: 18396339) revealed exaggerated 8-oh-dpat-provoked hypothermic response. thus, the objective of the present study was to substantiate the contribution of postsynaptic 5-ht 1a receptors to thermoregulation, more precisely to the hypothermic effect of 8-oh-dpat, in mice. methods: we used radio telemetry technique to monitor the basal body temperature and the hypothermic effect of different doses of 8-oh-dpat (0.1 mg/kg -4 mg/kg i. p.) in male transgenic mice in comparison to nmri wild-type males. additionally, we investigated whether reduction of serotonergic activity by pretreatment with the 5-ht synthesis inhibitor parachlorophenylalanine (pcpa; 100 mg/kg, i. p. on four consecutive days) would alter the effects of 8-oh-dpat on body temperature in transgenic mice postsynaptically overexpressing the 5-ht 1a receptor. results: 5-ht 1a overexpressing mice revealed lower levels of basal body temperature than wild types (transgenic mice: 36.0 °c; nmri wild-type mice: 37.4 °c). in both genotypes, systemic administration of 8-oh-dpat dose-dependently decreased body temperature, being significantly more pronounced in mutant mice (-2.8 °c compared to -1.5 °c in nmri wild types). dose response curves of 8-oh-dpat revealed an ed 50 = 0.4 mg/kg in transgenic and an ed 50 = 0.57 mg/kg in nmri wild-type mice. pcpa pretreatment did not alter the hypothermic response to 8-oh-dpat in mice. the dose-response curves indicate a higher potency of 8-oh-dpat in transgenic mice. the exaggerated hypothermic response to 8-oh-dpat in mutant mice implies that postsynaptic 5-ht 1a receptors could be involved in thermoregulatory function in mice. this assumption is further confirmed by the fact that 8-oh-dpatevoked thermal responses were not influenced by pretreatment with pcpa, most notably in transgenic mice overexpressing 5-ht 1a receptors postsynaptically. genetic variation within g protein-coupled receptors compromises the therapeutic application of drugs targeting these receptors. one of the most intensely studied variation is p.arg389gly in the human beta1-adrenoceptor (adrb1). the adrb1 carrying arginine at position 389 in helix 8 in the proximal carboxy terminus is more frequent among caucasians and is hyperfunctional. yet, the molecular basis for the differences between the beta1-adrenoceptor variants arg389-adrb1 and gly389-adrb1 is poorly understood. despite its hyperfunctionality, we found the arg389-variant of the adrb1 to be hyperphosphorylated upon continuous stimulation with norepinephrine when compared to the gly389-variant. using adrb1 sensors to monitor activation kinetics by fluorescence resonance energy transfer (fret), the arg389-adrb1 exerted faster activation speed and arrestin recruitment than the gly389-variant. both depended on phosphorylation of the receptor as shown by knockdown of g protein-coupled receptor kinases and by fret experiments using phosphorylation-deficient adrb1 mutants. point mutation of single serines and threonines in the carboxy terminus of the adrb1 finally revealed a variant-specific phosphorylation pattern that determines arrestin recruitment. taken together, these findings suggest that differences in receptor phosphorylation determine the differences in activation speed, efficacy and arrestin recruitment of adrb1 variants. opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (mor) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. there is substantial evidence suggesting that g protein-coupled receptor kinases (grks) and βarrestins play key roles in regulating mor signaling and responsiveness. following phosphorylation by grks, β-arrestins bind to phosphorylated mors, which prevents further interactions between the receptor and g proteins even in the continued presence of agonist resulting in diminished g protein-mediated signaling. we have previously shown that agonist-induced phosphorylation of mor occurs at a conserved 10-residue sequence, 370 trehpstant 379 , in the carboxyl-terminal cytoplasmic tail. morphine induces a selective phosphorylation of serine 375 (s375) in the middle of this sequence that is predominantly catalyzed by g protein-coupled receptor kinase 5 (grk5). by contrast, high-efficacy opioids not only induce phosphorylation of s375 but also drive higher-order phosphorylation on the flanking residues threonine 370 (t370), threonine 376 (t376), and threonine 379 (t379) in a hierarchical phosphorylation cascade that specifically requires grk2/3 isoforms. to investigate this mechanism further, we have developed novel β-galactosidase complementation assays to monitor agonist-dependent recruitment of grk2 and grk3 to activated mors. using this assay, we were able to show that activation of mor by high-efficacy agonists such as damgo results in a robust translocation of grk2/3. in contrast, activation by low-efficacy agonists such as morphine results in a much less pronounced recruitment of grk2/3 isoforms. interestingly, damgo-induced β-arrestin recruitment was strongly inhibited by sirna knock down of grk2 or grk3. conversely, the morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of grk2 or grk3. mutation of s375 to alanine strongly inhibited both grk and β-arrestin recruitment. however, mutation of all 11 carboxyl-terminal serine and threonine residues of mor was required to completely abolish its interaction with arrestins and grks resulting in a complete loss of mor internalization and desensitization. heterotrimeric g proteins are located at the inner leaflet of plasma membranes and are a major primary transducer of cell signaling initiated by g protein-coupled receptors (gpcrs). based on sequence similarity, heterotrimeric g proteins can be subdivided into four main classes, i.e. gi/o, gs, gq/11, and g12/13, which interact with distinct cellular effectors to shape the final cellular response 1 . identification of new selective and cell-permeable g protein inhibitors is of great interest as these may be beneficial in complex pathologies that involve signaling of multiple gpcrs 2 . mechanistically, small molecule g protein inhibitors may, for instance, block nucleotide exchange by inhibiting gdp release (i.e. guanyl nucleotide dissociation inhibitors, gdis) or allow gdp release but block gtp entry by stabilizing the g protein in an empty pocket conformation 3 . here, we set out a new approach to classify g protein inhibitors regarding their mechanism of action in radioligand binding experiments. in particular, we investigated the influence of the specific gα q/11/14 inhibitor fr900359 4 on agonist-radioantagonist binding experiments performed with membranes of cho cells stably expressing the muscarinic m1 acetylcholine receptor (cho-m1). agonistradioantagonist competition under these conditions is biphasic because agonists bind with higher affinity in the ternary complex consisting of agonist, receptor and nucleotidefree g protein compared with a g protein-free receptor 1,5 . we show that fr900359 can be classified as a gdi as it significantly reduced high affinity binding of carbachol in cho-m1 membranes. in contrast to this, bim-46187, a pan g protein inhibitor with a cell-type-dependent preference for gq, did not influence high affinity agonist binding and thus stabilized gq in an empty pocket conformation 3 . interestingly, inhibition of high affinity agonist binding by fr900359 was incomplete in agonist-radioantagonist displacement studies and also when a radioagonist was applied. to fully prevent high affinity agonist binding by fr900359, combined uncoupling of both gi and gs proteins from m1 was required by pre-treatment with pertussis toxin and cholera toxin, respectively. these data demonstrate that not only gq, but also gi, and gs, contribute to the high affinity fraction of m1 receptors. taken together, our findings show that radioligand binding experiments are an attractive approach to classify new g protein inhibitors according to their mechanism of action. universität, würzburg, germany g protein-coupled receptors (gpcrs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric g proteins [1] . gpcr-induced cell signaling can be rather complex as several gpcrs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other "allosteric" binding sites [2] . in the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (machrs). to this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [2] . therefore, application of different linker length may control the signaling outcome. here, we applied the human m1 machr which preferentially activates g proteins of the g q/11 type but can also promiscuously stimulate g s proteins. g q/11 -and g sdependent signaling pathways were analyzed by application of cho cells stably transfected with the human m1 machr in ip1 and camp accumulation assays, respectively. in comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both g q -mediated and g s -mediated signaling. our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. particularly, the compound iper-6-naph completely lost intrinsic activity for both g q/11 and g s activation at the m1 machr. moreover, g s -mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than g q -signaling. interestingly, longer linker length led to improved signaling for both pathways (g q and g s ) in both hybrid series. iper-7-phth seems to be an exception as it had a higher intrinsic efficacy for g s -dependent signaling than the other phth hybrids with longer linker chains. strikingly, only iper-fri-phth, which corresponds to iper-8-phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on m1 machr for both signaling pathways under investigation. taken together, these data strongly suggest that, in comparison to g q/11 -mediated signaling, activation of the g s protein in m1 machr is more sensitive to spatial restriction in the allosteric vestibule. thus, it appears to be possible to control signaling of the m1 machr by allosteric constraint of the receptor's conformational flexibility. for more than three decades 3-(1h-imidazol-4-yl)propylguanidine (sk&f-91,486 (1) [1] ) is known as the prototypic pharmacophore of highly potent histamine h 2 -receptor (h 2 r) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine. [2] in order to get more insight into the structure-activity relationships of alkylated analogues of sk&f-91,486, we characterized 78 newly synthesized derivatives including several bivalent compounds (e.g., 2) by using different pharmacological in-vitro methods. [3] the potential h 2 r agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of sf9 cells [4] (hh 1,2,3,4 r). compounds were also functionally characterized in the [ 35 s]gtpgs assay (hh 2 r, sf9 cell membranes). [5] selectivity vs. hh 1,3,4 r was determined for selected derivatives also using this technique. organ bath studies (gph 1 r (ileum), gph 2 r (right atrium)) yielded functional data in a more physiological environment. the major part of the new sk&f-91,486 analogues displayed partial or full agonism via hh 2 r and gph 2 r, respectively. the most potent analogue, bivalent thiazole-type bisguanidine 2, was a partial agonist (e max = 88%) and 250-times as potent as histamine vis-à-vis the gph 2 r. attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pa 2 values for the antagonist (5.87 -6.38) which are in accordance with literature data. however, in the functional in-vitro assay on gph 2 r, the positive chronotropic response evoked by sk&f-91,486 was surprisingly resistant to antagonism by cimetidine and other typical h 2 r antagonists (ranitidine, famotidine), although the compound so far has been unanimously classified by others as a weak partial h 2 r agonist. this behaviour is unique within the large series of sk&f-91,486 analogues studied so far under similar conditions. probably the positive chronotropic effect of the lead compound is -at least in part -the result of a second molecular interaction which has been overlooked so far. [1] parsons, m.e. et al.; agents actions 1975, 5, 464. [2] buschauer, a.; j. med. chem. 1989 , 32, 1963 -1970 [3] pockes, s.; dissertation, univ. regensburg 2015. [4] seifert, r. ; j. pharmacol. exp. ther. 2003, 305 (3) , 1104-1115. the nociceptin/orphanin fq (n/ofq) peptide (nop) receptor is the fourth most recently discovered and least characterized member of the opioid receptor family (mor, kor and dor). nop receptor is widely distributed and modulates several physiological processes by its endogenous ligand nociceptin. the nop receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states such as chronic and neuropathic pain. consequently, there is increasing interest in understanding the molecular regulation of nop receptor. recently, we generated two phosphosite-specific antibodies directed against the carboxyl-terminal residues serine 351 (s351) and threonine 362 /serine 363 (t362/s363), which enabled us to selectively detect either the s351-or the t362/s363-phosphorylated forms of the receptor. our results show that nociceptin, mcoppb, sch221510 and ro64-6198 induce a stably phosphorylation at s351 and t362/s363 followed by a profound internalization of the receptor. the nociceptin-induced s351 and t362/s363 phosphorylation can be blocked by selective nop receptor antagonists such as j113397 or sb612,111. nnc63-0532, buprenorphine and norbuprenophine failed to induce a phosphorylation at these sites. in the presence of nociceptin, s351 phosphorylation occurred at a faster rate than phosphorylation at t362/s363 indicating that s351 is the primary site of agonistdependent phosphorylation. activation of pkc by phorbol 12-myristate 13-acetate facilitated receptor phosphorylation only at s351 but not at t362/s363, indicating that s351 can also undergo heterologous phosphorylation. using nop-gfp knock in mice, we detected nop receptors in brain, spinal cord and dorsal root ganglia (drg). we were also able to demonstrate a dose-dependent nop receptor phosphorylation at t362/s363 in mouse brain in vivo using western blot and mass spectrometry. in contrast, mcoppb and sch221510 failed to induce phosphorylation in vivo. together, these data provide new insights into the molecular regulation of the nop receptor in vitro and in vivo. several findings indicate that inflammatory diseases are initiated or maintained by an imbalance of receptor baised signaling; the latter referring to the ability of distinct ligands to endue individual receptors with qualitatively different g-protein-and/or b-arrestindependent signaling (1). chemokines and their receptors regulate a wide array of leukocyte functions, including chemotaxis, adhesion, and transendothelial migration, and thus play important roles in regulating inflammation (2) . in man, two cc chemokine receptors ccr2a and ccr2b are present that only differ in their carboxyl terminal portions; the latter known to interact with multi-protein complexes made up of heterotrimeric g proteins (pertussis toxin sensitive and -insensitive) and non-g-protein components, including b-arrestin (2) . interested in differential signaling of ccr2a and ccr2b we comparatively analyzed ligand-induced g-protein-regulated signaling pathways (e.g. activation of phospholipase c isoenzymes and rhogtpase-induced serum response factor [srf] activity) and b-arrestin-regulated pathways (e.g. internalization of receptors and phosphorylation of erk1/2) in the presence of ccl2, ccl8, ccl7, and ccl13. all these chemokines have been shown to interact with human ccr2 receptors. in addition, the structural requirements of the carboxyl terminal portions involved in determining specificity in g-protein-dependent signaling was addressed by using ccr2 receptor mutants. the comparative analysis revealed that differences in ligand-induced activation of g-protein-dependent (pertussis-toxin-sensitive versus pertussis-toxin-insensitive) and/or b-arrestin-dependent signaling exist. for example, activation of ccr2b receptors by ccl2 induced both rho-dependent srf activation and receptor internalization, while ccl8-stimulation resulted in srf but little if any receptor internalization. in contrast, ccr2a-expressing cells showed ccl2dependent srf-activation but any receptor/ligand internalization. analysis of the structural requirements of ccr2 receptors for coupling to g proteins revealed that arginine 313 within the putative 'eighth helix' of the carboxyl-terminal portions of ccr2a and ccr2b is not involved in ga i -mediated induction of erk1/2 and plays a minor role in ccr2b receptor internalization, but is specifically required for the ccr2a/ and ccr2b/ga q -mediated activation of srf. serotonin 5-ht 2c receptors (5-ht 2c r) are functional engaged with gq proteins and expressed in the central nervous system (cns). 5-ht 2c r significantly regulate emotion, feeding, reward or cognition and thus might serve as promising targets for drugs against psychiatric disorders or obesity. due to the technical difficulties in isolating cells from the cns and the hitherto lack of suitable cell lines that would endogenously express 5-ht 2c r, our knowledge about this receptor subtype is rather limited. recently established hypothalamic mhypoa2-10 cells show some characteristics of appetite-regulating hypothalamic neurons of the paraventricular nucleus, where 5-ht 2c r in vivo expression has been detected. thus, we tested mhypoa2-10 cells for putative 5-ht 2c r expression by performing single cell calcium imaging. we observed that serotonin and the specific 5-ht 2c r agonist, way161,503 induced robust calcium transients, which were strongly inhibited by two unrelated 5-ht 2c r-selective antagonist (sb206,553, rs102,221). serotonin and way161,503 also activated a camp response element-dependent reporter gene construct and induced significant phosphorylation of extracellularregulated kinases-1/2 in a sb206,553 and rs102,221 sensitive manner, providing further evidence for functional 5-ht 2c r expression in mhypoa-2/10 cells. intrinsic activity of way161,503 ranged between 0.3 and 0.5 compared to serotonin in all assays, defining way161,503 as a partial 5-ht 2c r agonist. in conclusion, we provide convincing data that hypothalamic mhypoa-2/10 cells endogenously express 5-ht 2c r and thus represent the first cell line to analyse 5-ht 2c r pharmacology, signaling and regulation in its natural environment. optical and electrophysiological methods allow detection and characterization of g i/o -protein coupled receptors j. straub 1 1 walther-straub-institut für pharmakologie und toxikologie, münchen, germany g-protein mediated signaling pathways are essential components of basic cellular functions. of note, g-protein coupled receptors (gpcrs) constitute one of the major drug targets in modern medicine. however, despite their clinical importance, fundamental properties of these receptors remain unknown. in particular, regulation of the major second messenger camp by g s -and g i/o -protein coupled receptors is of special interest. the classical biochemical method to detect receptor-mediated camp level changes uses pre-labeling with 3 h-adenine and calculation of the conversion rate to 3 h-camp. although, this multi-cellular method is highly sensitive and reproducible, it lacks time resolved and spacial assessment of camp formation in single living cells. to measure g s -protein induced increases of intracellular camp levels in single living cells in a time resolved manner, the fret-based camp-sensor epac is commonly used. however, it was unknown whether this sensor might be suitable to detect g i/o -protein mediated decreases of intracellular camp levels as well. in this study, we show that fret-based camp sensors can be deployed to reliably monitor g i/o -protein mediated camp level decreases. fret experiments with adrenergic α 2a or µ 1 opioid receptors in combination with different fret-based camp sensors showed a notable reduction of intracellular camp levels upon receptor activation which could be significantly reduced by selective receptor antagonists. of note, hek293 cells had to be pre-incubated with forskolin in submaximal concentration to increase basal camp levels. our findings suggest that fret based epac sensors are suitable to detect g i/o -protein activation similar to electrophysiological whole-cell measurements with g i/o -protein coupled receptors and trpc5 or heteromeric kir3.1/kir3.2 or kir3.1/kir3.4 channels coexpressing cells. hereby, agonist stimulations caused current increases with characteristic current-voltage relationships. altogether, our findings indicate that both optical and electrophysiological approaches allow time resolved detection and characterization of g i/o -protein coupled receptor activation in single living cells. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated and partially characterized transgenic mice (tg) which overexpress the human histamine h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), histamine increased heart rate and ejection fraction (ef) measured in vivo by echocardiography under isoflurane anesthesia. to investigate some aspects of the signaling pathway in these mice, we crossed the tg mice with pp2a mice leading to double transgenic mice (h 2 xpp2a = dt). pp2a mice (j biol chem 2004; 279:40827) overexpress the catalytic subunit of protein phosphatase 2a (pp2a) in cardiac myocytes and develop a cardiac hypertrophy. at an age of about 240 days we noted reduced ef in pp2a (33.1 ± 3.5 %, n=16) compared to wt (54.4 ± 4.5 %, n=10) and tg (59.8 ± 2.9 %, n=10). interestingly, in dt the ef (43.9 ± 4.9 %, n=11) was higher than in pp2a at similar heart rates. e´/a´ was elevated in dt compared to wt. relative heart weights were unchanged between these groups. in summary, we demonstrated that pp2a is involved in h 2 -receptor signaling and we tentatively conclude that the h 2 -receptor is able to ameliorate systolic but not diastolic cardiac function of pp2a mice. serotonin (5-ht) can exert positive inotropic and chronotropic effects in humans via 5-ht 4 -receptors. we have generated transgenic mice (tg) which overexpress the human 5-ht 4 -receptor selectively in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), serotonin induced increases in heart rate and ejection fraction. we treated the mice with 30 µg lps (lipopolysaccharide, i.p.; a standard model of sepsis) per g body weight or isotonic sodium chloride solution (as solvent control). echocardiography with isoflurane anesthesia was performed before and 3, 7 and 24 hours after lps treatment. lps led to a time-dependent deterioration of cardiac function in both tg and wt. the deterioration included systolic function (left ventricular ejection fraction=ef) as well as diastolic function (height of a and e waves through the mitral valve: e/a). for instance, ef amounted to 58.8 ± 20.7% seven hours after lps in wt and to 61. [4] [5] [6] [7] [8] [9] [10] [11] [12] p<0 .05 vs pre-lps value). however, 24 hours after lps, diastolic function, measured as e/a, amounted to 1.86 ± 0.43 in p<0 .05 tg vs. wt). moreover, after 24 hours a less pronounced decline in body temperature (probably due to superficial abdominal hyperemia) occurred in tg versus wt. in contrast, while all flow parameters declined after 3 and 7 hours of lps, they were not different between wt and tg. for instance, maximum flow (in mm/s) through the ascending aorta declined from 1158.3 ± 212.2 to 782.5 ± 154.3 in wt and from 1208.4 ± 235.1 to 798.8 ± 170.1 in tg (tg vs. wt, p>0.05, n=10-12; after 7 hours). we tentatively conclude: 5-ht 4 -receptors overexpression protects the heart against sepsis, putatively by interference with the intracellular biochemical pathway of lps in cardiomyocytes. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in tg, but not in wild type mice (wt), histamine (ec 50 = 34 nm) or amthamine (ec 50 = 10 nm), a more selective and potent h 2 -receptor agonist, induced positive inotropic effects (pie) and positive chronotropic effects (pce) in isolated left and right atrial preparations, respectively. in order to investigate the signal transduction for the pie in atrium, contractile studies using partially depolarized preparations were performed. therefore, left atrial preparations were equilibrated in the organ bath to 44 mm potassium chloride. thereafter, histamine (100 µm) induced a pie (31.1 ± 10.4 % of control, n=7) in tg but not in wt preparations whereas isoprenaline (10 µm) increased force in both wt and tg. the pie of histamine in potassium treated tg atrium could be blocked by cimetidine. compound 48/80, a releasing agent of endogenous histamine, increased force of contraction in tg left atria to a higher extent than in wt. furthermore, we tested whether analgetics known to release histamine were inotropically active in tg. however, morphine (10 µm) was unable to affect contractility in wt or tg, whereas ketamine and fentanyl increased left atrial contractility in both tg and wt. in summary, we demonstrated an involvement of the l-type calcium channel current in the h 2 -receptor mediated pie in tg atria. we failed to release inotropically active histamine using classical analgetics, arguing that a direct effect also in the human heart is unlikely to occur. the initial step in the homologous desensitization of g-protein-coupled receptors is their phosphorylation by one of the g-protein-coupled receptor kinases (grks). we demonstrate here measurement of the interaction of grk2, a ubiquitously expressed grk, with the μ-opioid receptor (µor) by fret and its dependence on agonist efficacy. hek293t cells transfected with yfp-tagged µor and mturquoise-tagged grk2 as well as non-fluorescent gα i1 , gβ and gγ subunits showed a robust increase in fret upon superfusion with 10 µm [d-ala 2 , n-mephe 4 , gly-ol]-enkephalin (damgo) which was reversible upon agonist washout. the partial agonist morphine (30 µm) also caused a fret increase but the amplitude of the fret signal was reduced to approximately 15-20% of that of the corresponding damgo signal. grk2 binds g-protein βγ (gβγ) subunits, and therefore we aimed to find out how cotransfection of grk2 affected the interaction of gβγ with the µor. however, we could not measure any damgo-induced fret changes between yfp-tagged µor and mturquoise-tagged gβ in the presence of non-fluorescent gα i1 and gγ. this was unexpected because we had previously successfully determined interactions between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor. this lack of fret was not due to an inability of the tagged gβ to interact with the µor as we could measure damgo-induced fret changes between yfp-tagged gα i1 and gβγ (gβ tagged with mturquoise) in the presence of non-fluorescent µor. moreover, when we attempted to establish an effect of grk2 on the interaction between the µor and gβγ, we could pick up fret between the µor and gβγ. comparison of the on-and off-kinetics of the µor-grk2 interaction with that of the µor-gβγ interaction in the presence of grk2 revealed similar time constants both for the on-and off-kinetics (grk2: k on 0.16 s ). this suggests that, in the absence of grk2, the orientation of the two fluorophores on the µor and gβγ may be unfavorable or the interaction may be too short-lived to produce an appreciable fret signal. in the presence of grk2, however, gβγ changes its position relative to the µor in a way that allows the interaction of the grk2-gβγ complex with the µor to be detected by fret. similarly, we measured fret between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor in the absence and presence of grk2 and compared the kinetics with the kinetics of grk2 binding and unbinding to these receptors. in both cases, we found that grk2 and gβγ in the presence of grk2 associate and dissociate from these receptors with comparable kinetics. our results suggest that ligand efficacy for µors is already apparent on the level of receptor-grk interaction. institute of pharmacology, university medical center göttingen, ag lutz, göttingen, germany introduction: the monomeric gtpase rhoa is dysregulated in heart disease and in vivo models provide evidence of rhoa signaling being involved in the progression of cardiac fibrosis and hypertrophy. how rhoa is regulated within this context on a cellular level is not defined. objective: the goal of this study was to analyze rhoa activation in adult cardiomyocytes (amcm) from normal and diseased mouse hearts in response to g protein-coupled receptor activation. this project also aimed at providing new insight into the dependence of rhoa localization and activation on the signaling organizing caveolae in neonatal as well as adult cardiomyocytes and engineered heart muscles. methods: cardiomyocytes from sham-operated c57bl/6 mice, from mice subjected to transverse aortic constriction (tac) or from neonatal rats were either directly fixed after isolation or cultured in the presence or absence of methyl-b-cyclodextrin (mβcd). for analyses of rhoa activation cells were treated with endothelin-1 (et-1) for 90 sec. cells were prepared for immunofluorescence analysis or lyzed for immunoblotting. imaging was performed using confocal microscopy. effects of mβcd were further studied in 3d engineered heart muscles (ehm) made from total neonatal rat cardiac cells. the contractile function of ehm was assessed in the organ bath and cells were studied in sections by immunofluorescence analysis. results: in amcm from sham mice active rhoa mainly localizes at the sarcolemma and is augmented in response to et-1 treatment. in tac-amcm the basal level of active rhoa is increased and surprisingly et-1 had no further effect. immunoblot analysis demonstrated that in tac-amcm rhoa expression was per se higher and the major caveolae protein caveolin-3 was reduced. to test the influence of caveolae on rhoa activation and expression, we treated nrcm with mβcd and found the expression of rhoa as well as of rhoa target genes ccn1 and ccn2 to be moderately up-regulated after 24h. in addition, an intensified longitudinal alignment of sarcomeric actin fibers was detectable, which could also be seen in mβcd-treated ehm. however, mβcd had no effect on ehm twitch tension but increased the resting tension compared to control. we further treated amcm with mβcd and found rhoa expression to be increased and its activity less sensitive to et-1 treatment. finally, we could show that the perinuclear localization of cav3 and rhoa was strongly reduced after mβcd treatment. whereas g-protein coupled receptors (gpcrs) have been long believed to signal through cyclic amp only at cell surface, our group has previously shown that gpcrs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent camp production (1). this phenomenon, which we originally described for the thyroid stimulating hormone receptor (tshr) in thyroid cells, has been observed also for other gpcrs (2) (3) (4) . however, the intracellular compartment responsible for such persistent signaling was insufficiently characterized. the aim of this study was to follow by live-cell imaging the internalization and trafficking of tshr, tsh and effector proteins in thyroid cells. mouse primary thyroid cells were transfected with fluorescent-protein tagged tshr, g-proteins, nanobody specific for active g-proteins and/or subcellular markers by electroporation, stimulated with fluorescently labeled tsh and visualized using highly inclined thin illumination (hilo) microscopy. our results suggest that tsh is internalized in complex with its receptor and they traffic retrogradely via the trans golgi network (tgn). while we could not find any evidence of internalized tsh/tshr complexes activating g-proteins in early endosomes, we show that tsh/tshr complexes meet the intracellular pool of gαs in the tgn and activate it, as visualized in real-time by a nanobody specific for active gαs. upon acute brefeldin a-induced golgi collapse, the retrograde trafficking of tsh/tshr via tgn is hindered. bulk tsh stimulations in primary mouse thyroid cells isolated from transgenic mice expressing the camp sensor, epac1-camps, also show a significantly reduced camp production in the presence of brefeldin-a. these data provide evidence that internalized tsh/tshr complexes meet and activate g-proteins at the tgn, which might serve as the main platform of persistent camp signaling after receptor internalization. objective: sphingosine 1-phosphate (s1p) is generated by sphingosine kinase (sk)-1 and -2 and acts mainly as an extracellular ligand at five specific g protein-coupled receptors, denoted s1p 1-5 . after activation, s1p receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration methods and results: here we demonstrate that dexamethasone treatment lowered s1p 1 mrna and protein expression levels in rat mesangial cells measured by taqman® and western blot analyses. this effect was abolished in the presence of the glucocorticoid receptor antagonist ru-486. in addition, in vivo studies showed that dexamethasone downregulated s1p 1 expression in glomeruli isolated from c57bl/6 mice treated with dexamethasone (10 mg/kg body weight). functionally, we identified s1p 1 as a key player mediating s1p-induced mesangial cell migration. using boyden chamber assays, we could show that dexamethasone treatment significantly lowered s1p-induced migration of mesangial cells. this effect was again reversed in the presence of ru-486. conclusion: we suggest that dexamethasone inhibits s1p-induced mesangial cell migration via downregulation of s1p 1 . overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells (koch et al., biol. chem. 2015; 396: 803-12) . the g protein-coupled receptor mrgd is a receptor for angiotensin-(1-7) involving g alphas , camp, and phosphokinase a rationale: angiotensin (ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental ang ii within the renin-angiotensin system. although it is well-accepted that the g-protein coupled receptor mas is a receptor for the heptapeptide, the lack in knowing initial signalling molecules stimulated by ang-(1-7) prevented final verification of ligand/receptor interaction as well as the identification of further hypothesized receptors for the heptapeptide. objective: the study aimed to identify a second messenger stimulated by ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. we identified camp as the second messenger for ang-(1-7). the heptapeptide elevates camp concentration in primary cells such as endothelial or mesangial cells. using camp as readout in receptor-transfected hek293 cells, we provided final pharmacological proof for mas to be a receptor for ang-(1-7). more important, we identified the g-protein coupled receptor mrgd as a second receptor for ang-(1-7). consequently, the heptapeptide failed to increase camp concentration in primary mesangial cells with genetic deficiency in both mas and mrgd. furthermore, we excluded the ang ii type 2 receptor at2 as a receptor for the heptapeptide, but discovered that the at2 blocker pd123119 can also block the mas and mrgd receptors. conclusions: our results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for the protective ang-(1-7) but excluding the at2 receptor, and by enforcing the revisit of such publications which concluded at2 function by using pd123119 as a specific at2 blocker. members of the g protein-coupled receptor (gpcr) superfamily are integral membrane proteins that are activated by extracellular ligands and induce cell signaling via g proteins and other adaptor proteins. rhodopsin, the prototypical gpcr that mediates vision, is activated by photons that isomerize its covalent ligand. spectroscopic analyses of the cognate agonist retinal allow a detailed description of rhodopsin dynamics at submillisecond resolution. using rhodopsin as a model, it has been demonstrated that receptor activation, i.e. a switch from the fully inactive to the fully active state, occurs within 1 ms 1 . activation kinetics of other receptors have been studied mainly using fluorescence resonance energy transfer (fret) which allows kinetic studies with high resolution in living cells 2 . in contrast to rhodopsin, activation constants of several gpcrs using agonist superfusion have been determined to range between 30-80 ms 3 . however, it is unknown if all gpcrs with diffusible ligands are really activated on a longer timescale or if ligand diffusion to the binding site is rate limiting. in this study, we intend to overcome ligand diffusion by using photodestruction of caged ligands. we monitor activation-related conformational changes of homodimeric metabotropic glutamate receptor 1 (mglur1) sensors by fret after uncaging of an inert glutamate derivative. 4-methoxy-7-nitroindolinyl-l-glutamate (mni-glutamate) is a caged derivative of glutamate that does not activate mglur1s. upon pre-incubation of hek-tsa cells expressing both cfp-and yfp-tagged mglur1 protomers with mniglutamate, a short uv laser pulse releases active l-glutamate close to the receptor binding site. we demonstrate very rapid mglur1 activation kinetics and this allows us to study the process of signal transduction of this homodimeric gpcrs opioids are still the mainstay of modern pain treatment. most of the clinically established substances primarily exert their effects via the µ-opioid receptor (mor). however, many side effects such as tolerance, constipation and respiratory depression limit their therapeutic use. the efficacy of mor agonists in the treatment of chronic pain is unsatisfactory. in general analgesic effects can be mediated by all four members of the opioid receptor family. the nociception receptor (nop) is the latest member of the opioid receptor family. there is a rapidly growing interest for the development of novel nop and combined mor/nop agonists. the aim of this developement is novel therapeutic agents with improved analgesic characteristics and less classical mor-mediated side effects. here we used buprenorphine as a clinically established opioid which exerts its effect on multiple opioid receptor subtypes. recently, nalfurafine, a potent kappa-opioid receptor (kor) agonist was granted by japanese authorities for the treatment of uremic pruritus. even though kor agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. rudolf-virchow-zentrum für experimentelle biomedizin, würzburg, germany g protein-coupled receptors (gpcrs) belong to a superfamily of cell surface signaling proteins that mediate many physiological responses to hormones and neurotransmitters. they represent the prime targets for therapeutic drugs in healthcare. however, due to the limited knowledge about the pharmacology of the majority of gpcrs, only few of them are employed as therapeutic target. in our lab, the activation kinetics of the α 2aadrenergic receptor, among others receptors, has been extensively studied in single cell assays (1) (2) . the activation kinetics of the labeled α 2a -adrenergic were monitored by förster resonance energy transfer (fret). the goal of our study is to design a sensor to monitor receptor activation kinetics in high throughput screening assays. for the proof of concept, we used the α 2a -adrenergic receptor as a prototype. to optimize the fret efficiency we exchanged the previous acceptor (yfp) with the halotag technology (3) . additionally, we used halotag in combination with the nanoluc (4) to explore the possibility of using bret as a high-throughput approach to monitor receptor activation kinetics. the fret-based sensor α 2a -halo/cfp showed an increase in fret upon application of the full endogenous agonist norepinephrine with an ec50 value in accordance with the previously published data. this suggests the functionality of the fret-based α 2a -halo/cfp sensor. similar results were obtained with the α 2a -adrenergic receptor bret-based sensor. in contrast to the full agonist norepinephrine, the inverse agonist, yohimbine, decreased the ratio in both, fret as well as bret-based α 2aadrenergic receptor sensors. this suggests the sensitivity of the methods to discriminate among agonist (increased ratio) and antagonist (decreased ratio) induced receptor kinetics. our results show the feasibility of using halotag to monitor receptor activation via fret in a single cells format and halotag, in combination with nanoluc, can be used to monitor receptor activation in high-throughput format. , c. hoffmann the homologous visual arrestins) that has recently been solved by x-ray crystallography. here we investigated both the interaction with gpcrs and β-arrestin conformational changes in real time and in living cells with a series of fret-based β-arrestin2 biosensors. upon stimulation, β2-adrenergic receptors bound β-arrestin2 with a time constant τ = 1.3 ± 0.17 s, indicating that β-arrestin2 binding rapidly terminates their gprotein signaling. we observed a subsequent receptor-mediated conformational change in β-arrestin2 with a τ = 2.2 ± 0.22 s. stimulation of β2-adrenergic vs. m2 muscarinic or ffa4 receptors resulted in different patterns of conformational changes in the various β-arrestin2 sensors and also in downstream kinase signaling, revealing receptor-specificity in β-arrestin2 activation. upon agonist removal, first the interaction (delay = 1.9 ± 0.51 s) and only then the active state of β-arrestin2 (delay = 4.2 ± 0.85 s) were reversed. accordingly, β-arrestin localization at the cell membrane lasted much longer than the direct interaction with β2-adrenergic receptors. our data indicate a rapid, receptorspecific, two step binding and activation process between gpcrs and β-arrestins; they further suggest that β-arrestins remain active following dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. thus, gpcrs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signaling. patghogenic clostridium difficile produce two large glucosyltransferases, tcda and tcdb, which are the main pathogenicity factors. the cytotoxin tcdb is about 1,000 fold more potent than tcda. tcda and tcdb are a/b structure toxins exhibiting an enzymatically active (a) domain and a binding/translocation domain (b) to deliver the active glucosyltransferase domain into the cytosol of host cells. beside its glucosyltransferase activity by which the substrate proteins mainly of the family of rho gtpases are inhibited, tcdb has additional cytotoxic effects that are independent of rho glucosylation. to investigate the mechanism by which tcdb induces early cell death, we applied chimeras of tcdb from different toxinotypes where different glucosyltransferase domains were combined with different translocation domains. to this end we cloned tcdb from strain vpi10463 (historical strain), strain 1479 (serotype f, variant tcdbf), strain r20291 (hypervirulent strain, ribotype o27), and strain r9385 (hypervirulent strain with tcdbf characteristics). we were able to investigate the impact of the glucosyltransferase domains with different substrate specificity when translocated into the host cell by identical translocation domains. furthermore, we tested different translocation domains to deliver the same glucosyltransferase domain into host cells. we found that the glucosyltransferase domain of tcdbf (strain 1470) is less cytotoxic with respect to early cell death mediated by reactive oxygen species than that from reference strain vpi10463. in addition, the translocation domain also showed significant impact on cytotoxicity, probably by faster intracellular delivery of the gtd. by using glucosyltransferase deficient mutants where the highly conserved dxd-motif was changed to nxn, we were able to show that glucosylation of rho gtpases counteracts the cytotoxic effect, since the mutants were more cytotoxic than wildtype toxins. in conclusion, the cytotoxicity of tcdb mainly depends on the translocation efficiency into the host cell and on the kinetic of glucosylation of their substrate gtpases. thus, sensitivity of target cells towards cytotoxic effect also depends on receptor abundancy and intracellular status of rho gtpases, whereas the cytopathic effect, i.e. cell rounding, is predominatly determined by the substrate specificity. introduction: p63rhogef activates the g protein-coupled receptor (gpcr)-mediated induction of rhoa in different cells. however, its role in cardiac fibroblasts (cf) is not defined yet. thus we studied its localization and function in cf in 2d and 3d culture experiments. methods: neonatal rat cardiac fibroblasts (nrcf) and adult ventricular fibroblasts (amcf) from wild type mice and p63rhogef-knockout mice were adenovirally transduced for 48 to 72 h with recombinant adenoviruses or directly used. for 2d studies the cells were treated with angiotensin ii (ang ii). the location of the involved signaling components, rhoa activation and down-stream effects were studied by confocal microscopy and biochemical analyses. in addition, cf were used to prepare cf-containing engineered connective (ect) or muscle (ehm) tissues. results: we could show that p63rhogef locates at the plasma membrane, adjacent to the golgi apparatus and at the base of primary cilia. in accordance, p63rhogef regulates in response to ang ii the expression and secretion of the connective tissue growth factor (ctgf) in nrcf involving the serum response factor. in ect, p63rhogef increases the stiffness of these tissues and in ehm containing cf expressing gain-and-loss-of-function p63rhogef variants it influences the contractility. interestingly, the increase in ect stiffness was independent of p63rhogef's regulatory function of ctgf, as overexpression of ctgf in cf had no impact on ect properties arguing for a more general role of p63rhogef in auto-and paracrine signaling. moreover, our data on amcf with a genetic deletion of p63rhogef implies that p63rhogef is not only a transducer of gpcr-dependent rhoa activation as its loss led to an increase in rhoa expression accompanied by an increase in rhoa-dependent gene expression suggesting a role of p63rhogef in the feedback regulation of this signaling cascade. conclusion: in summary, our data show that p63rhogef regulates auto-and paracrine signaling in cardiac fibroblasts. the atrophic rhinitis is characterized by a drastic destruction of nasal turbinate bones in different animals. it leads to shortening and twisting of the snout and a growth retardation of young pigs. this bone degradation is induced by pasteuralla multocida toxin (pmt), a toxin produced by p. multocida serogroups a and d. this destructive effect indicates an interaction of pmt with bone cells like osteoclasts and osteoblasts. we demonstrated that pmt stimulates the differentiation of osteoclasts and inhibits the differentiation of osteoblasts in a gq-dependent mechanism. the underlying molecular mechanism of the toxin is the deamidation of an essential glutamine residue in the αsubunits of heterotrimeric g proteins, which results in the constitutive activation of the g protein. until now only the function and the pmt-dependent effects on osteoblasts and osteoclasts were studied in detail, but there is also a third important cell type in bone, the osteocytes. osteocytes are discussed as the regulator of the bone turn-over by interacting with osteoclasts and osteoblasts e.g. via secretion of several osteoclastogenic and osteoblastogenic cytokines. therefore, we studied the effects of pmt on the function of osteocytes in more detail. we utilized an osteocyte like cell line and primary osteocytes isolated from tibiae and femurs from mice. the susceptibility of primary osteocytes and the osteocyte like cell line towards pmt was demonstrated by detection of toxin-induced deamidation of g proteins. we also observed a pmt-induced secretion of different cytokines, like rankl, il-6 and tnf-α, which are known to induce osteoclastogenesis or inhibit osteoblastogenesis. furthermore, we studied the underlying signal transduction pathways and other pmtinduced effects on osteocytes, like morphological changes. in summary, we show that pmt acts on osteocytes by stimulating heterotrimeric g proteins. this might have impact on overall bone metabolism due to modulation of osteoblast and osteoclast activity. pasteurella multocida is an opportunistic pathogen often residing in the nasal pharyngeal space of animals. one virulence factor of p. multocida serogroups a and d is the protein toxin pmt (p. multocida toxin), which is the causative agent of atrophic rhinitis characterized by degradation of nasal turbinate bones in pigs and other animals. on the molecular level, pmt activates distinct members (g q/11 , g 12/13 and g i ,) of heterotrimeric g proteins leading to a modulation of bone metabolism: the toxin stimulates osteoclastogenesis but blocks osteoblastogenesis which results in bone loss. this mechanism of action of pmt might be exploited to counteract the human disease fibrodysplasia ossificans progressiva (fop), a rare and highly disabling disorder of extensive heterotopic bone growth. the underlying cause of fop is a point mutation in the activation domain of acvr1 (r206h), a bmp (bone morphogenic protein) type 1 receptor. this mutation leads to an inflated bmp-signaling and heterotopic osteoblastogenesis. here, we report that c2c12 cells, a mouse myoblast cell line often used as a fop model, are susceptible to pmt intoxication. pmt induces deamidation of g proteins in these cells. furthermore, pmt very efficiently inhibits bmp4-induced osteoblast differentiation in c2c12 cells. this has been shown by measuring alkaline phosphatase expression which is an early marker of osteoblast differentiation. additionally, the impact of pmt on acvr1 r206h induced osteoblastogenesis will be investigated and the involved cellular signaling pathways will be characterized in detail. the data indicate that activation of heterotrimeric g-proteins might be a rationale for pharmacological therapy of fop. p63rhogef is an activator of the monomeric gtpase rhoa and was shown to be expressed in the heart. in cardiac fibroblasts and smooth muscle cells, p63rhogef regulates rhoa in response to angiotensin ii and controls the actin cytoskeleton as well as protein expression and secretion. its role in cardiomyocytes, however, has not been elucidated so far. cardiomyocytes were isolated from neonatal rat hearts (nrcm), wild type mouse hearts (wt-amcm) and homozygous (ko-amcm) p63rhogef knockout mouse hearts. the cells were either directly fixed or adenovirally transduced for 48 h in culture. for activation of the g q/11 -signaling the cells were treated with endothelin-1 (et-1), angiotensin ii (ang ii) or phenylephrine (pe) for 90 s. rhoa activation was assessed by affinity binding or with a specific active-rhoa antibody. other proteins were detected by immunoblot or immunofluorescence analysis with subsequent confocal imaging. in nrcm p63rhogef is involved in the regulation of the et-1-induced rhoa activity and thus increases the expression and secretion of the rhoa target gene ctgf. in accordance, p63rhogef was found to be localized at the sarcolemma as well as in intracellular membrane compartments. the strongest co-localization was detected with the kdel-receptor 3 (kdelr3) which resides in the endoplasmatic reticulum membrane. next, we analyzed rhoa activation in wt and ko-amcm and could show that a loss of p63rhogef led to an increase in basal rhoa activity and an uncoupling from the gpcrs. interestingly, in the ko-amcm caveolin-3 the major component of caveolae, in which several gpcrs are clustered, was reduced in its expression and a shift in localization from transverse to longitudinal membrane tubules was found, arguing for a role of p63rhogef in intracellular protein transport. in accordance, golgi apparatus particles, which were demonstrated to play role in caveolae formation, were reduced in size in ko-amcm. to further address the role of p63rhogef in the transport of membrane proteins, we overexpressed p63rhogef in wt-amcm and could show that this led to an increase in the expression of the kdelr3 and its co-localization with p63rhogef in the perinuclear region and at the sarcolemma. no sarcolemmal localization of kdelr3 was found in control-transduced cells. further, p63rhogef was localized adjacent to golgi apparatus particles which were similar reduced in size as detected in the ko-amcm. finally, we expressed the dominant negative construct p63dn and detected similar changes with respect to kdelr3 localization and golgi structure suggesting that this regulatory function of p63rhogef is not dependent on its activity. conclusion: p63rhogef mediates the activation of rhoa from gpcrs coupled to g q/11 proteins. moreover, it has a function in intracellular transport and distribution of membrane proteins independent of its activity. universität bonn, pharmacology and toxicology section, bonn, germany g protein signaling is a means allowing cells to quickly respond and adapt to environmental changes. four major g protein classes (gs, gi/o, gq/11, g12/13) exist in mammals and these must suffice to convey signals from about 800 g protein-coupled receptors to the cell interior. as such, g proteins receive, interpret, and finally route the gpcr signals to diverse sets of downstream target proteins and thereby permit cells to respond to their ever changing environment. 1 understanding contribution of individual g protein classes or even isoforms to complex signaling networks in living cells requires capacity to activate or inactivate proteins with great precision and selectivity. one approach towards inactivation of g protein function is via chemical inhibition. however, "true specificity" of chemical inhibitors for their associated targets may often be debated. in this study we posit that fr900359, a cyclic depsipeptide isolated from the leaves of ardisia crenata, may clearly be designated as "truly specific" for inhibition of gq signaling. using a broad set of complementary methods based on label-free holistic cell sensing, classical endpoint assays, and bioluminescence resonance energy transferbased g protein biosensors we assign exceptional selectivity to fr for inhibition of gq/11/14 over all other mammalian isoforms ("on-target effects"). in holistic label-free recordings using hek293 cells that lack functional gq/11 alleles by crispr-cas9 genome editing, bona-fide gq stimuli were undetectable. however, reintroduction of gq into the knockout background was required and sufficient to fully restore both, agonist responses and their inhibition by fr. moreover, fr was completely ineffective in cells lacking gq/11 using phenotypic assays that examine basic cellular functions such as cell growth, viability, morphology and expression of housekeeping genes ("off-target effects") 2 . from these results we conclude that fr is of outstanding value as molecular probe to unravel contribution of gq signaling in complex biological processes in vitro, ex vivo and in vivo. just as pertussis toxin, applied world-wide by numerous laboratories to diagnose signaling of gi/o proteins, we anticipate fr to stand out at least equally for investigations into the biological relevance of gq. binary actin adp-ribosylating toxins like c. perfringens iota toxin and c. difficile transferase cdt cause depolymerisation of the cortical actin cytoskeleton, induce the formation of microtubule-based cell membrane protrusions and redirect rab-dependent intracellular traffic (schwan et al. 2009 ). here, we employed the model of toxin-induced protrusions to study the formation of cilia. we found that toxin-induced microtubule-based protrusion formation at the cell membrane depends on recruitment of septins, which are highly conserved, small gtpbinding proteins. similar to toxin-caused protrusions, septins are also recruited to the site of ciliogenesis. inhibition of septins by shrna-based knockdown inhibits ciliogenesis as well toxin-induced protrusion formation. septins are suggested to be involved in exocytotic processes, which are important for ciliogenesis and also for toxin-induced protrusion formation. accordingly, translocation of septins is accompanied by a recruitment of rab proteins and proteins of the exocytotic machinery. the data indicate that septins function as a scaffold at the base of cellular processes like cilia and toxin-induced protrusions, organizing the cross-talk between the actin cytoskeleton and microtubules to regulate the vesicle traffic-and exocytotic machinery. hypervirulent clostridium difficile strains are associated with increased morbidity and mortality. these strains produce the actin-adp-ribosylating clostridium difficile toxin cdt. cdt depolymerizes the actin cytoskeleton, causes formation of microtubule-based protrusions and increases pathogen adherence. septins are essential for cdt-induced protrusion formation. sept2, 6, and 7 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. the septins are a prerequisite for protrusion formation. the inhibitor forchlorfenuron or knock-down of septins inhibit protrusion formation. septins colocalize with active cdc42 and its effector borg which act as up-stream regulators of septin polymerization. microtubules interact with septin structures. precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to microtubule plus end tracking protein eb1 thereby guiding incoming microtubules. the data indicate that cdt hijacks conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, cdc42, borgs and restructuring of the actin cytoskeleton. the zebrafish danio rerio has become an important vertebrate model organism for a wide range of scientific questions [1] . current studies are mainly focused on development, genetics and disease for which the zebrafish is particularly well suited due to its small size, rapid development, short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [1] . hitherto, nothing is known about the composition and endogenous level of different cnmps in various developmental stages and organs of danio rerio. therefore, we used the zebrafish in our study as a vertebrate model to characterize systematically the temporal-and organspecific occurrence(s) of all cnmps including cump in vivo. cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. we observed specific cnmp patterns in developmental stages and different organs from adult zebrafish, which is in support of the hypothesis of a distinct cnmp signaling code [2] . camp, cgmp and cump were present in tissue samples of both developmental stages (embryos at 24 hours post fertilization, larvae at 5 days post fertilization) and within all harvested organs. remarkably, these three cnmps were the only ones detected in the brain. camp concentration of entrails as well as camp and cgmp concentrations in the brain were similar to those previously described in mouse tissues [3] . ccmp was detected throughout development and was present in all organs except the brain. the identity of ccmp and cump in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (hplc-ms/tof). thus, we unequivocally show for the first time that cump occurs in vertebrates. furthermore, we detected cimp in several developmental stages of the zebrafish, and observed the highest concentrations in testes and heart, but we were unable to unequivocally identify cimp via hplc-ms/tof. in the zebrafish, sac is evolutionarily not conserved and absent, since a search in the ncbi gene data base revealed no entry for sac (also referred to as ac10). therefore, sac can be excluded as cump-and ccmp generator in this system and sgc remains as the only bona fide cump-and ccmp generator in the zebrafish. to test this hypothesis, the effects of no donors, sgc stimulators and sgc activators on cump levels in zebrafish should be examined in future studies. recently, induction of apoptosis in mouse lymphoma cells by ccmp-am has been described [4] . thus, it would be interesting to examine the effect of ccmp-am on zebrafish embryos in future studies as well. [1] seifert, r.: ccmp and cump: emerging second messengers, trends in biochemical sciences 40, 8-15 (2015) . ccmp, 3',5'-cump and 3',5'-cimp were ineffective. to further characterize the action of 3',5'-cgmp on hut-78 cells, we determined apoptosis (propidium iodide/annexin v staining) and proliferation (carboxyfluorescein succinimidylester staining). 3',5'-cgmp significantly increased apoptosis (ec 50 = 75 µm) and inhibited proliferation (ec 50 = 63 µm) of okt3-activated hut-78 cells. interestingly, also 2',3'-cgmp exhibited comparable effects on apoptosis and proliferation with ec 50 values of 92 µm and 75 µm, respectively, while 2',3'-camp, 2',3'-ccmp and 2',3'-cump were ineffective. this indicates that the pro-apoptotic and antiproliferative action of cgmp does not depend on the position of the phosphodiester bond. we also tested 3',5'-cgmp degradation products under the same experimental conditions and found that both 5'-gmp and guanosine increased apoptosis and inhibited proliferation with ec 50 -values between 50 and 100 µm. by contrast, adenosine did not influence cell growth and viability, suggesting that adenosine receptors are not involved in the observed effects. our results suggest that the guanosine moiety is responsible for the pro-apoptotic and antiproliferative effects of 3',5'-cgmp, 2',3'-cgmp, 5'-gmp. it has been reported earlier that guanosine is toxic to jurkat cells, another t cell lymphoma cell line [1]. 3',5'-cgmp may be hydrolyzed by an ekto-phosphodiesterase on the cell surface of hut-78 cells (e.g. enpp1), yielding 5'-gmp, which could be further degraded to guanosine by the 5'-ekto-nukleotidase cd73. a similar pathway may lead from 2',3'-cgmp to guanosine. a previous analysis of phosphodiesterases (pdes) revealed that the dual-specific pde isoforms 3a and 3b as well as the cgmp-selective pde9a also degrade the emerging second messenger cump [1, 2] . we analyzed the enzyme kinetics of pde3b-mediated cump-hydrolysis using recombinant gst-tagged pde3b and a highly sensitive and specific hplc-ms/ms method. our data show that pde3b is a low-affinity enzyme for cump with a cump k m -value of >100 µm. the pde3-selective competitive inhibitor milrinone inhibited pde3b-mediated cump degradation, suggesting that cump binds to the catalytic center. pde3b is highly expressed in adipose tissue [3, 4] . thus, we differentiated murine 3t3-l1-mbx-fibroblasts into adipocytes and analyzed differentiation-dependent alterations of pde3b expression and basal cnmp-concentrations. in both differentiated and undifferentiated 3t3-l1 cells cump and ccmp were detected in addition to the established second messengers camp and cgmp. differentiation to adipocytes reduced camp and cgmp by 66 % and 60 %, respectively, while ccmp was reduced by 78 % and cump even by 85 %. these findings suggest that cump plays a distinct role in adipocyte differentiation. the cump-hydrolyzing pde3b was upregulated ~1000-fold on mrna level after adipocyte differentiation, which may contribute to the observed reduction of basal cump concentrations. we currently investigate the potential biological role of cump in differentiation and lipolysis experiments, analyzing the effects of the membrane-permeant cumpacetoxymethyl ester cump-am. in future experiments, we will also analyze the enzyme kinetics of pde9a-mediated cump hydrolysis. pde9a is the first example of a cgmp-"specific" cump-hydrolyzing pde. background: cgmp and camp are cyclic nucleotide messengers relevant to many physiological and pathophysiological conditions. live-cell imaging with fret-based biosensors is a powerful method to study the spatiotemporal dynamics of cgmp and camp under close-to-native conditions. however, with the existing biosensors it is difficult to resolve potential membrane-associated cgmp microdomains and to monitor cgmp and camp signals in parallel in the same cell. we have generated novel versions of the "green" cfp/yfp-based cytosolic cgmp biosensor, cgi500. they comprise a "green" membrane-targeted version (mcgi500) and a "red" variant (red cgi500) that contains the fluorophores tsapphire and dimer2. methods: the sensors were expressed and characterised in primary vascular smooth muscle cells (vsmcs). intracellular cgmp was elevated in intact vsmcs by application of a nitric oxide donor or natriuretic peptides, and the sensor's sensitivity to each stimulation and its signal-to-noise ratio were determined. to test each sensor's sensitivity and specificity for cgmp versus camp, sensor-expressing cells were permeabilised with β-escin and exposed to defined concentrations of cyclic nucleotides. results: the original cgi500 sensor showed a good signal-to-noise ratio, an ec 50 value of ≈1.1 µm for cgmp, and a high selectivity for cgmp over camp (>100-fold). flincg3, a non-fret-based cgmp sensor, showed similar properties as cgi500. the new membrane-targeted mcgi500 and the new red cgi500 displayed ec 50 values of ≈0.4 µm cgmp and a high selectivity for cgmp over camp (>300-fold). in vsmcs, the red cgi500 showed a better signal-to-noise ratio than the previously described "red" cgmp sensor, red cges-de5. the "green" fret-based camp sensor, epac1-camps, showed a signal-to-noise ratio comparable to that of cgi500, an ec 50 value of ≈3 µm for camp and a selectivity for camp over cgmp of ≈6-fold. finally, imaging of cells expressing both the epac1-camps and the red cgi500 demonstrated the feasibility of combined visualisation of camp and cgmp signals in the same cell. the new cgmp biosensors should be useful for a broad spectrum of applications requiring real-time monitoring of cgmp signals. for example, mcgi500 would be useful to investigate membrane-associated cgmp compartments and red cgi500 to study the crosstalk between cgmp and camp signalling in living cells and tissues. the cgmp-system is a major regulator of blood pressure. cgmp-dependent protein kinases (cgks), located in the smooth muscle layer of vessels, enable cells to dilate and therefore cause a decrease in blood pressure (bp). to the contrary, the reninangiotensin-aldosteron-system (raas) acts as opponent and causes an increase in bp; furthermore, it influences fluid-electrolyte balance. renin, which is secreted from reninproducing cells located in the juxtaglomerular apparatus (jga), is the key regulator enzyme in this system. pharmacological inhibition of the raas, e.g. via ace-inhibitors or at1-receptor-antagonists, is a powerful tool to treat hypertension, but chronically challenges this endocrine system, resulting in an enhancement of renin expression. this is caused by an increased number of renin-expressing cells (the so-called reninrecruitment), which derive from a reversible metaplastic retransformation of extraglomerular and smooth muscle cells of afferent arterioles. next to regulation of renin-function via camp/pka, it has been shown that enos-derived no supports this recruitment via activation of sgc and subsequent generation of cgmp [1] . whether this causes an activation of cgks is not known. these enzymes exist in 3 different isoforms, cgkiα, cgkiβ und cgkii. in contrast to the β-isoform, cgkiα (as well as cgkii) is highly expressed in the jga [2] , [3] . therefore, we analyzed, whether cgkiα also plays a role regarding renin synthesis, secretion or recruitment. to characterize the function of cgkiα in jga-cells, we generated renin-cell specific cgkiα-knockout mice (ren cre-cgki fl/fl) and stimulated renin recruitment via administration of a low salt diet (0.02% na + ) and enalapril (10mg/kg/d) for 3 weeks. we analyzed blood pressure, mrna and renal protein content of renin and cgkiα, plasma renin activity and renin recruitment. furthermore, we activated the cgmp-system in these mice using bay41-8543, a sgcstimulator, and re-analyzed the above-mentioned parameters. our results indicate that cgkiα could be an additional system supporting renin recruitment but is not a crucial pre-requisite. in contrast, the basal renin concentration and activity appears to be downregulated in ren cre-cgki fl/fl-mice, thus, cgki could be an important regulator of renin synthesis. universität regensburg, pharmakologie und toxikologie, regensburg, germany jaw1/lrmp (lymphoid-restricted membrane protein) is a type 2 membrane protein, localised to the cytoplasmic face of the endoplasmic reticulum. it encodes a 539 amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a cooh-terminal transmembrane domain [1] , [2] . jaw1 and irag have a limited homology throughout the length of the protein. the coiled-coil domain and the putative transmembrane anchor at the c-terminus of jaw1 and irag share the highest homology [3] , [4] . the coiled coil domains of irag and jaw1 are important for the interaction with ip 3 rs. as already known, irag forms a trimeric complex with cgkiβ and ip 3 r1 and gets phosphorylated by cgkiβ [5] . hence we examined if jaw1 is a new target protein of cgkiβ. the recognition site, where a substrate can be phosphorylated by cgki, is composed of the following amino acids: (k/r)(k/r)x(s/t). in the amino acid sequence of jaw1 possible phosphorylation sites can be found. our in vitro studies with jaw1 and cgki showed that jaw1 gets phosphorylated in a cgmp-dependent manner by cgkiβ. in contrast, jaw1 was not phosphorylated by cgkiα. furthermore, no stable interaction between jaw1 and cgkiβ was detected. to examine the importance of jaw1 in vivo, we generated a conditional knockout mouse. mating with a cmv cre mouse, resulted in an ubiquitous deletion of jaw1. mrna analysis and western blot analysis approved the deletion. the expression pattern revealed high expression in the thymus and weaker expression in the lung, spleen, colon, pancreas and the tongue. as already published by shindo et al., jaw1 was found in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. we confirmed these results by x-gal staining and mrna analysis. therefore, we decided to analyse if jaw1 influences taste perception. two bottle preference tests did not result in significant differences between wildtype and knockout mice, indicating that taste perception is not altered by jaw1. hence, the function of jaw1 in taste receptor expressing cells has to be further examined in future studies. the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (camp) and guanosine 3',5' cyclic monophosphate (cgmp) are well-characterized second messengers. both are generated by nucleotidyl cyclases and degraded by phosphodiesterases. several binding partners of camp and cgmp were already identified and functionally analyzed, e.g. camp-dependent protein kinase (pka) and cgmp-dependent protein kinase (pkg) as well as exchange protein activated by camp 1 and 2, hyperpolarization-activated cyclic nucleotide gated channels and phosphodiesterases. recent data indicate that the cyclic pyrimidine nucleotides cytosine 3',5'-cyclic monophosphate (ccmp) and uridine 3',5'-cyclic monophosphate (cump) also fulfill the criteria of second messengers [1, 2] . the interaction of ccmp with the regulatory subunits of pka (pkariα and pkariiα) has already been shown by using ccmpagarose [3] . additional ccmp-and cump-binding proteins such as calnexin (chaperone), myomegalin (phosphodiesterase-interacting protein) and akap9 (a-kinase anchoring protein) were identified by mass spectrometry analysis. to verify the interaction of ccmp and cump with these potential target proteins, ccmp and cump linked to biotin were used as another approach. the biotin-constructs exhibit lower steric interference than the ccmp-and cump-agarose matrices, which were previously used to confirm the binding of pkariα to ccmp and cump [4] . flag-tagged calnexin, flag-tagged myomegalin and myc-tagged yotiao (smallest splice-variant of akap9) were examined as potential ccmp and cump target proteins. hek293 cells were transiently transfected with the cdna of the respective proteins. the lysates of the protein-overexpressing cells were then incubated with ccmp-and cumpbiotin matrices and bound proteins were purified using strep-tactin® beads (iba). afterwards, the interaction of ccmp and cump with the potential binding partners was analyzed by western-blotting. a pkariα antibody was used as a positive control. analogous experiments were also performed using ccmp-and cump-agaroses. once the interaction between the cyclic pyrimidine nucleotides and the potential binding partners has been confirmed, deletion mutants will be cloned to localize the ccmp-and cump-binding area of the target proteins in further studies. axonal branching is essential for the correct formation of neuronal circuits and enables the simultaneous transmission of information throughout the body. in mice, the bifurcation of axons of sensory neurons at the dorsal root entry zone of the developing spinal cord depends on a cgmp signaling cascade that includes c-type natriuretic peptide (cnp), natriuretic peptide receptor 2 (npr2, also termed gc-b), and cgmpdependent protein kinase iα. in this study it was investigated, if a disturbance of cgmp signaling induced by manipulation of cgmp breakdown or cnp scavenging affects axon bifurcation of murine embryonic dorsal root ganglion (drg) neurons. rt-pcr screens, in situ hybridization, and fret-based cgmp imaging in living neurons revealed phosphodiesterase 2a (pde2a) as the major enzyme for degradation of cnp-induced cgmp in embryonic drg neurons. interestingly, cgmp measurements and dii labeling of pde2a knockout embryos indicated that a strongly elevated concentration of cgmp does not impair sensory axon bifurcation of drg neurons in vivo. the natriuretic peptide receptor 3 (npr3) was found to be expressed in the roof and floor plate of the spinal cord as well as in the dorsal roots of e12.5 embryos. because npr3 binds natriuretic peptides, but does not generate cgmp, it is thought to act as a natriuretic peptide clearance receptor. by scavenging cnp, npr3 could lower the activity of the cnp-npr2-cgmp signaling cascade in drg neurons. in the absence of npr3, the majority of sensory axons showed normal bifurcation, but »13 % of the axons turned only in rostral or caudal direction. this study shows (1) that pde2a is important for the degradation of cgmp in embryonic drg neurons, (2) that the bifurcation of sensory axons in the spinal cord can tolerate high levels of intracellular cgmp in the absence of pde2a, and (3) in the central nervous system, no-dependent cgmp signalling is associated with many different developmental processes and brain functions, and plays an important role in memory consolidation and cognition. to analyse cgmp signals in primary cells, a knock-in mouse was generated which stably and ubiquitously expresses a fret-based cgmp indicator (cgi500). cultured cortical and hippocampal neurons were found to respond to exogenously applied no (gsno). in these cell types, endogenous no is mainly generated by the neuronal no synthase (nnos) isoform which requires a rise in intracellular calcium for activation of the no/cgmp-signalling cascade. here, we show that ampa-type ionotropic glutamate receptors were capable to induce cgmp response in cultured cortical and hippocampal neurons. surprisingly, amparinduced cgmp signals were independent of nmdar activation, as inhibition of nmdars with the nmdar antagonist d-apv (d-2-amino-5-phosphonopentanoic acid) did not block ampar-induced cgmp response. however, cgmp accumulation depends on no synthase activation as the nos inhibitor l-nna (ng-nitro-l-arginine) completely abolished cgmp accumulation. whether ampar-induced nos activation depends on calcium influx via calcium permeable ampars, vgccs (voltage gated calcium channels) or calcium release from intracellular stores will further be investigated in detail. cyclic adenosine monophosphate (camp) is an important and ubiquitous cellular second messenger. a dogma in signaling is that camp is distributed homogenously in the cell and that its concentration changes equally upon stimulation. in contrast, a large body of evidence suggests the existence of concentration gradients (so-called microdomains) of camp. in this regard, phosphodiesterases (pdes), the only enzymes which can degrade camp, have been suspected to be responsible for maintaining those gradients. however, how pdes establish camp gradients is entirely unknown. here, we measure local camp levels in hek cells and cytosolic fractions thereof using the camp fretsensor epac1-camps fused to a phosphodiesterase (pde4a1). we demonstrate the existence of low camp concentrations in close proximity to pdes and show that this gradient is maintained by pde hydrolytic activity. further we establish that camp gradients cannot be maintained solely on the basis of pde activity as the camp turnover is very slow. we provide evidence that pdes are structurally organized in yet unspecified 'microstructures' in which camp diffusion must be considerably slowed down. taken together, we suggest that camp gradients are established by pde hydrolytic activity in cellular regions with slow diffusion of camp. our study sheds light on the organization and maintenance of signaling compartments in cells. the influence of pde hydrolytic activity on camp gradients universität würzburg, pharmakologie und toxikologie, würzburg, germany phosphodiesterases (pdes) are a family of enzymes that degrade cyclic amp (camp) and cyclic gmp (cgmp) to their respective monophosphates. although several pdes have been shown to play an important role in a wide variety of physiological and pathological processes, their complexity and function in cell signalling is only beginning to be understood. it is especially astonishing that eukaryotes express more than 100 different pde isoforms while their single function is to degrade camp, cgmp or both. in recent years, a large body of evidence has suggested that pdes (especially isoforms of the pde families 3, 4 and 5) are key players in establishing signalling compartments. these so-called microdomains are yet unspecified regions in cells where the concentrations of camp and cgmp are higher or lower than in the bulk cytosol. in a companion abstract (bock & lohse) we provide evidence that camp nanodomains, i.e. regions of low camp concentrations, exist in cells in the direct vicinity of pde4. however, the mechanisms by which pdes establish and maintain camp gradients are largely unknown. here we study if establishing camp gradients is a general role of pdes and, moreover, if the size of camp nanodomains mainly depends on pde hydrolytic activity. by fusing an ultra-fast pde2a3 (v max = 120 µmol/min/mg) to a camp fret sensor (epac1-camps) we monitor camp concentrations in direct vicinity of pde2a3. in comparison to pde4, we show that pde2a3, albeit displaying a high camp turnover, only establishes a small camp gradient in both cytosol preparations of transfected hek cells and in living cells. interestingly, this gradient can be increased by deleting the nterminal regulatory domains while maintaining fast camp turnover. biochemical mapping of the camp gradient gives an estimate of the size of nanodomains. taken together, our data suggest that establishing camp gradients does not exclusively depend on pde hydrolytic activity. arglabin is a plant-derived sesquiterpene lactone used for cancer therapy in kazakhstan and russia. signaling pathways targeted by arglabin are poorly understood. we have isolated arglabin by using high performance liquid chromatography from a methanolic extract of artemisia glabella, a plant endemic in kazakhstan. mass spectrometric analyses confirmed the chemical structure and the purity of the isolated arglabin. in j774 macrophages, arglabin strongly induced accumulation of lc3 type ii protein in the absence of inflammasome activators, and also in cells activated with lps and cholesterol crystals. in addition, arglabin induced clustering of lc3-ii at autophagosomal membranes, as evidenced from its punctuated pattern in confocal microscopic images of arglabin-treated macrophages, which is a characteristic sign for autophagy. since autophagy activation leads to increased degradation of nlrp3 and pro-interleukin (il)-1β, we further analyzed whether arglabin inhibits the nlrp3 inflammasome. arglabin reduced expression of nlrp3 and pro-il-1β, inhibited activation of caspase-1, and release of mature il-1β by lps-and cholesterol-crystal-stimulated macrophages consistent with inhibition of the nlrp3 inflammasome. intraperitoneal injection of arglabin into female apoe2.ki mice fed a high fat diet resulted in significantly decreased plasma levels of proinflammatory il-1β. moreover, arglabin markedly reduced mean lesion areas in the sinus and whole aorta in mice. thus, arglabin may represent a new promising drug to treat diseases associated with inflammasome activation, e.g. atherosclerosis. this work was supported in part by a grant from the nouvelle société francophone d'athérosclérose (nsfa). recently, we found that activation of the proteinase-activated receptor (par) 2 stimulates renin release in the isolated perfused kidney model. therefore, we determined in the current experiments the response of plasma renin concentration (prc) to acute intraperitoneal administration of the par2 activating peptide sligrl (100µg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), losartan (3 mg/kg) and furosemide (40 mg/kg) in conscious wild-type (wt) and par2-deficient mice. prc was measured in plasma obtained by tail vein puncture. renal renin expression was determined by quantitative rt-pcr. renal protein expression was measured by immunohistochemistry. on a control diet (0.6% nacl), plasma renin concentration (in ng angiotensin i per ml per hour) was significantly lower in par2-deficient mice than in wild-type mice (190 ± 35 versus 380 ± 91). renin mrna expression was 50 ± 9% of wt. renin-expressing cells were located at the juxtaglomerular position and renal renin protein expression was lower in par2-deficient mice. as measured by tail-cuff method, systolic blood pressure was not different between par2 (-/-) and wt mice. administration of sligrl increased renin secretion about 6-fold (p<0.01). acute stimulation of renin release by furosemide, isoproterenol, losartan and hydralazine caused significant increases of plasma renin concentration in both par2 (-/-) and wt mice. the absolute changes (delta prc) were similar (3780 ± 770, 2230 ± 400, 2780 ± 70, 1390 ± 460 in wt, and 2320 ± 270, 2530 ± 250, 3010 ± 210, 1230 ± 470 in par2 (-/-)). in conclusion, chronic absence of par2 reduces basal renin expression and renin release. however, par2-deficiency does not alter renin release in response to typical stimuli for renin secretion. therefore, par2 does not appear to be a mandatory and specific requirement for acute regulatory responsiveness. increased myofilament ca 2+ sensitivity could be the underlying cause of diastolic dysfunction. we evaluated acute effects of epigallocatechin-3-gallate (egcg), which has been shown to decrease myofilament ca 2+ sensitivity, on cardiac myocyte contractility and force-ca 2+ relationship of skinned cardiac muscle strips in an hcm mouse model with left ventricular hypertrophy and both systolic and diastolic dysfunction. methods: the hcm mouse model used in this study carries a point mutation in the cardiac myosin-binding protein c gene at the homozygous state (mybpc3-targeted knock-in; ki). we isolated ventricular myocytes from adult ki and wt mice and analyzed sarcomere shortening and ca 2+ transients at 37 °c under 1 hz pacing using the ionoptix system in the absence or presence of egcg (1.8 µm). furthermore, force-ca 2+ relationships of skinned cardiac muscle strips of ki and wt mice were obtained ±egcg (30 µm). results: in baseline settings and absence of fura-2, ki cardiomyocytes displayed higher sarcomere shortening ( type-1 serine/threonine protein phosphatase (pp1) comprises a family of enzymes that dephosphorylate cardiac regulatory proteins, thereby modulating ca 2+ handling and contractility. all pp1 heterodimers possess a catalytic subunit, which is selectively inhibited by inhibitor 2 (i-2). it has been shown by our group that the heart-directed overexpression of a truncated, constitutively active form of i-2 resulted in an improved basal ca 2+ handling and contractility. in contrast, chronic pressure overload by transverse aortic constriction exacerbated the progression of cardiac remodeling and heart failure in transgenic mice. in the present study, we tested whether the overexpression of a full-length form of i-2, regulated by gsk3-dependent phosphorylation at thr 72 , resulted in comparable functional alterations using a model of induced heart failure. for this purpose transgenic (tg) and wild-type (wt) mice were subjected to chronic application of isoprenaline (iso, 30 mg/kg/d) via osmotic minipumps. iso-stimulated mice were compared to mice treated with 0.9% nacl (n=5). after one week of iso administration, cardiac hypertrophy was comparable in tg and wt. ca 2+ transients were measured in isolated, indo-1-loaded myocytes. the peak amplitude of [ca] i was reduced by 39% in tg nacl compared to wt nacl (p<0.05), whereas chronic iso application was associated with comparable effects in tg and wt. [ca] i decay kinetics were comparable in nacl-treated groups but hastened by 25% in tg iso compared to wt iso (p<0.05). consistently, sr ca 2+ load was diminished by 28% in tg nacl compared to wt nacl (p<0.05). chronic iso stimulation led to an unchanged sr ca 2+ content in tg and wt myocytes. biochemical analyses revealed that chronic βadrenergic stimulation was accompanied by a more than 4-fold higher phospholamban phosphorylation at ser 16 in tg (p<0.05). thus, these findings suggest that overexpression of i-2 is able to reduce the progression of heart failure by an improvement of myocyte ca 2+ handling. the ligand-activated farnesoid x receptor (fxr) is a nuclear receptor highly expressed in gastrointestinal and metabolic tissues, such as the duodenum, jejunum, ileum, colon and the liver, but also in lower amounts for instance in macrophages. the endogenous agonists for this receptor are bile acids with the primary bile acid chenodeoxycholic acid as the most active one. activation of fxr regulates the transcription of target genes relevant in bile acid homeostasis, glucose and lipid metabolism, liver protection, inflammation, and cancerogenesis. agonists for fxr have been discussed as possible therapeutic options for the treatment of obesity and the metabolic syndrome. atherosclerosis is the main pathology underlying cardiovasular diseases and often occurs side-by-side with the metabolic syndrome. cholesterol deposition and the formation of cholesterol-loaded foam cells from macrophages lead to the formation of atherosclerotic plaques. this can be prevented by stimulation of cholesterol efflux from macrophages. based on leoligin, a lignan-type secondary plant metabolite, naturally occuring in leontopodium alpinum cass., 168 derivatives were synthesized and subjected to a fxr pharmacophore-based in silico screening. testing of 56 virtual hits in a luciferase-based fxr transactivation assay yielded one compound with promising activity on fxr. moreover, the heterodimer partner of fxr, rxrα, was not activated by this leoligin derivative in a luciferase-based rxrα assay. in addition, this compound was able to increase cholesterol efflux in thp-1 macrophages without affecting cell viability. western blot experiments revealed an increase in atp-binding cassette transporter a1 (abca1) expression in human thp-1 macrophages by this leoligin derivative. the transporters abcg1 and scavenger receptor class b (sr-bi), which also play a key role in macrophage cholesterol efflux, will be investigated. moreover, the effect of the leoligin derivative on liver x receptor activation, the nuclear receptor responsible for upregulation of these transporters, has to be studied. based on this data, further characterization of the molecular mechanism underlying the described effects will provide valuable insights in a possible crosstalk between macrophage cholesterol efflux and fxr activation. background: rho-associated kinases rock1 and rock2 are serine/threonine kinases that are downstream targets of the small gtpases rhoa, rhob, and rhoc. rock1 and rock2 are known to play a pivotal role in the pathogenesis of myocardial fibrosis. however, their specific function in cardiac fibroblasts (cf) remains unclear. remodelling of the diseased heart results in the transition of fibroblasts to a myofibroblast phenotype exemplified by an increased proliferation, migration rate and synthesis of extracellular matrix (ecm) proteins. therefore, we sought to investigate whether rock protein signalling intermediates have an impact on cellular characteristics, intracellular protein expression and mechanical properties in cf and engineered tissues. methods: neonatal cardiac fibroblasts were isolated from wild type rats and downregulation of rock1 and rock2 by 75% was achieved by lentiviral transduction or transfection. wild type fibroblasts were treated with 10 μm fasudil or 3 µm h1152p for general rock inhibition and 3 µm slx-2119 for inhibition of rock2. protein expression and modification was determined by immunoblot analysis, gene expression by qpcr analysis, cf morphology and the localisation of cytoskeletal proteins by immunofluorescence analysis, cell proliferation by automated nuclei counting, cell migration on a planar surface by life cell imaging, and rigidity of engineered tissues by rheological measurement. results: our results show that both rock1 and rock2 influence cf morphology, gene expression, proliferation and migration. the knockdown and inhibition of rocks was associated with changes in cf morphology accompanied by a disorganization of higher-order actin structures including stress fibers and geodesic domes. moreover, the knockdown of rock1 and rock2 in cf increased adhesion velocity, whereas proliferation was attenuated. interestingly, downregulation of rock2, but not of rock1 led to a significantly decreased migration velocity and distance suggesting an isolated principle role for rock2 in cardiac fibroblast migratory behavior. analysis of a three dimensional engineered tissue model composed of cardiac fibroblasts (engineered connective tissue, ect) suggested that rocks are involved in the regulation and turnover of the extracellular matrix (ecm) and thus influence viscoelastic properties of engineered tissues. destructive tensile strength measurement in ect treated with rock inhibitors showed that rigidity was significantly reduced when compared to control tissues. rna sequencing of ect treated with the rock inhibitor h1152p and qpcr analysis of cf with a downregulation of rock1 and rock2 showed that both rocks are involved in the regulation of ecm proteins, such as collagens 4a2, 6a, and 8a1, biglycan, decorin, elastin and its respective degrading enzyme mmp12. conclusion: this study demonstrates that rock signalling controls myofibroblast characteristics of cf via remodelling of the cytoskeleton and the ecm. background: regulation and fine-tuning of gene transcription in cardiomyocytes (cms) is a centerpiece of cardiac development, function, and disease. in order to obtain authentic data, cell type-specific analyses are indispensable. recently, high-purity isolation protocols for cm nuclei were established [bergmann, exp cell res, 2011] and employed for detailed genetic and epigenetic studies on cardiac gene transcription [gilsbach, nat commun, 2014] . however, corresponding protein analyses, which bridge from transcriptional control to cm function are still lacking. therefore, we aimed to map the landscape of nuclear protein expression in newborn and adult mice in order to complement and extend our epigenetic studies. methods and results: cardiac nuclei were isolated from homogenized adult and p1 mouse frozen hearts by sucrose gradient centrifugation. magnetic-assisted cell sorting (macs) with pcm-1 as a nucleus-specific marker was used to enrich cm nuclei to >95%. proteins were extracted from nuclear lysate with 2% sds. quantitative protein data were obtained from silac-based liquid chromatographytandem mass spectrometry (lc-ms/ms) experiments with an ltq orbitrap xl mass spectrometer after in-gel digestion with trypsin. nuclear protein extracts from 3 murine cell lines served as silac (lys8/arg10)-labeled internal standard. finally, protein data are correlated with corresponding mrna data obtained by rna-sequencing. we identified 1041 proteins, 688 of which are annotated to the nucleus. 21%/23% (adult / p1) of nuclear proteins are annotated as dna-binding. 1.5%/1.4% belong to transcription factor complexes; 7.3%/7.5% are able to bind transcription factors. 7.9%/8.3% have chromatin modification functions; 4.3%/4.4% modify histones. nuclearenriched go terms include mrna processing and transport, transcription, nucleosome assembly and protein degradation. 71% of proteins are shared between p1 and adult nuclei. 142 proteins are exclusive to p1, 34 are only found in adult. 93 proteins are enriched (1.3-fold increase in abundance) in p1 hearts, 89 proteins in adult proteins related to heart development, gene silencing, and dna replication are more prominent in or exclusive to newborn mice. adult nuclei strongly express proteins related to regulation of actin fibers and cm function, proteins involved in protein degradation, and chaperones. although high mrna expression increases the chance of protein identification, a significant correlation between mrna and protein level could not be observed on a genome-wide scale. conclusions: we present a comprehensive and specific protein landscape of newborn and adult cm nuclei. young cm nuclei appear as a developing tissue, show the ability for proliferation, and indicate ongoing alterations in gene expression. adult cm nuclei prominently display a focus on regulation of contractile fibers and cm function, as well as chaperones and proteasomal proteins indicative of its arduous function. background: fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. however, its relevance in the heart remains to be established. objective: to investigate the role of premature senescence in myocardial fibrosis. methods: murine models of cardiac disease and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. results: senescence markers p21 cip1/waf1 , senescence-associated ß-galactosidase (sa-ß-gal) and p16 ink4a were increased 2-, 8-and 20-fold (n=5-7; p < 0.01), respectively, in perivascular fibrotic areas after transverse aortic constriction (tac) when compared to sham-treated controls. similar results were observed with cardiomyocytespecific β1-adrenoceptor transgenic mice and human heart biopsies. senescent cells were positive for vimentin (92 ± 0.9%), platelet derived growth factor receptor α (94 ± 0.9%) and α-smooth muscle actin (71 ± 2.3%), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. conclusion: our data provide first evidence for an essential role of premature senescence of myofibroblasts in myocardial fibrosis. it is tempting to speculate that pharmacologic modulation of premature senescence might provide a novel therapeutic target for anti-fibrotic therapies in the heart. introduction: the endocannabinoid system is increasingly studied in cardiac research due to its role in fibrosis, inflammation and cell fate modulation. the deregulation of this system has been implicated in myocardial infarction (mi) and consequent heart failure development. a recent study suggests cannabinoid receptor 1 (cb1) inhibition to improve cardiac function and to reduce adverse remodeling after cardiac stress, but the exact underlying molecular mechanisms of these beneficial effects are still unknown. micrornas (mirnas, mirs) provide a complex layer of post-transcriptional regulation modulating key biological processes such as tissue remodelling in heart failure. the aim of the present study was to explore microrna pathways in the chronic effect of cb1 receptor inhibition after angiotensin-fibrosis induction and left ventricular remodeling. methods and results: adverse cardiac remodelling was induced in mice by chronic administration of angiotensin ii (angii, 1,5 mg/kg/day) with osmotic minipumps for 14 days. treatment with cb1 antagonist, or vehicle was performed every second day during the angii administration period. hemodynamic parameters were measured by echocardiography and cardiac pressure volume catheter and tissue samples were taken for molecular and histological analysis. after two weeks of angii infusion, left ventricular dysfunction was prevented by cb1 antagonist treatment. this was shown by significantly improvements of the myocardial performance index and end-diastolic pressure values. at the tissue level, anti-fibrotic effects of cb1 antagonist treatment was confirmed histologically and by expression analysis of pro-fibrotic genes. these beneficial effects were also observed in cb1 ko mice and in an aging mice model. the particular role of tissue fibroblast in aii-induced cardiac fibrosis was further explored. primary cardiac fibroblasts (cf) from each experimental group were isolated and analysed by next generation deep rna sequencing to identify differentially regulated micrornas. microrna-181a/b family was downregulated by in vivo angii delivery and vice versa upregulated after cb1 antagonist treatment and foxb1 (a direct target of mir-181a family) was differentially regulated, suggesting a possible mechanism of action for the benefits of cb1 receptor inhibition. conclusion: we found that in angii-induced cardiac remodelling, lv function is preserved by chronic cb1 antagonist treatment and that cardiac fibrosis is reduced with concomitant downregulation of fibrogenic genes. also, cf-enriched mir-181a/b family seems to be sensitive to cb1 antagonist treatment, thereby affecting cardiac fibrosis. the current study employs a novel concept regarding chronic cb1 inhibitor treatment and may provide important details and novel targets for anti-fibrotic approaches in heart failure. background: low homoarginine (harg) was recently identified as an emerging biomarker for stroke, myocardial infarction, and heart failure in clinical and epidemiological studies. harg competes with arginine as a substrate for nitric oxide (no) synthase and weakly inhibits arginase. both mechanisms might lead to increased no formation in vivo. the aim of this study was to investigate whether harg effects the development of atherosclerosis as a potential underlying mechanism of cardiovascular diseases. methods: harg-deficient agat-knockout (agat -/-) and wildtype (wt) mice were crossed with apolipoprotein e (apoe) deficient mice and fed with high fat diet (hfd) for three months to induce atherosclerosis. harg plasma concentrations were determined using mass spectrometry. en face preparation of aortae followed by red oil staining of atherosclerotic plaques and quantitative evaluation of plaque areas was performed for female mice. endothelial function of male mice was tested with acetylcholine (ach) and nitroglycerin (ntg) after contraction with prostaglandin f2α. background: the direct oral thrombin inhibitor dabigatran etexilate (dabigatran) is used for the prevention and treatment of venous thromboembolism. obese patients as well as patients with type 2 diabetes mellitus (t2dm) have an increased risk for thrombotic disease and show enhanced thrombin generation. besides its role in blood coagulation, thrombin is known to be involved in many pro-inflammatory processes. in obesity, adipose tissue (at) inflammation plays a crucial role in the development of insulin resistance and t2dm and contributes to atherosclerosis development. the aim of the present study was to analyse the effects of dabigatran on at inflammation in a mouse model of diet-induced obesity in the context of accelerated atherosclerosis. methods: 10-week-old female low-density lipoprotein receptor-deficient (lldr -/-) mice were fed a high-fat diet containing 5 mg/g dabigatran or respective placebo for 20 weeks. results: analysis of visceral at revealed a significant increase in adipocyte size in dabigatran-treated mice, although body weight, fat mass, glucose tolerance, and insulin resistance were unchanged between groups. this effects seemed to be directly mediated by thrombin, as treatment with another thrombin inhibitor (argatroban) also resulted in the development of adipocyte hypertrophy. accordingly, in vitro studies in 3t3-l1 cells revealed an inhibitory effect of thrombin on lipid accumulation in adipocytes. the amount of pro-inflammatory cd11c-positive macrophages (atms) in visceral adipose tissue was significantly reduced, and the secretion of pro-inflammatory il-6 from visceral at was significantly lower in dabigatran-treated animals. in vitro studies using 3t3 l1 cells and primary bone marrow-derived macrophages revealed that the changes in macrophage polarization were not directly mediated by thrombin, but indirectly by a change in the secretion profile of adipocytes. a similar reduction in proinflammatory macrophages as detected in at could also be observed in the aortic wall of dabigatran-treated mice. conclusions: the direct thrombin inhibitor dabigatran inhibits at inflammation and the accumulation of pro-inflammatory macrophages in vat but also the aortic wall of ldlr -/mice. these anti-inflammatory effects of dabigatran might contribute to the known atheroprotective effects of dabigatran. background: sarco/endoplasmic reticulum ca 2+ -atpase (serca2a) and its inhibitor phospholamban (pln) are critical determinants of cardiomyocyte calcium cycling and hence, cardiac contractility. pln exists in an equilibrium between mono-and pentamers. while monomeric pln has been implicated in direct serca2a inhibition, a functional role for the pentamers remains ambiguous. recently it has been shown that pln pentamers modulate pka-dependent phosphorylation of pln monomers in vitro. 1 using transgenic mouse models we now investigated the effects of pln pentamers on pln phosphorylation, myocyte ca 2+ cycling and contractility in cardiac myocytes. methods: phosphorylation patterns of pln were analyzed by western blot using phospho-specific antibodies as well as phosphate affinity sds-page. to assess the phosphorylation at baseline, pln knockout (pln-ko) mice expressing either wild type pln (tgpln) or the solely monomeric pln afa mutant (tgafa) transgene were deeply anesthetized, whereas pln phosphorylation by pka was induced using the betaadrenergic agonist isoproterenol. the consequences on myocyte ca 2+ kinetics were measured in isolated, fura2-loaded and electrically paced (0.5hz) cardiomyocytes as the time to 50% decay of the ca 2+ signal (t 50% ). the time to 50% baseline of sarcomere length (t 50% baseline) characterized the speed of myocyte relaxation. results: under basal conditions, we found stronger phosphorylation of pln pentamers than monomers, pointing at pentamers as the preferred pka target. pln afa monomers showed 3.3-fold stronger phosphorylation signals if pentamers were absent (p<0.0001). consistent with a higher basal phosphorylation of pln afa monomers, measurements of calcium kinetics revealed a faster decay of calcium signals in tgafa compared with tgpln cardiomyocytes (t 50% [ms]: 92±8 and 122±8, respectively, p<0.05). notably, t 50% of pln-ko myocytes was 79±5ms (p= not significant versus tgafa), indicating that the strong basal phosphorylation of monomers leads to near complete inactivation of pln in tgafa. upon stimulation of pka, pln monomer phosphorylation and calcium kinetics of tgpln and tgafa mouse myocytes were indistinguishable, because monomer phosphorylation and the speed of cytosolic ca 2+ clearance strongly increased only in tgpln. acceleration of sarcomere relaxation upon pka stimulation was also more pronounced in tgpln than in tgafa and pln-ko myocytes (increase of t 50% baseline [ms]: 32±8 in tgpln versus 7±4 in tgafa-pln and 5±7 in pln-ko, p<0.05). even high-dose isoproterenol induced phosphorylation of only about half of all protomers of pln pentamers suggesting a high capacity of pentamers to attenuate monomer phosphorylation by acting as a phosphate scavenger. conclusions: our data demonstrate that pln pentamers reduce basal phosphorylation of pln monomers in myocytes. nevertheless, pentamers allow strong phosphorylation of monomers during beta-adrenergic stimulation, thereby extending the range within which pln can modify diastolic ca 2+ kinetics and myocyte relaxation. therapeutic inhibition of micrornas is a promising field in cardiovascular research. vector-based overexpression of an inhibitor construct (e.g. microrna sponge) is one approach to achieve sustained inhibition with potential applicability in humans. yet the strength of expression achieved by the currently available gene therapy vectors (e.g. aavs) in humans remains a limiting factor, therefore inhibitory constructs with increased potency would provide an improvement of this approach and bring it closer to therapeutic application. micrornas are believed to discriminate between potential binding sites, based on additional factors provided by the endogenous untranslated regions at the 3' end of mrnas (3' utrs) and the proteins that are bound to them. aim of this project was to investigate whether selected endogenous 3' utrs can likewise increase the potency of microrna inhibitory constructs. to this end several known targets for a cardiac microrna were selected and their relative potencies of microrna inhibition was compared. to accurately assess changes in the activity of the respective micrornas we constructed dual-fluorescent reporter plasmids and established an automated fluorescent microscopy acquisition and analysis pipeline. among several tested utr constructs we found one which strongly increased the inhibitory potency of the microrna binding site in primary rat cardiac myocytes (nrcms). furthermore a similar effect was obtained when the binding site was exchanged for that of a different microrna and analyzed in the nih-3t3 fibroblast cell line. we therefore conclude that endogenous utr contexts can indeed be successfully applied to increase the potency of vector-based microrna inhibitors. the g-protein-coupled protease-activated receptor-2 (par2) regulates inflammatory responses including monocyte migration and cytokine release. par2 is activated by the coagulation factor-xa or by the tissue-factor (tf)/factor viia complex. the immunomodulatory lipid sphingosine-1-phosphate (s1p) is released from activated platelets and interlinks blood coagulation and inflammation. this study investigates the impact of s1p on the expression of par2, tf and of the anticoagulant protein thrombomodulin (tm) in human monocytes and after pma-induced differentiation into macrophage-like cells. monocytic thp1 and u937 cell lines were used as human monocyte models. primary monocytes were isolated from healthy volunteers using a magnetic bead-based monocyte isolation kit. expression of par2, tf and tm was measured by quantitative real-time pcr and western blotting. differentiation of monocytes into macrophage-like cells was induced by incubation with 50 ng/ml pma (phorbol 12-myristate 13-acetate) over 72 h. calibrated automated thrombin (cat) generation was determined in plateletrich plasma from healthy volunteers. in thp1 and u937 cells s1p induced a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of par2 mrna and total protein expression. par2 total protein was upregulated maximally (about 1.5-fold, n=7) with 1 µm s1p after 16 h incubation. comparable effects were seen in human primary monocytes. in comparison, tf mrna and protein were only marginally elevated in non-differentiated thp1 monocytes and tm was not regulated by s1p. after differentiation of cultured monocytic cells with pma into adhesive macrophage-like cells, incubation with s1p resulted in a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of tf expression within 3 to 6 h of incubation. conversely, par2 total protein expression was reduced by about 50% after 24 h s1p incubation. the expression of tm was again not affected. the generation of thrombin in platelet-rich plasma was determined using pma-differentiated thp1 cells as tf source. timedependent incubation with s1p (1 µm) in differentiated monocytes shortened the time to the onset (lag time) of thrombin generation in plasma from 8.0±1.6 to 6.5±1.4 min and elevated total the thrombin generation capacity from 814±130 to 1286±129 nm. peak thrombin formation was elevated from 66±31 to 130±30 nm/min (control versus s1p for 6h, mean±sd, n=5, respectively). these data suggest that s1p induces an enhanced expression of par2 in undifferentiated human monocytes while tf and tm are not regulated. in differentiated monocytes/macrophages, s1p does upregulate tf expression but attenuates par2 levels. since par2 is involved in regulation cell migration, s1p may stimulate a phenotypic switching from a migratory to a procoagulant phenotype during differentiation of monocytes into macrophages. the pro-inflammatory cytokine interleukin-6 (il-6) plays an important role in vascular inflammation. coagulation factors such as the activated factor-x (fxa) may regulate local inflammatory responses of the vessel wall. in this study we investigated whether fxa regulates il-6 expression and secretion in human vascular smooth muscle cells (smc) as well as in failed thrombosed vein grafts. also, we analysed its possible prothrombotic impact on monocytes. il-6 mrna expression was determined in primary human saphenous vein smc by taqman® real-time pcr. secretion to the cell culture media was measured by elisa. tissue factor (tf) expression in monocytic thp-1 cells was determined by western blot. immunostainings for il-6 and the smc marker smoothelin were performed on paraffin embedded tissue sections from failed thrombosed vein grafts and control veins. incubation of cultured human venous smc with fxa (30 nm) induced a time-dependent (3 -16 h) increase in il-6 mrna expression. maximum expression was observed within 6 h to a 7.2±3.3 fold increase (mean±sd, n=5, p<0.05). incubation with an inhibitor of p38 map kinase (sb203580, 10 µm) or pi3k (ly294002, 10 µm) significantly attenuated fxa-induced il-6 mrna expression (n=5). inhibition of p42/44 mapk, rho kinase or nf-kb had no significant effect. stimulation with fxa for 24h resulted in a markedly increased il-6 secretion into the smc culture media from 0.6±0.2 to 1.1±0.3 ng/ml (p<0.5, n=4). stimulation of thp-1 cells with il-6 (1 ng/ml) induced a time dependent (6 -24 h) increase of up to 2.1±0.6 fold (p<0.05, n=5) in tf protein expression. immunostainings of tissue sample of failed vein grafts revealed enhanced il-6 expression in smc-rich regions in vessel walls compared to non-thrombosed control veins suggesting an elevated il-6 regulation in thrombosed vein grafts in vivo. in conclusion, fxa induced il-6 expression and secretion in venous smc which may be regulated via p38 and pi3k signaling. il-6 enhanced tf expression in thp-1 monocytes and was found in smc-rich regions in failed thrombosed vein grafts. fxa-stimulated il-6 release may be involved in regulating local pro-thrombotic processes during vascular inflammation and possibly vein graft failure. rationale: the transcription factors camp-response element binding protein (creb) and camp-responsive element modulator (crem) bind to camp response elements (cres) and mediate a camp dependent gene regulation. suppression of cre mediated transcription is linked to atrial remodeling in genetic mouse models. inhibition of creb target genes is associated with atrial fibrillation (af) susceptibility in patients. creb and crem affect histone acetylation recruiting the creb-binding protein (cbp/p300). the histone acetyltransferase (hat) activity of cbp facilitates gene transcription by loosening chromatin structure. histone deacetylases (hdacs) catalyze the inverse reaction: histone deacetylation with consecutive gene silencing. mice with heart directed expression of the human cardiac isoform crem-ib∆c-x (tg) show atrial dilatation, morphological and physiological alterations in atria preceding spontaneous-onset af. the hdac inhibitor (hdac i ) valproic acid (vpa) reduced atrial weight and af incidence in tg mice. here we tested the hypothesis, that vpa attenuates the structural remodeling in tg atria by reversing changes in atrial gene regulation due to the transgene. methods and results: tg and wt mice were treated from week 5-16 with vpa (0.71% in drinking water, ad libitum) or vehicle (veh). atrial ultrastructure was studied by electron microscopy (em) (week 7 and 16). veh-treated tg atria showed a progressive dysorganisation of sarcomeres (sm) with less mitochondria and more collagen fibers between cardiomyocytes as compared to veh-treated wt atria. the fraction of sm structure in veh-treated tg atria was significantly reduced as compared to veh-treated wt atria (week 7: tg veh : 33±2%, wt veh : 47±1%; week 16: tg veh : 19±2%, wt veh : 44±1%, p<0.05). vpa led to a more organized ultrastructure and restored, at least partially, the degradation of the sm in the tg atria (tg vpa at week 7: 40±2%, tg vpa at week 16: 34±1%, p<0.05 vs. tg veh ). the structure of wt atria was not affected by vpa. we further analyzed the protein abundance profiles in the groups of all animals (wt veh , wt vpa, tg veh , tg vpa) by using lc-ms/ms. between veh-treated genotypes (tg veh vs. wt veh , p<0.05) 998 proteins were significantly changed while 854 proteins were differentially abundant between vpa-treated groups (tg vpa vs. wt vpa, p<0.05). 109 proteins were regulated by vpa in wt atria (wt vpa vs. wt veh , p<0.05), whereas vpa affected 525 proteins in tg atria (tg vpa vs. tg veh , p<0.05), out of which 43 proteins were common. 295 prominent changed proteins between veh-treated tg and wt atria were significantly regulated by vpa in tg atria in the opposite direction. a functional pathway analysis showed that pathways activated in tg atria such as cardiac fibrosis, mitochondrial dysfunction were inhibited by vpa treatment. conclusion: similar to human af, crem-tg mice present atrial dilatation, ultrastructural changes and impaired conduction and spontaneous af. while vpa had little to no effect in wt mice, valproate improved the tg phenotype by interfering with pathways involved in structural remodeling. this supports the idea that hdac inhibition by vpa antagonizes effects of crem expression in atria. in isolated mouse cardiac preparations, histamine is ineffective regarding inotropic or chronotropic effects, presumably because of lack of receptor protein expression. on the other hand, histamine can exert positive inotropic and chronotropic effects in humans via cardiac histamine h 2 -receptors. hence, we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes. in isolated left and right atrial preparations of these mice, we investigated the histamine metabolism on a functional level. preparations of wild type mice (wt) served as control. histamine induced positive inotropic effects (pie) and positive chronotropic effects (pce) in left and right atria of tg mice, respectively, but not in wt. interestingly, the inhibitor of histamine oxidation, aminoguanidine (1 mm), shifted the concentration response curves for the pie of histamine from ec 50 = 110 nm to 37 nm (p<0.05). furthermore, the unspecific inhibitor of mono amine oxidase, tranylcypromine (10 µm), shifted the pie of histamine from ec 50 = 70 nm to 38 nm and increased the efficacy of histamine for the pie (p<0.05). these data indicate that exogenously applied histamine is subject to degradation in the mouse heart by two different pathways namely via diaminoxidase and mono amine oxidase. drugs that inhibit theses enzymes could conceivably alter cardiac function also in the human heart. protein phosphorylation by kinases and dephosphorylation by protein phosphatases has a crucial function in cell signal cascades. it has been shown that cardiomyocyte specific overexpression of serine /threonine protein phosphatases pp1, pp2a, pp2b (calcineurin) and pp5 in mice leads to cardiac hypertrophy and alters cardiac function. to examine the function of another important protein phosphatase in the heart we established a mouse model overexpressing protein phosphatase 2cβ (pp2cβ) under control of the α-myosin heavy chain promoter. cardiac overexpression was demonstrated by western blotting. like other serine/threonine phosphatases, pp2cβ can lead to cardiac hypertrophy. in transgenic mice (tg), relative ventricular weight was increased (4.24 ± 0.14 mg/g) compared to wild type (wt) littermates (3.78 ± 0.20 mg/g; p<0.05) whereas weights of right and left atria were unchanged. therefore, relative heart weight was increased in tg (4.78 ± 0.17 mg/g) vs. wt (4.13 ± 0.22 mg/g; n=8-12; 10-11 months of age; p<0.05). left ventricular function, measured in vivo by echocardiography under isoflurane anesthesia was diminished in tg compared to wt (ejection fraction: 58.33 ± 3.16 % (tg) versus 76.94 ± 0.92 % (wt); n=12-16; 8-11 months; p<0.05 and fractional shortening: 30.84 ± 2.02 % (tg) versus 44.94 ± 0.87 % (wt); n=12-16; 8-11 months; p<0.05). the left ventricle was dilated (systolic diameter: 2.78 ± 0.21 mm (tg) versus 1.84 ± 0.06 mm (wt); n=12-16; 8-11 months; p<0.05; diastolic diameter: 3.97± 0.19 mm (tg) versus 3.33 ± 0.07 mm (wt); n=12-16; 8-11 months; p<0.05). in contrast, atrial function measured as response to β-adrenergic stimulation in isolated left and right atrial preparations was unchanged in tg vs. wt. in summary, our results indicate that pp2cβ overexpression can lead to ventricular dysfunction and hypertrophy. the underlying signal transduction pathways need to be elucidated. the insulin-like growth factor binding protein 5 (igfbp5) -a potential developmental gene is regulated upon cardiac stress m. wölfer background: cardiac remodeling is a complex biological adaptation process of the failing heart accompanied by a re-activation of embryonic gene expression, which so far has unclear pathophysiological relevance. we and others showed that insulin-like growth factor binding protein 5 (igfbp5) is expressed in the early pre-cardiac region in mouse embryos and its up-regulation impairs cardiac progenitor differentiation. igfbp5 functions as an extracellular growth factor binding protein for igf and also has igfindependent activities. the role of this factor in the context of cardiac remodeling is still unknown. the aim of this study was to investigate the relevance of igfbp5 in cardiogenesis and cardiac remodeling and its role as a potential target for ameliorating stress-induced cardiac remodeling methods and results: we investigated the expression of igfbp5 in murine cardiac tissue at different developmental stages by qpcr normalized to tpt1 (tumor protein, translationally-controlled 1). this analysis showed temporal changes of cardiac igfbp5 expression from developing to postnatal hearts, where a high expression was detected in early heart stages, which decreased during cardiac development and became low in the postnatal heart. the analysis of igfbp5 expression in different heart cells showed a very low igfbp5 in adult cardiomyocytes in contrast to a high expression in undifferentiated sca-1 positive cells. in a mouse model with cardiac specific wnt/βcatenin activation, which led to cardiac dysfunction, igfbp5 was found up-regulated (p<0.05). further we found an increased igfbp5 expression after pressure induced cardiac hypertrophy using mice with transverse aortic constriction (tac) (p<0.01). in line with this data, an in vitro model of human heart muscle hypertrophy using engineered cardiac heart muscle (ehm) showed an up-regulation of igfbp5 upon adrenergic activation via norepinephrine stimulation accompanied by a functional deterioration in comparison to untreated controls (p<0.05). all these findings were further supported by rna-sequencing analysis from human aortic stenosis patient samples, where igfbp5 expression was found increased in patients with compensatory hypertrophy and in a higher extent in patients with heart failure in comparison to non-failing heart samples. interestingly, the expression of igfbp5 in angiotensin 2 or norepinephrine stimulated neonatal murine cardiomyocytes, as well as in hearts of mice treated with angiotensin 2, showed the opposite results, namely a reduction in its expression (p<0.01; p<0.05, respectively). summary and conclusion: our results show active igfbp5 transcription in the early developing heart but a low expression in the postnatal heart. a re-activation of expression was found in the process of pathological heart remodeling in mouse and human, in vivo as well as in vitro, indicate the participation of igfbp5 in a conserved manner. we hypothesize that igfbp5 may participate in the developmental gene program becoming activated in the diseased adult heart again. the functional role and regulation of igfbp5 is under investigation. we have recently shown that perivascular adipose tissue (pvat) plays a crucial role in obesity-induced vascular dysfunction. in pvat-free aortas isolated from male c57bl/6j mice fed a high-fat diet (hfd) for 22 weeks, the endothelium-dependent nitric oxide (no)-mediated vasodilator response to acetylcholine remained normal. in contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when pvat was left in place. these results suggest that the reason for vascular dysfunction in diet-induced obese mice is a pvat dysfunction rather than an endothelial dysfunction. treatment of hfd mice during the last 4 weeks with crataegus extract ws® 1442 (150 mg/kg/day) completely normalized vascular function in pvatcontaining aorta. the expression of endothelial no synthase (enos) was not changed by ws® 1442, neither in pvat nor in aorta. phosphorylation at serine 1177 is the most important positive modulation of enos activity. hfd-induced obesity was associated with a reduction in enos phosphorylation at serine 1177 in pvat, but not in aorta. ws® 1442 treatment significantly improved enos serine 1177 phosphorylation selectively in pvat but had no effect in aorta. a major upstream kinase for enos serine 1177 phosphorylation is akt. the activity of this kinase is inhibited in the pvat of hfd mice, which was largely reversed by ws® 1442 treatment. in addition, ws® 1442 treatment enhanced the mrna expression of the nad-dependent deacetylase sirtuin-1 (sirt1), known also as a longevity gene. the activity of sirt1 depends, among others, on the intracellular content of its cofactor nad. ws® 1442 treatment led to an upregulation of nicotinamide phosphoribosyltransferase (nampt), a rate-limiting enzyme in the salvage pathway of nad biosynthesis. one of the non-histone substrates of sirt1 is enos. deacetylation of enos at lysine residues 497 and 507 by sirt1 enhances the activity of the enos enzyme. currently, we are studying the effect of ws® 1442 on nad synthesis, sirt1 activity, and enos (de)acetylation. in conclusion, crataegus extract ws® 1442 reverses obesity-induced vascular dysfunction by improving pvat function. the molecular mechanisms may involve enos phosphorylation at serine 1177 and upregulation of sirt1. the raf kinase inhibitor protein (rkip) inhibits g-protein-coupled receptor kinase (grk2) and the raf-erk1/2 pathway. these two functions of rkip could counteract each other. while grk2 inhibition is cardio-protective, inhibition of the pro-survival erk1/2 axis promotes signs of heart failure in patients and experimental models. in view of this ambivalent nature, the function of rkip in vivo is not clear. furthermore, rkip could have a pathophysiological role because heart specimens from patients with heart failure showed rkip up-regulation (ref. 1). to investigate the impact of cardiac rkip upregulation in vivo, we generated transgenic mice with myocardium-specific expression of the human rkip gene (pebp1) under control of the alpha-mhc promoter. two different rkip-transgenic lines with 2.7-fold and 3.4-fold increased cardiac rkip protein level were generated (ref. 2, and jax id number 911819). we investigated the cardiac phenotype and found that tg-rkip mice developed cardiac hypertrophy with a significantly increased heart weight to body weight ratio and a decreased left ventricular ejection fraction relative to non-transgenic fvb controls, as early as 10 weeks of age. histology analysis revealed progressive atrial and ventricular enlargement of tg-rkip hearts. ecg abnormalities, a lower maximum rate of left ventricular pressure rise, and a strongly decreased left ventricular ejection fraction of 32.9±2.3 % (n=6; ±s.d.) were documented at an age of 8 months. down-regulation of the transgenic rkip by lentiviral transduction of an rkip-targeting mirna retarded the cardiac phenotype of tg-rkip mice. thus, dual-specific inhibition of the grk2 and raf-erk1/2 axis by the human rkip gene (pebp1) triggers signs of heart failure in vivo, and the documented upregulation of the cardiac rkip in heart failure patients could aggravate disease pathogenesis. these findings are in contrast to rodent rkip (pebp1), which does not seem to inhibit the raf-erk1/2 axis in vivo but instead confers grk2 inhibitionheterodimerization between the at1 receptor (at1r) for the vasopressor peptide, angiotensin ii, and the b2 receptor (b2r) for the vasodepressor peptide, bradykinin, enhances angiotensin ii-stimulated signalling in cells. in addition, at1r--b2r heterodimerization has a major pathophysiological role and contributes to the angiotensin ii hypersensitivity in women with preeclampsia hypertension. to analyse the vascular function of the at1r--b2r heterodimer in vivo, we generated a transgenic model of at1r--b2r heterodimerization (tg-b2r+) by transgenic expression of the b2r gene (bdkrb2) in the b2r-deficient tg-b2r-/-strain. fluorescence resonance energy transfer (fret) imaging was applied to analyse the interaction between different gprotein-coupled receptors in the aorta of transgenic mice. we report here that fret imaging detected the close interaction between the aortic at1r and b2r at a distance of less than 9 nm in tg-b2r+ mice whereas the at1r--b2r heterodimer was absent in tg-b2r-/-mice. in contrast, fret was not detectable between the endothelin eta receptor (etar) and the b2r in the aorta of tg-b2r+ mice, although immunofluorescence and immunohistology confirmed the aortic (co-)-localization of both, etar and b2r. the efficient at1r--b2r heterodimerization in tg-b2r+ mice was accompanied by an enhanced angiotensin ii at1r-stimulated vasopressor response relative to that of tg-b2-/-mice, which lack the at1r--b2r heterodimer. as a control, the endothelin-1-stimulated vasopressor response mediated by the etar, which did not dimerize with b2r, was not significantly different between tg-b2r+ and tg-b2r-/-mice. together these findings provide strong evidence that at1r--b2r heterodimerization occurs in vivo and enhances the angiotensin ii at1r-stimulated vasopressor response. dysfunction of the cardiac energy substrate metabolism is a characteristic feature of late-stage heart failure. the dysfunctional cardiac substrate metabolism contributes to insufficient energy generation and has limited treatment options. in search for a treatment approach, we investigated whether inhibition of g-protein-coupled receptor kinase 2 (grk2) could confer cardioprotection by targeting the dysfunctional cardiac substrate use. the impaired substrate metabolism of late-stage heart failure was reproduced in a transgenic model with myocardium-specific expression of fatty acid synthase (fasn), which is the major palmitate-synthesizing enzyme. experiments with a seahorse xf24 extracellular flux analyzer revealed that in an adult-like lipogenic milieu, fasn-transgenic cardiomyocytes reproduced the overall depressed substrate use of late-stage heart failure with a switch from fatty acid to predominant glucose utilization. the impaired substrate use was largely retarded by co-expression of a small peptide inhibitor of grk2, grkinh. the grkinh-mediated protection against cardiometabolic remodelling required an intact raf-erk1/2 axis and involved the erk1/2-dependent inactivation of the heart failure-promoting peroxisome proliferatoractivated receptor gamma (pparg) by phosphorylation of serine-273. as a consequence of erk-dependent phosphorylation of pparg on serine-273, the expression of heart failure-related pparg targets such as fatty acid synthase, resistin and adiponectin was decreased. the importance of pparg serine-273 phosphorylation was further shown in transgenic mice with myocardium-specific expression of the phosphorylation-deficient pparg serine-273a mutant, which was resistant to the cardioprotective activity of grkinh. taken together our experiments show that grk2 inhibition could target cardiometabolic remodelling by inhibition of the heart failure-promoting transcription factor pparg. the effect of sodium valproate on the action potential of atrial myocytes of crem ib∆c-x transgenic mice introduction: in mouse, cardiomyocyte directed over-expression of transcription factor crem (camp response element modulator) causes an atrial phenotype characterized by hypertrophy, reduced contractility and increased duration of the monophasic action potential (map). moreover, this animal model (crem ib∆c-x) showed spontaneous atrial fibrillation (af) episodes as early as 5 weeks of age in homozygous mice and 10-12 weeks of age in heterozygous mice (phenotype delayed towards adult stage). previous studies in heterozygous mice targeted hdac2 inhibition by sodium valproate (vpa, an anticonvulsant drug, acting also as inhibitor of hdac class i>ii). vpa treatment delayed significantly the development of atrial hypertrophy and the incidence of af episodes, without affecting cellular hypertrophy. our aim was to investigate the effect of chronic vpa treatment on the electrical activity of atrial myocytes isolated from crem ib∆c-x and wild type (wt) littermate mice. methods and results: atrial myocytes were isolated from 12 weeks old wild type mice (wt) and heterozygous crem ib∆c-x transgenic mice with enlarged atria (tg), treated for 7 weeks with vpa (0.4 mm in the drinking water) vs. water (vehicle control). action potentials (ap) were measured at room temperature using the patch-clamp technique. atrial myocytes of water treated tg mice had the ap amplitude significantly reduced by 7 mv compared to water treated wt, and, in line with previous results for the map, the tg cells depolarized with a slower slope of 90.2±8 v/s (tg: n=33 cells) vs. 109.8±5.1 v/s (wt: n=30, p=0.05). moreover, ap of atrial myocytes isolated from water treated tg mice had longer duration (apd) at 50% (tg: 11.6±1.4 ms, n=35 vs. wt: 6.9±0.5 ms, n=30, p<0.01), at 70% (in ms: 21.2±2.5 vs. 13.6±1, p<0.05) and at 90% (in ms: 46.8±4.5 vs. 34±2.5, p<0.05) repolarization. vpa treatment reduced ap amplitude in wt mice by 6 mv (n=22, p<0.05) vs. water treated wt, without altering the slope of depolarization or the apd. in vpa treated tg mice the apd was reduced (50%: 7.3±0.8 ms, 70%: 13.8±1.5 ms, 90%: 30.9±3.2 ms, n=21, p<0.05 vs. untreated tg), the amplitude was increased by 5 mv (n.s.) and the slope of depolarization was increased by 21% (p=0.16, n.s.). membrane capacitance evaluation, as an estimation of atrial myocyte size, showed that in untreated tg mice the cells were larger than in wt (tg: 104±7.2 pf, n=27 vs. wt: 55±5.4 pf, n=30, p<0.001), in line with the occurrence of cellular hypertrophy in tg atria. chronic vpa treatment did not change the cell size in either genotype (wt-vpa: 64.8±7 pf, n=17 n.s. vs. wt; tg-vpa: 88±7 pf, n=14, p<0.05 vs. wt-vpa; p<0.01 vs. wt; n.s. vs. tg). conclusions: in hypertrophied atrial myocytes of crem-ib∆c-x, ap were characterized by smaller amplitude, slower onset of depolarization and increased duration compared to wt cells. despite having no effect on atrial myocytes size, vpa treatment reduced the duration and showed a tendency to increase the amplitude and the slope of depolarization of the action potential in tg mice to values similar to wt. these data suggest that chronic treatment with vpa restored partially the electrical activity of atrial myocytes and may reverse the electrical remodeling via hdac2 inhibition. (supported by the dfg) of the transcription factor crem (camp response modulator) icer, smicer and crem-ib∆c-x are inducible by β-adrenergic stimulation and code for similar or even identical proteins. thus, these isoforms are able to repress expression of respective target genes in response to camp and might play a role in an arrhythmogenic remodeling during the development of chronic heart diseases. here we test this hypothesis in a mouse model with transgenic expression of crem-ib∆c-x (tg). these mice develop not only spontaneous onset atrial fibrillation but likewise arrhythmogenic alterations in the ventricle. patch clamp experiments revealed an increased na + -ca 2+ exchanger current (i ncx ) and decreased transient outward current (i to ) in tg ventricular cardiomyocytes (vcms) vs. wild-type controls (ctl). these alterations were associated with an increased arrhythmogenicity in tg vcms. action potentials were prolonged in tg vcms vs. ctls leading to an increased proportion of vcms displaying early afterdepolarizations. ca 2+ imaging revealed that the transduction rate of spontaneous sub-threshold ca 2+ -waves into supra-threshold transient-like ca 2+ -events which is mediated by the ncx was increased in tg vcms. at the same time the serca mediated ca 2+ transport rate (r serca ) was enhanced in tg vcms potentially limiting ca 2+ extrusion by the ncx. underlining the in-vivo relevance of our findings ventricular extrasystoles (ves) were augmented in ecgs of tg mice (ves/mouse during 10 -6 m isoproterenol challenge, tg: 3.65*, ctl: 0.4; n=20/condition). the increase in i ncx and r serca and the decrease in i to went along with an increase of ncx1, serca2a and decrease of kchip2 protein levels. however, the respective mrna levels (slc8a1, atp2a2 and kcnip2) were unaltered between groups pointing to a post-transcriptional regulation of these genes. in a mrnasequencing approach we identified the downregulation of precursor mirnas inter alia for mir-369 (fold change in tg: 0.04*) and mir-1 (fold change in tg: 0.13*) (n=10/condition). atp2a2 is a predicted target of mir-369 and mir-1 has recently been shown to regulate ncx1 and i to related potassium channel subunits. (*p<0.05 vs. ctl) our results demonstrate that transgenic expression of crem-ib∆c-x in mouse vcms leads to distinct arrhythmogenic alterations. they further indicate that the repression of micro rnas by short crem repressor isoforms may lead to the upregulation of genes in the context of an arrhythmogenic remodeling. since crem-repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of credependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease. ( chronic overstimulation of cardiac β-adrenergic receptors (β-ar) is a major trigger for the development and maintenance of cardiac hypertrophy and heart failure. although the camp activated proteinkinase a (pka) is known as a prominent downstream effector of β-ar signaling, its functional contribution to pathological cardiac remodeling is neither well understood nor directly studied so far. to address this issue we used mice carrying a point mutation in the regulatory pka subunit riα (pkariαb), which prevents binding of camp and consequently diminished kinase activity. this dominant negative mutation was controlled by a tamoxifen (tam) inducible αmhc promotor driven cre transgene which allows a selective expression in the ventricular myocardium. the inducible and tissue specific gene expression was analyzed and confirmed by pcr, rt-pcr and immunohistochemistry. furthermore diminished phosphorylation of several pka targets verifies impaired pka activity in tam treated double transgenic animals. hypertrophic response in ventricular pka mutants was studied in genetic, pharmacological and surgical mouse models of heart disease as well. genetically induced heart failure was observed following tam treatment in mice expressing an inducible myocardial-specific cre transgene. this deleterious cardiac phenotype develops independently of the presence of the floxed transgene. 8 days after tam treatment, controls displayed elevated heart weight to bodyweight ratio (hbr) and heart weight to tibia length (htr). hbr shifted from 6.2(mg/g) in untreated control animals to 10.9(mg/g) in tam injected mice. in contrast pka inhibited mutants displayed a minor increased hbr of 7.7 (mg/g). for the pharmacological induction of cardiac hypertrophy we implanted osmotic mini pumps, delivering a combination of isoproterenol and phenylephrine. control animals showed a significantly increased hbr (8.4 mg/g) compared to saline treated animals (6.8mg/g) and pka mutants (7.1 mg/g). paradoxically, all pka inhibited animals displayed a consistent elevation in important hypertrophic markers like anp. surgical constriction of the aortic arch (transverse aortic constriction tac) led to a pressure induced hypertrophic response (hbr: 6.5 vs 7.9 mg/g) followed by a pronounced elevation in several hypertrophic factors such as anp, myh6/7 ratio and myocytes size. in contrast pka mutants displayed an irregular progression of cardiac hypertrophy presented by two groups with either an unchanged (6.9 mg/g) or a strongly elevated hbr (13.5 mg/g). however, additional hypertrophic factors including anp, myh6/7 ratio and myocyte size were significantly increased in both groups. to our knowledge this is the first report, which directly studies the role of ventricular pka activity in cardiac hypertrophy in a genetically altered mouse model. our results suggest that in an early stage of cardiac remodeling pka inhibition alleviates cardiac weight gain but provokes a detrimental shift during further progression, which implicates a protective role of ventricular pka activity in cardiac disease. acetyl-coa carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-coa into malonyl-coa. acc-generated malonyl-coa functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. despite this interest in acc, no attention has as yet been given to the role of acc in endothelial cells. we aimed to investigate the role of acc in two functional key aspects of angiogenesis: endothelial cell proliferation and migration. we used the acc inhibitor soraphen a, a polyketidic natural compound isolated from the myxobacterium sorangium cellulosum, as well as an rnai-based approach to inhibit the function of acc. primary human umbilical vein endothelial cells (huvecs) were used as in vitro model. first, we analyzed the action of soraphen a on cell viability. the compound did neither lower the metabolic activity of huvecs up to a concentration of 100 µm after 24 and 48 h (ctb assay) nor increase in the apoptosis rate after 24, 48, or 72 h up to 100 µm. measuring adenosine triphosphate (atp) levels revealed that 30 µm soraphen a does not alter the atp levels in huvecs after 24 h treatment. in contrast, a 48 h treatment significantly lowered the atp levels by 12 %. also gene silencing of acc1 in huvecs attenuated the atp levels by 11 %. mitochondrial membrane potential (mmp) assays showed decreased mmp levels (10 %) in soraphen a-treated cells after 24 h. interestingly, the compound inhibited the proliferation of endothelial cells with an ic 50 value of 34 µm. cell cycle analysis showed that soraphen a decreases the amount of cells in the g 0 /g 1 phase by 26 % and increases the number of cells in the g 2 /m phase by 50 %. the compound also inhibited the activation of akt (western blot analysis). in a wound healing/scratch assay, 30 µm soraphen a lowered the migration of endothelial cells by 65 %. gene silencing of acc1 in huvecs strongly decreased endothelial migration, whereas a knockdown of acc2 had no influence. furthermore, boyden chamber assays revealed that soraphen a can also lower chemotactic migration by 34 %. since actin rearrangement is necessary for migratory processes, we analyzed the factin cytoskeleton (microscopy) and found that soraphen a decreases the number of filopodia by 60 % but did not influence stress fiber formation. surprisingly, soraphen atreated cells did not exhibit significant alterations in their capacity to form tube-like structures on matrigel. in summary, we could gather first hints that inhibiting acc has an immense impact on the proliferation and migration of primary endothelial cells. the mechanistic basis of this phenomenon will be investigated in future studies by analyzing the lipid profile and the transcriptome of endothelial cells. acknowledgement: this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2) . introduction: statins are among the best examined drugs with excellent efficacy and safety profiles. lowered low-density lipoprotein (ldl) cholesterol goals, new indications for treatment and new knowledge about their pleiotropic effect have promoted a considerable increase in statin use. but as statin use becomes more widespread, awareness of their adverse effects as well as the recognition of statin intolerance problems increase. statin intolerance is a significant problem in the treatment of dyslipidemia, understood as the inability to tolerate a dose of statin required to reduce individual cardiovascular risk sufficiently and could result from different statin-related side effects. muscle-related adverse events, elevation of liver enzymes, cognitive problems and new onset diabetes mellitus have all been described, especially at higher doses. although muscle symptoms are the common side effects observed, excluding other adverse events might underestimate the number of patients with true statin intolerance. these patients represent a target population for the newest lipid lowering drug category i.e. the proprotein convertase subtilisin/kexin type 9 (pcsk9) inhibitors. this work aimed to give an overview of published definitions derived from clinical studies, associations as well as major drug regulatory agencies. we discussed overlaps, differences and limitations in the current definitions. methods: literature based search included pubmed and uptodate publications in english and german language until october 2015. we performed hand searches of the references retrieved and performed an overview. results: a definition of statin intolerance of the european medicines agency (ema) or the us food and drug administration (fda) is not available. in clinical studies, different definitions are chosen and the results are not comparable. also different associations, such as the american heart association (aha), the european atherosclerosis society (eas), the canadian working group or the national lipid association (nla) are not able to agree on one common definition. statin intolerance definitions included different types of muscle symptoms, integration of ck levels and minimal requirements of statin doses. there are currently no validated questionnaires or specific laboratory parameters available. in addition, the term 'myopathy' is often considered as a synonym to statin intolerance. overall, only a few major studies have been conducted with statin intolerant patients so far using inconsistent definitions. discussion and conclusion: there is an unmet need to find a robust and clear definition of statin intolerance as overemphasizing it might hinder appropriate clinical use of this important drug class. thus, further work is required to develop a consensus definition on statin intolerance or a more focused definition regarding statin-associated muscle symptoms only. subsequently, these definitions could be implemented in patient care and their relevance being analyzed and tested in future studies. background: development of cardiac hypertrophy is characterized by reactivation of genes involved in cardiac development. wnt/β-catenin signaling is essential for embryonic cardiac development and is known to be dysregulated in pathological heart remodeling. our previous work suggested a cardiac specific protein complex regulating wnt/b-catenin/tcf transcription in the adult heart. we aim to identify and to characterize this complex in order to find potentially interesting targets for pharmacological therapy preventing maladaptive cardiac remodeling and the onset of heart failure. results: we previously demonstrated that the krüppel-like-factor 15 (klf15) is a βcatenin interaction partner and a cardiac specific nuclear inhibitor of the wnt/β-catenindependent transcription. because klf15 and β-catenin are ubiquitously expressed, we suggest the existence of cardiac specific co-factors responsible for cardiac specificity in this complex. we identified the basic leucine zipper and w2 domain containing protein 2 (bzw2), a phylogenetically conserved protein, as a β-catenin and klf15 interaction partner using yeast-two-hybrid screen. in vitro overexpression experiments and coimmunoprecipitation validated these interactions, which were also confirmed by mass spectrometry. in the developing mouse embryo bzw2 mrna expression is detectable in the heart, neuronal tissue, somites, limbs and branchial arches as shown by whole mount in situ hybridization. in the adulthood expression of bzw2 is confined to the heart, predominantly in cardiomyocytes and in cardiac progenitor cells compared to cardiac fibroblasts (*p<0.05, cm n=4, cfb n=4, cpc n=2), and skeletal muscle. bzw2 was localized in both the cytosol and in the nucleus. mutation analysis showed the importance of the n-terminus of bzw2, containing a putative bzip dna interaction domain, for the nuclear placement of the protein. bzw2 protein expression was significantly increased under cardiac wnt/β-catenin signaling activation in vivo in two mouse models (klf15 knockout (ko) mice **p<0.01 n=3, and in a cardiac specific β-catenin stabilized mouse model, **p<0.01 n=6). a mouse model with constitutively bzw2 loss of function (bzw2 ko) showed cardiac specific upregulation of β-catenin on rna level (***p<0.001, p<0.05, ctrl n=4, bzw2 ko n=5) and on protein level (**p<0.05, ctrl n=7, bzw2 ko n=9). echocardiography analysis in eight-weeks-old bzw2 ko mice showed increased left ventricle wall thickness indicating a hypertrophic phenotype at baseline. we also observed increased levels of bzw2 expression in angiotensin ii treated mice as a model for cardiac hypertrophy (*p<0.05 ctrl n=4, angii n=3) as well as in human samples derived from patients with dilated cardiomyopathy and ischemic cardiomyopathy (*p<0.05 ctrl n=3, dcm n=11, icm n=10). conclusion: these data demonstrated that bzw2 is associated with components of the canonical wnt cascade and suggest its relevance in the constitutive regulation of the wnt/β-catenin components specific in the heart. this study further contribute to the elucidation of the tuning of the wnt-off/-on states aiming to establish a proof-of-concept model for wnt-modulation as a therapeutic strategy in hypertrophic-induced heart failure. objective: sphingosine-1-phosphate (s1p) is involved in the regulation of cell growth, survival, migration and adhesion. it is formed by sphingosine kinases and degraded by phosphatases and s1p lyase [1] . mice that lack s1p lyase are characterized by the accumulation of s1p and sphingosine in their cells and tissues, and by lymphopenia, generalized inflammation, multiple organ damage, and a strongly reduced life span [2] [3] [4] . on the other hand, embryonic fibroblasts from s1p lyase-deficient mice (sgpl1 -/--mefs) are resistant to chemotherapy-induced apoptosis [5] , in part due to an upregulation of multidrug transporters of the atp-binding cassette (abc) transporter family [6] . interestingly, s1p lyase-deficient mice have elevated plasma levels of cholesterol and triglycerides, while suffering from strongly reduced body fat [7] . the aim of the present study was to analyze the link between s1p lyase deficiency and altered cholesterol homeostasis using sgpl1 background: cardiac gene expression changes during cardiac development and under pathophysiological conditions. these alterations in gene expression are regulated by several processes and the exact regulation of gene expression is essential for the proper development and function of the heart. crucial steps in transcription regulation are rna polymerase ii (pol ii) recruitment and changes in pol ii activity. pol ii activity is tightly linked with phosphorylation at serine-2 (p-ser2) of the carboxyterminal domain of pol ii. thus, the aim of the present study was to identify cardiomyocyte-specific genomewide pol ii and p-ser2-pol ii enrichments to get insight into pol ii activity and recruitment in development and disease. methods and results: to get insight into rna polymerase ii dynamics, genome-wide maps of rna polymerase ii occupancy were generated by chromatinimmunoprecipitation in cardiomyocyte nuclei purified from normal neonatal and adult mouse hearts. in addition, cardiomyocyte nuclei were obtained from adult hearts after 4 weeks of pressure overload induced by transverse aortic constriction (tac). cardiomyocyte nuclei were isolated by magnetic beads with an anti-pcm1 antibody. nuclei were used for pol ii chromatin-immunoprecipitation followed by deep sequencing (chip-seq). to test if pol ii marks correlate with nuclear mrna expression in cardiomyocyte nuclei all coding genes were ranked according to their expression level. genes expressed in cardiomyocyte nuclei (> 0.062 fpkm, gene expression rank < 12,653) showed high pol ii enrichment at promoters as well as in genomic regions. many of the gene promoters showed high levels of pol ii accumulation at the transcription start site, as compared to genic regions, which have been associated with pol ii pausing. in contrast, p-ser2-pol ii showed enrichment downstream of the transcription start site. the genomic region of troponin i type 1 (tnni1) which is expressed in cardiac muscle only during development but not in adult cardiomyocytes, was enriched for pol ii in neonatal cardiomyocytes. at the tnni1 gene, pol ii was absent in adult or pressure-overloaded cardiomyocytes. in contrast, pol ii enrichment at the alpha actin 1 (acta1) locus was only present in pressure-overloaded cardiomyocytes. these data are consistent with cellular rna-seq data showing an induction of acta1 after tac. furthermore no pol ii enrichment could be detected in the genomic region of biglycan (bgn), a matrix proteoglycan that is not expressed in cardiomyocytes. this confirms a high purity of cardiomyocyte chromatin. conclusions: this study provides, for the first time, cardiomyocyte-specific landscapes of rna polymerase ii occupancy in heart development and disease. cardiac myocyte maintenance dna methyltransferase 1 is essential for embryonic heart development but is dispensable for cardiac function and remodeling postnatally t. nührenberg background: recent studies have identified dynamic changes in dna methylation in cardiac myocytes during development, postnatal maturation and in disease. however, the enzymes involved in shaping the cardiac myocyte dna methylome are only partially known. here, we explored the role of maintenance dna methyltransferase dnmt1 in cardiac development and in remodeling after chronic left ventricular pressure overload. methods: in mice, deletion of the dnmt1 gene was accomplished by use of two different cre recombinases. crosses of homozygous dnmt1 fl/fl mice with heterozygous dnmt1 fl/+ mice expressing a cre recombinase under control of the atrial myosin light chain gene promoter (myl7-cre) resulted in embryonic deletion of dnmt1 (ko). embryos without myl7-cre served as controls (ctl). embryos were dissected and genotyped at e11.5, e12.5, e13.5 and e14.5. rna-seq and pyrosequencing of genomic dna was performed on e11.5 hearts, histology on e12.5 hearts and electron microscopic imaging on e13.5 hearts. for deletion of dnmt1 in adult mice, homozygous dnmt1 fl/fl mice expressing an inducible cre recombinase (myh6-mcm) were given tamoxifen i.p. over 4 days. homozygous dnmt1 fl/fl mice not carrying myh6-mcm as well as myh6-mcm carrying mice without dnmt1 fl alleles were also injected with tamoxifen and served as controls. cardiac phenotyping including histology, echocardiography and qpcr was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (tac). results: myl7-cre mediated loss of dnmt1 resulted in progressive embryonic lethality with absence of living ko embryos after e14.5. ko embryos displayed loss of cardiomyocyte gene expression patterns, decreased promotor cpg methylation of aberrantly expressed genes and ultrastructural features of wide-spread cardiac myocyte cell death. in contrast, tamoxifen-induced ablation of dnmt1 in adult mice did not affect survival of ko mice. cardiac phenotyping of adult mice revealed no significant differences between ko and ctl mice under sham and tac conditions. conclusion: dna methyltransferase dnmt1 in embryonic cardiac myocytes is essential for proper heart development. in adult cardiomyocytes, dnmt1 is dispensable for normal cardiac function and for adaptation to chronic cardiac pressure overload. background: recent studies showed that mice with general deletion of the oxidoreductase tet3 involved in dna demethylation are embryonic lethal, the underlying cause remaining unknown. this prompted us to investigate wether embryonic lethality is caused by cardiomyocyte-specific loss of tet3. methods: female mice homozygous for tet3 flox and male mice heterozygous for tet3 flox and heterozygous for myl7-cre were mated and offspring were genotyped after weaning. mice homozygous for tet3 flox and heterozygous for myl7-cre (ko) or homozygous for tet3 flox without myl7-cre (controls) were sacrificed at 12 weeks of age and ventricles were harvested. mrna of the ventricles was isolated and expression of cardiomyocyte-specific genes was evaluated by quantitative real-time pcr. cardiomyocyte-specific genomic dna from ko and control mice was obtained from facs-sorted cardiomyocyte nuclei and bisulfite-converted for analysis of dna methylation by pyrosequencing. results: ko mice showed embryonic lethality of nearly 50 %. born ko mice developed without phenotypic abnormalities (normal heart weight/tibia length ratio) and displayed compensatory upregulation of tet1 and tet2. cardiomyocyte genomic dna of ko mice showed significantly higher methylation levels in the body of the atp2a2 gene and at the binding site of the transcription factor gata4 but not near the promotor and the binding site of the transcription factor tbx5. higher methylation levels were not accompanied by changes in atp2a2 expression. however, both myh7 and nppb were upregulated in ko mice compared to control mice. conclusions: our findings suggest that tet3 is involved in dna demethylation in cardiomyocytes. loss of tet3 expression resulted in embryonic lethality. compensatory upregulation of tet1 and tet2 isoenzymes may contribute to the incomplete penetrance of this phenotype. further studies are ongoing to investigate the functional relevance of tet3 in cardiomyocytes. to identify novel proteins secreted by the myocardium, we have previously conducted a genetic screen, which led to the identification of protease inhibitor 16 (pi16). a recent gwas analysis showed an association of a genetic variant in the pi16 genomic locus (rs1405069) with chemerin plasma levels. here we tested the hypothesis that pi16 determines chemerin plasma levels through regulation of chemerin processing. we generated mice deficient for pi16, which did not display an overt phenotype under basal conditions. plasma levels of chemerin were found significantly lower in pi16-deficient animals compared to littermate controls. to investigate whether pi16 and chemerin interact, we performed co-immunoprecipitation experiments. indeed, we found pi16 to co-precipitate with chemerin from both murine plasma and cell culture supernatants. as chemerin is proteolytically processed and activated, we next asked whether the presence of pi16 would affect the processing of pro-chemerin to its processed forms in native tissue. western blot analysis on cardiac and adipose tissue lysates that detected both the unprocessed precursor and the processed forms of chemerin showed a significant shift towards the processed forms upon genetic deletion of pi16. when we assayed for the activity of the chemerincleaving protease cathepsin k, we found recombinant pi16 to potently inhibit cathepsin k activity. taken together, we propose pi16 to act as a regulator of chemerin processing. the transient receptor potential canonical 6 (trpc6) is a second messenger-gated cation channel, which mediates depolarization and ca 2+ entry. it is known to be activated by diacylglycerol derivatives (dag, 1-oleoyl-1-acetyl-sn-glycerol oag) [1] in a pkc-independent manner and plays important roles in lung and kidney physiology. gain-of-function mutations in the trpc6 gene can cause focal segmental glomerulosclerosis (fsgs), a kidney dysfunction leading to end stage renal disease. [2] thus, the discovery of potent inhibitors of trpc6 may help to develop new therapeutic strategies. urban et al. discovered that larixol, a natural product with a labdane skeleton found in the oleoresin of the european larch (larix decidua), blocks the oag-dependent activation of trpc6. [3] larixyl acetate, another component of the resin showed an even higher potency in trpc6 inhibition (ic 50 = 0.26 µm) and a 12-fold selectivity compared to trpc3. these findings led to the idea that further modifications of the larixol lead structure may reveal even more potent inhibitors. furthermore, changes in selectivity and efficacy of such compounds may also provide a deeper insight about relevant structural elements for channel binding. as larixyl carbamate was assumed to exhibit a higher metabolic stability as larixyl acetate, this compound was already investigated in previous studies. it showed a potent and subtype-selective inhibition of trpc6. hence, the development of further carbamates was a priority objective. as an alternative to the use of different isocyanates for the introduction of a carbamate function at the c1 position of the molecule we found an elegant way via formation of an active ester with carbonyldiimidazole. this precursor allowed the design of several isosteric compounds like larixyl methylcarbamate, larixyl hydrazide and larixyl methylcarbonate, which were all able to block trpc6 with similarly low ic 50 values. the introduction of more bulky side chains appeared to diminish the bioactivity of the compounds, the stereochemistry at the c1 position, however, seems to play no important role for the inhibition of trpc6 currents. larixyl methylcarbamate lead to trpc6 inhibition with an ic 50 value of 0.15 ± 0.06 µm. compared to larixyl carbamate and larixyl methylcarbonate, which are also very potent blockers of trpc6, this compound bears the benefit of high subtype selectivity towards trpc3. even with concentrations up to 50 µm of larixyl methylcarbamate, no complete inhibition of the ca 2+ influx via trpc3 channels could be achieved. this fact distinguishes this larixol derivative as a very promising compound for further studies of trpc6 in health and disease. poisoning by organophosphorus compounds (opc) including pesticides and highly toxic nerve agents is based on irreversible inhibition of acetylcholinesterase (ache) resulting in an excess of acetylcholine causing accumulation. the subsequent overstimulation at nicotinic and muscarinic receptors finally leads to respiratory arrest due to paralysis of the respiratory muscles. therapy focuses on competitive antagonism at muscarinic acetylcholine receptors and reactivation of inhibited ache by bisquarternary pyridinium oximes. thereby, nicotinic malfunction is not directly approached. for that reason, an alternative strategy appears rational using nicotinic acetylcholine receptor (nachr) active substances to counteract the effects of accumulated acetylcholine and thus to restore the loss of function of nachrs. different bispyridinium-non-oxime-compounds (bps) have been demonstrated to be able to serve as target structures for the identification of new positive allosteric modulators of nicotinic receptors. unlike nicotinic agonists, positive allosteric modulators can reinforce the endogenous cholinergic neurotransmission despite of acetylcholine accumulation in the synaptic cleft. to this end, the following electrophysiological in vitro study investigated the effect of twelve diversely substituted bps on human α7 nachr using whole-cell patch clamping under voltage-clamping conditions (-70 mv) performed with planar electrodes in an automatic system (nanion technologies gmbh, munich). cholinergic currents of hα7 nachr that have been expressed in stably transfected cho cells were activated by the agonist nicotine. measurements of the effect of various bps concentrations in the presence of nicotine were performed to establish concentration-response relations. cholinergic inward currents were generated by human α7 nachrs in response to low nicotine concentrations. at high concentrations of the drug the currents were decayed reflecting both, desensitization of the receptors and presumably block of the open channel by high agonist concentrations. four out of twelve bps co-applicated with nicotine showed a concentration-dependent enhancement of peak agonist-evoked currents and, most pronounced, 4-tert-butyl-substitued-bp, also demonstrated a marked elongation of the evoked response. this suggests a positive allosteric effect of these compounds on the nicotinic receptor. however, at high bp concentrations in the presence of agonist, responses were decayed significantly, presumably resulting from an open channel block induced by bps. hence, further compounds have to be synthesized to identify promising candidate compounds for improvement of effective therapy against nerve agent poisoning. the transient receptor potential channels (trp) are a family of tetrameric nonselective cation channels, which are involved in a variety of physiological and pathological processes (1). among the 28 mammalian trp channels, the canonical channel 5 (trpc5) is a ca 2+ -permeable ion channel, which is predominantly expressed in the brain (2) . many aspects of trpc5 function are still elusive although behavioral experiments with trpc5-knock-out mice suggest a role in innate fear-response (3) and some studies indicate a trpc5-mediated down regulation of neurite outgrowth in nerve cells (4, 5) . to elucidate trpc5 function on a cellular level, selective and potent compounds are required to acutely control channel activity. despite extensive research, trpc5 modulators often lack selectivity or exhibit toxicity, limiting their applicability in vivo (6, 7) . thus, there is still a need for identifying novel and efficient trpc5 modulators. we therefore screened a compound library (chembionet) and identified a benzothiadiazine derivative (btd) as a novel, potent, and selective trpc5 activator. hek293 cells heterologously expressing trpc5 upon tetracycline induction (hek trpc5 ) show a btd-induced concentration-dependent activation in both ca 2+ assays (ec 50 = 0.71 µm) and in electrophysiological whole cell patch clamp recordings (ec 50 = 1.1 µm). btd elicits currents with an n-shaped i/v curve, typical for trpc5. the resulting activation is long lasting, reversible and sensitive to clemizole, a recently established trpc5 inhibitor (8) . mtt assays revealed that incubating hek trpc5 cells for 24h with btd concentrations above 1 µm results in a concentration-dependent decrease in viability and cell proliferation, indicating a ca 2+ -mediated cytotoxic effect in consequence of sustained channel activity. non-induced control cells remain unaffected by btd at concentrations up to 10 µm. ca 2+ assays showed no influence of btd on closely related trpc4 channels, as well as trpc3/6/7 at concentrations up to 10 µm. the same applied to more distantly related trpv and trpm channels. besides a homotetrameric organization, trpc5 subunits can also assemble to heteromeric channel complexes with their closest relatives trpc1 and trpc4 (9) . trpc1/5 and trpc4/5 heteromers can also be activated by btd as evident from their typical i/v curves in patch clamp experiments, suggesting a high selectivity of btd for channel complexes bearing at least one trpc5 subunit. transient receptor potential canonical channels 3/6 and 7 are controlled by membrane lipids and highly expressed in neuronal and cardiac tissues. the involvement of these channels in development and (patho)physiology of these tissues is well documented, while our understanding of structure-function relations, specifically in terms of the lipid sensing machinery, in these channel proteins is still incomplete. using a homology model of trpc3, based on the recently available structural information on trpv1, we performed structure-guided mutagenesis and identified a single residue in transmembrane domain 6 (g652), which is conserved within the canonical family of trp channels. single point mutations at position 652 in trpc3 largely eliminated lipid sensitivity. trpc3 g652a expressed in hek293 cells was found resistant not only to activation via the phospholipase c pathway but also to direct administration of diacylglycerols. on the contrary, a synthetic agonist of trpc3/6/7 channels (gsk1702934a) activated wild-type trpc3 and trpc6 channels as well as the respective lipid insensitive mutants (trpc3 g652a, trpc6 g709a ). interestingly, the synthetic activator was found to generate substantially enhanced trpc conductances in cells expressing the lipid-insensitive mutants as compared to wild-type proteins. closer inspection of sensitivity of the wt and mutant proteins to various gsk derivatives argue against a contribution of g652 to gsk recognition by trpc3. our results demonstrate the existence of two different mechanisms of trpc3/6 activation supposedly involving distinct gating movements in the channel complex. we suggest that lipid gating of trpc3/6 involves a hinge-point and/or requires a certain level of flexibility within transmembrane segment s6 provided by g652. lipids and synthetic activators of trpc3/6 may be capable of initiating markedly different structural rearrangement in these channels. objective: organophosphorus compounds (opcs), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (ache). inhibited ache results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nachr) in the postsynaptic membrane is provoked. as the therapeutic efficacy of oximes is limited, e.g. poisoning by soman or tabun, the direct targeting of nachr may be an alternative therapeutic approach. studies with the non-oxime bispyridinium compound (bp) mb327 (1,1'-(propane-1,3-diyl) bis (4-tert-butylpyridinium) di(iodide)) demonstrated a therapeutic effect against soman in vitro and in vivo. consequently, studying the affinity of bps at muscle-type nachrs and functional effects are topics of interest. to identify potential candidates, homologous series of substituted and non-substituted analogues (linker c 1 -c 10 ) of mb327 were investigated by using binding and functional assays. experimental procedures: crude membranes from frozen electric organ of torpedo californica were purified by sucrose-gradient density centrifugation and used in both affinity and functional assays. in competition radio-ligand binding assays, the influence on [³h]epibatidine binding sites of torpedo muscle-type nachr was determined. functional assessments were carried out with a bilayer method to investigate the effect on the cholinergic signal induced by 100 µm carbamoylcholine. results: bispyridinium compounds bearing unsubstituted pyridinium rings and long alkyl linkers (> c 7 ) inhibited the binding of [³h]epibatidine and decreased the cholinergic signal of 100 µm carbamoylcholine in the functional assay. mb327 and several bispyridinium structure analogues (mainly c 2 -c 4 linker) exhibited no regular displacement curves at [ 3 h]epibatidine binding sites and enhanced the carbamoylcholine-induced signal. the results demonstrate that the described affinity and functional screening methods detected some structure-activity-relationships (sar). depending on linker length and substitution pattern, the investigated bispyridinium compounds seemed to interact as positive allosteric modulators. further research is necessary to verify this hypothesis. non oxime bispyridinium compounds with an effect on soman-blocked respiratory muscle function have no effect on normal muscle function the life threatening toxicity of organophosphorus compounds (op), like nerve agents or pesticides, lies in the inhibition of acetylcholinesterase (ache) which causes cholinergic crisis. the accumulated acetylcholine in neuromuscular synapses results in the desensitization of nicotinic acetylcholine receptors (nachr) and paralysis of respiratoric muscles. the 4-tert-butyl-substituted bispyridinium compound mb327 showed therapeutic efficacy in soman and tabun poisoned guinea pigs in vivo. partial restauration of neuromuscular transmission by bispyridinium compounds (bp), e.g. mb327 or mb420, could also observed in soman paralysed respiratory muscles in vitro and was partly attributed to an interaction of bp with nachrs. however, it is unknown, whether these bp might affect normal respiratory muscle function in the absence of cholinergic crisis. therefore this study investigated the effect of bp on physiological rat diaphragm muscle function. force generation of rat diaphragm hemispheres was determined after incubation with increasing bp concentrations (1-300 µm) and compare to sham treatment. the diaphragm hemispheres were stimulated every ten minutes by an indirect electrical field (20, 50, 100 hz). muscle force was analyzed as time-force integral and is expressed as percentage of the individual control values, measured at the outset of the experiment. the muscle force dropped during the experiment. the application of the bispyridinium compounds mb327 (1,1′-(propan-1,3-diyl) bis (4-tertbutylpyridinium) di(iodide)) and mb420 (1,1′-(propan-1,3-diyl) bis (2-ethylpyridinium) di(iodide)) in the tested concentration range (1-300 µm) did not change muscle force production compared to the sham treated muscle. this was equally true for low (20 hz) and high (50 and 100 hz) stimulation frequencies. this study showed that bispyridinium compounds which can partially reverse somaninduced neuromuscular block in rat diaphragms show no effect on respiratory muscle function in absence of the op-induced neuromuscular block. these results suggest that the bp tested in this study interacted with desensitised nachrs only, but do not affect physiological neuromuscular transmission. this effect needs to be investigated with further, promising bp compounds. interaction of recombinant pain-relevant atp-and proton-gated ion channels in an expression system; potentiation of the p2x3 receptor-induced current by the opening of asic3 channels g. stephan 1 , p. illes 1 1 universität leipzig, rudolf-boehm-institut für pharmakologie und toxikologie, leipzig, germany the p2x3 receptor (r) is a ligand-gated cationic channel, which is activated by extracellular atp. the acid sensing ion channel 3 (asic3) belongs to the enac/degenerin family and is gated by extracellular protons. despite their different amino acid sequences both ion channels share the same structure and pore architecture, by i.e. consisting of three identical subunits. besides, they are both located at partially overlapping subpopulations of dorsal root ganglia neurons and are implicated in acidic pain signaling. consequently, their physical interaction in the cell membrane or even the formation of heteromeric receptor channels from p2x3 and asic3 subunits has to be taken into consideration. we transfected rat (r)p2x3r and rasic3 constructs individually or together in a ratio of 1:1 into cho cells. we further used the whole cell patch clamp technique to analyze the current responses either elicited by the application of α,β-methylene-atp (α,β-meatp) or by a decrease in the extracellular ph value. the functionality of the individually transfected p2x3r-and asic3-constructs was verified by recording concentration-response curves for the agonists α,β-meatp and protons, respectively. after co-transfection of both ion channels, a ph-shift from 7.4 to 6.7 caused a rapidly desensitizing current response and a subsequent strong potentiation of the α,β-meatp-induced current. an even larger potentiation was achieved after a decrease of the ph value to 6.5. the opening of asic3 channels failed to facilitate the p2x3r current when 2-guanidine-4-methylquinazoline was used to stimulate a non-proton ligand-sensor of asic3. then, we substituted ca 2+ in the extracellular medium by ba 2+ or decreased the intrapipette concentration of egta, to modify the free intracellular ca 2+ concentration. in cells individually transfected with the receptor-channels, external ba 2+ increased the effect of α,β-meatp but decreased the effect of protons. the lowering of intrapipette egta modified p2x3r-and asic3-specific currents in a similar manner as external ba 2+ . in cells co-transfected with p2x3r/asic3, the ionic manipulations mentioned above abolished the potentiation of the α,β-meatp currents by asic3 activation. taken together, our results suggest that p2x3r and asic3 interact with each other, since the activation of asic3 had a marked impact on the p2x3r specific current response. further experiments are required to clarify the mechanism of this interaction, although it has been shown that extra-and intracellular ca 2+ and the proton sensor of asic3 appear to critically participate in this process. university of duisburg-essen, institute for anatomy, essen, germany background: the sperm acrosome reaction is an all-or-none secretion process, mainly following the conserved principles of calcium-regulated exocytosis in neurons and neurosecretory cells. however, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (naadp), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. in addition, the expression of two-pore channel (tpc) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of naadp were examined in mammalian spermatozoa. methodology/ principal findings: our results show that treatment of spermatozoa with naadp resulted in a loss of the acrosomal vesicle, which shows typical properties, described for tpcs: (i) registered responses were not detectable for its chemical analogue nadp, and (ii) where blocked by the naadp antagonist trans-ned-19. in addition, (iii) two narrow bell-shaped dose-response-curves were found, with maxima either in the nanomolar or low micromolar naadp concentration range. performing immungold-electron microscopy with a tpc1 specific antibody, a co-localization with naadp-binding at the acrosomal region was detectable. moreover, quantifying loss of the acrosomal vesicle in tpc1 null sperm upon application of different naadp concentrations, responsiveness to low micromolar naadp concentrations was completely abolished. conclusions/significance: our finding that two convergent naadp-dependent pathways are operative in driving acrosomal exocytosis and that zona pellucida induced acrosomal exocytosis is prevented by trans-ned-19 support the concept that both naadp-gated cascades match local naadp concentrations with the efflux of acrosomal calcium, thereby ensuring reliable and complete fusion of the large acrosomal vesicle. since the acrosome reaction shares the same basic sequence of events typical for the conserved process of calcium regulated exocytosis, such as tethering, docking, priming and final vesicle fusion, the sperm model system may also be useful to comparatively examine whether the same convergence of naadp-dependent pathways is also operative in cellular systems with many secretory vesicles. walther-straub-institut, münchen, germany trpv4 channels are members of the vanilloid family of trp proteins. the channel is nearly ubiquitously expressed and can be found in brain, kidney, skin, heart, blood vessels as well as in the lung. pulmonary expression of trpv4 has been identified in endothelial cells (1), epithelial cells (2) and arterial smooth muscle cells (3) . most interestingly, the channel is known to be involved in the development of several lung diseases such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (reviewed in 4). it is a matter of debate however if trpv4 activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for trpc6 (5) which is also expressed in both cell types. to analyze the potential role of trpv4 on ischemia-reperfusion-injury(iri)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. much to our surprise, we detected a significantly enlarged edema formation after 90 minutes of ischemia in trpv4-deficient mice in comparison to wild-type (wt) mice. this effect was observed by constant weight measurements as well as wet-to-dry ratio gain and was not dependent on the initial perfusion rate. most interestingly, edema formation of trpv4/trpc6 double deficient lungs was indistinguishable from wt lungs, indicating antagonizing effects of both channels, because trpc6 deficiency protected lungs from iri-induced edema (5) . moreover, we identified reduced expression levels of aquaporin 5 in trpv4-deficient lung lysates compared to wt lungs. these findings raise the intriguing possibility that trpv4 might be involved in the regulation of aquaporin expression in lung endothelial cells. endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. endolysosomal dysfunction can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic and neurodegenerative diseases, retinal and pigmentation disorders, trace metal dishomeostasis, infectious diseases, and cancer. endolysosomal ion channels and transporters are highly critical for the tight regulation of the multiple endolysosomal fusion and fission processes including endo-and exocytotic events as well as the regulation of proton and other ionic concentrations in the lumen of endolysosomal vesicles. methods to patch-clamp endolysosomal organelles are continuously improving. yet until now it has not been possible to selectively enlarge endosomal or lysosomal organelles with pharmacological tools for patch-clamp experimentation. we show here by using a combination of two small molecules that we can selectively enlarge early endosomes to a degree sufficient for patch-clamp experimentation. the ability to more selectively patch-clamp intracellular organelles will substantially improve the functional investigation of endolysosomal ion channels under physiological and pathophysiological conditions. the transient receptor potential (trp) channels are a superfamily of non-selective ion channels involved in a variety of physiological processes and in the pathgenesis of many disorders. in the kidney, trp channels have been implicated to be involved in diabetic nephropathy, focal, segmental glomerulosclerosis, polycystic kidney disease, hypomagnesemia with secondary hypercalcemia and idiopathic hypercalcuria. the melastatin-like trp channel subfamily 3 (trpm3) has been shown to be expressed in human kidney 1, 2 . using newly developed anti-trpm3 antibodies, we are able to visualize trpm3 protein in epithelial cells of proximal tubule as well as collecting ducts in the mouse kidneys. therefore, we compared renal function of male, five month old mice lacking trpm3 (trpm3 the atp-gated p2x7 receptor (p2x7r) is a non-selective cation channel widely expressed in epithelia, endothelia, and cells of hematopoietic origin. it plays a central role in cytokine release and studies in p2x7 -/animals indicate its involvement in inflammatory and neurodegenerative diseases. in addition, accumulating data suggests a functional role in neurons and its involvement in neurotransmitter release in the brain. however, despite its importance as a drug target, its precise localization and its molecular and physiological functions remain poorly understood. in particular, the location and function of p2x7r in neurons remain a matter of ongoing debate. to clarify the cellular and subcellular distribution of the p2x7r and to investigate its physiological and pathophysiological role in the brain we generated bac transgenic mouse models in which murine polymorphic variants of egfp-tagged p2x7r are overexpressed under the control of the endogenous p2x7 promoter. the egfp-tagged p2x7rs are efficiently overexpressed in the plasma membrane and can be directly visualized by green fluorescence or indirectly by anti-egfp antibodies. the obtained mouse lines show different expression levels but identical expression patterns with predominant expression in the cerebellum, hippocampus, and thalamus. using cell type-specific markers, p2x7-egfp was identified in almost all microglia and subpopulations of oligodendrocytes and astrocytes in the brain. in the spinal cord, numerous astrocytes in the white matter showed egfp immunoreactivity. so far, no egfp immunostaining was found on map2-and neun-positive cells indicating that under non-pathological conditions, the p2x7 receptor is not expressed in neurons of the cns. interestingly, a higher expression level of cd68 protein was observed in the p2x7r-overexpressing mice. these results suggests that overexpression of p2x7rs alone is sufficient to induce microglia activation, even under non-pathological conditions. since cd68 primarily localizes to lysosomes and endosomes, this further supports a role of the p2x7r in the regulation of phagocytosis. to validate these data, conditional knockout mice are generated. the current status of the project will be presented. the two-pore channels (tpcs) -tpc1 and tpc2 -are located in membranes of intracellular organelles of the endo-lysosomal system. the tpc-protein-monomer contains two homologous domains with six transmembrane α-helices each. a functional tpc probably consists of a dimer of two tpc-proteins resembling an ion channel architecture with the typical four domain organization like voltage gated na + or ca 2+ channels or such as trp channels. due to their biophysical properties, tpc1 and tpc2 are assumed to be involved in the efflux of ca 2+ from intracellular organelles and thereby contribute to fusion/fission processes of endosomes and lysosomes. thus, tpcs are supposed to be important regulators for vesicle trafficking, sorting and degradation/recycling processes. recently, it was shown that virus entry and replication of certain strains of filoviridae -such as ebola -depends on functional tpcs and that either block or genetic inactivation of tpcs reduces virus infectivity. a large family of bacterial protein toxins elicit their effects by modification of intracellular target proteins of host cells. these toxins are taken up by receptor mediated endocytosis and follow different endosomal routes to reach their final cytosolic destination. these toxins principally use two different intracellular routes: the first group uses an entry route via early or late endosomes (short-trip toxins), the second group takes a retrograde route via endosomes, golgi network and the endoplasmic reticulum (long-trip toxins) to get access to the cytosol. translocation of short-trip toxins -such as diphtheria toxin (dt), pasteurella multocida toxin (pmt) and bacillus anthracis lethal factor (pa/lf) -from early and late endosomes into the cytosol is driven by ongoing acidification. long-trip toxins -including cholera toxin (ct) -are retrogradely transported after endocytosis via the golgi apparatus to the endoplasmic reticulum (er). within the er a specific peptide-motif allows the translocation into the cytosol. due to the role of tpc1 for vesicle fusion & fission processes we investigated a potential impact of tpc1 on the uptake of bacterial toxins. first we determined the precise localization of tpc1 in intracellular compartments. to deduce its role for trafficking processes we performed co-localization and correlation studies with a whole set of established markers such as rab-gtpases and pips. second we intoxicated wild-type and tpc1 deficient cell lines with different bacterial protein toxins such as cholera (ct), diphtheria (dt) or pasteurella multocida (pmt) toxin. using cell viability and other intoxication assays we investigated the consequences of tpc1-deletion on bacterial toxin uptake, translocation and cytotoxicity. universität des saarlandes, institut für experimentelle und klinische pharmakologie und toxikologie, homburg/saar, germany trpm3 ion channels are considered to be involved in hormone release from pancreatic islets and the pituitary gland 1,2 . the trpm3 gene encodes a number of different splice variants that differ in their permeation properties and their activity in response to agonists 3, 4 . variations include the presence or absence of five stretches of 10 to 25 amino acid residues within the aminoterminus, long or short pore loops and long or truncated carboxytermini 5 . furthermore, three different aminotermini of human and mouse proteins have been described 5 , but presumably these differences are caused by the activity of different promoters. screening of a mouse pituitary gland cdna library identified 12 variants that differ in exons 8, 13, 15, 17 and 20 . however, only variants bearing the short, ca 2+ permeable pore loop were detected. 3´ rapid amplification of cdna ends (3´race) revealed that trpm3 proteins of the pituitary gland carry truncated c-termini exclusively. 5´race identified five independent regions of transcription initiation within the trpm3 gene implying the presence of five independent promotors. the different transcripts encode four trpm3 amino termini α, β, γ and δ of 1-155 amino acid residues including those described in humans (β,γ). however, the activity of these variants after stimulation with pregnenolone sulfate varied largely just as their frequency in the pituitary with shortened γ-variants being most abundant (~76 %). two-pore channels (tpcs) are a small family of ion-channels found throughout the endolysosomal system of eukaryotic cells. phylogenetically tpcs belong to the voltagegated ion channel superfamily sharing common traits with ca v / na v and trp channels. tpcs show a duplicated architecture with two homologous trans-membrane domains. each domain is build up by six membrane spanning alpha helices linked by short loops. it is very likely that tpcs form dimers maintaining the four-fold symmetry found in other members of the voltage-gated ion channel superfamily. due to their localization in the endolysosomal system tpcs are not accessible to conventional patch clamping. to investigate tpcs electrophysiological properties we use black lipid bilayer measurements. purified channels are integrated into an artificial phospholipid bilayer that separates two chambers enabling us to apply different buffers. upon activation by naadp ions flow through the channel and can thereby pass the diffusion barrier. the movement of charged molecules through tpcs results in currents which can be amplified and recorded. the controlled environment of the lipid bilayer setup allows testing of different ions as well as putative activating and inhibitory substances. one of the major drawbacks of conventional lipid bilayer setups are long preparation times between each measurement. stability of the phospholipid bilayer can pose another issue. often several membranes have to be established before a measurement can be performed making it very time consuming to achieve adequate experiment numbers. new multichannel systems resolve this issue supporting fast formation of bilayers while allowing measurement of up to 16 different bilayers at a time. here we utilize the "orbit" multichannel system with a meca16 chip by ionera to measure tpc1 channel activity. we will present data generated by the "orbit" system and a conventional bilayer setup using different channel constructs and charge carriers. cardiac action potentials are generated and propagated through the coordinated activity of multiple ion channels, including voltage-gated sodium channels (nav1) and potassium channels (like kv1, kv4). the voltage-gated na + channel nav1.5 initiates the cardiac action potential (ap), is essential for rapid depolarization, and is also known to control the ap duration in cardiomyocytes. the voltage-gated k + channel kv4.3 is responsible for the early repolarization of action potentials in human heart. similar to many membrane proteins, nav1.5 and kv4.3 have been found to be regulated by several interacting proteins. the transmembrane β subunit dipeptidyl aminopepidase-like protein (dpp) 10 is known to interact with the kv4.3 channel complex, modulating kinetics and voltage dependence. the overexpression of dpp10 in ventricular cardiomyocytes of rats revealed strong reduction of ap amplitude and significant slowing of ap upstroke velocity and ap duration, which could not be explained by the effects on cardiac kv4 channels. to study the potential influence of dpp10 on nav1.5 channels, we performed whole-cell patch-clamp analysis of transiently transfected cho-k1 cells, expressing scn5a alone or with dpp10. surprisingly, we observed significant effects of dpp10 on nav1.5 channel voltage dependence and kinetics. thus, the co-expression of dpp10 significantly shifted the half-maximal voltage of steady-state activation and steady-stateinactivation to more positive potentials compared to nav1.5 channels alone. in addition we analysed the effects of dpp10 on the kinetics nav1.5 currents. while time to current peak was not affected in cells co-expressing nav1.5 and dpp10 compared to nav1.5 alone, dpp10 slightly accelerated the inactivation. in addition, the time course of recovery from inactivation was clearly accelerated in cells expressing both nav1.5 and dpp10 compared to nav1.5 alone. in summary, we provide first evidence that dpp10 not only interacts with kv4 channels, but also influences nav1.5 channels modulating the depolarization as well as the early repolarization phase of the cardiac ap. therefore, it becomes likely that these ion channels are part of large, multi-protein complexes, and that the pore-forming subunits kv4.3 and nav1.5 behave very differently depending on the expression of its associated proteins like dpp10. cardiac fibroblasts (cf) comprise the most abundant cell type of the mammalian heart and it is known that they contribute to maladaptive cardiac remodeling processes. in response to pressure or volume overload, ischemia-reperfusion injury or myocardial infarction, cardiac fibroblasts proliferate and transdifferentiate into myofibroblasts which produce collagen and pro-hypertrophic cytokines influencing cardiomyocyte function and size. it was shown that β-adrenergic stimulation of cfs with isoproterenol leads to angiotensin ii (at-ii) production and autocrine stimulation of these cells (jaffre et al, 2009 ). activation of phoypholipase c triggered by at-ii leads to formation of inositol trisphosphate (ip 3 ) and subsequent release of calcium from intracellular stores as well as calcium entry across the cell membrane. the focus of our research is the identification of the plasmalemmal channel proteins such as trpc channels mediating this calcium entry, and whether these calcium entry pathways in cfs contribute to pathological remodeling. to date the precise role of calcium entry for these pathological processes is largely unknown. trpc channels are candidates for the analysis of calcium homeostasis in cf. recently, we showed that trpc1/c4-deficient mice are protected from maladaptive cardiac remodeling after neurohumoral stimulation or pressure overload, respectively, which can be explained by a significant reduction of a background ca 2+ -entry (bcge) pathway in cardiomyocytes; this bgce is enhanced by stimulation with agonists such as isoproterenol or angiotensin ii and it critically depends on trpc1 and trpc4 (camacho londoño et al., 2015) . nevertheless, the role of trpc1/c4 for calcium homeostasis in cfs has not been analyzed so far. we established an in vitro model that allows the analysis of calcium release and entry triggered by several (patho)physiological agonists in cultured primary adult cfs from mice. cfs were isolated using langendorff-perfusion and were cultured for maximal 6 days. our results show that there is no difference in 100 nm at-ii induced ca 2+ -release or ca 2+ -entry in trpc1/c4-deficient cf compared to wt. to evaluate whether the lack of trpc1/c4 can be compensated by other trpc channels we currently analyze cfs from trpc hepta ko mice lacking all seven trpc channel proteins concerning at-ii induced ca 2+ -release and ca 2+ -entry and we will also analyze the influence of other agonists on cfs which are known to evoke a longer lasting rise in the [ca 2+ ] i like isoproterenol, 5-ht, thrombin and endothelin-1. cardiovascular and metabolic diseases are currently the primary cause of morbidity and mortality in the western world and are spreading to the rest of the world following globalization. adipose tissue, in particular perivascular adipose tissue (pvat) is recognized as an important player in the development of these diseases. the release of relaxing factor(s) from the pvat has been a matter of interesting and highly spirited debates about its nature, the channels that govern its activities and its role in vascular dysfunction. data from our laboratory indicate that adipose-derived relaxing factor (adrf) is an important player, however the potential channels necessary for its downstream activities are still under study. our recent research primarily focuses on kv7.1 channels, which are known to be expressed in vascular smooth muscle cells. k v 7.1 voltage-gated potassium channels are expressed in vascular smooth muscle cells (vsmc) of diverse arteries, including mesenteric arteries. based on pharmacological evidence using r-l3 (k v 7.1 opener), hmr1556, chromanol-293b (k v 7.1 inhibitors), these channels have been suggested to be involved in the regulation of vascular tone. however, the specificity of these drugs in vivo is uncertain. we used kcnq1-/-mice to determine whether k v 7.1 plays a role in the regulation of arterial tone. we found that r-l3 produces similar concentration-dependent relaxations (ec 50 ~1,4 µm) of wild-type (kcnq1+/+) and kcnq1-/-arteries pre-contracted with either phenylephrine or 60 mm kcl. this relaxation was not affected by 10 µm chromanol-293b, 10 µm hmr1556 or 30 µm xe991 (pan-k v 7 blocker). the anti-contractile effects of pvat were normal in kcnq1-/-arteries. chromanol-293b and hmr1556 did not affect the anti-contractile effects of perivascular adipose tissue (pvat). isolated vsmcs from kcnq1-/-mice exhibited normal peak k v currents. the k v 7.2-5 opener retigabine caused similar relaxations in kcnq1-/-and wild-type vessels. we conclude that k v 7.1 channels are apparently not involved in the control of arterial tone by alpha 1 adrenergic vasoconstrictors and pvat. in addition, r-l3 is an inappropriate pharmacological tool for studying the function of native vascular k v 7.1 channels in mice. introduction: according to international guidelines [1] [2] [3] [4] [5] [6] [7] , both human and animal skin in vitro models have been used and validated to predict percutaneous penetration in humans. excellent correlations have been found for domestic pig as surrogate for human skin [8] [9] . material and methods: tissue the skin samples are descended from female pigs of german landrace (50 kg weight, approx. 4 month). process approved under german welfare law. after narcotization and euthanization the animals were shaved with an electric shaver, washed and dried. microbiological investigation swabs from 10 different skin area (fig. 1) were taken before next preparation step. skin areas were cut, stretched and subcutaneous fatty tissue was carefully removed. the skin was harvested at thickness of 1.000 µm by dermatome. after dermatomization, samples were taken for histological examination. skin disks of 30 mm were punched out from the frozen skin stripes and stored at -20°c. hplc and skin absorption waters corporation hplc containing 2767 sample manager, 2545 binary gradient pump, 2998 pda detector (optional: 3100 electron spray mass spectrometer); column: nucleodur® 100-5 c18 ec 50mm x 4.0 mm id. hanson microette™ vision® diffusion test system (hanson vision® autoplus™ autosampler/autofill™ collector, 6-cell drive system with vertical diffusion cell "standard". the permeation experiment was performed over a period of 48 h at 32°c. the dosage compartments of each cell were filled with approximately 300 µl of the caffeine solution (10 mg/ml). samples of 1 ml are taken after 0, 12, 24, 36 and 48 hours from receptor medium of each cell. aliquots from each vdc are analyzed by hplc in duplicate. microbiology staphylococcus spp. were found explicitly on pig skin surface. bacteria are facultative anaerobe, gram-positive bacteria that are physiologically colonizing the skin, oropharynx and the gastrointestinal tract. histology and skin thickness he staining and mechanical skin thickness determinations confirmed intact dermatomizing process of skin (fig. 2) . thickness was in the order of magnitude between 897 to 1.230 µm and intra variations were less than 10 %. caffeine skin absorption permeability coefficients and lag-phases recorded are in the same order of magnitude of previous work [10], demonstrating intact barrier properties of the membranes after 3 month storage process. until today, intra-assay variations are superior or equal to interregional and inter-animal variations. discussion: well characterized dps1000 provides ready to use research tool for locally and systemic skin investigations. ongoing experiments will cover skin structure analysis by raman spectroscopy, biophotonics and storage impact on skin barrier function. respirable biopersistent granular dusts (gbs) should fulfill the criteria of i.) a negligible solubility in physiological lung fluid that ii.) do not exhibit a specific surface chemistryrelated toxicity at volumetric non-overload conditions in lungs. in 2012, the mak commission derived a new threshold value of 0.3 mg/m 3 for gbs with a density of 1, recognizing that at this concentration a chronic inflammation and increase of the lung cancer risk will not occur. -the objectives of the project were i.) to determine an experimental dissolution value for 'low soluble' gbs using six candidate dusts; ii.) in addition, to measure the inflammatory response in lung lavage fluid and to decide on the criterion 'inert dust'; iii.) to investigate whether nanoscaled dusts could possibly fulfill the criteria to be included in the gbs class. -six micro-and nanoscaled dusts (one of them a well-characterised inert tio 2 dust (microscaled; rutile modification) were compared analysing the solubility in the lung fluid (day 3, 28 and 90) and the lung toxicity after intratracheal instillation in rats (day 3 and 28): tio 2 (rutile, micro), tio 2 (anatase, nano), eu 2 o 3 (micro-nano mixed), baso 4 (micro), zro 2 (micro) and amorphous sio 2 (nano). two doses of 0.5 and 1.5 µl per rat were administered to wistar rats; these volume doses resulted in a non-overload and moderate overload of lungs, respectively. -the differential cell count showed only slight inflammatory cell levels after treatment with tio 2 (rutile) and baso 4 (pmn < 5% after 3 days in the low dose group; < 15% in the high dose group; full recovery after 28 days). in contrast, the tio 2 (anatase) showed a stronger response (pmn > 30% after 3 and 28 days). the rare earth eu 2 o 3 (micro-nano) dust showed the strongest effect (approx. 40% pmn after 3 and 28 days) including a red-coloured lung lavage fluid. µ-zro 2 and amorphous sio 2 showed a strong acute response after 3 days, however, mostly complete recovery after 28 days. the low solubility criterion was met by the following dusts: tio 2 (both) and zro 2 . -similar volumetric lung burdens were deposited in a parallel validation experiment (14-day subacute inhalations). overall the physiological inhalation route confirmed the results obtained in the instillation study, thus suggesting the applicability of the latter as a tool for identification for gbs dusts. however, for nanoscaled dusts an individual toxicological characterization seems to be adequate. polycyclic aromatic hydrocarbons (pah) represent a complex mixture of compounds and occur in considerable amounts as contaminants in the environment and food. some pah have been demonstrated to be carcinogenic and mutagenic. benzo[a]pyrene (bp), the most known and studied member of the potent carcinogenic pah, is classified by iarc as group 1 carcinogen and is present in a wide variety of food items. however, other non-carcinogenic pah such as pyrene (pyr) and fluoranthene (fa) are also found in substantial amounts in the diet and are strongly suspicious to cause interactive effects. reporter gene assays were used for analyzing interactive effects of a ternary mixture including bp, pyr and fa in relative proportions occurring in food on the nuclear receptors aryl hydrocarbon receptor (ahr) and constitutive androstane receptor (car). the observed activations were verified at the gene expression level in the human hepatoma cell line heparg. beside the well characterized ligand bp, 25 µm of pyr and 20 µm fa also activated the ahr, even though to a much lesser extent. no significant higher activation over the level of bp alone was reached when testing different pah mixtures. however, in heparg cells the analysis of cyp1a1 gene expression as a model target gene of ahr showed synergistic effects after pah co-exposure. in addition, the activation of human car was analyzed. pyr and fa are each strong agonists, whereas bp was less potent. for the ternary pah mixture with bp a strong decrease of the induction was observed. this inhibiting effect was verified at the mrna level using the model car target gene cyp2b6 in heparg cells. in conclusion, really occurring mixtures with non-carcinogenic pah can modulate the effects of carcinogenic pah. such effects warrant to be investigated in more detail to enhance our knowledge of interaction of pah mixtures at the molecular level. cytochrome p450 enzymes and transporters are important for the turnover of pharmaceutical compounds. their expression levels and activity influence bioavailability and convey drug-drug interactions. moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. overexpression of export transporters in tumors can lead to multiple drug resistance. however, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. antibody -based analysis of cytochrome p450 enzymes and transporters is challenging due to their sequence homology. therefore, we developed a test system for protein quantification which combines the sensitivity of immunoprecipitation and the specificity of mass spectrometry: this method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. one peptide from each protein, which can be assigned unambiguously, is identified via tandem ms and quantified by means of an isotope labeled reference. prior to ms-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. these epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. the major advantage of this method is that whole cell or tissue lysates -without preparation of microsomal fractions -can be used for quantification by lc-ms. also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. physiologically based toxicokinetic modeling (pbtk) is an in silico tool to predict compound kinetics based on test substance related properties and physiological parameters of the organism. pbtk is a key element for inverse dosimetry to relate effect concentrations in vitro to external, e.g. oral doses. in our investigations, we use 8 compartment models for the rat including adrenals and testes or ovaries. test substance specific properties taken for pbtk modeling are molecular weight and logp o/w as well as ivis based tissue specific partition coefficients, hepatic clearance, intestinal permeability and plasma protein binding. berkeley madonna software was applied to solve consequent differential equations. here we present the above described model for the 3 test substances bisphenol a (bpa), fenarimol (fen) and ketoconazole (keto). using the lowest effect concentrations (loecs) of bpa, fen and keto from 1) an in vitro yeast based assays with human estrogen and androgen receptor combined with a reporter gene and 2) the interaction of steroidogenesis model calculations were made to relate in vitro concentrations to oral doses in the rat. model calculations, based on in vitro loecs of 10 µm (bpa), 3 µm (fen) and 0.01 µm (keto), for concentrations in target organs resulted in estimated oral loels of 16, 4 and 0.04 mg/kg. when calculations were made for plasma levels oral loels were estimated to be 608, 77 and 16 mg/kg for bpa, fen and keto, respectively when compared to existing in vivo data with endocrine related loels of 375 mg/kg bw day for bpa (1), 50 mg/kg day for fen (2) and 6 mg/kg day for keto (3) , it can be concluded that for the exemplary test substances addressed, ivis related risk assessment approaches based on target tissues seems overpredictive, whereas plasma related loels were closer to the in vivo situation, to study the activation of the nuclear receptor pxr a gal4/uas-based pxr transactivation assay was used. the pxr-mediated induction of cyp3a4 promotor activity was investigated using a pxr-dependent cyp3a4 reporter gene assay. cyp3a4 induction was analyzed at the mrna and protein levels in hepg2 cells using qpcr and western blot. to cover the most frequently occurring pa structures (retronecine, heliotridine and otonecine type as well as monoester, open-chain diester and cyclic diester) the four pa senecionine, heliotrine, echimidine and senkirkine were selected as representative pa for initial analyses. out of the four investigated pa only echimidine activated pxr. accordingly, pxrmediated induction of cyp3a4 promotor activity could only be detected for echimidine. cyp3a4 induction by echimidine was verified at the mrna and protein level in hepg2 wildtype and hepg2 pxr-overexpressing cells. taking heinrich-heine universität düsseldorf, institut für toxikologie, düsseldorf, germany introduction: in higher concentrations, the blood pressure regulating hormone angiotensin ii (ang ii), leads to vasoconstriction, hypertension and oxidative stress by activation of the renin angiotensin system (ras). here we investigate if nadphoxidases are responsible for ras-mediated oxidative stress in kidney and heart. nadph-oxidases (7 isoforms are known, nox 1-5, duox 1 & 2) are membrane-bound enzymes that produce reactive oxygen species (ros) for example during the immune response and cell signaling. material and methods: to clarify the role of nadph-oxidases, wildtype mice and nox 1-, nox 2-and nox 4-deficient mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600 ng/kg during 28 days to stimulate high blood pressure. kidney and heart were investigated for steady-state ros levels and dna damage (dna single and double strand breaks). results: in wildtype mice, ang ii leads to hypertension, a declined renal function, formation of ros in kidney and heart and to dna single and double strand breaks in the kidney. all nox-knockout mice exhibited ang ii-mediated hypertension and albuminuria. the lack of nox 2 and nox 4 could neither protect from the formation of oxidative stress in the kindey nor from dna double strand breaks in the kidney. initial findings from the nox 1-knockout mouse do not show an increase in dna double strand breaks in the kidney. discussion: contrary to published results of nox 1-knockout mice, we observed a constant rise in blood pressure over the treatment period compared to the control. this can possibly be due to different ang ii doses. in nox 4-knockout mice we observed increased oxidative stress and increased renal dna damage already in untreated control animals, which is in line with reports suggesting a protective effect of nox4. conclusion: separate eliminations of 3 nadph-isoforms did not allow the identification of the enzyme which is responsible for ang ii-induced oxidative stress. a possible explanation is that oxidative stress is caused by more than one nox-isoform or other enzymes like xanthine oxidase or nitrogen synthase take major part in the formation of ros. of mice (rats, cats, dogs, monkey) and men -how to measure kidney biomarkers across species introduction and objectives: there is a need for reliable biomarker assays to detect organ toxicity induced by drug candidates. in the last 50 years about 60 drugs were withdrawn from the market due to liver and/or kidney damage. for the detection of druginduced organ injury, safety-tox studies in rodents, dogs, non-human primates and humans are mandatory in the drug development process. currently, several protein biomarkers for kidney, liver and cardiovascular organ toxicitity are being clinically validated by international consortia like the safer and faster evidence-based translation (safe-t) or the predictive safety consortium (pstc). we are developing mass-spectrometry-based immuoassays suitable for the detection of these markers in animal models to support these efforts method: urinary proteins are proteolytically digested to peptides using trypsin. subsequently synthetic isotope-labelled peptide standards are spiked in at known concentrations. we employ multi-specific antibodies (txp-antibodies) targeting cterminal amino acid motifs for the enrichment of peptides derived from the protein biomarkers. finally, the protein biomarkers are quantified using nanolc-parallel reaction monitoring-ms. the use of our group-specific txp-antibodies allows protein analysis of samples from different species using the same antibody. results and discussion: we established an ms-based immunoassay platform for the analysis of kidney (diki) injury protein biomarkers in urine across 5 species; human, cynomolgus, mouse, rat and dog. we analyzed the potential diki biomarkers aquaporin 2, podocin, synaptopodin, retinol-binding protein 4, clusterin and osteopontin in urine samples from toxicity studies in cynomolgus monkeys, rodents and humans. conclusion: the application of group-specific txp-antibodies and mass spectrometry allows the quantification of biomarkers in urine of all relevant model organisms. the results strongly support the validation of translational drug-induced organ injury protein biomarkers. although effective anticancer-therapeutic regimen are available, they are accompanied by severe adverse effects on normal tissue, for instance chemotherapy induced peripheral neuropathy (cipn) caused by platinum compounds. the pathophysiology of this clinically highly relevant side-effect is still unknown and neither prophylaxis nor specific treatment is available. therefore, further research elucidating the underlying molecular mechanisms of platinating anti-tumor drugs leading to cipn is required as basis for future development of preventive or therapeutic strategies. in general, platinum compounds lead to cell death mainly via dna damage induction (mostly intrastrandcrosslinks) and through interference with the redox homeostasis of cells. here, we introduce and suggest the well-known nematode model organism c. elegans to elucidate mechanisms of neurotoxicity triggered by platinating agents. so far, we determined doses for cis-and oxaliplatin, which have only moderate effects on development, reproduction and body movement (muscular read-out). however, these doses are sufficient to trigger apoptosis in c. elegans and to induce a considerable amount of 1,2-intrastrand crosslinks in dna (measured by south-western blotting). even more important they lead to strong neurotoxicity in a functional read-out (pharyngeal pumping). with regard to redox homeostasis, we determined the oxidative stress resistance showing that e.g. cisplatin sensitizes c. elegans to reactive oxygen species (ros), which could be prevented if worms were co-or pretreated with n-acetylcysteine. furthermore we determined the level of ros in living c. elegans after treatment with platinating agents and also in combination with protective compounds. using the advantages of c. elegans as a genetic model system, we will further clarify the relevance of different defense mechanisms, including dna repair (nucleotide excision repair, base excision repair), detoxification systems (antioxidative stress factors, metallothioneins) as well as drug transporters and signaling proteins. this will be achieved by using rna interference approaches that allow targeting either the whole animal or specific tissues (i.e. neurons) only. first results of this approach will be presented. finally we aim to use this setup to identify neuroprotective compounds that prevent chemotherapy induced peripheral neuropathy induced by platinating anti-tumor drugs. clostridium botulinum c3 exoenyzme (c3) exclusively adp-ribosylates rhoa, b and c leading to reorganization of the actin cytoskeleton and morphological changes. in addition to the enzyme-based inhibition of rho-gtpases, c3 promotes in an enzymeindependent manner axonal and dendritic growth in neurons. as c3 lacks the canonical binding and translocation domains of bacterial protein toxins, cell entry is currently not well understood. based on overlay assays and mass spec analyses the intermediate filament vimentin was identified as the putative membrane receptor for c3. knock down of vimentin by sirna and application of the selective vimentin disruptor acrylamide led to a significantly delayed uptake of c3. moreover, addition of extracellular vimentin to cells induced an enhanced uptake of c3. proof of principle experiments in astrocytes and neurons from vimentin knock out mice showed c3-induced morphological changes (astrocyte stellation and axon growth) to a reduced extent and a significantly delayed uptake of c3 compared to wild type cells. as vimentin knock out did not completely inhibit c3 uptake into cells, an additional uptake mechanism or additional receptor for c3 is likely. nevertheless, our data reveals that c3 employs a specific endocytosis mechanism with involvement of the intermediate filament vimentin to gain access to host cells. the primary target organ of organic hg species-mediated toxicity is the central nervous system (cns). humans are exposed to organic hg mainly in the form of methylmercury (mehg) via the consumption of contaminated fish and other seafood products. in terrestrial food sources hg is mostly found as inorganic hg. thiomersal is a further organic hg compound which is used as a preservative in medical preparations. exposure to organic hg promotes primarily neurological effects. the understanding of transfer mechanisms regarding the cns is an important precondition for an evaluation of hg species-induced neurotoxicity. thus, primary porcine in vitro models of the bloodbrain barrier and the blood-cerebrospinal fluid (csf) barrier were used to investigate effects of mehgcl, thiomersal and hgcl 2 on the barriers as well as transfer properties into and out of the cns in vitro. the results show significant transfer differences of the various incubated species as well as in the different barrier systems. whereas the bloodbrain barrier seems to account for the transfer of organic hg species from the blood side to the brain side, these species are transferred in the contrary direction by the blood-csf barrier. inorganic hgcl 2 was not transferred across both brain barriers towards the brain side but was able to leave the brain side across the blood-brain barrier. additionally, cytotoxic effects of the hg species by themselves as well as the combination of organic and inorganic hg species have been investigated in human astrocytes and human differentiated neurons. differentiated neurons were much more sensitive towards all hg species. organic species exerted stronger cytotoxic effects in both cell types as compared to hgcl 2 . interestingly, a coincubation of organic and inorganic hg species led to an increased cytotoxicity in the astrocytes. this cocytotoxic effect is currently investigated in differentiated neurons. the species-specific differences with respect to both, effects on and transfer across the blood-brain and the blood-csf barrier in vitro as well as toxic effects in brain target cells, clearly emphasizes the necessity for comparative analyses. introduction: the neural crest is a multipotent stem cell population that arises at the neural plate border during early fetal development. neural crest cells (nccs) migrate to target sites in the periphery, where they differentiate into multiple cell types, including melanocytes, cranial bones and peripheral neurons. failure of ncc migration can lead to severe disorders, such as hirschsprung's disease. aim: to test whether toxicants interfere with human ncc migration, a high-throughput migration assay was established. this test system was used to screen an 80 compound library of potential developmental toxicants. methods: nccs were derived from human embryonic stem cells. the cells were allowed to migrate for 24 h before toxicants were added to the cells. migration and viability of the cells were then measured after another 24 h by high-content image analysis and a custom-developed software package. results: the screening library was assembled by the us national toxicology program (ntp) and consisted of different substance classes, e.g. organophosphates, organochlorines, drug-like compounds, pesticides and polycyclic aromatic hydrocarbons (pahs). out of the tested potential developmental toxicants, 26 compounds reduced ncc migration at non-cytotoxic concentrations. hit-confirmation testing confirmed 23 of the compounds as concentration-dependent inhibitors of ncc migration. among the potential developmental toxicants identified here, there were several organophosphates (e.g. chlorpyriphos) and drug-like compounds as well as polybrominated diphenyl ethers (pbdes) and organochlorine pesticides (e.g. ddt and dieldrin), while none of the tested pahs inhibited ncc migration. the negative controls in the screening library, like acetylsalicylic acid, acetaminophen and saccharin, proved to be non-toxic. conclusion/outlook: the newly established test system allows screening of potential developmental toxicants in a high throughput manner for interference with human ncc migration. confirmation in other types of migration assays is ongoing, and selected compounds from amongst the screen hits are undergoing mechanistic evaluation. oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. how oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. increasing evidence suggests, however, that oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein 1 (rip1) independently of death receptor activation. here, we show that erastin-induced ferroptosis which involves inhibition of the glutamate-cystein transporter (xc -), glutathione depletion and lethal formation of reactive oxygen species (ros) 1 , triggers mechanisms of regulated necrosis independent of tnfα-signaling. in hippocampal ht-22 cells erastin promotes activation of rip1 and subsequent rip1-rip3 necrosome formation which has been investigated as a hallmark of regulated necrosis 2 . in fact, silencing of rip1 by sirna or by the rip1 inhibitor necrostatin-1 prevents ferroptosis-induced cell death whereas the ferroptosis inhibitor ferrostatin-1 fails to protect cells against tnfα-induced classical necroptosis, a form of programmed cell death that is mediated by receptor interacting protein-1 (rip1) and rip3 kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor tnfr) 2, 3 . recently, a genome-wide sirna screen linked cylindromatosis (cyld) to rip1/rip3-dependent necroptosis 4 and also in the present paradigm of ferroptosis, cyld depletion promotes neuronal survival and decreases rip1-rip3 complex formation, suggesting a role of cyld in intrinsic pathways of regulated necrosis triggered by oxidative stress. the ns5a inhibitor daclatasvir is used in combination with other antivirals such as the polymerase inhibitor sofosbuvir for treatment of chronic infection with the hepatitis c virus. daclatasvir is embryotoxic and teratogenic in rats and rabbits at exposures at or above the clinical exposure. in contrast, no teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir, another ns5a inhibitor. we studied these compounds in the embryonic stem cell test (est) alone and in combination with sofosbuvir. the ns5a inhibitors were obtained from selleckchem, the main metabolite of sofosbuvir, psi-6202, was from medchem express. murine embryonic stem cells (es-d3) were obtained from atcc. they were kept in iscove's modified dulbecco's medium (imdm). substances were dissolved in dmso at a final dmso-concentration of 0.1% in the culture medium. a cytotoxicity assay as well as a differentiation assay were performed. after 10 days in culture the cells were evaluated. cytotoxicity was measured by an mtt test. differentiation into contracting myocardial cells was determined using direct phase contrast microscopy. the substances were tested at concentrations between 0.1 and 30 mg/l, which is a broad coverage of the therapeutically relevant concentrations reached in patients. at a concentration of 10 mg daclatasvir / l medium and higher the substance inhibited differentiation of cells. we observed contracting myocytes in 23, 22 and 2 wells out of 24 wells in total at concentrations of 1, 3 and 10 mg/l. at 30 mg/l no differentiation was observed. effects on cell viability were observed at 30 mg/l. unexpectedly, we found a higher potency with ledipasvir. at the low, therapeutically relevant concentration of 1 mg/l this nsa5-inibitor showed a clear impact on differentiation with 6 out of 24 wells affected and no differentiation at higher concentrations. addition of sofosbuvir or its main metabolite psi-6206 at concentrations up to 30 mg/l had no influence on the concentration effect curves established for daclatasvir or ledipasvir. this is the first indication of an embryotoxic potential of ledipasvir. the difference to the results from the routinely performed animal experiments is unknown. possibly, metabolic activity in the maternal organism is responsible for this discrepancy. dimoxystrobin is a european-registered pesticidal active ingredient. biologically it is acting as an inhibitor of the fungal respiratory chain. for the purpose of european registration a full set of toxicological studies has been conducted with dimoxystrobin, including reproduction toxicity studies (according to the most recent oecd tg 416) and developmental toxicity studies (oecd tg 414) in rats and rabbits. dimoxystrobin interferes with the iron transport in rats and mice. this leads to lower serum iron levels and anemia in rats after repeated exposure. this holds true for treated dams and offspring in reproduction toxicity studies. furthermore, offspring effects seen at the high dose of the 2-generation toxicity study were a hypochromic microcytic anemia, impaired body weight development, which only developed postnatally, and reversible cardiomegalies in some 21-days old pups. for all effects clear noaels were determined. in the 2-generation toxicity study no dose adjustment during pregnancy and lactation was performed, which resulted in considerably higher food and compound intakes in dams and offspring during these lifestages. as a result, it seemed, that pups were more severely affected by body weight effects compared to the parental generation. by performing a life-stage specific comparison of body weight and substance intakes, as well as benchmark dose calculations (bmd) for these parameters, it could be demonstrated that the point of departures (pods) and the loaels for direct dimoxystrobin-related effects were comparable for offspring and parents. the heart effects (cardiomegaly), which were reversible, occurred only after direct dimoxystrobinexposure and are considered to be secondary to the detected offspring anemia. both effects (lower body weights and offspring cardiomegalies) only occur postnatally and are not the consequence of in-utero exposure, as no respective effects at higher doses in rat prenatal toxicity studies were seen. two new mechanistic studies (1-generation toxicity study and a 3-week study in young and adult rats, additionally investigating serum iron levels and anemia) confirmed, that pups and young rats were not more sensitive than adult animals to develop anemia or decreased serum iron levels. in 2006, dimoxystrobin was classified with r63 (possible risk of harm to the unborn child) by the ecb, which was the european authority responsible for classification and labeling, before echa in helsinki was formed. the r63 (which has been translated into the ghs classification repr. 2, h361d) was based on offspring body weight and heart effects seen in the 2-generation toxicity study. based on a comprehensive re-evaluation of existing and on new data of dimoxystrobin, the conclusion can be drawn, that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. perfluorooctanesulfonic acid (pfos) and perfluorooctanoic acid (pfoa) are perfluorinated substances (pfas) which are used for the fabrication of surfaces with water-and dirt-repellent properties. due to their reprotoxic properties and their persistence in the environment, the use of pfos was restricted in 2009 and a restriction program for pfoa was initiated in 2013. therefore, industry switches to pfoa and pfos substitutes, which are predominantly pfas with a shorter carbon chain length, or structure-related compounds. in contrast to pfoa and pfos only few toxicological data are available for their substitutes. aim of this study was to examine endocrine effects of the substitutes perfluorohexanesulfonic acid (pfhxs), perfluorobutanesulfonic acid (pfbs), perfluorohexanoic acid (pfhxa), perfluorobutanoic acid (pfba) and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid (genx) in comparison to pfoa and pfos. a hek-293t cell-based dual-luciferase reporter gene assay was used to investigate the potential of these compounds to affect the activity of the human estrogen receptors herα and herβ. the reporter gene assay revealed no activation of herα or herβ by the pfas tested in this study. to investigate the potential inhibition of herα and herβ by pfas, a coincubation with the estrogen receptor agonist 17β-estradiol was performed. none of the tested pfas inhibited herα or herβ activity. however, in the case of herβ an enhancement of 17β-estradiol-stimulated activity was observed. thus, pfas do not directly activate or inhibit the human estrogen receptors but have an impact on herβ activity as they amplify the activation mediated by 17β-estradiol. further studies will be conducted to examine this synergistic effect in more detail. the xeer-reporter cell line: a novel dual-color luciferase reporter assay for simultaneous detection of estrogen and arylhydrocarbon receptor activation p. tarnow consumers are exposed to a multitude of anthropogenic and natural substances capable of activating or inhibiting ligand activated transcription factors, respectively. this in turn can lead to adverse health effects, particularly for substances acting on signalling pathways that are subject to regulatory crosstalk such as xenoestrogens and polycyclic aromatic hydrocarbons (pahs). xenoestrogens are known to activate human estrogen receptors (ers), whereas pahs or dioxins act on the arylhydrocarbon receptor (ahr). importantly, both receptor signalling pathways are interconnected by a complex crosstalk on multiple levels. this ranges from direct protein-protein interactions to competition for common co-factors. however, although this cross-talk has been long known we still lack a deeper understanding of its molecular mechanisms and physiological implications. one reason for this is a lack of tools to visualise and investigate receptor interaction in vivo. based on the breast cancer cell line t47d we thus developed a dualcolour reporter assay which allows time-resolved simultaneous monitoring of the activation of er and ahr in living cells. the assay uses two beetle luciferases emitting luminescence in the red (slr) and the green (eluc) spectrum, respectively. while eluc is expressed under the control of a 6-fold repeated xenobiotic response element (xre) slr is subject to transcription regulation by a 6-fold repeated estrogen response element (ere). both constructs were stably transfected into t47d human breast cancer cells, which endogenously express erα and ahr and are thus ideally suited for monitoring interactions with both receptors. the respective "xeer"-cell line has been successfully subjected to proof of principle studies, using prototypical er-and ahrligands as well as various phytochemicals and xenobiotics. besides e2 and tcdd ligands included various pahs, polychlorinated biphenyls, alpha-and betanaphthoflavone, cosmetic ingredients (butylparaben, benzophenone-2 and 4-mbc), bisphenol a, genistein, resveratrol, diindoylmethane as well as pharmacological antagonists of both receptors. asian women consuming soy rich food throughout life possess lower levels of 17betaestradiol (e2) in plasma (pl) than western women, whose diet is characterized by less soy consumption during early life and possible intake of soy based dietary supplements during adulthood. however, the impact of these soy exposure scenarios on estrogen (biotrans)formation and the consequence thereof in female mammary glands (mg) has not been investigated yet. thus, female august copenhagen irish rats were fed either isoflavone (if) depleted diet (idd, western exposure scenario without if supplement) or if rich diet (ird, asian exposure scenario) until the end of the study at postnatal day (pnd) 81. furthermore, rats fed idd until pnd74 were fed ird for 7 days (idd+ird, western dietary exposure scenario with if supplement). estrous was determined histologically. levels of transcripts were determined by qpcr and e2 and estrone (e1) in pl and mg were quantified by gc-ms/ms. statistical analyses of estrogens were performed by kruskal wallis and unpaired wilcoxon tests and of transcript levels by linear regression models considering the explanatory variables tissue levels of e2 and diet (idd vs ird and idd vs idd+ird). e2 levels in pl and mg did not coincide with those predicted by estrous. furthermore, median levels of e1 and ratios e2/e1 in mg and ratio of e2 levels in pl/mg were not affected by diet. in contrast, diet tended to affect e2 concentrations in pl (p=0.1211) due to an increase in the ird group (p=0.0056) whereas e2 levels in the idd+ird group only tended to be elevated (p=0.0788). in mg, ird and idd+ird increased e2 levels only weakly (p=0.0788 each). likewise, besides significant changes in transcript levels of cyp1a1 and 1a2, putatively decreasing oxidation of e2 to catechols, in the idd+ird group and (not significantly) also in the ird group, no changes in transcript levels putatively affecting e2 levels were observed. moreover, no decrease in levels of transcripts indicative for cellular (oxidative) stress (gclc, tp53, mt1a) was observed in the idd+ird group. e2 mg levels were significantly associated with an increase in transcript levels of areg and pgr, indicating activation of estrogen receptor (er). in contrast, ird was associated with a significant and idd+ird with a not significant decrease in pgr transcript levels. e2 levels but not diet were significantly associated with gata3 transcript levels, indicating tissue differentiation. furthermore, levels of transcripts involved in intercellular communication (egfr, wnt4) were significantly decreased by idd+ird and not significantly by ird and differed from that affected by e2 (increase in gdf15, hgf, igf1r, wnt5a). bmf, a marker transcript for apoptosis was increased by ird, but not affected by e2 and even decreased not significantly by idd+ird. taken together, despite an increase in e2 levels in pl, less er activation was observed after dietary exposure to if. whereas e2 and transcript levels of enzymes involved in e2 (biotrans)formation as well as er activation and cellular communication were affected similarly but to a different extend in both asian and western if exposure scenarios, differences in apoptosis were observed between ird and idd+ird groups. supported by dfg le 1329/10-1. august copenhagen irish (aci) rats with 17β-estradiol (e2)-releasing implants are an accepted model to study the etiology of breast cancer, but neither e2 (biotrans)formation in mammary gland tissues (mg) during tumorigenesis, nor the impact of isoflavones (if) shown to affect tumorigenesis in aci rats, has been investigated, yet. therefore at postnatal day (pnd) 75 and 175, placebo (-e2) or silastic implants containing 4 mg e2 were implanted in female aci rats exposed to either if depleted diet (idd) or if rich diet (ird) since conception until the end of the study at pnd 285. palpable mg tumors (pt) and 1-2 mg per animal without pt were characterized histologically and categorized into normal (-e2 group, n=12), hyperplasia and non-pt and pt with and without solid tumors (+e2 group, n=32). e2, estrone (e1), their hydroxylation products and methylation (meo-) products thereof, as well as conjugates of e1 and e2 in plasmas and mg were analyzed by gc-and uhplc-ms/ms, respectively. levels of 49 transcripts involved in (biotrans)formation of e2 and estrogen receptor (er) activation were determined by taqman®-pcr. without exogenous e2, plasma e2 as well as e1 and (borderline) e2 levels in mg were higher in ird. plasma e2 as well as e1 and e2 levels in mg were lower in the -e2 group than that in the +e2 group. e2 levels as well as e2/e1 and e2 mg/plasma ratios were elevated in pt, accompanied by a significant increase in transcript levels indicative for estrogen receptor activation (areg, pgr) and proliferation (mki67). ird increased e2/e1 ratio in pt and, although ird did not affect er activation (areg, pgr), ird increased differentiation (gata3) in normal and hyperplastic tissues and tended to decrease proliferation in hyperplastic (ccnd1) tissues. levels of e1 and 2-meo-e1 were highest in hyperplastic tissues, accompanied by an increase in transcript levels of hsd17b2 (conversion e2 to e1) and cyp1a1. transcript levels of gstm1 and gstm2 were decreased in the whole +e2 group and of gstt1 and gstt3 in hyperplastic tissues, possibly decreasing inactivation of electrophilic metabolites. accordingly, maximum transcript levels of tp53 and mt1a indicating cellular (oxidative) stress were observed in hyperplastic tissues. ird did neither affect levels of 2-meo-e1 nor cellular stress (gclc, mt1a, tp53). of note, neither 4-meo-e1, nor e1 catechols, nor e2 catechols nor methylation products of the latter were observed in any sample. furthermore, no conjugates of e1 or e2 were detected in plasmas and mammary gland tissues. thus, changes in transcript levels of conjugating enzymes induced by tumorigenesis and by ird were not related with detectable conjugate levels of e1 or e2. taken together, whereas hyperplastic tissues were characterized by maximum oxidative metabolism of e1 and cellular (oxidative) stress, pt exhibited highest e2 levels and er activation. ird increased differentiation and decreased proliferation in normal and hyperplastic tissues but increased e2/e1 ratio in pt. supported by dfg le-1329/10-1. level of 17beta-estradiol (e2) in human breast tissue is considered to affect breast cancer initiation, promotion and progression. although putatively beneficial and adverse effects of soy isoflavones (if) on the human mammary gland, in particular in western women, have been discussed extensively, the influence of if levels on estrogen formation in human mammary gland tissue has not been investigated yet. thus, glandular tissues were dissected from 37 mammoplasty specimen obtained from women (age 18-66 years old) not taking estrogen active drugs. 14 of these women had been exposed to if by their usual diet or by intake of a soy-based dietary supplement for 7 days prior to mammoplasty. information on soy consumption and lifestyle were collected by questionnaire and tissues were characterized histologically. genistein, daidzein their conjugates (n=12) and bacterial metabolites (n=7) as well as the estrogens estrone (e1)-sulfate, e1, e2 and 2-methoxy-e1 were determined by uhplcand gc-ms/ms, respectively and transcript levels of 19 enzymes involved in e2 (biotrans)formation were quantified by taqman®-pcr in glandular tissues. isoflavonoids were categorized into the if parameters aglycones (agl) and conjugates (con) of either genistein, daidzein or sum of both and were further statistically analyzed by spearman`s rank correlation analysis. a positive correlation of e2/e1 ratio with agl(+con) was observed in glandular tissues (r=0.49, p=0.002), accompanied by a significant negative correlation of e1 levels with agl (r=-0.35/p=0.032), possibly due to reduction of 17beta-hydroxysteroid dehydrogenase 2 (conversion of e2 to e1) expression as indicated by a weak negative correlation of transcript levels of 17beta-hydroxysteroid dehydrogenase 2 with agl+con (r=-0.25, p=0.080). further statistical analysis taking into account multiple variables using linear regression models will provide more insights into variables affecting e1/e2 ratio. taken together, estrogen profile in human glandular breast tissue seems to be affected by if levels. supported by dfg le-1329/10-1. allergic contact dermatitis (acd) is a widespread disease often caused by substances in consumables. the eu prohibits the testing of cosmetic ingredients in vivo. this urges the development of reliable in vitro testing strategies. activation of dendritic cells (dcs) represents a key step during sensitization as they are essential for selection and priming of allergen specific effector t cells. in an integrated omics approach we aimed to further elucidate the molecular mechanisms of dc activation using quantitative metabolomics and proteomics. monocytic thp-1 cells were used as a model system and treated with the sensitizer 2,4dinitrochlorobenzene (dncb; 5, 10 and 20 µm) and the irritant sodium dodecyl sulfate (sds; 100 µm). samples were taken after 4, 8 and 24 hours. thp-1 activation was analyzed by measuring the established activation markers cd86 and cd54 after 24 hours. a targeted lc-ms/ms approach was used to analyze 188 metabolites including amino acids and lipids. protein levels were quantified by nano-lc-maldi-ms/ms after stable isotope labeling by amino acids in cell culture (silac). data sets were examined by multivariate analyses for identification of biomarker candidates. regulated metabolites and proteins were subjected to pathway analysis. the data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. drug induced liver injury (dili) is one of the most frequent causes of acute liver injury and a main cause for drug withdrawals. currently there are no reliable models to test the dili potential of new compounds available. kupffer cells (kc) play an important role in hepatic cell stress mediated through chemokines and release of endogenous proteins. kc activation by damaged or stressed hepatocytes can lead to activation of the nf-κb signaling pathway transmitted by reactive oxygen intermediates (roi). we have recently established a liver model composed of primary human hepatocytes (phh) and kc which enables investigation of immune reactions after induction of hepatocyte stress (kegel et al., 2015) . aim of the present study was the kinetic investigation of hepatic cell stress induction and macrophage activation after treatment with subtoxic concentrations of hepatotoxic drugs. primary human hepatocytes (phh) and kc were isolated from human liver resectates using a two-step collagenase perfusion technique. initial kc activation was characterized by the dcf assay and immunofluorescence staining. phh were incubated with different concentrations of acetaminophen (apap) and diclofenac (dic) for different time intervals. cell stress was evaluated by measurement of oxidative stress (dcfassay) and viability (xtt-assay). in order to simulate macrophage activation following hepatocyte damage, kc and macrophages derived from the monocytic cell line thp-1 were incubated with supernatants of phh treated with hepatotoxic compounds. kc and thp-1-macrophage activation were investigated by measuring intracellular formation of roi using the dcf-assay and cell activity using the xtt-assay. the characterization of kc activation revealed a donor and disease dependent kc activation resulting in kc differentiation to pro-and anti-inflammatory macrophages. therefore, kc were substituted by macrophages derived from thp-1 cells. evaluation of hepatic cell stress showed the strongest effect on thp-1-macrophages when phh were incubated with apap or dic for 4 h.treatment of kc and thp-1-macrophages with supernatants of phh challenged for 4 h with hepatotoxic compounds indicates that thp-1-derived macrophages react similar to kc when treated with phh supernatants in terms of cell activity and roi-production. in conclusion, thp-1 derived macrophages might be a suitable alternative to kc concerning macrophage activation. the evaluated kinetic window of 4 h covering hepatic stress induction and immune reaction allows to perform these measurements in a since the use of cerium dioxide nanoparticles is known to be beneficial e.g. in terms of reducing fuel consumption when added to diesel fuel it has become a frequently used nanomaterial. to compensate the concurrent lack of information on its toxicology a 90day nose-only inhalation study was initiated. by comparing the results to a combined chronic inhalation toxicity and carcinogenicity study using the same test items and experimental conditions (basf, ludwigshafen, germany) early indicators for genotoxic and carcinogenic effects should be determined. rats were exposed to 0, 0.1, 0.3, 1 and 3 mg/cm³ ceo 2 as well as 50 mg/m³ baso 4 nanoparticles (6 h/day, 5 days/week, 13 weeks). animal dissections were conducted at five time points (exposure day 1 and 28; recovery day 1, 28 and 90) aiming for endpoints mandatory according to oecd guideline 413. additionally, gene expression analyses in isolated pneumocytes type ii were performed using pathway arrays for inflammation, oxidative stress, genotoxicity, apoptosis and lung cancer. the given results intend the identification of marker genes displaying modulated expression in response to nanoparticle exposure. investigations on ceo 2 and baso 4 retention in the lung are also included in this project. in bronchoalveolar lavage fluid (balf) a time and dose-dependent increase of inflammatory cells has been detected up to the end of exposure. the amount of inflammatory cells decreased during post-exposure; however, in the high dose group a persistent inflammation up to 90 days was detected by balf and histopathology examination. based on our current results effects of ceo 2 nanoparticles on the respiratory system are suggested. its relevance in the context of long term effects such as tumor development needs to be estimated considering all investigations included in this study. the inhalt-90 project is funded by the german federal ministry of education and research (bmbf) -03x0149a. core or coating material? what dictates the uptake and translocation of nanoparticles in vitro? nanoparticles are becoming increasingly important role in consumer-related products. understanding the interactions between nanoscaled objects and living cells is therefore of great importance for risk assessment. in this context, it is generally accepted that nanoparticle size and shape are crucial parameters regarding the potential of nanoparticles to penetrate cell membranes and epithelial barriers. current research in this field additionally focuses on the particle coating material. in order to distinguish between core-and coating-related effects in nanoparticle uptake and translocation behavior, this study investigated two nanoparticles equal in size, coating and charge but different in core material. silver and iron oxide were chosen as core materials to ensure similar nanoparticles characteristics after particle synthesis. nanoparticles were coated with poly (acrylic acid) (pas) and extensively characterized by tem (transmission electron microscopy), saxs (small-angle x-ray scattering), zetasizer tm and nanosight tm . for uptake and transport studies the widely used human intestinal caco-2 model in a transwell tm -system with subsequent elemental analysis (aas) was used. for evaluation and particle visualization transmission electron microscopy (tem) and ion beam microscopy (ibm) were conducted. although similar in size, charge and coating material, the behavior of particles in caco-2 cells was quite different. the internalized amount was comparable, but pas-coated iron oxide nanoparticles were additionally transported through the cells. by contrast, pascoated silver nanoparticles remained in the cells. our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. in summary, a core-dependent effect on nanoparticle translocation was revealed. both the uptake and transport of nanoparticles in and through cells should be considered when discussing nanoparticle fate and safety. nanotechnology is having a great impact not only on basic research but also on many sectors of industry opening the market for numerous new applications ranging from electronics to the health care system. besides their great innovative potential, the large variety of existing synthetic nanomaterials used in the last decade represents a major challenge for scientists and regulators in terms of measuring and assessing the potential hazard caused by the materials or the products themselves. equally, consumers often miss reliable and easy-to-understand information on nanomaterials and nanotechnology and do not know where to get such information. therefore, the international dana 2.0 expert team brings together its expertise and knowledge from different research areas dealing with all aspects of nanosafety research in order to create and provide easy-to-understand, up-to-date and quality-approved nanomaterials' knowledge base on www.nanopartikel.info. this information platform covers the 25 market-relevant nanomaterials focusing on their effects on the safety of humans and the environment.in order to manage and asses the rapidly increasing number of publications related to nanosafety issues, the dana 2.0 project developed a customised methodology «literature criteria checklist», which includes mandatory and desirable assessment criteria covering physico-chemical characterisation, sample preparation and (biological) testing parameters. this checklist facilitates the discrimination between high-and low quality publications and all positively evaluated literature is then fed into the dana knowledge base. accounting for the need to harmonise experimental practices, the dana team also developed a template for standard operation procedures (sop) to support careful scientific practice. validated protocols generated within the german bmbf-funded nanosafety research projects are presented together with results from the swiss ccmx project v.i.g.o. and available for download. another unique feature of the dana knowledge base is the integrated application-based database that provides a unique link between nanomaterials in real applications (e.g. environmental remediation or medical products) and their potential impacts/ toxicological effect(s) that can be easily accessed by the interested visitor. additionally, dana2.0 provides a list of faqs, a link platform with contact data to other information portals and the opportunity to directly pose questions to our experts via e-mail. dana 2.0 is also present on twitter, follow us @nano_info. background: particulate matter of combustion processes enhances cardio-vascular diseases and increases associated mortality rates. around 13% of total pm10 emissions are emitted by wood burners (uba 2006) . how wood combustion aerosols (particles and gasses) can affect human lung cells and how such cellular responses depend on the usage of different wood types and burners is widely unknown. methods: in an exposure chamber imitating the human respiratory tract human alveolar cells (a549) were exposed at an air-liquid-interface (ali) to gasses and particles of wood combustion aerosols. log wood of beech, birch and spruce was burnt in a conventional oven and compared to the combustion of wood pellets in a modern pellet burner. the combustion aerosols were diluted 1:40 and directly delivered to the exposure chamber. after 4h exposure the lung cells were lysed and rna was isolated. in an array based transcription analysis of the whole genome the effects of the aerosol exposures on lung cells was assessed. in parallel, physical and chemical parameters of the combustion aerosols were analyzed. results: the combustion aerosol of wood pellets contained less organic substances than the log wood aerosols, but was higher in its zinc content. genome-wide we found a higher number of regulated genes with combustion of pellets compared to combustion of log wood. the gas phase alone (filtered aerosol) showed comparable gene regulatory activity as the particle-containing total aerosol. aerosol from log wood burning induced mainly genes of the xenobiotic metabolism and cellular signaling. pellet aerosols additionally regulated apoptosis and dna repair processes. conclusions: modern pellet burners reach better combustion efficiencies than conventional log wood ovens, but their emissions seem to stress human lung cells stronger. one reason might be the higher zinc content of wood pellet aerosols. multiwalled carbon nanotubes (mwcnts) may pose as a risk similar to asbestos in causing cancer, notably mesothelioma, which is a malignant tumor originating from mesothelial cells. to identify molecular cues leading to mesothelioma development, we performed genome-wide transcriptome analysis using microarrays in primary human peritoneal mesothelial lp9 cells treated with two different tumor-inducing tailor-made mwcnts (rat model; rittinghausen et al. 2014 part fibre toxicol 11:59), or amosite asbestos at 3 µg/cm2 for 24 h. specifically, we determined how the transcriptomic changes of the highly tumorigenic mwcnt a would differ from another tumor-inducing mwcnt with albeit lesser potency (mwcnt d), long amosite asbestos as positive control, and milled mwcnt a as material control. initial analysis using bioinformatic tools, revealed 3788 significantly differentially regulated genes for mwcnt a, 1680 for mwcnt d, 145 for amosite, and 4 for milled mwcnt. further analyses with ingenuity pathway analysis comparing the two different mwcnt types and amosite, found common as well as exclusive biomarkers. interestingly, we identified many differentially regulated genes implicated in cellular senescence, a growth arrest in response to different stressors including dna damage, disrupted chromatin, and strong mitogenic signals. paradoxically, cellular senescence can represent both tumor suppression and tumor promotion mechanisms. more important, we found differential expression of genes associated with senescence-associated secretory phenotype (sasp) such as inflammatory cytokines, chemokines, proteases, and growth factors, which were manyfolds up-and down-regulated in mwcnt a, compared to mwcnt d and amosite. the mechanisms leading to mesothelioma induction by mwcnts are from far clear, but the key information emerging from the present transcriptomic data, together with our previously identified senescence markers, indicate that cellular senescence has a likely role. nanotechnology offers great advantages for the food industry despite its partly unknown risks, whose enlightenment is the main target of nanotoxicology. due to variability in terms of size, material, shape, surface texture and several endogenous influences, the toxicity of most frequently used and ingested nanomaterials is difficult to estimate. therefore, the aim of this study was the in vitro investigation of toxicological endpoints such as cell viability, dna integrity and the induction of apoptotic processes in human colon carcinoma cells (ht29). for this purpose ht29 cells were exposed for 24 hours with metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, titanium dioxide, zinc oxide) in concentrations of 2-10 µg/ml. at first the cellular uptake of the nanoparticles by means of an icpms was determined. the influence of cell viability was demonstrated by the trypan blue staining and the mtt assay. the alkaline comet assay gave information about possible dna damages and the use of the repair enzyme formamidopyrimidine dna glycosylase (fpg) additionally allowed the detection of oxidized bases. the induction of programmed cell death was examined using by annexin v fitc assay. the icp-ms data showed a maximum particle content of 1.39 pg per cell for the used concentration range. the metal oxide nanoparticles resulted in a significant reduction of cell viability with a decrease up to 40 % after copper oxide and zinc oxide treatment. for metal particles, only for silver a reduced cell metabolism of about 50 % was detectable by the mtt assay. low genotoxic effects could be determined for silver nanoparticles (tail intensity about 12%; control about 6%), while for titanium dioxide the amount of oxidized bases was additionally increased (tail intensity about 20%; control about 6%) for concentrations above 8 µg/ml. induction of apoptosis was determined for silver particles (up to 24% early apoptotic and 20% late apoptotic cells) as well as for titanium dioxide and zinc oxide (10% each early apoptotic cells), whereby the most significant increase in late apoptotic cells was detected for zinc oxide (up to 90%). the results obtained in our studies indicate a clear particle-dependent influence on cell viability and apoptosis-triggering processes, depending on the used material or the concentration deployed, while only minor changes of dna integrity were detected. the evolution in the field of nanotechnology led to a variety of novel materials at the nanoscale. among them are different carbon materials like buckyballs, graphene nanoplates and carbon nanotubes (cnts). cnts are hollow carbon fibres with either one (sw) or multiple sidewalls (mw). mwcnts usually show a diameter of up to 100nm and can be several micrometres long. because of their nanoscale diameter cnt-uptake can take place directly through the plasma membrane of cells by the so called nanoneedle effect [1] . additionally cnts, like most nanomaterials, show a high surface to volume ratio and, because of their micro scale length, a potentially high loading capacity. these properties make cnts interesting for the potential use as drug delivery carriers (ddcs). mwcnts, produced via chemical vapour deposition with a diameter of 45nm and 16µm length, were used in three different forms, unmodified, acid oxidized (ox_cnt) and ground. cytotoxicity testing was performed in human umbilical vein endothelial cells (huvec). the cells were seeded in 48-well plates and exposed to doses of 1, 5, 10 and 25µg/cm² growth area of the respective cnt type for 24h. the wst-8 assay was applied for testing cell viability and the ldh cytotoxicity assay to identify potential damage to the plasma membrane and to calculate overall cytotoxicity. the results show that an increased oxidation time for the ox_cnts, in a h 2 so 4 /hno 3 mixture, leads to decreased cytotoxicity in huvec, compared to unmodified mwcnts. during the oxidation reactive oxygen groups are formed on the cnt surface [2] . these groups lead to a reduced hydrophobicity of the cnt surface which could be responsible for the decline in cytotoxicity. future investigations will include the toxicological analysis of mwcnts functionalized with polyethylene glycol (cnt-peg). the hydrophilic polymer peg will be covalently bound to the cnt surface and is expected to further reduce the cytotoxic effect. for these investigations different analytical methods will be used. among others, cell cycle analysis, the brdu assay, pathway arrays and qrt-pcr for the investigation of gene expression and cytokines will be measured. these methods will involve a co-culture model of huvec and human umbilical vein smooth muscle cells (huvsmcs) for a better approximation to the cellular in vivo situation. additionally the peg modified mwcnts will be tested for their loading capacity and efficacy with the anticancer drug doxorubicin for a potential use as an intravenous drug delivery carrier in vivo. although aluminium is one of the most common elements in the biosphere, up to now little is known about its impact on human health. aluminium and its chemical derivatives are highly abundant in food, food contact materials and consumer products what leads to an exposition via the gastrointestinal tract (gi tract), the lung and via skin contact. recently, aluminium is hypothized to cohere with cancer and neurodegenerative disorders. lately, due to an increasing attentiveness on this topic, limiting values for food additives have been tightened by the eu commission. however, cellular effects of aluminium and especially aluminium-containing nanomaterials, are in the focus of our research activities, for example in the international solnanotox project. we established an in vitro simulation system of the gi tract, where nanomaterials undergo the different physiological, chemical and proteinbiochemical conditions of saliva, gastric juice and the intestine. the artificially digested nanomaterials, as well as soluble aluminiumchloride as ionic control substance, were subjected to several analytical and biochemical methods to characterize their change of appearance and their cytotoxic effects on intestinal cellular models. we observed the fate of the nanomaterials during typical ph-values of saliva, gastric and intestinal juice with dynamic light scattering measurements and icp-ms in the single particle mode. after observable disappearance at ph 2 the particles recovered in the simulated intestinal fluid. the simulation of the gi tract, mainly the change of ph settings, may lead to a certain chemical activation of aluminium that can increase bioavailability in the intestine after oral uptake of aluminium-containing food products. in vitro assays like ctb, mtt and cellular impedance measurements showed that there were no acute cytotoxic effects measurable after a period up to 48h after incubation, comparable to undigested particles. in contrast, high amounts of aluminium ions showed additional effects on cell viability compared to non-digested aluminium ions. although toxicological potential of al ions to healthy tissue appears to be low, increased hazardous potential cannot be ruled out to pre-damaged tissue and can have a relevance for special consumer groups with for example chronical intestinal inflammation or dietary eating behavior combined with high exposure to al-containing food products. in the eu, there is a strong need for solutions to substitute halogenated flame retardant (hfr) additives, employed in the fabrication of fr thermoplastic and thermoset materials. these materials are used in diverse commercial products, applications and markets, such as electrical/electronic devices, low-voltage wires or household appliances. the phoenix project, funded by the european union 7 th framework program (grant agreement no. 310187), therefore investigates e.g. several tailor-made, nano-layered hybrid particles as alternatives to hfr additives. considering "safer-bydesign" strategies, potential fr nanomaterials (nm) were physico-chemically characterized (e.g. particle size distribution, zeta potential) and screened early in development for their (geno)toxic and pro-inflammatory potential to timely reject nm with high health hazard. as inhalation is the most important exposure route for nm, lungrelevant cells were used as in vitro screening models. to better enable detection and differentiation of the biologic effects of the most promising nm, screening was started with primary rat lung alveolar macrophages (am; cells of first contact for inhaled nm), at a high concentration of 50µg/cm 2 (24h of incubation), using membrane damage (lactate dehydrogenase release assay), direct dna-damage (alkaline comet assay), and il-8 liberation (elisa) as primary endpoints, and quartz dq12 and al 2 o 3 as particulate positive and negative controls, respectively. in this screening system, biologically inert nm could be differentiated from more active ones. thereby, mg(oh) 2 nanoplatelets (mg1; mean lateral size: 1,5-2 µm; mean thickness: 15-20 nm) represented the least, and pristine few layers graphene nanoplatelets (gr1; mean lateral size: 2 µm; mean thickness: 3 nm; graphene layers: 8 ± 0.5) the most biologically active nm. clear concentration dependencies were detected for gr1 in follow-up experiments. mg1 and gr1 were further tested in other lung-relevant cell types, i.e. mrc-5 primary human lung fibroblasts and a549 lung adenocarcinoma epithelial cells. interestingly, mrc-5 cells were less sensitive towards biological effects of gr1, compared to am, whereas a549 cells showed nearly no effect, keeping in mind that lung epithelial cells are the target cells of lung tumor development. to test the hypothesis that the observed cellspecific differences in sensitivity might in part be based on cellular uptake, cells were exposed for 24 h to 12.5 or 25 µg/cm 2 of gr1 on chamber slides. slides were finally stained with dapi and analyzed by dark field microscopy. cells indeed demonstrated differences in uptake capacity and also showed unique pattern of cellular localization of gr1, i.e. with tight perinuclear agglomeration in am, a more scattered cytoplasmic distribution in mrc-5 cells and limited uptake in a549 cells. additionally, biological activity of the diverse nm seemed also to correlate with cellular uptake, as determined by light and dark field microscopy in am and mrc-5 cells. in conclusion, an am-based screening system was able to differentiate biological activity of diverse nm, with morphology, physico-chemical characteristics, and related cellular uptake most likely to be key for nm-and cell type-specific hazard. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) for two years; a control group was exposed to clean air. after one year exposure, 42 µg/lung was found in animals exposed to 0.1 mg/m³ and 2.6 mg/lung in animals exposed to 3 mg/m³. histological examination of lungs revealed several adverse and non-adverse effects in the lung. the non-adverse effects comprised accumulation of particle-laden macrophages in alveolar/interstitial areas and in the balt, particle-laden syncytial giant cells in the balt and bronchiolo-alveolar hyperplasia (alveolar bronchiolization). the adverse effects included (mixed) alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, interstitial fibrosis and alveolar lipoproteinosis. the incidence and severity of the effects were concentration-related. alveolar lipoproteinosis was not observed at low concentrations of 0.1 and 0.3 mg/m³ ceo 2 . neither pre-neoplastic nor neoplastic changes were observed after 12-months exposure. a no observed adverse effect concentration could not be established in this study. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. this project is part of the eu project nanoreg. moreover, german federal ministry for the environment, nature conservation, building and nuclear safety, german federal institute for occupational safety and health, german federal environment agency funded this project. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) and barium sulfate (nm-220; 50 mg/m³) for two years; a control group was exposed to clean air. lung burdens and burdens in extrahepatic tissues were measured at various time-point. the two year exposure period was successfully terminated and 50 animals per dose group were examined for organ burden and histopathology. the remaining animals currently are kept exposure-free for maximally 6 additional months. up to two years exposure to both nanoparticles did not lead to body weight reduction compared to control animals. the mortality rates were in an acceptable range. macroscopically evident tumors were not detected after two years. the ceo 2 lung burdens were maximally 3.5 mg/g lung tissue at the highest exposure concentration of 3 mg/m³. in comparison, highest ceo 2 burdens in organs remote to exposure were liver and spleen with maximally roughly 1 x 10 -3 g/g tissue. in brain, maximum ceo 2 levels were 7x10 -6 mg/g lung tissue. baso 4 lung burdens were comparatively low (1 mg/g) within the first 13 weeks of exposure and steeply increased to 6 mg/g lung tissue after one year. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. the european centre for ecotoxicology and toxicology of chemicals (ecetoc) 'nano task force' proposes decision-making framework for the grouping and testing of nanomaterials (df4nano) that consists of 3 tiers to assign nanomaterials to 4 main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. the df4nanogrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and systemdependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. use (including manufacture), release and route of exposure are applied as 'qualifiers' within the df4nano to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. the four main groups encompass (1) soluble nanomaterials, (2) biopersistent high aspect ratio nanomaterials, (3) passive nanomaterials, and (4) active nanomaterials. the df4nano aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. this is eventually directed by a nanomaterial's intrinsic properties. however, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the df4nano uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. the df4nano is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nano-technological products. it ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources. the the grouping decisions of df4nano for 24 nanomaterials were validated against grouping by results of existing in vivo data and demonstrated 23 concordant grouping decisions. serum concentrations in cell culture medium influence the effect of cerium oxide nanoparticles on human lung a549 cells: cytotoxicity, proinflammation, cellular uptake, and particle properties k. burchardt the wide use of cerium oxide (ceo 2 ) nanoparticles (np), e.g. as fuel additive and for industrial and biomedical applications, evoked intense studies to understand the effects those particles might have on living organisms. contradictory results of ceo 2 nanoparticle toxicity have been published as they depend on many variables like shape, size, diluent and others. in our work we used an in vitro model of ceo 2 nanoparticles and lung carcinoma cells to investigate the role of serum content in the cell culture medium on cellular toxicity, particle uptake, proinflammation and particle characteristics. proinflammatory ceo 2 np with an average diameter of 15-30nm and different concentrations were diluted in cell culture medium with different fetal calf serum (fcs) concentrations (0.01, 0.1, 1 and 10%) and were used to expose human lung adenocarcinoma cells (a549) for up to 24 hours. at 100µg/ml ceo 2 np showed little to no toxic effecton growth arrested a549 cells at fcs concentrations of 1% or below, but cell viability was decreased to about 80% in proliferating cells in cultures with 10% fcs. the proinflammatory effect of ceo 2 np was investigated through measurement of il-8 mrna expression after 3 hours and cellular il-8 secretion after 24 hours. the qrt-pcr showed that the expression of il-8 mrna in cells treated with 100 µg/ml ceo 2 np in 10% fcs medium was three times higher than in cells treated with lower fcs concentrations. this finding correlated with the cellular il-8 secretion, which showed a stronger increase by cells treated at 10% fcs. differences in cellular uptake of ceo 2 np was determined by fluorescence-activated cell sorting (facs) after 2 and 24 hours of exposition. after 2 hours, the cells treated with ceo 2 np in 10% fcs medium showed a lower mean granularity (∆gmean) as measure for cellular particle uptake than those with less fcs in medium. after 24 hours all probes showed about the same granularity. to examine the effect the fcs concentrations on ceo 2 np characteristics in cell cultures we used dynamic light scattering (dls) and phase contrast microscopy (pcm). dls measurements revealed an increasing hydrodynamic diameter of the particles with decreasing fcs concentrations (about 1030nm (10% fcs) to 4090nm (0.01% fcs)), which was correlated by an increasing particle agglomeration shown by pcm. our results show that the fcs concentration in cell culture medium has a direct or indirect impact on the cytotoxicity, the proinflammatory effect, the facs parameter for cellular particle uptake as well as on particle properties, which should be taken into account when designing, performing and interpreting in vitro experiments to investigate the toxicity of nanoparticles. toxic and inflammatory effects of shape-engineered titanium dioxide nanoparticles in nr8383 rat alveolar macrophages. knowledge about the contrasting toxicity of nanoparticles (np) of different chemical composition has steadily increased over the past decade. however, available literature often reveals considerable differences in effects within a specific type of nanomaterial. these contrasts have been contributed to different handling and testing protocols as well as to sample-specific differences in physico-chemical properties of np that could affect their mode of interaction with cells. within the nanometrology project setnanometro, the highly controlled generation and characterisation of a large set of shape-engineered tio 2 np allows us to investigate the potential role of subtle shape-and surface structure changes on np toxicity. as inhalation represents the most relevant uptake route of np, the nr8383 rat alveolar macrophage cell line was selected for in vitro toxicological testing. since oxidative stress and inflammation are considered as key biological pathways in nanotoxicity, we evaluated the expression of the oxidative stress marker genes heme oxygenase-1 (ho-1) and g-glutamylcysteine synthetase (g-gcs) as well as the pro-inflammatory genes interleukin (il)-1β, il-6, il-18 and inducible nitric oxide synthase (inos) by qrt-pcr. protein levels of il-1β and tumour necrosis factor-α (tnf-α) were measured by elisa. cytotoxicity testing of the tio 2 np by wst-1 assay overall revealed only minimal toxicity in comparison to sio 2 np which were used as reference material. ho-1 and g-gcs mrna analyses indicated that specific tio 2 np triggered a moderate induction of oxidative stress. il-6 was only induced after sio 2 treatment, whereas il-18 was not affected by any of the tested np. in contrast, various tio 2 np caused a significant induction of il-1β mrna expression. however, no significant induction of il-1β and tnf-α protein secretion was observed for any of the tio 2 np. the results obtained from these and ongoing investigations will be linked to the physico-chemical database as being developed for all tio 2 np within the setnanometro project, with the overall aim to build and model nano-structure activity relationships (nsar) for this widely applied type of nanomaterial. acknowledgements: the setnanometro project is supported by the eu-fp7 programme. specific types of tio 2 particles were obtained by solaronix (switzerland), evonik and cristal. potential of silver and silver nanoparticles to reduce n-acetyltransferase 1 (nat1) activity j. lichter 1 , b. blömeke 1 1 trier university, department of environmental toxicology, trier, germany humans are exposed to various kinds of engineered nanoparticles including silver, which is frequently used in consumer and biomedical product due to its bactericide properties. despite their widespread usage, knowledge about influences on cellular functions is still incomplete. n-acetyltransferase 1 (nat1), an enzyme which is ubiquitously expressed in human tissues, catalyzes the transfer of an acetyl group to its substrates and although its endogenous function is not clear yet, it is well known to be involved in the n-acetylation of arylamines. in addition nat1 enzyme activity is known to modulated by non-substrates including metals and certain nanoparticles, however, the influence of silver on nat1 has not been analyzed yet. to address whether human nat1 is a target of silver nanoparticles and released irons on protein, purified nat1 was exposed to silver ions (agno 3 ) and silver nanoparticles (ag10-cooh, average size 10 nm, carboxyl functionalized), and nat1 enzyme activity was analyzing the n-acetylation of the nat1 substrate para-aminobenzoic acid (paba). therefore, purified nat1 (1 ng/µl) was co-exposed for 20 min to paba (1 mm) and agno 3 or ag10-cooh (0.01, 0.1, 1, 10 and 100 µg/ml each), resulting in a nat1:silver relation of 1:0.01-100 (w/w). both, agno 3 and ag10-cooh inhibited the n-acetylation of paba in a concentration dependent manner. using equal amounts of silver and nat1 (w/w, 1 µg/ml) enzyme activity was reduced about 98±0.2% (agno 3 ) and 82±0.6% (ag10-cooh). the lowest concentration analyzed (0.01 µg/ml) reduced nat1 activity about 24±5% (agno 3 ) and 17±5% (ag10-cooh). fifty percent activity reduction was caused by 0.11 ± 0.01 µg/ml of agno 3 and 0.21 ± 0.04 µg/ml of ag10-cooh, which is 10-fold lower compared to the published ic50 values for other metal oxide nanoparticles (3-15 µg/ml). these data indicate that both chemical silver species are able to modulate paba acetylation. further studies will be performed to clarify whether silver ions and/or silver nanoparticles could affect the specific n-acetylation of arylamines in human cells. colloidal silver has been used in medicine for centuries and nanosilver is present in many consumer-related products. however, despite of intense research in the past few years, the potential of nanosilver to induce effects different from ionic silver in vivo and in vitro, is still under debate. in this study, we compared proteomic effects of nanosilver (agpure™) and ionic silver (silver acetate) in the kidney of male rats after repeated oral delivery in a rat 28-days toxicity study. in order to avoid overt signs of toxicity, silver was dosed moderately in amounts of 60 and 6 mg/kg body weight for nanosilver and corresponding amounts of silver acetate (9.3 and 0.93 mg/kg). accordingly, no pathologic effects, including results from clinical chemistry and hematology, were reported. kidney tissue protein crude extract was separated by 2-d gel electrophoresis and differentially expressed spots were identified by maldi-ms. 374 unique proteins, showing a log 2 ratio of ≤ -0.3 for downregulation and ≥ 0.3 for upregulation were identified in all treatment groups. protein lists were analyzed with ingenuity pathway analysis (ipa). when comparing effects of particulate and ionic treatments, similar alterations were indicated for canonical pathways associated with glycolysis, gluconeogenesis and tricarboxylic acid cycle. regarding inflammatory responses, stronger effects were derived for ionic treatments. for both types of silver exposures, changes of protein expressions were linked to changes of fatty acid metabolism and nrf2-mediated stress. mitochondrial dysfunction was highlighted for both nanosilver treatments only, as well as activation of the insulin receptor. in the top-scored network of the higher dose nanosilver treatment, upregulated 14-3-3 protein zeta (ywhaz) displays a central position. ywhaz, an important regulator of cell cycle and apoptosis, interacts with the insulin receptor and is well known to be envolved in many types of cancer. overall, both forms of silver treatment revealed similar patterns of affected cellular and molecular functions in rat kidney, supporting common and overlapping mechanisms of particulate compared to ionic silver. because of the widespread application of nanomaterials and the fact that for some nanomaterials effects on different organisms where shown, nanomaterials are still in focus of interest. moreover the fate of nps is only partiallyassessed over the lifecycle of products containing nanomaterials. while general toxicological properties of nps are well described in diverse in-vitro and in-vivo experiments, the distribution of these particles during the whole and complex process of waste incineration shows big knowledge gaps. in the "nanoemission"-project the entire route from the residual material via incineration, filtering of the exhaust gas up to a possible release into the environment are considered together with the toxicological evaluation of effects on humans and the environment. in these experiments the influence of thermal waste treatment on the toxicological profile of nanoparticles, contained in the waste, will be described. after a complete characterization of the two types of baso 4 -nps from two different manufacturers by scanning electron microscopy/ energy dispersive x-ray analysis (sem/edx), measurement of the specific surface area (bet) and dynamic light scattering (dls) we investigated the impact of the pure baso 4 -nps on primary cells (normal human bronchial epithelial cells (nhbec) and peripherial lung cells (plc)). both materials show statistically significant cytotoxic effects in the resazurin-assay (decreased viability below 40 % for 1 mg/ml after 72 h). in general the effects of both nps were almost similar. additionally the effects of the baso 4-nps were compared more in detail. uptake of the baso 4 was quantified by icp-ms after 24 h and 72 h as well as the release of ba-ions into the cell culture medium after centrifugal separation. the incubation with baso 4 of plc and nhbec to 0.1 mg/ml over 24 h and 72 h leads to ~200 µg baso 4 /1 mio cells. the uptake is dose dependent but not time dependent. the impact on the secretion of inflammatory cytokines was determined by bead-based multiplex-elisa flow cytometry. tnf-α, il-8 and il-33 could be detected in nhbec and plc after np incubation. the possible induction of apoptosis was measured by flow cytometry as well. first investigations showed no induction of apoptosis for both materials. the impact of both nps on the intracellular glutathione level was measured by hplc and showed a decrease of gsh after 72 h. summing up baso 4 -nps showed toxic effects in primary human lung cell cultures after 72 h for concentrations under 1 mg/ml. c3 exoenzyme from c. botulinum is an adp-ribosyltransferase that inactivates selectively rhoa, b and c by coupling an adp-ribose moiety. rho-gtpases represent a molecular switch integrating different receptor signalling to downstream transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton, cell proliferation and apoptosis. previous studies with the murine hippocampal cell line ht22 revealed a c3-mediated inhibition of cell proliferation and a prevention of serum-starved cells from apoptosis (rohrbeck et al., 2012) . former results of studies on map kinase signalling indicated c3-induced modulations of downstream signalling modules. therefore, ht22 cells treated with 500 nm c3 for 48 h were applied for screening of the activity of 48 various transcription factors followed by luciferase reporter assays. five transcription factors namely sp1, atf2, e2f-1, cbf, stat6 were identified as significantly regulated in their activity. for validation of identified transcription factors, studies on the protein level of certain target genes were performed. western blot analyses exhibited an enhanced abundance of sp1 target genes p21 and cox-2 as well as a raise in phosphorylation of c-jun. in contrast, the level of apoptosisinducing gadd153, a target gene of atf2, was decreased. our results suggest that c3 is able to modulate the activity of transcription factors whose target genes are involved in the regulation of cell proliferation and apoptosis. via covalent binding of chemical compounds to dna, adducts can be formed. as a consequence, mutations may occur, which represent stages of chemical mutagenesis and carcinogenesis. the isolation of leucocytes represents an essential field of work within dna-adductomics of blood cells, in which adducts serve as markers of exposure for biological monitoring. peripheral mononuclear blood cells (pbmc) comprising monocytes and lymphocytes can be separated from other blood cells like granulocytes, erythrocytes (and dead cells) by isopycnic density gradient centrifugation of buffy coats (bc´s). bc´s are blood concentrates rich in leucocytes and thrombocytes, prepared from whole blood samples thorough removal of plasma and erythrocytes. for the experiments only bc´s from healthy donors were used. while erythrocytes contain no dna, lymphocytes, which constitute a subcategory of leucocytes, carry genetic information. a variety of commercially available fluids with a density of 1.077g/cm 3 , based on different polymers, exists. these materials form the gradient required for the centrifugation. furthermore, there are several methods available, which differ in the ratio blood vs. fluid, duration of the different work up steps, centrifugation parameters and others. the aim of this work was to compare the effectivity of the different fluids and optimize the workflow. for isolation of pbmc the fluid was put in a tube and then covered by a thin layer of blood. after centrifugation, five layers were obtained (figure 1 ). the interphase was removed carefully, washed and the cells were counted. the yield is expressed as percentage of isolated vs. total leucocytes in the bc, which was based on the leucocyte count of the whole blood sample. as separation fluids, ficoll® paque plus (ge healthcare), histopaque® (sigma aldrich) and lsm® (ge healthcare) were tested. no significant differences between the different fluids were observed. although dilution is often recommended in literature, it was found that dilution had no effect on the yield. similarly, using different fluids (rpmi or pbs) for the washing steps did not reveal any differences. increasing centrifugation speed from 400 to 500 xg resulted in higher yields. in general, large variations in yield (46.8% ± 17.1%) among the various bc´s arose. this result is due to the different parameters such as age, storage, leucocyte and erythrocyte counts of the different bc´s used. therefore, the test conditions were optimized using the same batch of bc. the results show that the low priced separation fluids are comparable in performance tothe more expensive ones. by the direct lamination with bc (without dilution) the wastage could be minimized, the yield increased and thus the isolation made more efficient. the franz cell is a well-established model in lots of skin research fields. we adapted the diffusion cell system to additives and contaminants of consumer products which are designated for skin contacts. we were aimed to simulate real exposure as realistic as possible. to meet requirements like longevity, haptic properties and factory costs, different polymers are used as the raw material of choice and modified by a variable number of additives in the majority of commodities. besides additives with a defined function such as plasticizers, stabilizers, colorants and vulcanization accelerators, contaminants as well as decomposition products or so-called non-intentionally added substances (nias) can be part of the material, among them potentially harmful substances. in a first step, polymers of consumer products like flashlights or tools have been characterized concerning additive composition. possible breakdown products were identified by means of gc-ms/ms or pyrolysis-gc-ms. we focused on analytes of toxicological relevance including antioxidants such as n-phenyl-2-naphthylamine (neozon d), which is suspected of causing cancer, and on degradation products like cresol and its derivatives (e.g., mesitol or 2-tert-butyl-4-methylphenol). subsequently, analytes of interest were brought into direct skin contact using porcine, human and artificial skin models in the franz cell chamber assay. the analytes were either followed up layer by layer using tape stripping or examined utilizing cryo sections. for visualization purposes, analytical evaluation has been completed by use of imaging techniques like he staining or atr-ftir (attenuated total reflectance fourier transform infrared) microscopy. the latter was used with intrinsic markers for tissue specific distribution. our project provides evidence for the potential of polymer components to overcome the natural epidermal barrier and, in part, to enter the viable layers of the epidermis. during skin contact with consumer products several substances migrate out of the matrix and penetrate the skin, among them substances which are hazardous to health such as neozon d. depending on its dose, the blister agent sulfur mustard (sm) may lead to painful erythema, blistering with complicated wound healing, pulmonary edema, pulmonary bleeding and temporary blindness. sm is listed as a schedule 1 chemical in the chemical weapons convention and thus its production, stockpiling and use is prohibited. sm still represents a serious threat for civilians and military forces, especially in asymmetric, terroristic and accidental scenarios. after exposure, the highly reactive molecule alkylates nucleophilic sites in endogenous biomacromolecules forming the characteristic and stable hydroxyethylthioehtyl (hete) residue. hence, bioanalytical methods targeting theses adducts in forensic post-exposure verification analysis are of high interest. herein, we present an optimized, accurate and comparably simple method to detect adducts of sm and human serum albumin (hsa) alkylated at its cysteine 34 -residue. since albumin extraction from human plasma is a time-consuming and expensive step of an established procedure, an alternative method for direct proteolysis of human plasma was developed. plasma samples were cleaved directly with pronase resulting in the alkylated dipeptide hete-cysteine-proline (hete-cp) which is detected by micro-liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µlc-esi hr ms/ms). in order to optimize reproducibility and yield of proteolysis, kinetics were investigated for different kinds of plasma (edta-, citrate-and heparin-) as well as serum. two different mass spectrometers, a triple quadrupole system (4000 qtrap) and a hybrid quadrupole time-of-flight instrument (tt5600 + ), were compared. the latter one proved to be the more selective and sensitive system. the method was successfully applied to in vitro and in vivo samples of real cases of sm poisoning. organophosphorus compounds (op), which were originally intended to be used as pesticides to increase agricultural yields at the onset of the 20 th century, still represent a considerable threat to human health. by irreversible inhibition of acetylcholinesterase op lead to cholinergic crisis due to uncontrolled increase of acetylcholine in the synaptic cleft. finally, sudden death by respiratory failure may result if medical countermeasures are lacking. therefore exceptionally toxic compounds were designed und synthesized as chemical warfare agents (cwa), among which v-type nerve agents, i.e. vx, chinese vx and russian vx, belong to the most toxic artificial substances. recent events like the first gulf war, terrorist attacks in tokyo and the conflict in syria underline the need for ongoing and strict surveillance of cwa prohibition by the chemical weapons convention. unambiguous evidence of such substances (verification analysis) plays an important role with great political and legal impact. a variety of such bioanalytical methods have been established at the bundeswehr institute of pharmacology and toxicology in munich, which is accounted for medical chemical defence in germany. exhibiting quite short half-lives in vivo nerve agents can hardly be detected days or even weeks after exposure. accordingly, there is a great need for additional long-term biomarkers like specific protein adducts. consequently, the current work focuses on examination of adducts between nerve agents and human serum albumin (hsa) as its high abundance and stability in vivo provide relative ease of sampling. after incubation of hsa with v-type nerve agents in vitro the protein was subjected to proteolysis. subsequently resulting peptides were separated using microbore high-performance liquid chromatography (µlc) and detected on-line by modern high-resolution tandemmass spectrometry (hr ms/ms). this allowed unambiguous identification of already known phosphylated tyrosines as well as novel adducts between cysteine-proline dipeptides and the thiol-containing leaving group of v-type nerve agents. simultaneous detection of both biomarkers was realized by a new method, which was applicable even at the very low toxicologically relevant concentrations of v-type agents. therefore, this method represents a valuable and novel supplement of existing methods for verification. fatty acid esters of glycidol (glycidyl esters) are processing contaminants formed as byproducts of industrial deodorizing of plant fats or during other heating processes. following oral intake, glycidyl esters are mainly cleaved to release the reactive glycidol in the gastrointestinal tract. according to the national toxicology program (ntp), glycidol is carcinogenic, genotoxic and teratogenic in rodents. it is classified as probably carcinogenic to humans (iarc group 2a). the exposure assessment of the oral intake of glycidyl esters in humans is difficult, because the current data set for glycidyl ester contents in food is incomplete. we developed a method for the determination of the internal exposure to glycidol by mass spectrometric quantification of a hemoglobin adduct reflecting the total glycidol burden over approximately three months. a modified edman degradation was adapted for the cleavage of the valine residues from the n-termini of hemoglobin by fluoresceinisothiocyanat (fitc) (von stedingk et al. (2011 ) chem res toxicol 24, 1957 resulting in the formation of dihydroxypropyl-valine-fluorescein thiohydantoin (dhp-val-fth). the target analyte is purified with mixed-mode anion-exchange solid-phase extraction and analyzed by lc-ms/ms. a major advantage of the technique is the application to whole blood samples, which renders the time-consuming isolation of erythrocytes unnecessary. we synthesized dhp-d 7 -val-fth as an internal standard for the quantification of the glycidol adduct by lc-ms/ms multiple reaction monitoring. a limit of detection of 5 fmol per injection (5 pmol adduct/g hemoglobin) was achieved. the application of this method will possibly allow future monitoring of the internal exposure of glycidol in human studies. glycidol and 3-monochloropropane-1,2-diol (3-mcpd) are carcinogenic food contaminants, which are present in heat-processed oils and fats mainly in form of fatty acid esters. the risk assessment concerning human consumption of these substances is complicated by various reasons. for example, the data on the occurrence in food stuffs is incomplete. also, the amounts of the proximate carcinogens released from ester hydrolysis in the gastrointestinal tract in humans are not known. monitoring of the internal exposure would be an alternative strategy to support the assessment of possible health risks related to the intake of glycidol and 3-mcpd and their fatty acid esters. for short-term monitoring of the internal exposure, urinary metabolites are suitable biomarkers. we study the potential use of two different substances as descriptors of the oral intake of glycidol and 3-mcpd. the metabolite 2,3-dihydroxy mercapturic acid (dhpma) is generated following glutathione conjugation of both compounds. the second target analyte is 3-mcpd itself, which may also be formed from glycidol in the reaction with hydrochloric acid in the stomach. a method for the quantification of urinary 3-mcpd by gc-ms is currently developed. an lc-ms/ms multiple reaction monitoring technique was devised for the quantification of dhpma in urine samples with the isotope-labeled reference compound 13 c 2 -dhpma. the limit of quantification is 10 µg dhpma/l. related to creatinine, the analyte was detected to be in a relatively small concentration range in urine samples from humans. the average concentration in urine samples (n = 45) of one male volunteer collected over ten days was 154 ± 21 µg dhpma/g creatinine. a meal of a highly contaminated, commercially available frying fat (containing 1.1 mg of glycidol equivalents) did not lead to a visible increase of the urinary concentrations. the considerable background levels of dhpma in urine of humans and also in urine samples of other mammals support the hypothesis that dhpma may be also be formed from an endogenous c 3 -metabolite, as already reported by eckert et al. furfuryl alcohol is a common food contaminant formed by acid-and heat-induced dehydration from pentoses. it induced renal tubule neoplasms in male b6c3f1 mice and nasal neoplasms in male f344/n rats in a study of the national toxicology program (ntp). the neoplastic effects may originate from sulfotransferase (sult)-catalyzed conversion of furfuryl alcohol into the dna reactive and mutagenic 2-sulfoxymethylfuran. the incomplete data set of furfuryl alcohol contents in food does not allow estimating the human exposure. thus, we sought a method for the determination of the internal exposure. recently, the dna adduct of furfuryl alcohol n 2 -[(furan-2-yl)methyl]-2′deoxyguanosine was detected in specimen of human lung tissue. however, human biomonitoring of dna adducts has various disadvantages. for example, dna adducts are removed by various repair systems and human dna samples are usually not accessible in sufficient quantities. we decided to develop a biomarker for the internal exposure to furfuryl alcohol using blood proteins as dosimetric targets. bladder cancer (bc) is a smoking and occupation related disease showing a substantial genetic component. though the prognosis is generally good, a major problem is the frequent relapses affecting about half of the patients. n-acetyltransferase 2 (nat2) is well-known to modulate bc risk in persons heavily exposed to carcinogenic aromatic amines. we aim to investigate the impact of nat2 genotypes, in particular, the ultraslow genotype, on relapse-free time after first diagnosis in 772 bladder cancer cases. we used follow-ups of three case-control studies from lutherstadt wittenberg (n=207), dortmund (n=167) and neuss (n=398). nat2 was genotyped using seven characteristic polymorphisms (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208, rs1799931). haplotypes were reconstructed using phase v.2.1.1. we compared slow to rapid acetylators. additionally, we differentiated between the most frequent slow nat2*5b/*5b and *5b/other slow haplotypes as well as between the ultra-slow *6a/*6a and *6a/other slow haplotypes compared to rapid acetylators. chi-square tests used to check the frequency of relapses in ultra-slow, slow and rapid acetylators. genotype differences in relapse-free time up to 5 yr after first diagnosis of bc were analysed using cox proportional hazards models adjusted for age, gender, smoking habits, invasiveness and study group. a total of 371 (49%) patients showed a relapse within the first 5 yr after bc diagnosis. slow acetylators show a higher frequency of relapses than rapid acetylators (51% vs. 46%, p=0.154). this frequency is even higher in ultra-slow acetylators (61%, or=1.81, p=0.019) but not in slow *5b/*5b genotypes (49%, p=0.609). ultra-slow acetylators had a significantly shorter relapse free time within 5 yr after bc diagnosis than rapid acetylators (median 0.66 vs. 0.94 yr, hr=1.57, p=0.009). this trend was not that pronounced in all slow acetylators combined (0.78 yr, hr=1.19, p=0.127) nor in the subgroup of nat2*5b/*5b genotypes (0.79 yr, hr=1.20, p=0.255). the effect of ultraslow nat2 is even more pronounced in smokers (hr=1.78, p=0.003) but absent nonsmokers (hr=0.89, p=0.781). ultra-slow nat2 seems to be associated with a higher recurrence risk and a shorter relapse-free time, especially in smokers. slow nat2 in general seems to have less impact on recurrence. aalborg university, department of biotechnology, chemistry and environmental engineering, aalborg, denmark xenoestrogens with the potential for endocrine disruption like bisphenol a (bpa) may bind to the estrogen receptors (ers) and modulate expression of er target genes mimicking the natural ligand 17β-estradiol (e2). the potential for endocrine adversity is still predominantly assessed in vivo as existing in vitro tests have only limited value for an exposure-based risk assessment. thus, the development of reliable bioassays for the detection of endocrine disruptors is one of the paramount challenges faced by modern toxicology. a targeted metabolomics approach in mcf-7 cells treated with e2 or bpa revealed potential biomarkers for the estrogenic potency of the studied compounds. among them were several phosphatidylcholines and amino acids. we further addressed proline levels that were found to be strongly increased. investigations of proline levels over time showed a clear proliferation correlated concentration dependency after both e2 and bpa stimulation. furthermore, sirna knockdown experiments suggested an influence of the oncogenic transcription factor myc and the dependency of erα activation on the estrogen-mediated proline increase. our study demonstrates metabolomics as a powerful tool for biomarker identification and hypothesis generation. the results could be used further to develop bioassays for the detection of endocrine disruptive chemicals. children are considered to be more sensitive to most chemicals than the general population due to a variety of factors, including dynamic growth and developmental processes as well as physiological, metabolic and behavioral differences [1]. however, only a few data are available on the magnitude of preschool children's exposure to most chemicals present in many consumer products. several of these chemicals are linked to endocrine disrupting effects in animal studies and are suspected to have also adverse effects e.g. on development and function of the reproductive organs as well as on neurological and behavioral development in humans. among of the chemicals that have been a major focus of discussion in the last years are phthalates, dinch, parabens, bisphenol a, and triclosan due to their suspected health effects. therefore we aimed to investigate exposure levels to metabolites of different phthalates and parabens as well as to bisphenol a and triclosan in urine samples collected from preschool children in german day-care centers from north rhine-westphalia (lupe iii; 2011/12). urine specimens from children aged from 20 to 80 months from 23 different day-care centers were analyzed. in total, 253 preschool children were recruited with mean age of 54 months. our study results show that nearly all children (>95 %) of the study population had urine concentrations equal to or above the limit of quantification for five most common phthalates metabolites (mnbp, mibp, 5oh-mehp, 5oxo-mehp, 7oxo-minp), for bisphenol a and methylparaben. triclosan was detected in 16 % of the study population. in general, the median urinary concentrations of the above mentioned phthalate metabolites were about 5-50 µg/l in spot urine samples. the highest amount among the phthalate metabolites was observed for mibp with maximal values of about 1000 µg/l. median urinary concentration for methylparaben and bisphenol a were about 48 µg/l and 2 µg/l respectively. the maximum methylparaben, bisphenol a and triclosan level found were 1770 µg/l, 72.4 µg/l and 55,6 µg/l respectively. in conclusion, our study shows a widespread exposure of young children to various phthalates, parabens and bisphenol a in north rhine-westphalia, germany. a follow-up human biomonitoring study (2014/2015) has finished recruitment and is in the process of analyzing data. polycyclic aromatic hydrocarbons (pahs) represent a large group of organic compounds that are common environmental contaminants. they are formed by incomplete combustion of organic matter such as coal or crude oil and are often known to be carcinogenic, mutagenic and teratogenic. the acute toxicity of pahs is rather low, but because of their stability and lipophilic character those compounds can accumulate in the human body and cause severe chronic effects. additionally pahs may enter the food chain when preserving meat or fish by exposure to smoke. in the european union maximum levels of 2 µg/kg benzo[a]pyrene and 12 µg/kg as the sum of benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene in the meat of smoked fish and smoked fishery products are set, respectively. smoked fish is often handmade in small fishery stores in schleswig-holstein, where self caught fish is prepared in smoke houses. this technique implies the danger of pahs to accumulate in smoked fishery products above allowed maximum levels. here, we report our findings of pahs in smoked fishery products bought in local convenience and fishery stores in schleswig-holstein and give a brief overview about actual contaminant levels. hplc with fluorescence detection was used to determine the quality and quantity of several toxic pahs in smoked fishery products made locally. pahs may constitute risks for human health when exposed to hazardous levels and therefore it is important to have knowledge about given contaminant levels. colorectal cancer (crc) is one of the most frequent cancers worldwide and is tightly linked to dietary habits. epidemiological studies provided evidence that the intake of red meat is associated with an increased risk to develop crc [1]. red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. the underlying molecular mechanism, however, remains elusive and may involve increased cell proliferation and dna damage induction by heme-iron. in this study, we set out to analyze the genotoxic and cytotoxic effects of heme-iron in human colonic epithelial cell lines. we used hemin (fe iii ) as commercially available heme source, which was compared to inorganic iron chloride (fecl 3 ). first, the time-dependent internalization of hemin and fecl 3 into hct116 cells was determined using icp-ms/ms analysis. treatment of cells with inorganic iron resulted in a maximum of intracellular iron content after 1 h at all doses tested, while hemin particularly at high doses caused an iron accumulation up to 24 h. hemin catalyzed the formation of reactive oxygen species (ros) in a dose-dependent manner in caco-2 and hct116-cells as shown by flow cytometry. consistent with this finding, hemin dose-dependently induced the oxidative dna lesion 8-oxoguanine (8-oxog) as revealed by slot blot analysis and fpg-modified alkaline comet assay. using a pharmacological inhibitor of mutt homologue 1 (mth1), which protects the nucleotide pool by hydrolysis of 8-oxogtp, 8-oxog dna adduct levels in hemin-treated cells were further enhanced. in contrast, inorganic iron hardly affected the cellular ros level and only slightly increased oxidative dna damage. subsequently, a time-and dose-dependent activation of the dna damage response (ddr) by hemin was shown in hct116 and caco-2 cells using western blot analysis, which was followed by a reduction in cell viability at high doses after 72 h. finally, the cytotoxic effects of hemin and inorganic iron were tested using an ex vivo model of intestinal crypt organoids. preliminary results indicate that hemin is highly cytotoxic in organoids, whereas inorganic iron does not impair their viability. taken together, this study demonstrated that hemin induces oxidative stress and dna damage, resulting in the activation of the ddr and subsequent cytotoxicity. in contrast, inorganic iron displayed only modest effects. further in vivo studies using dna-repair deficient and proficient animals will shed light on the contribution of specific dna lesions to hemeassociated colorectal carcinogenesis. red and processed meat consumption is known to be a crucial risk factor in the development of colon cancer, which is one of the most common cancers worldwide. a diet rich in red and processed meat increases n-nitrosation within the colon leading to an increase in endogenously formed nitroso compounds. these compounds are able to alkylate dna of gastrointestinal tract cells, resulting in the formation of dna adducts such as -meg is repaired by mgmt, the potential of this enzyme to repair o 6 -cmg in cells is not well characterized. additionally, adenomas containing a k-ras gc-at transition mutation have lower mgmt levels than adenomas without this mutation. therefore, the aim of this study is to determine the role of mgmt in protecting colorectal cells from genotoxicity by repairing o 6 -cmg adducts. for this purpose, an mgmt-deficient non-transformed human colon epithelial cell line was established by stable transfection via rna interference to inhibit the expression and therefore the activity of mgmt. the transfected cell line was analyzed for complete mgmt gene silencing by activity and expression analyses and cell characterization. results confirmed that there was neither expression nor activity for mgmt in the transfected cells, and the cell characterization data showed that mgmt deficiency did not lead to differences in growth behavior or cell morphology or malignant cell transformation. cytotoxicity experiments performed in the transfected and nontransfected cell lines by treatment with dna alkylating agents suggest that the mgmtdeficient cell line is more sensitive to dna alkylating agents than the non-transfected cell line. these results were also supported by dna damage analysis via comet assay. asarones are secondary plant constituents mainly occurring in acorus calamus l. and guatteria gaumeri. the essential oil of the rhizome of acorus calamus l. is used for flavoring of food and alcoholic beverages. the concentration of β-asarone (ba) in these oils varies between 0.3% and 95%. the bark of guatteria gaumeri, which is rich in αasarone (aa), is used as cholesterol-lowering drug and to treat gallstones in traditional mexican medicine. both, aa and ba are carcinogenic in rodents and mutagenic in the ames test after metabolic activation and thus classified as genotoxic carcinogens. [1, 2] previously, the major metabolites in microsomal incubations with aa and ba were identified and synthesized in our laboratory. side-chain epoxides, the corresponding diols, side-chain alcohols and aldehydes were identified as the major metabolites of aa and ba. [3] the investigation of the mutagenic potency in the ames fluctuation test showed positive results in the salmonella strain ta 100 for aa and ba with metabolic activation and for aa-and ba-epoxide without metabolic activation. while it is known that epoxides are reactive against nucleophiles we set up the hypothesis of dna-adduct formation by epoxides to explain the mutagenic effect. this adducts are currently isolated, characterized by nmr-spectroscopy and used to quantify adducts in cells. at the same time primary rat hepatocytes were incubated with non-cytotoxic concentrations of aa and ba for 24 h. after harvest and lysis of the cells, the dna was isolated by chloroform/phenol extraction and enzymatically hydrolyzed. [4] the residual hydrolysates will be used to identify the dna-adducts formed in cells and to determine the adduct formation rate. preliminary experiments indicate that both, aa and ba also form dna-adducts in intact cells in a concentration-dependent manner. [1] göggmann, schimmer (1983) . mutagenicity testing of α-asarone and commercial calamus drugs with salmonella typhimurium. mutation research. 121, [191] [192] [193] [194] wiseman et al. fatty acid esters of 3-chloro-1, and of 2-chloro-1, are food process contaminants that are formed, e.g., during refinement of vegetable oils. after ingestion, the esters are hydrolyzed in the gut, thereby releasing free 3-mcpd and 2-mcpd. 3-mcpd has been identified by the international agency for research on cancer (iarc) to be possibly carcinogenic to humans (class 2b) and is therefore in the focus of food safety authorities. to elucidate the toxicological properties of these compounds at the molecular level (mode of action) a proteomic study was conducted in which 10 mg/kg b.w./day of either 3-mcpd or 2-mcpd were orally administered to rats over a period of 28 days. total protein was extracted from different tissues of the animals and separated via twodimensional gel electrophoresis (2de). among others, the redox sensor protein dj-1 was identified to be deregulated in liver, kidney, testis, and heart of rats treated either with 3-mcpd or 2-mcpd. up to six different isoforms of dj-1 were identified by 2de-western blot analysis, all of them having the same molecular weight but different pi values, indicating protein modifications of low molecular weight but with a strong impact on the protein charge. treatment of the animals with either 3-mcpd or 2-mcpd predominantly led to a shift of the abundance between two dj-1 isoforms in the rat tissues. this effect was more pronounced with 3-mcpd compared to 2-mcpd. mass spectrometric analysis of these two isoforms identified an oxidation of a conserved cysteine residue (cys106) of dj-1 to a cysteine sulfonic acid to be the protein modification induced by treatment of the rats with either 3-mcpd or 2-mcpd. dj-1 is discussed to participate in a number of biological processes such as proteolysis, protein folding, or redox regulation. oxidation of cys106 was shown to be crucial for the activity of dj-1, and the irreversible oxidation of cys106 to a cysteine sulfonic acid as observed in the present study was shown to result in a loss of protein function and was correlated with diseases related to oxidative stress such as parkinson's disease. thus, the potential impact of 3-mcpd and 2-mcpd on cellular oxidative stress and on associated neurodegenerative diseases has to be considered in the ongoing risk assessment of these food contaminants. pyrrolizidine alkaloids (pa) are secondary plant compounds and widely spread in plant kingdom. humans can therefore be regularly exposed via direct or indirect contamination of food, like herbal teas, honey, wheat or salad. 1,2-unsaturated pa are known for their potentially harmful effects. an acute intoxication may cause venoocclusive disease leading to hepatomegaly, ascites as well as liver hardening resulting in high mortality. on the other hand, chronic pa intoxications due to regular consumption of small amounts of pa are characterized by hepatic necrosis, fibrosis and cirrhosis. an initial whole genome transcriptome study in hepatocytes revealed that molecular pathways related to cancer development, cell cycle regulation and cell death are regulated by the four structurally different pa echimidine, heliotrine, senecionine and senkirkine in short-term exposure (24h). additionally, lipid and bile acid metabolism was affected. however, the uptake of pa by food is more likely to be continuous than singular. therefore, the aim of this study was to investigate molecular effects of longterm exposure (14d) comparatively to short-term exposure (24h) in the hepatoma cell line heparg with the four structurally different pa. in this context we analyzed selected cellular parameters like cytotoxicity and morphology. in contrast to short-term exposure, structure-and concentration-dependent cytotoxicity was found for the long-term exposure (sn>he>em/ski). furthermore, obvious morphological changes such as destructuration and perforation of the heparg cell monolayer were seen after 14d of incubation. based on these findings, a possible induction of apoptosis or necrosis by pa was examined. effects of long-term exposure to pa on gene expression were investigated for a set of genes found to be regulated in the short-term whole genome transcriptome study. the identified regulation of gene expression was confirmed for both terms, with the strongest regulation for cyp7a1 (down-regulation), a gene involved in cholesterol metabolism. therefore, the effects of pa on various parameters related to cholesterol metabolism were analyzed, showing pa effects on cholesterol levels and bile acid secretion. short-term exposure to pa did not affect cell viability. however, repeated doses of pa resulted in severe effects on hepatic cells, concerning viability and morphology. at the mrna level, short-and long-term incubation with pa seem to affect a wide range of signaling pathways. in conclusion, we show for the first time that heparg cells can serve as an in vitro model for hepatotoxicity following chronic intake of pa. shiga toxin-producing e. coli (stec) strains cause a diversity of enteric symptoms in humans, ranging from mild diarrhea to severe diseases such as the hemolytic uremic syndrome (hus). hus is a life threatening disease with microvascular endothelial damage in the gastrointestinal tract and the kidneys, which often leads to haemolytic anemia, thrombocytopenia and acute renal failure. shiga toxin plays a major role for the pathogenesis of hus but the subtilase cytotoxin, which was identified during an hus outbreak in 1998 in stec strains, might contribute as an additional potent enterotoxin. like shiga toxin, subab is an ab 5 protein toxin. the pentameric binding/transport subunit (subb) delivers the enzymatic active subunit (suba), a protease, into the endoplasmic reticulum (er) of human target cells. in the er, suba cleaves the chaperone bip/grp78, which results in cell stress and caspase 3/7dependent cell death. recently, we discovered that higher concentrations of suba (10 mg/ml) causes cytotoxic effects in human epithelial cells (hela, in the absence of subb. the cytotoxic effects were investigated in hela cells in more detail. here, suba binds in a concentration dependent manner to the cell surface and induces dramatic morphological changes as well as caspase-dependent cell death [1] . in contrast to hela and caco-2 cells, cho fibroblasts did not respond to suba. currently, further cell types including macrophages are tested for suba effects and the molecular mechanisms underlying the cytotoxic effects caused by suba and the cell type selectivity are investigated. although there are strong cytotoxic effects caused by suba on some human epithelial cells in vitro, the situation in vivo and the pathogenic relevance of the newly observed suba effect are not known. thermal treatment of fat-containing foodstuff in the presence of salt leads to formation of 3-mcpd and its esters. high amounts of 3-mcpd esters detected in food raised toxicological concern. recent studies revealed that food may also contain significant amounts of structurally related 2-mcpd and its fatty acid esters. toxicological studies indicate genotoxicity in vitro and a carcinogenic potential of 3-mcpd, pointing to kidney and testes as main target organs. 3-mcpd esters were shown to cause similar, but milder effects. few unpublished data exist for 2-mcpd, showing similar organ toxicity compared to 3-mcpd and identifying heart as additional target organ. no such data exist for 2-mcpd diesters. in consequence, further toxicological data were required in order to complete risk assessment for 3-mcpd, 2-mcpd and their esters. hence, an oral 28-days study was performed in male rats in order to fill data gaps and provide essential information for risk assessment. a proteomic analysis was included in order to compare molecular effects induced by 3-mcpd and 2-mcpd and its dipalmitic ester in rat liver, kidney, testes and heart. in order to avoid overt toxicity, moderate doses of 3-mcpd + 2-mcpd (10 mg/kg body weight), and 2-mcpd-dipalmitate (53 + 13.3 mg/kg body weight) were applied. accordingly, no pathologic effects were reported. here, we present proteomic results obtained after analysis of heart tissue. after separation of heart tissue protein crude extract by 2-d gel electrophoresis , differentially expressed spots were identified by maldi-ms. a total of 270 unique proteins deregulated at a log 2 ratio of ≤ -0.5 for downregulation and ≥ 0.5 for upregulation at p ≤ 0.05 were identified. comparing deregulated spots induced by different treatments revealed that a higher number of spots was deregulated by 2-mcpd versus 3-mcpd. dipalmitate treatment even caused more deregulation than 2-mcpd. upregulated cytochrome b-c1 complex subunit rieske (ucri) and downregulated acetyl-coa acetyltransferase (thil) were among the top deregulated proteins after 2-mcpd and 2-mcpd dipalmitate treatment. pronounced upregulation of respiratory chain protein ucri, not deregulated after 3-mcpd treatment, indicates an effect on energy metabolism. downregulation of thil, involved in ketone body metabolism, was only weakly affected after 3-mcpd treatment. protein dj-1 (park7), a multifunctional redoxsensitive chaperone and protease protecting the cell against oxidative stress, was significantly downregulated after treatment with 3-mcpd and the higher dose of the 2-mcpd diester. tropomyosin beta chain (tpm2) was commonly upregulated after 3-mcpd and 2-mcpd treatments, possibly indicating a tgf-beta induction of actin stress fibers. for rat heart, data show that similarities but also some significant differences of 2-mcpd-and 2-mcpd dipalmitate-induced proteomic changes exist compared to 3-mcpd, indicating different mechanisms of toxicity for this structural analogues. oxidation products (oxy) of cholesterol (chol) such as 7α-hydroxy-chol (7α-ho-chol), 7β-hydroxy-chol (7β-ho-chol), 7-keto-chol (7-o-chol), 5,6α-epoxy-chol (α-epoxy-chol) and 5,6β-epoxy-chol (β-epoxy-chol) are formed by autoxidation of chol and are discussed as biomarkers for inflammation and oxidative stress in human tissues to be used in the identification of risk factors for disease. however, oxy-chols are also present in processed foodstuff where 7β-ho-chol (milk) and 7-o-chol (fish, meat, and egg) tend to represent the main oxychols, whereas epoxy-chols, are generally minor constituents. thus, levels of oxychols in tissues may result from both endogenous formation as well as dietary exposure. since quantitative profiles of oxy-chols have not been determined in human adipose tissues yet, levels of oxychols and chol were quantified using gc-ms/ms (internal standards: deuterated oxychols) and gc-fid (internal standard: 5α-cholestan-3β-ol), respectively in breast adipose tissues of 48 healthy women undergoing mammoplasty. furthermore, tissue levels of 25 fatty acids in adipose tissues were determined by gc-fid (internal standard: undecanoic acid) to assess relative levels of pentadecanoic acid, docosahexaenoic acid and elaidic acid, indicative for consumption of dairy products, fish, and processed fats, respectively. all oxychols were detected in all breast adipose tissues. the most abundant oxychol was β-epoxy-chol (median: 0.36 nmol/g tissue, range: 0.06-1.55 nmol/g), followed by α-epoxy-chol > 7-o-chol > 7α-ho-chol> 7β-ho-chol (0.009 nmol/g, range: 0.002-0.11 nmol/g). tissue levels of chol (2.8 micro mol/g, range: 1.88-3.87 micro mol/g) did not correlate (spearman's rank analysis) with that of epoxy-chols and correlated even negatively with that of 7α-ho-, 7β-ho-, and 7-o-chol (r = -0.29, p=0.024-0.044) median oxychol/chol ratios ranged from 0.0119 (5, to 0.0003 (7β-ho-chol). 7-o-chol and 7-ho-chols correlated strongly with each other (r=0.81-0.91, p oxy-chol levels did not correlate with relative amounts of pentadecanoic acid and docosahexaenoic acid, whereas total oxychol, 7β-ho-chol, and β-epoxy-chol levels correlated with relative amounts of elaidic acid (r=0.30, 0.30, and 0.31, respectively, p=0.037, 0.034, 0.028, respectively) . no correlations of oxychol levels, individual or total oxychol/chol ratios with age or bmi were observed. taken together, oxychol profiles in breast adipose tissues were different from that usually present in food but could be affected by dietary habits. classification and labelling of hazardous substances and hazardous consumer products (1) has proven to be a very efficient tool for risk communication. consumer products, such as glue, varnish, or washing and cleansing products need to be classified and labelled if they contain dangerous ingredients that render the mixture hazardous. personal care products, however, need not be classified and labelled if they contain dangerous substances above the thresholds for classification. they are excluded in the clp regulation. what would happen without this exception? when i applied the criteria for classification and labelling to a selection of cosmetic product formulas in a conservative approach, most products would have to be labelled and classified, mainly due to hazardous effects to the eye and to the skin (2) . benefits of personal care products can go along with unwanted properties such as the hazards for the human health, and consumers should be informed about them (3) . risk communication for every day products like personal care products should be clear, easily and quickly understandable. according to the cosmetic regulation (4) the ingredients must be listed on the containers. it must be questioned whether the listing of the ingredients without hazard pictograms on the products could be considered as a clear, easily and quickly understandable risk communication instrument (3). the results show that it is urgent to inform consumers better about the potential dangers of personal care products, because cosmetics need to be applied even with more care than any other consumer product. it is strongly recommended to delete the exception provision in the clp regulation for personal care products. the infochemical effect describes that anthropogenic substances can interfere with the chemical communication and influence organisms so that they perceive their chemical environments differently (1,2). infochemicals play a role in life history, habitat search, food related aspects and survival which shows that disturbed communication (infodisruption) could affect population vulnerability at various decisive points (3). the classical ecotoxicological standard tests do not allow to observe the infochemical effect. systematic analyses are needed to find out more about the relevance of this new chapter in ecotoxicology for natural ecosystems. the first crucial step is to select suitable test substances. repellents (substances used to keep away target organisms, e.g. invertebrates such as midges or fleas via olfaction) enter surface waters mainly indirectly via wastewater discharges from sewage treatment plants or directly by being washed off from the skin and clothes of bathers. there are various indications that repellents which are not toxic for aquatic animals could induce effects like organismic downstream drift of non-target species (downstream dislocation of e.g. crustacean and insect larvae in streams). in a literature study, three repellents were identified to be suitable test compounds for proof of concept of the infochemical effect. deet (cas 134-62-3), icaridine (cas 119515-38-7) and ebaap (cas 52304-36-6) (4). these substances are investigated in new test designs in a subsequent experimental part of the project which are designed to detect and quantify the infochemical effect. persistent, bioaccumulative and toxic (pbt) substances or very persistent and very bioaccumulative (vpvb) substances are compounds with hazardous properties. the non-biodegradability (persistence) and high accumulation in organisms (bioaccumulation) may elicit long-term adverse effects in the environment. persistent and bioaccumulative substances concentrate in the environmental compartments (water, sediment, soil, air) and can be distributed in the food chains. ecotoxicological effects are strengthened by bioaccumulation and appear often in remote areas like marine and polar regions. in the framework of pbt assessment, contrary to the risk assessment, the substances are evaluated regardless of the emission into the environment. an evaluation of medicinal active ingredients under assessment in the german federal environment agency (uba) identified less than 10% as potential pbt candidates. due to data lacks in many cases a definite pbt classification is not possible. the poster presents results of an extensive literature research on the global occurrence of pharmaceuticals in the environment. data on measured environmental occurrences from more than 1,000 international publications have been transferred to a database, with more than 120,000 entries. according to the database, pharmaceuticals have been found in the environment of 71 countries worldwide in all five un regions. most published data are for the compartments surface water and sewage effluent; less information is available for groundwater, manure, soil, and other environmental matrices. more than 600 active pharmaceutical substances (or their metabolites and transformation products) have been detected in the environment. most findings have been published for industrialized countries. monitoring campaigns are increasingly being conducted in developing and emerging countries. these have revealed the global scale of the occurrence of pharmaceuticals in the environment. for example, diclofenac, a non-steroidal inflammatory drug, has been detected in the aquatic environment in 50 countries worldwide. a number of globally marketed pharmaceuticals have been found in both developing and industrialized countries. the available ecotoxicological information indicates that certain pharmaceuticals pose a risk to the environment at measured concentrations. options for cooperative action to address the risk of are also presented. the aim of the research presented was to support the discussion of the proposed emerging policy issue pharmaceuticals in the environment under the strategic approach to international chemicals management (saicm), which is a global initiative of united nation environment programme (unep). the european chemicals' legislation reach aims to protect man and the environment from substances of very high concern (svhc). chemicals with (very) persistent, (very) bioaccumulative and toxic properties (pbt and vpvb compounds), substances that are carcinogenic, mutagenic and toxic to reproduction (cmr compounds), as well as chemicals of comparable concern like endocrine disruptors (eds) may be subject to authorization. the identification of eds is limited as yet, because specific experimental assessments are not required under reach. evidence is currently based on a combination of few experiments, expert judgement and structural analogy with known eds. structural alerts for the identification of potential eds: predictions of properties and effects from chemical structures are based on similarities with either active or inactive substances. structural alerts for the identification of potential estrogen/androgen-ed activities are relevant parts of the structures of compounds that are known to interact with estrogen and androgen receptors as either agonists or antagonists. in addition to the backbones of the chemical structures (pharmacophore) for steric fit to the receptors, modulating factors may be small substructures for local interactions with receptor binding sites and physicochemical properties related to the strength of binding to the receptor. comparison of evidence from in silico, in vitro and in vivo assays for potential eds: identification of potential eds based on findings from mammalian long-term reproduction studies, fish life-cycle tests, receptor assays, and chemical alerts were compared and differences analysed. agreement is limited because in vivo, in vitro and in silico methods address different aspects of potential effects on endocrine systems regarding toxicological targets, modes of action and functional similarity of chemical structures. a combination of toxicological and chemical assays can provide comprehensive and complementary information to support evidence-based identification of potential eds among the chemicals released into the environment. application of structural alerts for the identification of potential eds to the einecs inventory: more than 33000 discrete organic einecs compounds are within the applicability domain of the structural alerts for potential estrogen/androgen-ed activities. among them, 3585 chemicals (ca. 11%) are indicated as potential candidates for endocrine effects based on structural alerts. due to the possibility that these chemicals may interact with estrogen/androgen receptors they should be subject to further investigations regarding their potential for endocrine effects, eventually leading to regulatory actions. within the imi (innovative medicine initiative) project "intelligence-led assessment of pharmaceuticals of the environment " (ipie;http://i-pie.org/), a more intelligent environmental testing and tools for prioritisation of legacy compounds shall be developed. regarding the evaluation of fish toxicity, screening approaches in fish embryos are pursued. while the standard fish embryo toxicity (fet) test is restricted to lethal parameters more relevant as substitutes for acute effects, additional sublethal endpoints may provide expanded applicability of the fet assay for chronic effect assessment in fish. in this respect, the analysis of the metabolome could provide additional insights into biochemical processes elicited by pharmaceutical compounds and could potentially support the extrapolation from fish embryo to chronic fish toxicity as displayed in the standard early life stage (els) test. in the context of ipie a pilot study was performed with the aim to quantify and comparatively assess changes in the metabolic signatures of fish and fish embryos induced by the reference compound amikacin, an aminoglycoside antibiotic, in order to identify metabolic patterns applicable as biomarker profiles that can be linked to apical endpoints in terms of an integrated approach. therefore, toxic effects in fathead minnow embryos and els fish were investigated following a 4 and 32 days exposure, examining conventional endpoints and additionally using a combined direct injection and lc-ms/ms assay for metabolite identification and quantification. metabolic endpoints were found to be at least as sensitive as standard apical endpoints such as growth and mortality as detected in the longer-term fish study. furthermore, multivariate data analysis (pca-x, opls-da) revealed substance induced metabolic perturbations specific for fish and fish embryos, respectively. beyond that, the statistical approach of shared and unique structure (sus) identified some metabolites from the classes of amino acids, biogenic amines and lipids to be similarly changed in both developmental stages related to amikacin treatment, representing shared biomarker candidates. in this first pilot study, the integrated metabolomics approach yielded insights into the molecular consequences of amikacin exposure and provided indications for biomarkers for common effects in fish embryos and fish. due to the different exposure levels in the fet (1 -100 mg/l) and els study (0.0003 -0.03 mg/l), more definitive conclusions regarding the predictivity of metabolic responses in fish embryos for apical endpoints in chronic fish tests are yet not possible. further studies are ongoing with a range of pharmaceutical compounds of different chemical classes which will reveal more substantial information on the applicability of this technology in the prediction of longterm effects in fish. the human cationic amino acid transporter hcat-1 (slc7a1) represents the major uptake route for arginine and other cationic amino acids (such as the essential amino acid lysine) in most mammalian cells. it thus provides these amino acids for protein synthesis as well as for essential metabolic pathways. in endothelial cells, special attention has been given to the role of hcat-1 for supplying arginine to nitric oxide synthase. in spite of its wide distribution, hcat-1 expression is highly regulated both, on the transcriptional and post-transcriptional level. however, the genetic elements involved in transcriptional regulation a largely unknown. here we studied the expressional regulation of hcat-1 in human ea.hy926 endothelial cells. starvation of these cells from cationic amino acids led to a pronounced induction of both, hcat-1 mrna and protein. the mrna induction was almost completely abolished by the transcription elongation inhibitor drb (5,6-dichloro-1-β-dribofuranosylbenzimidazole), suggesting the involvement of transcriptional regulation. we thus aimed at identifying the promoter elements in the hcat-1 gene responsible for this regulation. to our surprise and in contrast to data published by others 1 the chromosomal region up to 5 kb upstream of the first hcat-1 exon exhibited no promoter activity in either endothelial or dld-1 colon carcinoma cells that exhibit a very strong endogenous hcat-1 expression. we could however identify a promoter element within the first intron of the hcat-1 gene. transcriptional activity of this element increased upon amino acid starvation in a similar way as endogenous hcat-1 expression. our results indicate starvation-induced transcriptional regulation of hcat-1 through newly identified promoter regions distinct from those published previously. the transport of a multitude of drug molecules into the cell is mediated by multispecific organic cation transporters (octs), belonging to the solute carrier group (slc). one of these families within this group of membrane transport proteins is the slc47-family consisting of the two multidrug and toxin extrusion transporters mate-1 (slc47a1) and mate2-k (slc47a2). while mate-1 is highly expressed in several different tissues like kidney, liver, skeletal muscle, adrenal glands, testes and heart, mate2-k exclusively occurs in the apical membrane of proximal tubular epithelial cells within the kidney. both transport proteins translocate organic cations in exchange of protons into as well as out of the cell. to define the affinity of both transporters for the anti-diabetic drug metformin and to investigate their interactions with 26 different anti-neoplastic agents comparative transport experiments with the model substrate 1-methyl-4-phenylpyridinium (mpp) have been carried out. therefore stably transfected hek293-cells expressing mate-1 or mate2-k transport proteins generated by portacelltec biosciences gmbh and vector transfected hek293-cells were used. the interaction analyses were carried out by determining the uptake of [ 14 c]-metformin and the inhibition of [ university of basel, basel, switzerland drug transporters play a pivotal role in pharmacokinetics by modulating the cellular entry or efflux of compounds. one transporter facilitating the transport of bile acids, steroid hormones, and statins is the organic anion transporting polypeptide (oatp) 2b1 that is highly expressed in placenta, liver, and small intestine. especially its activity in small intestine and liver is assumed to be basis for specific drug-drug interactions. understanding mechanisms involved in pharmacokinetics is a prerequisite in drug development. to test whether there are species differences in transport activity we compared the expression and function of the human and rat orthologue. first, we determined the transport activity of the known oatp2b1 substrate estrone 3sulfate (e 1 s), and observed a significantly lower k m for mdck-hoatp2b1 compared to .00 ± 10.23 µm, *p<0.05 student´s t-test) whereas there was no difference in v max (mdck-hoatp2b1 119.4 ± 11.3 fmol/min/µg protein; mdck-roatp2b1 102.3 ± 12.8 fmol/min/µg protein). the human oatp2b1 exhibits multiple binding sites for its substrates that may explain specific drug-drug interactions [1] . to identify whether rat oatp2b1 owns the same characteristics, the cellular accumulation of 50 µm e 1 s (low affinity site) or 0.005 µm e 1 s (high affinity site) was determined in presence of specific inhibitors/substrates of oatp2b1. as observed for atorvastatin, a known inhibitor of both affinity sites, the rat transporter failed to exhibit the low affinity site. in detail, while atorvastatin reduced the accumulation of e 1 s in mdck-hoatp2b1 cells (110.0 ± 14.6 fmol/min/µg protein vs. 60.7 ± 7.5 fmol/min/µg protein, *p<0.05 student´s t-test), there was no inhibition of e 1 s accumulation in mdck-roatp2b1 cells (53.2 ± 13.2 fmol/min/µg protein vs. 49.0 ± 17.4 fmol/min/µg protein). additionally, we observed a different membrane localization of both transporters as assessed by immunofluorescent staining showing an apical localization for rat oatp2b1 while human oatp2b1 is localized at the basolateral pole of the cellular model. absolute quantification of mrna (copy number per 1000 ng of total rna) in different tissues of rat revealed a high expression of oatp2b1 in liver (159.6 ± 45.5), a moderate expression in small intestine (14.9 ± 4.0) and colon (57.0 ± 12.3), and a low expression level in kidney (2.3 ± 0.8) . the latter is in accordance with previous findings showing that oatp2b1 is abundant in rat kidney as quantified by absolute proteomics technics [2] . our data demonstrated species differences in localization and activity of the drug transporter. further studies are warranted to proof whether this knowledge will help in future drug development and which molecular cause is responsible for the observed data. background: intestinal drug absorption depends on various factors including aqueous volume, site-specific ph and intestinal motility. moreover, the expression of efflux-and uptake-transporters vary in dependence of the anatomical localization making the gut a complex barrier for drug transfer into the body. in a recent study, site-specific protein and mrna expression levels of 10 drug transporters were determined along the entire length of the human gut. interestingly, discrepancies between mrna expression and protein levels were observed. moreover, there were quantitative differences between small intestine and colon. as a consequence the question arose if this observation could be explained by small non-coding rnas acting as highly tissue-specific posttranscriptional regulators of gene expression. hence, in our current study, we aimed to investigate the impact of mirnas on site-specific transporter expression along the human intestine. methods: total rna was isolated from biopsies obtained from six disease-free organ donors. tissue samples were acquired from the duodenum, the upper and lower jejunum, the upper and lower ileum, and the transversal or descending colon. the expression of 754 mirnas was measured using rt-pcr based low density arrays. expression of all detected mirnas was correlated with transporter protein data recently determined by lc-ms/ms-based targeted proteomics. mirnas and transporter genes showing an inverse pearson's correlation between mirna and protein expression underwent an in-silico search (microcosm targets v.5, targetscan7.0) for putative mirna/mrna interaction. to investigate those interactions in more detail, reporter gene assays and site directed mutagenesis were conducted. results: out of 754 mirnas, 248 were detected in all tissue types. out of ten transporters five showed significant inverse correlations with 10 putatively targeting mirnas (e.g. pept1 and hsa-mir-27a, r= -0.498, p=0.002). reporter gene assays indicated interactions of mir-193a/b with pept1 3'-utr (p = 0.0025 and p = 0.0012) as well as of mir-27a with abcb1 3'-utr (p = 0.0006). the site-specific abundance of intestinal drug transporters is significantly affected by microrna-mediated post-transcriptional regulation under physiological conditions as exemplified for pept1 and abcb1. background: the human uptake transporter nact [gene symbol slc13a5; also known as mindy, the human orthologue of the drosophila indy (i´m not dead yet) transporter] is expressed in liver and brain. nact is important for energy metabolism and brain development and mediates the uptake of tricarboxylic acid (tca) intermediate such as citrate and succinate. reduced expression of this transporter, as studied in knock-out mice, mimics aspects of dietary restriction, promotes longevity and protects against insulin resistance and adiposity. furthermore, mutations in the human slc13a5 gene are associated with epileptic encephalopathy with seizure onset in the first days of life, possibly due to the reduced uptake of tca intermediates into neurons. to gain more insights into the role of nact in drug transport and into structure-function relationships, we studied the inhibition of nact-mediated citrate transport by various drugs and analyzed the effect of known mutations in the slc13a5 gene on nact-mediated transport. methods: drug inhibition studies were performed using hek cells stably expressing human nact with citrate as prototypic substrate. twenty-four drugs were used as potential inhibitors of nact-mediated uptake. the effects of eight mutations, three of them (nactp.g219r, -p.t227m and -p.l488p) associated with epileptic encephalopathy, were analyzed using a transient transfection approach. furthermore, the first computational-based structural model of the nact transporter was established and the impact of all mutations on substrate transport was modeled. results: inhibitions studies demonstrated that only a few drugs (three out of 24 tested) inhibited nact-mediated citrate transport at the tested drug concentration of 100 µm. from these, benzbromarone shows the strongest inhibition with an ic 50 value of 83.2 µm. furthermore, citrate transport was also slightly inhibited by pioglitazone and rosiglitazone. citrate transport of the mutated proteins nactp.g219r, -p.g219e,p.t227m, -p.l420p and -p.l488p was totally abolished and the effect of these mutations could be explained on the basis of the structural model. conclusion: inhibition studies demonstrated that simultaneously administered drugs can inhibit nact-mediated uptake of the prototypic substrate citrate. nact-mediated uptake is abolished by mutations in the slc13a5 gene associated with epileptic encephalopathy. the effect of these mutations can be explained on the basis of the first structural model of this uptake transporter. the atp-binding cassette subfamily b member 1 (abcb1) is a drug efflux pump responsible for the classic multi-drug resistance phenotype in cancer cells. increased activity of abcb1 in cancer cells contributes to protection against a wide range of chemotherapeutic agents. this dramatically decreases therapeutic options and the chance of patient survival. knowledge of the underlying mechanisms for abcb1 deregulation is a critical step for the reversal of this unfavorable condition. of note, phosphatidylinositol-4,5-bisphosphate (pip 2 ) is a known regulator of abcb1. the protein "myristoylated alanine-rich c-kinase substrate" (marcks) is known for its ability to bind and sequester the phospholipid pip 2 . in various forms of colorectal cancer the deregulation of marcks protein goes hand in hand with an increase in malignancy and therapeutic resistance. in this study, we characterized the enigmatic marcks as a modulator of abcb1 activity. we focused on a subgroup of colon cancers, where marcks resides in a hyperphosphorylated state for which the established cell line ht-29 served as a model. we employed various in-vitro methods for the measurement of abcb1 activity, in combination with imaging experiments, assays for cellular viability and classical methods of molecular biology. we combined these approaches with pharmacological inhibition of marcks phosphorylation state or rnai-mediated depletion of marcks. with these interventions we could modulate endogenous abcb1 activity and re-sensitize our cell model against chemotherapeutic agents like 5-fluorouracil which are known to be substrates of abcb1. taken together, our findings suggest a new way how a cancer cell can gain a state of therapeutic resistance. the exploitation of marcks as modulator of abcb1 might be a new approach to target resistant tumors without interfering with the natural function of abcb1 in non-malignant tissue. background: in several large clinical studies low blood concentrations of homoarginine were identified as independent risk marker for stroke, cardiovascular events and mortality. experimental data suggest an active role of homoarginine deficiency in disease development. interference with l-arginine-dependent pathways and signaling has been implicated as a possible mechanism. it was the aim of the present study to identify transport mechanisms involved in the cellular uptake and tissue distribution of homoarginine, which is poorly understood, so far. the experiments focused on cationic amino acid transporters (cats) and possible interactions with known cat substrates. methods: uptake assays were performed using [ 3 h]-labeled homoarginine as substrate and human embryonic kidney (hek293) cells stably overexpressing human cat1 [gene: slc7a1 (solute carrier family 7)], cat2a (slc7a2a) or cat2b (slc7a2b). cells transfected with an empty vector were used as control. unlabeled known catsubstrates l-arginine and asymmetric dimethylarginine (adma) were investigated as inhibitors. results: compared to the uptake into control cells, uptake of homoarginine was significantly higher in hek cells overexpressing cat1 (7-fold), cat2a (1.6-fold) or cat2b (2.2-fold) after 2.5 min using 100 µm substrate (each p<0.001). apparent k m values for cellular net uptake of homoarginine were 174.6 µm for cat1, 3640 µm for cat2a and 522.8 µm for cat2b. homoarginine uptake by the three cats could be inhibited by addition of l-arginine and adma. conclusion: the protective biomarker homoarginine is a substrate of the human cationic amino acid transporters cat1, cat2a and cat2b. compared to other cat substrates, the cat1-and cat2b-mediated homoarginine transport is characterized by relatively high affinity. the uptake of homoarginine by all three cats can be inhibited by l-arginine and adma. taken together these findings make cat-mediated transport of homoarginine a possible target for interactions and pharmacological interventions aimed at homoarginine homeostasis. this project is supported by the doktor robert pfleger-stiftung. background and aim: organic cation transporter 1 (oct1, alternative name slcc22a1) is a polyspecific membrane transporter, which has been suggested to play a role in absorption, distribution and elimination of drugs and toxins. besides endogenous compounds like thiamine (vitamin b1), known oct1 substrates are toxins like mpp + as well as drugs like metformin, o-desmethyltramadol, ranitidine, and sumatriptan. tissue distribution of oct1 has been shown to have strong inter-species differences. in rodents oct1 is strongly expressed in both the sinusoidal membrane of hepatocytes and the basolateral membrane of kidney epithelial cells. human oct1 is strongly expressed on the sinusoidal membrane of hepatocytes, but not in the kidney. furthermore, substrate specific differences have been observed between the human and rodent oct1 orthologs. the aim of this study is to characterize the mechanisms causing substrate specificity between rodent and human oct1 orthologs. methods: overexpression of oct1 orthologs in mouse, rat and human cells was performed by targeted chromosomal integration of t-rex™ 293. the cells were characterized in detail for their ability to transport the model substrates tea + , mpp + , and asp + , the drugs ranitidine, sumatriptan and fenoterol. results: mouse and rat oct1 orthologs showed similar transport activity for all the model substrates and drugs tested. however, significant differences were observed when rodent orthologs were compared to the human oct1. human oct1 showed a 5fold higher v max for the uptake of asp + and 2-fold increase of v max for sumatriptan in comparison to the rodent oct1 orthologs. conversely, human oct1 showed a 4-fold lower v max for the uptake of fenoterol compared to rodent oct1s. there was no difference between rodent and human oct1 in the uptake of ranitidine. these differences were further characterized in detail using chimeric mouse-human oct1 constructs and by comparing the effects of key point mutations in mouse and human oct1 orthologs. conclusions: these data demonstrate strong differences in the substrate specificity between rodent and human oct1 orthologs. therefore oct1 pharmacokinetic data obtained in mouse or rat models could not be directly extrapolated for use in human. furthermore, comparative functional analyses of orthologs may help reveal the mechanisms underlying polyspecificity of oct1. ranitidine is a histamine h 2 -receptor antagonist which is commonly used without prescription for the treatment of pyrosis and gastric ulcers. approximately 30% of the systemic clearance of ranitidine is via hepatic metabolism. ranitidine is known to be a substrate of the hepatic organic cation transporter oct1 [1]. oct1 is expressed on the sinusoidal membrane of human hepatocytes and is highly genetically variable. sixteen major oct1 alleles are known [2] . thereof 12 alleles confer partial or complete loss of oct1 activity. the observed loss of activity was highly substrate specific and should be analyzed substrate by substrate. in this study we analyzed the effects of genetic polymorphisms in oct1 on the uptake of ranitidine. we used hek293 cells stably transfected to overexpress wild type or variant oct1 isoforms. the variant oct1 alleles oct1*1a (met408val), oct1*1c (phe160leu), oct1*1d (pro341leu/met408val), oct1*2 (met420del), oct1*3 (arg61cys), oct1*4 (gly401ser), oct1*5 (gly465arg/met420del), oct1*6 (cys88arg/met420del), oct1*7 (ser14phe), oct1*8 (arg488met), oct1*9 (pro117leu), oct1*10 (ser189leu), oct1*11 (ile449thr), oct1*12 (ser29leu) and oct1*13 (thr245met) were analyzed. we characterized in ranitidine uptake determined k m and v max for the different polymorphic oct1 isoforms. wild type oct1 showed a time and concentration dependent uptake of ranitidine with a k m of 62.91 ± 4.32 µm and a v max of 1125.41 ± 86.12 pmol/min/mg protein. variants oct1*5, oct1*6, oct1*12 and oct1*13 completely lacked ranitidine transport activity. variants oct1*2, oct1*3, oct1*4 and oct1*10 showed v max values decreased by 64, 77, 91 and 50 %, respectively. oct1*8 variant showed an increase of v max by 25 %. there was no significant changes in ranitidine uptake by variants oct1*1a, oct1*1c, oct1*1d, oct1*7, oct1*9 and oct1*11 compared to the wild type. there were no significant differences in the k m values between the wild-type and variants. in conclusion, we confirmed ranitidine as an oct1 substrate and demonstrated that genetic polymorphisms in oct1 can strongly affect ranitidine uptake. the effects of oct1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. otto-von-guericke university, institute for pharmacology and toxicology, magdeburg, germany serotonergic hallucinogens ( s hgs), such as lysergic acid diethylamide (lsd), induce profound alterations of human consciousness, which are thought to be mediated by activation of 5-ht 2a receptors. with repeated application, the mind-altering effects of most s hgs rapidly are undermined by tolerance. the only exception seems to be dimethyltryptamine (dmt), whose mind-altering effects for reasons unknown even with repeated application do not decrease. assuming that dmt differs from other s hgs in its capacity to regulate 5-ht 2a receptors, we here compare lsd and dmt with regard to processes of 5-ht 2a downregulation. in heat-exposed rats, lsd and dmt induce a marked increase in body core temperature (hyperthermia) accompanied by "defensive hypersalivation". both effects are mimicked by the 5-ht 2 selective agonist dimethoxybromoamphetamine (dob) and can be blocked by the selective 5-ht 2a antagonists ketanserin and mdl100907. after repeated application, the temperatureregulatory effects of lsd are subject to tolerance, whereas the ones of dmt are not. tolerance to lsd (as measured by dob induced [ 35 s]gtpуs binding) is paralleled by a desensitisation of frontocortical 5-ht 2 receptors; for dmt, there is no such decrease. applying techniques of immunocytochemistry, transphosphatidylation, and quantitative real-time pcr to (ha-5-ht 2a transfected) hek293 and (endogenously 5-ht 2a expressing) c6 glioma cells, respectively, we furthermore demonstrate that dmt in contrast to lsd fails to internalise 5-ht 2a receptors, fails to activate the phospholipase d (which is needed for 5-ht 2a internalisation), and fails to inhibit the synthesis of 5-ht 2a receptors. given that dmt unlike lsd turns out to be inactive as to all processes of 5-ht 2a downregulation investigated, our data suggest that the differential tolerance development noted for dmt and lsd indeed might be accounted for by differential regulation of 5-ht 2a receptors. lsd and dmt both have recently regained scientific attention as potential therapeutics in the treatment of depression and/or anxiety disorders. providing mechanistic insights into their action, thus, is of timely clinical relevance. increased gaba release in human neocortex at high intracellular sodium and low extracellular calcium -an anti-seizure mechanism? ] e , this reduction might induce an anti-seizure mechanism by augmenting gat-mediated gaba release, a mechanism absent in rats. aging is complex on the systems as well as on the molecular level. the process of aging is characterized by a progressive loss of physiological functions and accumulation of cellular damage. one hallmark of aging is an impaired protein homeostasis. the imbalance of the quality control of both de novo protein synthesis and protein degradation, therefore, is likely to contribute to the phenotype of aging. we investigated protein turnover rates with the state-of-the art techniques funcat (dieterich et al., 2010) and sunset (schmidt et al., 2009) in aging neuronal cell cultures . using these techniques we show a prominent decrease in protein synthesis and degradation that progressed gradually in aging neuronal cells cultured up to div 60. in order to rejuvenate the protein turnover in aged neuronal cells we applied the selective eukaryotic elongation factor-2 kinase inhibitor a 484954 and the polyamine spermidine and observed protein translation utilizing funcat and sunset. whereas both a 484954 and spermidine had no effect on de novo protein synthesis in juvenile neurons (div 20) , both substances increased the de novo protein synthesis to a juvenile level in aged neuronal cultures (div60). this effect is seen in neuronal somata and dendritic spines. the molecular function of spermidine as an "anti-aging agent" is not defined yet. thus, additional pharmacological interventions are used for further examination of specific molecular spermidine targets. in conclusion, the described experimental setup is used to investigate impaired protein homeostasis as one hallmark of aging. agents with a presumed "anti-aging" effect can be tested for a potential rejuvenating effect on the level of protein homeostasis. a screening approach to test tolerability of multitargeted drug combinations for antiepileptogenesis in mice a large variety of brain insults can induce the development of symptomatic epilepsies, particularly temporal lobe epilepsy. in the latent period after the initial insult multiple molecular, structural, and functional changes proceed in the brain and finally lead to spontaneous recurrent seizures. prevention of these developments, called antiepileptogenesis, in patients at risk is a major unmet clinical need. several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. in the majority of studies, drugs were given as monotherapy. however, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. recently, multitargeted approaches were proposed ("network pharmacology") to interfere with epileptogenesis. developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis is a strategy, which would allow a relatively rapid translation into the clinic. we developed an algorithm for testing such drug combinations in a screening approach, modelled after the drug development phases in humans. tolerability of four repeatedly administered drug combinations was evaluated by a behavioral test battery: a, levetiracetam and phenobarbital; b, valproate, losartan, and memantine; c, levetiracetam and topiramate; and d, levetiracetam, parecoxib, and anakinra. as in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful use in preclinical studies on antiepileptogenesis and translation of these preclinical findings to the clinic. based on previous studies, we expected that tolerability would be lower in epileptic mice than in nonepileptic mice. therefore nonepileptic mice were used as a first step, followed by epileptic mice and mice during the latent period shortly after status epilepticus. except combination b, all drug cocktails were relatively well tolerated. in contrast to our expectations, except combination c, no significant differences were determined between nonepileptic and post-status epilepticus animals. as a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy. major depression is one of the most common mental disorders worldwide, with serious social and economic consequences. there are many different hypotheses concerning the pathophysiology of this disease. the complex brain serotonin system and particularly the serotonin 1a receptors (5-ht 1a r) apparently play a pivotal role in the development of depression. the involvement of an altered, 5-ht 1a r-mediated signalling in adult neurogenesis is also discussed. however, in this context the effects of pre-and postsynaptically located 5-ht 1a rs have not been clarified yet. mice with a permanent overexpression of postsynaptic 5-ht 1a rs (oe mice) represent a unique tool to elucidate the effects of postsynaptic 5-ht 1a rs in adult neurogenesis and depressive-like behaviour. previous studies demonstrated an increased proliferation and survival of newborn cells in the adult dentate gyrus of female oe mice in comparison to controls. in the present study, we investigate the proliferation and survival of adult born cells after chronic treatment (15 days) with the selective 5-ht 1a r agonist 8-oh-dpat (0,5 mg/kg/day) in young adult oe and wt mice. on the last three days of treatment, newly generated cells of oe and wt mice are labelled by injections with bromodeoxyuridine (brdu; 50 mg/kg/day). mice are sacrificed either one day (proliferation) or 21 days (survival) after the last injection. we hypothesise that the data we will present will confirm our previous results, with possibly more pronounced proneurogenic effects and differences in male mice. further immunohistochemical studies post-exercise and behavioural analyses are in progress to identify the relation between chronic postsynaptic 5-ht 1a r stimulation, depressive-like behaviour and hippocampusdependent learning. dystonia is a common movement disorder characterized by intermittent and prolonged muscle contractions resulting in involuntary movements and/or abnormal postures. the lack of knowledge of the pathophysiology of dystonia hampers the development of effective therapeutics. although benzodiazepines can improve dystonic symptoms, tolerance and side effects limit their use. there is evidence for striatal dysfunctions in human dystonia. gabaergic striatal interneurons (in) are important for the regulation of striatal signaling. in the dt sz mutant hamster, a model of paroxysmal dystonia, immunoreactive in were reduced at the age of maximum severity of dystonia (33 days), but not after spontaneous remission (age 90 days). as indicated by unaltered homeoboxprotein nkx 2.1 (cell density, mrna), the age-dependent deficit seems not to be related to a disturbed migration, but to a retarded maturation of in in mutant hamsters. here we further determined the maturation of striatal gabaergic neurons in the dt sz hamster compared to healthy controls. kcc2 and cavii mrna, used as markers for the gaba-switch, were unchanged in 18 and 33 day old mutant hamsters, indicating that there is no general delay in gabaergic maturation. as a retarded maturation seems to be specific for in, we used another marker for gabaergic maturation: the expression of specific gaba a receptor (gaba a r) subunits (mature striatal in express the alpha1 subunit). by stereological determination, we found a 46% decrease in alpha1 subunit expressing neurons. a lower immunoreactive intensity was restricted to the somata of dorsomedial striatal in (32%) of dt sz hamsters, indicating both a reduced density as well as a delayed maturation. these findings prompted us to examine the effects of the αlpha1 gaba a r preferring compound zolpidem in comparison with the benzodiazepine clonazepam. zolpidem (2.0 and 10.0 mg/kg i.p.) only exerted moderate antidystonic effects compared to the benzodiazepine clonazepam (0.5 and 1.0 mg/kg i.p.) in the dt sz hamster. examinations of αlpha2 gaba a r preferring compounds are ongoing. in summary our studies indicate that there is no general defect in striatal gabaergic maturation in the dt sz mutant but a specific alteration of striatal gabaergic interneurons which express αlpha1 gaba a r subunits. changes in αlpha1 gaba a r subunit expression and differences in the antidystonic efficacy of zolpidem and clonazepam indicate that further investigations on the role of gaba a r subunits could lead to new therapeutic approaches for the treatment of dystonia. 2005) . therefore, the hypothesis of the present study was that pregnenolone attenuates the inhibition of synaptic transmission elicited by cannabinoids. methods: 250 µm-thick slices containing the cerebellum and the nucleus accumbens were prepared from the brains of mice and rats. spontaneous and electrically evoked gabaergic inhibitory postsynaptic currents (sipscs and eipscs) and evoked glutamatergic excitatory postsynaptic currents (eepscs) were analyzed in superfused brain slices with patch-clamp electrophysiological techniques. results: a) the synthetic cannabinoids jwh-018 (5 x 10 -6 m) and jwh-210 (5 x 10 -6 m) inhibited the spontaneous gabaergic synaptic input (sipscs) to purkinje cells in mouse cerebellar slices. the inhibition by jwh-018 was not affected by pregnenolone (10 -7 m), the inhibition by jwh-210 was only marginally attenuated. b) the depolarization of the purkinje cells induced suppression of the gabaergic input to purkinje cells (dsi); pregnenolone (10 -7 m) did not affect this endocannabinoid-mediated form of synaptic suppression. c) in rat nucleus accumbens slices, gabaergic and glutamatergic synaptic input to medium spiny neurons was activated by electrical stimulation of axons. ∆ 9 -tetrahydrocannabinol (2 x 10 -5 m) suppressed the gabaergic and glutamatergic synaptic transmission in the nucleus accumbens. these suppressive effects of ∆ 9tetrahydrocannabinol were not changed by pregnenolone (10 -7 m). d) finally, we tested whether we can observe neurosteroid-mediated effects in our brain slice preparations. tetrahydro-deoxycorticosterone (thdoc, 10 -6 m) markedly prolonged the decay time constant (τ) of spontaneous gabaergic postsynaptic currents (sipscs), similarly as in previous experiments (ej cooper et al., j physiol 521: 437-449, 1999) . the results show that inhibition of gabaergic and glutamatergic synaptic transmission by synthetic-, endogenous,-and phyto-cannabinoids is not changed by pregnenolone. therefore, it is unlikely that interference with cannabinoid-induced inhibition of synaptic transmission is the mechanism by which pregnenolone attenuates behavioural and somatic effects of ∆ 9 -tetrahydrocannabinol in vivo. the hypothalamus is one of the key players in the regulation of the energy homeostasis. cold stress leads to an activation of neurons in the paraventricular hypothalamic nucleus (pvn) and increases thermogenesis. the thyrotropin-releasing-hormone (trh) neurons have an important function in this effect. however it is hardly understood which role the trh neurons exactly play and how they are connected to other regions of the brain. we have transduced neurons in the pvn of mice with a recombinant adeno associated virus which contains an activating "designer receptors exclusively activated by designer drugs" (dreadd) system under the control of a shortened trh promotor. two weeks after transduction the animals were injected with clozapine-n-oxide (cno). to analyse the physiological function of this neurons we performed indirect calorimetry, measured rectal temperature and thermogenesis in the brown adipose tissue (bat), analysed drinking feeding behaviour and the home cage activity. after stimulation we measured the expression of genes in bat as well plasma hormone levels of pituitary hormones. propranolol and the specific β3-antagonist sr59230a were used to analyse the relevance of the sympathetic system. to further characterise the transduced neurons and their projections we used immunohistochemistry methods. after stimulation with cno the energy expenditure and body temperature were increased. these effects were mostly driven through an activation of the brown adipose tissue (bat). in dreadd transduced trh-receptor 1 (trh-r1) knockout mice this effects were abolished. in parallel the plasma levels of tsh, the ucp1 mrna level in the bat, the home cage activity as well the food and water intake were increased. after the treatment with propranolol and sr59230a the effects on the thermogenesis were reduced, but the home cage activity was not affected. sr59230a treatment normalised the food intake and increased in parallel the plasma leptin concentrations after cno stimulation. transduced neurons project into the raphe nucleus, the medial part of the thalamus and the spinal cord. with our experiments we could provide strong evidence for a sympathetic connection of the transduced neurons in the pvn to the bat and for the involvement of thr neurons in these effects. therefore, this system is a suitable tool to investigate the metabolic relevance of trh neurons in detail. background & objective: during obesity development, tissue factor signalling contributes coagulation-independently to inflammatory and metabolic dysfunction of adipose tissue. adipogenesis involves proliferation and differentiation of preadipocytes, apoptosis and hypertrophic growth of differentiated adipocytes, angiogenesis and extracellular matrix reorganisation. the coagulant protease thrombin promotes similar processes in various cell types, through activation of protease-activated receptors par-1, par-3 and par-4. in human adipose tissue, par-1 is found in vascular stromal cells and par-4 in preadipocytes and differentiated adipocytes. thrombin stimulates mitogenic kinase signalling and induces inflammatory cytokine and angiogenic growth factor secretion in adipocytes. we have examined the contribution of thrombin receptor activation to adipogenesis processes in 3t3l1 cells. results: differentiation of 3t3l1 preadipocytes with insulin, dexamethasone and isobutylmethylxanthine increases leptin and pparg gene expression and accumulation of triglycerides and oil red o-stained lipids. par-4 is time-dependently upregulated in maturing cells while par-1 expression is detectable but not altered. in preadipocytes, thrombin (3 u/ml) activates the mitogenic kinase erk1/2, promotes cell proliferation and induces gene expression of the maturation markers leptin and pparg and the inflammatory marker tumor necrosis factor alpha (tnfa). repeated stimulation of differentiating adipocytes with thrombin suppresses induction of leptin and pparg and attenuates lipid accumulation, while expression levels of the proliferation marker ki67 and the inflammatory cytokine interleukin (il)-6 are increased compared to differentiated control cells. similar proliferative and anti-adipogenic effects are seen with the selective par4-activating peptide (gypgkf, 100 µm) and cathepsin g, a proteolytic par-4 activator released from neutrophils and mast cells. repeated exposure of maturing 3t3l1 cells to conditioned medium from degranulating mouse peritoneal mast cells (mccm) augments lipid accumulation and il-6 expression. pretreatment of mccm with a par-4 inhibitor further drives lipid accumulation, the induction of il-6 by contrast is suppressed. conclusion: par-4 activation by thrombin or inflammatory cell-derived cathepsin g appears to suppress adipogenesis, possibly by maintaining proliferative capacity and preventing the growth arrest essential for initiating matuation. increased par-4 expression in maturing adipocytes may instead support inflammatory changes, thereby promoting the onset of insulin resistance. the chemokine receptor cxcr4 antagonist amd3100 exerts deleterious effects in endotoxemia in vivo s. seemann 1 , a. lupp the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). although a blockade of the cxcr4/cxcl12 axis revealed beneficial outcomes in chronic inflammatory diseases, its importance in acute inflammatory diseases remains contradictious and not well characterized. as cxcr4 seems to be part of the lipopolysaccharide sensing complex, cxcr4 agonists or antagonists may have a positive impact on tlr4 signaling. additionally, cxcr4 is involved in the production of pro-inflammatory cytokines, suggesting the receptor to be a promising target in terms of mitigating the cytokine storm. therefore, we aimed to investigate the impact of a cxcr4 blockade on endotoxemia by applying a sublethal lps dose (5 mg/body weight) in mice. the selective cxcr4 inhibitor amd3100 was administered intraperitoneally shortly after lps treatment to ensure an immediate effect after endotoxemia onset. 24 hours after lps administration, the clinical severity score, the body temperature and the body weight of the animals were determined. afterwards, the mice were sacrificed and serum tnf alpha as well as ifn gamma levels were measured. furthermore, the oxidative stress in the brain, liver, lung and kidney tissue was assessed. in addition, the biotransformation capacity of the liver was evaluated and finally, the expression of gp91 phox as well as of heme oxygenase 1 in the spleen and liver were determined by means of immunohistochemistry. the mice of the amd3100 plus lps treatment group displayed a significantly impaired general condition, a reduced body temperature and a decreased body weight in comparison to the control and to the lps treated animals, respectively. tnf alpha levels were significantly increased by more than 200% or 35% when compared to the control or to the lps group, respectively, whereas ifn gamma levels were elevated by about 11% in comparison to mice which had received lps only. in all investigated organs, but especially in the liver and in the kidney, co-administration of amd3100 and lps caused massive oxidative stress. furthermore, the protein contents and the activities of several cyp enzymes in the liver were significantly reduced. immunohistochemistry revealed gp91 phox to occur above average, whereas heme oxygenase 1 expression was distinctly decreased. our results indicate that a blockade of the cxcr4 in endotoxemia is disadvantageous and even worsens the disease. co-administration of amd3100 and lps impaired the health status of the animals, caused massive oxidative stress and diminished the biotransformation capacity. thus, handling acute systemic inflammation with a cxcr4 antagonist cannot be recommended, hence indicating the activation of cxcr4 to be an attractive treatment option. toll-like receptors (tlrs)recognizemicrobial pathogens and trigger inflammatory immune responses to control infections. in acne vulgaris, activation of tlr2 by propionibacterium acnes contributes to inflammation. although glucocorticoids have immunosuppressive and anti-inflammatory effects, acne can be provoked by systemic or topical treatment. enhanced tlr2 expression by glucocorticoids has been reported in undifferentiated keratinocytes, however, human skin cells of different epidermal and dermal layers have not been investigated. in this study, the modulation of tlr expression by dexamethasone was assessed in monolayer cultures of primary human keratinocytes and dermal fibroblasts, as well as the immortalized keratinocyte cell line hacat. constitutive tlr2, tlr1 and tlr6 mrna and protein expression was confirmed in basal keratinocytes, calcium-induced differentiated keratinocytes, hacat cells and fibroblasts by qpcr and western blotting. dexamethasone induced tlr2 expression in a time-dependent and concentration-dependent manner and reduced tlr1/6 expression in keratinocytes but not in hacat cells or fibroblasts. stimulation with dexamethasone in the presence of the pro-inflammatory cytokines tnfα or il-1β further increased tlr2 mrna levels. gene expression of mapk phosphatase-1 (mkp-1) was also upregulated by dexamethasone. glucocorticoid-induced tlr2 expression was negatively regulated by p38 mapk signalling under inflammatory conditions through mkp-1 induction which functions to deactivate mapks. as expected, dexamethasone inhibited the immune responses linked to tlr2 signalling as demonstrated by reduced il-8, il-1β, mmp-1 and mcp-1 levels. however, the expression of traf6, a critical cytosolic regulator of tlr-and tnf family-mediated signalling, was further upregulated by the tlr2 agonist hklm (heat-killed lysteria monocytogenes) in dexamethasonepretreated basal keratinocytes.conclusively, our results provide novel insights intothe molecular mechanismsof glucocorticoid-mediatedtlr expressionand function in human skin cells. psoriasis is a cutaneous chronic inflammatory disease characterized by increased amounts of il-1 cytokines and t helper 17 (th17) related cytokines in lesional psoriatic skin. treatment with beta-adrenoceptor antagonists is associated with induction or aggravation of psoriasis, however, the underlying mechanism is poorly understood. previously, we could demonstrate a pivotal role for langerhans cells and dermal dendritic cells in antimalarial-provoked psoriasis by maintaining a potent th17 activity. in the present study, we investigated the effect of propranolol on human monocyte-derived langerhans-like cells (molc) and dendritic cells (modc) under inflammatory conditions. in the presence of il-1β, propranolol induced the th17 priming cytokines il-23 and il-6 in a concentration-dependent manner. the increased cytokine release was not mediated by camp suggesting gpcr-independent pathways. in contrast, il-36γ and lps failed to increase il-23 release in molc and modc in the presence of propranolol but further induced secretion of il-1β. autophagy has been linked with the secretion of il-1 family cytokines that are upregulated in chronic inflammatory disorders such as psoriasis. propranolol upregulated the expression levels of the autophagy marker p62 and lc3-i to lc3-ii conversion, induced accumulation of lc3 positive vesicles, as well as expression of il-1 signalling downstream adapter molecule traf6, indicating a late-stage block in autophagy. in summary, our results suggest a prominent role of cutaneous dendritic cell subtypes in psoriasis-like skin inflammation mediated by propranolol and possibly other beta blockers. langerhans cells (lcs) represent a highly specialized subset of epidermal dendritic cells (dcs), yet not fully understood in their function of balancing skin immunity. in the present study, we investigated in vitro generated langerhans-like cells obtained from the human acute myeloid leukaemia cell line mutz-3 (mutz-lcs) to study tlr-and cytokine-dependent activation of epidermal dcs. mutz-lcs revealed high tlr2 expression and responded robustly to tlr2 engagement, confirmed by increased cd83 and cd86 expression, upregulated il-6, il-12p40 and il-23p19 mrna levels and il-8 release. tlr2 activation reduced ccr6 and elevated ccr7 mrna expression and induced migration of mutz-lcs towards ccl21. similar results were obtained by stimulation with pro-inflammatory cytokines tnf-α and il 1β whereas ligands of tlr3 and tlr4 failed to induce a fully mature phenotype. despite limited cytokine gene expression and production for tlr2-activated mutz-lcs, co culture with naive cd4+ t cells led to significantly increased ifn-γ and il-22 levels indicating th1 differentiation independent of il-12. tlr2-mediated effects were blocked by the putative tlr2/1 antagonist cu-cpt22, however, no selectivity for either tlr2/1 or tlr2/6 was observed. computer-aided docking studies confirmed non-selective binding of the tlr2 antagonist. taken together, our results indicate a critical role for tlr2 signalling in mutz-lcs considering the leukemic origin of the generated langerhans-like cells. the stagnation in the development of new antibiotics during the last decades and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that pep19-2.5, a synthetic antimicrobial and lps-neutralizing peptide (salp), efficiently neutralizes pathogenicity factors of gram-negative and gram-positive bacteria and protects against sepsis. in the present study, we investigated the potential of pep19-2.5 and the structurally related compound pep19-4lf for their therapeutic application in bacterial skin infections. primary human keratinocytes responded to tlr2 (fsl-1) but not tlr4 (lps) activation by increased il-8 production, as determined by elisa. western blot analysis showed that both salps inhibited fsl-1-induced phosphorylation of nf-κb p65 and p38 mapk. furthermore, the peptides significantly reduced il-8 release and gene expression of il-1β, ccl2 (mcp-1) and hbd-2 as assessed by qpcr. no cytotoxicity (mtt test) was observed at salp concentrations below 10 µg/ml. in lps-stimulated monocyte-derived dendritic cells, the peptides blocked il-6 secretion, downregulated expression of the maturation markers cd83 and cd86, as analysed by flow cytometry, and inhibited ccr7-dependent migration capacity. similarly, monocyte-derived langerhans-like cells activated with lps and pro-inflammatory cytokines showed reduced il-6 levels and cd83/cd86 expression in the presence of salps. in addition to acute inflammation, bacterial infections often result in impaired wound healing. since re-epithelialization is a critical step in wound repair, we tested whether pep 19-2.5 affects keratinocyte migration using the scratch wound assay. the peptide markedly promoted cell migration and accelerated artificial wound closure at concentrations as low as 1 ng/ml and was equipotent to tgf-β. conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. recently, we and others have shown that the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2), a major regulator of the cellular antioxidant defence system, is activated by mechanical ventilation. during ventilator-induced lung injury, nrf2 exerts a protective role by interaction with the stretch-induced growth factor amphiregulin. in the current study, we aimed to investigate the role of nrf2 in acid-induced lung injury, a model for aspiration-induced ards. methods: nrf2-deficient (nrf2 -/-) mice and wild type (wt) littermates were tracheotomised and ventilated for 30min (v t =16ml/kg, f=90min -1 , peep=2cmh 2 o, fio 2 =0.3), before 50 µl hydrochloric acid (hcl) with ph=1.5 or ph=1.3 were instilled intratracheally, controls received nacl. mice were then ventilated for further 6h under monitoring of lung mechanics and vital parameters. blood gases as well as proinflammatory mediators, neutrophil recruitment and microvascular permeability were examined to assess lung injury. results: instillation of hcl ph=1.5 induced mild lung injury, indicated by hypoxemia (po 2 /fio 2 ~300mmhg) and continuously increasing lung tissue elastance (stiffness), from which nrf2 -/mice were protected. pulmonary inflammation, characterized by liberation of cytokines, chemokines and oedema formation, was attenuated in nrf2 -/mice. in contrast, hcl ph=1.3 caused more severe lung injury (po 2 /fio 2 ~200mmhg) with a steeper incline in elastance and more severe inflammation in both wt and nrf2 -/mice. conclusion: we conclude that the presence of nrf2 augments mild acid-induced lung injury, but plays no role in more severe injury. these discrepant results will be elucidated in future investigations. uniklinik rwth aachen, institut für pharmakologie und toxikologie, aachen, germany 2 fakultät für maschinenwesen der rwth aachen, werkzeugmaschinenlabor, aachen, germany rationale: reproducibility is key to science. in recent times, the reproducibility of biomedical research has been questioned increasingly. this reproducibility crisis also affects complex animal experiments, which -if not reproducible -might also be regarded as unethical and lose public acceptance. part of the problem is frequently that the provided documentation is not sufficient for reproduction. therefore, in this study we analyzed the potential of conventional quality management tools -used as standard in machine production -as an approach to improve the documentation and ascertain the quality of complex animal experiments. methods: quality management tools were transferred to an experimental animal set up -the mouse intensive care unit (micu) -which we use for lung injury studies. the tools included visualization of the experimental set-up, transfer of the experimental procedures to an event-driven process chain (epc) and statistical process control (spc) of all crucial pulmonary and cardiovascular parameters. data from ventilator-and acidinduced lung injury studies acquired in the micu were analyzed retrospectively. results: schematic visualization of the micu resulted in a chart comprising medical components, hardware, software and generated data types. the customized epc included all important activities and the resulting events for preparation of the mouse and the workplace, the actual animal ventilation experiment and sample-taking. in addition, checklists were provided for these activities and events, to ensure standardization of every work step. lung impedance and cardiac functions from ventilator-and acid-induced lung injury models were analyzed by spc and correlated with events in the epc. the spc proved to be suitable to identify outliers, predict processes and thereby validate the lung injury models. conclusions: conventional quality management tools were successfully adapted to analyze the quality of lung injury experiments in the micu. we suggest that this new approach is suitable to standardize animal testing procedures and increase the reproducibility of animal studies. background: a dysfunctional endothelial l-arginine-nitric oxide (no) pathway is a key pathomechanism of idiopathic pulmonary arterial hypertension (ipah) that can be provoked by hypoxia in cell culture models [1] [2] [3] [4] . the small peptide apelin is involved in the maintenance of pulmonary vascular homeostasis and angiogenesis although its precise mechanism of action is still unclear [5] . asymmetric dimethylarginine (adma) is known to be an endogenous inhibitor of endothelial no synthase and is associated with several cardiovascular diseases [6] . adma is degraded by dimethylarginine dimethylaminohydrolase 1 and 2 (ddah) enzymes [7] . objective: to determine the effect of apelin on the l-arginine/no pathway in human pulmonary microvascular endothelial cells (hpmecs). methods: hpmecs were cultured under normoxic and ph-related hypoxic conditions and treated with apelin. the expression of regulators of the l-arginine/no pathway were analysed using real-time pcr. the effect of apelin on the phosphoinositide-3 kinase (pi3k)/akt signalling pathway was determined using immunoassays and specific inhibitors[lh1] . apelin and adma concentrations were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and a liquid chromatography-tandem mass spectrometry assay. results: treatment with apelin resulted in a reduced expression of the apelin receptor (aplnr) on hpmecs suggesting a negative feedback mechanism. apelin directly influenced the l-arginine/no pathway by increasing the expression of ddah1 and ddah2 enzymes. thus, the concentration of adma was decreased in hpmecs supernatant following treatment with apelin. the effect of apelin could be abrogated by modulation of the pi3k/akt pathway. conclusion: apelin modulates the l-arginine/no pathway and mediates enhanced degradation of adma via an upregulated expression of ddah 1 and 2 enzymes. the pi3k/akt pathway might play a decisive role in regulation of the effect of aplein. an apelin receptor agonist could be a novel and promising therapeutic option for ipah treatment. background and purpose: there is presently no proven pharmacological therapy for the acute respiratory distress syndrome (ards). recently, we and others discovered that the heptapeptide angiotensin (ang)-(1-7) shows significant beneficial effects in preclinical models of acute lung injury (ali). here, we aimed to identify the best time window for ang-(1-7) administration to protect rats from oleic acid (oa) induced ali. experimental approach: the effects of intravenously infused ang-(1-7) were examined over four different time windows before or after induction of ali in male sprague-dawley rats. hemodynamic effects were continuously monitored, and loss of barrier function, inflammation, and lung peptidase activities were measured as experimental endpoints. key results: ang-(1-7) infusion provided best protection from experimental ali when administered by continuous infusion starting 30min after oa infusion till the end of the experiment (30-240min). both pretreatment (-60-0min before oa) and short-term therapy (30-90 min after oa) also had beneficial effects although less pronounced than the effects achieved with the optimal therapy window. starting infusion of ang-(1-7) 90min after oa (late-term infusion) achieved no protective effects on barrier function or hemodynamic alterations, but still reduced myeloperoxidase and angiotensin converting enzyme activity, respectively. conclusions and implications. our findings indicate that early initiation of therapy after ali and continuous drug delivery are most beneficial for optimal therapeutic efficiency of ang-(1-7) treatment in experimental ali, and presumably accordingly, in clinical ards. airway epithelium functions as a physicochemical barrier against dust, air pollutants and other pathogens and plays a critical role in physiological and pathological processes including modulation of the inflammatory response, innate immunity and airway remodeling such as in human asthma, copd and equine recurrent airway obstruction (rao). models of the airway epithelia are, indeed, missing for the horse; thus, we established long-term equine bronchial epithelial cell cultures using the rock inhibitor y-27632 and cell growth and differentiation was characterized. bronchial epithelial cells (ebec) from adult horses were cultured in the presence and absence of 10 µm y-27632 under conventional and air-liquid-interface (ali) culture conditions. cell proliferation and differentiation were analyzed. formation of a functional epithelial barrier was investigated by transepithelial electric resistance (teer) measurement and immunocytochemical staining of the tight-junction-protein zonula occludens-1 (zo-1). under conventional culture, y-27632 induced higher growth rate of primary ebec and increased the passage number up to 5 passages with retained epithelial cell behavior. in the presence of y-27632, ebecs under ali showed higher teer values. expression of zo-1 correlated with the increase in teer, but in y-27632-treated ebec tight-junctionformation was more rapid, indicating accelerated differentiation, as well h/e-staining and scanning electron microscopic imaging showed a higher amount of cilia and microvilli and pas-positive cells. in conclusion, the data suggest that the rock inhibitor y-27632 facilitates long-term culture of equine bronchial epithelial cells which can be used to study airway disease mechanisms and to identify pharmacological targets. leishmaniasis is a neglected disease of tropical and subtropical regions with millions of people at risk of infection with severe consequences including death. current antileishmanial drugs exhibit serious side effects and also development of resistances is rising. this disease is caused by protozoal organisms from the genus leishmania. in their insect vector they exist in the promastigote form, while in the mammalian host they survive as amastigotes inside the phagolysosomes of macrophages. this makes a specific pharmacotherapy complicated. due to the success of artemisinin in malaria therapy, it was of interest whether endoperoxides are also useful to treat leishmaniasis. in a previous study we demonstrated that ascaridole, an endoperoxide from chenopodium ambrosioides, can cure cutaneous leishmaniasis in a mouse model and exhibited ic 50 values for the viability in the low micromolar range [1] . even though in chemical model systems some basic ideas about the mechanism of activation of these endoperoxides exist, in biological systems including leishmania parasites this activation step has never been demonstrated. therefore, we set up experiments to identify primary drug intermediates formed from ascaridole by activation in leishmania tarentolae promastigotes using electron spin resonance spectroscopy in combination with spin trapping methods. ascaridole was activated in a cell-free system by fe 2+ . the radicals were trapped by 2-methyl-2-nitrosopropane (mnp). the resulting esr spectra consisted of the triplet of duplets. spectral simulations revealed coupling parameters of a n = 16.8 g, and a h = 1.8 g. these coupling constants are compatible with iso-propyl radicals as primary intermediates. in the cellular system, consisting of leishmania tarentolae promastigotes, instead of mnp the less cytotoxic 5,5-dimethyl-1pyrroline-n-oxide (dmpo) was used for spin trapping. without addition of fe 2+ a six line esr signal was observed. spectral simulations of the dmpo spin adduct revealed coupling constants of a n = 16.1 and a h = 24.6 g. according to previously published data [2] from other spin trapping experiments, this corresponds to the formation of carboncentered radicals from ascaridole by leishmania parasites. additional experiments using iron chelators and antioxidants as well as a comparison with the endoperoxide artemisinin were performed. in summary, this study for the first time demonstrated the activation of the endoperoxide ascaridole by a protozoal organism to its active intermediate as a prerequisite to understand its mechanism of action. [1] l. monzote, j. pastor, r. scull, and l. gille. antileishmanial activity of essential oil from chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in balb/c mice. phytomedicine 21:1048-1052, 2014. nitric oxide (no), produced by the inducible nitric oxide synthase (inos) has many functions in physiological and pathophysiological pathways. after induction of inos expression by cytokines and other agents the enzyme produces high amounts of no in a ca 2+ -independent way. this high no production can have beneficial microbicidal, antiparasital, antiviral and antitumoral effects. in contrast, aberrant inos induction may have detrimental consequences and seems to be part of many diseases such asasthma, arthritis, multiple sclerosis, colitis, psoriasis, neurodegenerative diseases, tumor development, transplant rejection or septic shock. analysis of the human inos-mrna structure revealed the existence of an upstream open reading frame (µorf) and a putative internal ribosome entry site (ires) in the 5' untranslated region (5'utr) in front of the start codon of the inos coding sequence (cds). to analyze the function of the µorf and the putative ires we cloned different egfp and luciferase reporter constructs and transfected them into the human colon carcinoma cell line dld1. using a plasmid construct with the µorf fused with the egfp cds, we could show that the µorf can be translated. however, compared to the positive control plasmid less egfp was produced, which can be explained by a weak kozak sequence of the µorf. blocking the mrna cap-dependent translation by cloning a stem loop structure in front of the inos 5'utr within a luciferase reporter plasmid led to a remarkable loss of luciferase production. thus, the expression of inos seems to be cap-dependent. furthermore, transfection experiments with dld1 cells using constructs coding for a bicistronic renilla-firefly luciferase mrna showed that there is no ires in front of the inos cds. taken together, the inos expression seems to be cap-dependent and without influence of an ires, while the µorf is translatable. therefore we speculate that inos expression is only possible due to a leaky scanning mechanism depending on the weak kozak sequence of the µorf. objectives: vascular oxidative stress is considered a pathophysiologic factor promoting cardiovascular diseases such as coronary artery disease, heart failure, diabetes and hypertension. there are several sources of superoxide in vascular smooth muscle and endothelial cells but whether an impairment of the catalytic function of enos and thus generation of oxidative stress is involved in blood pressure (bp) regulation and/or the development of hypertensive disease states is unknown. methods: we generated a mutant enos in which one of the two essential cysteines required for the coordination with the central zn-ion, correct dimer formation and normal activity is replaced by alanine (c101a-enos). normal enos (enos-tg) or a novel dimer-destabilized c101a-enos described previously (antioxid redox signal. 2015 sep 20; 23(9) :711-23) were introduced in c57bl/6 in an endothelial-specific manner. mice were monitored for enos expression and localization, aortic relaxation, systolic blood pressure, levels of superoxide and several post-translational modifications indicating activity and/or increased vascular oxidative stress. some groups of mice underwent voluntary exercise training for 4 weeks or treatment with sod mimetic tempol. results: c101a-enos-tg showed significantly increased superoxide generation, protein-and enos-tyrosine-nitration, enos-s-glutathionylation, enos 1176/79 phosphorylation and amp kinase (ampkα) phosphorylation at thr172 in aorta, skeletal muscle, left ventricular myocardium and lung as compared to enos-tg and wild type (wt) controls. the localization of enos-c101a-tg was restricted to endothelium as evidenced by immunohistochemically staining for enos and an endothelial-specific marker cd31. exercise training increased phosphorylation of enos at ser 1176/79 and of ampkα at thr 172 in wt but not in c101a-enos-tg. aortic endothelium-dependent and endothelium-independent relaxations were similar in all strains. in striking contrast, c101a-enos-tg displayed normal blood pressure despite higher levels of enos, while enos-tg showed significant hypotension. tempol completely reversed the occurring protein modifications and significantly reduced bp in c101a-enos-tg but not in wt controls. conclusions: by means of a novel transgenic mouse model we demonstrated that vascular oxidative stress generated by endothelial-specific expression of a dimerdestabilized variant of enos selectively prevents bp reducing activity of vascular enos, while having no effect on aortic endothelial-dependent relaxation. these data suggest that oxidative stress in microvascular endothelium may play a role in the development of essential hypertension. the herbal medicinal product myrrhinil-intest ® consists of myrrh, chamomile flower dry extract and coffee charcoal. clinical data prove the effectiveness of this herbal preparation for inflammatory intestinal disorders. to further investigate the anti-inflammatory potential of the single components as part of a multi-target principle, an ethanolic (my) and aqueous (mya) myrrh extract, ethanolic chamomile flower extract (ka) and aqueous coffee charcoal extract (cc), were examined in an in vitro tnbs inflammation model using rat small intestinal preparations. the effect of the plant extracts on tnbs induced inflammatory damage was characterised based on tnfα-gene expression analysis, isometric contraction measurement and histological analysis. furthermore, tnfα-release from lpsstimulated thp-1 cells was determined. budesonide was used as positive control. additionally, microarray gene expression analysis was performed in lps/ifnγ stimulated native human macrophages to determine potential underlying mechanisms. the tnbs-induced overexpression of tnfα-mrna was reduced after ka (0.1 mg/ml) and mya (1 mg/ml) treatment down to 24% and 16% resp.; tnbs-induced loss of contractility and reduction of mucosal layer thickness was inhibited after ka (3 mg/ml) treatment by 26% and 25% resp.; after mya (0.1-1mg/ml) treatment by 17% and 44% resp. lps-induced tnfα release from thp-1 cells was inhibited concentrationdependently by my (ic50 = 60.65 μg/ml; 97% inhibition), ka (ic50 = 439 μg/ml; 71% inhibition) and cc (ic50 = 1886 μg/ml; 44% inhibition). furthermore, ka (200 µg/ml) and cc (500 µg/ml) inhibited the lps/ifnγ-induced expression of genes associated with chemokine signalling up to 100-fold (for cxcl13). the presented study demonstrates further evidence for anti-inflammatory properties of the herbal components which contribute to the reported clinical effectiveness. introduction: the purine nucleoside adenosine, which is involved in a variety of physiological functions, regulates immune and inflammatory responses and acts as a modulator of gut functions. although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to micromolar range. by activation of different receptor subtypes adenosine is able to induce anti-inflammatory or pro-inflammatory impacts. aim: the current study examined the impact of adenosine a2a receptors (a2ar) and adenosine a2b receptors (a2br) to regulate contractility in untreated and inflamed rat colon preparations using a specific a2ar agonist (cgs 21680) and an a2br antagonist (psb-1115) on acute inflammation in rat colon preparations. further it focused on interactions of the multi-herbal drug stw 5 with a2ar as a possible mechanism of the protective effect of stw 5 in gastrointestinal disorders. methods: inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (tnbs). contractions were measured isometrically in an organ bath set up. gene expression was determined using rt-pcr. radio ligand binding assays (competition experiments) were carried out with rat brain homogenates. morphological changes were estimated after van gieson staining. results: all four adenosine receptor subtypes were expressed in untreated colon preparations. activation of a1, a2b, and a3 receptor with specific agonists reduced the acetylcholine (ach, 10µm)-induced contractions, while activation of a2br enhanced it. after incubation with tnbs morphological damages in colonic mucosa and muscle walls were detectable followed by reduced ach-contractions. the tnbs-mediated decrease of ach-contractions as well as the morphological damages were partially normalized by co-incubation of tnbs with cgs 21680 (10µm) or with psb 1115 (100µm). the same effects with smaller intensity were found for stw 5 (512 µg/ml) in female but not in male colon preparations. these results are in accordance with ligand binding studies indicating that stw 5 interact with the a2ar. conclusion: anti-inflammatory mechanisms and cell protective actions of stw 5 are partly due to the interaction with adenosine receptors. the results give a clear-cut correlation with symptom improvements in clinical trials and thereby highlight the relevance of stw 5 as a therapeutic approach in ibs. (allescher 2006) . therefore, a multi-target approach is a promising therapeutic strategy, as is exemplified by stw 5(ottillinger et al. 2013) . stw 5 (iberogast®) is a fixed combination of nine plant extracts with iberis amara (stw 6) as one of its components. it is successfully used for treatment of functional dyspepsia and irritable bowel syndrome (ibs). to allow an overview of targets addressed by stw 5 and the role of its components in relation to the different forms and causes of functional gi diseases, an evaluation of the data, which have been gained from more than 150 pharmacological tests, is needed. all data from studies including stw 5 alone, or stw 5 and its components, were retrieved and sorted according to types of study models (human and animal systems, animal disease models, gi-preparations, cell cultures, in vitro-systems) and respective etiologic mechanisms related to fgds and then visualized in the form of 2d histograms (lorkowski et al. 2015) . results: more than 150 pharmacological tests indicated anti-oxidative activity, electrophysiological effects, ulcer protection, anti-inflammatory actions, pro-kinetic and spasmolytic effects as well as reflux and acid reduction. moreover, the analysis indicated that the components of stw 5 contribute differently to the overall effect of stw 5. altogether, the evaluation of the data shows that stw 5 is active in response to multiple etiologic factors involved in fgds, especially functional dyspepsia and irritable bowel syndrome, and to which extent the herbal extract components of the combination are relevant for the different mechanisms of action and their translation to clinical efficacy. conclusion: multi step clustering allows the transformation of complex data sets. it makes the allocation of specific actions to the different components of stw 5 manageable, so also giving support to its clinical use in patients with different symptoms. introduction: stw 5 ii has been recently developed in an effort to reduce the number of active extracts in the mother multi-component herbal preparation, stw 5 (iberogast ® , steigerwald arzneimittelwerk gmbh, darmstadt, germany) without affecting the overall therapeutic efficiency. stw 5 consists of a mixture of 9 standardized extracts: bitter candytuft (iberis amara), lemon balm (melissa officinalis), chamomile (matricaria recutita), caraway fruit (carum carvi), peppermint leaf (mentha piperita), liquorice root (glycyrrhiza glabra), angelica root (angelica archangelica), milk thistle (silybum marianum) and celandine herb (chelidonium majus), whereas stw 5 ii lacks the last 3 components. stw 5 was shown to be effective clinically to treat functional dyspepsia (1) and irritable bowel syndrome (2) and was shown experimentally to be effective to guard against the development of radiation induced intestinal mucositis (3) and in the management of ulcerative colitis (4) . the present study was initiated to show whether stw 5 ii with the reduced component extracts would also be as effective in the latter condition. this was induced in wistar rats by feeding them with 5% dextran sodium sulfate in drinking water for 1 week when lesions were observed in the colon evidenced by histological examination as well as colon shortening and reduction of colon mass index. this was associated with a rise in myeloperoxidase and a fall in reduced glutathione, glutathione peroxidase, and superoxide dismutase in colon homogenates as well as a rise in tnfα in serum. oral administration of stw 5 in doses of 2 and 5 ml/kg or stw 5 ii in a dose of 2 ml/kg for 1 week before and continued during dss feeding tended to normalize all the changes in a fashion comparable to sulfasalazine, used as a reference drug in a dose of 300 mg/kg. conclusions: the modified preparation, stw 5 ii thus proved to be as effective as stw 5, thereby reflecting its potential usefulness in ulcerative colitis possibly by virtue of its anti-inflammatory and anti-oxidant properties. (1) schmulson mj (2008) the emetic pathways include the action of neurotransmitters dopamine, serotonin and substance p in the emetic centers localized in the brainstem, area postrema and vagal nerve afferents. previous in vivo studies in beagle dogs revealed that the plant alkaloid lycorine potentially induce nausea and emesis. though antagonists of the tachykinin receptor 1 (maropitant) and serotonin receptor 3 (ondansetron) prevented lycorinemediated emesis, the molecular mechanism of nausea and vomiting remain still unknown. to study the mechanism of action of the emetic agents, we analyzed the effect of lycorine (direct activation of nk1) and channel opening (activation of 5ht3) on the intracellular calcium homeostasis (using fluorometric ca 2+ analysis) and cell proliferation rates in endogenously nk1 and 5ht3 receptor expressing cell lines as well as in cho and hek cells stably expressing the receptors. neither endogenously receptor expressing nk1 or 5ht3 cells nor receptor overexpressing cells showed calciumflux or calcium mobilization after stimulation with lycorine. furthermore, we are measuring the receptor number and subtypes using radioligand binding studies. it is planned, moreover, to obtain fluorescent labeled constructs of the nk1 receptor to gain insights into the involvement of receptor internalization which might mediate emesis. by characterizing these molecular principles of the nk1 and 5ht3 receptors, we are attempting to obtain more information in predicting drug-induced side effects such as nausea and emesis. the intestinal epithelium is completely renewed every 4-5 days. this process is driven by stem cells, which reside within specialized niches in the intestinal crypts and give rise to several differentiated cell types, including enterocytes, paneth, enteroendocrine, goblet and tuft cells. however, the molecular mechanisms that establish and maintain differentiated cell numbers and proportions remain largely unknown. here, we systematically analyzed the intestinal expression of semaphorins and plexins, which constitute a ligand-receptor system that plays central roles in cell-cell communication in various biological contexts. we identified plexin-b2 and its semaphorin ligands to be highly expressed in intestinal epithelial cells. genetic inactivation of plexin-b2 in intestinal organoids strongly reduced the number of enteroendocrine cells. our data suggest that semaphorin-plexin-b2 signaling promotes differentiation of intestinal epithelial cells towards the enteroendocrine lineage. the gastric epithelium contains several types of differentiated cells, including foveolar cells that produce mucus, parietal cells that secrete gastric acid and intrinsic factor, chief cells that synthesize pepsinogen and gastric lipase, and enteroendocrine cells that release different hormones. these differentiated cell types all originate from multipotent stem cells, yet little is known about how this differentiation process is regulated on a molecular level. the gap protein rasal1 controls the activity of small gtpases of the ras family, and its expression levels have been shown to inversely correlate with progression of stomach cancers. however, functional studies on the physiological role of rasal1 in the gastric epithelium are lacking. here, we established and characterized a mouse line with inactivation of the rasal1 gene. we observed that these mice showed increased numbers of enteroendocrine cells in the gastric mucosa. conditional inactivation of rasal1 in enteroendocrine cells, using a mouse line in which cre expression is driven by the atoh1 promoter, further corroborated that rasal1 expression in enteroendocrine cells determines enteroendocrine cell numbers. these findings identify rasal1 as a regulator of gastric epithelial cell differentiation. ). the present study investigates the impact of two faah inhibitors (arachidonoyl serotonin [aa-5ht], urb597) on a549 lung cancer cell metastasis and invasion. lc-ms analyses revealed increased levels of faah substrates (aea, 2-ag, oea, pea) in cells incubated with either faah inhibitor. in athymic nude mice faah inhibitors were shown to elicit a dosedependent antimetastatic action. in vitro, a concentration-dependent anti-invasive action of either faah inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (timp-1). using sirna approaches, a causal link between the timp-1-upregulating and anti-invasive action of faah inhibitors was confirmed. moreover, knockdown of faah by sirna was shown to confer decreased cancer cell invasiveness and increased timp-1 expression. inhibitor experiments point toward a decisive role of cb 2 and transient receptor potential vanilloid 1 in conferring the anti-invasive effects of faah inhibitors and faah sirna. finally, antimetastatic and anti-invasive effects were confirmed for all faah substrates. collectively, the present study provides first-time proof for a pronounced antimetastatic action of the faah inhibitors aa-5ht and urb597. as underlying mechanism of its anti-invasive properties an upregulation of timp-1 was identified. regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (mscs). the present study focused on inhibitors of the enzyme fatty acid amide hydrolase (faah), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (n-oleoylethanolamine, n-palmitoylethanolamine). in boyden chamber assays, the faah inhibitors, urb597 and arachidonoyl serotonin (aa-5ht), were found to increase the migration of human adipose-derived mscs. lc-ms analyses revealed increased levels of all four aforementioned faah substrates in mscs incubated with either faah inhibitor. following addition to mscs, all faah substrates mimicked the promigratory action of faah inhibitors. promigratory effects of faah inhibitors and substrates were causally linked to activation of p42/44 mitogen-activated protein kinase (mapk), as well as to cytosol-to-nucleus translocation of the transcription factor, peroxisome proliferator-activated receptor α (pparα). whereas pparα activation by faah inhibitors and substrates became reversed upon inhibition of p42/44 mapk activation, a blockade of pparα left p42/44 mapk phosphorylation unaltered. collectively, these data demonstrate faah inhibitors and substrates to cause p42/44 mapk phosphorylation, which subsequently activates pparα to confer increased migration of mscs. this novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by faah inhibitors. background: the hematopoietic disorder chronic myeloid leukemia (cml) is one of the most extensively studied neoplasms. it is caused by translocation between chromosomes 9 and 22 leading to the formation of the philadelphia chromosome and the bcr-abl fusiongene. first-line targeted therapy is still the tyrosine-kinase inhibitor imatinib (im), which led to tremendous success in treatment. however, the amount of therapeutic resistances is increasing, caused either by bcr-abl-dependent mechanisms (e.g. bcr-abl amplification/overexpression, point mutations) or bcr-ablindependent mechanisms. these might be linked to alterations in drug transporter expression or particularly, microrna-expression levels. in our previous study, we analyzed the changes of microrna expression profiles during the development of imresistances in the leukemic cell line k562. an inverse correlation of mir-212 expression and protein levels of the efflux transporter atp-binding cassette transporter g2 (abcg2) was observed in cells resistant to different im-concentrations, pointing to a relation of mir-212 to im-resistance. hence, we investigate in current studies, how the influence of mir-212 on im-sensitivity could be explained. methods: we transfected k562 cells, sensitive treatment-naïve cells and cells resistant to various im-concentrations, either with mir-mimic pre-mir-212 or inhibitory anti-mir-212, challenged them with im and analyzed effects on cell viability, activation of apoptosis and cell death using wst-1-, caspase glo 9-assay and cell counting. in addition, we analyzed changes in abcg2 expression using flow cytometry and qrt-pcr and investigated alterations in im-efflux using hplc and hoechst efflux assay. results: under im-treatment, sensitive k562 showed an effect of mir-212-inhibition using anti-mir-212. this led to a significant promotion of cell survival apparent on the level of respiratory chain function (p<0.01) and cell membrane integrity and reduced capase-9 activity (p<0.05). furthermore, these mirna-effects are dose-dependent as confirmed in concentration row-experiments. regarding transport and abcg2 expression, we found that 2 µm im-resistant k562 do not express higher amounts of abcg2, but showed higher transport rates of im or the abcg2-substrate hoechst 33342. conclusions: overall, these experiments indicate that mir-212 does not only affect abcg2-expression, but also influences cell sensitivity to im in a more direct manner. further analysis will now be performed to reveal the underlying mechanism, how cell sensitivity to im is altered and if these effects occur due to a direct regulation of abcg2. in summary, these findings could be relevant in cml-therapy, overcoming imresistances with a better understanding of mirna-and drug transporter alteration in cml. acknowledgments: we would like to thank all the authors for their contribution to this project. this work was funded by the university hospital schleswig-holstein. oxidized silicon nanoparticles and iron oxide nanoparticles for radiation therapy s. klein radiation therapy often combined with surgery and/or chemotherapy is applied to more than 50 % of patients at some point of their treatment. the cytotoxic effects of ionizing radiation occur from their ability to produce dna double-strand breaks through the formation of free radicals within cells. however, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. to overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. in our studies we used amino functionalized, oxidized silicon nanoparticles (sinp), superparamagnetic iron oxide nanoparticles (spion) and iron doped silicon nanoparticles (fe(1%)-sinp) to increase the formation of reactive oxygen species (ros) in cells. cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of 1-3 gy using a 120 kv x-ray tube. after irradiation, the formation of the different ros species including superoxide, hydroxyl radicals and singlet oxygen was investigated. sinps with sizes around 1 nm can easily cross the cell and nuclear membrane. the positively charged amino functionalized sinps stick in all membranes as well in those of the mitochondria. irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. amino functionalized sinps, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the x-ray radiation on the mitochondria and therefore increase the concentration of superoxide. [1] oxidized sinps with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation. spions enter the cells via endocytosis, whereas the uncoated spions remain in the vesicles and the citrate coated spions accumulate in the cytoplasm. cells loaded with citrate coated spions show no higher ros concentration than in media-cultured cells. but after irradiation, the ros formation increased drastically. this enhancing effect is explained with the impact of x-rays onto the surface of spions which is due to the destruction of surface structures. the freed spion surface contains easier accessible iron ions. this ions can participate in the fenton and haber-weiss chemistry and thus, catalyze the hydroxyl radical formation. [2] 1 to 5 % iron doped sinp increase the formation of hydroxyl radicals as well as the generation of singlet oxygen after irradiation. chronic pain in response to tissue damage (inflammatory pain) or nerve injury (neuropathic pain) is a major clinical health problem, affecting up to 30% of adults worldwide. currently available treatments are only partially susceptible and are accompanied with therapy limiting side effects. thus it is important to elucidate molecular mechanisms of pain signaling in detail to obtain new insights in potential future therapies. recent data indicate that hydrogen sulfide (h 2 s) contributes to the processing of chronic pain, however pro-as well as antinociceptive effects have been described so far. moreover the sources of h 2 s production in the nociceptive system are only poorly understood. here we investigated the expression of the h 2 s releasing enzyme cystathionine g-lyase (cse) in the nociceptive system and characterized its role in chronic pain signaling using cse deficient mice. paw inflammation and peripheral nerve injury led to upregulation of cse expression in dorsal root ganglia. however, conditional knockout mice lacking cse in sensory neurons as well as global cse knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to wt littermates. thus, our results suggest that cse is not critically involved in chronic pain signaling in mice and that sources different from cse mediate the pain relevant effects of h 2 s. this work was supported by the deutsche forschungsgemeinschaft (sfb815-a14) and in part by loewe-schwerpunkt "anwendungsorientierte arzneimittelforschung". heinrich-heine-universität, institut für toxikologie, düsseldorf, germany 2 heinrich-heine-universität, urologie, düsseldorf, germany background: cisplatin (cispt) is frequently used in the therapy of advanced stage urothelial cell carcinoma (ucc). yet, inherent and acquired drug resistance limits the clinical use of cispt. here, we comparatively investigated the response of epithelial-like (rt-112) and mesenchymal-like (j-82) uc cells following cispt treatment. methods: upon selection with equitoxic doses of cispt for months, we obtained cispt resistant variants (rt-112 r , j-82 r ). cell viability was measured using the alamar blue assay. cell cycle distribution was analysed by flow cytometry. immunocytochemistry was used to quantify the number of nuclear γh2ax and 53bp1 foci representing dna double strand breaks (dsbs), while western blot was used to unravel the role of dna damage response (ddr) to acquired cispt resistance. qrt-pcr was performed to analyse the mrna expression of genes associated with cispt resistance. j-82 and j-82 r cells were treated with different concentrations of lovastatin and selected ddr inhibitors to elucidate their influence on cell viability. results: untreated rt-112 cells showed an about 2-3-fold higher resistance to cispt than j-82 cells. both cell lines differed in the expression pattern of genes that are associated with cispt resistance. rt-112 r and j-82 r revealed a 2-3-fold increased cispt resistance as compared to the parental cells. during the selection procedure, we observed that acquired cispt resistance goes along with morphological alterations that resemble epithelial mesenchymal transition (emt). cell cycle analysis of rt-112 r cells disclosed a reduced apoptosis and enhanced g2/m arrest following cispt exposure as compared to rt-112 wild-type cells. by contrast, induction of cell death was similar in j-82 and j-82 r cells. notably, j-82 r cells showed a reduced formation of cispt-induced dsbs. correspondingly, the related ddr was diminished in j-82 r as compared to their parental cells. this was not found when ddr was comparatively analysed between rt-112 r and rt-112 cells. data obtained from qrt-pcr analysis indicate that different mechanisms contribute to acquired drug resistance of j-82 r and rt-112 r . unexpectedly, j-82 r and rt-112 r shared the upregulation of xaf-1. treatment of j-82 r cells with statins and protein kinase inhibitors revealed an enhanced sensitivity to pharmacological inhibition of chk-1 and, moreover, re-sensitization to cispt by chk-1 inhibitor. based on the data we suggest that mechanisms of acquired cispt resistance of epithelial and mesenchymal uc cell lines are different with apoptosisrelated mechanisms appear to be more relevant for epithelial-like rt-112 cells and ddr-related mechanisms dominating cispt susceptibility in mesenchymal-like j-82 cells. furthermore, our findings indicate that chk-1 might be an appropriate target to deal with acquired cispt resistance in ucc. in many patients, gastric cancer treatment with conventional cytostatic agents shows only limited clinical response. novel therapeutics, which inhibit rtk signaling by targeting c-met or her family receptors, have demonstrated some efficacy; however, primary resistance of gastric cancer cells against these inhibitors is still a major problem. in the present study we investigated the mechanism of heregulin (hrg)-promoted survival of gastric cancer cells after treatment with c-met inhbitors or sirna-mediated downregulation of c-met. we found that hrg treatment of gastric cancer cells with a c-met amplification partially rescued the cells from the antiproliferative effects of pharmacological c-met inhibition or sirna-mediated downregulation of c-met. moreover, c-met inhibition or downregulation led to an induction of her3 expression on mrna and protein level, whereas other her family receptors were unaffected. downregulation of her3 impaired the hrg-mediated rescue of cell survival upon c-met inhibition. in other tumor entities the chromatin organizer special at-rich sequence-binding protein 1 (satb1) has been described as a regulator of her family receptor expression involved in adaptive responses of tumor cells. thus, we investigated the contribution of satb1 in the upregulation of her3 after c-met inhibition. of note, c-met inhibitors as well as c-met-specific sirnas markedly induced satb1 expression in gastric cancer cells, and the downregulation of satb1 by sirnas completely prevented the induction of her3 upon c-met inhibition. in contrast, her1 or her2 expression levels were not affected by satb1-specific sirnas. the function of satb1 as transcriptional regulator is controlled by its phosphorylation status, which in turn is modulated by pkc activity. thus, we also tested the effect of pkc inhibitors on her3 expression after c-met inhibition. interestingly, the upregulation of her3 in gastric cancer cells was significantly reduced by pkc inhibitors. to summarize, satb1 and pkc are critically involved in the regulation of her3 expression in gastric cancer cells after treatment with c-met inhibitors and the oncogene her3 plays a crucial role for tumor cell survival in this context. thus, inhibition of pkc or satb1 may help to overcome resistance against c-met inhibition in this tumor entitiy. in the rising field of nanomedicine, development of new approaches in diagnosis and treatment of cancer is a challenging task. typically, a nanocarrier is synthesized and linked to functional compounds displaying either diagnostic or therapeutic effects in cancer models. recently, nanomaterials combining both diagnostic and therapeutic properties, so-called 'theranostics', became of primary interest. here we used a human albumin-polyethylene glycol (peg) copolymer (hsa) as a theranostic platform for molecular integration of the chemotherapeutic drug doxorubicin (dox) and the magnet resonance imaging (mri) contrast agent gadolinium (gd) yielding gd-hsa-dox nanoparticles. besides in vitro testing, which demonstrated cytotoxic efficacy of gd-hsa-dox, we used the chorioallantoic membrane (cam) of fertilized chick eggs as a preclinical xenotransplantation model. the cam assay, which in legal terms does not represent an animal experiment, allows testing of compounds in an in vivo setting. this model is particularly helpful to narrow the gap between in vitro and in vivo applications in rodents, because it can help to reduce number of elaborate experiments with typically nude mice, and it reduces or even avoids exposure of those animals to adverse effects and distress. treatment-resistant mda-mb-231 breast cancer cells stably transfected with luciferase were xenotransplanted onto the chorioallantoic membrane. after formation of solid breast cancer xenografts, gd-hsa-dox was injected intravenously and its antiproliferative effect was evaluated by ivis imaging of luciferase activity and by immunohistochemical analysis of the tumor xenografts for the ki-67 proliferation antigen. in comparison to conventional dox, gd-hsa-dox showed increased antiproliferative efficacy and reduced general toxicity in the cam assay. on the basis of these findings, a rodent model was established, where the mda-mb-231 breast cancer cells were orthotopically xenotransplanted into the mammary fat pads of female nmri nu/nu mice. in this model, we further investigated biocompatibility, as well as diagnostic and therapeutic properties of the engineered nanomaterial. after repeated administration of gd-hsa-dox into the tail vein of the animals, biocompatibility of gd-hsa-dox was confirmed by uncompromised liver, kidney and hematopoietic parameters. to warrant diagnostic properties, accumulation of the nanomaterial in tumor tissue is indispensable. by small animal mri of gd, kinetics of intravenously applied gd-hsa-dox in tumor tissue was monitored. an enhancement of the engineered nanomaterial in tumor tissue was detected for up to 47 h after injection indicating successful enrichment of gd-hsa-dox within the tumor tissue, which can be ascribed to the enhanced permeability and retention (epr) effect observed in the microenvironment of many solid tumor tissues. we are currently investigating the antitumor efficacy of gd-has-dox in this mouse model and preliminary data seem to indicate a dose-dependent anticancer effect. supported by the volkswagenstiftung. tubulin-binding agents are the most important anti-tumoral drugs. due to the side effects and the development of resistances, the discovery of new agents is still of importance. recently, pretubulysin (pt), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound. in the dfg research group for 1406, pt was characterized as anti-tumoral, antiangiogenic and vascular-disrupting compound. moreover, pt was also found to inhibit the formation of metastases in vivo. aim of the present study was to gain first insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of pt on the interaction of endothelial and tumor cells in vitro. pt treatment of primary human endothelial cells (huvecs) strongly increased the adhesion of breast cancer cells (mda-mb-231) on huvecs, but limited their transmigration through the endothelium (transwell assay). based on this data, the gene expression of presumably involved adhesion molecules was determined by qrt-pcr: icam-1, vcam-1, e-selectin, n-cadherin, and galectin-3. moreover, the chemokine system cxcl12/cxcr4 was analyzed. it could be demonstrated that the mrna level of endothelial n-cadherin is upregulated by pt. while total protein expression of ncadherin was enhanced in pt treated huvec, its surface expression was not largely influenced by pt (western blot, flow cytometry). in line with this, blocking endothelial ncadherin by a neutralizing antibody revealed that this protein is not involved in ptevoked tumor cell adhesion. interestingly, pt strongly augmented the mrna and protein expression of cxcl12 in huvecs (qrt-pcr, western blot), whereas its endothelial secretion was not affected by pt (elisa). an autocrine action of cxcl12 could be excluded, since blocking the cxcl12 receptor cxcr4 on endothelial cells with plerixafor did not influence cancer cell adhesion. by microscopic analyses, we observed that pt treatment causes transient gaps in the huvec monolayer, where tumor cells prefer to adhere. since β1-integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. both the pt-evoked increase in cell adhesion and decrease in transmigration was completely abolished when β1-integrins were blocked on mdas by a neutralizing antibody. these results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2). introduction: tyrosine kinase inhibitors (tkis) for the treatment of non-small cell lung cancer (nsclc) patients harboring activating mutations in the epidermal growth factor receptor have shown prominent success. nevertheless, patients treated with tkis eventually acquire resistance and relapse (1). based on an evolutionary cancer model (2) , weekly high dose-pulsed tki regimens were proposed to delay resistance. using data from nsclc bearing mice treated with erlotinib at different dosing regimens, we developed a semi-mechanistic pharmacokinetic/pharmacodynamic model for erlotinib effects on tumor killing and resistance development. methods: data was available from experiments in xenograft mice bearing nsclc tumors (pc9 and hcc827 cell lines; both erlotinib sensitive) (3). plasma concentrations from two single-dose groups, 30mg/kg and 200mg/kg, were used for pharmacokinetic modeling. relative tumor volume changes in mice randomized to five dosing regimens (15mg/kg daily, 30mg/kg daily, 200mg/kg every 2 days, 200mg/kg every 4 days, or vehicle) was the pharmacodynamic endpoint. a tumor growth inhibition model was developed by testing linear, exponential and logistic models to account for the tumor growth kinetics, as well as fitting an emax model to explain the effect of exposure on killing the sensitive tumor cells, and resistance development. analysis was performed using nonmem 7.3. results: absorption was dose dependent, and a precipitate compartment accounted for dissolution limited absorption for the 200mg/kg dose. a 1-compartment model with first order elimination kinetics described distribution and elimination. to describe tumor volume changes, a tumor was assumed to be a mixture of sensitive and resistant cells (represented by distinct compartments and ordinary differential equations). exponential kinetics best described natural growth (doubling times: 13 and 52 days, for sensitive and fully resistant cells, respectively). a tumor was found to transit through a less sensitive phase before acquiring full resistance. an e max model (less than linear) best described effect on the sensitive cells (ec 50 =0.53μm for both cell lines), and on the partially sensitive transit phase (ec 50 =1.24μm and 3.00μm, for hcc827 and pc9 cell lines, respectively), urging to provide adequate trough erlotinib concentrations for optimal effects. conclusions & future perspectives: an exposure-driven tumor growth inhibition model accounting for the kinetics of resistance development was developed. the model emphasizes the need for establishing an adequate trough erlotinib concentrations to delay disease progression. extracts of the stem bark of ficus platyphylla (fp) have been used in traditional nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. previous studies have revealed the analgesic and anti-inflammatory effects of fp in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. in this study, we assessed the effects of the standardised extract of fp on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. we also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (cox) and 5-lipoxygenase (5-lo). fp (i) increased the hot plate nociceptive threshold and vocalisation threshold. the increase in hot plate nociceptive threshold was detectable over a period of 30 min whereas the increase in vocalisation threshold persisted over a period of 90 min. (ii) fp showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) fp inhibited the activities of cox-2 and 5-lo but not of cox-1. we provided evidence supporting the use of fp in nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. it is interesting to note that the dual inhibition of cox-2 and 5-lo appears favourable in terms of both efficacy and side effect profile. despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in first world countries underestimated its significance for a long time. the alarming prevalence of multidrug-resistant gram-negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. 2,5 million deaths per year were actually caused by acute diarrheal infections. the most common causative agents of acute diarrheal infections, amongst others, are yersinia enterocolitica, campylobacter jejuni, salmonella spp., shigella spp., escherichia coli, vibrio cholerae, and clostridium difficile. the established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. in either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogenspecific treatment. one very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. due to the fact that many decisive virulenceassociated outer membrane proteins (omps) of gram-negative enteropathogens are substrates of the periplasmic chaperone sura exclusively, we developed a new assay format to determine sura in vitro chaperone activity. previous publications by behrens et al., 2001 and buchner et al., 1998 documented an assay to determine sura in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (hts). we now developed a luciferase-based screening assay. this highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z-factor of > 0,6. in cooperation with the hzi braunschweig (germany) and the hzi saarbrücken (germany), we were able to screen over 7000 purified compounds and over 500 extracts of myxobacteria. during the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of 0,05 %. additionally, we developed an elaborate follow-up strategy to validate positive hits, which includes a well-established mouse infection model. we are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure sura. the potential atypical antipsychotic and dopamine d 2 receptor partial agonist 2bromoterguride antagonizes phencyclidine-and apomorphine-induced prepulse inhibition and novel object recognition deficits in rats e. tarland objectives: schizophrenia is a disabling mental disorder affecting more than 21 million people worldwide. available medical therapies are effective in the treatment of psychosis and other positive symptoms, however come with considerable side effects and often fail to ameliorate cognitive deficits and negative symptoms of the disorder. the dopamine d 2 receptor partial agonist 2-bromoterguride (2-bt) has recently been shown to exhibit antipsychotic effects in rats without causing adverse side effects common to antipsychotic drugs [1]. to determine its atypical character in vivo, the ability of 2-bt to antagonize the disruptive effects of phencyclidine (pcp) and apomorphine on sensory motor gating was determined in the prepulse inhibition paradigm. the effect of 2-bt on cognitive deficits was assessed in the novel object recognition (nor) test after object recognition memory deficits were induced by pcp treatment. method: 10 week old male sprague-dawley rats were injected with 2-bt (0.1 or 0.3mg/kg; i.p.) followed by pcp (1.5mg/kg; s.c.) or apomorphine (0.5mg/kg; s.c.). prepulse inhibition was measured in two sound-proof startle chambers. the attenuating effect of 2-bt (0.1 or 0.3mg/kg; i.p.) on visual learning and memory deficits following subchronic administration of pcp (5.0mg/kg; i.p. twice daily for 7 days) was assessed in the nor task consisting of a 3min acquisition trial and a 3min retention trial separated by a 1h inter-trial interval. clozapine (5.0mg/kg; i.p) or haloperidol (0.1mg/kg; i.p) were used as positive controls. results: the dopamine d 2 receptor partial agonist 2-bt (0.3mg/kg) and the typical antipsychotic haloperidol successfully antagonized apomorphine-induced ppi-deficits. interestingly 2-bt also ameliorated the pcp-induced ppi-deficits to the same extent as the atypical antipsychotic clozapine. preliminary data from the nor test indicate that 2-btreduces subchronic pcp-induced cognitive deficits in novel object recognition analogous to clozapine. the disrupting effects of pcp on ppi are mediated by non-competitive antagonism at nmda sites indirectly influencing a series of neurotransmitter systems. our results indicate that 2-bt mediates actions at multiple neurotransmitter receptors as it successfully ameliorated both the pcp-and apomorphine-induced ppi disruptions in rats, showing an atypical antipsychotic character. furthermore, our preliminary results support the potential atypical antipsychotic effect of 2-bt as it restored performance in the nor test, a test with good predictive validity. due to the previously shown properties and antipsychotic-like effects of 2-bromoterguride [1], this substance may be a promising candidate for treatment of schizophrenic patients. ongoing experiments investigate the potency of 2-bt to improve social deficits following a sub-chronic pcp regime in rats. background and objectives: cannabinoid-1 receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cb2 possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. pro-and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. methods: to specifically address the role of cb2 for age-associated obesity we analyzed metabolic, cardiovascular, immune and neuronal functions in 1. 2-1.8 year old cb2 -/and control mice, fed with a standard diet and assessed effects of the cb2 agonist, hu308 during high fat diet in 12-16 week old mice. results: the cb2 -/mice were obese with hypertrophy of visceral fat, immune cell polarization towards pro-inflammatory sub-populations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. they also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. the cb2 agonist hu308 prevented hfd-evoked hypertension, reduced hfdevoked polarization of adipose tissue macrophages towards the m1-like proinflammatory type and reduced hfd-evoked nociceptive hypersensitivity but had no effect on weight gain. conclusion: cb2 agonists may fortify cb2-mediated anti-obesity signaling without the risk of anti-cb1 mediated depression that caused the failure of rimonabant. leishmaniasis is a neglected tropical disease caused by leishmania, eukaryotic protozoal organisms, which infect humans and other mammals. this disease is transmitted by sandflies of the genus phlebotomus. due to global warming the endemic region of these vectors expands further to northwards and threatens south european countries as well. the treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. the so far unexplored inhibition of mitochondrial functions in leishmania by natural products or even food ingredients seems to be an interesting alternative. two food ingredients, resveratrol (res) and xanthohumol (xan), were widely studied in mammalian cells but little is known about their actions on protozoal parasites. therefore, we compared the influence of res and xan on the function of leishmanial and mammalian mitochondria. anti-leishmanial activities of xenobiotics were assessed in cell culture of leishmania tarentolae promastigotes (ltp), leishmania amazonensis amastigotes (laa) and compared to peritoneal macrophages from mouse (pmm) using viability assays. furthermore, mechanistic studies regarding mitochondrial functions were conducted in ltp, mitochondrial fractions isolated from ltp and bovine heart submitochondrial particles using oxygen consumption measurements, assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation by photometry, fluorimetry and electron spin resonance spectroscopy. in ltp, xan inhibited the viability more effective than res (ic 50 : xan 23 µm, res 161 µm). likewise, xan and res demonstrated anti-leishmanial activity in laa (ic 50 : xan 7 µm, res 14 µm) while had less influence on the viability of pmm (ic 50 : xan 68 µm, res > 438 µm). in contrast to res, xan strongly inhibited oxygen consumption in leishmania. further studies demonstrated that this is based on the inhibition of the mitochondrial electron transfer complex ii/iii by xan which was less pronounced with res. however, xan also demonstrated inhibitory activity on mammalian mitochondrial complex iii. in addition, xan caused no decrease of the membrane potential in leishmanial mitochondria, while res resulted in mitochondrial uncoupling. neither xan nor res increased mitochondrial superoxide release in ltp. these data show that res, a major polyphenol from red wine, and xan, an ingredient of hop-containing beer, may have selective anti-leishmanial activity. tryptophan hydroxylase (tph) is the rate-limiting enzyme in serotonin (5-ht) biosynthesis. its two existing isoforms are exclusively expressed in the periphery (tph1), or the raphe nuclei of the brainstem (tph2) and the respective 5-ht populations are distinctly separated by the blood-brain barrier, offering the possibility to pharmacologically modulate central and peripheral functions in an independent manner. peripheral 5-ht is mainly produced by tph1-expressing enterochromaffin cells of the gut and taken up into platelets and transported in the blood stream. upon platelet activation, 5-ht is rapidly released and locally induces multiple effects, such as vasoconstriction, cell proliferation or fibrosis and is furthermore involved in the regulation of e.g. vascular tone, gut motility, primary hemostasis, insulin secretion and t-cell-mediated immune response. following the classical early drug development pathway, we developed a fluorescencebased tph activity assay and performed a high-throughput screening of about 37000 small chemical compounds. we discovered a novel class of tph inhibitors, which was thoroughly validated in a variety of in vitro assay setups. combining medicinal chemistry and x-ray crystallography, we further aimed to develop these inhibitors into preclinical drug candidates. to date we were able to generate and patent a series of novel tph inhibitors with optimized affinity and an in vitro ic 50 in the low nanomolar range. this novel class of tph inhibitors could potentially be used to treat a variety of disorders with aberrant peripheral 5-ht signaling, such as gastrointestinal disorders (e.g. irritable bowel syndrome, crohn's disease, various forms of diarrhea), cardiac valve diseases, pulmonary hypertension, chronic respiratory diseases and some neuroendocrine (carcinoid) tumors. primary hepatocellular carcinoma (hcc) is the most frequent type of liver cancer. therapeutic options are rare. beside sorafenib, a tyrosinkinase inhibitor, which is only used in end stage liver cancer, the surgical intervention is the only successful clinical treatment option. hence there is an urgent need to develop new therapeutic strategies and to identify new drugs for therapy of hcc. hcc often arises in fibrotic or cirrhotic liver, which is accompanied by a change of the extracellular matrix (ecm) composition. in addition it was shown that hepatoma cells express different integrins, which interact with ecm and intracellular cell signaling, compared to hepatocytes. snake venoms have gained increased attention, as it was shown that some of their enzymes and peptides directly act on tumor cells and their multicellular arrangement or indirectly by influencing the stroma environment of the tumor. aim of the present study was to investigate the effect of snake venoms on liver cancer related cell lines as well as their specific action on the ecm-integrin axis. the effects of the snake venoms vipera palestinae (vp), calloselasma rhodostoma (cr) and echis sochureki (es) on a cellular level (mtt, ldh release), on cell-cellconnections (caco2 permeability assay) and on cell-matrix-interactions (adherence test) were investigated. cell-matrix interactions were tested with an adhesion assay using collagen i (c-i), collagen iv (c-iv), fibronectin (fn) and laminin (lm) as ecm compounds. in our in vitro models we used hepg2 as a hcc tumor cell line and the fibroblast cell line fi301 as stroma simulation. additionally caco2 cells were used, a colon carcinoma cell line representing colorectal liver metastasis. the toxicity of snake venom on liver cancer related cell lines was determined in the range of 0.01 -100 µg/ml and plotted into dose response curves. the noaels were calculated from these dose response curves: vp: 0.5 µg/ml -cr: 1 µg/ml -es: 5 µg/ml. performance of the caco2-transwell permeability assay revealed no influence of the tested venom concentrations on the integrity of the cellular arrangement. investigations for integrin inhibition revealed that the venom from vp reduced adherence on lm coated plates and the venom of ec reduced adherence on lm and fn coated plates compared to untreated cells. there was no effect on the adherence on any matrix from the venom of cr observable. co-incubation of the snake venoms of vp and es (below or near noael concentrations) with 5-fluorouracil (5fu), which is used as a chemotherapeutic agent, caused a reduction of its ic50 values. the results indicate that components of vp and ec inhibit the formation of cell-matrixinteractions possibly acting as disintegrins. the co-incubation experiments demonstrated a synergistic effect of 5fu and snake venoms. further experiments should enable the isolation of therapeutic active venom compounds, identification of disintegrins and their role in synergistic mechanisms in liver cancer therapy. modulation of the blood-brain barrier with peptidomimetics to improve drug delivery s. dithmer after decades of research, the blood-brain barrier (bbb) still remains a major problem for successful delivery to the brain for the vast majority of drugs. the main component forming the bbb is the brain microvascular endothelium. the paracellular permeation is limited by tight junctions (tjs), a multiprotein complex composed of the members of the claudin family claudin-1, -3, -5, -12. claudin-5 is known to be the key tj protein tightening the bbb. therefore, claudin-5 has been selected as target to modulate the bbb. for this reason, drug enhancer peptides (peptidomimetics) were designed to modulate transiently claudin-5 and, thereby, permeabilize the bbb. by combining biochemical protein/peptide interaction and tissue culture methods, we identified, validated and optimized peptide sequences modulating claudin-5 containing barriers. the claudin-5 targeting peptides decreased the transcellular electrical resistance and increased the permeability through mdck-ii cell monolayers stably expressing yfpclaudin-5 and immortalized brain endothelial cells (bend.3). the peptides decreased the amount of claudin-5 and zo-1 at cell-cell contacts and changed the cell morphology from spindle-shaped to more round-shaped. all tested peptides showed no signs of toxicity on cell cultures and in vivo (intravenous injection). permeability measurements in mice proved enhanced permeation of na-fluorescein (376 da) through the bbb, which was confirmed by magnet resonance imaging of contrast agents (gd-dtpa, 547 da). in summary, we identified new peptides with the potential to enhance cerebral delivery of small molecules through the bbb. treatment of cerebral diseases is limited by the capability of pharmacologically active agents to penetrate the blood-brain barrier (bbb). this paracellularly tight diffusion barrier is formed by brain capillary endothelial cells. the paraendothelial cleft is sealed by tight junctions (tjs), a multiprotein complex. cerebral tjs predominantly consist of claudin-5 (cldn5) which tightens the bbb for molecules <800 da. consequently, cldn5 is a potential target for transient and size-specific modulation of the bbb to improve cns penetration for pharmaceutically active agents. in high throughput screening using a cldn5 assay, the barrier opener 1 (bo1) was identified as a cldn5 modulator. initially, a significant removal of cldn5 from the plasma membrane was shown by confocal microscopy using epithelial and endothelial cell lines. measurement of transcellular electrical resistance and of paracellular permeability using lucifer yellow (mw 521 da) demonstrated the effect of bo1. concentration dependent treatment (50-150 µm) of cell monolayers with bo1 reduced tightness of the tjs between some hours and 24 h. applying 2-hydroxypropyl-ß-cyclodextrin as a solubilizer, opening activityof bo1 became detectable in mice. due to short stability (< 2 h) of bo1 in the bloodplasma repeated administration (1.5 mg/kg i.v.) was required to induce significantly increased permeability of the bbb for na-fluorescein(mw 376 da). the small molecule bo1 is a promising new approach for transient opening of the bbb in vivo. further modification of the stability and solubility of bo1 is necessary to optimize its applicability. the complex of tight junction (tj) proteins is located between opposing epithelial or endothelial cells. tjs restrict the paracellular permeation of ions and other solutes. tricellulin (tric) tightens tricellular tjs (ttjs) and regulates bicellular tj (btj) proteins like claudins and occludin (occl). current data suggest an important role of ttjs at the blood-brain barrier (bbb). a main pharmacological problem is modulation of the bbb to improve drug delivery to the cns. therefore, tricsi has been developed as a peptide taken from tric to open tissue barriers specifically and transiently.initially, a recombinant protein was generated based on a sequence of an extracellular loop of tric, tagged with maltose binding protein. the fusion protein caused down-regulation of tric, internalization of both tric and occl (confocal laser scanning microscopy), and a significant decrease in transcellular electrical resistance (ter) of a human epithelial colorectal adenocarcinoma cell line. then, studies with the synthetic peptide tricsi indicated its capacity of cell barrier openingafter about 16 h of incubation with concentrations varying from 100 to 150 µmaffecting the membrane localization of tricand occl. barrier opening was proven by decreasing ter, increasing permeability coefficient of lucifer yellow (457 da) and fitc-dextran (10 kda); the localization of tric elongated from ttjs towards btjs and cldn1 was weakened at btjs.physiochemical properties of tricsiexamined by circular dichroism spectroscopy suggested ß-strand structure and no helical propensity. taken together, a tric-derived peptide has been identified increasing the paracellular permeability of tissue barriers and redistributing the cellular localization of tj proteins. tricsi is a novel, promising tool to overcome cerebral barriers with the potential to improve drug delivery to the cns. further experiments are needed to better understand the role of tric in tissue barriers as well as to clarify the mode of action of tricsi. introduction: lung transplantation has become an established treatment option for a variety of end-stage lung diseases, but the long-term survival is often disappointing. the leading cause of death is generally chronic rejection which is characterized by inflammation and fibrous obliteration of the small airways, progressively leading to a reduction of the airflow. the mouse heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease. despite its widespread application, the heterotopic transplantation model does have a number of limitations, as for example the lack of airflow. the present study provides a description of the orthotopic tracheal transplantation mouse model, which shares more similarities with transplant situation in humans, and provides the analysis of airway obliteration via micro ct and histological evaluation. methods: a seven-ring donor trachea from balb/c mice was implanted into the recipient c57bl/6 mice. c57bl/6 mice without transplantation were used as normal controls. donor c57bl/6 mice to recipient c57bl/6 mice were served as the isograft group. 42 days after transplantation, mice were scanned using an in vivo small animal µct (skyscan 1176). tracheal tissue was harvested and fixed in formalin, embedded in paraffin, cut and stained with hematoxylin and eosin (h&e) as well as sirius-red/fast-green. results and conclusions: histologic evaluation showed luminal narrowing with subepithelial inflammatory cell infiltrates and fibrosis, as well as partially damaged and flattened epithelium. the aerated volume of the allogeneic grafts, analyzed by micro ct was significantly reduced compared to the isogenic control grafts and normal controls. non-invasive imaging via micro ct may offer an option for longitudinal monitoring of the progression of obliterative airway disease as well as response to treatment. c. elegans is a well-established model organism to study the aging process as well as effects of various substances in vivo. its lifespan is regulated by multiple signaling pathways (e.g. insulin or mtor signaling), which are well conserved up to humans. the insulin/igf-1 pathway was the first pathway shown to effect ageing in animals. mutations that decrease the activity of daf-2 (igf1r) lead to a significant increase of lifespan accompanied by a decrease of age pigment accumulation in c. elegans. the relevant effector of the insulin/igf-1 pathway is the transcription factor daf-16 (hfoxo3a). inhibition of hmg-coa reductase (enzyme of mevalonate pathway) by statins, which are frequently used as cholesterol-lowering agents in the clinic, has been shown to attenuate protein prenylation and glycosylation. notably, prenylated-, membrane-bound small gtp-binding proteins are important for the regulation of the afore mentioned age-related signaling pathways like the insulin/igf-1 pathway. recently, a cohort study showed that a decreased mortality rate in humans between age 78 -90 correlates with statin treatment, but is independent of total cholesterol levels. as c. elegans harbors the mevalonate pathway, but the branch leading to cholesterol synthesis is missing, it is a well-suited model to study cholesterol -independent effects of statins on aging-associated phenotypes and the underlying molecular mechanisms. here, we show that exposure of c. elegans to statins substantially decelerated the accumulation of age pigments. while the level of age pigments roughly doubled in control animals, there was only a slight increase in the lovastatin group. the use of atorvastatin gave comparable results indicating a more general effect of the inhibition of the hmg-coa reductase. the retarded accumulation of age pigments could be partly phenocopied using an inhibitor of the small gtpase rac1 or using rnai against the hmg-coa reductase. a reduced level of age pigments is prognostic for an elevated mean lifespan (about 20%) in c. elegans. a post reproductive treatment with lovastatin, mimicking the use of statins in patients of advanced age increased the mean lifespan in c. elegans even further. in addition, we could show a mild reduction of fertility and a developmental delay as well as a marked increase in acute thermal stress resistance mediated by lovastatin. besides the reduced accumulation of age pigments and the increased lifespan these are phenotypes which are usually observed under accumulation of daf-16 overactivity. consequently we found an increased nuclear localization of daf-16 in the presence of lovastatin and lovastatin completely failed to reduce age pigments in a daf-16-ko mutant background. rt-qpcr brought jnk-1, a known activator of daf-16, into play as a possible effector induced by statins. this is currently under investigation. in summary, statin exposure induces a longevity phenotype in c. elegans, which might be daf-16 dependent. this findings indicates that a product of the mevalonate pathway might influence the insulin/igf-1 pathway and particularly the transcription factor daf-16. the high-fat diet (hfd)-fed, streptozotocin (stz)-treated rat model is one of the experimentally-induced animal models of diabetes. this model is often used to evaluate the antidiabetic activity of several agents. according to srinivasan et al. (2005) , prolonged exposure of high-fat diet leads to insulin resistance, and the development of diabetes occurs only in insulin-resistant hfd-fed rats following low dose stz, because the hfd-fed rats are already mildly hyperglycemic due to insulin resistance (1). in hfd/stz model, the rats are fed with high-fat diet for 2-4 weeks or for a relatively long time (≥ 3 months) in order to simulate the insulin resistance and/or glucose intolerance. after induction of diabetes with multiple or single low-dose of stz (30-35 mg/kg), some of the diabetic rats receive treatment (2) . in this way, the impact of treatment can be determined by comparing the differences between groups. despite the lack of methodological information concerning the feeding time in some studies, all rats should be allowed to continue to feed on their respective diets until the end of the study. but what would happen if the hfd was switched to normal pellet diet in these diabetic rats? in our experience, the feeding of npd for 4 weeks significantly decreased fbg in diabetic rats compared to hfd-fed diabetic rats (234.40 ± 42.71 mg/dl vs. 464.00 ± 23.88 mg/dl, p < 0.05). although diet regulation could not restore normal blood glucose, such a decrease was unexpected. in addition, the body weights of the npd-fed diabetic rats were significantly lower than the body weights of the hfd-fed diabetic rats (249 ±6.00 gvs. 288.00 ±4.41 g, p < 0.05). there was no significant difference in body weight between nondiabetic control rats and diabetic rats fed npd for 4 weeks. further details can be found in table 1 . diet regulation and weight loss may prevent, control and reverse diabetes. however, at later stages of the disease, it is difficult to improve blood glucose control without medication, because the disease progresses from insulin resistance to insulin deficiency (3) . according to some diabetes researchers, the amount of residual functional betacells mass is an important issue, and another important question is whether hfd/stz rat mimics an early or late stage of type 2 diabetes (4). these preliminary findings suggest the possibility that hfd/stz rat model may simulate the characteristics of early stage more than the final stage of type 2 diabetes, and hyperglicemia in the experimental model can partially reverse with diet regulation. references: 1. srinivasan, k., viswanad, b., asrat, l., kaul, c. l., ramarao, p. (2005) . combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. pharmacol res 52 (4): 313-320. 2. oztürk z, gurpinar t, vural k, boyacıoglu s, korkmaz m, var a. (2015) . effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet. biotech histochem 90 (7): 506-515. 3. franz, m. j. (2007) . the dilemma of weight loss in diabetes. diabetes spectr 20 (3) animal models are pivotal for studies of pathogenesis and treatment of movement disorders. dystonia, characterized by sustained or intermittent muscle contractions causing twisting movements/postures, is regarded as a basal ganglia disorder. the pathophysiology is however poorly understood. in mouse models of dyt1 dystonia, which is caused by a gag deletion in tor1a that encodes for the protein torsin a, ex vivo electrophysiological studies have shown an abnormal d2 receptor mediated release of acetylcholine from striatal interneurons. in these models, which do not exhibit a dystonic phenotype, the functional relevance of the increased d2 receptor mediated acetylcholine release has not been examined yet. the aim of present study was to (1) generate more powerful tests to detect behavioural alterations in the dyt1 knock-in mouse and to (2) examine the behavioral effects of the d2 receptor agonist quinpirole. for this purpose, a sequence of cognitive, motoric and sensorimotor tests were performed in this mouse model. only the adhesive removal test that explores sensorimotor connectivity revealed significant impairments in the dyt1 knock-in mice compared to controls. to induce a more characteristic and stronger phenotype, the "rotating beam test" was developed. this motoric test measures motor coordination and balance. interestingly, dyt1 knock-in mice showed significant motor deficits in the rotating beam test. based on these results, the acute effects of quinpirole (0.25 -1 mg/kg i.p.) were tested in dyt1 knock-in and wildtype mice. subsequent to the injections, mice were tested in the open field, the rotating beam test and the adhesive removal test, respectively. in the open field test, dyt1 knock-in mice showed increased thigmotaxis at a dose of 0.5 mg/kg quinpirole. in the rotating beam test, both groups showed a dose-dependently reduced performance. in the adhesive removal test, quinpirole improved the reaction time in dyt1 mice independently of dosage, while no effects were observed in the wildtype littermates. however, in vehicle follow-up (post-drug control), this effect remained consistent in the dyt1 model, suggesting a habituation effect. in conclusion, we generated a new test, i.e., the rotating beam test which improves the detection of mild motor impairments in dyt1 knock-in mice. furthermore, the adhesive removal test revealed sensorimotor dysfunctions in this animal model. these results represent an important step for our ongoing optogenetic examinations on the role of abnormal neuronal plasticity in dyt1 dystonia and for pharmacological studies. the first data on the effects of quinpirole do not indicate a critical role of d2 dysregulated acetylcholine release, but this has to be clarified by ongoing local striatal injections of quinpirole and by pharmacological manipulations of the cholinergic system. renal fibrosis is characterized by decreased nitric oxide (no) bioavailability and pronounced transforming growth factor β (tgfβ) signalling subsequently excessive extracellular matrix (ecm) deposition. here, the effects of the soluble guanylate cyclase (sgc) stimulator bay 41-8543 after unilateral ureter obstruction (uuo) have been studied. kidney fibrosis was induced by unilateral ureter obstruction (uuo) in wild type (wt) and cgki knock-out (cgki ko) mice. starting one day after uuo, the sgc stimulator bay 41-8543 was (4mg/kg/daily) i. p. injected for seven days. biomarkers indicating remodelling processes in the kidney were analysed via mrna expression and protein expression. bay 41-8543 administration influenced the activity of the ecm degrading matrix metalloproteases (mmp2 and mmp9) and their inhibitor timp-1, the expression pattern of extracellular matrix proteins (e.g. collagen and fibronectin) of profibrotic mediators (e.g. connective tissue growth factors (ctgf) and plasminogen-activator inhibitor-1 (pai-1)) and the secretion of cytokines, e.g. il-6. thereby, bay 41-8543 increments the cgmp pool among others via modulation of endothelial no synthase (enos) expression. agents, which enhance no and cyclic guanosine monophosphate (cgmp) ameliorate the progression of fibrotic tissue. however, the molecular mechanism by which cgmp via cgki affects the development of kidney fibrosis has not fully been elucidated. accordingly, the present study investigates the functional role of sgc stimulation in regulating the fibrotic process, the signalling pathway and the underlying mechanisms involved. we hypothesize that the antifibrotic potential of bay 41-8543 might be related to the increased cgmp pool and the inhibition of the mapk and smad signalling pathway. the elucidation of the signalling allows the development of new therapeutic options. infection of mice with listeria monocytogenes (lm) results in a strong t-cell response that is critical for an efficient defense. here, we demonstrate that the adapter protein sly1 (sh3-domain protein expressed inlymphocytes 1) is essential for the generation of a fully functional t-cell response. the lack of sly1 leads to reduced survival rates of infected mice. the increased susceptibility of sly1 ko mice was caused by reduced proliferation of differentiated t cells. ex vivo analyses of isolated sly1 ko t cells displayed a dysregulation of forkhead box protein o1 (foxo1) shuttling after tcr signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the t-cell population. foxo1 shuttles to the cytoplasm after phosphorylation in a protein complex including 14-3-3 proteins. interestingly, we observed a similar regulation for the adapter protein sly1, where tcr stimulation results in sly1 phosphorylation and sly1 export to the cytoplasm. moreover, immunoprecipitation analyses revealed a binding of sly1 to 14-3-3 proteins. altogether, this study describes sly1 as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during lm infection, and provides a model of how sly1 regulates t-cell proliferation (schäll et al., eur j immunol. 2015) . the catalytical isoforms p110γ and p110δ of phosphatidylinositol-4,5-bisphosphate 3kinase γ (pi3kγ) and pi3kδ play an important role in the pathogenesis of asthma. two key elements in allergic asthma are increased eosinophil and ige levels. whereas dual pharmacological inhibition of the catalytical subunits p110γ and p110δ reduces asthmaassociated eosinophilic lung infiltration and ameliorates disease symptoms, it has been shown that dual genetic deficiency in pi3kγ and pi3kδ in p110γ ko δ d910a mice increases serum ige and basal eosinophil counts in mucosal tissues and blood. this suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. here we analysed p110γ/δ -/mice and determined ige and eosinophil counts in a basal state and the immune response to ovalbumin (ova)-induced allergic asthma. we found that serum concentrations of ige, il-5 and eosinophil numbers in blood, spleen and bone marrow were significantly increased in p110γ/δ -/mice in comparison to single knock-out (ko) and wildtype (wt) mice. nevertheless, p110γ/δ -/mice were protected against ovainduced infiltration of eosinophils, neutrophils, b cells and t cells into the lung tissue and the bronchoalveolar space. moreover, p110γ/δ -/mice, but not single ko mice, showed a reduced bronchial hyperresponsiveness as measured with the isolated and perfused lung. we conclude that although the dual deficiency of p110γ and p110δ causes eosinophilia and ige hyperproduction, p110γ/δ -/mice are not prone to develop ovainduced allergic asthma. an increase of plasma extravasation induced by activation of constitutively expressed endothelial bradykinin type 2 receptors (b2) has been shown to contribute to the development of angioedema occurring as a sometimes life-threatening side effect of angiotensin-converting enzyme inhibitors such as enalapril (new engl j med 2015:372; 418-425) . these drugs inhibit the degradation of bradykinin and increase its vascular steady-state concentration. hence, it is reasonable to assume that bradykinin may destabilise the endothelial barrier, i.e. may increase physiologic extravasation. while the commonly used miles assay provides a useful and relatively easy tool to study the effect of permeabilizing mediators in-vivo, it does not distinguish between intravascular and interstitial evan's blue dye. likewise, extravasation can only be quantified at one particular time point per animal, usually 20-30 min. furthermore, evaluation of physiologic extravasation is not possible. in contrast, non-invasive twophoton laser microscopy may allow separating the intravascular from the interstitial compartment and thereby investigations of changes of the physiologic endothelial barrier induced by drugs or transgenes. therefore, we have evaluated this methodology for its suitability to study endothelial permeability in mice in vivo. to establish this, we used two different fluorescent dyes of different molecular weight. a 200,000 kda dextran equipped with a green fluorescent chromophore which cannot leave vascular lumen was injected intravenously to visualize small dermal blood vessels of the mouse ear located approximately 200 µm below the surface. after stabilization of the green fluorescent signal, a 10,000 kda dextran equipped with a red fluorescent chromophore which easily traverses the endothelial barrier was applied by intravenous injection. the red fluorescence permeates into the interstitium during physiologic extravasation and accumulates in the interstitial space. this process can be followed by measuring the decrease of intravascular red fluorescence over various time periods. using this methodology we have studied whether endothelial-specific overexpression of b2 changes physiologic endothelial permeability. this newly developed transgenic mouse line (b2 tg ) was established using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment. we observed that b2tg showed a significantly stronger extravasation than their transgene negative littermates as evidenced by the more rapid extravasation of the 10,000 kda dextran at each time point (fig. 1) .we conclude that two-photon laser microscopy is suitable to study endothelial permeability non-invasively in-vivo and that this methodology allows to study the effects of drugs and transgenes on the endothelial barrier under non-inflammatory conditions. furthermore, our results suggest that endothelial-specific overexpression of b2 increases physiologic extravasation. non-allergic angioedema such as angioedema induced by angiotensin converting enzyme inhibitors (acei) develops as a consequence of increased activation of bradykinin receptor type 2 (b2). using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment a transgenic mouse line harbouring an endothelial-specific overexpression of b2 was generated and backcrossed to c57bl/6 for more than 10 times (b2 tg ). lung mrna using primers specific for the human or the mouse b2 cdna revealed a 12.5-fold stronger expression of human b2 in b2 tg (n=6), while the expression of murine b2 mrna was unchanged and similar to transgene negative littermates (b2 n ). we have evaluated the specificity of several antibodies directed against b2 and found that a rabbit monoclonal anti b2 antibody appears to be reliable, i.e. there was just a faint staining in lung tissue of b2 -/mice. however, this antibody primarily stains rodent b2 and has only little cross-reactivity to human b2. hence, we were not able to detect a significant increase of b2 protein in tissues of b2 tg . previous experiments have shown that bradykinin induced concentration-dependent constrictions of aortic rings with a maximal effect at 1 µm of bradykinin. the contraction due to bradykinin was completely inhibited by icatibant or diclofenac indicating that it is mediated by endothelial b2 activation and dependent on cyclooxygenase activity. in striking contrast to their transgene negative littermates b2 n , we found a significant icatibant sensitive aortic dilation in b2 tg following preincubation with diclofenac which indicates functional overexpression of b2 in conductance vessels of b2 tg . to evaluate whether this applies to dermal micro vessels we used the miles assay to quantify dermal extravasation of the albumin-bound dye evans blue following intradermal injection of 30 µl of either vehicle, bradykinin, labradimil and histamine (control). increasing concentrations of bradykinin caused a significant increase of extravasation reaching 4.41±0.11 fold at 18.9 nmol bradykinin in c57bl/6 (n=6 each, p<0.0001 vs. vehicle). a similar increase was found in b2 n (4.45±0.25 fold, n=7, p<0.0001 vs. vehicle), while there was a stronger response in b2 tg (5.50±0.16 fold, n=7, p<0.0001 vs. vehicle) which was significantly different to b2 n (p<0.01) and c57bl/6 (p<0.01). in another set of experiments the specific and ace-resistant b2 agonist labradimil (1.89 nmol) was used instead of bradykinin. labradimil increased extravasation by 3.736±0.121 fold in c57bl/6 (n=6 each, p<0.0001 vs. vehicle), by 4.51±0.11 fold in br2 n (n=7 each, p<0.0001 vs. vehicle) and 4.88±0,21 fold in b2 tg (n=6, p<0.0001 vs. vehicle) which was significantly different to c57bl/6 (p<0.01) but not to b2 n (p>0.05). the effects of bradykinin and labradimil were largely blocked by 10 nmol icatibant (i.v.) in c57bl/6, b2 n and b2 tg mice (p<0.0001) and hence mediated by activation of b2. these data suggest that overexpression of b2 in b2 tg is functionally active in endothelial cells of large conductance and small dermal vessels. therefore, b2 tg represents a new animal model suitable for cardiovascular and non-allergic angioedema research. pharmacokinetic pharmacodynamic modeling of irreversible effects: the rituximab example f. keller 1 1 universitätsklinikum, innere 1, nephrologie, ulm, germany background: for pharmacokinetic-pharmacodynamic modeling usually the sigmoid emax model is used as described by the hill equation. however, treatment regimens exist where the effect is only exerted as long as the drug concentration increases whereas decreasing concentrations produce no longer an effect. examples are the pulse anti-cancer therapy such as originally proposed by the devita protocols. methods: here, the new model for irreversible drug action is derived from the time dependent change of the concentration that must be larger than the time-dependent growth of the number of target cells (tumor or bacteria). the irreversible effect can be assumed if the is no further growth of the target cells occurs. de/dt = + dc/dt -dn/dt dn/dt = 0 a solution for the above differential equation can be obtained by use of the integral exponential function iec based on the euler-mascheroni constant (gamma = 0.5772 …). this model of an irreversible effect was applied.to the example of rituximab where the initial effect on cd19+ and cd20+ b-cells completely persists for 6 month. to obtain a numerical solution, the following parameters are needed to be determined: the target concentration ctarget = 100 mcg/ml and the infusion time t = 2 hours. results: it can be shown that a plausible result for rituximab can be estimated only under the condition that a short distribution half-life is assumed of t1/2 = 2 hours (not shorter than the time t of infusion). with the terminal half-life of 460 hours no plausible solution is obtained. under these conditions two observations are made: there is a negative effect both, initially for low concentrations and after cessation of the infusion when concentrations decrease (in reality this means no effect in both cases). the irreversible effect is proportional to the target concentration. the shorter the half-life comes out relative to the infusion time (t1/2 < t) the stronger is the effect (figure) . occasionally there are specific questions occurring on the ruminant xenobiotic metabolism: 1) are the observed metabolites ruminant specific and formed directly in the rumen? 2) are ruminants able to cleave plant specific metabolites like glycosides to the respective aglycon? in the past new additional in vivo goat metabolism studies with at least one animal were performed. the aim of the project was to elucidate an alternative in-vitro method to replace the existing in vivo method in order to address robustly specific questions on xenobiotic metabolism in ruminants for registration of ppp. fresh sheep rumen fluid was incubated in-vitro >7 days by using rumen simulation technology (rusitec). the conservation of the physiological conditions were proven by measurement of ph (~ph 6.6) and redox potential (~-300 mv). the microflora composition and their viability (bacteria, protozoa and fungi) of the rumen were monitored by microscopy, incubation on agar plates and performing several viability tests (e.g. glycosidase-test, nh3 and short fatty acids). all the tests showed that the rusitec is a successful tool to maintain sheep rumen fluid for at least 7 days in -vitro. the metabolic behavior and performance of the rumen fluid was tested by e.g. incubating 14c-triazol derivative metabolites (tdm) like triazol-alanin (ta); triazol-acetic-acid (taa) and triazol-lactic-acid (tla), which are usually formed in plants after application of triazol-containing fungicides. it was shown that ta was cleaved within 72 h to 1.2.4.-triazol, while taa and tla were stable under these conditions. these data are in a good accordance with available in vivo data in cows. moreover glycosides (12c-polydatin, octyl-14c-β-d-glucopyranosid) were cleaved within 1 hour completely. all these data show, that the rumen fluid maintained its metabolic performance by using rusitec. basf identified the rusitec method, which is usually used in different areas (e.g. investigation of methane production in-vitro) as suitable and adapted this method for the purpose of investigation of ruminant xenobiotic metabolism. it was shown that rusitec is a robust method to analyze rumen xenobiotic metabolism and therefore can clearly substitute in vivo animal studies on ruminant metabolism studies beyond oecd503 and contribute significantly to animal welfare (3r: replacement). results: by using the training set, physicochemical (e.g. lipophilicity) and pharmacokinetic characteristics of mtx (e.g. v max for active tubular secretion) were slightly adjusted. using the gfr formula of morris et al. (1982) and including an empiric correction factor, mrd for the training set was 2.49 whereas bias was 2.80 µmol/l. by applying the developed pbpk model to the test set the respective values were mrd=3.92 and bias=1.43 µmol/l. for the covariates "at least one potentially interacting co-medication" and "trimethoprime/sulfamethoxazole" a significant impact on the prediction quality was found. conclusions: using the developed pbpk-model, a good prediction of the pharmacokinetics of hd mtx in severely ill children was found. by including additional factors influencing the prediction of mtx characteristics (e.g. co-medications) an improved prediction of mtx-sl might be reached. in prospective clinical trials, those more complex models should be evaluated and might be helpful to predict hd mtx pharmacokinetics and reduce unwanted side effects. hypericin is a natural polycyclic quinone found in hypericum perforatum. although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is poorly soluble in physiological medium. recently we have shown that quercitrin and isoquercitrin, but not hyerosid, quercetin or rutin increased the uptake of hypericin in caco-2 cells. the major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the caco-2 cell system under different experimental conditions. the permeation characteristics of hypericin (5 µm) in absence or presence of hypericum extract 145, 62.5 µg/ml) were studied in the absorptive direction. following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. the amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of the extract (from 0 to 7.5 %). the majority of hypericin was found after cell extraction (44% in absence and 76% in presence of the extract). the recovery was in the range of 90 %, and significant amounts of hypericin found after cell extraction. fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. the precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. however, our experiments demonstrated that the permeation characteristics of hypericin significantly improved in presence of the extract. background: the combination of gamithromycin (gam), a novel drug with the big advantage of a once weekly administration, and rifampicin (rif) is used in the treatment of lower airway disease in foals. both are effective in the therapy of infections with rhodococcus equi, a gram-positive coccobacillus bacterium, which is known to survive and reproduce within alveolar macrophages. macrolides are combined with rif to prevent resistance developing with single agent therapy. both drug classes reach high concentrations in the lung, penetrate into phagocytes and kill intracellular pathogens. methods: a controlled, single-and multiple dose study with four-periods was conducted in 10 healthy foals (5 ♂ and 5 ♀, age: 42-63 days, body weight: 100-177 kg) which were treated once with rif alone (10 mg/kg s.i.d., p.o., a) followed by the administration of gam (6 mg/kg once weekly, i.v., b) for 3 weeks. study period 3 ("rif-gam acute", c) includes the administration of gam and rif for 7 days with an administration interruption after the first rif dose for blood sampling. for the last study period ("rif-gam chronic", d) both gam and rif were coadministered for 2 weeks. all periods were completed with blood sampling for pharmacokinetic analysis for 48 ( . rif is also accumulated in the lung, but to a much lower degree than gam (elf/c 24 h : 1.2 ± 0.5; balc/c 24 h : 2.01 ± 1.24). conclusion: pharmacokinetic data of the present study provides surprising results. in previous studies coadministration of clarithromycin and rif show a dramatic decreased plasma exposure of the macrolide, whereas the balc/c 24 h -ratio was unaffected (peters et al. 2011) . in contrast, systemic exposure of gam increase significantly in case of the combined therapy and the balc/c 24 h -ratio was nearly halved. both macrolides have in common, that they are intensively accumulated in the lung (elf << balc). at the moment there is further research required (e.g. in vitro studies) for a better understanding of the very interesting in vivo data. 1 1 institut für klinische pharmakologie, göttingen, germany background and aim: desvenlafaxine is a selective serotonin and norepinephrin reuptake inhibitor, which is approved in the usa (but not in europe) for treatment of major depressive disorder. desvenlafaxine is the major active metabolite of the antidepressant venlafaxine. desvenlafaxin is produced by o-desmethylation via cyp2d6. direct administration of desvenlafaxine should bypass the variability in venlafaxine pharmacokinetics caused by the highly polymorphic cyp2d6. however, desvenlafaxine is less lipophilic than venlafaxine and may require carrier-mediated transport to penetrate cell membranes. based on our in vitro data, desvenlafaxine is a substrate of the hepatic organic cation transporter oct1 and common genetic polymorphisms abolished desvenlafaxine cellular uptake. about 9% of caucasians are compound heterozygous carriers of loss-of-function oct1 polymorphisms. therefore, oct1 polymorphisms may cause substantial inter-individual variability in the hepatic uptake and plasma concentrations of desvenlafaxine. in this study we evaluated the influence of genetically-determined loss of oct1 function on the pharmacokinetics and pharmacodynamics of desvenlafaxine. primary aim was dependence of desvenlafaxine plasma concentrations (represented by auc as primary endpoint) on the number of active oct1 alleles. methods: 50 mg desvenlafaxine (pristiq®) was orally administered to 41 healthy subjects preselected according to their oct1 genotypes. oct1*1 allele was regarded full active, oct1*2 to *6 alleles were regarded loss of function. plasma concentrations of desvenlafaxine and its main metabolite didesmethylvenlafaxine were quantified in plasma sampled up to 60 hours after administration using lc-ms/ms. pupillographic measurements were performed as possible surrogate markers for desvenlafaxine pharmacodynamics. results out of the 41 subjects 14 carried two active, 13 one active, and 14 zero active oct1 alleles. age, height and weight were 26.9 ± 6.4 years (mean ± standard deviation), 1.75 ± 0.11 m and 70.9 ± 12.1 kg with no significant differences among the oct1 genotypes. there were strong variations in the pharmacokinetics of desvenlafaxine and its metabolite didesmethylvenlafaxine. the auc 0-infinity of desvenlafaxine varied between 52.8 and 282.2 min*mg/l and auc 0-infinity of didesmethylvenlafaxine between 3.5 and 30.7 mg*min/l. however, neither desvenlafaxine nor didesmethylvenlafaxine pharmacokinetics significantly differed among the three oct1 genotypes. concerning pharmacodynamics of desvenlafaxine, pupil diameters at maximal constriction after a standardized light exposure were on average 14% greater around the time of t max than before administration. in line with the pharmacokinetic results there were no significant differences in maximal constriction or other pupillographic parameters among the oct1 genotype groups. conclusions: our results suggest that oct1 genotype does not affect the pharmacokinetics of desvenlafaxine and therefore no dose adjustment in respect to oct1 genotype should be considered. other factors like renal transporters or polymorphic glucuronidation may explain the great variability in desvenlafaxine pharmacokinetics. background: cardiovascular disorders and medication are highly prevalent in elderly (1). due to age related changes in the body, the elderly are particularly vulnerable to side effects and adverse drug reactions. some psychotropic drugs are linked with reports of cardiac side effects. additionally, some cardiac drugs may also cause psychiatric symptoms. of these, angiotensin converting enzyme inhibitors, beta blockers, methyldopa and calcium channel antagonists can induce or exacerbate symptoms of depression (2) . the aim of this study was to provide information on the concomitant use of cardiovascular drugs among elderly patients who took psychotropic medication. methods: we conducted a single-center, retrospective study between september 2013 and december 2013 using the medical records of elderly patients (≥65 years of age) admitted to basin sitesi polyclinic, izmir ataturk research hospital, turkey. demographic characteristics of patients, diagnoses, prescription drugs were evaluated, and spss 16.0 statistical software was used for data analysis. number, percent, mean and standard deviation were used as descriptive statistics. results: a total of 541 elderly patients with psychiatric disorders were identified. one in four patients receiving psychotropic medication took at least one cardiovascular agent concomitantly (n=135). median age was 72 (min:65, max:98), 84 patients were female (62.2%). according to medical records of 135 patients, the most commonly used drugs were escitalopram, sertraline, mirtazapine, quetiapine, mianserin and risperidone. the proportion of the concomitantly use of cardiovascular drugs was higher among the patients who took more than one psychotropic drug (69.6% vs. %30.4) compared to patients taking psychotropic monotherapy. a higher percentage of women used diuretics (65.4% vs. 33.3%) and angiotensin receptor blockers (36.9% vs. 23.5%) concomitantly with psychotropic drugs when compared to men. the proportion of men using angiotensin-converting enzyme inhibitors and lipid-modifying agents was higher than women (58.8% vs. 38%, 68.6% vs. 20.2%, respectively). conclusions: the world's population is ageing rapidly. according to world health organization, over 20% of elderly suffer from a psychiatric or neurological disorder. our data showed that use of cardiovascular drugs among elderly patients with psychiatric disorders was extensive. the effects and interactions of these drugs should be discussed and carefully evaluated before starting treatment in the elderly. further studies focusing on drug use in elderly will increase the success in geriatric pharmacotherapy. since the adoption of the ich e14 guideline [1], the thorough qt (tqt) study has become a standard element of clinical drug development. however, with the iq/csrc study [2] the ability to detect qtc-prolongations of about 10 ms in a phase i setting has been demonstrated. as a consequence, regulatory agencies have begun to grant waivers for a tqt study based on negative qt findings obtained from first-in-man studies. a concentration-response model is the key tool that gives sufficient power to an analysis based on data from single or multiple ascending dose studies. this power has been investigated in subsampling studies that simulate situations comparable to those encountered in first-in-man studies [3, 4] . other topics to be addressed are the assurance of sufficient quality of the ecgs obtained, in particular in doses that cause adverse reactions, and a replacement for the active control that is part of a tqt study. if a model based statistical analysis is used for confirmatory inference, it must be specified in advance. this pre-specification includes tests to ascertain that the model assumptions are met and alternative methods to be used in case they are violated. in particular linearity and the absence of a hysteresis, i.e. a delay between the drug concentration and the observed qt effect need to be tested. this is an area of active research. in this contribution, i will share current experience from a statistical perspective, both based on real data and on simulated studies. i will also discuss critical points in the design of first-in-man studies that are intended to be used to obtain a waiver for a tqt study. human platelets express the g-protein-coupled angiotensin receptor-like-1 (apj) receptor. apj is activated by apelin, which is produced as pre-apelin and cleaved into several bioactive peptides such as apelin-12, -13 and -17. apelin and apj are expressed in a variety of tissues such as the heart, the vessel wall, several tumor types, and in platelets. to date, there is no description or a suggested function of the apelin/apj system in platelets to date. here, we investigate apj expression and function in human platelets. apelin and apj expression were determined in platelet-rich plasma from healthy donors by immunofluorescence, western blotting and flow cytometry. in a pilot study apelin and platelet apj expression were analyzed in 23 patients with nstemi, 4 stemi patients and 14 controls. here, platelet aggregation was analyzed by light transmission aggregometry (lta); platelet cd62 and apj expression by flow cytometry and circulating plasma apelin by elisa. in resting human platelets, apj receptor expression was observed predominantly in the outer cell membrane, as determined by immunofluorescence staining and flow cytometry. activation with a selective thrombin receptor-activating peptide (ap1) resulted in decreased apj protein levels determined by western blotting in platelet lysates compared to untreated controls. preincubation of platelets with different apelin isoforms for 30 sec to 5 min reduced platelet aggregation in lta studies by up to 20 % for apelin-17. this effect was inhibited by preincubation of the platelets with the enos inhibitor l-name (300 µm), suggesting the involvement of a no-dependent mechanism. in patients with myocardial infarction the expression of platelet apj was significantly reduced compared to the control group (56,84% ± 9,28 % in mi versus 100 % ± 19,35 %in controls; p = 0.029). this reduction in apj expression on platelets was accompanied by decreased plasma levels of apelin-17 in patients with mi (14.95 ± 0.6 pg/ml versus 16.98 ± 0.6 pg/ml; p = 0.035). interestingly, the decreased apj expression on platelets in mi patients significantly correlated inversely with the troponin t plasma levels (r = -0.46; p = 0.03). this may suggest an association of apj expression with lower plasma levels of troponin t and possibly tissue damage. in conclusion, our study shows for the first time the expression of apj and a possible function in human platelets. apj may act as an endogenous inhibitor of platelet aggregation in response to certain apelin isoforms, predominantly apelin-17. upon platelet activation, apj is internalized and surface expression is reduced by about 50 %. in mi patients, plasma levels of apelin-17 and platelet apj expression were reduced. this correlated inversely with troponin t levels. reduced circulating apelin-17 levels and platelet apj expression may be associated or partly account for platelet hyperactivity in mi patients. anticholinergic drug use, m 1 receptor affinity and dementia risk-a pharmacoepidemiological analysis using claims data f. thome background: dementia is characterized by cumulative cognitive decline and progressive inability for independent living. the lack of suitable therapies for terminating the progression of this disease underlines the importance of the detection of risk factors. anticholinergic drugs have been shown to enhance cognitive decline in the elderly. the classification of anticholinergic drugs according to their anticholinergic burden, however, is inconsistent. since cholinergic transmission is mainly mediated by the m 1 muscarinic acetylcholine receptor in the brain, we classified anticholinergic drugs from anticholinergic risk lists according to their affinity for the m 1 receptor subtype and calculated the risk for the onset of incident dementia. methods: our analyses are based on claims data of the public health insurance fund aok. data include information of inpatient and outpatient diagnoses and treatment from 2004 to 2011. inclusion criteria comprised the initial absence of dementia and age of 75 years or older in 2004. anticholinergic drugs were taken from three anticholinergic risk lists. the pdsp-database and literature search were used to define k i -values for the substances. hazard ratios were calculated using time-dependent cox regression including covariates like age, gender, and several comorbidities. results and conclusion: anticholinergic drug exposure increases the risk for dementia. we found that anticholinergics with small k i -values are at a higher risk than those with greater k i -values. furthermore, conventional risk factors for dementia (e.g. age, depression, stroke) could be confirmed. in conclusion, the intake of anticholinergic drugs increases the risk for incident dementia in the elderly. taking into account the m 1 receptor affinity may be a contribution in determining the anticholinergic load in view of the risk for incident dementia. safety signal detection in a large german statutory health insurance databasefirst results of a feasibility assessment f. andersohn 1,2 , s. schmiedl 3, 4 , k. janhsen 3, 5 , p. thuermann 3, 4 , j. walker background: during the last years, approaches to routinely screen health care databases based on electronic medical records or claims to identify drug safety signals were proposed. to evaluate the performance of such methods, reference sets of index drugs have been compiled consisting of (1) drugs with a known association to a certain adverse event (=positive controls) and (2) drugs without any evidence to cause this adverse event (=negative controls). the best possible signal detection method would identify a safety signal for 100% of the positive control drugs, and for none of the negative controls. ryan et al. 2013 developed drug reference sets for four adverse events of interest (acute myocardial infarction=ami, acute kidney injury =aki, acute liver injury=ali, and upper gastrointestinal bleeding=ugib) and have shown feasibility of using these reference sets in us health care databases. if the use of these us specific drug lists for evaluation of signal detection methods is also feasible within german health care databases, is unknown. aims: to evaluate if the drug reference sets developed by ryan et al. (drug saf. 2013 ;36 suppl 1:s33-47) could be used for testing signal detection methods in a large german statutory health insurance database. methods: data source was the health risk institute (hri) database, an anonymized healthcare database with longitudinal health insurance data from approximately six million germans. new users (initiators) of index drugs in 2010 to 2013 were identified and followed-up for one year from their first prescription. exposed person-time to the respective index drug was assessed to estimate for which of these drugs an increase in risk of 50% (relative risk 1.5) compared to the background incidence of the respective adverse event (ami, aki, ali or ugib) could be identified with 80% statistical power. results: from a total of 182 index drugs in the reference sets of ryan et al., 142 (78.0 %) were also available on the german drug market and were used by at least one insurant in the hri database during the study period. a total of 5,485,722 index drug initiators were included in the analysis. for a total of 16 index drugs, a relative risk of 1.5 could be detected with 80% power. the numbers of index drugs for each of the outcomes of interest were: ami (3 positive controls; 6 negative controls); aki (3 positive controls; 1 negative control); ugib (2 positive controls; 1 negative control). as the background incidence of ali was low, no positive or negative control with sufficient power was identified for this outcome. conclusions: using the set of reference drugs proposed by ryan et al., the number of drug-event pairs with 80% power to detect a relative risk of 1.5 was low, despite the magnitude of the database used. this may be attributable to differences of drug exposure in germany and the us. hence, an adaptation of the drug list to the german drug market and consumption data might be relevant for future evaluations of signal detection methods using german databases. correlation of sativex™ doses to steady state concentrations of 11-nor-9-carboxyadministration of the oromucosal spray sativex™ represents a therapy option for treatment of spasticity in patients with multiple sclerosis. sativex™ is an extract containing equal amounts of the cannabis-derived cannabinoids ∆ 9tetrahydrocannabinol (thc) and cannabidiol (cbd). in cases of cannabis abuse a long elimination half life of some thc metabolites is known. therefore, in patients receiving sativex™, the long elimination half life of these metabolites should allow a drug monitoring under conditions of steady state. due to the fact that immunologically based methods for thc determination are very common in medical chemistry, a monitoring might be simply performed even in patients under sativex™ therapy. in a preliminary observational study 11-nor-9-carboxy-∆ 9 -thc (thc-cooh) concentrations were measured with a commercial immunoassay in urine samples of 16 patients with multiple sclerosis obtaining sativex™. in addition, thc-cooh, thc, cbd as well as the hydroxy metabolites of thc and cbd were measured by gc/ms in urine and blood samples. using this analytical technique, only an excessive dosing (as compared to the declaration by the patient) can be detected. as a result of this approach, thc-cooh concentrations determined by the immunoassay were found not to correlate to the daily applicated amount of sativex™ as indicated by the patients (spearman rang order test: p > 0.05). two patients mentioned not to have taken sativex™ on the day the samples had been taken, and one patient predicated an additional cannabis abuse. in three patients the immunological thc-cooh determination was negative or nearly negative. interpretation of the data is hampered by the fact that an incorrect declaration of sativex™ applications by the patients cannot be excluded. introduction: learning analytics seeks to enhance the learning process through systematic measurement and analysis of learning related data to provide informative feedback for students and lecturers. however, which parameters have the best predictive power for academic performance remains to be elucidated. objective: to analyze the potential of different learning analytics parameters to predict exam performance in undergraduate medical education of pharmacology. methods and results: hypertext preprocessor (php) as server-side scripting language was used to develop a learning analytics platform linked to a my structured query language (mysql) database for storage and analysis of data (www.tumanalytics.de). the database consisted of 440 lecturer-authored multiple choice questions that were made available to a cohort of undergraduate medical students enrolled in a pharmacology course (winter term 2014/15) at technische universität münchen (tum). the course consisted of a 28-day teaching period, followed by a 12-day self-study period and a final written exam. students' assessment data of tumanalytics was collected during the self-study period and correlated to the individual exam results in a pseudonymized manner. a total of 224 out of 393 (57%) students participated in the study. the coefficient of multiple correlation (r) was calculated for different parameters in relation to exam results as a measure of predictive power. of different parameters investigated, the total score and the score of the first attempt in tumanalytics had the highest positive correlation with exam performance (abb. 1). no sex-specific differences were observed. summary and conclusion: in this study we systematically investigated the potential of different learning analytics parameters to predict learning outcome and exam performance. total score and score of the first attempt were identified as parameters with the highest predictive power. in conclusion, our study underscores the potential of learning analytics as valuable feedback source in undergraduate medical education of pharmacology. in educational settings, tests (e.g., written or oral exams) are usually considered devices of assessment. however, a recent and intriguing line of evidence from basic cognitive psychological research suggests that tests may not only help to assess what students know, but may also help to improve the learning and long-term retention of information. the goal of the present study was to apply such test-enhanced learning to pharmacological teaching. after the last lecture of a pharmacology class (n=194 3 rd -year medical students, n=213 4 th -year medical students: basic or clinical pharmacology, respectively), one week prior to the final exam, students were given the opportunity to voluntarily participate in online exam. because pilot work from previous semesters had revealed relatively low levels of participation in such formative exams, students were offered bonus points for (successful) participation as an incentive. the online exam consisted of 60 items (i.e., selected pieces of information from the lectures and seminars) and was provided on the e-learning platform ilias. twenty of the 60 items were presented as statements for restudy, 20 items were tested using single-choice questions, and 20 items were tested using short-answer questions. randomly a third of the students were assigned to different sets of questions. the summative final written exam for each group consisted of 30 single-choice questions, 15 questions of which had not been used before (as a standard of comparison). the remaining 15 questions of the final exam were taken from the previous online exam, but were slightly reworded to avoid ceiling effects. each of 5 of these reworded 15 questions from the final exam corresponded to restudy items, single-choice items, and short-answer items from the online exam, respectively. the main points of interest were (i) whether the re-processing (rewording and asking for transfer of knowledge) of information in the online exam affected participants' performance on the final exam, and (ii) whether any effect depended on the specific type of re-processing (restudy vs. single-choice test vs. shortanswer test). if previous findings from basic cognitive psychological research on testenhanced learning can be generalized to more applied settings and educationally more relevant materials such as pharmacological information, students' performance in the final exam should be better for questions corresponding to previously tested items than for questions corresponding to previously restudied items. moreover, if more difficult tests lead to more test-enhanced learning than less difficult tests, as is suggested by recent findings from cognitive psychological research, performance for questions corresponding to (supposedly more difficult) short-answer items should even exceed that for questions corresponding to (supposedly less difficult) single-choice items. the present findings bear direct implications for educational practice. safe and rational prescribing is one of future physicians' key skills [1] . in order to address the persisting prescribing deficiencies [2] , we set out to develop a learning tool for pharmacotherapies of the most important diseases worldwide. the format, scope, information architecture, and functionalities of the app were identified through assessment of existing apps, literature analysis, app simulation-based student surveys, and expert advice. a fully functional offline app format for smartphones was selected based on the trends in using digital technologies for educational purposes [3] and on the unreliable internet and power availability in many learning settings. a relational database based on semantic relationships was chosen to minimize information redundancy and to enable the retrieval of drug-related information in the context of mechanisms, contra-and indications, adverse drug reactions, interactions, and common prescribing situations. the usability was optimized using a simulation of the app evaluated by medical students from germany and tanzania, and by experts. a list of 67 indications was assembled beginning with disease burden data for the seven who world bank regions. each disease accounts for at least 0.3% of life years lost due to premature death or lived with ill-health or disability (daly) in at least one region. the list was further complemented according to expert recommendations. therapeutic recommendations are based on current guidelines, considering cheaper treatment alternatives provided in the who list of essential medicines. a novel dual-scale classification system lists drug mechanisms according to the affected physiological process and to the resulting therapeutic effect [4] . contraindications, adverse drug reactions, and interactions were compiled using drug monographs of the european medical agency, the us food and drug administration, and health canada. unexpectedly, we found significant differences among these sources in respect of adverse drug reactions. this necessitated the ongoing verification through surveying general practitioners and specialists in internal medicine. during the dgpt meeting we will present the results of testing of the cardiovascular section comprising 8 indications, 28 drugs representing 25 mechanisms, and up to 120 adverse drug reactions. the european certified pharmacologists (eucp) programme was lauched in july 2014 by the federation of european pharmacological societies with the intention to identify experts in the field of pharmacology whose competency profile, in addition to their personal specialised scientific expertise, covers expert knowledge in all major fields of the discipline. seventeen ephar member societies have declared their active participation in the eucp programme so far (austria, croatia, czech republic, finland, france, germany, greece, hungary, italy, the netherlands, norway, poland, portugal, serbia, slovenia, spain, turkey) . eacpt, the european association of clinical pharmacology and therapeutics, has also recently decided to participate in the eucp programme. national programmes must meet all requirements of the eucp guidelines including a clear catalogue of criteria with respect to knowledge, practical awareness and skills, as well as general rules including rules for final assessment of candidates. such programmes may be based on existing diplomas or training schemes or may consist of a set of rules how applicants may submit credentials for their expertise with respect to the eucp criteria. so far, three ephar member societies have submitted a national eucp program: austria, italy and the netherlands. the programmes differ in structure and reflect the flexibility of the eucp programme with respect to the respective national conditions. while the italian programme is based on a catalogue of criteria where applicants have to certify and document their expertise on the basis of this catalogue and the dutch program is based on a structured phd training course, the eucp scheme submitted by the austrian pharmacological society aphar is based on the legally regulated diploma medical specialist (facharzt) in pharmacology and toxicology (aphar also plans to submit separate regulations for specialists in clinical pharmacology and for nonmedically qualified pharmacologists). the aphar eucp scheme has been approved by the eucp committee in november 2015 and its regulations are available on the eucp website (www.eucp-certification.org). the differently structured programs of the italian and dutch pharmacological societies will also be available at this website, once approved by the eucp committee and may thus serve as 'case studies' for other ephar and eacpt member societies wishing to take part in the eucp programme. introduction: the outstanding importance of pharmacovigilance (pv) for the safe use of medicines has increasingly been recognised during recent years. the multidisciplinary character of pv requires know-how in topics as different as pharmacology, clinical medicine, pharmacoepidemiology, information technology, pharmaceutical manufacturing, legal aspects, public health policies, and medical traditions in different regions of the world. in this complex situation there is a growing need for pv capacity building, in particular by professional training through high quality pv courses with different focuses and different levels of detailing. against this background, the world health organization (who) and the international society of pharmacovigilance (isop) have co-operated to create a curriculum for teaching pv which can be used for a wide range of audiences and in very different settings and situations. the purpose was to provide an inventory and systematically structured overview of pv including recent developments of topics like pharmacogenomics, consumer reporting of adrs, risk management and who-led international projects, and to propose a range of tasks for practical training. we made use of several relevant already existing packages of pv topics and concepts of pv teaching from national and international institutions. we also drew from extensive printed material as well as comprehensive reviews, textbooks and guidelines developed by international organisations which are often available online. results: the curriculum includes a main component consisting of a major part for theoretical lecture-based training and a minor component with suggestions for hands-on exercises. the theoretical part has a three-level hierarchical and modular structure with evenly divided tiers. there are 15 chapters. each of them is divided into four sections and each section into four to six sub-sections. the practical part consists of twelve times three or four proposals for practical tasks which are related to the theoretical lectures. since its launch in 2014 1 it has successfully been used in several international courses. currently a pilot project is under way to explore its use for 'crowd sourcing': it is placed on the isop homepage with a programme allowing for institutions or persons experienced in pv teaching to upload any relevant presentations they may have with a link to related chapters, sections or subsections. these presentations will be offered for downloading by interested users. the curriculum provides a comprehensive coverage of almost all areas of pv. the structure and content allows almost every kind of focusing on specific issues and going into depth, while maintaining the overall context. it offers opportunities of tailoring courses specifically to the needs of different audiences and can be applied to various forms of training, such as broad, comprehensive and intensive courses, short overviews or focuses on specific narrow topics in perspective. according to the reach regulation (ec) no. 1907/2006 chemicals produced, marketed or used within the european union have to undergo a registration process, wherein the registrants have to provide information on hazard and potential risks presented by the substances. however, the standard information requirements defined in annexes vii to x of the regulation might be waived or adapted by the registrants if adequate documentation and justification according to criteria specified in annexes vii to xi are provided. to evaluate the data availability in registration dossiers of high tonnage substances (above 1000 tpa) and their compliance with the reach regulation, the federal institute for risk assessment (bfr) in cooperation with the federal environment agency (uba) developed a systematic web-based scheme. in total, 1932 dossiers were checked for selected human health and environmental endpoints such as repeated dose toxicity, genetic toxicity and ecotoxicity. a remarkable high rate, 43% to 82 % depending on the endpoint, of the evaluated dossiers included waiving or adaptations from the standard information requirements. therefore, those dossiers were not concluded, but categorised as 'complex' (springer et al., 2015) . the use of waiving and adaptations in 'complex' endpoints were part of a follow-up project. herein, it was evaluated whether the given justifications were in accordance with the criteria set out in the respective reach annexes. the results will show the frequency and pattern of waiving/adaptation approaches for the human health as well as the environmental endpoints. besides this general overview, specific problems regarding the application of the reach regulation were identified and their significance with regard to remaining data gaps will be discussed. the german commission for the investigation of health hazards of chemical compounds in the work area has re-evaluated dimethylformamide, and classified it in the carcinogen category 4. this category is for chemicals with carcinogenic potential for which a non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the mak and bat values are observed. under these conditions no contribution to human cancer risk is expected. dmf was identified as a substance of very high concern by european commission. the amount of dmf manufactured and/or imported into the eu is, in the range of 10000-100 000 t/y. n,n-dimethylformamide is a hepatotoxin in humans and rats. the carcinogenicity studies in both mouse and rat were conducted with test material of an acceptable purity and physical form. the critical study involved administration of dmf via inhalation, which is relevant to human exposure. there is conclusive evidence that dmf induces significant increases of hepatocellular carcinomas in rats after exposure to 800 ml/m³ and in mice in response to 200 ml/m³ and higher. several in vitro and in vivo studies have indicated that dmf is not genotoxic. the results of the long-term studies reveal that the tumors develop in the liver only after chronic toxic inflammatory and degenerative changes have developed in this organ. the commission concluded that the tumors are a result of chronic liver damage, occurring at high exposure concentrations. the available evidence therefore suggests that there is a threshold dose for the carcinogenic effects of dmf. accordingly, dmf was classified in carcinogen category 4 with a mak-value of 5 ml/m³, an exposure concentration which does not induces liver toxicity and as a consequence is not associated with an increased cancer risk. today, a large majority of people is constantly exposed to electromagnetic radiation. many studies have been performed to investigate whether this type of radiation has a potential to affect biological systems at low intensity levels. even though no complete consensus has been reached so far in this issue, most of the investigations do not indicate a harmful potential of this radiation. two questions remain open until today, i. e. long-term effects and specific effects on children. it has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. these absorptions occasionally exceed the recommended safety limits. the aim of this study was to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. as biological systems, two cell types were investigated, hl-60 cells as an established cell line, and human hematopoietic stem cells. the radiation was modulated according to the two major technologies, gsm (900 mhz) and umts (1950 mhz). additionally, lte (2.535 mhz) modulation was applied because this technology is used worldwide already but has not been studied sufficiently. cells were exposed for a short and a long period and with different intensities ranging from 0 to 4 w/kg. studied endpoints included oxidative stress, differentiation, dna repair, cell cycle, dna damage, histone acetylation, and apoptosis. appropriate negative and positive controls were included and three independent replicate experiments were performed. exposure to radiofrequency radiation did not induce any alterations of cell functions, measured as oxidative stress and cell cycle. cell death in the form of apoptosis was not observed. primary dna damage was not induced and dna repair capacity for nucleotide excision repair was not changed. epigenetic effects (measured as histone acetylation) were not observed. finally, differentiation was not affected. the effect of treatment with various chemicals as positive controls was different in the two cell types. all in all, mobile phone radiation did not induce effects on human hematopoietic cells. in 2014 who published guidance on evaluating and expressing uncertainties in human health hazard characterisation (hc). in this approach, the outcome of hc is expressed as an interval or distribution rather than a "traditional" deterministic point estimate, such as a reference dose (rfd), thereby communicating potential uncertainties more clearly. risk management protection goals, such as the acceptable magnitude of effect (m) and incidence (i) in the population, are made explicit quantitatively along with the confidence with which they are achieved, e.g. by an rfd. specifically, the goal of this approach to hc is to estimate the "hd m i" , i.e. the "true" human dose associated with m and i (e.g. body weight decreased by ≥ 10% (m) in 5% (i) of the population). if uncertainties are expressed by providing estimates of the hd m i as confidence intervals or uncertainty distributions, both the "degree of uncertainty" (ratio of upper and lower limit of the interval/relevant distribution segment) and the "coverage" (statistical confidence) associated with a given rfd value can be characterised. alternatively, one may start from a chosen coverage and calculate the associated "probabilistic rfd". uncertainty in each hc aspect, e.g. inter-/intraspecies or time extrapolation, can be characterised by a "generic default uncertainty distribution" which has been derived from historical data, but may be replaced by a substance-or effect-specific distribution (analogous to a chemical-specific adjustment factor in the "traditional" deterministic approach), where available. the uncertainty distributions for the individual hc aspects can then be combined into an overall uncertainty interval/distribution in (1) a simple non-probabilistic way, (2) a more refined "approximate probabilistic analysis" (aproba, a free spreadsheet tool for easy implementation also by non-statisticians is available from the who website), or (3) a fully probabilistic monte carlo analysis. the hc aspects contributing most to overall uncertainty are also identified and may be prioritised for refinement in a next assessment tier. the who approach uses a tiered strategy which may start with evaluating the uncertainties in the outcome of a "traditional" deterministic hc. in this way it represents a unified framework integrating deterministic and probabilistic hc methodologies. moreover, the concept can be easily combined with exposure uncertainty assessment. risk managers may use the additional information in better weighing potential health effects against other interests. when they consider the overall uncertainty larger than desirable in view of the problem formulation, they may decide to ask for a more refined (higher tier) assessment. if all possibilities for refinement are exhausted, the new approach can also aid in the selection of new data which might need to be generated. due to a constant improvement in analytical methods an increasing number of substances are found in drinking water. the joint project toxbox aimed for the development of a reliable test battery, allowing for a rapid evaluation of single substances in water. eleven partners either from the research (nine) or the business sector (2) formed the project. the attention was focused on genotoxic, neurotoxic and endocrine effects, which are considered to be of most concern to the consumer. by the end of the project a set of guidelines is published that describes the analytical methods in detail. the project was based on a theoretical concept, called "health-related indicator value" (hriv), which was developed by the german federal environment agency (uba) for the assessment of substances with incomplete toxicological data. depending on the type of effect a hriv between 0.1 and 3.0 µg/laws derived for the substance which had to be evaluated. during the years an increasing amount of substances as well as an increase in finds was observed in drinking water. this called for the creation of an evaluation scheme that offers rapid and at the same time reliable evaluations of chemicals for which there are no data available. the concept is in accordance with tox21, which envisages the trustworthy evaluation of relevant endpoints by two or three in vitro assays. in the context of toxbox this was provided for the endpoints genotoxicity, neurotoxicity and endocrine effects. in all cases a hierarchic strategy is applied that enables a first assessment via relatively simple test assays and only when these test give a hint towards an effective more elaborate techniques are applied for a final assessment. the ames test and micronucleus assay in combination with the umu test will form the panel for genotoxicity testing. neurotoxicity will be assessed by comparing necrotic and apoptotic effects as well as the development of reactive oxygen species in human nervous cell with human liver cells. additionally neuron specific assay like the neurite outgrowth test are performed. this is complemented by measuring the activity of acetyl choline esterase activity and the development of the side line organ in zebra fish (danio rerio). the test battery for endocrine effects consists of hormone specific reporter gene s81 assays in addition to the h295r assay. when necessary a reproduction assay in the mud snail potamopyrgus antipodarum is carried out. during the project some 60 substances were evaluated. this allowed for the development of a reliable test strategy. currently the guidelines for performing the required tests are in the making. metabolomics has gained increasing interest over the last years with numerous possible applications ranging from strain optimization for industrial production over drug discovery to improved toxicity testing. however, regulatory acceptance of this promising approach is still not reached, mostly because standardization and evaluation of reproducibility are still mostly lacking. the metamap®tox database has been developed by basf over the last ten years containing toxicity and metabolome profiles of more than 750 different compounds. to ensure maximum reliability, data was gained from plasma samples of highly standardized 4 week rat studies. animal maintenance and treatment, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized including thorough documentation, the compliance with sops and safe data storage. data from more than 80 control groups with each 10 males and females were analyzed to assess variability. an in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. after artificially splitting the groups of 10 control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was located to the left of the exact (gaussian) center, but tailed off to the right more than expected under the normal distribution. from this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. during the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (kamp et al., 2012) . comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = 0.1, the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. the high quality standards applied as well as the examination of control data increase the robustness of this approach, going also hand in hand with improved data quality. this significantly facilitates decision making based on the gained data. due to these improvements a new level of transparency is reached, which might allow inclusion of metabolome data in a regulatory environment. hydroxycitric acid (hca) is a fruit acid naturally occurring in fruits of the tropical plant garcinia cambogia. a number of dietary supplements intended for weight loss contain hca, which is added in form of g. cambogia extracts. the composition of these extracts is often not clearly specified. health concerns about safety of the hca-containing supplements have been raised, based on results from animal studies, which observed toxic effects on the testis and on spermatogenesis after administration of preparations containing high hca doses. in the current risk assessment, the possible health risks associated with consumption of hca-containing dietary supplements (hca doses of approximately 1000 to 3000 mg per day) were evaluated based on relevant animal and human studies with the focus on testicular toxicity as a critical endpoint. in several published animal studies, repeated (short-term or subchronic) ingestion of certain hca-preparations (g. cambogia-extract or ca 2+ -hca salt) induced testicular atrophy (i. e. atrophy of seminiferous tubules, degenerative changes of sertoli cells at histological examination) and impaired spermatogenesis (i. e. decreased sperm counts) in male rats at high doses (noael and loael of 389 and 778 mg hca/kg body weight & day, respectively). animal studies with other hca-preparations (ca 2+/ k + -hca salt) found no such effects at the highest hca-doses tested (noael: 610,8 mg hca/kg bwt & day). human intervention studies which addressed the safety of hca in healthy test persons reported no substancespecific adverse effects after ingestion of hca doses up to 3000 mg per day over the period of up to 12 weeks. however, the question of possible adverse effects of hca on the human testes was not adequately addressed in studies with human volunteers. in a single clinical study with 24 male test subjects, no significant changes in endocrinologically relevant parameters such as serum inhibin b or fsh were observed after consumption of 3000 mg of hca for 12 weeks. however, no investigations of direct parameters that might inform on potential effects on spermatogenesis, such as sperm quality and sperm count, were conducted in this study. considering the serious adverse effects on the testes observed in several animal studies as well as in view of lack of the adequate human data on the safety of the long-term use of hca-preparations, it is concluded that knowledge gaps and substantial uncertainties exist regarding the safety with respect to human health of high amounts of hca found in commercially available food supplements, particularly with regard to the human male reproductive system. a critical look on the passing-bablok-regression b. mayer 1 1 universität ulm, institut für epidemiologie und medizinische biometrie, ulm, germany background: the passing-bablok (pb)-regression is a commonly used approach to prove the equality of different analytical methods when studying quantitative laboratory data. it is based on the assumption that the measurements of two methods are linearly related. if then one method is regressed onto the other and the respective confidence intervals of the intercept and the slope include 0 and 1, respectively, it is assumed to have a proof of methods equality. however, this conclusion is problematic in respect of an essential principle of statistical hypothesis testing. methods: in this talk the general idea behind the pb-regression is discussed critically. although the method makes use of confidence intervals a decision is made, which is why it is important to discuss how the results of a statistical hypothesis test have to be interpreted. moreover, alternative statistical approaches to investigate agreement in biometrical practice are pointed out by means of a practical example and their advantages and limitations are addressed. results: all approaches applied to a sample data set led to the same conclusions. demonstrating methods equality though necessitates an a-priori definition of an appropriate equivalence margin. conclusion: the pb-regression may give useful advice when comparing two measurement methods towards equality. however, its results are statistically inconclusive, since the pb-method does not follow the principle of equivalence testing. alternative measures of agreement should be applied instead to ensure results which are not attackable and serve as a statistical proof. insulin is an important parameter both in toxicology (toxicity to the endocrine pancreas) and pharmacology (models of diabetes and metabolic syndrome). currently available elisa and ria methodologies for insulin often require up to 100 µl plasma or serum for a single measurement. in order to meet the general trend to include more relevant parameters in animal studies and restrictions through animal welfare requirements to limit the volume of interim blood draws we explored the rat/mouse insulin singleplex assay of meso scale discovery (msd) as an alternate assay consuming only 10 µl serum or plasma or less for a single measurement. the assay is a sandwich immunoassay, whereby insulin in the sample binds to the capture antibody immobilized on the working electrode surface at the bottom of each well and recruitment of the labeled detection antibody (anti-insulin labeled with electrochemiluminescent compound, msd sulfo-tag™ label) by bound analyte completes the sandwich. voltage applied to the plate electrodes then causes the label bound to the electrode surface to emit light the intensity of which is a quantitative measure of insulin. the msd insulin assay was characterized by a robust calibration and only small variations within repeated measurements. the assay presented a broad dynamic range and differences in insulin levels of normal and rats suffering from metabolic syndrome could readily be demonstrated. furthermore, the high sensitivity may even allow the use of smaller sample volumes. these features render this assay an attractive alternative for the measurement of insulin. the lack of corresponding quality control samples for internal quality control may be considered as a relative drawback. however, the cross reactivity of the assay with human insulin provides the opportunity to use qcs designed for human assays and to possibly participate in ring trials for human insulin for external quality control if needed. surfactants are main constituents of different consumer products, e.g. detergents or cosmetic cleansing products. since surfactants show an intrinsic skin irritation potential, dilutions are used in the final products to avoid adverse effects like irritant contact dermatitis from product use. in addition, mixtures of different surfactants are typically formulated, as it is a long-standing experience that those mixtures exhibit much lower acute irritation potential than expected from the mere summation of their individual irritation potential, an effect coined surfactant antagonism. only few studies were performed to gain a more fundamental understanding of the effect, and it's mechanistic basis remains unclear. however, a thorough understanding of the surfactant antagonism is not only of value for the formulation of products that are considered 'mild to the skin'. it is also important for the classification of products according to the clp regulation in cases when data of the mixtures is missing, because summation of the ingredients' irritating effects usually results in over-classification as skin irritant. due to the progress in the development of alternatives to animal testing, different in vitro methods have become available to determine skin irritating properties of substances. methods like the oecd tg 431 and 439 especially aim at deriving a classification for skin irritation/corrosion effects according to the clp regulation. however, even though these methods became the preferred test methods for skin irritation testing, to our knowledge hitherto isolated investigations on the surfactant antagonism were only performed either by human patch test studies or by non-standard in vitro assays. in this study, the irritation potential of binary mixtures of sodium dodecylsulfate (sds), linear alkylbezene sulfate (las), cocamidopropyl betaine (cabp) and alkylpolyglucosid (apg) compared to the single compounds was investigated using open source reconstructed epidermis (os-rep) models. combinations of sds or las with cabp and apg, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants, even though the total surfactant concentration was higher in the mixtures. in addition, the effect of surfactant antagonism was also observed in a mixture of cabp and apg. the reduced irritation potential of mixed surfactants came along with both reduced skin penetration of fluorescein and reduced release of ldh. since no surfactant antagonism is observed in monolayer cultures of keratinocytes that were exposed to mixtures of surfactants, it is assumed that keratinocytes in the viable parts of the reconstructed epidermis are promptly damaged by the surfactants once the model's barrier is destroyed. hence, surfactant antagonism appears to be primarily driven by the mixture's lower ability to damage the skin model's barrier. the micronucleus (mn) test is a reliable method for the detection of cytogenetic damage in proliferating cells. in recent years, substantial progress has been made on automated, thus faster and more objective scoring of mn test samples, i.e., methods based on flow cytometry. the aim of the present study was to use the adherently growing human bladder cancer cell line rt4 to carry out a comparison between traditional (fluorescence microscopy) and automated (flow cytometry) mn scoring. for this purpose, different substances which are known to be positive controls were used. rt4 cells were either continuously incubated for 72 h (approximately 1.5 cell cycles duration) with methyl methanesulfonate (mms; 50-200 µm), benzo[a]pyrene (b[a]p; 0.1-1 µm), vincristine (3-20 nm) , and colcemid (10-20 nm) or cells were irradiated with xrays (0.5-2 sv) and then cultured for 72 h. for standard mn scoring, cells were harvested, subjected to hypotonic treatment, fixed with methanol/acetic acid, placed on glass slides, stained with acridine orange and observed by fluorescence microscopy. for the flow cytometric method, harvested cells were stained in two sequential steps. intact cells were subjected to ethidium monazide bromide followed by photoactivation (75 w, 20 min) to label dead or dying cells. then, cells were lysed and stained with sytox green for a pan-dna labelling and analyzed on a flow cytometer. both, chemically-and radiation-induced treatment led to a dose-dependent induction of mn when evaluated by fluorescence microscopy. when the flow cytometry-based method was applied, clearly positive results including a dose-dependent induction of mn, however, were obtained only for 3 out of the 5 treatments (vincristine, colcemid and x-rays); whereas, treatment with mms and b[a]p led to only minor increases in relative mn frequencies (≤2-fold), even at the maximum concentrations. in summary, flow cytometry-based mn scoring has been successfully applied in rt4 cells. however, our initial results suggest that flow cytometry-based mn scoring is less sensitive than microscopic scoring when rt4 cells are used. so far, only few adherently growing cell lines have been applied to flow cytometry-based mn scoring. further substances (positive and negative controls) and possibly other adherent cell lines need to be tested to expand our knowledge on the effectiveness of automated mn scoring in vitro and compared to traditional approaches. background: the platinating agent cisplatin is commonly used in the therapy of various types of solid tumors, especially urogenital cancers. its anticancer efficacy largely depends on the formation of bivalent dna intrastrand crosslinks, which impair dna replication and transcription. these crosslinks stimulate mechanisms of the dna damage response (ddr), thereby triggering checkpoint activation, gene expression and cell death. the clinically most relevant adverse effect associated with cisplatin treatment is nephrotoxicity, which mainly results from damaged tubular cells. here, we analyzed the influence of the hmg-coa-reductase inhibitor lovastatin on the cisplatin-induced geno-and cytotoxicity in the rat renal proximal tubular epithelial cell line nrk-52e. methods: cell viability was determined by using the alamar blue assay, as well as by electrical impedance measurements via the icelligence system. alterations in cell cycle progression were assayed by flow cytometric analysis. the formation of pt-(gpg) intrastrand crosslinks was determined via southwestern blot. the amount of dna double-strand breaks (dsbs) was quantified by measuring the level of s139 phosphorylated h2ax (γh2ax) via immunocytochemistry as well as by western blot. additionally, neutral and alkaline comet assays were performed to determine the amount of dna single-and dna double-strand breaks. mechanisms of the ddr were analyzed by western blot as well as by quantitative real-time pcr. results: the data show that pretreatment of nrk-52e cells with a subtoxic dose of lovastatin reduced the cytotoxicity evoked by high doses of cisplatin by protection from cisplatin-stimulated apoptotic cell death. moreover, lovastatin had extensive inhibitory effects on cisplatin-induced ddr, as reflected on the level of p-atm, p-p53, p-chk1, p-chk2 and p-kap1. furthermore, activation of mitogen-activated kinases (mapks) was also reduced. the lovastatin-mediated mitigation of cisplatin-induced ddr was independent of the initial formation of dsbs as well as of pt-(gpg) intrastrand crosslinks. lovastatin protects nrk-52e cells from cisplatin-induced cytotoxicity by interfering with proapoptotic mechanisms of the ddr independently from initial dna damage formation. with respect to the clinic, the data indicate that lovastatin might be useful to mitigate cisplatin-induced nephrotoxicity. the influence of oxidant tert-butylhydrochinone (tbhq) on endothelial cell migration in wrn-deficient cells k. laarmann 1 , g. fritz 1 1 institut für toxikologie, düsseldorf, germany introduction: wrn is a dna helicase and possesses a 3´-5´exonuclease and atpase activity as well as a single strand annealing activity. it is involved in dna repair, by interacting with proteins of base excision repair (ber) and nucleotide excision repair (ner). defects of wrn are marked by genome instability which, in turn, is caused by defects in dna damage repair. patients with a mutation in the wrn gene show premature aging and early mortality. the latter is mainly caused by arteriosclerosis. furthermore, wrn participates in the regulation of genotoxic stress responses stimulated by reactive oxygen species (ros) and alkylating agents. the aim of this study was (i) to investigate whether endothelial cell migration and adhesion were effected by sub-toxic (ic 10 ) and moderate toxic (ic 40 ) concentrations of the oxidant tertbutylhydrochinone (tbhq) and (ii) whether wrn influences migration and adhesion in the presence or absence of tbhq. methods: endothelial-like ea.hy926 cells were treated with different concentrations of the redox cycling and thus ros producing oxidant tbhq. viability was measured by the alamar blue assay. ic 10 and ic 40 were determined after 24h permanent treatment. to investigate the influence of wrn on endothelial cell migration and adhesion, a wrn knock-down was performed in ea.hy926 cells using rna interference. to measure migration, a confluent cell monolayer was scratched using a pipet tip, 24h after permanent tbhq treatment. pictures were taken at the time points 0h, 4h and 8h after performing the scratch. the non-closed area was measured. in a second part, adhesion of the calcein-labeled colon adenocarcinoma ht-29 cell line on the ea.hy926 monolayer was investigated. wrn-deficient or non-deficient cells were treated with 100 µm and 160 µm tbhq or with tnfα. results: for ea.hy926 cells, 100 µm and 160 µm tbhq were determined as ic 10 and ic 40 , respectively. performing the migration assay, ea.hy926 cells showed 75% gap closure, whereas wrn-deficient cells showed a closure of only 35% after 8h. the gap was closed of 65% and 55% after 100 µm and 160 µm tbhq treatment. in wrndeficient cells no remarkable effect on migration was observed after 100 µm tbhq treatment, whereas the treatment with 160 µm tbhq showed a slight decrease in migration of about 10% compared to wrn-deficient cells. no effect on adhesion was observed after tnfα treatment. after 160 µm tbhq treatment a slight increase of adhesion was detected in ea.hy926 cells. the influence of moderate tbhq concentration on adhesion was reduced in the absence of wrn. conclusion: wrn influences endothelial cell migration. in contrast to wild-type ea.hy926 cells, no significant effect of tbhq was observed on migration of wrndeficient cells. furthermore, the moderate toxic concentration of tbhq showed slightly increased ht-29 adhesion to ea.hy926, which was not found in wrn-deficient cells. outlook: in forthcoming studies we analyse the effect of alkylating agents on migration and adhesion. data will be presented and discussed. the aim of the present work was to compare the sensitivity of leukemia cell lines (hl60, jurkat and tk6) and hematopoietic stem cells with regard to the response to genotoxic agents. chromosomal damage was analyzed by evaluation of the micronucleus frequency. furthermore, changes in the proliferation index and the frequencies of apoptotic and mitotic cells were assessed. several cytostatic drugs with different mechanisms of action were used as genotoxic agents. doxorubicin was used as an intercalator, radical producer and topoisomerase ii inhibitor. also, the effects of vinblastine, a mitosis-inhibiting drug and of methyl methanesulfonate, which forms dna-adducts and stalls replication forks, were analyzed. in general, a difference in sensitivity between the different substances was observed. with regard to the formation of micronuclei after treatment with doxorubicin, jurkat and tk6 cells showed similar increasing trends, whereas hl60 cells showed a much higher increase in micronucleus frequency. a clear decrease in proliferation and the frequency of mitotic cells was observed at the highest concentration (100 nm doxorubicin) investigated, and only a slight increase in the number of apoptotic cells could be shown. the biggest differences in formation of micronuclei could be detected after treatment with vinblastine. hl60 cells showed only a slight increase of micronuclei, but the effect on jurkat cells was stronger. the highest micronucleus frequency after vinblastine treatment was detectable for the tk6 cells. the results for the highest investigated concentrations (31.6 nm and 100 nm vinblastine) showed a significant reduction of the proliferation index. this effect is reflected by the increasing numbers of apoptotic cells in all cell lines. the results for methyl methanesulfonate demonstrated only a small increase in micronucleus formation for the jurkat cells, but higher values for the tk6 cells. in contrast the hl60 cells did not lead to a concentration-dependent effect with methyl methanesulfonate. these results are complemented by preliminary findings in hematopoietic stem cells at selected compound concentrations. the different results between the leukemia cell lines and the stem cells might possibly originate from the different p53 status of hl60 (null), jurkat (multiple mutations), tk6 (wild type) and hematopoietic stem cells (wild type). this difference might also cause differences in cell cycle control or repair mechanisms, and needs further investigations. hyperinsulinemia is thought to enhance cancer risk. a possible mechanism is induction of oxidative stress and dna damage by insulin, here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. the rational for this were reported antioxidative properties of metformin and the aim to gain further insights into mechanisms responsible for protecting the genome from insulin mediated oxidative stress and damage. comet assay, micronucleus frequency test and a mammalian gene mutation assay were used to evaluate the dna damage produced by insulin alone or in combination with metformin. for analysis of antioxidant activity, oxidative stress and mitochondrial disturbances, the cell-free frap assay, the superoxide-sensitive dye dihydroethidium and the mitochondrial membrane potential-sensitive dye jc-1 were applied. accumulation of p53 and pakt were analysed. as an in vivo model, hyperinsulinemic zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic euglycemic clamp, were treated with metformin. in the rat kidney samples, dhe staining, p53 and pakt analysis, and quantification of the oxidized dna base 8-oxodg was performed. metformin did not show intrinsic antioxidant activity in the cell free assay, but protected cultured cells from insulin mediated oxidative stress, dna damage and mutation. treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. metformin may protect patients from genomic damage induced by elevated insulin levels. this may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia. the human skin is the primary barrier against environmental and chemical impacts. as such it shields us against a plethora of xenobiotics such as potentially carcinogenic polycyclic aromatic hydrocarbons (pahs). at the same time it is the second most densely populated organ, harbouring more than 1000 bacterial species and population densities of up to 10 7 cfu per cm 2 . yet little is known about this microbiome's potential to metabolise and toxify pahs such as benzo[a]pyrene (b[a]p). previous work at the bfr showed that degradation of b[a]p and other pahs is a universal feature of the skin's microbiome (sowada et al., 2014) . the corresponding metabolites only partly overlap with those known from eukaryotic metabolism and possess cytotoxic as well as genotoxic properties. excretion of these metabolites will lead to exposure times of 20-30 hours or longer for full and partial metabolisers, respectively. while in vitro studies show the corresponding substances to exert their effects synergistically, an assessment of their potential impact on human carcinogenesis is pending. one obvious mode of action would be direct genotoxicity. however, another option is interference with uv-damage repair. ultraviolet radiation (uvr) from sunlight is regarded the main causative factor for the induction of skin cancer. it induces two of the most abundant mutagenic and cytotoxic dna lesions, that is cyclobutane-pyrimidine dimers (cpds) and 6-4 photoproducts (6-4pps). these lesions are repaired primarily by nucleotide excision repair (ner), a system that is also responsible for the removal of pah-derived dna adducts. we therefore wanted to know whether and to what extent bacterial b[a]p metabolites have the capacity to interfere with ner, potentially contributing to uv-induced dna-damage. to investigate this selected genotoxic metabolites were examined for their potential to affect the dna repair capacity of skin cells (hacat). following treatment with uva/b and bacterial b[a]p-metabolites the skin's repair capacity was assessed using a modified comet-assay. ionizing radiation (ir) is a well-established model to induce dna double-strand breaks (dna-dsbs), but it also generates a broad range of other dna lesions including dna single-strand breaks as well as oxidative dna base modifications. furthermore, ir is able to modify membrane components and triggers the activation of epidermal growth factor receptor. a more specific dsb-inducer is cytolethal distending toxin (cdt), which is produced by a variety of gram-negative bacteria and harbours an intrinsic dnase-like endonuclease activity [1] . dsbs are potent cytotoxic lesions and promote genomic instability, e.g. by formation of chromosomal aberrations. a cellular mechanism to prevent genomic instability and maintain cell homeostasis could be autophagy. this process is highly regulated involving the lysosomal degradation of damaged organelles and proteins. here, we study autophagy induction following dsb generation in human colorectal cancer cells as well as in primary human colonic epithelial cells (hcec) and analyzed regulatory mechanisms. first, the autophagy-specific marker lc3b was shown to increase in a dose-and time-dependent manner after treatment with both cdt and ir as assessed by confocal immunofluorescence microscopy and western blot analysis in hct116. similar results were obtained in sw48 and hcec cells via western blot. these findings are in agreement with the enhanced formation of autophagosomes and the dose-dependent decrease of the autophagy substrate p62 as observed by flow cytometry and western blot analysis in hct116, sw48 and hcec. cdt-and irinduced autophagy rates in hct116 increased over time correlating well with the dsb induction. importantly, a dnasei-defective mutant of cdt did neither cause dsbs nor induce autophagy. additionally, the time-dependent accumulation of the lysosomal associated membrane protein 1 (lamp-1) was observed by confocal immunofluorescence microscopy. dsb-induced autophagy was blocked by chemical inhibitors. next, we showed that both ir and, to a lesser degree, cdt induce the phosphorylation of akt at ser473. pharmacological inhibition of akt in hct116 cells enhanced the cdt-and ir-induced autophagy shown by accumulation of lc3b and lamp1 after 48 h and increased autophagosome formation. upregulation of dsbinduced autophagy by akt inhibition resulted in a decreased cytotoxicity after 72 h and significantly lower apoptosis/necrosis rates after 48 h, which were determined by mts cell viability assay and annexin-v/pi staining. ongoing studies will evaluate the impact of other dna damage response pathways and the potential protective role of autophagy against genomic instability. mustard agents are potent dna alkylating agents. among them, the bi-functional agent sulfur mustard (sm) was used as a chemical warfare agent due to its vesicant properties. although the use of sm in warfare has been banned in most countries of the world, its use in terroristic attacks or asymmetrical conflicts, such as the syrian civil war, still represents a realistic and significant threat. on the other hand, especially nitrogen mustards, such as cyclophosphamide or melphalan, have been used as chemotherapeutic agents due to their cytostatic properties. thus, mustard-induced dna damage, in particular dna crosslinks, can trigger complex pathological states, as it is observed in sulfur mustard exposed victims, but on the other hand also lead to the chemotherapeutic effects of clinically-used nitrogen mustards. mass spectrometric monitoring and quantitation of mustard-induced dna adducts can help to unambiguously identify and verify sm-exposed victims and to monitor the efficiency, as well as potential side-effects of mustard-based chemotherapy. up to now, the verification of mustard-induced nucleic acid damage is mainly based on immunohistochemical methods, which have several drawbacks such as limited specificity, sensitivity, and low dynamic range of quantitation. with this project, we aim to develop a (hplc/uplc)-ms/ms-based platform for the quantitation of the most common mustard-induced dna adducts including bis(n7-guanine-ethyl) sulfide dna crosslinks. up to date, we established methods for the quantitation of the several common dna adducts induced by the mono-functional sulfur-mustard derivative 2chloroethyl ethyl sulfide ("half mustard", cees). for that reason purification protocols, chromatographic conditions and mass spectrometric settings were developed to detect n7-ethylthioethyl-2´desoxyguanosine (n7-ete-dg) and n3-ethylthioethyl-2´desoxyadenosine (n3-ete-da) and their thermal hydrolysis products n7ethylthioethyl-guanine (n7-ete-gua) and n3-ethylthioethyl-adenine (n3-ete-ade), respectively, and the sensitivity was compared to immunohistochemical methods. additional non-radioactive isotope-labelled standards are being synthesized, which will be spiked into samples to account for technical variability during sample work-up and to improve ms-based quantitation. this procedure requires minimal cellular material and therefore should be transferred to quantitation of dna adducts in human blood samples. this will allow to monitor dna adducts as biomarkers of exposure in potential smexposed victims as well as in mustard-based chemotherapy. this method also sets a basis to investigate specific mustard-induced dna repair mechanisms and their cellular consequences. the γh2ax assay vs. comet assay for genotoxicity testing universitätsmedizin mainz, institut für toxikologie, mainz, germany dna damage leads to activation of the cellular dna damage response (ddr). this signalling network results in activation of various dna repair proteins and chromatin structure modulators. a frequent manifestation of ddr is the phosphorylation of histone 2ax (gh2ax), which can be visualised as gh2ax foci by immunocytochemistry. in the present study, we tried to assess if gh2ax is a reliable biomarker for detecting the cellular response to dna damage. we selected 14 well-characterised genotoxic compounds and compared them with 10 non-genotoxic chemicals in the wellcharacterised cho cell system. we measured quantitatively γh2ax by manual and automatic scoring of γh2ax foci, and by flow cytometry counting of γh2ax positive cells. the cytotoxicity dose-response was determined by the mtt cell proliferation/viability assay. we show that a) all genotoxic agents were able to induce dose-dependently γh2ax in the cytotoxic range whereas no induction was observed after treatment with non-genotoxicants; b) manual scoring of γh2ax foci and automated scoring gave similar results, with the automated scoring being faster and more reproducible; c) data obtained by foci counting and facs analysis of γh2ax positive cells showed a significant correlation. further we compared dna damage induced by 4 selected genotoxins at the time-points using the alkaline and neutral comet assay. significant correlation with the alkaline and neutral comet assay was observed for some but not all genotoxins and, predominantly, at earlier time points. we suggest that comet assays detect mainly primary dna damage, whereas γh2ax assay detects a specific response to dna damage which can persist longer. the γh2ax foci and flow-cytometry assays allow for a rapid and reliable determination of genetic damage in mammalian cells and can be used as additional genotoxicity assays. available in vitro methods to investigate the genotoxic potential of drugs fall short of throughput, specificity and mode of action information. a set of mechanistic biomarkers for clastogenic, aneugenic or apoptotic effects may help to overcome these limitations. thus, a staining assay amenable to flow cytometric analysis is being developed by litron laboratories, rochester, ny, supported by international collaborators. the experimental design of this assay consists of 3 stages. the objective of this work is the evaluation of this assay in the laboratories of bayer pharma ag. the biomarkers covered by the assay are associated with dna damage response pathways that have potential for class discrimination (clastogen/aneugen/cytotoxicant) of in vitro genotoxicants: dna double strand breaks (γh2ax), nuclear division (phospho-h3, dna content), apoptosis (cleaved parp). based on the pilot work at litron laboratories, tk6 cells were introduced to the genetic toxicology of bayer pharma ag. cells were exposed for 4 and 24 hrs in triplicates on a 96 microwell plate to one reference clastogen (etoposide, eto), aneugen (vinblastine, vb) and cytotoxicant (carbonyl cyanide 3-chlorophenylhydrazone, cccp). after staining, the samples were analyzed with the flow cytometer bd accuri c6 (bd biosciences, heidelberg, germany). the reference substances yielded the responses expected from the pilot study at litron laboratories: vb showed distinct increases of phospho-h3 events at 4 and 24 hrs and polyploidy at 24 hrs time point. eto induced a clear increase of yh2ax with a simultaneous reduction of phospho-h3 at 4 and 24 hrs. finally, cccp caused a reduction of phospho-h3 events, increased cleaved parp events and did not influence γh2ax. moreover, benchmarking experiments under pilot work conditions were performed with high content imaging analysis. we compared yh2ax and phsopho-h3 pilot study results as well as cleaved parp with caspase 3/7. in addition, the tunel assay (click-it ® tunel alexa fluor, thermofisher) was executed to benchmark cleaved parp. the benchmarking results support the selected biomarkers of the multiplexed assay. in stage 2, additional reference compounds (three aneugens/clastogens/cytotoxicants) were investigated. so far, the chosen biomarkers of dna damage response appear useful for class discrimination and provide additional information to existing genotoxicity tests. cell-cell contacts are involved in keeping a physiological balance between proliferation, differentiation and apoptosis. far less is known about the role of cell-cell-contacts in regulating necrosis, for instance in response to oxidative stress. previous findings of our group demonstrated that, in contrast to semi-confluent proliferating cultures, confluent murine fibroblasts (nih3t3, mef) and human keratinocytes (hacat) are protected against necrosis induced by tert-butyl hydroperoxide (t-booh). comparison of confluent cells (g0/g1 = ~70 %) and semi-confluent cultures, similarly arrested in the g0/g1 phase by serum-starvation or the mek inhibitor u0126, ascertained that the resistance against t-booh is mediated by cell-cell contacts and not by cell cycle arrest. we further revealed that confluent cultures are protected against t-booh-induced dna double strand breaks as assessed by the neutral comet assay and against mitochondrial damage detected by flow cytometric analysis of dioc6 staining. to better understand the protective role of cell-cell-contacts in ros-mediated necrosis, we started characterizing the signaling cascade induced by t-booh in semi-confluent proliferating cultures. in accordance with the observed formation of dna double strand breaks in response to t-booh, we detected phosphorylation of the checkpoint kinase chk2. however, inhibition of atm, the kinase responsible for chk2 activation, did not influence t-booh-induced cell death. interestingly, first experiments gave a hint for the participation of rip1, since the chemical rip1 kinase inhibitor necrostatin-1 (nec-1) blocked cell death up to averagely 50 %, what is described as a specific marker for regulated necrosis. in line with this observation, t-booh-induced cell death could not be blocked by the pan-caspase inhibitor z-vad-fmk strongly indicating that caspase activity is not required. moreover, parp-1 and p53 are probably not involved. deeper analyses could give evidence that nec-1 did not block formation of dna double strand breaks nor mitochondrial damage indicating that the kinase blocked by nec-1, possibly rip1, acts downstream of dna double strand breaks and / or mitochondrial damage. in the end, we could identify a crucial role of ca 2+ signaling for t-booh-mediated toxicity. as the calcium chelator bapta-am was able to completely block not only cell death, but also mitochondrial damage and dna double-strand break formation, there is a strong need for further investigations of the possible interplay between regulated necrosis and calcium, regulated by cell-cell contacts among oxidative stress. the work was supported by the hoffmann-klose-stiftung, the promotionsförderung rheinland-pfalz, the johannes gutenberg-university and the university medical center of the johannes gutenberg-university. the mammalian target of rapamycin (mtor) forms two multiprotein complexes (mtorc1 and mtorc2) and influences cell growth, proliferation, survival and metabolism. constitutively activated mtor was found to be deregulated in several cancer types, which makes it an interesting target for therapeutic cancer strategies. rapamycin is able to inhibit mtor and its downstream targets and is currently studied for its anticancer properties in clinical trials. despite previous evidence, there are studies that show an adverse effect in cancer treatment causing tumour growth, evolving the question of the effectiveness of the drug in cancer treatment. therefore, we examined the transformational potential of rapamycin in a balb/c cell transformation assay (cta) as well as markers of proliferation and protein synthesis. the balb/c 3t3 cell transformation assay mimics different stages of in vivo carcinogenicity (initiation, promotion, post-promotion phase) and is a promising alternative to rodent bioassays. balb/c fibroblasts are treated for 3 days with the tumour initiator mca (3-methylcholanthrene) followed by 13 days with the promotor tpa (12-o-tetradecanoylphorbol-13-acetate). upon treatment with these chemicals cells are transformed into morphologically aberrant foci and can be visualized after six weeks by giemsa staining. it is possible to apply additional substances during the whole assay or in several phases of transformation and evaluate the colony formation. furthermore, our improved protocol allows additional westernblot or immunofluorescence analysis. the influence on cell proliferation of different concentrations of rapamycin was investigated by cell counting (living and dead) to choose a suitable concentration for the cta. performances of balb/c ctas with 10 nm rapamycin showed, contrary to expectations, an increase in cell transformation. by administration of rapamycin only in the promotion phase we could detect an increase in colony formation, whereas a treatment with rapamycin in the post-promotion phase with already established foci, seemed to reveal its therapeutic properties. to better understand the role of mtor in our cell transformation system we used another mtor inhibitor called osi-027. surprisingly, an incubation with 3 µm osi-027 led to a decrease in colony formation. we are now able to investigate the underlying mechanism with westernblot and immunofluorescence analysis and can compare regulations of downstream targets like the marker of protein synthesis p-s6. our investigations revealed different cell transformation outcomes by comparing the two known mtor inhibitors rapamycin and osi-027, which need to be further evaluated. in the ongoing project we want to detect differences between rapamycin and osi-027 by protein analysis and identify key proteins, which are involved in this opposed colony formation of the balb/c cells. these results can be helpful to better understand mtor inhibition in matters of tumour therapy. introduction: over the past 50 years, the biguanide compound metformin has been widely prescribed as an insulin sensitizer in type 2 diabetes mellitus. interestingly, recent meta-analyses of epidemiological studies have shown that metformin might be involved in risk reduction of carcinogenesis. in vitro studies have described amp-activated protein kinase (ampk)-dependent, by inhibition of the respiratory chain complex i, as well as ampk-independent actions of metformin. however, the detailed molecular mechanisms by which metformin affects cell proliferation and carcinogenesis have not been well identified up until now. method: to evaluate the protective potential of metformin, balb/c 3t3 cell transformation assays were performed. this valid toxicological method is an alternative to in vivo carcinogenic testing and mimics the different stages of cell transformation during carcinogenesis. in detail, mouse fibroblasts are treated with metformin and/or the tumour initiator 3-methylcholanthrene (mca) and the tumour promotor 12-otetradecanoylphorbol-13-acetate (tpa). in the first experiment several metformin concentrations (0.1-1 mm metformin) were applied answering the question of an effective metformin concentration. next, metformin treatment during the different phases of carcinogenesis (initiation, promotion, post-promotion phase) was done determining the most effective phase for an intervention, i.e. chemopreventive or chemotherapeutical properties of metformin. additionally, the effect of metformin on the energy metabolism of the cells was analysed using various methods like immunoblot and oxygen measurement by clark electrode. results/discussion: analysis of different metformin concentrations revealed a concentration-dependent effect of metformin. in detail, decreased colony forming potential of balb/c cells was most prominent using 1 mm metformin. this effect was not caused by growth inhibition of metformin itself since 1 mm metformin showed no growth inhibitory properties in a cellular growth pretrial. interestingly, the 2 phase cell transformation assay showed that the metformin effect is more pronounce in the postpromotion phase than in the initiation and promotion phase pointing to a chemotherapeutical potential. investigating several energy metabolism parameters, the results indicate that metformin may affect cell respiration as well as energy-dependent mechanistic markers like ampk. the presented results support rather the idea of the chemotherapeutic potential of metformin than a chemopreventive, using 1 mm metformin. the initial analysis of energy metabolism markers discovered interesting starting points for further investigations. johannes gutenberg university, institute of toxicology, 55131 mainz, germany nvp, widely used e. g. as a monomer for polyvinylpyrrolidones (pvp) with applications in food technology or cosmetics is a known hepatocarcinogen in rats after inhalative exposure to 5, 10, and 20 ppm for 2 years. nvp is tested in a battery of genotoxicity assays (e.g. ames, hprt, mouse lymphoma, uds, chromosome aberration, cell transformation, micronucleus test (mnt) in mice bone marrow) [1]) that all yielded negative results. however, nvp induces cell proliferation in liver (loaec: 0.5 ppm) after whole body exposure to vapor [2] . to confirm the absence of genotoxicity in the context of a potentially non-genotoxic mode of action, a five day whole body inhalation study to nvp vapor with concentrations of 0, 5, 10, 20 ppm was conducted in wistar rats (six animals per gender and group, ethyl methanesulfonate 200 mg/kg bw p.o. as positive control). genotoxicity was investigated by the mnt in bone marrow and the comet assay (± fpg) in liver and lung. further investigated endpoints related to possible non-hepatogenotoxic moa were: enzyme induction (erod, prod, brod), oxidative stress (gsh-, gssg-, non-protein sulfhydryl group level), and peroxisome proliferation (cyp4a, cyanide-insensitive palmitoyl-coa-oxidase). at carcinogenic inhalative doses, the results of this study proved the absence of genotoxicity in lung, liver and bone marrow as neither the tail intensity in the comet assay nor the number of micronuclei in the mnt was increased compared to the controls. however, also the non-genotoxic parameters (cyp-enzyme activity, glutathione levels, cyanide-insensitive-palmitoyl-coa-oxidase) were not affected by nvp-treatment. as potential metabolic activation cannot be excluded and may essentially contribute to the understanding of the carcinogenic mechanism, in vitro investigations in rat liver systems (subcellular fractions, hepatocytes, precision cut liver slices (pcls)) were performed additionally. up to now, 2-pyrrolidone is the only identified in vitro metabolite. as these results cannot mimic the in vivo situation of two described, ring-and vinylmajority containing unidentified metabolites [3] detailed investigations on metabolism may be a future perspective to approach the overall understanding of the carcinogenic mechanism of nvp. introduction: in ischemic conditions such as wound healing and myocardial infarction, new vessels are generated by vasculogenesis and angiogenesis. these processes are stimulated by the signalling peptide vascular endothelial growth factor which therefore has been proposed as a promising compound for the treatment of ischemic conditions. however, results of respective clinical studies have not been fully convincing yet. here, we investigated principles underlying the selforganization of newly formed vessels to functionally adequate microvascular networks indispensable for proper tissue substrate supply. intravital microscopy of the chick chorioallantoic membrane (cam), a non animal model as defined by the american national institutes of health's office for protection from research risks, was used to study peripheral expansion of existing arteriolar and venular trees by recruiting segments of the dense polygonal capillary mesh. this process we call "emerging angiogenesis". methods: white leghorn chicken eggs were put into incubators on embryonic day 0 (e0) at 37.5°c and 82% humidity. on e3, the eggs were cracked open and transferred into petri dishes. on e10, cam microcirculation was recorded using time-lapse intravital videomicroscopy at discrete time points for up to 48 hours. to improve the visibility of the capillary mesh, videorecordings were processed offline by generating coefficient of variation images of pixel grey values over time. changes of network topology during the observation time were investigated. results: in the cam, a sequence of specific events leading to extension of existing vessel trees was observed: in a capillary mesh region near terminal branches of existing vessel trees, homogeneous flow distribution is transferred to inhomogeneous flow distribution: preferred flow pathways through the mesh evolve carrying most of the blood. over time, these flow pathways exhibit diameter increase, straighten and connect the mesh to arteriolar and venular trees. in contrast, less perfused parallel mesh flow pathways and transversal mesh segments exhibit progressive decrease of flow and diameter resulting in vessel regression. as a result, hierarchical vessel tree structures are extended into the mesh region. while newly generated tree extensions are located above the mesh at the beginning, they sink to a lower level at later stages until they are finally covered by a reconstituted mesh network. the cam ex ovo model is well suited for studying emerging angiogenesis. vessel tree extension occurs via parallel processes of vessel maturation and capillary mesh segment regression. at later stages, newly formed vessel tree branches sink and the capillary mesh is reconstituted above. in the next step of our project, we will implement these phenomena in a computer simulation and use theoretical modeling to further investigate and better understand principles underlying microvascular network maturation. this will allow us to derive effective therapeutic strategies which could be tested in the cam model. chemicals are able to induce cancer in a wide range of organs. therefore, it is very important to investigate the toxic properties of chemical substances, especially their carcinogenic potential. in this context the number of animal experiments will drastically increase in the future. in order to avoid the use of expensive and time consuming animal experiments for long-term carcinogenic studies, the development of an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is imperative. by combination of the well-established balb/c cell transformation method with the soft agar colony formation assay, we developed a high-throughput in vitro system to identify effects of chemicals on cell transformation for the first time. balb/c mouse fibroblasts are treated with 3-methylcholanthrene as a tumour initiator and 12-o-tetradecanoylphorbol-13-acetate as promotor for several days, whereby foci of transformed cells are developed. after the promotion phase of the common balb/c cell transformation assay, cells are transferred into soft agar to further monitor the anchorage independent growth of transformed cells only. the established soft agar transformation assay reproduces the foci growth of previous experiments and is performed in 96-well plate format. hence, we can analyse the carcinogenic potential of several chemical substances in parallel and are also searching for alternative endpoint analysis, e.g. the usage of fluorescing cells stably expressing irfp, instead of the former time-consuming microscopic assessment. the here presented new technique is a high-throughput and low priced alternative for the evaluation of the carcinogenic potential of chemical substances in a short period of time without animal testing. the effort to develop new or refine established in vitro test systems rises due to animal welfare, scientific and/or regulatory reasons (e.g. the animal testing ban concerning the risk assessment of cosmetic product ingredients in march 2013). this progress, among others, leads to an increased performance of cell-based assays. the majority of model cell lines are routinely cultured using medium supplemented with fetal bovine serum (fbs) in amounts between 5-10%. the application of serum-substitutes will provide a reduction of the animal number needed, which corresponds to the guiding principles of the three r's (3r), described by russel and burch in 1959. in addition, chemically defined serum-substitutes have the potential to reduce the inter-experimental variability of test conditions caused by the inherent differences in chemical composition across fbs batches 1 , resulting in a refinement of in vitro testing. in this study, human tk6 cells were gradually adapted to serum-free conditions, where they show comparable growth gradients at the exponential phase. for cells under serum-free conditions a mean doubling time of 19.3 (± 1.7) h was observed while fbs supplemented cells showed a doubling time of 13.5 (± 0.8) several non-animal test methods addressing key events in the sensitization process have passed formal validation and oecd (draft) test guidelines are available. one of these methods is the direct peptide reactivity assay (dpra) assessing the ability of a chemical to bind to proteins to form a complete antigen (oecd tg 442c). the test is used to obtain a yes/no answer on whether the substance has a protein-binding potential. for a complete risk assessment, however, an estimation of a chemical's potency is also needed. in this study we examined if an assessment of potency could be achieved by 1) determining reactivity class cut offs based on published data on 199 substances for the dpra performed according to oecd 442c to predict un ghs sensitizer classes, 2) a variant of the dpra assessing reaction kinetics (time and concentration) for 12 substances or 3) an extended protocol testing several test substance concentrations for 50 reference substances and estimating the concentration of a test substance that is needed to cause a peptide depletion of 6.38% (ec6.38%). results of the first approach indicated that cut offs to differentiate the un ghs sensitizer classes 1a and 1b could indeed be defined. secondly, evaluating the reaction time based assay in which several time points between 5 min and 24 hours were assessed, it was found that not all reactions followed ideal kinetics. hence further investigations are needed to eventually derive a reaction time based prediction model. the results of the 3 rd approach (the standard protocol of the dpra was amended by testing three concentrations i.e. 1, 10, and 100 mm) indicated that potency classes could be assigned using the ec6.38% value to assess potency. in summary, using quantitative information derived from the dpra in particular using ec6.38% value may support the assessment of the skin sensitizing potency. identification of pre-and pro-haptens with non-animal test methods for skin sensitization since pro-haptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (xme) activities in cell lines used for testing of sensitization in vitro is of special interest. metabolic activity of e.g. n-acetyltransferase 1 (nat1) and esterase in the keratinocyte (keratinosens tm and lusens) and dendritic cell-like cells (u937 and thp-1) was previously demonstrated. aldehyde dehydrogenase (aldh) activities were found in keratinosens tm and lusens cells. activities of the investigated cytochrome p450-dependent alkylresorufin o-dealkylases, flavin-containing monooxygenase, alcohol dehydrogenase as well as udp glucuronosyl transferase activities were below detection in all investigated cell lines. a set of 27 putative pre-and pro-haptens (no obvious structural alert for peptide reactivity but positive in vivo) was routinely tested using the above mentioned cell lines as well as in the direct peptide reactivity assay (dpra). 18 of the compounds were unexpectedly positive in the dpra und further analyzed by lc/ms techniques to clarify the reaction mechanism leading to true positive results in this assay. oxidation products like dipeptide formations or the oxidation of the peptide-based sulfhydryl group led to positive results for benzo[a]pyren or 5-amino-2-methylphenol, respectively. in contrast, covalent peptide adducts were identified for 12 putative pre-haptens, indicating the dpra to be suitable for compounds requiring abiotic oxidation to get activated. for some dpra negatives, the keratinocyte and dendritic cell based assays provided true positive results. a combination of dpra, keratinosens tm and h-clat within a '2 out of 3' prediction model provided a high sensitivity of 81% for the set of the pre-/pro-haptens. the sensitivity of this combination of non-animal test methods in the '2 out of 3' prediction model in a set of 95 direct haptens was comparable (sensitivity = 87% when compared to llna skin sensitization testing is mandatory for all substances produced or marketed in volumes larger than 1 tonne per year under the european reach legislation. with reach supporting in vivo testing only "as a last resort" and the marketing ban for finished cosmetic products with ingredients tested in animals, attention has been given to developing integrated testing strategies combining in vitro, in silico and in chemico methods. key challenges are which tests to select and how to combine non-animal methods into testing strategies. this study suggests a bayesian value of information (voi) approach for developing non-animal testing strategies, which consider information gains from testing, but also expected payoffs from adopting regulatory decisions on the use of a substance, and testing costs. the 'value' of testing is defined as the expected social net benefit from decision-making on the use of chemicals with additional, but uncertain information from testing. the voi is calculated for a set of individual nonanimal methods including dpra, oecd qsar toolbox, are-nrf2 luciferase method covered by keratinosens and lusens, and hclat, seven battery combinations of these methods, and 86 two-test and 360 three-tests sequential strategies consisting of nonanimal methods. their voi is compared to the voi of the local lymph node assay (llna) as the animal test. we find that battery and sequential combinations of nonanimal methods reveal a higher voi than the llna. in particular, for small prior beliefs (i.e. a chemicals is, prior to testing, assumed to be a non-sensitiser), a battery of dpra + lusens reveals the highest voi. if there are strong beliefs that a chemical is a sensitizer, a sequential combination of the battery dpra + lusens, followed by keratinosens + hclat at the second stage and by the oecd qsar toolbox at the third stage performs best. for given specifications of expected payoffs the voi of the nonanimal strategy significantly outperformed the voi of the llna, for the entire range of prior beliefs. this underlines strong economic potential of non-animal methods for skin sensitization assessment. a chemical series to predict the proarrhythmic potential of drugs with low solubility for which no reliable purkinje fiber results could be obtained. these validation results showed that this cardiosafety in silico model can successfully be applied in r&d to predict the proarrhythmic potential of drug candidates within the model ad. introduction: the use of p-phenylenediamine (ppd) and derivatives (tab. 1) in oxidative consumer hair dye products is considered as key in hair dye allergic contact dermatitis [1] [2] [3] [4] . in recent supplement, 2-methoxymethyl-ppd (me+) shows significantly reduced sensitizing properties [5, 6] . since overcoming the skin barrier is a prerequisite for sensitization, numerous in vitro an in vivo studies on skin penetration of ppd and derivatives have been performed. the aim of the present study is the in silico prediction of the penetration of ppds, because such computations may help in understanding the processes involved in sensitization. for the first time, software dskin [7] is challenged to simulate this class of compounds. in silico results are retrospectively compared to previously published experimental data and may assist in future tailoring of in vitro experiments. material and methods: the permeabilities, lag-times and the time-dependent accumulated amounts of ppds were computed using dskin. input parameters for the latter were a concentration of 1 mg/ml (1%), finite dosing and 30 min in use incubation periods. molecular structures were optimized ab initio and the condensed fukui functions (ff) were estimated from mulliken population analyses [8] and electrostatic potentials using gamess [9] . results: initial results agree with experimental results using ppd in white petrolatum, demonstrating the applicability of dskin to ppds. the four ppds exhibit only small differences in permeabilities in silico (tab. 1). toluene-2,5-diamine shows a higher accumulated mass due to increased lipophilicity (fig. 1) . in general, the ff were very similar for all ppds and indicated that the n atoms would be the preferred targets for radical and electrophilic attack. discussion and outlook: in silico methods may be used to model the permeation of ppds despite their low molecular weight and low lipophilicity. the low amounts of ppds under in use conditions result from oxidative conditions. computed me+ permeation was not different to other ppds, therefore other properties account for the reduced sensitization potential. the very similar ff values hint at similar reaction pathways. furthermore, ppd and its derivatives are prone to n-acetylation in living skin resulting in metabolites exhibiting higher molecular weight and greater lipophilicity than the parent compounds. the effects of n-acetylation and reactions of ppd and its derivatives with histidine and cysteine residues are subject of upcoming computations. dermal absorption is an important factor in regulatory science regarding the registration of chemicals, agrochemicals and cosmetics. the issue has gained importance since it has been realized that the skin is not completely impenetrable for chemical substances. [1] the different ways to assess dermal absorption range from qsar models to complex in vivo studies including a complete toxicokinetic examination. the choice of method depends on the question that has to be answered as different systems give different results: absorption as % of applied dose in in vivo studies or permeability coefficient and lag time in infinite dose in vitro studies [1] . ideally both data would be available. since the oecd has adopted a guideline for assessing dermal penetration in vitro in 2004 the number of in vitro studies is rising continuously. depending on the chosen method results may vary in reliability and in acceptance by regulatory authorities. the skinab database [ba1] contains data for about 600 substances on dermal absorption which has been found through the echemportal [3] and extended with data from the edetox database. for selected substances with a broad spectrum of data available further analysis has now been started. 165 chemicals have been investigated in a comparable test system; from these 79 were shown to have a low dermal absorption of less than 1% and 51 compounds showed a high absorption rate of more than 50%. for the assessment of dermal exposure either the absorbed dose in percent or the flux can be measured. data analysis showed that only for 15 substances both is available: flux data from in vitro studies and absorption data from in vivo studies. this data could be used to clarify which parameter would be most useful for exposure assessment regarding dermal exposure. seven substances in the dataset were conspicuous for their range of absorption rates in different studies: less than 1% to more than 50%. an in depth analysis revealed the complex influence that different exposure parameters have on the results of dermal absorption studies. for some chemicals the influence of exposure time on increasing absorption values could be clearly demonstrated. beside other factors such as the chosen vehicle, and the (non-)occlusion of the site of exposure especially the choice species introduced a high variability; this holds even for the most common laboratory animals t. a review published by jung in 2015 [5] which comes to the conclusion that i.e. hairless species are usually not a good model to predict dermal absorption in humans. [1]who (2006) ehc 235 dermal absorption [2] scholz et al (2014) naunyn-schmiedeberg´s arch pharmacol 3 (suppl 1):s86 [3] www.echemportal.org [4] http://edetox.ncl.ac.uk [5] jung et al (2015) in-silico methods have evolved to indispensable tools in various areas of life sciences. several stages in drug development including hit identification and lead optimization, for instance, highly benefit from an accurate estimation of binding free energies associated with biological host-guest systems. as a consequence, the need for laboratory experiments including in-vivo experiments and animal testing is considerably reduced. another field profiting from free energy calculations is human as well as ecotoxicology. upon the development and risk assessment of new chemicals, transformation products arising from biotic or abiotic degradation of the parent substance have usually been neglected. however, since few years, the risk assessment of new chemicals often includes transformation products probably causing more harm than the parent substance itself. such studies as well are mostly carried out on the basis of in-vitro and in-vivo tests. moreover, many metabolites can be detected but neither enriched nor synthesized in amount sufficient for toxicological evaluations. at this stage, computational methods come into play. using classical molecular dynamics simulations in combination with an empirical linear prediction model, we have investigated several metabolites of the drugs sulfamethoxazole and carbamazepine and prioritized them according to their estimated binding affinities to potential biological target proteins. consequently, a couple of metabolites were identified that bind to one or more human cytochrome p450 variants and the bacterial enzyme dihydropteroate synthase, respectively, which are known to be sensitive to the two drugs. the investigations were carried out in the framework of the bb3r poject funded by the german government through bmbf. instituto superiore di sanità, environment and primary prevention dept., rome, italien introduction: in vitro methods have been increasingly used to characterize pharmacological and toxicological properties of substances. to address the problem of nominal versus actual concentrations, in vitro biokinetic studies were recently undertaken (truisi et al., toxicol lett 233:172-86, 2015) . we use those data as input into a physiologically based human kinetic model (pbhkm) to model the in vivo doses leading to the in vitro measured concentrations. methods: a pbhkm was used to simulate the concentration time profile of ibuprofen in the hepatic vein after oral administration. the details of the model and the physiological parameters used have been described elsewhere (abraham et al., arch. toxicology 79: 63-73, 2004) . we modelled the concentration time profile exploring the dose which would lead to a concentration at 1 hour and at 24 hour as similar as possible to the concentration measured in the supernatant of human freshly prepared cell cultures after dosing the culture with ibuprofen. we parametrized the pbhkm with the parameters which have been estimated from the in vitro kinetic studies (clearance between 3 and 15 µm 3 /sec (truisi et al., 2015) and an absorption of 100% and an absorption rate of 1/h (cristofoletti and dressman, j pharm sci 103: 3263-75, 2014). results: the data of the in vitro study with 100 µm ibuprofen could well be modelled. when assuming a clearance of 15 µm 3 /sec the dose of 1480 mg resulted in an 1 hour concentration of 66.5 µm in the hepatic vein of pbhkm equal to 133.0 nmol/well (volume of the well = 2 ml) in the in vitro study in which the measured concentration was 138.75 nmol/well. the concentration at 24 hours of 8.7 µm (equal to 17.4 nmol/well) corresponded with the in vitro concentration (16.5 nmol/well). the modelling approach was less successful with in vitro dosing of 1000 µm. the 10 fold higher dose of 14800 mg lead to nearly double the concentration at 1 hour than measured in vitro. with a dose of 8500 mg/kg an approximation was feasible resulting in 396.7 µm in the hepatic vein at 1 hour which is equal to 793.4 nmol/well whereas the measured concentration in vitro was 782.85 nmol/well. even with a clearance value as low as the 2.5 percentile (3 µm 3 /sec), the concentration at 24 hours was modelled to be lower than the in vitro measured value (in vivo model: 98.7 µm which corresponds to 197.4 nmol/well; measured in vitro concentration: 979.2 nmol/well). discussion: this is the first attempt to use kinetic data obtained in vitro to feed in in a pbhkm for reverse dosimetry finding the dose which corresponds in vivo to the in vitro situation. in the case presented here, the in vitro dose assumed to be low in vitro (100 µm) corresponds to a dose of 1480 mg (note: the highest approved daily dose is 2,400 mg). for the high in vitro dose modelling was successful only for the concentration 1 hour after dosing and a dose of 8,500 mg. conclusions: in vitro kinetic parameters, such as clearance, can successfully be used for parametrizing a pbhkm. it is of utmost importance for the relevance of in vitro finding to assure that the concentrations used in vitro can be obtained with relevant in vivo doses. in this case, the in vitro concentrations were within (low dose) and 3.5 fold above (high dose) the in vivo relevant therapeutic concentration range. introduction: a variety of drug residues have been detected in sewage plant run-offs, rivers and lakes, but also in groundwater and tap water samples. studies have yet to identify a risk for human health from these contaminants, but adverse health effects have been reported for various species, including fish and birds. it has recently been suggested that for a comprehensive risk assessment toxicologists should also consider transformation products (tps) of such water contaminants that may arise from abiotic and biotic (metabolic) reactions. with aciclovir (acv), a well-known antiviral drug, as the parent drug we tried an in-silico approach to identify tps that might be of interest due to some mutagenic or carcinogenic toxicophores. methods: from a literature and database search we picked up 12 acv-tps. predicted acute toxicities and mutagenic / carcinogenic properties for these tps were derived from an expert system analysis using the lazar portal (http://lazar.in-silico.ch/) as front end. results: two of the identified acv-tps could not be handled by lazar because of insufficient training data in one out of eight queried categories. the highest score (4 positive out of 8 possible genotoxicity categories) was assigned to 9 of the 12 tps, including acv itself. this is a rather low score when compared to other water-borne drug residues, e.g. carbamazepine. cofa, an imidazole derivative of acv seen in advanced oxidation processes, had shown antiproliferative effects in several ecotoxicologic screening assays, e.g. [1], but was unremarkable in our tests. additionally, a computer-based simulation of the respective tps interacting with human cyp isozymes did not support concerns that these tps may pose a risk for human health. conclusions: our in-silico analyses of 12 acv-tps did not provide evidence for any adverse health effects in the micromolar concentration range. further studies are needed to clarify if the biological activity of some acv-tps in ecotoxicological assays may eventually affect yet unidentified biological targets in the human body. sulfur mustard (sm) is a chemical warfare agent which was first used in world war i, but has found use in several conflicts afterwards. although sm is prohibited by geneva protocol, terroristic attacks cannot be ruled out. latest news give rise to concern that is may be in the possession of sm and is willing to deploy it. even 200 years after the initial synthesis of sm its mode of action is not fully unraveled. thus, no antidote does exist. however, chemosensing ion channels have been shown to be activated by highly toxic chemicals and might represent a specific therapeutic target. previous studies have shown that the sm-surrogate cees (mono-functional alkylating agent) is able to activate transient receptor potential ankyrin 1 (trpa1) channels that are known to affect mapk cell signaling. mapk-pathways, especially perk1/2, are known to increase protein biosynthesis through activation of transcription factors binding to the serum response element (sre). it is unknown whether alkylating agents have also impact on mapk signaling mediated through trpa1 activation. our results demonstrate that aitc resulted in phosphorylation of the mapk perk1/2 and increased protein biosynthesis of sre-regulated genes in hek293 cells overexpressing htrpa1. cees increased perk1/2 levels already after 2.5 min which could be prevented by the trpa1 blocker ap18. activation of target genes through perk1/2 signaling was also evident, but less pronounced compared to aitc. our results demonstrate that alkylating agents have impact on cell signaling through trpa1 channel activation. thus, trpa1 might represent a promising target for counteracting sm toxicity. sulfur mustard (sm) is a chemical warfare agent that provokes severe inflammation and blistering upon exposure to the skin accompanied by disturbed wound healing. the potential use of sm in terroristic assaults amplified the interest in understanding the underlying cellular and molecular pathomechanisms in order to improve therapeutical intervention. autophagy is a highly conserved catabolic pathway in eukaryotes that ensures the degradation and recycling of cellular components through the lysosomal machinery. autophagy is important for cell survival in physiological and pathological stress situations. emerging knowledge indicates that imbalanced regulation of autophagy disturbs basal cell functions including proliferation, differentiation and migration, thus contributing to the pathophysiology of various diseases. after penetration into skin cells, sm alkylates and thereby modifies nucleic acids and proteins thus forming aggregates of dysfunctional proteins destined for autophagic disposal. in our studies, we analyzed the influence of sm on protein expression (western blotting) of autophagy-related (atg) genes as well as proliferation (wst-1) of primary normal human keratinocytes (nhek) and primary normal human dermal fibroblasts (nhdf). preliminary results demonstrate that sm strongly dysregulates the biosynthesis of atg proteins that may contribute to the diminished cell migration and proliferation under these conditions. our findings suggest that sm affects autophagy in correlation with an impairment of physiological functions in keratinocytes and fibroblasts that are essentially required for normal tissue regeneration. thus, application of pharmacological modulators of autophagy might be useful in the treatment of the delayed wound healing in skin upon exposure to sm. exposure of the respiratory tract to airborne particles is a major risk to human health. due to the ubiquitous application of these particles in the field of pharmacy, industry and in daily life, there is a strong necessity to investigate the toxic properties and the underlying pathomechanisms of these inhalable substances. in addition, the eu chemicals regulation requires not only that all substances placed on the market have to undergo a toxicological characterization, including the identification of potential toxic inhalation hazards (reach), but also that animal testing shall be undertaken only as a last resort ("3rs" principle) and the promotion of the development of alternative methods. thus, the development, establishment and validation of alternative in vitrobased test systems for the assessment of pulmonary toxicity are in the focus of current research. until now, most of the available in vitro cell culture models are limited to some extent as in those studies the exposure is either done under submerged conditions, not resembling the exposure conditions in vivo, or a homogeneous particle distribution is not guaranteed. the cultex ® radial flow system (rfs) is a specially designed in vitro modular exposure system that overcomes these limitations. it enables the homogenous exposure of human lung epithelial cells at the air-liquid interface (ali), thereby mimicking the physiological conditions of the alveolae. however, further optimizations are needed for the enhancement of the cultex® methodology. aim of this study was first the optimization of the test methodology in general (i.e. focus on clean air controls of the human lung epithelial cell line a549), and second the improvement of cultivation conditions. parameters such as handling of the cultex® device (proper closing and opening operation of the cultex® rfs module; improved washing conditions and media supply), treatment of the incubator controls, adjustment of clean air pressure and flow rates, and integration of two additional filters were sequentially adjusted in order to enhance the methodical setup. our results show that the test parameters for clean air exposure of the a549 cells were successfully optimized resulting in more accurate and robust data. cultivation conditions were improved by changing from closed-wall cell culture inserts to open-wall cell culture inserts. the openwall inserts turned out to be more suitable for exposure experiments as they provided a better medium supply and preserved humidity. deductive, the change of the cell culture inserts was identified as the deciding factor for the improvement of cell morphology. hence, we have successfully optimized the cultex ® rfs methodology for clean air exposure of a549 cells. human primary hepatocytes represent the gold standard in in vitro liver research. due to their low availability and high costs, alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. the human hepatocarcinoma cell line hepg2 is often used as a model for liver toxicity studies. however, under two-dimensional (2d) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers is very low. cultivation for 21 days in a three-dimensional (3d) matrigel culture system has been reported to strongly increase the metabolic competency of hepg2 cells. in our present study we extended previous studies and compared hepg2 cell cultivation in three different 3d culture systems: collagen, matrigel and alvetex culture system. cell morphology, albumin secretion, cytochrome p450 monooxygenase (cyp) enzyme activities, as well as expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. our results show that the previously reported increase of metabolic competency of hepg2 cells is not primarily the result of 3d culture but a consequence of the duration of cultivation. hepg2 cells grown for 21 days in 2d monolayer exhibit comparable biochemical characteristics, cyp activities and gene expression patterns as all 3d culture systems used in our study. however, cyp activities did not reach the level of heparg cells. in conclusion, the increase of metabolic competence of the hepatocarcinoma cell line hepg2 is not due to 3d cultivation but rather a result of prolonged cultivation time. in vitro assessment of the neurotoxic potential of arsenolipids arsenolipids are organic, lipid-soluble arsenic compounds, which occur mainly in marine organisms. major human exposure routes are fatty fish including herring or fish oil-based food supplements. about 55 different arsenolipids have been identified so far. thereby, arsenic-containing hydrocarbons (ashc) and arsenic-containing fatty acids (asfa) represent two subgroups of the arsenolipids [1]. our in vitro studies have demonstrated high cellular bioavailability and a high cytotoxic potential of ashcs in human liver and bladder cells [2] , whereas asfas were less toxic [3] . a substantial transfer across an intestinal barrier model (caco-2) indicated that ashcs are highly intestinal available. in comparison, asfas showed lower intestinal bioavailability and underwent a presystemic metabolism [4] . moreover, in drosophila melanogaster ashcs exerted late developmental toxicity and accumulated in the fruit fly's brain. these results suggest that ashcs might pass the blood-brain-barrier due to their amphiphilic structure [5] . in order to assess the neurotoxic potential we currently investigate the toxicity of several arsenolipids in differentiated, human neurons (luhmes). after 48 h incubation with ashcs or asfas, cell number (hoechst) as well as cellular dehydrogenase activity (resazurin) were measured, with the latter endpoint turning out to be more sensitive. ashcs showed substantial cytotoxic effects (ic 50 ~ 7-12.5 µm) in a concentration range comparable to that of arsenite (ic 50 ~ 7.5 µm), whereas asfas were less cytotoxic (ic 50 > 100 µm). after incubation with ashcs the cellular arsenic concentrations increased 10-20fold as compared to incubation with arsenite. further studies indicated that one possible toxic mode of action of arsenolipids could be a disruption of the cellular energy level. therefore, the mitochondrial membrane potential was investigated after incubation with the arsenic compounds in differentiated neurons. whereas arsenite did not exert an impact, ashcs reduced the mitochondrial membrane potential significantly. this might be due to interactions of the amphiphilic ashcs with mitochondrial membranes. currently we investigate the impact of the arsenolipids on neurite outgrowth as a developmental toxicity endpoint. standard treatment of poisoning by organophosphorus compounds (op; e.g. nerve agents and pesticides) consists of co-administration of atropine and an oxime-based reactivator of inhibited cholinesterases. due to lack of efficacy of clinically used oximes against various op-inhibited human acetylcholinesterase (ache) (e.g. soman) research started focusing on new therapeutic approaches. several research groups conducted in silico screenings [1, 2] in order to identify new non-oxime reactivators, presenting amodiaquine as a promising candidate for paraoxon-inhibited hache. for decades, antimalarial drugs like amodiaquine and chloroquine have been closely investigated regarding their side effects, thereby discovering interaction with cholinesterases, which could pose a new potential therapeutic benefit for inhibited cholinesterases. therefore, in this study interactions between antimalarial agents in presence or absence of ops were examined spectrophotometrically by a modified ellman assay. reversible inhibition of cholinesterases was observed with both antimalarial agents. amodiaquine had higher inhibitory potency for hache than human butyrylcholinesterase (bche), being confirmed by ic 50 values of 0.67 ± 0.02 µm for hache and 81.28 ± 0.04 µm for hbche. ic 50 values with chloroquine were 28.37 ± 0.02 µm for hache and 55.62 ± 0.02 µm for hbche, thus representing a weaker inhibition of hache than amodiaquine. furthermore, reactivation of paraoxon-(pxe), sarin-(gb), cyclosarin-(gf), and vxinhibited hache and hbche by amodiaquine and chloroquine was determined. after 60 minutes, only paraoxon-inhibited hache (50%) and cyclosarin-inhibited hbche (10%) were reactivated by 500 µm chloroquine. on the contrary, 10 µm amodiaquine reactivated all tested ops after 60 minutes in the following order: pxe > vx > gf > gb. in contrast, with hbche the highest reactivation was generated with 100 µm amodiaquine in the following order: vx > gb > gf > pxe. due to the high reversible inhibitory potency of amodiaquine, an increased concentration does not result in a higher reactivation of op-inhibited hache. in summary, our results show that amodiaquine is a reactivator of op-inhibited cholinesterases. in the future, non-oxime reactivators that are structurally-related to amodiaquine should be further investigated. [1] bhattacharjee, a.k., marek, e., le, h.t., gordon, r.k., eur. j. med. chem., 2012, 49, 229-238. [2] katz, f.s., pecic, s., tran, t.h., trakht, i., schneider, l., zhu, z., ton-that, l., luzac, m., zlatanic, v., damera, s., macdonald, j., landry, d.w., tong, l., stojanovic, m.n., chembiochem., 2015 , 16, 2205 -2215 bundesinstitut für risikobewertung, lebensmittelsicherheit, berlin, germany development of mammary gland tumors is connected to a deregulation of breast epithelial cell differentiation, a complex process which cannot be reproduced in vitro under standard cell culture conditions. however, cultivation of cells in a tissue-like environment in an in vitro three dimensional (3d) model can mimic general architecture, function and differentiation of mammary bulks. in this project, a 3d model was used consisting of the permanent breast epithelial cell lines mcf10a (er -, estrogen receptor negative) and mcf12a (er + , estrogen receptor negative) grown in matrigel tm , which mimics the complex extracellular matrix in vivo. the 3d culture of mcf10a and mcf12a cells in matrigel tm results in the formation of growth-arrested, polarized spheroids with a lumen (acini-like organoids). in order to perform a semi-quantitative estimation on the influence of substances on the differentiation of the breast cells for the identification of non-genotoxic carcinogens a scoring method was developed. this scoring method provides information about substance-induced morphological changes of the spheroids during differentiation based on the following parameters: size of the spheroids, the formation of the lumen, and the degree of polarization. furthermore, the model allows distinguishing between erdependent (mcf12a) and er-independent (mcf10a and mcf12a) effects. the 3d in vitro model is a useful tool for toxicologists to study substance effects on differentiation processes. the system will be used to examine the potential of e.g. food contaminants such as phthalates or perfluorinated substances (pfas) to disrupt the differentiation process of breast epithelial cells and will therefore serve as a valuable in vitro tool to assess their carcinogenic potential. inflammatory episodes occur erratically throughout life and are likely to play a critical role in the alteration of the individual susceptibility of a person to idiosyncratic druginduced liver injury (idili), a particular severe form of drug-induced liver injury (dili). in concordance with the inflammatory stress hypothesis, modest inflammatory stress can lower the threshold for hepatotoxicity and make an individual susceptible to develop liver injury during exposure to therapeutic doses of a drug. in order to evaluate the role of immune cells and its secreted factors during drug therapy, we established an in vitro test battery consisting of two cell culture systems in presence or absence of proinflammatory factors (lps, tnfα): (a) the monoculture of human hepatoma (hepg2) cells and (b) co-culture systems of human monocytic or macrophage-like (thp-1) and hepg2 cells. with these different test settings we aimed to identify whether the introduction of inflammatory immune cells and/or pro-inflammatory factors could increase the sensitivity of liver cells towards idili compounds. three reference substance pairs were tested, namely troglitazone -rosiglitazone, trovafloxacin -levofloxacin, and diclofenac -acetylsalicylic acid, each of them being composed of a compound that is known to induce idili and a partner compound of the same substance class that does not induce idili. first, all compounds were tested for cytotoxicity towards the single cell systems using the wst-assay. co-culture experiments with hepg2 and thp-1 monocytes or macrophages as well as co-exposure experiments with lps or tnfα were then done at about 20% cytotoxicity of the respective substance in the most sensitive cell type. subsequently the results were compared to the experiments in the monoculture of hepg2. we observed that every idili compound showed a significant increase in cytotoxicity in a minimum of one exposure combination while this effect was not observed with the corresponding non-dili partner compound. in conclusion, a combination of different culture systems and co-exposures with proinflammatory factors is needed for a valid differentiation between non-dili and idili compounds. this test battery could provide a useful tool for the prediction of inflammation-associated idiosyncratic drug-induced hepatotoxicity. furthermore, our results support the inflammatory stress hypothesis and points to an involvement of proinflammatory factors in the development of idili. extensive animal models of carcinogenicity ensure a safe usage of chemicals. to elucidate fundamental molecular mechanisms of carcinogenicity these methods are expensive, time consuming and above all too complex. in contrast, most in vitro methods are rather simple and detect only selected endpoints, like dna damage, mutations or changes in proliferation. the balb/c cell transformation assay is a validated toxicological method to identify potential tumour initiators and promotors. first, balb/c mouse fibroblasts form a monolayer culture and get contact-inhibited after reaching confluence. upon treatment with a tumour initiator (3-methylcholanthrene) and promotor (12-o-tetradecanoylphorbol-13-acetate) transformed cells do not stop proliferation and grow as morphologically aberrant foci over the monolayer of normal cells. after fixation with methanol at day 42, morphological aberrant foci can be visualized with giemsa staining. because the balb/c assay mimics different stages of the malignant cell transformation process (initiation, promotion and post-promotion phase) and detects with the colony formation a late endpoint of carcinogenicity we improved this method for mechanistic cancer research. using the example of insulin-signalling pathway we can show that (1) several substances have a different impact on the transformation process, (2) it is possible to identify for each substance the phase with the greatest effectiveness and (3) we can detect additional endpoints to elucidate the mechanistic mode of action. therefore we used several compounds (linsitinib, metformin, rapamycin, …) to manipulate the insulin-signalling pathway on different levels (insr, ampk, mtor, …) and analysed a number of characteristic endpoints of carcinogenesis. changes on protein level and signalling (westernblot, immunofluorescence, flow cytometry) or parameters of energy metabolism (oxygen consumption, glucose or atp measurement) are measurable and enable new insights into the process of cancer origin. summing up, the balb/c 3t3 assay proves to be a cheap and short-time alternative to rodent bioassays. although this method does not mimic the whole in vivo neoplastic process, it can be used to provide essential information regarding key proteins and their signalling, during the different stages of transformation. is there hope to correctly classify severe ocular irritant agrochemical formulations using in vitro methods: a proof of concept using the isolated chicken eye test, two modified bcop protocols and an epiocular™ et50 protocol while some in vitro methods addressing ocular irritancy have gained regulatory acceptance, to date the draize rabbit eye test (oecd tg 405) is the only world-wide regulatory accepted test for the determination of the full range of eye irritation potential. further although several in vitro methods for the severe eye irritation have gained regulatory acceptance, agrochemical formulations are nor explicitly included nor excluded from the applicability domain to predict severe ocular irritant formulations. systematic analyses are only available for e.g. the hen's egg test-chorioallantoic membrane (het-cam), and bovine corneal opacity and permeability (bcop, oecd tg 437) assays both showing that the used protocols do not provide sufficient sensitivity to reliably predict severe ocular irritating formulations. the purpose of this study was to evaluate whether the regulatory accepted isolated chicken eye (ice, oecd tg 438) test including corneal histopathology (as suggested for evaluation of the depth of injury), as well two modified protocols of the bcop and/or an et50 (exposure time reducing viability of treated tissue to 50%) protocol using the reconstructed cornea model epiocular™ are useful to predict severe ocular irritant agrochemical formulations. a proof of concept comprising the testing of ten to twelve agrochemical formulations with available in vivo data in each assay was conducted. in summary, based on the ice evaluation described in oecd tg 438, one of the five severe ocular irritant formulations (un ghs cat 1) was predicted correctly. using both modified protocol versions of the bcop the result for one of the four tested un ghs cat 1 formulations was just above the un ghs cat 1 classification border for using one of the modified protocols. lastly and most promising, the epiocular™ et50 predicted four of five tested un ghs formulations correctly with the fifths being close to the classification border. additional agrochemical formulations will be tested to further evaluated the epiocular™ et50 protocol to identify severe ocular irritant agrochemical formulations. drug-induced pancreatic toxicity comprises effects on the exocrine and/or the endocrine pancreas, which both can have serious clinical implications, e.g. acute pancreatitis or diabetes mellitus. adverse effects on the pancreas are occasionally observed during drug discovery and development and often prohibit further development. hence, there is a need for reliable in vitro models to early on identify the pancreas-toxic potential of drug candidates. permanent cell lines and primary cells have many shortcomings, e.g. loss of cell-to-cell and cell-to-matrix relationships or changes in cell physiology due to the isolation procedure. pancreas tissue slices are a potential alternative, circumventing most of these limitations. their preparation is rather elaborate which may explain its rare use. so far, pancreas tissue slices have predominantly been used to address physiological or pharmacological questions, although they might also serve as valuable in vitro model for toxicological applications. therefore, this work aimed to establish and characterize rat pancreas tissue slices as in vitro model for studying drug-induced pancreatic toxicity. results will be compared to the responses of the permanent endocrine (ins-1e) and exocrine (ar42j) pancreatic cell lines to evaluate a potential added value. rat pancreas tissue slices were prepared by a protocol adapted from marciniak et al. (nat protoc, 2014 . 9(12): p. 2809 . briefly, pancreas was infused and embedded with agarose. tissue sections of app. 200 µm were prepared using a vibratome and maintained in cell culture medium for up to 6 days. cell viability was determined by daily measurement of lactate dehydrogenase (ldh) in medium supernatants and by microscopic evaluation following fixation in 10 % formalin and h&e staining. functional integrity of acinar and beta cells were assessed by cell-type specific secretory responses (i.e. insulin, amylase, lipase) to physiological stimuli. moreover, the effects of the pancreas toxins streptozotocin (stz), alloxan (all), and the cholecystokinin (cck) analogue cerulein on the viability and functional integrity of tissue slices were compared to the respective responses of the cell lines. we were able to establish an optimized isolation and cultivation procedure for rat pancreas tissue slices applying minor modifications to the original protocol. cell viability declined over the cultivation period. stimulation of the cell lines with glucose or cerulein increased secretion of insulin (ins-1e cells) or amylase/lipase (ar42j cells), respectively. the pancreas slices responded to both stimuli, demonstrating functional integrity of endocrine and exocrine cells. treatment of ins-1e islet cells with the betacell toxicants all or stz only slightly affected islet cell viability, whereas treatment of ar42j acinar cells with cerulein at supraphysiological concentrations had no effect. this set of experiments is currently completed by investigating the effects of all, stz and cerulein on the viability of acinar and islet cells in pancreas slices. our preliminary data demonstrate feasibility to prepare and cultivate rat pancreas tissue slices over a period of 6 days thereby maintaining functional integrity to some extent. coculture of human monocytes with the keratinocyte cell line hacat in serumcontaining medium leads to higher sensitivity to weak contact allergens: an improvement for the loose-fit coculture-based sensitization assay (lcsa) a. sonnenburg 1 , j. the loose-fit coculture-based sensitization assay (lcsa) has proved reliable for the in vitro detection of contact sensitizers in the past. however, the use of primary human keratinocytes has some disadvantages. to facilitate high throughput screening of chemicals, we replaced primary keratinocytes from the original assay setup (setup a) by the human keratinocyte cell line hacat. these cells were cocultured with monocytederived dendritic cells in serum-free medium (setup b) or fetal calf serum (fcs)containing medium (setup c). upregulation of the dendritic cell maturation marker cd86 assessed by flow cytometry served as endpoint. we have tested four substances known as sensitizers and four non-sensitizers in both new setups as well as in the original setup with primary cells. three out of four sensitizers (2,4-dinitrochlorobenzene, 2-mercaptobenzothiazole, and coumarin) , and three out of four non-sensitizers (glycerol, monochlorobenzene, and salicylic acid) were correctly assessed under all culture conditions. the weak sensitizing potency of resorcinol was only detected by setup b with fcs supplemented medium. a false positive reaction to caprylic (octanoic) acid in all three setups confirms earlier results from our laboratory that some fatty acids are able to induce cd86 on dendritic cells in vitro. culture in fcs supplemented medium led to generation of dendritic cells showing a more pronounced upregulation of cd86 after application of substances with rather high sensitization potency compared to dendritic cells which are formed under serum-free conditions. therefore, we characterized dendritic cells from setups b and c by flow cytometric measurement of additional dendritic cell surface markers. dendritic cells from the original setup a had been characterized extensively before (schreiner et al., toxicology 2008; 249:146-1529) . dendritic cells generated in fcs supplemented medium were cd1a+/cd1c+, whereas dendritic cells from serum free culture conditions were cd1a−/cd1c− regardless whether cocultured with primary human keratinocytes or hacat. populations with cd1a+/cd1c+ dendritic cells in coculture seem to show a higher sensitivity to weak sensitizers, which proved beneficial for the identification of resorcinol. in conclusion, modification of the lcsa protocol led to an increased sensitivity of the assay. due to ethical and social reasons, in vitro assays are being developed to replace animal tests for addressing e.g. toxicological questions. for the induction of skin sensitization by chemicals, resulting in tolerance or allergic contact dermatitis after repeated exposure, prerequisites are the induction of inflammatory responses in keratinocytes supporting maturation of dendritic cells (dc), which is needed for the t cell response. although related in vitro assays consisting of one single cell type have good hazard prediction capacities, they have limitations in predicting sensitization potency. one drawback could be the lack of communication between keratinocytes and dc. with respect to the activation of keratinocytes and maturation of dc, intercellular communication between these two cells may include the release of danger molecules such as cytokines, damage-associated molecules such as atp, and metabolized chemicals. beside this, microrna (mirna), among them those that can regulate dc activation or maturation, can be differentially expressed upon stimulation but can also be transferred between cells. for skin sensitizers, we reported already that cross talk between hacat keratinocytes and thp-1 cells, as model for dc, enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol, belonging to a subgroup of chemicals (prohaptens) whose sensitizing potential depend on prior metabolic activation e.g. via cytochrome p450 (cyp) enzymes. furthermore, coculture clearly increased the upregulation of the cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens and also several other skin sensitizers. in this study we further elucidate the cross talk between thp-1 cells and hacat cells by analyzing the impact of hacat cells on the expression of mirnas in thp-1 cells by microarray technology. we identified 6 differentially expressed mirnas in cocultured thp-1 cells compared to monocultured thp-1 cells irrespective of the treatment (medium, 0.2% dmso as solvent control, b[a]p). in the presence of dmso and b[a]p (after 48h) 8 additional mirnas are differentially expressed. up to now it is not clear whether the cross talk between hacat and thp-1 cells comprises the exchange of mirna between the cocultured cells or whether it influences the expression of these mirna in thp-1 cells, or both. given that one mirna has several gene targets these results illustrate that the cross talk between thp-1 and hacat cells also impacts on the mirnome. walther-straub-institut der lmu-münchen, münchen, germany transient receptor potential (trp) proteins represent a large superfamily of nonselective cation channels sensing toxic stimuli in the human body. trpa1 expresses a high number of aminoterminal ankyrin repeats and is the only member of the trpa family. channel monomers form homotetramers in the plasma membrane with six transmembrane segments (tm) and a pore forming loop between tm5 and 6. trpa1 has been extensively described in sensory nerve endings as an important cellular detector for toxic stimuli and as an oxygen sensor (reviewed in 1). although recently two reports identified trpa1 in pulmonary epithelial and endothelial cells (2, 3) , its expression in non-neuronal tissues is still a matter of debate. after isolation and identification of different murine lung cells we were able to identify murine trpa1 protein in primary endothelial cells, pneumocytes type ii (atii) and fibroblasts by using specific antibodies in a western blot analysis, but not in cells from trpa1-deficient mice. atii cells were identified by specific cell markers such as surfactant protein c and were further differentiated to ati cells characterized by their specific expression of podoplanin. quantitative trp expression patterns will now be evaluated by quantitative reverse transcription (rt)-pcr as well as utilizing nanostring ® technology in different lung cells. to characterize trpa1 on a cellular level we cultured a hek293 cell line stably expressing trpa1 (4) . allylisothiocyanate (aitc) a specific activator as well as hypoxia and hyperoxia was able to induce ca 2+ -influx in this cell line, which was blocked by the specific inhibitor a96079. in the future, we will utilize the isolated perfused lung model (5) to quantify toxin-induced edema formation in ex vivo lungs from wt and trpa1-deficient mice after exposure to potential toxic inhalation hazards (tih see 6) to challenge the hypothesis of trpa1 as an important toxin sensor in the lung. by this strategy we hope to understand trpa1 function in lung cells and to evaluate trpa1 proteins as potential pharmacological targets for a specific therapeutic intervention during toxin-induced edema formation. metabolism by the intestinal microbiota is likely to contribute essentially to the plasma metabolite profile of the mammalian host organism and it requires adequate identification of effects of the microbiome on the endogenous plasma metabolite patterns. the current investigations present insights in the mammalian-microbiome cometabolism of endogenous metabolites. antibiotics have a profound effect on the micro-organism composition of the microbiome and hence on the mammalian-microbiome co-metabolism. the consequences, however, on the functionality of the microbiome (defined as the production of metabolites absorbed by the host) and which of these changes are related to the microbiome are not well understood. to identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have employed a metabolomics approach. to this purpose broadspectrum antibiotics belonging to the class of aminoglycosides (streptomycin, neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were administered orally for 28 days to male rats including blood sampling for metabolic profiling after 7, 14 and 28 days. fluoroquinolones and tetracyclines can be absorbed from the gut whereas aminoglycosides cannot. to distinguish between metabolite changes caused by systemic toxicity of the antibiotics and microbiome related changes, the metabolites identified in the metabolome pattern were compared to a list of metabolites known to be produced by the gastro-intestinal micro-organisms. beside changes mainly concerning amino acids and carbohydrates, hippuric acid and indole-3-acetic acid were identified as key metabolites being affected by antibiotic treatment. for each class the following gut metabolites were found to be unique: indole-3-propionic acid for aminoglycosides, taurine for fluoroquinolones, 3indoxylsulfate, uracil and allantoin for tetracyclines. for each class of antibiotics specific and selective metabolome patterns could be established. the results suggest that plasma based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight in the mammalian-microbiome co-metabolism of endogenous metabolites. drug-induced liver injury (dili) is still a major reason for termination of clinical trials and thus is an important concern in drug development. identification and prediction of dili in the clinic and in preclinical safety testing still relies on the classical clinical chemistry panel and histopathology with known limitations in sensitivity and specificity. in the last years bile acids (bas) have been studied as potential biomarkers to better characterize drug-induced liver injury with promising results (ellinger-ziegelbauer et al., 2011; luo, schomaker, houle, aubrecht, & colangelo, 2014; yamazaki et al., 2013) . to evaluate whether a targeted bile acid profiling via lc-ms/ms in plasma and liver tissue can improve assessment of liver injury, methapyrilene (mpy) a known hepatotoxin, or corresponding vehicle, was administered daily to male wistar rats at a low (30 mg/kg) and a high (80 mg/kg) dose. rats were sacrificed following 3, 7, or 14 consecutive daily doses, or after recovery 10 days following 14 consecutive administrations of mpy or vehicle. in addition to bile acids which were determined both in plasma and tissue, conventional preclinical safety endpoints (histopathology and clinical chemistry) assessment and gene expression profiling was performed in liver to obtain mechanistic information about potential changes in regulation of bile acid levels. conventional findings included periportal necrosis, inflammation and biliary hyperplasia, and increased liver enzyme activity and bilirubin levels during the treatment phase. the bile acid pattern showed increased levels of conjugated and unconjugated bile acids in low dose and high dose groups compared to the controls after administration of methapyrilene. furthermore, although liver enzyme activity and bilirubin levels in serum were decreased again in the recovery groups, suggesting recovering liver injury, bile acid concentrations remained elevated with no signs of recovery. analysis of transcriptomics data revealed decreased levels of mrna encoding α-methylacyl-coa racemase (amacr) 4 and 15 days after dosing, a gene responsible for bile acid synthesis. membrane transport systems for bile acids like sodium/taurocholate co-transporting polypeptide (ntcp) and organic anion transporting polypeptide 1 (oatp1) expression were down regulated as well, indicating that the increased bile acid concentrations in plasma and tissue could be attributable to reduced uptake by the hepatocyte. in summary the data suggest that targeted bile acid profiling could be used as potential biomarkers to enhance assessment of drug-induced liver injury. photorhabdus asymbiotica is an entomopathogen and emerging human pathogen causing soft tissue infections in humans. photorhabdus asymbiotica produces the bacterial protein toxin patox, which is cytotoxic for various cell lines and kills insect larvae. previous studies have established that patox harbors two enzymatic active domains, a glycosyltransferase and a deamidase domain. the glycosyltransferase domain inactivates host gtpases of the rho family by glcnacylation of a tyrosine residue in the effector binding loop, which results in the disassembly of the actin cytoskeleton. the deamidase domain deamidates a crucial glutamine residue in heterotrimeric gα i and gα q/11 proteins, which renders the g proteins constitutive active. sequence and structural homology analyses of patox revealed a third domain (patox p ) resembling peptidases of the c58 protease family. patox p contains the conserved catalytic triade (c/h/d) of papain-like cysteine proteases and shares sequence similarity with effectors from yersinia pestis (yersinia outer protein yopt) and pseudomonas syringae (avirulence protein avrpphb). transient expression of patox p in hela cells induces cell rounding and indicates a cytotoxic potential of patox p . incubation of patox p with linearized bovine serum albumin (bsa) results in cleavage products of bsa assuming proteolytic activity of patox p . mutation of the catalytic cysteine in patox p prevents cleavage of bsa and blocks cytotoxicity. we were not able to observe autocatalytic cleavage of patox constructs under various conditions. the intracellular activity of the protease domain is most likely involved in the pathogenicity of patox. vitamin d metabolism -involved in triazole fungicide toxicity? a. lehmann 1 1 background: in a 28-day rat feeding study with the azole fungicides cyproconazole (c), epoxiconazole (e), propiconazole (p), tebuconazole (t), prochloraz (pz) as well as combinations c+e and c+e+pz, a reduction of vitamin d (vitd) receptor mrna levels was reproducibly observed in adrenals for c, e and p. transcription of various enzymes related to vitd homeostasis (including cyp2r1, gc, cyp3a, ugt1a) in liver was also affected, while initial indications for modulation of renal cyp24a1 and renal and hepatic cyp27b1 could not be confirmed. a possible induction of parathormone (pth) was noted for the high dose of c, but statistical significance could not be shown. we have now performed supporting analyses for serum vitd levels, measured additional transcript levels and will provide a framework for the interpretation of the findings. methods: male wistar rats (n=5 for single substances, n=10 for combinations) were treated for 28 days at dose levels tested based on noaels from 90-day subchronic feeding studies and ranged from noael/100 to noaelx10. quantitative rt-pcr analyses were performed on organ samples obtained at sacrifice. serum vitd levels were determined using the total (25-oh) vitamin d elisa (drg instruments gmbh, marburg, germany). results: the elisa established for diagnostic analysis of human serum and plasma samples could be applied to rat serum. vitd levels in control animals (n=30) were 64.7±8.3 ng/ml (min/max: 48/78 ng/ml), i.e. in the range of values reported previously for rats. for the high dose of c (1000 ppm in food, n=5), there was a statistically nonsignificant reduction of vitd levels to 71.3±17.6% of the concurrent control (n=5). however, for 4 of 5 animals of this group, measured vitd level were below the range observed in pooled controls (n=30). an according follow-up is ongoing. qrt-pcr analysis of adrenal tissue showed deregulation of apoptosis related genes (p21 for c, e and pz; cdk1 and gadd45a for e; cdkn1c for c), which is in agreement with an involvement of vitd in the autocrine/paracrine regulation of cell proliferation. conclusion: reduction of circulating vitd levels would be plausible as a result of induction of hepatic cyp3a1/2 and ugt1a. however, this could not be confirmed by elisa as a general mechanism for all azole fungicides under investigation. only for rats fed with 1000ppm cyproconazole, there were indications for a moderate reduction of 25-oh vitamin d, which would correlate with the previously reported moderate increase in serum pth for this group. hansen's disease during pregnancy and lactation: two babies born to a mother using antileprosy drugs z. ozturk hansen's disease, also known as leprosy, during pregnancy has been rarely reported in europe and united states. early diagnosis is important, and medication can decrease the risk of those living with leprosy patients from acquiring the disease. this report presents a case of multidrug antileprosy therapy during pregnancy and lactation. a 26-year-old multiparous woman with a known case of multibacillary leprosy presented with unplanned pregnancy. her pregnancy was discovered in the 9th week, and she has been taking a multidrug therapy (dapsone 100 mg/day, rifampicin 600 mg/month, clofazimine 50 mg /day and clofazimine 300 mg/month) for the past 8 months. diagnosis of leprosy was established in her previous pregnancy. the patient was informed about the risks of drugs used in pregnancy. the treatment was continued unchanged during pregnancy. a detailed fetal ultrasonography was offered to scan the development of the fetus at about 20 weeks. in the 8th, 22nd, 28th weeks of pregnancy, prenatal sonographic examinations revealed normal fetal growth and amniotic fluid volume. at 28 weeks pregnant, she was diagnosed with gestational diabetes. diabetes did not cause any symptoms during pregnancy, and it was controlled with a reduced-calorie diet in a week. the patient delivered a healthy baby girl by vaginal birth in the 39th week of gestation without perinatal complications. the baby was also healthy (apgar 8-9,3300 g,51 cm), and its growth and development were normal during a 6-month follow-up period. the patient decided to breastfeed while taking medication. she had a previous experience with use of anti-leprosy drugs while breastfeeding, her other child was 15 months old and healthy. as well as in the first child, skin discoloration was observed in newborn due to clofazimine during lactation. after 3 months, she stopped breastfeeding, and the infant's skin changes were reversed. for pregnant women and practitioners, treatment of leprosy in pregnancy can be complicated. physical and neurological damage may be irreversible even if cured. multidrug therapy consisting dapsone, rifampicin and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discolouration of the infant due to clofazimine. therefore, multidrug therapy for leprosy patients should be continued unchanged during pregnancy and lactation. methods: individuals included in the analysis were participants of the berlin initiative study (bis). bis is a population-based prospective cohort study initiated in 2009 in berlin, germany, to evaluate kidney function in people ≥ 70 years. medication was assessed through personal interviews and coded using the anatomical therapeutic chemical classification system. for estimation of glomerular filtration rate (egfr) we used the ckd-epicr equation. predictor analysis was conducted via logistic regression. results: figure 1 illustrates the percentage of drug use for the three noacs and phenprocoumon, the most common vitamin k antagonist in germany, over the course of four years. table 1 shows the characteristics of patients for each oral anticoagulant group during the four-year follow-up visit (from january 2014 until april 2015). the probability of dabigatran use rose with increasing age (+12%), and the probability of phenprocoumon use rose in case of egfr < 60 ml/min/1.73m 2 (+54%) or male sex (+82%). discussion: our data show that also in the elderly noac use increased over the past years. characteristics such as age, sex or kidney function had an impact on the choice of oral anticoagulation. objective: orthostatic hypotension (oh) is an important factor in determining cardiovascular mortality especially in older age. different factors were discussed to influence oh. arterial stiffness, medication and frailty were demonstrated as modifying factors of oh. the aim of this study was to assess prevalence of and influencing factors on oh in nursing home residents (nhr) in germany. methods: systolic (sbp) and diastolic (dbp) blood pressure as well as pulse pressure (pp) and pulse wave velocity (pwv) as markers of arterial stiffness were measured in nhr aged ≥ 65 years in 12 nursing homes in berlin, germany. measurements were first performed in the sitting position and then repeated after standing up. oh was defined as a sbp decrease of > 20 mmhg and/or dbp decrease of > 10 mmhg within 3 min after standing up. hypertension was defined as the presence of diagnosis arterial hypertension, the prescription of at least one antihypertensive drug, or mean sbp values > 139 mmhg and/or mean dbp >89 mmhg. information about antihypertensive medication was received from interviews and medical records. frailty was determined by geriatric assessments, e.g. "timed up and go test" (tug) or barthel scale. results: oh testing could be performed with 96 nhr (mean age = 84.5 ± 7.3 years). in total, 15 subjects (15.6%) had oh. the mean change in sbp from sitting to standing was 19.2 ± 15 mmhg (range +8.5 to -52.5 mmhg) in patients with oh and 1.5 ±10.9 mmhg (range +44.5 to -17 mmhg) in patients without oh. mean sbp was significantly higher (143.6 ± 17.1 mmhg) in people with oh than in those without (131.5 ± 20.1mmhg). all of the nhr with oh were hypertensive compared to 89% of the nhr without oh. sex, mean age, pwv and pp was not significantly different between individuals with or without oh (p>0.05). medication data was available for 89 patients. all individuals with oh and 60 nhr without oh (80%) had antihypertensive medication. more than 2 different antihypertensive drugs were present in 11 patients with oh (78.5%) and in 43 patients without oh (57.3%). the intake of beta-blockers had no impact on oh development. geriatric assessments did not differ significantly between the oh group and the non-oh group. more than 75% of patients in both groups reached 80 points as maximum in barthel scale defining a need for assistance and tug analyses demonstrated that around 50% of patients with oh as well as patients without oh needed more than 19 sec showing a motor slowing. conclusion: we found a relatively low prevalence of oh in our very old patient cohort and the overall bp control was good. similar to earlier publications mean sbp was significantly higher in nhr with oh. all of the other investigated factors were not associated with the occurrence of oh. the small cohort size might have limited the detection of cardiovascular, epidemiological or geriatric associations. in addition, important confounding factors such as the inability to stand of some nhr and the lack of standardized fraility assessments must be addressed. impact of reticulated platelets on the initial antiplatelet response to thienopyridine loading in patients undergoing elective coronary intervention c. stratz 1 , t. nuehrenberg are known to be involved in cell metabolism pathways and therefore ccrcc is supposed to be a metabolic disease. in order to facilitate a better understanding of cancer metabolism and to support tumor classification on the metabolite level we have developed a novel analytical approach for comprehensive metabolomic profiling of small molecules and lipids in kidney tissue. the method was established and validated based on porcine tissue and, as proof of concept, applied to a small cohort of human normal and ccrcc tissue samples for molecular tissue differentiation. methods: five fresh frozen ccrcc samples and corresponding normal tissue were used for cancer-specific metabolomic profiling and were derived from patients who underwent partial or radical nephrectomy. metabolites and lipids were recovered from tissue samples by a two-step extraction protocol. tissue homogenization and extraction of polar metabolites was performed in methanol/water (aqueous extract) by a beadbeating approach. lipids were recovered by consecutive extraction of the pellet with methanol/methyl tert-butyl ether (organic extract). metabolites in aqueous extracts were separated by hydrophilic liquid interaction chromatography whereas compounds in organic extracts were separated by reversed phase chromatography prior high resolution mass spectrometry. results: reproducibility of tissue extraction and metabolite analysis was assessed by the analysis of multiple individually prepared porcine kidney samples. more than 1000 metabolic features including amino acids, nucleotides, small organic acids, phospholipids, sphingolipids, glycerolipids and fatty acids could be reproducible (cv ≤ 30 %) analyzed with the novel non-targeted metabolomics approach. the validated protocol was applied for metabolomic profiling of kidney tissue derived from ccrcc patients. based on unsupervised multivariate statistics, a clear differentiation between cancerous and normal tissue for the small metabolites profile as well as for the lipid profile could be observed. a first subset of differentially regulated metabolites responsible for tissue differentiation could be tentatively identified. conclusion: metabolomic profiling of kidney tissue extracts enables differentiation between ccrcc and normal kidney tissue samples based on the lipid and small molecule metabolomic profiles. further studies on larger and independent sample groups are necessary to confirm and validate our preliminary findings. in summary, the presented approach provides a first basis for comprehensive metabolomics studies in human kidney tissue and thus offers great potential for the metabolic characterization of ccrcc with important prognostic and therapeutic implications in the future. introduction: clomiphene (clom) citrate as mixture of trans-and cis-isomer (60:40) is the first line therapy for the treatment of infertility caused by the polycystic ovary syndrome. treatment schedule includes dose escalation from 50 mg/d clom citrate to up to 150 mg/d in case of non-ovulation. however, therapy outcome is variable and approximately 10 -30% of patients do not benefit from clom treatment. the pro-drug clom is bioactivated via 4-hydroxylation of trans-clom by the highly polymorphic cytochrome p450 (cyp) 2d6 leading to the major active metabolite trans-4hydroxyclomiphene (trans-4-oh-clom) [1] . recently, we identified a less active trans-3-oh-clom which is also formed by cyp2d6. besides the formation of the active metabolites, their plasma concentrations are influenced by their clearance e.g. via glucuronidation and sulfation. here we investigated the glucuronidation and sulfation of both hydroxyl-metabolites. methods: isoforms of udp-glucuronosyl-transferase (ugt) and sulfotransferase (sult) responsible for conjugation of oh-clom were identified using commercially available supersomes. glucuronidation and sulfation kinetics were determined in pooled human liver microsomes. conjugated clom metabolites were quantified in plasma and urine samples obtained from healthy female volunteers who received a single dose of 100 mg clom citrate. results: incubations with human liver microsomes revealed an almost 60-fold higher glucuronidation rate for trans-3-oh-clom, which is exclusively catalyzed by ugt2b7, compared to the more potent trans-4-oh-clom. for the latter a pattern of multiple ugts was identified. in contrast, the intrinsic clearance of trans-4-oh-clom to its sulfate is 16-fold higher compared to 3-oh-clom. for both metabolites a participation of sult1a1 and sult1e1 was identified. these results were in line with previous studies, which identified the same sults [2] and ugts [3] responsible for the conjugation of the structurally related trans-4-hydroxytamoxifen. in addition, in vivo data from plasma and urine samples confirmed the reverse regioselective glucuronidation and sulfation of trans-3-oh-clom and trans-4-oh-clom. overall, concentrations of clomglucuronides were significantly higher than those of sulfates. highest concentrations in plasma and urine samples were measured for trans-clom-3-o-glucuronide. conclusion: our results suggest a new metabolic route via trans-3-oh-clom which appears to be a potential inactivation pathway of clom. 1 1 institut für pharmakologie und toxikologie der bundeswehr, münchen, germany for decades the biological effect of sm has been investigated. it is well known how sm interacts and destroys cells. unfortunately, it is still unknown if and how a cell can become resistant against sm. within the here described experiments we investigated a new approach adapting cells to the presence of sm. over a time period of nearly three years the cells were cultivated in presence of sm with increasing concentrations. before starting the initial sm sensitivity was investigated. at the beginning cells were cultivated with a concentration of 0.07 µm sm (ic 10 ). today the cells are able to tolerate a concentration of 7.2 µm sm (ic 90 ), which reflects to a concentration of which 90% of the original cells would have died. to determine cellular characteristics, the resistant cells were compared with wildtype cells. the following cell characteristics were investigated: proliferation, apoptosis, clonogenicity, size of nuclei and cytoplasm, cell-cell contacts, dna adducts formation, secretome, screening of mirna expression, next generation sequencing, vital observation and scratch assay, nad(p) + /nad(p)h, h 2 o 2 , glutathione, ca 2+ -influx, mdrchannels, resistance to other alkylating agents and the reversibility of the resistance. the resistant cells demonstrate smaller nuclei and cytoplasm, less dna adducts, a higher clonogenicity as well as proliferation and less apoptosis. the secretome analysis showed an up-regulation of anti-apoptotic acting cytokines timp and ang and the proproliferative acting cytokines timp and pdgf-aa. in contrast, immunologically active cytokines were down-regulated. concerning cell-cell contacts no differences were seen. in the mirna screening 49 significant up-regulated and 20 significant down-regulated mirnas have been observed. noteworthy was the regulation of various members of 11 different families. during vital observation and in a scratch-assay the resistant cells were show to have disadvantages. the observed resistance was not unique for sm but also towards other alkylating agents and cytostatic drugs. by analyzing the reversibility cells stayed resistant over more than 35 weeks. in conclusion, many aspects investigated in this study have an influence on the sm resistance, pointing out that it is a combination of various effects that are involved to switch on resistance. more likely, there are many aspects working together. the present results are an important step in the characterization of the sm-resistant cell line and further studies may be able to directly use these as a start for target identification in antidote or prophylactic agent discovery. the arylhydrocarbon receptor (ahr) is localized in a cytosolic complex that contains several co-chaperones and associated factors. the protein is shifted into the nucleus in response to endogenous and xenobiotic ligands. however, a transient nuclear transport does also occur in the absence of any ligands, while the predominant cytoplasmic compartmentalization is maintained by parallel export. we have analyzed the interplay between this basal nucleo-cytoplasmic shuttling and ligand induced transport in hepg2 cells, using a yfp-tagged fusion protein that is capable to respond to ligands and to trigger the induction of cyp1a1 expression. basal import was assessed in cells that had been treated with leptomycin b (lmb), an inhibitor of crm1-mediated nuclear export. interestingly, the apparent ahr import rate in lmb-treated cells was comparable with nuclear import as trigged by xenobiotic (b-naphthoflavone) or endogenous (kynurenine) ligands. this observation was confirmed for endogenous ahr in hepg2 cells, since both ligands and lmb showed comparable effects on nuclear compartmentalization. however, the basal nuclear import rate in lmb-treated cells was strongly increased by ahr ligands. ligand-induced nuclear transport was therefore confirmed as an import step in receptor activation. interestingly, lmb did also accelerate nuclear import of ahr after pretreatment of cells with ahr ligands. these data suggest that nuclear export of the ahr is maintained in the presence of ligands. receptor activation might therefore comprise several rounds of shuttling, thereby involving both accelerated import and continued export of the ahr protein fraction that has not already undergone interactions with arnt or dna. we suggest that nuclear export provides an additional kinetic control of ahr activation and function. mitochondrial toxicology: rescuing mitochondria in wilson disease avoids acute liver failure h. zischka 1 1 institut für molekulare toxikologie und pharmakologie, ag zischka, neuherberg, germany in wilson disease (wd) functional loss mutations in the hepatocyte atp7b gene cause dramatic copper overload leading to acute liver failure, posing an unmet therapeutic issue. we find that the pathology of severe wd cases is mirrored in lpp (-/-) rats carrying a functional loss atp7b mutation. this is especially apparent in the hepatocyte mitochondrial compartment. a progressive copper deposition increasingly harms the lifesustaining mitochondrial membrane integrity. thus, depleting this devastating mitochondrial copper burden is a core requirement for a treatment strategy against acute liver failure in this wd animal model. preparation for the master degree program in toxicology started in 2006 as a cooperation of charité universitätsmedizin berlin with the university of potsdam and other institutions of the region. first enrollment of students was done in 2008. the program was accredited in 2011 by the central evaluation and accreditation agency. it offers a modern curriculum encompassing a wide variety of scientific aspects with an interdisciplinary character. this training program in toxicology is organized in modules and ends with the degree "master of science" (m.sc.). the goal of this program in toxicology is to teach the basis of the interactions between substances at toxic concentrations and living organisms, as well as the molecular mechanism of the adverse effects of chemicals. the understanding of the mechanism of a toxic action is an important prerequisite for the scientifically based evaluation of a hazard associated with a substance. furthermore, only with the knowledge of the mechanism of action and a deduction of structure activity relationships it is possible to predict toxic effects of new substances. this knowledge should enable students to perform a risk evaluation of chemicals or to predict the adverse effects of chemicals with the aim that human beings and the environment can be protected from harmful consequences of chemical exposure. the program allocates 30 places per year to an average of 60 applicants. most applicants have a basic training in the fields biology, chemistry, pharmacy, veterinary medicine and nutritional sciences. about 75% of the students are female. the majority of them have a bachelor's degree before starting the master program, other degrees are diploma and state examination as pharmacists or physicians. ninety percent of the students pass the final examination consisting of the master's thesis and disputation at the end of the four semesters. afterwards, most of the graduates aim to obtain a phd degree. the program is well established in the education of toxicologists in germany. respiratory injury due to chlorine developed from consumer products. still an issue in germany u. stedtler 1 , m. hermanns-clausen 1 1 uniklinikum freiburg, vergiftungs-informations-zentrale, freiburg, germany objective: in the last decades strong effords have been took to improve product safety, especially in products intended for domestic use. hypochlorite-containing cleaners may develop chlorine gas when acidified e.g. by adding an acid sanitary cleaner. usually these cleaners contain sodium hydroxide or other strong alkalines to avoid this reaction. we analysed reports to our poisons center concerning inhalation exposure to chlorine developed from hypochlorite-containing mixtures. method: retrospective search in the case database of the poisons center. human inhalative exposures to chlorine released from mixing hypochlorite as well as human inhalative hypochlorite exposure alone were analysed. frequency and symptoms were compared. results: from 2010 to 2015 in total 85 cases of human exposures to chlorine developed from mixtures of hypochlorite and acids (0.8 of 1000 cases) were registered. in 55 cases the exposure was due to mixtures of products intended for domestic use. 94 % of the exposed patients reported symptoms. only in two cases the symptoms were not considered to be caused by the inhalation accident. most frequent symptoms reported were (percent of symptomatic patients): cough (45 %), dyspnea ( 33 %), irritated upper airway (26 %), abdominal discomfort (pain, nausea, vomiting) (21 %), thoracic pain (20 %), irritated eyes (11 %), dizziness (8%), and bronchospasm (6 %). further symptoms were malaise, headache, irritated nose, sweating, muscle pain, and others. in 12 patients (14 %) the symptoms were graded as moderate severe. main symptoms in this group were dyspnoea ( 83% ), cough, and irritated airway. one third of the patients experienced bronchial obstruction. all symptomatic patients developed symptoms while exposed or shortly after exposure. there were no severe or fatal cases (especially no lung edema) and all symptoms were expected to resolve completely. because hypochlorite containing procucts sontanously release "chlorine-like" smelling gases, we additionally analysed inhalation exposures to hypochlorite solutions alone in the same period. there were 42 patients in the same period exposed to hypochlorite evaporation alone. 36 of them (86 %) had symptoms of which in 30 cases these were considered to be caused or possibly be caused by the hypochlorite. most frequent symptoms were irritated upper airway (33 %), nausea or vomiting (30 %), cough (23 %), irritated eyes (20 %). dyspneoa was less fequent than in the mixture group (10 %). all symptoms were considered mild. there was no bronchospasm or thoracic discomfort. conclusion: respiratory injuries by chlorine from hypochlorite-containing solutions still occur despite clear warning on the label. the majority of cases was due to products for domestic use. symptoms develop shortly after exposure. the γh2ax assay for genotoxic and nongenotoxic agents: comparison of h2ax phosphorylation with cell death response perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? regulation of chromatin by histone modifications transcriptomic alterations induced by ochratoxin a in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model in vitro gene expression data supporting a dna non-reactive genotoxic mechanism for ochratoxin a fragment ion patchwork quantification for measuring site-specific acetylation degrees combinatorial patterns of histone acetylations and methylations in the human genome inroads to predict in vivo toxicology -an introduction to the etox project value of shared preclinical safety studies -the etox database acknowledgements: support of the bfr through grant 1322-530 is gratefully acknowledged. acknowledgement: supported by the robert bosch foundation, stuttgart, germany. [1] mürdter t, et al. hum mol genet, 2011, 21:1145-54 [2] nishiyama t. et al., biochemical pharmacology, 2002, 63:1817 -1830 [3] sun d. et al., drug metabolism and disposition, 2007, 35:2006 background: infections are a major problem in patients with burn diseases (bd). due to severe injuries of their total body surface area (tbsa), burn patients have altered pharmacokinetic characteristics. therefore, insufficient plasma concentrations may be achieved, when standard dosing schedules are applied for antibiotics such as piperacillin. for time-dependent antibiotics, the duration how long drug concentration exceeds the minimal inhibition concentration (mic) is crucial for their antibacterial effects. since pseudomonas spp. is the main problematic pathophysiological bacterium for bd patients. the aim of the present study was to monitor the plasma concentrations of piperacillin during piperacillin/tazobactam treatment in bd patients. patients from intensive care units (icu) served as controls. methods: 10 bd patients (5/5 m/f, 43.4±5.3y, tbsa 40 .9±5.9%) and 5 patients (74.4±3.9y) from the icu were included in this observational study. blood samples were taken within the 3 rd interval of the 8h-lasting dosing period of piperacillin/tazobactam (4/0.5g within 0.5h) at 1, 4 and 7.5h after the end of infusion. total and free piperacillin concentrations were determined in plasma using hplc-uv after deproteinisation with acetonitrile and by ultrafiltration, respectively. pharmacokinetic parameters and dosing simulations were calculated by tdmx (www.tdmx.eu). free plasma concentrations of piperacillin exceeding at least 1xmic but preferably 4xmic over the whole dosing interval were considered to be sufficient for antibiotic efficacy (mic 16 mg/l for pseudomonas spp.,www.eucast.org). results: the pharmacokinetic parameters of total piperacillin, calculated for each bd or icu patient using the concentrations at 1, 4, and 7.5h, were as follows: c max 69.6±7.9 vs.116.3±10.4 mg/l, p<0.05, half-life 1.8±0.3 vs. 2.3±0.3h, p>0.05, clearance 12.6±1.9 vs. 6.5±0.4 l/h, p<0.05, volume of distribution 27.8±2.0 vs. 20.9±1.3l, p<0.05. free concentrations (which were included in tdmx calculations) were 87±2 vs. 81±2% (p<0.05) of total concentrations. duration per day while concentrations exceeded 1xmic (15.6±1.9 vs. 22.0±1.1h, p<0.05) or 4xmic (5.4±1.3 vs. 9 .4±0.7h, p<0.05) were lower in bd than in icu patients. moreover, tdmx simulations predicted that the duration per day for 4xmic could be enhanced to 16.2±1.5h if the piperacillin amount will be increased to 4x8 g/d and the infusion duration to 3h. pharmacokinetic parameters have, however, to be determined in a pilot study with bd patients to ensure predicted values. conclusions: standard dosage regimens for piperacillin/tazobactam could result in suboptimal plasma concentrations of piperacillin in bd patients as well as in icu patients. drug monitoring and tdmx simulation of kinetic parameters may easily help to improve piperacillin treatment in bd patients. background: high dose methotrexate (hd mtx), defined as >1000mg mtx/m 2 bodysurface-area (bsa) is used in children to treat a variety of malignant diseases since the 1950s. clinicians observe relevant rates of severe unwanted side effects. identifying patients having an increased risk for toxicity due to altered mtx pharmacokinetics is urgently needed. we aim to develop and evaluate a physiology-based pharmacokinetic (pbpk) model for hd mtx in children using pk-sim® (bayer technology services gmbh, leverkusen, germany) with a special emphasize on relevant covariates. methods: in this non-interventional observational study, children receiving hd mtx intravenously at two major german pediatric oncology departments during the years 2004-2009 were included if at least one mtx serum level (mtx-sl) was determined during clinical routine. 29 patients aged 2-18 years (male = 19, female = 10) with following diagnoses were included: acute lymphoblastic leukemia, non-hodgkin lymphoma, burkitt lymphoma, brain stem glioma and glioblastoma multiforme. in total, 103 mtx treatment cycles corresponding to 300 mtx-sl were used in this study. patients were randomized into two patient sets (training set and test set). based on literature data, mtx pbpk-models were developed and slightly adapted taking into account mean relative deviation (mrd) and bias of predicted versus observed mtx-sl of the training set. the pbpk model with the lowest mrd and bias was chosen and finally evaluated using the test set. the impact of the covariates urine ph <6.5, trimethoprime/sulfamethoxazole, proton-pump-inhibitors, non-steroidal anti-inflammatory drugs and ß-lactam antibiotics on the prediction quality was assessed using the mann-whitney u test. ochratoxin a (ota) is a wide-spread food contaminant and one of the most potent renal carcinogens [1] . recent data by our group demonstrate that ota inhibits histone acetyltransferases (hats), thereby causing a global reduction of lysine acetylation of histones and non-histone proteins [2] . based on these findings and the importance of specific histone acetylation marks in regulating gene transcription [3] , we speculated that repression of gene expression as the predominant transcriptional response to ota [4, 5] may be linked to loss of histone acetylation. in this study we therefore used a novel mass spectrometry approach, which is based on chemical acetylation of unmodified lysine residues of histones using 13 c-labeled acetic anhydride and subsequent calculation of the degree of acetylation based on the measured intensities of heavy and light acetylated isotopologues [6] , to identify and quantify site-specific alterations in histone acetylation in human kidney epithelial (hk-2) cells treated with ota. our results demonstrate ota-mediated loss of acetylation at almost all important lysine residues at histones h2a, h2b, h3 and h4. we further selected acetylation at histone h3 lysine 9 (h3k9), a well-known euchromatic hallmark that is elevated at promoter regions of transcriptionally active genes [7] and which was reduced from ~ 3% in controls to < 0.1% in response to ota, to establish a link between loss of h3k9 acetylation and expression of genes consistently shown to be down-regulated in response to ota [4, 5] . using chromatin immunoprecipitation followed by quantitative real-time pcr (chip-qpcr), we observed ota-mediated loss of h3k9 acetylation at promoter regions of the selected genes (% of controls: amigo2: 45%, clasp2: 60%, ctnnd1: 54%). overall, these data provide first evidence for a mechanistic link between h3k9 hypoacetylation as a consequence of ota-mediated inhibition of hats and repression of gene expression by ota. a new paradigm to assess the proarrhythmic potential of drugs is proposed by the cipa (comprehensive in vitro pro-arrhythmia assay) initiative combining a suite of a priori in vitro assays (7 most important ion channels for cardiac activity) coupled to in silico reconstructions of cellular cardiac action potential (ap). the etox consortium has developed a multiscale simulation in silico model based on o'hara/rudy incorporating the principles of this new paradigm. the core model simulates the effects of drugs on a virtual cardiac tissue composed by different types of cardiomyocytes. the input of this model, the blockade of a set of 3 ion channels (ikr/herg, iks, ical), can be obtained experimentally or predicted using advanced 3d-qsar models. the system predicts the % change of the qt interval at different drug concentrations in order to facilitate risk assessment. this in silico model was validated using purkinje fiber assay results (input: ap prolongation and arrhythmogenic risk assessed by early after-depolarisation occurrence) from 500 in-house drug candidates. the validation showed that predictivity is highly dependent on the model's applicability domain (ad): for some chemical series the proarrhythmic potential could not be identified, for others, however, most of the positive drugs were correctly predicted with sensitivities up to 80-90% (average prediction accuracy was 70%). retraining of this model with additional internal data should help to improve the model ad and predictivity. it is important to note that ap prolongation was correctly predicted for many proarrhythmic drugs with only low (> 30 µm) in vitro herg inhibition. furthermore, the model showed high additional benefit for read-across within bayer pharma ag, investigational toxicology, berlin, germany etox [1] [2] started in 2010 and is a public-private partnership project within the european innovative medicines initiative (imi) [3] . the etox project is building a toxicology database relevant to pharmaceutical development and to elaborate innovative strategies and software tools. the overall goal is to better predict the toxicological profiles of new chemical entities in early stages of the drug development pipeline based on existing in vivo study results contributed by the participating efpia * companies in the consortium. the etox database is a relational database with a specifically designed schema to store complex and comprehensive preclinical safety data like the study design, toxicokinetics, adme data, clinical chemistry, hematology, gross necropsy, histopathological findings and general toxic effects. in addition relevant data from public sources has been included into the database. the primary focus for data collection are systemic toxicity (up to 4 week) repeated dose studies, mostly in rodent. overall more than 7000 study reports for approximately 1400 investigated compounds. in order to optimize the usage and mapping of data from different sources the development of common ontologies was a key task within the project. this timeconsuming step was necessary to make a high quality read-across analysis possible and valuable. therefore the ontobrowser [4] tool was developed to curate and harmonize the verbatim terms to standardize terms which are used within the etox database. until now more than 13 million verbatim terms were curated. additionally to the toxicology database, a web-based user interface called etoxsys was developed to allow the retrieving of toxicity information, as well as the prediction of toxic endpoints for chemical compounds. due to the complex search capabilities, the database can be queried for structural similarity, similar target classes and specific toxicological endpoints. approximately 150 prediction models based on public data are available and first models based on in vivo data are in development. the etox database therefore represents a valuable tool for early animal-free assessment of drug candidates [5] . * european federation of pharmaceutical industries and associations cell lines background: consumers are constantly exposed to chemical mixtures e. g. to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. since substances are tested for regulatory purposes on an individual basis at generally high dose levels, there is only limited data available on potential mixture effects especially in the low dose range. with more than 400 active substances approved for being used in pesticides and over 100000 chemicals registered under reach there are more possible combinations than one could test with classical animal experiments. the development of in vitro tools for assessment of mixture effects consequently is of tremendous importance. methods: as a first step in the development of such in vitro tools we used a group of fungicides, (tri-)azoles, as model substances in a set of different cell lines from known target tissues, basically liver (human: hepg2, heparg, rat: h4iie) and adrenal gland (human: h295r). concentrations were taken from measured tissue concentrations in vivo to ensure that used concentrations of the (tri-) azoles reflect realistic effect levels. the cell lines were exposed with the triazoles cyproconazole and epoxiconazole as well as with the azole prochloraz as individual substances and in binary or ternary combinations of these substances at three dose levels and three different time periods. the effects of the substances were subsequently analysed by transcriptomics and metabolomics. a support vector machine will be utilized to integrate the data from the different sources to gain a complete picture of affected adverse outcome pathways and mechanistic information about the applied fungicides. first results indicate combination effects of the substances also at the omics level depending on the specific endpoint and the concentration used. some of these are comparable to effects found with similar methods in a standard toxicity test, a 28-day feeding study in the rat, thus raising hope for the development of in vitro methods suitable to detect combination effects. background: plant protection and biocide products are chemical mixtures, which contain one or more active substances as well as several co-formulants (e.g. solvents, wetting agents, thickener or preservatives). nevertheless, to this day extensive toxicological testing is performed only with the individual active substances, while the plant protection products are only evaluated for acute toxicity, ie, a single dose group experiment with rats is performed as well as testing for skin-and eye-irritation. current pesticides regulation foresees testing of potential harmful mixture effects but only when adequate methods are available making the development of such methods a high priority. several published studies both in vitro and in vivo have shown fortified toxic effects of plant protection products compared to individual active substances. methods: here we present effects of plant protection products as a whole as compared to the individual active substances or co-formulants in a set of human cell lines of hepatic and renal origin (hepg2, heparg, hek293). cytoxicity has been analysed by wst-1 and nru assay as well as gene expression of several marker genes involved in xenobiotic metabolism. additionally reporter gene assays have been conducted for nuclear receptors such as ahr and car. results: while some active substances showed lower toxicity as compared to the respective products, this cannot be confirmed as a general rule for all endpoints for all of the analysed fungicides or herbicides containing active substances such as epoxiconazole, cyproconazole, azoxystrobin or glyphosate. chemical compounds may induce skin sensitization in humans, resulting in tolerance or allergic contact dermatitis after repeated exposure. mechanistically, the activation of dendritic cells is one of the prerequisites for the induction of skin sensitization. a subgroup of sensitizing chemicals, prohaptens, need metabolic activation, e.g. via cytochrome p450 (cyp) enzymes. thus, xenobiotic metabolism may crucially impact on a chemical's potential for the induction of skin sensitization by activation, but also deactivation of reactive molecules via conjugation, which determines the concentration and the chemical species available for protein haptenation and cell activation. we established a coculture model consisting of hacat keratinocytes and thp-1 as surrogate dendritic cells for the detection of sensitizing chemicals and found enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol as well as clearly increased expression of cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens (hennen et al., 2011) . here, we studied the impact of intercellular cross talk on activation and conjugation capacities in more detail. treatment of thp-1 with b[a]p and eugenol in coculture with hacat cells augmented cyp1a1 and/or cyp1b1 mrna levels, while this was not found for thp-1 monoculture. augmentation of cyp1a1 mrna needed continuous presence of hacat cells. in coculture, levels of 3-oh-b[a]p as exemplary cyp-dependent metabolite were increased compared to single cultures. in contrast to this, total glutathione contents as well as n-acetyltransferase 1 enzyme activities in both cell types were not modulated in coculture, furthermore the capacity for sulfation/glucuronidation of 3-oh-b[a]p was maintained in coculture. additionally, the decrease of the total glutathione content in thp-1 cells by 2,4-dinitrochlorobenzene (dncb) was much less pronounced when exposed in coculture with hacat cells, showing that hacat cells provide additional targets for cysteine-reactive chemicals such as dncb, diminishing the total amount of chemicals available for thp-1 cells.overall, results indicate that the cross talk between keratinocytes and antigenpresenting cells enhances their capacities for metabolic activation of chemicals, while hacat cells also provide supplementary capacities for phase ii reactions. references: hennen j et al. cross talk between keratinocytes and dendritic cells: impact on the prediction of sen-sitization. toxicol sci 2011 123:501-510.toxicology -toxic pathway analysis/aop 395 background: reticulated platelets are associated with impaired antiplatelet response to thienopyridine treatment. this interaction might be caused by intrinsic properties of reticulated platelets or a decreased drug exposure due to high platelet turnover reflected by reticulated platelets as surrogate. we investigated the impact of reticulated platelets on antiplatelet response to thienopyridines and if this effect is linked to platelet turnover. methods: this study randomized elective patients to loading with clopidogrel 600mg or prasugrel 60mg (n=200). adp-induced platelet reactivity was assessed by impedance aggregometry 30 to 120 minutes and day 1after loading but before intake of the next dose of thienopyridines. immature platelet count (ipc) was assessed as marker of reticulated platelets by whole blood flow cytometry. results: platelet reactivity increased with rising tertiles of ipc (figure) . this effect was more pronounced in patients on clopidogrel as compared to patients on prasugrel. overall, ipc correlated well with on-treatment platelet reactivity at 120min (r=0.21; p<0.001). this correlation did not change over time indicating an effect independent of platelet turnover (comparison of correlations 120min/day 1: p=0.57 for clopidogrel, p=0.76 for prasugrel). conclusion: a high immature platelet count is associated with impaired response to thienopyridine loading. this effect is independent of platelet turnover indicating a relation to intrinsic properties of reticulated platelets. introduction: one of the biggest drawbacks of protein-based therapeutics with intracellular targets is their inability to enter the cytosol. targeted toxins are known to be used in drug delivery. aim of the study was to target epidermal growth factor (egf) receptor overexpressed on pancreatic carcinoma using a novel well-defined targeted toxin consisting of egf fused to the toxic plant ribosome-inactivating protein dianthin and a glycosidic triterpenoid (so1861) as efficacy enhancer. methods: the enzymatic activity of dianthin-egf was verified by an adenine release assay. the kinetics of cytotoxicity were evaluated in pancreatic adenocarcinoma bxpc-3 and miapaca-2 cells in comparison to the non-target cell line nih3t3 with an impedance-based real time cell analyzer (xcelligence) and final cytotoxicity analyses with conventional end-point mtt assays. acute toxic of dianthin-egf was studied in male balb/c mice. a xenograft solid tumor model was developed in male nude mice by injecting bxpc-3 cells into the dorsal part subcutaneously. dianthin-egf was administered at the vicinity of the tumor and so1861 by subcutaneous injection at the neck. after the tumor reached a diameter of 2 to 3 mm in size 6 treatments were given in total. tumor volumes and body weight shifts were observed twice weekly to determine the potency of dianthin-egf when given alone and in combination with so1861 in comparison to placebo. immunohistochemical detection of egf receptor was performed according to the manufacturers's advice (dako, glostrup, denmark, k1492). complete blood count analysis was done by labor 28 gmbh, berlin. results: the adenine release mediated by dianthin-egf was 47.8 pmol adenine/pmol toxin/h. the in vitro efficacy of the targeted toxin was proven by an ic50 value of approximately 1 nm for egf receptor expressing miapaca-2 and bxpc-3 cells as compared to 100 nm for non-target nih3t3 cells. real time measurement of the cytotoxicity showed a dose-dependent decrease in cell viability from 10 pm to 1 µm. toxicity studies in balb/c mice revealed 0.4 µg/mouse to be non-toxic and maximum tolerated dose (mtd) whereas 40 µg caused moribundity accompanied with white ocular discharge. efficacy studies were performed for a period of 28 days. the combination therapy showed that the average tumor volume measured by a digital vernier caliper was found to be 80% less than for placebo whereas single therapy using dianthin-egf alone caused a further increase in tumor volume which was although yet 50% less when compared to placebo. immunohistochemistry slides showed egf receptor expression in each of all untreated xenograft tumors, which further confirms the presence of egf receptor overexpression in the target bxpc-3 cell line. enlarged spleen was only observed in untreated xenografts. no significant change in various blood parameters (rbc counts, wbc counts, hgb, hct, mcv, mch and mchc) were observed on hematological analysis except for the platelet (plt) counts in comparison to healthy male nude mice. conclusion: combination therapy with so1861 proves to be a promising approach for the targeted delivery of toxins instead of single therapy administering targeted toxin alone. the strategy is specific for egf receptor overexpressing tumors such as pancreatic cancer. introduction: moringa oleifera (mo) is a popular herbal supplement used for treatment and management of diverse diseases in sub-saharan africa. its intake among individuals infected with hiv/aids has increased recently due to the purported immune boosting property. limited information, however, is available regarding its potential to cause interactions with commonly prescribed medications that are substrates of cyp3a4 and p-glycoprotein. methods: the methanol extract and four fractions of mo were tested on recombinant cyp3a4 at different concentrations with and without nadph to determine the ic 50 shift reduction. the crude methanol extract of mo was incubated with testosterone (tst) and cryopreserved hepatocytes to evaluate its influence on clearance of tst. effect of mo on the efflux transporter, p-glycoprotein was investigated by incubating the methanol extract with mdr1 -mdckii cells. virtual screening was conducted to predict physicochemical properties, bioavailability and interaction potential of phytochemical compounds unique to mo using combination of molinspiration version 2014.11 and admetsar. results: fractions (f1-f3) indicated ic 50 shift reduction ≥5 post-incubation with and without nadph. mo showed moderate interaction (auc i /auc = 2.46) with tst in cryopreserved hepatocytes. also, mo mildly inhibited the transport of digoxin (ic 50 = 35.45 µg/ml) across mdr1 -mdckii cells. niaziminin indicated 85.57% bioavailablity via the human intestinal membrane with 61% chance of inhibiting cyp3a4. βsitostenone showed strong p-gp inhibition (83.27%) with 100% absorption via the intestine. conclusions: mo has the potential to inhibit the metabolism or excretion of other medications that are eliminated by cyp3a4 or p-glycoprotein, respectively, if adequate amounts of the active constituents such as niaziminin and β-sitostenone enter the circulation. background: herb-induced liver injury (hili) has attracted attention in the past years due to an increasing number of publications reporting cases of hepatotoxicity associated with use of phytotherapeutics. here, we present data on hili from the berlin case-control surveillance study fakos. methods: fakos was initiated in 2000 to study serious toxicity of drugs including hepatotoxicity. potential cases of liver injury were ascertained in more than 180 departments of all 51 berlin hospitals from october 2002 until december 2011. through a standardised face-to-face interview and review of medical charts information on all previous intakes of drugs or herbals, on co-morbidities, and demographic data was ascertained. inclusion criteria were an elevation of alanine aminotransferase or aspartate aminotransferase threefold above the upper limit of normal or an elevation of total bilirubin higher than 2 mg/dl. excluded were patients with underlying liver disease (e.g., alcoholic fatty liver disease). drug or herbal aetiology was assessed based on the updated council for international organizations of medical sciences (cioms) scale. results: of all 198 cases of hepatotoxicity included into the fakos study, herbs were involved in ten cases (5.1%). demographic, clinical, and laboratory characteristics of these ten cases are illustrated in table 1 . among the six patients with available liver biopsy results, five patients showed signs of necrosis, either disseminated or predominantly near the central vein. portal inflammation was more common than lobular inflammation, and the infiltrates contained mostly lymphocytes, neutrophil or eosinophil granulocytes. herbal aetiology was judged two times as probable (ayurvedic herb in patient 1, pelargonium sidoides in patient 6), and eight times as possible (valeriana in patients 3, 4, 8, 9, 10 , mentha piperita in patient 5, hypericum perforatum in patient 2, eucalyptus globulus in patient 7). in nine cases other non-herbal drugs were also suspected as potentially hepatotoxic (exception: patient 6). seven cases occurred in the ambulatory setting requiring hospitalisation, three cases occurred during hospital stay. discussion: this case series provides further information on laboratory and clinical aspects of hili. it corroborates known risks for valeriana and ayurveda treatment, and suggests that further herbals rarely or never associated with liver injury before such as pelargonium sidoides, hypericum perforatum or mentha piperita could also exhibit a hepatotoxic potential. clinical routine often requires to evaluate the cause of a newly occurring adverse event. if this event is regarded to be iatrogen, further information of the association between the drugs in the current medication list and the adverse event is needed. this information should ideally reflect the true risk and allow ranking of the drugs according to this risk to identify which drug to discontinue first. we discuss the summary of product characteristics (spc), the sider side effect resource and openvigil 2 as possible sources of information. spcs are becoming more and more a vindicative charter for pharmaceutical companies that contain misleading information which is not based on evidence (ref. 1). since it relies on the spcs, sider inherits these shortcomings and flags warnings that result from confounding factors (ref. 1, fig. 1 ). furthermore, if any rates are given, they are not easily comparable since they stem from different studies. pharmacovigilance data are biased by the very nature of the data and the collection method. however, once confounders are eliminated, pharmacovigilance offers better information om how to rank the drugs than spcs/sider. we present decision-guiding information obtained by sider and by openvigil 2 for one of our patients ( fig. 2 & 3 ) and discuss how this information was used to modify the therapy. institut für naturheilkunde und klinische pharmakologie, universität ulm, ulm, germany background: differences (polymorphisms) in target genes or genes encoding drug transport proteins or drug metabolizing enzymes may be responsible, among other factors, for observed variation in patients' response to medications. pharmacogenetics aims at identification of patients at higher, genetically determined, risk of adverse drug effects or ineffective medication, to modify dosage or switch to alternative therapy. there is, however, a lack of awareness of pharmacogenetic-based clinical practise guidelines. methods: a systematic literature review was conducted which focused on published guidelines on genotype-based (germ-line genetic variants) dosage modification or selection of drugs. we serched the medline and the pharmacogenomics knowledgebase (pharmgkb) databases. prescribing information was also screened for pharmacogenetic guidance. results: the systematic review revealed recommendations for 61 drugs (table) that enable the translation of genetic test results into actionable prescribing decisions. for 20% of these drugs the respective german drug labels recommend or even require pharmacogenetic testing (table, 3rd column). although pharmacogenetic testing is recommended, the prescribing information not always provides guidance on how to adjust the drug dosage based on the pharmacogenetic test result. compared with the german or european drug labels, the fda drug labels povide more detailed information on pharmacogenetic dose modifications. conclusions: academic working groups have a front-runner role in the development of prescribing recommendations based on genetic markers. to date, drug labels rarely contain detailed guidelines how available genetic test results should be used to adjust drug dosage. because pharmacogenetics has a growing role during drug development and pre-prescription genotyping will become more widespread, it is expected that specific pharmacogenetic guidance for the treating physicians will become increasingly important. bisphenol a (bpa) is a high production volume compound mainly used as a monomer to make polymers for various applications, including food-contact applications. people are exposed to low levels of bpa because very small amounts of bpa may migrate from the food packaging into foods or beverages. however, other potential sources of exposure, such as dermal contact have also been identified (efsa, 2015) . a substance evaluation process (corap) was initiated for bpa by the european chemicals agency (echa). as part of the safety evaluation of bpa, a study was required by echa to assess absorption and metabolism of bpa following dermal exposure to human skin. an in vitro study with human skin was requested according to oecd tg 428 under consideration of the scientific committee on consumer safety (sccs) criteria for the in vitro assessment of dermal absorption. to investigate potential dermal bpa metabolism fresh human skin was used. abdominal skin was obtained fresh from surgery from 4 different donors. split-thickness human skin membranes were mounted into flow-through diffusion cells (n=4 per dose and donor) and the receptor fluid was pumped underneath the skin at a constant flow rate. the skin surface temperature was maintained at 32°c throughout the experiment and electrical resistance barrier integrity testing was performed at the start (0 h) and end of the experiment (24 h). four test preparations at final bpa concentrations of 2.4, 12, 60, and 300mg/l were investigated. the highest concentration was chosen based on the maximum solubility of bpa in water and the lowest concenration was chosen based upon the specific activity of the radiolabelled [ 14 c]-bpa that could be used for mass balance. percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (dmem), containing ethanol (ca 1%, v/v), uridine 5'-diphosphoglucuronic acid (udpga, 2 mm) and 3'-phosphoadenosine-5'-phosphosulfate (paps, 40 µm)), at multiple time points througout the experiment. at termination the skin was removed from the cells and the stratum corneum was removed with 20 successive tape strips. the exposed epidermis was separated from the dermis using a scalpel. metabolism was investigated for the highest concentration (300 mg bpa/l) only, using a hplc with in-line radiodetection and confirmed bpa-glucuronide (bpa-g) and bpa-sulfate (bpa-s) standards for comparison. no metabolism was observed in any of the epidermis samples, however some metabolism is observed in dermis and receptor fluid samples. metabolites were identified with retention consistent with bpa-g and bpa-s, and also some more polar components. the mean total absorbed dose (receptor fluid + receptor chamber wash + receptor rinse) was between 1.7 and 3.6% of the applied dose and the mean dermal delivery (epidermis + dermis + total absorbed dose) was between 16 and 20% of the applied dose, with the majority of the radioactivity associated with epidermis samples compared to dermis and receptor fluid samples. a linear dose-response relationship is observed over the whole concentration range. anastrozole is a well-known non-steroidal aromatase-inhibiting drug approved for the second-line treatment of breast cancer after surgery and for treating postmenopausal women. treatment with the only available dosage form, anastrozole film-coated tablets for oral administration, is frequently associated with concentration-dependent unwanted side effects like hot flashes, fatigue, joint pain, joint stiffness, vaginal dryness, hair loss, skin rash, nausea, diarrhea and headache. in order to minimize the local gastrointestinal as well as systemic side effects, a system for transdermal anastrozole delivery has recently been developed. in this study, we describe the first experimental in vivo application of a transdermal therapeutic system (tts) to beagle dogs and, as a necessary prerequisite for the analysis of the time course of anastrozole release and uptake, a simple, sensitive and accurate lc-ms method for quantifying anastrozole in plasma. the detection of fragment ions at m/z 225 and 237 instead of the molecule ions (m/z 294 and 306) generated from the elevated collision energy, and the use of a deuterated internal standard resulted in increased relative abundances and improved signal-to-noise ratios.the lower limit of quantification and the limit of detection were 1.4 ng/ml and 0.5 ng/ml, respectively. the developed method was successfully applied in a pharmacokinetic study of anastrozole plasma levels in beagle dogs, measuring percutaneous drug absorption from an experimental, newly designed glycerol-based patch / tts. a distinct time course was observed, with an initial linear increase over 24 hours and a plateau thereafter. this offers promising strategies for the transdermal application of anastrozole with improved pharmacokinetics. background: the monocarboxylate transporter 4 (mct4), encoded by the slc16a3 gene, mediates h + -coupled transport of lactate across the plasma membrane. for cells with high glycolytic activity lactate export is of major importance for the maintenance of the glycolytic metabolism and for the prevention of intracellular acidification. in glycolytic tumor cells, the acidic extracellular environment resulting from export of lactate and h + , furthermore promotes anti-apoptotic effects and metastasis. clear cell renal cell carcinoma (ccrcc) is the most common subtype of renal cell carcinoma (rcc) and is characterized by a metabolic shift towards enhanced aerobic glycolysis and hence, increased lactate production. mct4 and its epigenetic regulation by slc16a3 promoter methylation has previously been identified as prognostic marker for ccrcc outcome and as target for ccrcc treatment. since metastatic ccrcc is associated with poor overall survival and represents a major challenge for treatment, mct4/slc16a3 might represent a promising prognostic marker and a target for therapeutic intervention also for metastatic disease. methods: mct4 protein expression was analysed in 130 paraffin embedded tissue samples of distant metastases derived from different organs by immunohistochemical staining of tissue microarrays. protein expression was evaluated semi-quantitatively using tissue studio v.3.6 (definiens ag). dna methylation in the slc16a3 promoter, specifically at the previously identified cpg site with prognostic potential in primary ccrcc, was analysed in 82 paraffin embedded metastasis samples by maldi tof-ms. mct4 protein expression data and dna methylation at the specific cpg site in the slc16a3 promoter were correlated with clinicopathological parameters and outcome data. results: distant metastases of primary ccrcc showed high mct4 protein expression irrespective of the affected organ. the most frequently affected organs like lung or bone, with approximately 28% and 14% in our cohort respectively, showed similar expression levels as less frequent metastatic sites such as thyroid gland or spleen. accordingly, dna methylation at the identified cpg site in the slc16a3 promoter was low in metastatic tissue in all investigated organ sites. an association of low promoter dna methylation level at the previously identified prognostic cpg site in metastases with poor tumor-specific survival of the patients was observed. conclusion: from these results we hypothesize that dna methylation at specific cpg sites in the 5'-regulatory region of mct4 may not only serve as a predictor for patient outcome and as potential novel target for therapeutic intervention in primary, but also for metastatic disease. tamoxifen is used to treat pre-and postmenopausal women with estrogen-receptor (er) positive breast cancer. as a prodrug, tamoxifen undergoes extensive hepatic metabolism resulting in a complex mixture of metabolites with estrogenic and antiestrogenic effects. while endoxifen and (z) 4-hydroxytamoxifen are the most potent antiestrogenic metabolites, bisphenol and both isomers (e) and (z) of metabolite e are the most potent compounds with estrogenic properties at the er. the mixed antagonist/agonist pharmacodynamic effects of the selective estrogen receptor modulator tamoxifen at the er have been mainly attributed to tissue specific action of er coregulators, yet little is known about agonistic metabolites contributing to its estrogenic actions. the aim of the present study was to clarify whether there is a genetic component for interindividual differences in the formation and clinical effect of agonistic tamoxifen metabolites. a genome-wide association study (gwas) was conducted on steady-state agonist plasma levels in 390 postmenopausal breast cancer patients of european origin who were treated with 20 mg/day of tamoxifen for at least 6 months. plasma concentrations of estrogenic metabolites bisphenol, (e), and (z) metabolite e were quantified using a recently established lc-ms/ms method 1 . promising snps for an association between genotype and either plasma metabolite concentration or clinical outcome were confirmed for their relevance in an independent patient cohort of 313 premenopausal breast cancer patients mainly of european descent 2, 3 . twelve snps close to or above genome-wide significance (p <5e-08) were found to be associated with allele-dependent variable (e) or (z) metabolite e plasma levels, while no genomic hit was found for the tamoxifen metabolite bisphenol. here, positive intergenic or genic regions mapped to chromosomes 1, 2 and 16 for (e) metabolite e and to chromosomes 15 and 18 for (z) metabolite e. upon genotyping of the validation cohort, two genetic loci with minor allele frequencies < 5% were confirmed as putative candidates: rs662106 was associated with a 21-39% variant allele-dependent increase of (e) and (z) metabolite e isomers (p< 0.05), and rs3731872, mapping to a gene encoding zinc finger protein znf124, was associated with increased risk of reccurrence or death (hr carriers 2.6, 95% ci: 1.3 -3.4; p < 0.005). these findings suggest the existence of genetic loci that may contribute to the formation and clinical effect of estrogenic tamoxifen metabolites and therefore could explain therapeutic failure of tamoxifen and/or the occurrence of adverse events during treatment. introduction: metabolomic monitoring of endogenous biomarkers is of increasing importance for the assessment of drug safety and efficacy during clinical drug development. myrcludex b, a novel lipopetide-based entry inhibitor for the therapy of hepatitis b and d, exerts its function through inhibition of the hepatic bile acid transporter na + -taurocholate cotransporting polypeptide (ntcp). in order to assess a myrcludex binduced metabolomic response in humans, lc/ms-based monitoring of endogenous metabolites was performed in blood and urine samples from healthy individuals before and during treatment with myrcludex b. methods: plasma and urine samples were collected from healthy volunteers participating in clinical phase i trials to evaluate safety, tolerability, and pharmacokinetics of single doses of the ntcp inhibitor myrcludex b. using quadrupole time-of-flight mass spectrometry coupled to reversed-phase chromatography (lc-qtof-ms) a set of 15 known ntcp substrates (bile acids) was quantified by targeted metabolomics. protein precipitation was performed in the presence of deuterium-labeled internal standards (istds) which allowed absolute bile acid (ba) quantification in low amounts of plasma. ba profiling in urine was performed after dilution with methanol/water (1:1) in the presence of istds. both methods were validated according to fda guidance and applied to monitor the effect of myrcludex b treatment on human bile acid homeostasis. results: dynamic quantification in plasma and urine was achieved in the range from 7.8 nm to 10000 nm depending on the ba species analyzed. intraday-and interday accuracy and precision were in the 15% tolerance range for all analytes in all matrices. matrix effects were between 39-104% (plasma) and 31-95% (urine), apparent recoveries in plasma were above 97%. basal plasma ba level (mean ± sd) in fasting healthy subjects were 667 ± 574 nm (unconjugated bas), 935 ± 629 nm (glycine-conjugated bas) and 104 ± 62 nm (taurine-conjugated bas). urinary ba level (nmol/g creatinine) were 193 ± 225 nm (unconjugated bas), 89 ± 29 nm (glycine-conjugated bas) and 6 ± 2 nm (taurine-conjugated bas). myrcludex-induced ntcp inhibition resulted in significantly elevated amounts of conjugated ba species demonstrating a spillover of ntcp substrates into the systemic circulation. furthermore, higher urinary ba level were observed during treatment indicating accelerated elimination of excessive bas from the body. conclusion: lc/ms-based monitoring of endogenous biomarkers has been successfully established and applied to study the effect of myrcludex b treatment on human ba metabolism. the results obtained by our assay demonstrate that a myrcludex-induced ntcp inhibition drastically affects human ba homeostasis. this observation provides valuable insights into the drug´s mode of action and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. further studies are required to assess a possible role of ba modification (e.g. sulfation) in the process of ba detoxification during myrcludex treatment. key: cord-006230-xta38e7j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: 2012-02-22 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-012-0736-0 sha: doc_id: 6230 cord_uid: xta38e7j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, 2.4 mg/kg/d 4 days) via minipumps. chronic iso increased mrna levels of ndpk c by 9.4±1.9-fold and its protein levels by 2.1±0.11-fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within 3 hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a 1.5-fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~50%. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. 1, 2 , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin-3',4',5'-trimethylether in the human colon carcinoma cell line hct116 and the human hepatoma cell line hepg2 as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin-3',4',5'-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h 2dcf-da assay in hct116 cells) decreased with increasing methyl groups in the b-ring. myricetin-3',4',5'-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct116 cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly389arg variant of the β1-adrenergic receptor (β1ar) on receptor conformation we used β1ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β1ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg389-β1ar did not show altered activation kinetics after prestimulation, the gly389-β1ar became slower compared to the initial stimulation suggesting that β1ars possess a memory of previous activation. we then permeabilized β1ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β1ar variants did not display receptor memory, suggesting that the β1ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna-145 or mirna-33a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. 1 micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir-145, which shows decreased levels in colon carcinoma. in contrast, while mir-33a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir-33a and identify the protooncogenic kinase pim-1 as a target of mir-33a. pim-1 harbours a highly conserved mir-33a binding site within its 3'-utr, and seed mutagenesis of this target sequence abolishes the mir-33a-mediated downregulation of pim-1. the knockdown of pim-1 by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir-33a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna-145 through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk5. likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir-33a. this is due to the mir-33a-mediated downregulation of pim-1 expression and resembles the pim-1 knockdown through rnai / pim-1 sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir-145 and mir-33a may be promising mirnas in colon carcinoma therapy. md 288, a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md 288 to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md 288 was also investigated. the potential of md 288 to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md 288 to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v79 lung fibroblasts (v79 cells). v79 cells were treated with 0.4 µm, 0.8 µm and 1.5 µm md 288 or the positive control, the direct mutagen 4-nitroquinoline-n-oxide (nqo, 1 µm) for 24 h. on day 6, mutants exhibiting loss of hprt function were selected with 6-thioguanine . whereas at 1 µm chloroquine, a 38% decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, 10 µm md 288 were needed to observe a similar decrease (28%) in fluorescence intensity of eb. therefore, md 288 is 10fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells was 9 ± 2. as expected, 1 µm nqo caused a significant increase in the mutant frequency (mf, 168 ± 9) . in contrast, mf was not significantly affected by treatment with md 288 at both noncytotoxic (0.4 µm: 5 ± 4) and cytotoxic (0.8 µm: 9 ± 7) concentrations. in conclusion, md 288 is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md 288 does not cause gene mutations in the hprt test. since current studies show that various metabolites of md 288 are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca 2+ entry or depolarisation. recently, we showed that trpm4 acts as a ca 2+ -activated non-selective cation channel and critically determines the driving force for ca 2+ influx in mast cells following fcεri-stimulation (1) . in addition to trpm4 we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc2, trpc3, trpc5, trpc6 and trpm7. in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc1, trpc4, trpc5, trpc6, trpm2, trpm4 and trpm5. to identify the functional role of those trp channel proteins for mast cell activation we analysed ca 2+ signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc6 channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp-9 and flufenamic acid (ffa), but could not evoke a rise in the [ca 2+ ]i in both pmc and bmmc. sphingosine 1phosphate and lysophosphatidylcholine, which were reported to activate trpc5 channels, induced only minor rise in [ca 2+ ]i in bmmcs, respectively. here, we will present our analysis of ca 2+ signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca 2+ -dependent mast cell activation such as adenosine, endothelin-1, substance p and compound 48/80 in bmmcs and pmcs derived from mouse lines with inactivation of trpc1, trpc3, trpc4, trpc5 or trpc6 since specific antagonists are still lacking for these trp channels. the α2a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α2a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α2a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α2a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α2a-ar activation with very high temporal resolution, we took advantage of the previously described α2a-ar flash/cfp sensor [1] . the results indicate that in our system the efficacy of ne in eliciting α2a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ 70 ms. the ec50 values decrease in an exponential manner and arrive at ~ 2 µm with a half-life of ~ 330 ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α2a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [2] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ 700 ms and ~ 3,000 ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine-1-phosphate (s1p) is an immune modulator produced by sphingosine kinase 1 (sphk1) and sphingosine kinase 2 (sphk2) and de-phosphorylated or degraded irreversibly by s1p phosphatases and a lyase, respectively. we recently showed that tlr4-induced il-12p70 is selectively counter regulated by sphk1, s1pr1 and its extracellular ligand s1p. on the other hand, spiegel et al. have demonstrated that specific, sphk1-dependent, binding of s1p to traf2 enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s1p. in a first approach we focused our investigations on sphk1 effects on both traf2 and traf6 stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr4 or cpg-tlr9 signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il-12p70 secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf2 activated via tnf-alpha interacted with the traf6 pathway to reduce il-12p70. further series with dcs derived from sphk1-deficient mice confirmed our former results that il-12p70 in contrast to other cytokines is specifically sensitive to sphk1-s1p feedback, but did not change the effects of tnf-alpha on wt dcs il-12p70 release. in comparison, cpg-tlr9-induced il-12p70 release reached only 20% of lps-induced il-12p70 levels and was less sensitive to tnf-alpha costimulation. however, sphk1-deficiency strongly augmented cpg-dependent il-12p70 production. in ongoing experiments we started to analyze the details of traf2/rip1 and traf6/tak1 activation by ubiquitination blots in wt, sphk1-and s1plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s1p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. 1 , besche v. 2 , bros m. 2 , li h. 1 , handler n. 3 , bauer f. 3 , erker t. 3 , behnke f. 4 , mönch b. 5 , förstermann u. 1 , dirsch v. m. 6 , werz o. 5 , kleinert h. 1 , pautz a. university of vienna department of pharmacognosy, althanstr. 14, 1090 wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p38 mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin 1 (sirt1) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld-1 cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna-155 function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p38 mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p38 mapk activation or activity. in addition we have evidence that sirt1 is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. 1 the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety (1) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all 16 nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude 61, 66421 homburg, germany tmem2 proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit 6 to 10 membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek293 cells. tmem2 -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura-2 revealed that the elevation of the cytosolic ca 2+ concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem2-deficient acinar cells. tmem2 is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem2 fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek293 cells the tmem2-eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem2-eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem2-eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) 0,79 ± 0,03, n= 4 using lysotracker ® dye). analysis of subcellular localization with independent tmem2 fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem2 and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem2 -/mice. the small molecule bcl-2/mcl-1 inhibitor tw-37 shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. 1 , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl-2 family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl-1 or bcl-2, with high expression correlating to high risk phenotype. co-expression can be detected in approximately 10% and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl-2 and other proteins, but not or to a lesser extend mcl-1, elucidated the importance of mcl-1 inhibition for cytotoxicity in nb. tw-37 is a small molecule inhibitor that showed almost equal affinity to bcl-2 and mcl-1. to explore the effect of combined bcl-2/mcl-1 inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr-5, sy5y and kelly) were treated with tw-37 and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw-37. using sirna, we investigated the functional relevance of mcl-1 and bcl-2 in kelly cells. for in vitro cell viability we observed ic50 values of 0.59 ± 0.39 µmol/l. on treatment with 1 µmol/l dose of tw-37, all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl-2 as well as mcl-1 knockdown induced apoptosis in kelly cells. interestingly, tw-37 was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl-2 and mcl-1 using tw-37 exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl-2/mcl-1 may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. 1 , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e1s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein 1 (oscp1) and the organic anion transporting polypeptides oatp6a1 and oatp1c1 are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek293 cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek293 cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ 3 the hepato-intestinal induction of the detoxifying enzymes cyp3a4 and cyp3a5 by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp3a4, cyp3a5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp3a5 promoter we demonstrate that the cyp3a5 expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang 1 (yy1)-binding site from the cyp3a5 promoter which occurred in haplorrhine primates. this yy1 site is conserved in cyp3a4, but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy1 binding site from promoters of the cyp3a5 gene lineage during primate evolution may have enabled the utilization of cyp3a5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp3a5 and cyp3a4. serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. 1 , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap-1) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant 0315370d) . barlow s. harrington house, 8 harrington road, brighton, bn1 6re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of 1 in a million of developing cancer during a lifetime. in 1995, from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of 0.5 micrograms/kg of diet (0.5 ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of 0.5 ppb is equivalent to 1.5 micrograms/person per day, assuming that 1500 g of food and 1500 g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in 2004, kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of 0.15 micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova (1 mg/ml) was dissolved in sodium borate buffer (0.1m, ph 9.4) . for chemical treatment various amounts (50-200 mg) of 1-fluoro-2,4dinitrobenzene (dnfb; sensitizer) or 2,4-dichloro-1-nitrobenzene(dcnb; non-sensitizer) was added and stirred for 2 hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with 100 µg/ml ova and blocked with 10% fcs in pbs. ova samples (native or conjugated; 0.6 -10 µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for 30min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace 50% of ab to plate-bound ova (ic50) was calculated (minimum of n=3 independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic50 value, whereas mock treatment resulted in comparable ic50 values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic50 value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. 2´-deoxy-camp in human cell lines: another second messenger? beckert c. 1 , hinz c. we have shown that 2´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~10 was observed. as a remarkable exception, in hl-60 promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than 3-fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek293 cells after 24 hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek293 cells ac assays (mn 2+ /forskolin-or mn 2+ /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde3 and pde4 hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek293 cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h1r -h4r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h1r and h4r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h1r and h4r. in order to analyze these signaling pathways, we established a cellular model using transfected hek 293 cells which stably express recombinant mh1r or mh4r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca 2+ concentration ([ca 2+ ]i) in cells expressing either of both, mh1r (pec50 = 8.2) or mh4r (pec50 = 6.9). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh1r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh4r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h1r-expressing cells were blocked by the h1r antagonist mepyramine ([ca 2+ ]i: pkb = 8.6) and those in the h4r-expressing cells by the h4r antagonist jnj7777120 ([ca 2+ ]i: pkb = 8.8) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj7777120, which behaves as a partial mh4r agonist in the steady-state gtpase assay using membranes of infected sf9 cells, was without effect on [ca 2+ ]i and forskolin-induced camp-accumulation in the mh4r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h1r and the h4r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk1/2, p38, jnk, creb, pkb (akt), and mkk 3/6 were detected in cells expressing either of both, mh1r and mh4r. in summary, the hek 293 cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β3 regulatory subunit of voltage-activated calcium channels belkacemi a. 1 cavβ subunits of voltage-activated ca 2+ channels are required for trafficking the poreforming cavα1 subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih 3t3 fibroblasts do express cavβ2 and cavβ3 subunits, but we could not detect any voltage-activated ca 2+ influx. whereas in mouse cardiomyocytes or hek 293 cells coexpressing cavβ3 and cavα1 subunits a dihydropyridin-sensitive voltage-activated ca 2+ influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca 2+ influx. among the proteins potentially interacting with cavβ3 are the inositol 1,4,5-trisphosphate receptors (ip3rs) [1, 2] . we therefore coexpressed mouse cavβ3 and mouse ip3r type 1, 2 or 3 in cos-7 cells and found coimmunoprecipitation of ip3rs using an antibody for cavβ3 and vice versa. to study the release of ca 2+ from ip3-sensitive stores we performed fura-2 measurements on fibroblasts isolated from wild type and cavβ3-deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par-1, lpa or bradykinin. receptor-activated ca 2+ release was more pronounced in β3-deficient mefs and cfs, whereas thapsigargin-induced ca 2+ release was the same in cells from both genotypes. in addition, ip3 production measured by a radioreceptor assay was already increased in β3-deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β3 knockout may result, in part, from the increased amount of ip3-releasable ca 2+ . [1] berggren, yang, murakami, et al., removal of ca channel β3 subunit enhances caoscillation frequency and insulin exocytosis. cell 119, (2004) 273-284. [2] müller, haupt, bildl, et al., quantitative proteomics of the cav2 channel nanoenvironments in the mammalian brain. pnas 107, (2010) 14950-14957. bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc42 by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat-1-mediated arginine transport by pma (vina-vilaseca et al, j biol chem 2011 286:8697) . this mechanism requires nedd4 e3 ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u373 glioblastoma cells overexpressing hcat-2a-egfp. in addition, sirna-mediated knock down of cdc42 prevented the decrease of hcat-2amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc42 is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. 1 parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase 1. by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase 1 has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp1-dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap1. whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap1 is a specific target of the kinase pink1 (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink1 is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase 1. we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table 1) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek293 cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac1, mac3, mac9, soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca 2+ homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca 2+ -channels and the expression of ca 2+ -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation 14 minipigs underwent ventricular pacing at 120 beats/min (ddd mode) for one year. 7 minipigs were paced from the rvfw and 7 minipigs from the lva, respectively. 7 minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca 2+ currents. western-blots were carried out to investigate the expression of the ca 2+handling proteins l-type ca 2+ -channel, serca2 and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca 2+ -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca 2+ -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca2-expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca 2+ -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. 1 the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like 3d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd 431) and irritation (oecd 439) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp 1a, 2b and 3a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo 1/3 activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [1] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [2] or propanal (aldh) oxidation [3] . nat1 activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt1 activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [1] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project (0315226d). signalling pathway indicates that in b104 cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about 1.5 µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to 58% of the tolerable weekly intake (twi) of 2.5 µg/kg bw for cd defined by the european food safety authority (efsa) in 2009. beverages and vegetables are the food groups most relevant for exposure to pb. about 3.7 µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects (4.41 µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is 0.49 µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with 94%. for pb a weekly intake of 5 µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα12/13 and gαq via specific rhogefs. the p63rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p63rhogef and characterize the dynamics of p63rhogef-gαq-interaction in single living cells. the fusion of p63rhogef with venus resulted in a functional gαq-regulated p63rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h1and cholinergic m3-recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p63rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p63rhogef was verified by introducing point mutations rendering p63rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p63rhogef and gαq upon cotransfection of rgs2, suggesting a very short lifetime of the p63rhogef-gαq-complex or the ability of rgs2 to bind to p63rhogef-associated gαq. taken together, fret-based imaging of the interactions between p63rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs2. toxicity of silver nanoparticles in intestinal cells boehmert l. 1 the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco-2 is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco-2 cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a4f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of 7.2 nm stabilised with tween-20 und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco-2 cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine-1-phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. 1 , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp2a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06112 halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase 2a (pp2a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n=3) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein 25 (hsp25) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp25 protein was increased (p<0.05, n=7-8) but not camkii (p>0.05). protein phosphatase type 5 (pp5) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p<0.05, . in contrast, pp5 protein was upregulated (p<0.05, in tg compared to wt. for comparison the regulatory a-subunit of pp2a and hsp90 were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp2a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h2s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. 1 , hassan m. there is increasing evidence that hydrogen sulfide (h2s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e2-related factor 2 (nrf2) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf2 -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h2s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy-1-induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf2 protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf2 may constitute a protective mechanism during glomerular inflammatory disease. rac1 knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. 1, 40225 düsseldorf, germany rac1 belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac1 is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap1. anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac1 in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac1 may be involved in the ddr. to study the in vivo function of rac1 we used an inducible cre-based knockout mouse model (rac1 flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh2ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone 3 as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac1 the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp1 mrna expression was reduced in the liver of rac -/animals as compared with rac1 proficient animals. in addition we found more apoptotic cells in rac1 -/mice. 96 hours after treatment with the anthracycline derivative doxorubicin the number of gh2ax foci in rac1 -/animals was reduced in comparison to rac1 +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac1 -/mice. none of these protective effects resulting from rac1 deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of 21 days. there were no significant differences in the number of gh2ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac1 -/animals. furthermore the number of mitotic events was almost two times higher in the rac1 -/mice compared to the rac1 +/+ mice. summarizing, our findings show that impaired hepatic expression of rac1 protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse 56, 72074 tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine 3t3 cells with tbhq in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf2 pathway. s14 056 angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type 1 receptor brand s. 1 , amann k. 2 , schupp n. 1 1 universität würzburg institut für pharmakologie und toxikologie, versbacherstr. 9, 97078 würzburg, germany 2 universität erlangen-nürnberg institut für pathologie, krankenhausstraße 8, 91054 erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c57bl/6 mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c57bl/6 mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c-3t3 cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm 15, 30173 hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c-3t3 cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c-3t3 cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c-3t3 cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c-3t3 cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s9. in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b 1 and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c-3t3 cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac5 and ac6 being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac5 and ac6 share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac5, as well as overexpression of ac6 both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac5 ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from 16-20 week old homozygote ac5 knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine 5'-triphosphate (gtp) and 5'-o-(3thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac1-9, gs-, gi-a and β1-, β2-ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac5 knockout, mrna expressions analysis of ac1-9, gs-, gi-a and β1-, β2-ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac5. whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac5 and ac6, or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk-1/2 and lkb-1 in hypothalamic gt1-7 cells breit a., ellen d., gudermann t. goethestrasse 33, 80336 münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin-4 receptor (mc4r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt1-7 cells) and monitored ampk phosphorylation at thr 172 which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr 172 and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser 79 . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases-1/2 (erk-1/2), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk-1/2 was blunted by pka inhibitors, we propose that erk-1/2 serves as a link between pka and ampk in gt1-7 cells. furthermore, down-regulation of liver kinase b-1 (lkb-1), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase-1 decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk-1/2 and lkb-1 in gt1-7 cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than 100 candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav1.2 gene (cacna1c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell 2004; 119:19-31) . functionally similar biophysical effects can be induced by structural variation β1-and β2-subunits of the voltagedependent calcium channels (herzig et al., faseb j. 2007; 21:1527-38; jangsangthong et al., pflugers arch. 2010; 459:399-411) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. 2006; 11:29-36) , we are investigating a function-based candidate gene hypothesis linking the β2 subunit gene (cacnb2) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb2 in 155 patients with asd. we found three rare missense mutations in asd patients, but not in 259 unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav1.2 subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β2 subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav1.2. β2 subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. (2010) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta (1) subunits. pflugers arch 459, 399-411. herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. (2007) . mechanism of ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. faseb j 21, 1527-1538. splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. (2004) . ca(v)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell 119, 19-31. trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. (2006) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry 11, [29] [30] [31] [32] [33] [34] [35] [36] background: brain serotonin (5-ht) has been implicated in the regulation of food-intake. the ingestive effects of 5-ht are mediated by a number of different receptor subtypes under which the 5-ht1a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by 5-ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei or by postsynaptic 5-ht1a heteroreceptors in serotonergic terminal structures. methods: the effect of the 5-ht1a receptor agonist 8-oh-dpat (0.1, 0.5 or 1.0mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l35 mice. l35 mice are characterized by an overexpression of postsynaptic 5-ht1a receptors. results: the administration of the 5-ht1a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the 5-ht1a receptor in food intake. further, we make the assumption that the hyperphagic effect of 8-oh-dpat is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei which decreases 5-ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic 5-ht1a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc2 (mrp2) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc2 expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) 1β on abcc2 mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc2 expression while inflammation. three different cell lines a) hepg2 cells (liver tissue) and b) caco2 (colon tissue) both without naïve il1β expression and c) skhep1 cells representing physiological liver tissue with naive il1β expression, were stimulated with different concentrations of il1β (range 10 pg/ml to 10 ng/ml). over a period of 48h samples were taken at defined time points. abcc2 mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p38 mapk, akt, erk1 and jnk) we analysed the signal transduction pathways associated with il1β-mediated transcriptional abcc2 regulation. on abcc2 mrna level an up-regulation in caco2 cells (1, 35 fold) and hepg2 cells (3, 6 fold) within the first hour after stimulation with 1 ng/ml il1β was shown. in contrast skhep1 cells demonstrated a decreased abcc2 mrna expression (0,59-0,62 fold) in comparison to unstimulated controls. the abcc2 protein expression exhibited a time and il1β dependent regulation as well. the analysis for the signal transduction showed for p38mapk a moderat time dependet down regulated phosphorylation (15%) in hepg2 cells whereas it showed no effect in caco2 cells. concluding, the expression of abcc2 is regulated moderately by il1b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc2 expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from 21 cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of 667 micrornas. statistical analysis using the 2 -∆ct method was performed. micrornas showing a p-value<0.01 and a fold change>2 were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan 1 , mirdb 2 , pictar 3 , microcosm 4 , diana microt 5 ), selected putative target genes were further functionally investigated by the david bioinformatics database 6 . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target 97 genes, especially transcription regulators (21%). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad4, nras, rb1, raf1). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators (19%), but also cellular transporters (18%, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and 12 micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta2-adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. 1 , buschauer a. activation of the β2-adrenergic receptor (β2ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [1] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β2ar ligands on cyclic adenosine 3',5'-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o2 •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o2 •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o2 •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o2 •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o2 •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β2ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [2, 3] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β2ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β2ar antagonists in the camp assay and the o2 •assay are different. such differences were previously reported for β2ar antagonists in other test systems [4] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o2 •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human 5-ht2a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße 44, 39120 magdeburg, germany the serotonin 2a (5-ht2a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of 5-ht2a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of 5-ht2a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged 5-ht2a receptor by internalization. using immunocytochemical techniques in stably transfected hek293 cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the 5-ht2a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized 5-ht2a receptors colocalize with a488-labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of 5-ht2a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces 5-ht2a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the 5-ht2a internalization to occur. overall, we conclude that the internalization of the human 5-ht2a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of 5-ht2(a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of 5-ht2a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung 4, kurt-georg-kiesinger-allee 3, 53175 bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma 2007). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma 2010) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/100). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method 4.1.1) and for atropine (ph. eur. method 3.1.1 or 4.1.1) based on lhrd leads in each case to a suggested fsd of d7 related to 10 g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method 3.1.1 or 4.1.1) based on food legislation emerged a proposed fsd of d6 related to 10 g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether in c. elegans büchter c. 1 , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin (100 µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h 2dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives (100 µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf-16, the nad + -dependent protein deacetylase sir-2.1 and the heat-shock transcription factor hsf-1, respectively. lifespan extension by myricetin disappeared in daf-16 and sir-2.1 loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf-1 loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf-1. in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf-16 and sir-2.1, probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma 2 bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany activation of phospholipase c-γ2 (plcγ2) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ2 is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology 2 (sh2) domains and one src homology 3 (sh3) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ 2 is also regulated by autoinhibitory elements within its specific array (walliser et al., 2008; everett et al., 2011) . our recent data demonstrated that plcγ2 is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh2 (sh2c) and the sh3 domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ2. plcγ2 has been shown to be phosphorylated at tyrosine residues 753 and 759 upon bcr stimulation (kim et al., 2004) . both tyrosines are located in the linker between the sh2c and the sh3 domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ2. interestingly, a novel phosphorylation site in plcγ2 was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue 733 (rikova et al., 2007) . in this work, we demonstrate, for the first time, the activation of plcγ2 by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr733 is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ2 in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ2 at tyr733. molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. 8-10, 10589 berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg2 was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between 5 µm and 25 µm. at concentrations higher than 25 µm the substance was cytotoxic to the cells (ic 50 47µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of 100 µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor 4α (hnf4α) by incubating the cells already with moderate concentrations of pfoa at a level of about 1 µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s17 070 human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. 1 , bumke scheer m. (kao et al., 1995) . two daughter cell lines were developed from m13sv1 after x-ray irradiation (m13sv1 r2) and an additional transfection with a mutated erbb2 oncogene (m13sv1 r2-n1), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., 2010) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the 3 hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using 2d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. (1995) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h1-receptor (h1r) shows no ameliorating effects on asthmatic symptoms, h4r antagonists may be new drugs for asthma therapy. in addition to the h3r-and h4rselective antagonist thioperamide, the selective h4r antagonist 1-[(5-chloro-1h-indol-2yl)carbonyl]-4-methylperazine (jnj7777120) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj7777120 as well as the selective h3r antagonist 1-({4[3-(piperidin-1-yl) propoxy]phenyl}methyl)piperidine (jnj5207852) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and 0.2 m znso4 using 30 µl of plasma. analyte extraction from lung tissue was achieved by treating 150 -200 mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp 80a mercury column (10 x 2mm; 4µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph 5) and methanol at a flow rate of 0.4 ml/min. the analytical run-time was 5 minutes. for plasma samples the assay was linear over a concentration range of 0.078 -40 µg/ml for jnj7777120 and jnj5207852, and 0.313 -40 µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of 0.008 -2 µg/sample, jnj7777120 and jnj5207852 in a range of 0.004 -2 µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk5 plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk5, one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk5-dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk5 is already expressed during embryogenesis and we can detect it in the developing heart. however, grk5 has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk5 on cardiac signalling and development. tools for grk5 specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk5 in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk5 in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk5 results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk5 may potentially serve as a candidate gene for congenital heart disease. identification of a hcn3 interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn1-4) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn1, hcn2 and hcn4 channels have been studied in quite some detail there is only little information on the particular role of the hcn3 channel. as an important step towards achieving a better understanding of hcn3 function we set out in this study to identify proteins that are assembled with hcn3 in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn3 c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn3 using heterologous coexpression in hek293 cells. here, we provide an in-depth analysis of the functional interaction between hcn3 and one of the identified interacting proteins (hip3.1). we show that hip3.1 physically binds to hcn3 channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc1 and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn1 and generated a series of tpc1 antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc1 knock-out model by our self-generated tpc1 antibodies. tpc1 knockout mice are viable and do not show any obvious deficits. to isolate tpc1 containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc1 containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc1 by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc1. foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf-7 breast cancer cells and mcf-7 cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo4 overexpressing cells was a20 a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo4-dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a20 is a direct target of foxo4, a luciferase reporter containing a 1.2 kb of the a20 promoter was co-transfected with different amounts of foxo4 wild-type expression construct. foxo4 induced a dosedependent increase in a20 promoter activity, supporting the assumption that a20 is a direct transcriptional target of foxo4. overexpression of foxo4 led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf-7 cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a20, an ubiquitin modifying enzyme, as a novel foxo4 target gene. our data implicate that sustained foxo4 expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm7 is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm7 is essential for motility, proliferation and cell growth. up-regulation of trpm7 function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm7 channel is inhibited by the known modulators of sk1-3 channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns8593, ska31, ucl 1684. the most potent of these compounds, ns8593 (ic50 1.6 µm), interferes with the regulation of trpm7 by cytosolic mg 2+ . here we show that ns8593 (10 µm) fully and reversibly inhibits native trpm7-like currents in hek 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm7 currents by ns8593 would impact cellular processes known to be affected by a genetic inactivation of trpm7. we found that ns8593 (10-30 µm) suppressed motility of hek 293 cells without a detectable effect on cell viability. taken together, our findings indicate that ns8593 is a potent and reversible inhibitor of endogenous trpm7 currents and may be a good candidate drug for pharmacological targeting of trpm7. sulfur mustard (2,2'-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance 2-chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed 10 minutes after treatment with 3 mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp-1 becomes activated upon treatment with mustards. parp-1 is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp-1 is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape1 and ercc1, i.e. endonucleases involved in ber and ner, respectively. the reduction of ape1 expression had no effect on parp activity. surprisingly, the knockdown of ercc1 almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc1-parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt-888. expression and activity of g protein coupled receptor kinase 2 (grk2) are elevated in several conditions of compromised heart function. although grk2 inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk2inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf1 but it also acts as a physiological inhibitor of grk2 upon phosphorylation by pkc at serine153. a detailed understanding of the rkip/grk2 interaction may help to identify inhibitory compounds for grk2. since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf1-into a grk2-inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s153 phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk2, we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk2 association. this implicates, that dimerization of rkip is essential to bind grk2. to determine whether rkip dimers consequently inhibit grk2 activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk2 inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk2 binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk2 interaction and will help to develop specific grk2 inhibitors. expression and function of trpm3 ion channels in epithelial mdck2 cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. 1, 66421 homburg, germany trpm3 proteins build ca 2+ permeable cation channels [1] activated by steroids [2] and sensitive to increased temperatures [3] . trpm3 channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [2] or as nociceptors of noxious heat, respectively [3] . however, northern blots and in situ hybridization experiments revealed that trpm3 is also expressed in epithelial cells of the choroid plexus and the ciliary body [1] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm3 is also expressed in madin-darby canine kidney2 (mdck) cells. quantitative analysis of trpm3 expression by qrt-pcr revealed a ~ 5 fold upregulation in mdck2 cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm7, a related ion channel described as regulator of proliferation in other cell types, remained constant. hek293 cells overexpressing trpm3 channels did not proliferate in the presence of the trpm3 agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck2 cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck2 epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca 2+ imaging experiments using fura2 revealed that pregnenolone sulphate induces ca 2+ entry in well separated mdck2 cells but not in cells growing in confluency. we hypothesize that trpm3 might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck2 cells. inhibition of grk2 by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase 2 (grk2). grk2 initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk2 are upregulated in human heart failure, it has been proposed that grk2 inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk2 inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r9c ). pln r9c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r9c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r9c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r9c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi2 and gαi3 with gαi2 as the predominant isoform. gene-targeted mice lacking either gαi2 or gαi3 were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi2-deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi2-deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi2-deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi2deficiency. to assess the pathophysiological consequences of platelet gαi2 function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi2-deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi2-deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi3deficient mice were strongly protected from injury. thus we suggest that gαi2 and gαi3 play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b16 melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog 8-br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase 5. anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b16 melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk1/2 and p38 phosphorylation, growth and migration of b16 melanoma cells. similar results were obtained with wm1205 human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav2.3 (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) 1 , which is associated with absence epilepsy. cav3.2 is one of the two t-type calcium channels known to be expressed in the rt 2 . it has been demonstrated that ablation of either cav2.3 or cav3.2 reduces susceptibility to experimentally induced epilepsy 3;4 and in addition that both channels share several pharmacological properties [5] [6] [7] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav2.3(-|-), cav3.2(-|-) and cav3.2(-|-)xcav2.3(+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav2.3 and cav3.2 calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip1/rip3-dependent necroptosis in neuronal cells diemert s. 1 , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip1/3-dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase 1 dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring 15, bldg. n, 54286 trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat1 enzyme activities after incubation of thp-1 cells with cyanamide for 24h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat1 protein inhibition was observed for 100 and 1000 µm cyanamide using over-expressed human recombinant nat1 (insect cell cytosol containing recombinant human nat1*4). however, no inhibition was found in the presence of recombinant nat2*4. as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat1 or nat2 cytosol, by thp-1 cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat1. further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat1, leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat1, which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat1 dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [1] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc6 a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca 2+ influx and the subsequent contraction of pasmc [2] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc6 channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [3] . trpc-expression in freshly isolated and passaged pasmc cultured in low (5%) and high fetal bovine serum (25%) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc1 and trpc6 the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek293 cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc6 and inducing acute hpv in pasmc will be presented. references [1] staub, n. c. (1985) . site of hypoxic pulmonary vasoconstriction, chest 88, 240s-245s. [2] weissmann, n. et al. (2006) . classical transient receptor potential channel 6 (trpc6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. 103, 19093-19098 trpv5 is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv5 is assumed to contribute to the maternal-fetal calcium transport. most probably trpv5 is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv5 protein in the placenta that might contribute to the regulation of the trpv5 protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv5 (aa 1-330 and 571-723) as trpv5-gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv5 interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv5 protein is calpain 6. in contrast to the classical calpains (calcium activated cystein proteases), calpain 6 is unique in that it lacks the cysteine residue in the active site. calpain 6 is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv5 and calpain 6 was confirmed in reciprocal pulldown experiments and the trpv5 binding region for calpain 6 was narrowed down by using overlapping n-terminal trpv5-gst fusion proteins. after injection of trpv5 crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain 6 crna. in further experiments we want to study potential regulatory effects of the trpv5 protein on the calpain 6 function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. 45, 48149 münster, germany it has been demonstrated that chronic ingestion of 50-200 µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. 2011 , j toxicol. 2011 we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g2/m-and s-phase arrest as well as subg1 peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg1, caspase 3 activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h2o2 induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h2o2 induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp-1 gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse 27, 4012 basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr5/app/ps2) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. at 12 months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases 1 . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c57/bj6-thy1-appsl (ad-mice) mice that were fed with 600 mg olesoxime/kg feed for 3 months. drug plasma levels reached approx. 600 ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca 2+ -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca 2+ -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca 2+ -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the 7th framework program for rtd -project mitotarget -grant agreement health-f2-2008-223388. several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µm) for 10 min. thereafter, the cell lysates were subjected to diagonal 2d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine 157 of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf2-keap1-antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf2 pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq1. the lusens assay was in house validated with a panel of 54 chemicals and cosmetic ingredients including the 22 performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of 83% compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. 1 ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il-5 and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße 1, 30625 hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d4 in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il-5 concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd16antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of 1.5x10 6 eosinophils. we stimulated eosinophils with varying concentrations of il-5, eotaxin-1 (ccl11), eotaxin-2 (ccl24), and eotaxin-3 (ccl26). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of 2-4 pm/1.5x10 6 eosinophils. neither il5 (50-500 pg/ml), nor any of the eotaxins (50 ng/ml either alone or in varying combinations) did stimulate anandamide production after 30min of incubation. our results suggest that chemotactic molecules like eotaxin and il-5 are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse 38, 67056 ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg 427 or 428. however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. 10 µl of test substance preparation are applied to the skin preparation. after 6h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above 80%. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p53 tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p53 is an attractive target in cancer therapy and several approaches were pursued to restore p53 function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p53 protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a549 lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p53 was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p53 into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p53 was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a549 and saos osteosarcoma cells. the internalized biotin-p53 also partially co-localized with early endosomes. importantly, the delivery of biotin-p53 into p53-deficient saos cells resulted in impaired cell viability and upregulation of caspase 3/7 activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p53 into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. 1 , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb126. activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb126. thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb126 leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a (1, 2) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb126. already 6 h after treatment, 69 genes were found to be upregulated and 76 genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the 69 genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch-223191 and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq1as a novel ahr target protein in rat liver oval cells. although the function of foxq1 is poorly understood, it has been shown very recently that foxq1 is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h1 and h3 receptors in the brain can be easily deduced since h1 receptor antagonists of the first generation have a sedative effect and an inverse h3 agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h2 and h4 receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked 3 h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h2 agonist impromidine 10 µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was 50 %, the pec50 was 6.9 and the pa2 of the h2 antagonist ranitidine against impromidine was 7.2. with respect to h4 receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h4 receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h4 agonist 4-methylhistamine 10 -30 µm but inhibited by histamine 10 µm via h3 receptors by 28 and 55 %, respectively. in mouse cortex membranes, 4-methylhistamine 30 µm also failed to affect 35 s-gtpγs binding although r-α-methylhistamine 3 µm, acting via h3 receptors, increased it by 20 %. in conclusion, h2 receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h4 mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of 35 s-gtpγs binding (mice), could not be shown. murine cx40 promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany connexin 40 (cx40) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx40 function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx40 gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx40 down-regulation, suggesting that creb related transcription factors are involved in cx40 gene regulation. in order to study the functional role of creb in the regulation of the cx40 promoter we have generated a luciferase based murine cx40 promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx40 promoter activity (cacreb 46% ± 5% vs control 100% ± 3%; p<0.05 vs control , n=15), dncreb 45% ± 2% vs control 100% ± 3%; p<0.05 vs control, n=18). the activity of the murine connexin 40 promoter is modulated by creb. both cacreb and dncreb led to cx40 down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf1 transcription factor family, which in turn could suppress cx40 promoter activity. (supported by the dfg) results: fxa increased par-2 mrna, protein and cell-surface expression and augmented par-2-mediated mitogenesis. par-1 expression was not influenced. the regulatory action of fxa on par-2 was concentration-dependent and mimicked by a par-2 selective activating peptide. the thrombin inhibitor argatroban or par-1 gene silencing did not influence fxa-stimulated par-2 expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox-1 in smc. nox-1 gene silencing prevented fxa-stimulated par-2 regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par-2 expression and mitogenic activity. fxa induced nuclear translocation and par-2 dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par-2 expression. in separate studies, fxa promoted par-2 mrna stabilisation through increased human antigen r (hur)/par-2 mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par-2 expression. conclusion: expression and mitogenic activity of vascular par-2, but not par-1, is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox-1-containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par-2 may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor 4 aim: we recently reported that functional expression of par-4 thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par-4. methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par-4 mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose (25 mm vs 5.5 mm) slowed par-4 mrna degradation in the presence of actinomycin d. par-1 mrna decay was not affected. hur binding to par-4 mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par-4 mrna. hydrogen peroxide (h 2o2) also induced cytosolic hur shuttling and increased par-4 mrna and total protein expression. the role of endogenously generated h2o2 in the regulatory effect of high glucose on par-4 expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h2o2 generation) and cell-permeant catalase (to degrade cellular h2o2). both approaches prevented the stimulatory effect of high glucose on par-4 expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par-4 transcript stability. cicaprost also attenuated high glucose-induced hur binding to par-4 mrna and as a consequence normalised par-4 expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par-4 thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h2o2, increase par-4 expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par-4 expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. 72h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day 0) and on postoperative days 3, 7, 14, 21, 28, and 35 . a significant reduction of recovery was observed in the ndpk b depleted mice at days 3 and 7. thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than 50% in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for 4 hours to constant atmospheric et concentrations of 5, 20, or 50 ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was 5.5 ± 1.4 nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of 2 between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was 1.5 ± 0.08 nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw11 on human dendritic cells fink c. 1 , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw 11 contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw11 on the expression of maturation related genes (cd1a, cd83), cytokines (tnfα, il-4, il-12), chemokines (ccr7), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr2, tlr4). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in 6 well plates. non-adherent cells were eliminated. to induce idc development, 50 ng/ml il-4 and 50 ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day 6, maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of 1µg/ml and cultured for additional 3 days. after incubation with different concentrations (0. in idc, compared to control, we found a significantly increased expression of cd83 (2.1-2.6 fold) and tnfα (2.6-3.3-fold) genes after treatment with 0.1-5% stw11, respectively, but no effect was found on the expression of cd1a, il-4, il12, adam19, cd11c, cd40,tlr2, tlr4, mhc-ii and ccr7. in summary, these data demonstrate a stimulatory effect of stw 11 in idc and especially in mdc, concerning an increase of various genes related to maturation (cd83), immunomodulation (tnfα, cd40), adhesion (ccr7) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately 1 µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring 20a, 76131 karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of 17-beta-estradiol (e2) and estrone (e1). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p450-catalyzed metabolic activation to catechols (wang et al., chem res toxicol 23, 1365 . like e2 and e1, zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res 53, 1123 . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of 8-oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp1a2, and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of 8-oxo-dg could be demonstrated with each extract. the levels of 8-oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. 15-hydroxylated zen/zel, which is the major catechol, was more reactive than 13-hydroxylated zen/zel to form 8-oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e2 and e1. the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me 574/32-1) and "food and health" of kit. thrombin regulates expression of sphingosine kinase-1 (sphk-1) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk-1 expression and atherosclerotic burden in vivo flößer a. 1 results: thrombin induced a time-and concentration-dependent (1-100 nmol/l) increase in sphk-1 mrna and protein expression in human saphenous vein smc, n=6-7. this was mimicked by a synthetic par-1 ligand. inhibition of sphk-1 attenuated thrombin-induced smc proliferation but not smc migration (n=5). the regulatory action of thrombin on sphk-1 expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk-1 mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk-1 mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk-1 expression by 50% (n=5) and plaque size by 35% compared to control animals (n=10). conclusions: thrombin induces sphk-1 expression and s1p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk-1 expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v79 cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to 0.4 µg/g don and 0.5 µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t-2 toxin, ht-2 toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to 6 µg/g don, 7 µg/g ennb, 16 µg/g ergotamine, 50 ng/g ht-2 toxin) exert pronounced cytotoxicity in v79 cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp1a1. recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp1a1 and proinflammatory cox-2, respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct 6-formylindolo [3,2b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc 2544) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 3'methoxy-4'-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase 1 (chk1), an important cell cycle regulator that arrests the cell in g2/m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk1 in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk1 client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for 90 days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: 50.0 ± 5.8 min. vs. mpa: 33.4 ± 4.2 min., n = 6 -9, p < 0.05). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: 53.7 ± 5.4 min. vs. drospirenone: 47.5 ± 4.8 min., n = 7 -8) and levonorgestrel (placebo levonorgestrel: 47.0 ± 3.2 min. vs. levonorgestrel: 41.8 ± 2.1 min., n = 6) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase 9 (mmp-9) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp-9, a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg 18, 42096 wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a 96-well plate format using glutamate/malate (20 mm/0.5 mm) and succinate (25 mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. (2008) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., 2007) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), 1-octyn-3-ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd86 expressed on dcrc (ec50 = half maximal effective concentration). a pronounced induction of cd86 at low concentrations was seen with la, lna and oa (ec50: 3, 5 and 5 µmol/l, respectively). ua exhibited an intermediate response (ec50: 307 µmol/l). with oc and ma, we observed effects at higher concentrations only (ec50: 1340 and 2293 µmol/l). no significant increase of cd86 was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd86 was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd86 at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd86 (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd86 was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter 1 (oct1) and oct2, respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph 7.4 and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of 50 most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least 4 million ddd each) for their inhibitory interaction with oct1 and oct2. human embryonic kidney (hek) cells stably overexpressing oct1 or oct2 and the prototypical oct substrate 1-methyl-4phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at 20 µm, 59% and 51%, respectively, of the tested compounds significantly decreased oct1-and oct2-mediated uptake of mpp+. the most potent inhibitors (inhibition >75%) of oct1 were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct2. in contrast, neither at 20 µm nor at 200 µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct1 or hek-oct2 cells. there was a significant correlation between the degree of oct1 and oct2 inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between 30 and 80 µm in humans, inhibitory interactions of these compounds with hepatic oct1 have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: 58±3 %, n=3) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: 63±7 %, n=3) and failing patients (ef: 28±1 %, n=3). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation450 beadchip (illumina). this microarray allows analysis of more than 485,000 methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified 1280 cpg sites in hypertrophic samples and 1365 cpg sites in failing samples which were differentially methylated compared to control specimens (delta >15%; p<0.05). 523 cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, 496 sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and 3`utr regions. specifically 9 probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase 1 (srpk3) showed diminished cpg-methylation in hypertrophic (-11.5±0.02%) and failing (-11±0.02%) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh6 or myh7. these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only 15 months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln18, u87-mg and gl261. the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic50 values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase 3 and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. 1 brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat 1 . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora1, a2a, a2b and a3. they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora1 or a2a, respectively 2 . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. 3 abundance of adora1, a2a, a2b and a3 mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora2b being the most abundant. adora1, adora2a and adora3 are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora1 is upregulated 4.4 fold (+/-0.3 fold) and 11 fold (+/-0.49 fold) at day 4 and at day 7, respectively, as compared to preadipocytes (n=5). adora2a is 23 fold (+/-1.96 fold) upregulated at day 4 and 57 fold upregulated (+/-2.48 fold) at day 7, respectively (n=4). adora3 was found upregulated 3.6 fold (+/-0.46 fold) at day 7 (n=4). in contrast to this, ador2b was downregulated to 0.87 fold (+/-0.09 fold) at day 4 and to 0.69 fold (+/-0.04) day 7 compared to preadipocytes (n=5). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora2a activation by cgs21680 significantly increased lipolysis by 88% (+/-0.33%) compared to untreated control. moreover, adora1 antagonist psb36 increased lipolysis by 35% (+/-0.05%) (n=4). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora2a agonist or adora1 antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio 2 or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified 39 peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin-2 (myoz2) and g protein signaling modulator 1 (gpsm1, also termed ags3) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa (4-methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: 10 cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below 10 µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of 40-50 µg/ml or prolonged maa elimination half-life of up to 12 hours. in one case with manifest hepatic insufficiency an maa concentration of more than 200 µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than 10 µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b 0+ amino acid transporters recognize fet as substrate (lat1 and 2, y + lat2, and b 0+ at, respectively). in contrast, y + lat1 and atb 0,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat1 is the dominant amino acid transporter in all glioblastoma cells investigated (ln229/u373/u87mg/u251/a172/t98g). a strong lat1 expression was also shown on the protein level. to find out whether lat1 is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln229 glioblastoma cells. [ 3 h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat1 in ln229 cells led to a concomitant decrease of lat1 mrna and tyr transport (down to 3% and 20%, respectively). these results indicate that tyr is exclusively transported by lat1 in ln229 cells. we are currently performing transport studies using [ 18 f]fet to investigate whether fet transport is also exclusively mediated by lat1 in glioblastoma cells. a further question is if lat1, a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat1 substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at1 receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type 2 diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan (10 mg/kg), valsartan (25 mg/kg) or valsartan (50 mg/kg) from 8 weeks of age for 17 weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac2-positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht-29 cells, and their doxorubicin ic50 was four times higher. comparative protein expression levels showed that the primary cells had less rad 51 and 52 as well as less topoiiα, while ku70 and 80 levels were similar. another very interesting protein is the mrn (mre11-rad50-nbs1) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre11. the toxicity of mirin in ht-29 cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of 100 µm and incubation times of 24 hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [1] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [2] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e1 and hdac 1 expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin-3-gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e1 protein level in the human colon carcinoma cell line ht29 after 24h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ 50 µm. hdac 1 expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac-1 and sumo e1. these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin-3-gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. 1, 35034 marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like 2 and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia2 rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into 17-oh-pregnenolone, the placenta tissue highly depends on the supply of c-19 steroids for their conversion into c-18 estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e 1) and estradiol (e2), while 16αoh-dheas supplied by the fetus contributes to over 90% of placental estriol (e3) synthesis. soat, a member of the slc10 family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e1s), and pregnenolone sulfate (pregs) [1] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg-3 as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg-3 cell line that transformed dhea into e2 and 16αoh-dhea into e3. by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg-3 cells. currently we investigate the transformation of dheas of these soat-jeg-3 cells by lc-ms-ms. we could demonstrate transport of 16αoh-dheas for stably transfected soat-hek293 cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [1, 2] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [3] for polymer toxicity testing. fertilized chicken eggs were incubated and after 72 h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after 1 -48 h (fig.1 ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative (0.9 % nacl) and positive controls (1 % sodium dodecyl sulphate (sds) and 0.1 n naoh) were investigated. additionally ld 50 values for different cationic polymers have been determined. within the selected incubation times (1 -48 h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps (1) hazard identification and characterisation, based on substance-specific toxicological hazard data, (2) estimates of the level of exposure toward the substance and (3) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires (1) information on human exposure, for which it is essential that exposure is fully captured and (2) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally 613, based on toxicological testing in animals (munroe et al., 1996) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., 1996) by classifying the chemicals into three toxicity classes using a decision tree based on a series of 33 questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., 1978) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the 95th percentiles and dividing them by the default uncertainty factor of 100. it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the 95th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn 2008) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning 3627 bp of the 5'flanking region of kibra into the pgl3-vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy5y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 5'cdna ends (5'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p1 was separated by ~1060 bp into two distinct regions, promoter p1a and p1b. deletion constructs harbouring promoter p1b were transcriptionally active only in ihke cells. 5'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_015238). exclusively in ihke cells, two additional tss were detected in intron 1, resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p2 and p3) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf7l2 (transcription factor 7-like 2 [t-cell specific, hmgbox]) resulted in a ~3-fold increase of promoter p1a and intron promoter p2 ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p1 and three alternative promoters p1b, p2 and p3. the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon 3 could result in truncated kibra protein isoforms. tcf7l2 is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg 428 and a corresponding technical guidance document (gd 28) give technical guidance how to perform valid experiments 1, 2 . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: -0.07 -6.2). finite dose experiments using rat and human skin were performed with 14 c-labeled testosterone, caffeine, mcpa (4-chloro-2-methylphenoxyacetic acid) and its 2-ethylhexyl-ester mcpa-2ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h2o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the 14 c-penetrants. teer, tewl and ³h2o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h2o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. 1 background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from 13 patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status 5'-upstream of dna regions encoding for selected candidate mirnas. results: out of 754 mirnas, 150 were detected in both tissue types. the expression of one mirna was 7.2 fold higher (q=0.01) and another was 3.8 fold lower (q=0.01) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of 5'-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox11, mecp2, bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm61390. recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il-21-and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c57bl/6 b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/2, cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt1 mice hamann m. 1 early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt1 gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. (2005, j. neurosci. 25 [22] , 5351-5355) initially described a transgenic mouse model (dyt1 mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine (75, 100 and 125 mg/kg) as well as a long-term treatment over 21 days (100 mg/kg/d) did not induce pronounced effects in dyt1 mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt1 mice compared to controls after repeated local striatal applications of pilocarpine (25 and 50 µg/0.5 µl/hemisphere) let presume an altered synaptic plasticity in dyt1 mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt1 mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt1 and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt1 mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt1 mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd332991 which inhibits cdk4 and cdk6 is currently tested in patients with solid tumors such as glioma. we found that pd332991 suppressed il-1-induced expression of il-8 suggesting that cdk4 or cdk6 may have unknown anti-inflammatory properties. to study the effects of cdk6 on the il-1-signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk6, cdk6 s178p, in asynchronized hela cells. cdk6-expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk6 s178p enhanced il-1-induced il-8 and il-6 mrna expression. moreover, shrna-mediated suppression of endogenous cdk6 confirmed a role of this kinase in regulation of maximal il-1-induced gene expression of il-8. these data also revealed that the contribution of cdk6 to inflammatory gene expression is highest in g1, when activity of endogenous cdk6 is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g1, g1/s, g2 or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk6 in hela fucci or inhibition by pd332991 suppressed inducible il-8 expression. microarray experiments identified many additional genes that required active cdk6 for maximal il-1-or tnf-inducible gene expression. we also found that cdk6 co-immunoprecipitated with p65 nf-κb, colocalized with p65 in the nucleus and was recruited together with the p65 subunit to the proximal il-8 promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc6 channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc6 mutations. since the first descriptions at least 17 different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc6 mutations, we have analysed all mutated trpc6 channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc6 in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring 20a, 76131 karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h4r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. 1, 30625 hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h4-receptor (h4r) is involved in acute inflammation and th2 cytokine production. consequently, we intended to analyze the role of h4r in a murine th2 lymphocyte transfer-based model of asthma. specifically the ability of h4r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd4 + t cells were polarized in vitro under th2-favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h4r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il-4 production in t cells polarized in the presence of h4r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h4r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th2 phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h4r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about 40% eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h4r -/-dcs yielded only about 10-20% eosinophils in bal fluids. in summery, the h4r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h4r on dcs reduced their ability to stimulate proper th2 polarization of cd4 + t cells, we conclude that ha via the h4r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf2 (skn-1) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box 101007, 40225 düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf2 signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn-1 is the nrf2 homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf2::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf2 in cells, it has no effect on skn-1 localisation. further the effect on the nrf2 protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf2 signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are 36 and 26 µg/(kg d), resp. for children, food accounts for 36% of the total exposure, followed by mouthing (30%) and house dust (19%). the adult exposure is almost entirely (82%) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter (8%) and dressings (mayonnaise) (14 %) as well as highly consumed foods e.g. bread and bakery (16%) and vegetables (6,8 %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed 0,9 µg/(kg d). high exposures were estimated (95th percentile) up to 10,8 µg/(kg d). on average people in germany are exposed to dehp below the current tdi of 50 µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) 3707 61 201). on the basis of the available measurements of dehp, the exposure assessment has been focused on 37 food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the 37 food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the 2008 who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier 1) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier 2) was then used to rank the cumulative probability distributions of the exposure assessments of 37 food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the 37 food groups to 10 groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm 2 ; mean±sem; creb-ko 4871±258 vs. wt 3396±171; n=68/69 cells, n=4 mice, p<0.01 vs. ctr.). the nuclear factor of activated t-cells c3, nfatc3, is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc3 signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc3 on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an 8.0±1.5 fold increase of the nfat model promoter activity (n=36; n=6 transfections), but had no impact on kv4.2 promoter activity which is known to be regulated by nfatc3 (1.2±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). nfatc3 overexpression led to a 4.5±0.7 fold increase of the nfat-dependent model promoter activity (n=12; n=2) and to an inhibition of kv4.2 promoter activity (0.8±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc3 might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p2x7-rezeptors -perazine-type neuroleptic drugs are potent modulators of p2x7 receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany p2x7 receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p2x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p2x7 is a promising target for pharmacological intervention. accordingly, p2x7 blockers are currently tested in phase ii clinical trials. in an attempt to identify p2x7-modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek293hp2x7 cell line. with ic50 values of 1-2 µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp (1 mm)-triggered increases in the intracellular ca 2+ concentration ([ca 2+ ]i) that was mediated by human p2x7 (hp2x7). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp2x7mediated ca 2+ influx. whole-cell inward currents, measured at -60 mv, were blocked by more than 60% by 3-10 µm prochlorperazine. the inhibitory effects of perazines developed within about 200 ms, hinting to a direct mode of action by binding to the p2x7 protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro-1 uptake when preincubated before p2x7 stimulation with 1 mm atp or when applied subsequent to the agonist. interestingly, when added to a hek293 cell line expressing the rat p2x7, perazines potentiated the atp-induced increase in [ca 2+ ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca 2+ ]i, changes in yo-pro-1 permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p2x7 activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p2x7. similarly, pre-treatment with lov also lowered dox-induced stabilisation of p53 and phosphorylation of chek1 and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h9c2), it decreased dox-and eto-induced phosphorylation of histone h2ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il6, ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p21, wee1, cjun/fos and hmox-1 following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore 42 genes from the 156 significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb1 and abcg8 transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change 1.80 and 1.73). moreover, cyp7a1 mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change 1.9). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc2a9, abcg2, npt1, and urat1 as predicitive for the serum levels of urate 1 . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate 2, 3, 4, 5 . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc2a9 have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc2a9. therefore, the aim of our study was to investigate which sequences in the slc2a9 gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc2a9 gene. dna from human kidney samples was then genotyped for rs6449237 being part of this region. next total slc2a9 mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs6449237 showed lower slc2a9 mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs6449237 were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc2a9, urat1, npt1, and oat10, respectively was observed. conclusion. our data suggest that the slc2a9 snps rs6449237 and rs74794351 might influence slc2a9 mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc2a9 and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. 1 methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p450 enzymes yielding 1´-hydroxymethyleugenol (1´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)-1´-ohme (separated into its enantiomers) served as test compound. we could show that hsult1a1, standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult1a1-proficient and sult-deficient bacteria was examined after incubation with 7 µm of (+)-or (-)-1´-ohme. for selective detection and quantification of me-derived 2´-deoxyadenosine (da) and 2´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult1a1. the concentration dependence of adduct formation in hsult1a1-proficient bacteria was examined for (+)-1´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered 54 mg/kg bw (±)-1´-ohme (i.p.) to mice carrying the hsult1a1/1a2 gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy926) and in human kidney cell lines (hek293t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy926 cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha1 and beta1 and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx1) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse 17, 91054 erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca 2+ -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx1) to pacemaking. ncx1 is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca 2+ -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx1 in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx1 knockout (cpncx1ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx1ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx1 results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx1ko animals as compared to controls. in conclusion, these initial results establish ncx1 as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg2). the ultimate carcinogenic phase i bp metabolite anti-bp-7,8dihydrodiol-9,10-epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco-2 cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco-2 monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc1, abcc2 and abcc3 knockdown cell lines were generated allowing to demonstrate that abcc1 mediates the basolateral, abcc2 the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco-2 cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. 31, 93053 regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura 2-am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of 8-br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without 8-br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. 1 tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after 4 hours of stimulation with forskolin (fsk) in immortalized rat vsmcs (13.7 ± 0.93; n=12 from 3 isolations). in a7r5 rat smooth muscle cells the icer promoter showed a maximum stimulation of 3.2 ± 0.16 fold after two hours of fsk stimulation (n=20 from 4 isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of 8 members (tmc1-tmc8) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc1 cause deafness in human and mice whereas tmc6 and tmc8 (also referred to as ever 1 and 2) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc6 and tmc8 in hek293 cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc6 or tmc8, stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc6-or tmc8-expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d1er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc6 or tmc8. recently, it has been reported that tmc6 and tmc8 might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc6 and tmc8 mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc8, hacat cells revealed the same phenotype as described above for the hek293 cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between 0.1 thz and 10 thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around 100 ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, 0.106 thz, 0.380 thz and 2.520 thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at 0.106 thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around 0.1 thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [1] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t-84 by resazurin fluorescence and compared it with the placebo supplement. additionally, the t-84 cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il-1-beta. after the cytokine stimulation the mrna expression of ip-10, il-8 and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t-84 cells increased the expression of ip-10 (gamma-interferon inducible protein 10), tnf-alpha and il-8. by preincubation with fn at 10 µm the mrna expression of ip-10 was strongly reduced (log2-ratio -14). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [1] h. hoensch, b. groh, l. edler, w. kirch (2008) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, 14, 2187-2193. the cxcr7 c-terminal domain mediates efficient cxcl12 uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. 1, 07747 jena, germany cxcl12-signaling mediated by the g protein-coupled cxcr4 receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl12-receptor cxcr7 modulates cxcl12/cxcr4-signaling by acting as a cxcl12-scavenger. given the distinct functions of cxcr4 and cxcr7, we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek293) with respect to trafficking, stability, 125 i-cxcl12 radioligand degradation, and g protein-coupling. we found that the 24 c-terminal residues of cxcr7 were sufficient for cxcr7 to undergo rapid spontaneous internalization. replacement of the cxcr7 c-terminal domain with that of cxcr4 (cxcr7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr4 c-terminal domain by that of cxcr7 caused ligand-independent internalization of cxcr4. receptor-mediated 125 i-cxcl12-uptake, release of 125 i-cxcl12-degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr7 c-terminus. while the cxcr7 c-terminus was sufficient to abolish g protein coupling in the cxcr4-7tail mutant, replacement of the cxcr7 c-terminus, cxcr7 second intracellular loop or both domains with the corresponding cxcr4 domain did not generate a g protein-coupled cxcr7 chimera. taken together, we provide evidence that the cxcr7 c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr7-c-terminal domain may effectively control cxcr7 functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. 1 brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, 2009 ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ 1 -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap2 and bat marker genes ucp1, pgc1α, cidea. the α2 and β1 isoforms of sgc were highly expressed in bat whereas α1 sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of 8-pcpt-cgmp. in contrast, staining was lower in sgcβ1 -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ1 -/brown adipocytes contain approximately 50% less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in 56-40% decrease in c/ebpα, pparγ and ap2 expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ1 -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately 80% in sgcβ1 -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. 1 carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. 2004 , muller et al. 2005 , ma-hock 2009 , pauluhn 2010 . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to 10 mg/m 3 . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. 165 synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße 26, 80336 münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of 3-pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the 3-pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, 5-methyl-1,3-bispyridin-3-ylmethyl-1h-pyrimidin-2,4-dion and 3-(3''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of 3-pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( 1 h, 13 c, cosy, hmqc, hmbc). for synthesis of 3-(3''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and 4dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with 5-([4,6,-dichlorotriazin-2-yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty 81/1-1). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon1, pon2 and pon3. while pon1 is found predominantly in the circulation, pon2 and pon3 are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon2 and pon3 are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon2 and pon3 were shown to interact with coenzyme q10 which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon2 and pon3 reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon2 caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon2-knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon2 and pon3 as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. 5, 64295 darmstadt, germany stw 5 (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw 5 on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw 5 are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw 5 and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco-2 cells after stimulation with lps (10 ng/ml) for 2 hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps (100ng/ml) stimulation of differentiated thp-1 cells was measured using a commercially available elisa. stw 5 (62.6 -500.5 µg/ml) reduced lps (10 ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of 50.0 %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw 5 to different extents. the maximum inhibition differed in a wide range between the components. stw 5 (500.5 µg/ml) reduced significantly the release of tnf-α by 87 % in lps (100 ng/ml)-stimulated differentiated thp-1 cells while having no effect in untreated cells. in concentrations equivalent to stw 5 caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw 5, indicating a possible synergistic effect. our results indicate a multi-target effect of stw 5. the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw 5. immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. 1 , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis (1) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp11a1, encoding p450ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd3 antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized 3 hduring the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of 365 mirnas in placentas of different gestation times revealed a significant expression of 277 mirnas by comparing placentas of early gestation (<10.week), preterm (<30.week) and term (>37.week). when comparing the analyzed groups, 106 of these mirnas expose a continuous up-(including mir-20a, mir-379) or downregulation (including mir-9, mir-200a). while comparing placentas of early gestation to term placentas, 30% (e.g. mir-9, mir-429) were up-and 70% (e.g. mir-126, mir-373) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, 60% of mirnas showed higher (e.g. mir-107) and 40% lower expression (e.g. mir-627, mir-191). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir-9 and mir-429. specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir-9 (93%) and mir-429 (94%) in term placentas, while crh is upregulated 114fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the 3´-utr sequence complementary to crh was exhibit for the predicted mir-9 binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~40%, whereas the decreased luciferase activity for the predicted mir-429 binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides 1-3%), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a1) and experimental (a2,a3 ) of rats weighing 350-450g local application of dichloromethane extract of s.i in vehicle olive oil preparations of 50 and 100mg were used. animals were anaesthetized ( ether pro narcosi ), trauma 2 cm long and 2mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for 4 days with 60λ of each preparation. group a3 showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a2. the macroscopic ulcus dimensions were better in group a3 while the microscopic view were similar in a2 and a3 and better in comparison to control a1. a tendency to trauma remodelling was observed, without statistical significance (t =0.3) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the 5xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female 5xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~1 mg particles/m 3 ) or clean air (controls) for 3 or 13 weeks (5 days/week and 6 hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the 5xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf (9772-365), the dfg graduate school grk1033 and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi3k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi3k/akt, as activation of pi3k/akt using a pi3k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase-3 activation in tcsl-treated fibroblasts. conclusions: 1. rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. 2. the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi3k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a4 (anxa4) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca2+ binding anxa4 undergoes conformational changes, which lead to oligomerization of anxa4 to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa4 on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa4, there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic50 value of 6.787 µm [5.745 to 8 .017] which is much higher than ic50 values of sirolimus described in literature [0.1nm to 1nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf9 and ifitm1 was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße 1, 07747 jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine 363 (s363) threonine 370 (t370) and serine 375 (s375) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s363a/t370a/s375a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s363a/t370a/s375a/t376a/t379a/t383a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t376 and t379 which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s375 was the primary phosphorylation site, whereas t370, t376 and t379 were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s375 but not of t370, t376 of t379. our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. 1 methylene dianiline (mda; cas no. 101-77-9) is on the usepa list 2 for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of 0.01 mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at 0.2 mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at 0.2 mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral 13-week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: 7.5 mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over 2 years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: 9 mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp1 controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. 1 background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein 1 (arfrp1) is a member of the arf-family and controls the arf-like 1 (arl1)-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp1 in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp1 flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp1 expression was suppressed by sirna in caco2-cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco2-cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp1 vil-/was 43.3±5% lower than that of control mice at the age of 28 days) arfrp1 vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp1. in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp1 vil-/mice. arfrp1 vil-/enterocytes as well as arfrp1 mrna depleted caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp1 vil-/epithelium and was predominantly colocalized with rab2. our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp1 is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. 1 the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about 50% resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the 2d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by 2.2-fold and in perimeter by 1.5fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about 20% of the rhoa knockdown cells reattach, but only 5% of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by 33%. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung 1, albertstraße 25, 79104 freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the 146-kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα13 and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. 2007; 3: 707) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor 16 (pi16). here we report the generation of a mouse line where pi16 can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi16 floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi16 allele. global pi16 deficiency (pi16 lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi16 +/+ = 5.4 ± 0.2 (n = 6), pi16 lox/lox = 5.9 ± 0.7 (n = 4), p > 0.05; fibrosis (%): pi16 +/+ = 3.3 ± 0.2 (n = 7), pi16 lox/lox = 3.9 ± 0.8 (n = 5), p > 0.05; fractional shortening (%): pi16 +/+ = 29.8 ± 0.7 (n = 7), pi16 lox/lox = 26.4 ± 2.5 (n=5), p > 0.05). in addition we carried out an immunohistochemical analysis of pi16 expression using pi16-deficient mice as negative controls. pi16 localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi16 preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi16 and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [1, 2, 3] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [4] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec 3.1.1.10) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [3] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [3] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [3] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [5] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β1-adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase 2 (grk2) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk2 and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on 7-week-old c57/bl6 mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice 3 weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after 3 weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c18 eps 2, c18 sh 2, phenyl 2, cn 2, and c30 were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a1, an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp90 as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [1] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp90 and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp90 and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp90 and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp90/cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk506-binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [1] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. (2011) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h441) on top and microvascular endothelial cells (iso-has-1) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about 10 days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, 30 nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin-1 and -2 labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, 30 nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il-8 increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp-1 (prestimulated with 8 nm pma for 4d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h441/iso-has-1 with thp-1) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il-8 release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [1] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [2] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [3, 4] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, 16 pro-and retrospective studies with 10 herbal medicinal products containing ethanol at doses of 40 to 240 mg per single dose, depending on the age group, have been analyzed, covering 49 816 patients of 0-12 years of age. in these studies, altogether 15 adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other 6 herbal medicinal products it was shown that during the past few years, more than 764 mio daily doses have been sold, corresponding to more than 33 mio of patients (data obtained from manufacturers; figures available partly from 1993 onwards, partly from 2003/4 onwards). from the packages sold in germany in the years between 2005 and 2009, 48.1 mio were attributable to self-medication, 10.8 mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on 9 may 2010. she has initiated this work. prucalopride was introduced as a new selective 5-ht4 receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that 5-ht4 receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other 5-ht4 receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human 5-ht4a receptor. isolated electrically driven (1 hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven (1 hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr113808, a 5-ht4 receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec50 of -6.8 and -7.1, respectively (p<0.05 vs. wt, n=3). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by 10 µm gr113808. we could demonstrate that prucalopride acts as an agonist at the 5-ht4 receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases (1, 2) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw 5 (a 9 component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome (3, 4) ). in one model, maternal separation (5) was performed on weanling rats starting from postnatal day 2 for 3 h each day for 3 weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) (6, 7) . adult animals were restrained for 90 min/ day for 1 week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw 5 in restoring the deranged parameters. a group of animals received stw 5 orally once daily starting treatment 1 week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw 5 was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw 5 in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the 8-oxoguanine dna glycosylase (ogg1), which generates harmful repair intermediates [1, 2] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodg) varies between the opposing dna strands of the gene [3] . we now have addressed the question, to which extent the transcription inhibitory potential of 8-oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single 8-oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single 8-oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for 8-oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of 8-oxodg present in the 5′-versus 3′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of 8-oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single 8-oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg1 protein. the results thus support the initiatory role of ogg1 in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg1 suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg 5, 55128 mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [1] . an efficient method for incorporation of a single 8-oxo-7,8-dihydroguanine (8-oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing 8-oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg1 [2] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing 8-oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a 5′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites (5′-gcaatgnn). adapters containing the restriction sites for directional cloning into the 5′-or the 3′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single 8-oxog in the same sequence context into opposing dna strands in the 5'utr and in the 3'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv3, for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above 33°c. less is known about the influence of cholesterol on trpv3 signalling. we modified the cholesterol content of hek293 cells stably transfected with trpv3 and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv3 by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv2, another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv3 -/mice, our results imply that a cholesterol-regulated trpv3 signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc6 blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution (100 ml min -1 ). after equilibration in a stable perfusion and ventilation mode for 30 min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for 4 h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po2 was adjusted to 120 mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po2 of 50 mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc6 blocker larixol acetate (5 µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during 2 h of reperfusion from 22.1 ± 1.32 g to 35.4 ± 4.23 g. this weight gain was inhibited by supplementation of the trpc6 blocker (from 21.4 ± 0.91 g to 27.4 ± 2.42 g 2 h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from 17.5 ± 1.51 g to 21.5 ± 2.29 g 2 h after reperfusion), and the trpc6 blocker had no additional effect (initial 19.5 ± 1.28 g, 2 h reperfusion 21.3 ± 1.22 g). we conclude that a trpc6-blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc6 in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc6 blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. 67, 55101 mainz, germany the transcriptome analyses of human cells showed that additionally to the 30.000 protein coding sequences the human dna codes for much more (450.000 ?) non-coding rnas 1 . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il-1β-induced inos expression by an antisense rna (as-3-utr-rna) in rat hepatocytes 2 . a promoter located on the antisense strand 3' to the last exon (exon 27) of the rat inos gene drives the expression of an as-rna complementary to the 3'-utr of the rat inos mrna. using different sense primers with homology to the 3'-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as-3-utr-rna in human cells. in addition, transient transfection analyses using constructs containing a 2.7 kb fragment of the 3'-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld-1 cells revealed no promoter activity of these sequences. korneev et al. described the expression of a 2 kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells 3 . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld-1 cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld-1 cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna 14, 2030 rna 14, -2037 rna 14, (2008 . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse 190, 8057 zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises 15 peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst2 somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst2 activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s341, s343, t353, t354, t356 and t359, which in turn leads to a robust endocytosis of the sst2 receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s341 and s343. somatoprim and ke108 are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke108 on sst2 somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke108 were tested for functional selectivity at sst5 receptor mutants, which possess a carboxyl-terminal sst2tail. this approach allows detection of receptor activation by phospho-specific sst2 antibodies. compared to octreotide, somatoprim activates sst2 but has a higher activity on sst5. ke108 and pasireotide are partial agonists at the sst2 receptor. pasireotide, ke108 and somatoprim show comparable effects on sst5 receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst5 receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. 1, 2 , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where 17beta-estradiol (e2) and cdcl2 modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi 10 .1007/s00204-011-0787-x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium (2mg/kg b.wt cdcl2 (cd2)) or steroid estrogen (0.1 mg/kg b.wt. 17alpha-ethinylestradiol (ee2)) on ahrassociated gene expression (i.e., ahr, cyp1a1, gsta2, nqo1) in the small intestine of rats after oral exposure (3 days gavage). the animals were also co-treated with cd2 and pure anti-estrogen (2.5 mg/kg b.wt zk191703 (zk)) or ee2, to asseess whether ermediated processes are involved. we also measured cyp1a1 mrna expression in two estrogen receptor negative colon cancer cell lines (ht-29 and caco2) treated for 5 days with cdcl2 (1µm) and e2 (0.01µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for 28 days (0.4-9 mg/kg b.wt cdcl2 equivalent to 5, 50, and 150 ppm) and ee2 (0.08 mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o 6 -methylguanine-dna methyltransferase (mgmt), o 6 meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o 6 meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u-87 mg and ln-229 glioma cell lines. the maximum amount of autophagy was observed several days (96 h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o 6 meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o 6 meg, are repaired preferably by homologous recombination (hr) ln-229 cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor 3-methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o 6 meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf (02nuk016). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb1a) have been found in patients suffering from rp. we used cngb1-deficient (cngb1 -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb1a cdna (approx. 4 kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype 8) into the subretinal space of 2-week-old cngb1 -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb1 -/mice expressed full-length cngb1a and cnga1, which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb1 -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb1 replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb1 -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h295r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on 14 reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category 1) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg 405) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of 50 agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [1] . for many nanomaterials published genotoxicity studies did not give a consistent picture [2] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [3] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [4] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco-2 human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco-2 cells by activated pmn and this effect increased with increasing pmn to caco-2 cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco-2 cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h 2o2 was increased in buthionine sulphoximine (bso) pre-treated caco-2 cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco-2 cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p450 monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg2 cells incubated with oa in the absence and presence of s9 mix. pure oa, as well as oa pre-activated with liver homogenisates (s9 mix) were used to treat human hepg2 cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s9 fraction plus cofactors of phase i enzymes. the cell viability was measured after 4 h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by 2´,7´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s9 mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s9 mix was toxic at 75 nm oa. strong effects could be observed when oa was pre-activated with human s9-mix at a concentration of 50 nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg2 cells treated with oa in the presence of all investigated s9-mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg2 cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse 10, 10589 berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in 2004 the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october 2011. here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. 44, 39120 magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p38 mapk and phospholipase d2. in line with this, we observed marked phosphorylation of p38 mapk and activation of phospholipase d2 induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to 100 fold induction of il-4, which was dependent on p38. in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il-4. by inducing il-4 opioids significantly modulate the t helper cell balance into the type 2 direction, which influences various immune responses, e. g. the antiviral, t helper cell type 1-mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse 150, 44780 bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde5, the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde1, the ca2+/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca2+ concentrations. and lastly pde3, the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde3. cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has1-3). the has3 gene is alternatively spliced. has3 transcript variant 2 (has3v2) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has3 transcript variant 1. the aim of the present study was to investigate whether has3v2 is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has3v2 mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has3v2 fusion protein and a ddk-tagged has3v2 construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has3v2 mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has3v2 protein expression in vsmc and platelets. in vsmc has3v2 mrna expression was strongly up-regulated in response to interleukin-1β (il-1β, 10 ng/ml) whereas stimulation with interleukin-10 (10 ng/ml), platelet-derived growth factor-bb (10 ng/ml), transforming growth factor β (10 ng/ml), tumor necrosis factor α (10 ng/ml) and interferon-γ (10 ng/ml) had no effect. transfection of hek cells with eyfp-has3v2 fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has3v2 proteins were precipitated together suggesting formation of multimeric has3v2 complexes. transfection of has3v2 did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has3v2 is present in vascular cells and responds to inflammatory cytokines such as il-1ß. because of the intracellular localization and the lack of ha secretion in has3v2 transfected cells, has3v2 may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th2 cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp 12 hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of 1 mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p15, in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig)2-vpgkg-(vpgig)2, were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p15 peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after 1 h adhesion (60%) and an enhanced proliferation (63%, 1 h adhesion, 24 h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. (1) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde2a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure (2-3-fold, p<0.05, n=8). notably, pde2a protein abundance is 4-fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p<0.05, n=7). to this end we tested whether pde2a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde2a overexpression induced α-sma expression (3.2-fold p<0.05, n≥8) and to a lower extent ctgf synthesis (1.5-fold, p<0.05, n≥5). mechanistically, pde2a showed preferential subsarcolemmal localisation with diminished total cgmp levels (-56%, p<0.05, n≥5). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde2a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde2a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (-60%, p<0.05, n≥5). these data implicate pde2a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde2a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. 2005) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: 12, 80 or 250 nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at 3 mg/m3, all three tested polymers -including the np (12 and 80 nm) -did not cause any changes in lavage fluid and in histopathology at 10 mg/m3. the organic pigment was a poorly soluble pyrrol with an intense orange color. the np (10 to 50 nm width and 30 to 400 nm length) and the coarse pigments (70 to 200 nm width and 0.3 to 3 µm length) are both needle-like. they were tested at 1, 3, 10 and 30 mg/m3 for the np and 3, 10 and 30 mg/m3 for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso4, zno, ceo2, al-doped ceo2, zro2, amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio2 and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, 6 h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after 3 days and 3 weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso4 as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the 16 substances ranged between < 0.1 and >50 mg/m 3 . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the 16 materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. 1 , wächter t. the eu reach regulation no 1907/2006 requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go3r, accessible free of charge at www.go3r.org, has been created to allow quickly finding relevant hazard information and data. go3r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go3r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid 5 chapters. hereby, the user can browse directly through the entire 21 million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go3r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h2-receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. 1, 30625 hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin 2 has been approved as orphan drug for the consolidation treatment of aml (1) . it is assumed that histamine exerts its effects by activating the histamine h2-receptor (h2r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation (2) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml (3), we initiated a study to explore the possibility that h2r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl-60 cells. in hl-60 cells, histamine and various h2-receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h2r conformations. h2r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h2r agonists showed no signs of cytotoxicity. intriguingly, following h2r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) 4, indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure (30 minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h2r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/11 protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb1 receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca 2+ release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca 2+ imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/11 proteincoupled metabotropic glutamate receptor 1 (mglur1) was activated by superfusion of (rs)-3,5-dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca 2+ concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur1 antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb1 receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb1 receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca 2+ stores by thapsigargin, cyclopiazonic acid and ip3. the results indicate that after activation of the gαq/11 protein-coupled metabotropic glutamate receptor 1 (mglur1) of the postsynaptic neuron ca 2+ is released from the endoplasmic reticulum. this ca 2+ release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb1 receptors. the guanine nucleotide exchange factor dock9 controls reelin dependent cdc42effects on radial migration pichler m. 1 the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi2-deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi2-deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi2deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low (3 mm) and high (16 mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi2 proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi2-deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi2-deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi2-targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi2 is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. 11689, 1992) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r7260 (molecular probes, 0.125 mg/ml, excitation 505 nm, emission 530 nm). the affinity of compounds in radioligand binding was slightly higher in sur2b than in sur2a-type channels, but the enantiomeric ratio in sur2a channels matched that one determined for the sur2b-type indicating some conformity of the binding pockets of sur2a and sur2b-proteins. surprisingly, however, the membrane potential tests revealed that the (3r,4s)-enantiomer acted as agonist (a) whereas the (3s,4r)-enantiomer acted as antagonist (b): (3r,4s)-bms-191095 induced membrane hyperpolarisation whereas (3s,4r)-bms-191095 repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms-191095 is not selective for sur2a as compared to sur2b-type k atp channels. its enantiomers activate and block sur2-type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur2b-type katp channels act as partial agonists at cardiac sur2a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. 1, 40225 düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur1-type katp channels (nielsen et al., j med chem 45: 4171, 2002) were characterized in sur2b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal 375, r45, 2007) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek 293(kir6.2/sur2a)-cells and compared to hek 293(kir6.1/sur2b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation 505 nm, emission 530 nm) using 0.125 mg/ml of the dye r7260 (molecular probes). standard-kco induced hyperpolarisation with ~10-fold smaller potency (pec50) in sur2a. ttd-compounds with ch3-cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur2a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur2a and sur2b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca2) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca2 channels in nonneuronal cells. the aim of this study was to investigate the expression of kca2 channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca2 channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca2.2 and kca2.3 channel activator cyppa (25 µm) and specific inhibitory peptides (50 µm) were applied to distinguish effects mediated by the kca2 channel subtypes. all kca2 channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps (200 ng/ml) induced microglial proliferation. the kca2.2/kca2.3 channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca2.3 channels, but not of kca2.2 channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il-6, but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il-6 production while tnf-a production was not affected. moreover, using inhibitory sk3/kca2.3 channel peptides, we demonstrated that sk3/kca2.3 channels modulate lps-induced cytokine il-6 production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca2.3 channel stimulation reversed microglial activation. thus, kca2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. (3r,4s)-(3s,4r)intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. 1, 30625 hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r.8 to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is 10 in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase 5 (cdk5) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk5 in cancer. cdk5 was shown to regulate tumor growth, and our group discovered that cdk5 regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk5 is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk5 in hcc. histology of tissue micro arrays indicates an increased expression of cdk5 in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk5 in hcc, we analyzed the impact of both pharmacological inhibition of cdk5 and specific downregulation of cdk5 with rna interference on hcc cells. pharmacological inhibition of cdk5 with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g2/m cell cycle arrest and cell death, and reduced motility of huh7 and hepg2 cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk5 also reduced proliferation, clonogenic survival, migration and invasion of huh7 cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat3 at ser727 that is directly phosphorylated by cdk5, and tyr705. in line with this, the expression of the antiapoptotic protein mcl-1 is reduced by inhibition of cdk5. our results point to an important function of cdk5 in hcc and suggest cdk5 as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c3 toxin lillich m. 1 , chen x. clostridium botulinum produces the adp-ribosyltransferase c3, which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c3 toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [1] . thus, c3 toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c3bot mutant (c3mut) and streptavidin. the c3 portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c3mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j774a.1 macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c3mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c3mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j774a.1 macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c3mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j774a.1 macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c3mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h4iie rat hepatoma cells. in this context cw60 (a diphosphane ligand with azoyl substituents r2p(ch2)2pr2, r= thiazol-2-yl) turned out to be the compound with the highest cytotoxic potential with an ic50 value of 6,5mm (24h incubation). further investigations revealed that cw60 induced an apoptotic cell death in h4iie demonstrated by the activation of caspase 3/7 (48h incubation with 10mm cw60). in addition the induction of apoptosis was confirmed by the dna ladder formation (24h incubation with 5mm cw60). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after 1h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi3k/akt but after 24h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw60 shows strong toxic effects in h4iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: 3-4 hrs vs. 1 hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for 923, biedersteiner str 27, 80802 münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc6-, and trpc3-, and trpc3/c6-double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc6-, and trpc3/c6-double knock-out mice as compared to aorta from ctr and trpc3-knock-out mice indicating a functional link between cgki and trpc6 channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by 8-br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc6-, trpc3-, and trpc3/c6-double knock-out mice. no effect of 8-br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc3-, and trpc6-knockout mice. trpc3 could be detected in both smooth muscle and endothelial cells whereas trpc6 was only detected in endothelial cells. the results suggest that absence of cgki or trpc6 impairs the vasodilator tone induced by endothelial no production but that cgki and trpc6 channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc6 -/-, and trpc3 -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb 153 in human hepatic cell models lohr c. 1 , neser s. after the treatment with tcdd, however, a total of 281 genes were more than 2-fold up regulated in hepg2 e.g. cytochrome p450 1a1 (cyp1a1) (32-fold) a sensitive marker for ahr activation. additional up regulated genes in hepg2 were; arylhydrocarbon receptor repressor (ahrr) 15-fold, aldehyde dehydrogenase 3 a1 (aldh3a1) 11-fold, and cytochrome p450 1b1 (cyp1b1) 10-fold. 44 genes were more than 2-fold down regulated in hepg2 cells e.g. -proprotein convertase subtilisin/kexin type 9. markedly different findings were obtained in hheps, i.e., 117 genes were up regulated, the highest up regulated gene was cyp1b1 with a 95-fold increase in gene expression, followed by cyp1a1 (41-fold) and aldh3a1 (21-fold). only a small group of genes were significantly down regulated (17 in total), e.g., solute carrier family 2 (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only 30 in common genes were up regulated, including cyp1a1, cyp1a2, cyp1b1 and aldh1a3. only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg2 and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg2 cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. 1 , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb 815/a14). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. -206, -213 and -214) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac 1, expressed in sf9 insect cell membranes, with cam, cam-206, -213, -214 and -215 by measuring the catalytic activity of ac 1. cam-mutants show a 3-4-fold lower potency than cam, but they are more efficacious than cam. most prominently, cam-215 was 133 % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam-215, it is more hydrophobic than cam and this leads to a better ppi with ac 1. in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac 1 interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac 1. because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac 1 interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee 114-116, 14558 potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type 2 diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type 2 diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the 16 th week of age and total pancreatic insulin content indicated diabetes prevalence of 68% in males and 25% in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type 2 diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = 0.69) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type 2 diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about 20-30% non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after 1, 2, 4, 6 or 12 weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p450 2e1 and 4a1 isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep3 receptors for prostaglandin e2 convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was (1) the pharmacological characterization of ep3 receptors in human pulmonary arteries and (2) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l-826266 served as the ep3 antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep1/ep3 agonist sulprostone (1 nm -100 mm) concentrationdependently contracted human pulmonary artery rings (pec50 and emax; 6.89±0.12 and 106.5±5.2%, relative to the contraction induced by kcl 60 mm). the concentrationresponse curve of sulprostone was not affected by the ep1 antagonist sc 19920 (10 µm) but shifted to the right by l-826266 (10 µm) (apparent pa2 6.22). extending the exposure time to l-826266 from 0.5 to 3 h increased its antagonistic potency to 7.39 (schild plot-based pa2; concentrations 0.1, 1 and 10 µm). in pithed rats electrical stimulation (0.66 hz, 1 ms, 50 v for 5 s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline (0.15 nmol/kg) increased heart rate (hr) by 55 beats/min. sulprostone (10 -1000 nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by 80%. l-826266 (3 µmol/kg) diminished the inhibitory effect of sulprostone 1000 nmol/kg on the neurogenic tachycardia by 20%. in conclusion, ep3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. -5-bromo-n[3-(5voltage-gated ca 2+ channels of the central nervous system control a multitude of ca 2+ dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav2.1 ca 2+ channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca 2+ -channels. ca 2+ influx via cav2.1 ca 2+ channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav2.1 ca 2+ channels play a crucial role in synaptic transmission. the global cav2.1 knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav2.1 ca 2+ channels for learning and memory. therefore, we crossed a floxed cav2.1 mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav2.1 in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav2.1 channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav2.1 knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav2.1 for learning and memory. helicobacter hepaticus-infected rag2 -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., 2009 , pnas 106: 1027 -1032 . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag2 -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f4/80-positive macrophages, by 10 wks postinfection (pi), progressing into colon carcinoma by 20 wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of 16 different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by 20 weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as 8-oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. 1 recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [1] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c90rf7, which shares 26,3% amino acid sequence identity with the drosophila protein. it covers 171 amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c90rf7 and the ca v2.2 channel in xenopus oocytes reduced the amount of the α1b and cavβ3 subunits of the ca 2+ channel in the plasma membrane but did not affect the gating properties of the cav2.2 channel. expression of c90rf7 alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c9orf7-fusion in e.coli (yield 2mg at 1mg/ml). we are currently preparing the c9orf7 part of the his-sumo-c9orf7fusion protein by ulp1-protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c9orf7 sequence. we could not identify any homologues of c9orf7 in the mouse genome and to analyze its function we are currently generating c9orf7 deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c9orf7 by expression of the bgalactosidase gene under the control of the endogenous c9orf7 promoter. by southern blot analysis we´ve already identified 210 homologous recombinant embryonic stem cell clones out of 300 analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c90rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of 17β-estradiol in cultured v79 cells expressing human cytochrome p450 1a1 martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany oxidative metabolism of the female sex hormone 17β-estradiol (e2) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e2 undergoes metabolic activation by cytochrome p450-dependent monooxygenase (cyp) isozyme 1a1 to 2-hydroxy-(2-ho) and to a lesser extent to 4-ho-e2. if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e2 was determined in male chinese hamster lung fibroblasts (v79 cells) expressing human (h) cyp1a1 and (ii) the metabolite profile of e2 in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp1a1 and dimethylsulfoxide as solvent control. v79 hcyp1a1 were treated with 100 nm e2 for 3 weeks and the resulting 6-thioguanine (6-tg) resistant mutants selected at weeks (w) 2 and 3. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells ranged from 5 ± 1 (w2) to 9 ± 4 (w3). as expected, 0.25 µm bap induced a significant increase in mutant frequency (mf, 266 ± 13, w2 and 132 ± 46, w3) . treatment with 100 nm e2 resulted in a 3-fold (17 ± 2, w2) and a 2-fold (23 ± 4, w3) increase in mf, suggesting slight mutagenic activity. in culture medium of v79 hcyp1a1 treated with 100 nm e2, 2-ho-e2, 2-methoxy-(meo)-e2, 3-o-methyl-2ho-e2 and 4-meo-e2 (suggesting intracellular formation of 4-ho-e2) were detected. while 4-meo-e2 concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w3 for methylcatechols and at w2 for 2-ho-e2, correlating with the maximum increase in mf, observed after 2 weeks as well. in conclusion, e2 possessed a slight mutagenic potential after hcyp1a1-mediated activation to 2-, 4-ho-e2 and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., 10589 berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis (17-β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg-3 and transactivation via estrogen receptors α and β in stably-transfected hek 293 cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of 17β-estradiol and progesterone in cell culture supernatants of jeg-3 cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and 17β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of >1.7 µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at 5 µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [1ng -1µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. 1 trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm4 as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm4 and trpm5 have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm4 gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm4 serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm4 in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm4 regulates catecholamine release. besides trpm4 we recently identified transcripts encoding additional trp channels including trpc5 and trpc6 in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation (10µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc1/c5/c6 triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp4 (ec 50 0.32 vs. 2.8 µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t0070907 but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at1a and at1b receptors as mechanosensors in myogenic vasoconstriction blodow s. 1 , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/11-proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/11-coupled receptors such as angiotensin ii at1b, vasopressin v1a, endothelin eta and etb and α1a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v1a receptor and α2a and α2ab adrenoceptors showed no differences of myogenic tone, blocking of at1a and at1b receptors by losartan and candesartan, eta receptor by bq123 and α1a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at1a -/mice with and without additional blocking of at1b receptors by candesartan suggested that especially at1b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at1b -/mice. these findings suggest that mechanosensitive gq/11-protein coupled receptors, especially at1b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm3 ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm3 gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [1] . they encode a substantial variety of isoformes and so far we have identified more than 20 distinct trpm3 proteins in mouse and rat each varying in exons 1, 2, 8, 13, 15, 17 and 24 . these variants differ enormously in their biophysical properties. for example splicing within exon 24 affects the channel pore and causes significant changes of the ionic selectivity of trpm3 channels [2] , whereas splicing of 54 nucleotides encoded by exon 13 leads to dormant trpm3 proteins. however, the frequency of these different isoformes in trpm3 expressing tissues is completely unknown. to get insight into the significance of the different trpm3 isoformes we investigated the abundance of alternative trpm3 transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon 13 ranges from 5 up to 20 % in different cell types and tissues. furthermore we analyzed the trpm3 transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm3 transcripts are most abundant. for that purpose we sequenced more than 120 clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon 13. in cells of the choroid plexus nearly all (124 /126 clones) carried the short ca 2+ permeable pore. furthermore, we identified seven variants spliced in exon 20 encoding truncated trpm3 proteins. however, the composition of the trpm3 transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm3 function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of 1800, 540 or 90 µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of 0.15 µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of 90 µg/person/day, which corresponds to a dose of 1.5 µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has1, -2, -3), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at 14 weeks coincided with ha deposition and induction of has3 in aortic root plaques. in human symptomatic carotid artery plaques has3 was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has3 was specifically induced via activation of nfkb by interleukin-1β (il-1β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay 11-7082. has3 was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il-1β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f4/80 positive macrophages as shown by immunohistochemistry. to study the effects of has3 mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has3 were employed. overexpression of has3 resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has3 were mediated by both pi3k signaling and mapk signaling via hyaluronan receptors cd44 and rhamm. conclusion: the present results suggest that has3-dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has3-mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for 12 weeks and were pretreated by inhalation of the arginase inhibitor (2)s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il-8 levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn3-deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. 5-13, 81377 münchen, germany hcn3 encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn3 -/and hcn3 +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn3 in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn3 knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn3 -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn3 knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn3 -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn3 in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. 1 , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, 2008) silva et al.,1993) . we simulated 1. the alcohol concentration in a breastfed neonate and a 3-month-old suckling infant after the nursing mother had consumed alcohol,2. the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman 3. the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was 0.59 ‰ after consuming 0.25 l of wine, peak concentration was 0.0033 ‰ in the newborn, 0.0038 ‰ in the 3-month-old infant and 0.38 ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was 0.015‰ in the neonate and 0.015‰ in the 3-month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation "1 to 2 glasses of wine on occasion" (agence nationale d'accréditation et d'évaluation en santé, assante 2002) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. (2002) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. (1993) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type 1 (gαi1) and adenylyl cyclase type v (ac5). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a2a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai1-cfp and yfp-ac5. after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. 2003) . to further analyze the properties of the dissociation between gai1 and ac5 we measured in parallel the offset kinetics of the interaction between gai1-yfp and gβg-cfp (gi1-fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs4 accelerated the deactivation of gi proteins and the dissociation of gai1-cfp from yfp-ac5. these experiments revealed that in the absence of rgs4 the dissociation of gai1 from ac5 after agonist withdrawal takes about 3 times longer than the deactivation of gi proteins. in the presence of rgs4 this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai1 from ac5 was only marginally accelerated by rgs4. these observations lead us to hypothesize, that ac5 might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai1-ac5 interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi0/0) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by 65% ± 1% in pkgi0/0 as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha (19% ± 4.8%), ppargamma (66% ± 2.9%) and ap2 (37% ± 10.6%) expression in pkgi0/0 cells as compared to pkgifl/fl. treatment of 3t3-l1 cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in 3t3-l1 cells. concomitant activation of pkgi in 3t3-l1 preadipocytes and treatment with the demethylating agent 5-aza-deoxycytidine significantly increased expression of uncoupling protein-1 (ucp-1) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser-188 in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp-1 promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex 1 and 2, mtorc1 and 2. under amino-acidrich conditions, activated mtorc1 promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc1 function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc1 function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc1 translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl 2 in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc1.2 bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the 1,4-bz flunitrazepam (flu), clonazepam (clo) and 4chlorodiazepam (4-cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas 4-cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc1.2 cells with 4-cd, flu and clo for 1, 6 and 24 hours up to 712 genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at 6 hours revealed that the expression of 217 genes was regulated by both flu and clo but only 8 genes were regulated by both 4-cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of 4-cd. the difference between the gene regulation by flu and clo on the one hand and that of 4-cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the 1,4-bz could be mediated by the recognition sites targeted by the 2,3-bz, i.e. the gria2-encoded ionotropic glutamate receptor ampa2, we investigated by quantitative pcr whether hmc1.2 cells express gria2, tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra3, gabrb3, gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra2, gabra4, gabrb2, gabrg3 and gabrr2. expression of gria2 was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee 114-116, 14558 nuthetal, germany 5-hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v79 cells genetically engineered for expression of human sult1a1 suggesting that hmf is converted into the reactive 5sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n 6 -((5-formylfuran-2-yl)methyl)-2'deoxyadenosine (n 6 -ffmda) and n 2 -((5-formylfuran-2-yl)methyl)-2'-deoxyguanosie (n 2 -ffmdg). these adducts were also detected in dna from v79-sult1a1 cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about 500 ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta100 engineered for expression of human sult1a1. the putative mutagen 2-sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta100-sult1a1 and in dna of liver, lung and kidney of fvb/n mice that had received about 390 mg ffa/kg body weight per day via the drinking water for 28 days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp2 (methyl cpg binding protein 2) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp2 is known to be a target gene for several micrornas including the cluster mir-132/212 in the brain. recently, our group could show that mecp2 expression is downregulated in human heart failure suggesting that mecp2 might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp2 by the cluster mir-132/212 in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir-132/212 expression, we treated cultured nrcms with 40 µm norepinephrine for 72 hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir-132 (1.79 ± 0.14-fold of untreated cells, p<0.01) and of mir-212-3p (1.73 ± 0.26-fold of untreated cells, p<0.05) and downregulated mecp2 mrna and protein levels (0.80 ± 0.04-fold of untreated cells, p<0.05). to check whether mecp2 downregulation also occurs by direct mir-132/212 activation we increased levels of mir-132 and mir-212 in cardiac myocytes by transfecting precursor mir-132 and mir-212-3p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp2 mrna and protein downregulation (0.75 ± 0.05-fold of control, p<0.05) after mir-132 overexpression. similar results were obtained by overexpression of mir-212-3p. to test the effects of adrenoceptor activation on the mir-132/212-mecp2 axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps (30 mg/kg/day each). after 7 days, cardiac ventricles were analyzed. nppa gene expression (2.81 ± 0.32 -fold of control animals), mir-132 and mir-212-3p levels (3.67 ± 0.5 and 2.62 ± 0.4 -fold of control animals, p<0.01 and p<0.05, respectively) were increased while mecp2 protein levels decreased to 77% (p<0.05) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir-132/212 expression and to downregulation of mecp2 in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at-1 receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at-1 receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a 30% increase of control (pec30%) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec30%=8.6±0.2 (mean±sem, n=18); maximum increase=110±8%]. neither rosiglitazone nor pioglitazone (0.3-3 µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec30%=8.8±0. 2, 9. 2±0.2 and 9.3±0.3; maximum increase=118±14%, 146±13% and 148±16%, in the presence of 0.3, 1 and 3 µm of rosiglitazone, respectively, n=4-6, each). in contrast, pioglitazone in concentrations up to 3 µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer 1 (dlc1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc1 loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia 1 and 2 (mkl1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc1. moreover, dlc1 loss and mkl1 nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl1 in dlc1-deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl1 phosphorylation. dlc1 loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr61, myl9 and myh9. furthermore, we identified a novel target gene, integrin a5, with a key role in cell migration and metastasis, that exhibited a dlc1-and mkldependent regulation. depletion of mkl1/2 suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc1 loss. our data provide insight into the mechanism by which dlc1 loss initiates tumorigenesis. as mkl1 and 2 have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, 53121 bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p2 receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p2y receptors (p2y1, 2, 4, 6, 11, 12, 13, 14) , and homo-or heterotrimeric ligand-gated ion channel or p2x receptors (subunits: p2x1-7). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p2y and p2x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p2y and p2x receptors [1] [2] [3] [4] [5] [6] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm2.5 has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm2.5 concentration in a telephone booth (1,75 m3 volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with 6 sec time resolution; laser scatter allowed a size resolution from 0,25 µm to 32 µm. for the pm2.5 concentration the following values were calculated: cumulative pm2.5 concentration as auc-pm2.5 (µg/m3/sec), peak pm2.5 concentration as cmax-pm2.5 (µg/m3) and average pm2.5 concentration cmean-pm (µg/m3). in closed door condition both cigarettes produced particulate auc-pm2.5 values of 59 000 ± 15 000 µg/m3/sec (rc) to 85 000 ± 31 000 µg/m3/sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin-6 (il-6) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has 1-3) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il-6 participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il-6. therefore, the aim of the present study was to elucidate whether il-6 regulates the expression and function of ha-matrix in cfs. cells were isolated from c57bl/6j mice and used during passage 2-3 for experiments. cfs were stimulated with il-6 or hyper-il-6 which is a fusion protein of il-6 and soluble il-6 receptor (sil-6r). after 10 and 30 min signal transducer and activator of transcription 3 (stat3) was phosphorylated in response to hyper-il-6 but not in response to il-6. rt-pcr revealed rapid upregulation of has 1 (5.94 ± 2.49 fold of unstimulated control, 1h) in response to hyper-il-6. has2 was induced to a lesser degree (2.04 ± 0.55 fold of unstimulated control, 1h) whereas has3 was not responsive (1.49 ± 0.42 fold of unstimulated control, 1h). in contrast, il-6 had no effect on transcript levels of has isoenzymes. in turn, expression of has1 and has2 in response to hyper-il-6 was inhibited by ag490, which indicates the involvement of stat signaling. interestingly, despite induction of has1 and has2 the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il-6. this may indicate that il-6 regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il-6 trans-signaling (hyper-il-6) via the soluble il-6r and subsequent stat3 signaling with increased ha-synthesis. the fact that il-6 had no significant effect suggests that the expression of the non-signaling membrane-bound il-6 α-receptor (il-6r) in cultured murine cardiac fibroblasts is not sufficient to induce has 1 and -2 gene expression. therefore, il-6 trans-signaling mediated by il-6 and the circulating sil-6r might be necessary to mediate the il-6-induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by 3-aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße 25, 53127 bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura-2 fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ 3 h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified 3-dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec50 165 µm). fura-2 fluorimetry showed an increase in intracellular ca 2+ levels by 3-dl-aminoisobutyric acid (300 µm). moreover, 3-dlaminoisobutyric acid reduced the forskolin-induced camp production by up to 65 % (ec50 195 µm). in addition to 3-dl-aminoisobutyric acid, we identified 3-dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of 4.9 (nfat assay), thioridazine with an apparent pkb of 5.4 (nfat assay) and 5.3 (camp assay) and rimcazole with an apparent pkb of 5.2 (nfat assay) and 5.2 (camp assay). in conclusion we show for the first time that 3-dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf-1-chemokine receptor cxcr4 plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr4 is involved in disease states including inflammation and cancer. it is well established that sdf-1-stimulated cxcr4 receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr4 c-terminal domain contains 15 serine and 3 threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr4. here, we analyzed the phosphorylation pattern of cxcr4 at 3 c-terminal sites after stimulation of the receptor with sdf-1 and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s324/325, s338/339 and s346/347 in immunoblot analyses, we showed that the 3 sites were phosphorylated after stimulation with sdf-1 or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf-1-induced phosphorylation at s324/325, s338/339 and s346/347 was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s346/347 and then at s324/325 and s338/339. taken together, these results indicate that the c-terminus of cxcr4 is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. 1 in [1] ), but information on transfer from maternal blood to milk is scarce: published data [2] indicate that levels of ota in milk are roughly one tenth (0.1) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, 18 lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [1] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was 235 ± 129 ng/l, and no major variations were observed over time (p = 0.19). on the other hand, ota levels found in colostrum (85 ± 60 ng/l) were higher than in mature milk (p < 0.05). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p 0.43 ± 0.26) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [3] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [1] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [2] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= 28) and turkey (n=10, only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [3] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [4] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of 5 ng/kg-bw/day set for adults [1] . in both cohorts, most of the urine samples tested positive for ota (chile 72%, turkey 80%). ota levels observed in urine samples from the turkish infants (range: 116 -1,361 ng/l) were 10 fold higher than levels found in chilean samples (range positive samples: 17 -320 ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to 30% of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p450 system. using the estrogen receptor response assay (e)-4-hydroxyclomiphene and (e)-4-hydroxy-n-desethylclomiphene (ec50: 2.5 and 1.4 nm, respectively) turned out to be 100 times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp2d6 revealed to be the major isoenzyme involved in the formation of 4-hydroxlated metabolites. n-deethylation was catalysed by cyps 3a4/5, 2d6, 2c19 and 2c8. rates of 4-hydroxylation in microsomes from 30 human liver donors correlated with the number of functional cyp2d6 genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp2d6 genes (poor metabolizers) cmax of (e)-4-hydroxyclomiphene and 4-hydroxy-ndesethylclomiphene was 8 and 12 times lower, respectively, when compared with subjects with at least one fully functional cyp2d6 allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was 10 and 50-fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound 1 proved to be the most active derivative, showing a significant toxicity at a concentration of 2,5 µm. compounds 2 and 3 showed significant toxic effects at a concentration of 5 µm. the compound 4 showed no toxicity up to a concentration of 100 µm. all derivatives 1, 2 and 3 have a ec50 between 10 and 15 µm. we further proved the induction of apoptosis by apo-one assay (caspase 3/7 activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb 153 using whole genome microarray analysis neser s. 1 , lohr c. 1 , van ede k. i. 2 , andresen k. 3 interaction with the aryl hydrocarbon receptor (ahr), with 2,3,7,8-tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p450 isoenzymes (cyps), i.e., cyp1a1, 1a2, and 1b1. one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c57bl/6 mice were treated with single doses of six dl-congeners (tcdd, pcb 118, pcb 126, and pcb 156) , and the 'non-dioxin-like' (ndl) pcb 153. quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array (4x44k) system. the quantity of genes affected (≥ 2fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated 89 genes, and down-regulated 66, 4-pncdf-treatment had impact on 3208 (up), and 2396 (down). treatment with pcb 118 led to marginal numbers of 16 up and 6 down-regulated genes. with 64 (up), and 38 (down) genes shared, the most extensive overlap occurred between tcdd-and 1-pncdd-treatment. no overlap was found due to treatments with the ndl pcb 153 (21 up, 1 down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of 19 up, and 5 down-regulated remain, most of them being related to drug metabolism. while pcb 118 regulated only genes involved in drug metabolism, omission of pcb 118-regulated genes resulted in consistently 51 (up), and 24 (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb 153 was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years 2009 and 2010 were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, 4454 enquiries were registered in total. in 4.7% of those enquiries systemic antibiotics were named with a total number of 283 drugs. 50.9% of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions (50.7%), diagnosis/treatment (35.4%), drug application (29.2%) and drug-druginteractions (19.4%). the majority of the requesting patients (61.5%) was born before 1970. a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged 40 years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and 14 c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer 344 rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over 120 min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter 2, oat2 (slc22a7), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like 5-fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat2 to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat2 localization in human liver. because interindividual variability of oat2 expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat2 expression. methods: an expression profile of oat2 for 20 human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat2 mrna expression was analyzed in well-characterized human liver samples from 150 caucasians that were accompanied by detailed demographic and clinical data. oat2 was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat2 protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc22a7 gene region. results: oat2 mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat2 was identified in human liver. hepatic expression of full-length oat2 mrna and oat2 protein varied 37-fold and 41fold, respectively. oat2 mrna and protein levels did not correlate with each other. oat2 was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the 9 exons, the 5'-flanking region, or the 3'-untranslated region of the slc22a7 gene were identified. univariate analysis showed that oat2 mrna is reduced in patients diagnosed for cholestasis (p=0.0012) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat2 expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat2 expression. this indicates consequences for hepatic drug elimination of and response to oat2 drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb327 demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [1] . we investigated the interaction of mb327 and several structure analogous at the orthosteric binding site of human α7 nachr (hα7 nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα7 nachrs were investigated with radioligand binding experiments performed as high-throughput method [2] . membrane preparations of gh 4c1 cells stably expressed hα7 nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb442, mb456 and mb770 exhibited ki values at micromolar concentrations while three other compounds, mb327, mb583 and the pharmacological inactive mb424 (negative control) did not show any interaction with the orthosteric binding site of hα7 nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb442 which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku80 mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase-3 and -7, and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh2ax and 53bp1 foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku80 formed at early times after acnu treatment less γh2ax and 53bp1 foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku80 mutant cells show foci levels similar to the wt indicating restitution of h2ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh2ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh2ax foci increased significantly in the s-phase and remained at a high level during g2 where a fraction of cells remained arrested. rad51, atm, mdc-1 and rpa-2 foci were also formed and shown to co-localize with γh2ax. these foci were ameliorated significantly in s-and g2-phase, which was similar to the time course of γh2ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk2 and less phosphorylation of chk1 in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb1 receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb1 globally knock-out animal as background, we induce the expression of cb1 specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb1 analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb1 receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c5a, fmlp, and leukotriene b4 are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi2 and gαi3 isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice 4 days after i.p. injection of 4% thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi2-deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi2 was upregulated in gαi3-deficient macrophages. vice versa gαi3 was upregulated in gαi2-deficient macrophages. concerning gβ isoforms, both gβ1 and gβ2 were strongly reduced in the gαi2-deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi3-deficient macrophages showed reduced gβ1 protein levels only which caused a change in the gβ2/ gβ1 quotient in favour of gβ2. we are currently establishing an in vivo infection model with l. monocytogenes in gαi2and gαi3-deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin2 biosensors reveal conformational changes upon binding to the β2-adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr.9, 97078 würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor (7tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin2, in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin2. upon β2-adrenergic receptor (β2ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin2flash2), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin2 relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin2/β2ar interaction on a timescale of seconds. a β2ar mutant that was previously shown not to interact with β-arrestin2 was utilized as control and did not induce a conformational change in the β-arrestin2 molecule. our data provide evidence that β-arrestin2 changes it`s conformation upon binding to the activated β2ar in living cells. the β-arrestin2flash2 sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a4 in the heart nunes f. 1 the calcium binding protein annexin a4 has been examined in the context of heart failure in the past. annexin a4 expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res 2000). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a4 gene trap model (gt) in which the annexin a4 protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of 0.5 hz, 1hz and 2 hz as well as an increased sarcomere shortening at 1 hz and 2 hz in anxa4 gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt (0,2±0.013 vs. 0.17±0.012, *=p<0.05 vs. wt; n=14-18/5). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca 2+ -atpase (serca2a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n=7). however, co-immunoprecipitation experiments suggest, that anxa4 is able to interact with hax1, which acts as a repressor of serca2a (n=4). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations (10 -9 m-10 -5 m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at 10 -4 m iso [%]: wt: 433±76; gt: 699±63 *= p<0.05 vs wt; n=8-10). in conclusion, annexin a4 contributes to the regulation of cardiomyocyte contractility. the anxa4 up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. 2000 mar;45 (4) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr1i subfamily of nuclear receptors. based on a 3-dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr1i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix 3 is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of 250 µl serum with oasis hlb cartridges allowed a reliable quantification down to 5 ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp18 column (55 mm x 2 mm; 4 µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai 7, 60590 frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl11(4-79) has antagonistic function for cxcr3, cxcl12(p2g2) is able to inhibit cxcr4 and the herpesvirus 8 encoded protein vmip-ii interferes with ccr1, -2 and -5 as well as with cxcr3, -4 and cx3cr1. their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl11(4-79), cxcl12(p2g2) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th-1 cell clone if12 with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl11(4-79), cxcl12(p2g2) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type-1-diabetes and contact dermatitis. small heterodimer partner 1 (shp-1) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp-1 is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp-1 expression, suggesting that shp-1 is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp-1, including hnf4α, lrh1, lxr, fxr, srebp1c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp-1 and to determine their impact on the transactivation of shp-1. 120 dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely -594t>c (rs71636795), -413g>c, -423 c>t (rs78182695) and del-195ctga (rs145613139). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh1, lxr, fxr, srebp1c and pparγ) of shp-1 expression. only the transactivation by hnf4α was decreased in the presence of the -423 c>t polymorphism to 69% and the -413g>c polymorphism to 75%. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about 1 in 20.000 individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [1] . mutations in the pkhd1 gene cause arpkd. more than 300 different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [2] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [1] [2] [3] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [2, 3] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [4] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda 1-1874 ) and tcdb (tcdb 1-1852 ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda 1-1874 and tcdb 1-1852 induced time and concentration dependent cell rounding and rac1-glucosylation. however, depending on the cell line, truncated toxins exhibit up to 10-fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda 1-1874 predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh3-only protein bid, a member of the bcl-2 family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi6c9 in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi-6c9 were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of 80 compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht-22 cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified 10 novel molecules that significantly prevented glutamate-induced toxicity in ht-22 cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. 1 , selinski s. in the 1990s, an uncommonly high percentage of glutathione s-transferase m1 (gstm1) negative bladder cancer cases (70 %) was reported in the greater dortmund area (golka et al., 1997) . the question arose whether this uncommonly high percentage of gstm1 negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, 15 years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the 1990s. in total 196 bladder cancer patients from the st.-josefs-hospital dortmund-hörde and 235 controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm1, gstt1 and the n-acetyltransferase 2 (nat2) tag snp rs1495741. the frequency of the gstm1 negative genotype was 52 % in bladder cancer cases and thus much lower, compared to a previous study performed from 1992-95 in the same area (70%). nat2 genotypes were distributed equally among cases and controls (63% slow acetylators). less gstt1 negative genotypes were present in cases (17%; controls 20%). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht-22 neurons. silencing of aif expression by sirna (20nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone (20nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht-22 cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca 2+ influx through cav1.4 channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav1.4 cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type 2a (csnb2), aland island eye disease (aied) and cone-rod dystrophy type 3 (cordx3). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav1.4 function we analyzed the visual phenotype of cav1.4-deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav1.4-deficient mice. heterozygous cav1.4 mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav1.4 channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. 25, 79104 freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase 2b15 enzymes (ugt2b15) revealed that ugt2b15.2 and ugt2b15.5 had markedly lower intrinsic clearance as compared to ugt2b15.1 (hanioka et al., 2011) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, 2009, mielke et al., 2011) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt2b15.2 variant (v2) was 5 fold and those of the ugt2b15.5 variant (v5) was 7 fold higher than that of the ugt15.1 variant. with dermal exposure at a relevant exposure level, the cmax values were 1.4 (v2) and 1.6 fold (v5) of ugt15.1 variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in 2.4 fold (v2) and 3.2 fold (v5) higher cmax values as compared to ugt15.1 variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose 5.7 fold (v2) and 8.6 fold (v5), for relevant dermal exposure 1.4 fold (v2) and 1.6 fold (v5), and for combined exposure 1.9 fold (v2) and 2.5 fold (v5) of ugt15.1 variant. from the results we conclude: (1) polymorphism of udpglucuronyltransferase (2b15.2 and 2b15.5) has an impact on the blood concentrations which, however, is less than 10 fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. (2) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase 2b15 polymorphism on bisphenol a glucuronidation. arch toxicol. 2011, 85(11) :1373-81. mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. 2009 8; 190(1) :32-40. mielke h, partosch f, gundertthe heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway (1) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde5 specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation (1) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. 2), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion (2) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde5) is expressed in cm (2). sil may act on other pdes, such as pde1c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase (7days of angii infusion at 2mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (t333) and 347 (t347), which enabled us to selectively detect either the t333-or the t347-phosphorylated form of sst5. we show that agonist-mediated phosphorylation occurs at t333, whereas t347 is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst5-selective ligand l-817,818 but not octreotide or ke108 were able to promote a clearly detectable t333 phosphorylation. however, none of these compounds was able to stimulate t333 phosphorylation and sst5 internalization to the same extent as the natural somatostatin. agonist-induced t333 phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase 2 (grk2). like that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. however, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were complete within minutes. we also identify protein phosphatase 1g (pp1g) as sst5 receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t333. in addition, we identify grk2-mediated phosphorylation and pp1g-mediated dephosphorylation of t333 as key regulators of rapid internalization and recycling of the human sst5 receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß2 adrenergic receptor, d1 dopamine receptor, parathyroid hormone receptor 1, thromboxane a receptor and the vasopressin receptor 1. however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst2a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase 1beta (pp1ß) as the phosphatase for the cluster of phosphorylated threonines (353ttetqrt359) within the sst2a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase 1gamma (pp1γ) as mor phosphatase that catalyzed t370 and s375 dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst2a chimera with the rsst2a c-terminal tail and a rsst2a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c7-carbonyl function of pat. the exocyclic amino group of the dna base is linked to c1 of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of 10 top prescribed psychiatric drugs. for 7 drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in 40 spcs 2220 individual data points (55.5±17.4 per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of 18.5 (range: 6-134) months. the respective spcs covered on average 71.4±30.3% (range: 12.5-100%) of all mentioned indications and 70.1±24.4% (range 15.4-100%) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average 59.5±17.1% (range: 12.5-93.3%) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the 28 spcs of the 7 drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. protein kinase ck2 (former name 'casein kinase 2') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [1, 2] . elevated ck2 activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [3] . ck2 is a heterotetramer consisting of two catalytic subunits (ck2α) attached to a dimer of regulatory subunits (ck2β) [4] . ck2β stabilizes ck2α against denaturation and modulates the substrate specificity of the catalytic subunit [5] . due to the relatively small and hydrophobic ck2α-ck2β interface (832 å²) [4] , low molecular weight inhibitors are able to interfere with the ck2 subunit interaction and thus affect the kinase activity [6] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [3] . we have developed an elisa-based ck2α-ck2β binding assay using recombinant human ck2 subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck2 subunit interaction. primary hits were further characterized by determination of the parameters ic50 and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < 0.1 µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy926 were exposed for 14 days to 100 ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab 1 . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for 24 hrs on top of matrigel-coated inserts. differential expressions of 20 phosphoproteins were found in cb-treated cells while an altered expression of 33 phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [1] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, 75, 2011, [375] [376] [377] [378] [379] [380] [381] [382] [383] 302 application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße 8-10, 10589 berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in 1996. since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in 2007 by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: 1. applicability domain. the chemical space of the ttc dataset (> 600 compounds) has to be compared with that of cosmetic ingredients (> 10 000 compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz 1, 44789 bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene -7,8-diol-9 ,10-epoxide (anti-bpde)-dna adducts as well as cytochrome p450 (cyp) 1a1/1b1 enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a549 lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after 24 h and 48 h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp1a1/1b1 activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of 340.5 ± 39.1 and 599.6 ± 132.4 anti-bpde/10 8 nucleotides after incubation with 1 µm (24 h) and 3 µm b[a]p (48 h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after 24 h than after 48 h. increased cyp1a1/1b1 activities were observed at > 0.1 -1 µm (24 h) and > 0.1 -3 µm (48 h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than 10% cytotoxicity in relation to the negative control were found after 24 h incubation with ≥ 10 µm b[a]p and after 48 h with ≥ 1 µm b[a]p. overall, incubation of a549 cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a549 cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav1.2 channel complex has been shown to play a key role in cav1.2 channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb2 gene coding for cavβ2, exon 14, contains several potential phosphorylation sites, e.g. for protein kinase a or ca 2+ /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav1.2. potential phosphorylation sites are ser478 and ser479 (in the β2 subunit in rat, perez-reyes et al. 1992 a ) and ser1928 in the c-terminus of the poreforming α1c subunit (lemke et al. 2008 b ) . in cardiomyocytes camkii regulates ca 2+ release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav1.2 channel complex and camkii are thr498 in the β2 subunit (in rat, grueter et al. 2006 c ) and ser1512 and ser1570 in the cterminus of the α1c subunit (blaich et al. 2010 d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon 14 after aa pro501. this mutation prevented translation of the cavβ2 c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ2stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ2stop mice with s1928a mice (lemke et al. 2008 b ) lacking the pka phosphorylation site s1928 in the α1c subunit (s1928aβ2stop mouse) or with sf mice (blaich et al. 2010 d ) lacking the camkii phosphorylation sites s1512 and s1570 in the α1c c-terminus (sfβ2stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s1928aβ2stop mice. electrophysiological investigations on cardiomyocytes of s1928aβ2stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav1.2 channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing 10% fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: 43.6 ± 4.6 pf, n=33; caf: 54.7 ± 5.1 pf, n = 17) and membrane potential (sr: -21.0 ± 4.3 mv, n = 14; caf: -27. 4 ± 4.8 mv, n = 16) . in both groups, we observed fast activating outward currents with a mean threshold at -20 mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: 23.8 ± 4.2 pa/pf, n = 15; caf: 6.1 ± 1.0, n = 6; p < 0.05). when maintained in culture for 3-5 weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: 38%; sr: 15%). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: 6.1 ± 2.0 pa/pf, n = 5; caf: 17.4 ± 4.4 pa/pf, n = 6; p < 0.05). na + currents were not altered by 100 nm tetrodotoxin (ttx), but 10 µm ttx reduced current amplitude to 42% of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav1.5. conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha2a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., 35043 marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha2a-adrenergic receptor. hek293t cells were transfected with yfp-tagged alpha2a-adrenergic receptor and the three subunits of gi1. the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha2aadrenergic receptor recruits gi with a half-life of around 150 milliseconds. when arrestin3 and grk2 were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around 2 seconds. there seemed to be no effect of grk2+arrestin3 cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk2 alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha2a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin3-cfp to yfp-tagged alpha2a-adrenergic receptors in the presence of grk2. upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin3, confirming previous observations that the alpha2a-adrenergic receptor recruits arrestin3. co-transfection of the three subunits of gi1 had no effect on the kinetics of arrestin3 binding to the alpha2a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha2a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b1, b2, b3) in detrusor and urothelium. we have shown previously that only b3-ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with 1 µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment 10 µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b3-ar in the absence of urothelium. but in intact detrusor strips b2-ars seem to have an additional inhibitory effect. affinity of the selective b3-ar antagonist l748,337 is unchanged, therefore the intact urothelium does not interact with the function of b3-ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator 1 (crtc1) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc1 is involved in this process, we investigated the expression of crtc1 in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc1 protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc3 gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc3 mutant mice the expression of crtc1 was significantly higher than in the controls (2.1-and 1.9-fold, respectively; n=3). in both forms of human hypertrophy, we found a significantly 5-fold upregulation of crtc1 protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n=7-10 for each group). conclusions: crtc1 is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc1 in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse 190, 8057 zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (<30%). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥2-fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at1-receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for 6 months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for 3 months with telmisartan (tel 8mg/kg/d) or amlodipine (aml, 12 mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after 6 months by more than 60 g. tel normalized sbp whereas it remained >200 mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first 4 weeks, but raised beyond control levels during the last 4 weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β2-adrenoceptor -camp mediated, immediate stimulation of β2-adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., 53105 bonn, germany based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β2-adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β2-adrenoceptor expression was addressed here. mrc-5 human lung fibroblasts were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mrna determination by qpcr. β2-adrenoceptor mrna decreased with a half-life of 25 min after inhibition of mrna synthesis with actinomycin d (30 µm), but increased by 333±85%, 502±52% and 640±165% (means±sem) within 1.5, 4 and 6.5 h, resp. after inhibition of protein synthesis by cycloheximide (30 µm). the β2-adrenoceptor agonists formoterol and olodaterol (1-100 nm) induced a rapid increase in β2-adrenoceptor mrna (maximally within 1 h by 100±19% and 110±19% at 10 nm, resp.). however, after 4 h exposure to 10 nm formoterol or olodaterol a reduction in β2-adrenoceptor mrna by 59±8% and 58±6%, resp., was observed. both, the stimulatory and inhibitory effects of β2adrenoceptor agonists were mimicked by forskolin (10 µm, increase by 88±14% and inhibition by 49±4%) and cholera toxin (5 ng/ml, increase by 76±12% and inhibition by 77±7%). the formoterol-induced up-regulation of β2-adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β2adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β2adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β2-adrenoceptor gene. et-1 appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et-1 and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et-1 expression in hlf was explored. mrc-5 hlf were cultured for up to 24 h in absence or presence of test substances, followed by prepro-et-1 (ppet-1) mrna determination by qpcr. the muscarinic agonist oxotremorine (10 µm) induced an increase in ppet-1 mrna by 180%, an effect prevented by 10 nm tiotropium. the β2-adrenoceptor agonist olodaterol (up to 100 nm) caused a reduction of ppet-1 mrna expression by 45%. the effect of 10 nm olodaterol was prevented by ici 118,551 (1 µm), but not affect by cgp 20712 (3 µm) . the pka agonist 6-bnz-camp (500 µm) caused a reduction in ppet-1 mrna expression by 65%, whereas the epac agonist 8-cpt-2'-o-me-camp (100 µm) caused only a marginal inhibition by 22%. olodaterol (10 nm) strongly opposed the stimulatory effect of 10 µm oxotremorine. an increase in ppet-1 mrna expression by 185% caused by 0.3 ng/ml tgf-β was effectively opposed by 10 and 100 nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet-1 mrna caused by a maximally effective concentration of tgf-β (1 ng/ml, increase by 620%) was not significantly affected by 10 or 100 nm olodaterol. likewise, the pkaagonist 6-bnz-camp (500 µm) opposed the increase in ppet-1 mrna expression caused by 0.3 ng/ml tgf-β, but not that caused by 1 ng/ml tgf-β. tgf-β caused, with an ic 50 of 0.3 ng/ml, a marked down-regulation of β2-adrenoceptor mrna expression, maximally by 90% within 6 h. et-1 expression in hlf is stimulated by muscarinic receptors and inhibited by β2adrenoceptors. the effect of β2-adrenoceptors may be mediated via pka. et-1 expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β2-adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β2-adrenoceptors. since et-1 can promote pro-fibrotic features in hlf, inhibition of et-1 expression could contribute to long-term beneficial effects of long-acting β2-adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h9c2 embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv4.3 and kv4.2, the cytosolic β-subunit kchip2 and the transmembrane subunits dpp6 and dpp10 using rt-pcr. treatment with doxorubicin (2 µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h9c2 cardiac myoblasts. while kv4.3 was detected in h9c2 cells and hearts from human and rat, kv4.2 mrna was only expressed in adult rats. during hypertrophy kv4.3 was downregulated in human tissue as well as in doxorubicin-treated h9c2 cells compared to the controls. in rat hearts kv4.2 expression was increased after doxorubicin treatment. interestingly, kv4.2 was also found to be up-regulated in rat heart tissues and h9c2 cells after treatment with doxorubicin. as previously shown, kchip2 mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip2 mrna was upregulated in hypertrophic rat hearts and h9c2 cells. the expression of dpp6 and dpp10 was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp6 and dpp10 were not expressed in the adult rat heart or h9c2 cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h9c2 cells, the mrna of dpp6 and dpp10 was up-regulated. h9c2 cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h9c2 cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp6 and dpp10 in doxorubicin-induced hypertrophic h9c2 cells. in preliminary experiments specific short-hairpin rna, targeting dpp6 and dpp10, has been designed and tested in heterologous expression systems. the nonsynonymous c.521t>c germline genetic variation in the liver-specific organic anion transporter slco1b1 is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. 1 background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc2, slco19a1, and slco1b1, have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc2, slco19a1, and slco1b1 contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c24h, mtx auc24-48h, and mtx clearance cl) in an unselected population of 419 children with all from the all-bfm 2000 trial (clinicaltrials.gov: nct00430118) who received 1676 courses of mtx at 5 g/m 2 as 24 h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. (1998) . abcc2 c.-24c>t (rs717620), slco19a1 c.80g>a (p.his27arg, rs1051266), slco1b1 c.521t>c (p.val174ala, rs4149056) and slco1b1 388a>g (p.asn130asp, rs2306283) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c24h (50.95 ± 24.15 µmol/l), auc24-48h (57.44 ± 37.52 h*mg/l), and cl (390.72 ± 223.95 ml/min/m²). the rgc values of c24h, auc24-48h, and cl ranged from 0.61-0.71 suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco1b1 c.521t>c snp was significantly associated with c24h (p<0.001), auc24-48h (p<0.001), and cl (p=0.011) of mtx. compared with the wildtype genotype, in patients with the tc genotype c24h and auc24-48h increased by 18% (p=0.009) and 28% (p=0.003), respectively, whereas cl significantly decreased by 15% (p=0.012). pharmacokinetic variables significantly changed with increasing number of variant c.521t>c alleles (p<0.02, jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc2 c.-24c>t, slco19a1 c.80g>a, and slco1b1 388a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c.521t>c polymorphism in slco1b1 contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase-2 (cox-2) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a549, h358, h460), the present study investigates the contribution of cox-2 and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner (10 -50 µm), whereas structurally-related cox-2 inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox-2 inhibitor ns-398, the pparγ antagonist gw-9662 and by sirna targeting cox-2 or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox-2 and pparγ at both mrna and protein level. using an established cox-2 activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns-398 was shown to suppress celecoxib-induced cox-2 activity when added prior to arachidonic acid. among the cox-2-dependent pgs analyzed, pgd 2 and its dehydration product 15-deoxy-∆ 12,14 -pgj2 were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns-398 which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox-2-and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox-2 and pparγ and a subsequent nuclear translocation of pparγ by cox-2-dependent pgs. uncoagulated ppp contained 40-60 nm thrombin throughout. fxa was initially measured in clots at 10 nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for 24h only marginally influenced smc apoptosis but increased mitogenesis over 15-fold. this was reduced by all 4 inhibitors. clot-stimulated induction of tnfα and interleukin-6 mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx9065a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef17 mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. 1 , stephan-schnatz k. the guanine nucleotide exchange factor rhogef17 is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef17 by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef17 influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef17 depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first 24 hours after seeding. cell counting revealed that 6 to 12 hours after seeding the percentage of adherent cells was significantly lower in the rhogef17 knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef17 was paralleled by a loss in rhoa and cadherine expression. as rhogef17 mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon (8-pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first 12 hours. in contrast, the adhesion of rasmc after rhogef17 knockdown was no longer stimulated by cgmp. as these data indicate that rhogef17 mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef17 depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef17, we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef17 knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef17 is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h2 receptors (h2r). activation of fpr leads to a release of reactive oxygen species (ros). h2r activation results in an increase of intracellular 3'-5'-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h2r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and 5-methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites (100 mg) were incubated in gc vials with 1 ml dest. water or 1 ml methanol, each at 37 °c for 72 hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites 18 different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n=4)( fig. 1 ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram (5 ± 2 µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr4 is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr4 is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr4 in tumor metastasis is discussed. subsequently, detection of cxcr4 expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr4 expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr4 antibody umb-2. methods: in comparison to negative and positive control samples (cxcr4-knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr4 was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr4-expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr4 positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb-2 may prove to be of great value in the assessment of cxcr4 expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β2-adrenoceptor-mediated inhibition of formyl-peptide-induced o2 β2-adrenoceptor (adrb2)-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb2 gene contains a total of 49 polymorphisms. this variability could cause part of the ~70 % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb2 can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb2agonists for each individual. (ortega et al. 2007; chung et al. 2011) our present study connects sequence data with pharmacological data of prototypical adrb2-ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o2 .production and its inhibition by the agonists are examined in a 96-well cytochrome-c assay. characteristic pharmacological values (pic50, emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb2-sequence variant is determined by sanger-sequencing. complete determination of a 1,490 bp sequence, including the entire adrb2 exon and part of the flanking 5'-and 3'-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. 1 recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine 3',5'-monophosphate (camp) and cyclic guanosine 3',5'-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine 3',5'-monophosphate (cump). 2 these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using 2'-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde3a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. 1 moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine 3',5'-monophosphate (cimp). 2 using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde3a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde3a. this finding is consistent with data published by hardman and sutherland 3 who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde3a. as cump has furthermore been proven to be present in mammalian cells 4 , to differentially activate camp-and cgmp-dependent protein kinases 5 and to be synthesized from utp by mammalian soluble guanylyl cyclase 6 , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde3a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat3 and identify signal proteins involved in this pathway. rhoa-dependent stat3 stimulation requires rock and junkinase activation as well as ap1-induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl1/i-309 as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat3-dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. 1 christian-albrechts universität institut für immunologie, michealisstrasse 5, 24105 kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about 50%. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt=8ml/kg and f=180min -1 with fio2=0.3 and peep=2cmh2o while lung mechanics were followed. half of the mice received 50µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at 37°c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability 6 hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. 1 cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht22 neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht-22 cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high 4525 µmol; medium 2219 µmol; low 1053 µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium) and 14.6 ± 6.8% (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: 551.5 ± 93.8 nm*h -1 (high); 299.3 ± 79.6 nm*h -1 (medium) and 222.8 ± 91.3 nm*h -1 (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p2x3 ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany the homomeric p2x3 receptor (p2x3r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p2x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p2x3 receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p2x3 receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p2x3 wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek 293 cells, heterologously expressing the human p2x3 receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p2x4r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p2x3 agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany purinergic p2x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p2x receptor family, the p2x3 receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p2x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p2x3 receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek293 cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp2x3 model that we developed from the known zebra fish p2x4 crystal structure in the closed state. voltage-dependent modulation of alpha2a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. 1, 35034 marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α2a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek 293 cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations (500 nm: 60 % inhibition) but almost absent at saturating agonist concentrations (100 µm: 9 % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine (1 µm, vm=-90 mv) resulted in a fret response that was inhibited by 40 % at +60 mv. in contrast to ne, strong receptor inhibition at +60 mv was present even at super-saturating concentrations of clonidine (100 µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a2a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = +60 mv. therefore we conclude that negative membrane potentials promote active conformations of the a2a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has3, in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal1,2) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. 1 , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e2) mediated effects on ha-rich ecm during actinic aging. 10 weeks of uvb irradiation (3 x 1 med (80 mj/cm 2 ), weekly) of hairless skh-1 mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e2) by means of controlled release pellets abolished these effects confirming the stimulatory role of e2. the increase of dermal ha correlated with induction of ha synthase has3 by e2. in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e2. however in cultured skin fibroblasts e2 reduced the expression of versican and had no effect on has3. therefore, direct upregulation of has3 and versican in fiborblasts by e2 was excluded. however, e2 increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has3 and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e2 correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e2 treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e2 during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. 1 , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno3 solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio2 nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar 18 strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio2 samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between 2 and at least 72 hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno3 solutions with 0.64 and 0.95 mg/cm 2 , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. 2, 10115 berlin, germany c3 exoenzyme (c3bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c3 is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c3bot to the hippocampus-derived ht22 cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c3bot. the binding assays established that c3bot bound in a concentration-dependent manner to ht22 cells. in the overlay assay we detected one clear band of 55 kda. to elucidate whether glycosylation is important for the c3bot-protein interaction, ht22 cells were incubated with glycosidase f resulting in a decreased binding of c3 to the 55 kda band. to explore the involvement of phosphorylation in the binding of c3 to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c3bot. pretreatment greatly reduced the c3bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c3botprotein interaction in the following overlay. thus, interactions between c3bot and ht22 cell proteins may require phosphorylation. to further characterize the 55 kda band as binding target of c3bot, the 55 kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this 55 kda single gel band 141 proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c3bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c3bot-protein interaction. s76 335 solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. 24, 50931 köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide (1% to 4%) on the assessment of k m, vmax and clint with regard to the 1-hydroxylation of midazolam via cyp3a4 and the cyp1a2 catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er81 rommel c. 1 , rösner s. introduction: the transcription factor er81 (ets related 81) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er81 is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er81 mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er81 expression. thus, the aim of the present study was to investigate the cardiac function of er81 in genetically modified mouse models. we previously generated transgenic mice overexpressing er81 under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day 5 after birth (p5) and in adult mice (3 months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day 5 after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er81 αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh477 and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er81 αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine 16 of phospholamban (pln) was reduced in er81 αmhc mice. in addition, protein phosphatase 1 (pp1) expression was significantly increased in er81 αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca2a protein in er81 αmhc atria. electron microscopy revealed the significant structural remodeling of er81 αmhc atria at 3 months of age. conclusion: increased cardiac expression of the ets-transcription factor er81 leads to structural and electrical remodeling of the atria. thus, er81 may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06097 halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p2-purinoceptors which are further divided in p2x1-7 and p2y1-14-receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. 100 µm atp as well as utp transiently increased phosphorylation of erk 1/2 and p38 mapk with a maximum effect at 5 to 10 minutes after application of atp and utp in neonatal cardiac myocytes (n=3 preparations each). the maximum phosphorylation of p38 increased with atp to 284% ± 68% (p<0.05) at 10 minutes and with utp up to 204% ± 21% (p<0.05) at 5 minutes. the phosphorylation with erk 1/2 mapk increased with atp to 234% ± 46.5% (p<0.05) at 5 minutes and to 337% ± 27% (p<0.05) with utp at 10 minutes of basal values, respectively. after 20 minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk 1/2 and p38 mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. 1 , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students (5 th or 10 th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving 8 -12 previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of 60 points could be obtained as a sum of both tests. results: in the means 40% of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr188 phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk1/2 ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany background and aims: the extracellular regulated kinases 1 and 2 (erk1/2) play an important role in cardiac hypertrophy and cell survival. erk1/2 are phosphorylated at the so-called tey motif, which in turn activates erk1/2. hypertrophic stimuli lead to an additional autophosphorylation threonine188 (thr188). this autophosphorylation of erk1/2 stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr188 phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk1/2 activity and prevention of erk thr188 phosphorylation, we either inhibited erk activity with pd98059 or overexpressed a mutant of erk2, which cannot be phosphorylated at thr188 phosphorylation. while inhibition of overall erk activity with pd98059 attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr188 phosphorylation by overexpression of the phosphorylation deficient mutant (erk2 t188a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk2 t188a significantly reduced tac-induced hypertrophy compared to wild-type erk2 overexpressing mice. in line with the in vitro experiments, erk thr188 inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr188 phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk1/2. therefore, specific targeting of erk thr188 phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde10, a pde in principle capable of hydrolysing camp as well as cgmp. pde10 contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde10 was mostly based on the analysis of mrna distribution, we generated antisera against pde10 to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde10 displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde10 inhibitor papaverine as well as specific pde10 inhibitors suggest pde10 as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde10 to camp degradation in striatum, to identify the physiological pathways pde10 is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse 31, 93053 regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ 100 amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp 3r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp3r-ii and the insp3r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the 52 amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca 2+ -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp3r interactions. the insp3r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp3r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp3r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp3r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp3r-i and insp3r-iii but not with insp3r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type 1 enhancer binding protein 1 (hivep1) is regulated by proinflammatory stimuli and statins salomon a. 1, 2 , schmitz b. objective: hivep1 binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned 90 kb upstream (rs169713) and another in exon 4 (rs2228220) of the hivep1 gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, 2010; plos one, 2011) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy926) with proinflammatory cytokines or statins (24h). serial hivep1 promoter deletion constructs were cloned into the pgl3-basic vector, a potential enhancer fragment, harbouring rs169713c/t, into the pgl3-promoter vector. in ea.hy926 cells and thp1 monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy926 cells, endogenous hivep1 expression was increased by proinflammatory cytokines tnfα and il-1β. simvastatin (1.2 and 2.4 µm) and atorvastatin (9 µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep1 expression. the construct harbouring rs169713t exerted significantly higher transcriptional activity (ta) compared to rs169713c (p<0.001). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep1 expression in ea.hy926 and thp1 cells. cotransfection of sp1 and egr1 led to an increase in ta, while wt1 exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp1 to the 5'-flanking region and the intronic modulator of hivep1. conclusion: increased hivep1 expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep1 expression is regulated by sp1 combined in a transcription factor module with egr1 and wt1 under basal and/or inflammatory conditions. the rs169713 site harbours potential activational capacity for hivep1 gene transcription and may communicate with the sp1/egr1/wt1 module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv7.2/3 channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv7.2/3 channels, we examined the acute effects of the preferred kv7.2/3 channel opener ica 27243 on lid in this animal model. four weeks post 6-ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with 10 mg/kg l-dopa and 15 mg/kg benserazide for 20 days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from 0 to 4 over 180 min. for drug testing, ica 27243 (5, 10 and 15 mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined 20 min after drug administration using the block and the stepping test. ica 24273 reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first 20 min after the application of 5 mg, it lasted up to 110 min in rats treated with 10 mg ica 27243. a higher dose of 15 mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v7 channel opener retigabine was based on its action on striatal kv7.2/3 channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n=4-5). myotoxic cerivastatin (0.01, 0.1, 1 µm for 5 days) caused a concentration-dependent decrease of contractile force (p<0,05, n=17-20) paralleled by an increase in inos transcript (mean±sem: 0.01 µm 4±0.9-fold, n=3 p<0.05; 0.1 µm 9.3±2.8-fold, n=3 p<0.05) and protein (0.01 µm 5.1±2-fold, n=4 p<0.05; 0.1 µm 7.8±0.8-fold, n=3 p<0.05). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with 1400w, a specific inos inhibitor. we applied 5 µm of 1400w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n=9-15). also, l-name (10 mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n=6-13, p<0.05). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp (10 µm) caused a reduction of contractile force. combined treatment with cerivastatin and 0.1 µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with 3m syndrome and mutations in the cul7 gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany 3m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< -4 sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul7 (76%) or obsl1 (16%) gene, respectively. cul7 constitutes an e3 ubiquitin ligase that is involved in the regulation of the insulin-like growth factor 1 (igf-1) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate 1 (irs-1). to investigate the role of cul7 mediated irs-1 degradation in the pathogenesis of 3m syndrome. primary skin fibroblasts of seven 3m syndrome patients (six with cul7 mutations, one with a obsl1 mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs-1 protein concentrations and activation of the igf-1 signaling pathway (western blot). the proliferation rate of 3m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs-1 protein levels and activation of the pi3k/akt and erk mapk pathway were only increased in a subset of 3m cells that carried cul7 mutations, but not in cells from a patient with the obsl1 mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in 3m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs-1 levels to the observed phenotype, human imr90 fibroblasts were stably transfected with retroviral vectors encoding irs-1. despite 20-fold overexpression of irs-1 compared to empty vector controls, no significant effect of igf-1 stimulation on proliferation rate or pi3k/akt and erk mapk signaling was observed. skin fibroblasts of 3m patients with cul7 mutations displayed an increased proliferation rate and enhanced activation of the igf-1 signaling pathways. despite accumulation of irs-1 in fibroblasts from a subset of 3m patients with cul7 mutations, no pathomechanistic role for irs-1 could be demonstrated. collectively, our data indicate that a dysregulated igf-1 signaling may contribute to the pathogenesis of 3m syndrome, yet in an irs-1 independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present (11/2011) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was 10 days. to date, the website consists of 49 pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has 1000 visitors per month with the following evaluation results: "excellent": 76%, "good": 15% and "satisfactory": 9% (n=33). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak1 sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. 43, 61231 bad nauheim, germany tgf-β activated kinase 1 (tak1) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin-1 or tnfa to ikk, p38 and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak1 in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t2 with animals carrying floxed alleles of the tak1 gene. adipoqcreer ; tak1 fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak1 knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak1 knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin-6. stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak1 deficient adipocytes show reduced activation of jnk and p38 which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak1 knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/11 and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/11-regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk2 and subsequently the erk1/2 cascade. the role of pyk2 for the growth of sclc cells was further supported by the fact that inhibition of pyk2 using a sirna approach or a novel specific inhibitor, pf431396, exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g 12/13 signaling by sirna-mediated g(alpha)12 or g(alpha)13 knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk2 versus the role of calcium-independent signaling via g12/13, we tested the effect of pyk2 inhibition in cells with impaired g12/13 signaling. notably, pyk2 and g12/13 double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk2 activation or g12/13-dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp-1 were used for 7 days and stimulated with various cytokines (il-4, gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd11c, cd209, cd83) and by immunfluorescence compared to monocytes. the bronchial tract contains up to 800 dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for 4 weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between 16 to 20 hz and the transepithelial resistance values were stable between 600 to 800 ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git1 and cavb3 schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, 66421 homburg, germany high-voltage activated ca channels are assembled from pore-forming α1 subunits and two distinct types of auxiliary subunits, cavβ1-β4 and, maybe, α2δ1-δ4. by a cavβ3-specific antibody based affinity chromatography the cavβ3 protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ3 were identified by mass spectrometry (lc-esi-ms/ms) and include α1-subunits, α2δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ3 protein, including the g protein-coupled receptor kinase-interactor 1 (git1). the 770aa git1 is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git1 and cavβ3 proteins were co-immunoprecipitated by the antibodies for cavβ3 and git, respectively. we cloned the git1 cdna from mouse brain and co-expressed it with the cavβ3 subunit in hek cells. like in brain lysates the git protein was retained by cavβ3 precipitated by the antibody for cavβ3 and cavβ3 was retained by the git1protein precipitated by the antibody for git1. both proteins, cavβ3 and git1 are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ3-git1 interaction in the absence of functional cav channels. in addition, using mefs from cavβ3-deficient mice enables us to control the impact of cavβ3 on git1 function. vice versa down-regulation of git1 by specific sirnas might allow to control the impact of git1 on cavβ3 function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine-1-phosphate and fty720 on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine 1-phosphate (s1p) is a mediator that modulates various physiological functions of skin cells. s1p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s1p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s1p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s1p, but not fty720 reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s1p via the s1p2 receptor, which is not activated by fty720. there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s1p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s1p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s1p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s1p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h441) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il-6). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp-1) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd206) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo-1) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above 50 µm are accompanied with inhibition of nat1 activity in human keratinocytes [1] . in the following we investigated the impact of ppd on nat1 activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp-1 cells. measured nat1 activity of thp-1 was comparable to those found in primary keratinocytes. a 24h treatment of thp-1 cells with physiologically relevant concentrations of ppd (10-200 µm) led to a 47% reduction of nat1 activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already 8h after application of ppd or mappd while nat1 mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat1 activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after 24h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat1 protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat1 protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat1 protein after 2h. the greatest inhibition was found for oxidised ppd (up to 50%). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat1 enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat1 in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec 3.2.1.22). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened 4011 bp of the 5'-flanking region of gla in 60 patients with fabry's disease and 60 controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy926) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 5'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs2071225; rs3027580; rs3027579; rs59647857; rs3027575; all minor alleles p<0.0027). we identified two regions, a proximal one between -110 and -425 and a distal region between 1106 and -1421 with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to 5.3-fold (p<0.001). in ihke cells, insertion of the minor t allele (rs2071225) significantly enhanced basal ta of the proximal promoter region (p=0.0006), while insertion decreased basal ta (p<0.0001) of the distal promoter portion. the combined insertion of the minor c alleles (rs3027580; rs3027579), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p=0.0037). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c16:0-cer levels were 1.9 fold increased in ms patients. this translates into the finding that c16:0-cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c16:0-cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) 6 in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers6 and its product c16:0-cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c16:0-cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers6 plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers6 and therefore to limit the inflammatory effects of c16:0-cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. 1 cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc1 or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm22 promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm22 promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a549 lung epithelial cells, hepg2 liver epithelial cells and hk-2 proximal tubule epithelial cells. the used nanomaterials incorporated five tio 2 samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from 0.3 to 80 µg/cm 2 for cytotoxicity and from 06 to 40 µg/cm 2 for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst-1 assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp7 project enpra (fp7-nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v79 cells expressing human cytochrome p450 1b1 schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v79 lung fibroblasts (v79 cells) represents a widely-used mammalian test system to detect gene mutations. since v79 cells do not express any cytochrome-p450dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v79 cells expressing human (h) cyp isozymes have been commercialized. to test these v79 cells for their use in the hprt test, v79 h1a1 and h1b1 cells were characterized regarding (i) spontaneous frequency of 6-thioguanineresistant clones per 10 6 clonable cells (smf), (ii) the stability of which over 4 weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v79 cells (w2:13±4; w4:2±1) and v79 h1a1 (w2:17±4; w4:3±0) only varied within the range of historical controls, smf of v79 h1b1 increased continuously over time (w2: 36±6; w4: 68±13). (iii) although the smf of v79 and v79 h1a1 were similar, the mutational spectrum of v79 cells was characterized by as many transversions as transitions and deletions of exon 4 or exon 4+5, whereas the mutational spectrum of v79 h1a1 was characterized exclusively by transversions and deletion of exon 7+8. surprisingly, with 57 out of 59 cdnas derived from v79 h1b1 mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v79 h1b1, cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between 0.7±0.6 and 1.2±0.0. yet its smf was unstable reaching up to 104±6. in conclusion, the mutational spectrum differed between the v79 cell lines. furthermore, h1b1 expression seemed to enhance smf in v79 cells. even though a temporary reduction of the smf by cloning was possible, smf of v79 h1b1 cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with 20µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient elution open cc on silica gel. the most active fractions 11-17 were separated again yielding 10 subfractions. this afforded 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol-3-o-β-dglucopyranoside, the structures of which were determined by esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats (10-12 weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca 2+ selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai1 and trpc3 in plasma membrane microdomains of rbl-2h3 mast cells. orai1-mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc3. this negative impact of trpc3 on icrac was independent of channel function as the trpc3 pore dead mutant (e630k) inhibited icrac to a similar extent as wild type trpc3. importantly, despite a reduction in icrac, nfat translocation in trpc3 overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc3e630k but substantially reduced by trpc3 mutants with either i) eliminated fkbp12/calcineurin binding (p704q) or ii) deficiency in pkc phosphorylation (s712a). moreover, inhibition of pkc phosphorylation by (gfx109203x; 3 µm) strongly suppressed nfat signaling. we suggest trpc3 as a scaffold that links orai-mediated ca 2+ -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. 1 , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf96365 were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to 39±26% of the initial airway area (%-iaa) and 63±21%-iaa, respectively. in frequency response curves half maximal response was found at 7.0±1.2 hz for guinea pig pcls and 8.1±0.8 hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to 18±15%-iaa. 60% of human pcls were responsive to capsaicin and airways contracted to 72±10%-iaa, respectively. in gp ruthenium red and skf96365 blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk1/2-pathway is involved in pkc-induced nox4 up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55131 mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox4 is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy 926 endothelial cells with phorbol 12myristate 13-acetate (pma) for 48 h leads to an up-regulation of nox4 expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö 6983 and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox4 up-regulation can be attenuated by pd 98,059 (an erk1/2 inhibitor), but not by sp 600125 (a jnk inhibitor), indicating in the involvement of erk1/2. consistently, pma treatment leads to a sustained activation of erk1/2, and sirnamediated knockdown of erk1/2 markedly reduces the pma-induced nox4 up-regulation. h89, an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox4 expression, indicating that msks are not the target molecules of erk1/2 in this scenario. on the contrary, knockdown of the transcription factor elk-1 by sirna significantly reduces the pma-induced nox4 up-regulation. in conclusion, erk1/2 and elk-1 are involved in the pkcα-induced nox4 up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, 97074 wuerzburg, germany annually, over 57,000 women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p53 gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron 6 of p53 gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with 2 g of normal mammary gland tissue a smf of 2.2±1.4x10 -7 per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (il-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il-8 mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p65 nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac1 expression, but did not affect epac2 and pka expression. importantly, epac1 expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il-8 release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac1. polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt4) exposed to 0.5 µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt4) were exposed to 0.5 µm bap for 24 h (n=5), 4 wk (n=7) and 8 wk (n=3). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. 1) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. 45 % of the differentially expressed proteins after 24 h of treatment are involved in the processing of pre-mrna (20 %) and protein metabolism (25 %). this pattern changed after 8 wk of treatment. then, 48 % of the proteins affected the cytoskeleton whereas still 26 % were categorized to protein metabolism and only 7 % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after 8 wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after 8 wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins 1 and 2, are currently being validate by pcr. fused master gel : representative 2-de gel of rt4 cells exposed to 0.5 µm bap for 8 wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. 1 , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd (0.01 -3 µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am-630 (cb2 receptor antagonist) and by o-1602 (g protein-coupled receptor [gpr] 55 agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) 1/2. blockade of erk activation by pd98059 prevented cbd-stimulated msc migration, whereas inhibition of p38 mapk by sb203580 was inactive in this respect. furthermore, am-630 and o-1602 were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ 9 tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c57bl/6 wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c57bl/6 wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. 1 hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße 1, 91054 erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr1i2) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco1a2 and slco1b3 and a significantly decreased expression of transporters such as slco2b1, slco2a1 and abcc3 in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco2b1 mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ 2 =6.59; p=0.010; n=34). bisulfite sequencing revealed that promoter cpg islands of abcc3 and slco2a1 were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc-1 and scc-15 cell lines, transcript expression of transporters (e.g., abcc3, slco2a1; p<0.001) and pxr (nr1i2; p<0.001) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc3 and slco2a1 expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco2b1 may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc-1 and scc-15 cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn2 in neuropathic and inflammatory pain schnorr s. 1 , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn1-4) and contributes to cellular excitability in the heart and the nervous system. hcn1 and hcn2 are the two most abundant hcn subunits in peripheral sensory neurons with hcn2 being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn2 for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn2 by using a nociceptor specific cre-transgene driven by the nav1.8 promoter. the nociceptor-specific knockout of hcn2 in drg neurons (nosphcn2ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn2 was mainly restricted to small sized drg neurons. the conditional loss of hcn2 resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn2 did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd7288 nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn2ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn2 protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn2 might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec50 values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy5y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa1 channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv1, trpv2, trpv3, and trpv4 and the cold receptor trpm8 were insensitive towards apomorphine treatment, trpa1 could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa1 channels in a stably transfected cell line (hek293-trpa1), as well as natively expressed trpa1 in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa1 activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa1 expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [1] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [2] , agonists of the muscarinic m2 receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m2 receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m2 receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m2 receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m2 receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for 1 h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day 15 the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp1a1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p450 (cyp) 1a1 and 1a2. the most prominent regulator of cyp1a1 is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp1a1 induction in murine hepatoma cells (hepa-1c1c7). indeed, aoh and ame enhanced the levels of cyp1a1 in hepa-1c1c7 cells but not in cells with inactivated ahr (hepa-1c1c12) or arnt (hepa-1c1c4). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp1a1 expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than 20,000,000 scientific abstracts, and 700,000 are newly added every year. the protein sequence database uniprotkb stores over 10,500,000 sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go3r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go3r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β1-adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca 2+ -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age 16-20 weeks) and subsequently used for experiments within 6 hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca 2+ cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. 150, 44781 bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca 2+ concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca 2+ concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc1, with angiotensinii (1.44 mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc1 ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study 72 hours after a single instillation of 18 µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size (2-, 20-and 200-nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to 20-nm and 200-nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. 43, 61231 bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc1), b16f10 melanoma cells or human neuroblastoma cells (sh-sy5y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc13-4 deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc1 and b16 cells was indistinguishable between wild-type mice and animals lacking munc13-4, the number of metastases in the lung was strongly reduced in munc13-4-deficient animals. the strong decrease in formation of metastases in munc13-4 deficient mice was also observed after i.v. injection of llc1 and b16f10 cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient 1methoxy-3-indolylmethyl glucosinolate and its break-down product 1-methoxy-3indolylmethyl alcohol schumacher f. 1 , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that 1-methoxy-3-indolylmethyl (1-mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [1] . we have identified and synthesized the major dna adducts n 2 -(1-mim)-dg and n 6 -(1-mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated 1-mim glucosinolate. the peak levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in the murine large intestine were reached 8 h after a single administration of 600 µmol 1-mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite 1-mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered 1-mim alcohol generated significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [1] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., 192 (2011) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. 1, 2 , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb1) and multidrug resistance protein 2 (abcc2), and the nuclear pregnane x receptor (pxr/nr1i2), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n=178) dgf occurred in 27.5 %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs2276707 8055tt genotype was significantly associated with dgf (recessive model: or, 9.0; 95%ci, p=0.004 unadjusted) , even after correction for multiple testing (p=0.035 holm-adjusted). when we performed multivariate analysis including genetic and 16 clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il-2 receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs2276707 tt genotype of the donor (recessive model: or, 16.08; 95% ci, 2.0-129.46; p=0.0046 unadjusted) which held true after correction for multiple testing (p=0.04). for abcc2 variants only the donor rs17222723 3563t>a genotype correlated with dgf by univariate (or, 4.65; 95%ci, p=0.005 unadjusted) as well as by multivariate analysis (or, 5.5; 95%ci, p=0 .01; table 4 ) but not after correction for multiple testing (p=0.12). for variants in the abcb1 gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant 03is2061c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs1/fe-10). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. 25, 79104 freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas 2011) . in the cytosol, cdt adp-ribosylates actin at arg 177, thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (>150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog 2009 ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp2 and acf7. besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim1 that usually functions as a calcium sensor in the er and activates the store operated orai1 calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd-1 mice, and dialysates were obtained while a monofilament was inserted for 60 min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma 600 analyzer -rose extensively during ischemia to more than 2000% of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after 60 min. when bilobalide (10 µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only 500% of baseline level (p<0.01). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to 60-70% of sham-operated mice. in the ischemic hemisphere, several values were lower at 40% of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of 16% of baseline. direct addition of bilobalide (10 µm) to post-ischemic mitochondria caused a 2-fold increase of complex i activity in vitro. pretreatment of mice with bilobalide (10 mg/kg i.p.) one hour before mcao caused a significant, 3-fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as 2-hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod-1), catalase (cat) or glutathione peroxidase (gpx1/2). raw246.7 mouse macrophages were exposed to hema (0-8mm) for 24h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine (50µm bso), or enhanced by 2-oxothiazolidine-4-carboxylate (5mm otc) and n-acetyl cysteine (10mm nac). expression of sod-1 found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod-1 expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx1/2 was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h2o2 decomposition. the inhibitory effect of hema on gpx1/2 expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h2o2 is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h2o2 production leads to the activation of cat expression and a feed-back inhibition of sod-1 expression. the hema-induced reduction of gpx1/2 expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e 14-15) , fixed to coverslips and incubated in roller tubes for about 2-4 weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a 12 chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx (0.75 µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime (10 µm) was added spontaneous muscle activity in the co culture recovered from 0.45 ± 0.24 hz to 1.67 ± 0.24 hz (control 1.79 + 0.22 hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to 69.9 ± 21.3 % by obidoxime compared to control. muscle force production after indirect stimulation was stable for 6 hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a 1.3 fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n=10 from 6 isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling 5 (rgs5) and peptidylprolyl isomerase a (ppia) were 1.5-2.5 fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n=6). transcripts of rgs5 (2.6 fold, ) and ppia (1.8 fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n=10-12). we conclude that crem deficiency is associated with transcriptional changes of rgs5, pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p101 and p87 subunits in regulating phosphoinositide 3kinase γ by gβγ and h-ras shymanets a. 1 phosphoinositide 3-kinase γ (pi3kγ) controls a plethora of cellular responses. pi3kγ, a heterodimer formed by non-catalytic p101 or p87 and catalytic p110γ subunits, is regulated by gβγ and ras. earlier we speculated that p101 binds to gβγ to translocate cytosolic pi3kγ to the plasma membrane, enabling direct activation of p110γ (brock et al., j. cell biol. 2003) . however, the p87 subunit does not function as gβγ adapter (kurig et al., pnas 2009) . since the impact of each non-catalytic subunit in regulating p110γ by gβγ and ras still remains elusive, we studied their role in detail. gβ1γ2 variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf9 cells and tested for their ability to activate pi3kγ. we observed that p101, but not p87, was able to rescue the stimulatory activity of gβ1γ2 mutants incapable to activate p110γ (shymanets et al., biochem. j. doi:10 .1042/bj20111664). to further study the functional impact of the non-catalytic subunits on pi3kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi3kγ variants. although p87/p110γ exhibited stronger sensitivity to gβ1γ2 than p110γ, the activity of vesicles-associated p101/p110γ was significantly higher as compared to vesicles-associated p87/p110γ or p110γ in the absence and in the presence of gβ1γ2. to study an effect of ras proteins on pi3kγ variants, recombinantly expressed h-ras was purified from sf9 cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p87/p110γ and p101/p110γ differed, which may explain integration of pi3kγ variants in different signalling pathways. taken together, p87 and p101 subunits implement discrete functions in respect to (i) membrane recruitment of pi3kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive 1999/45/ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, 2008) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive 1999/45/ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, 2010) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. 50 % of all saxonian citizens are inscribed to this service) for the years 2005 to 2007 was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/-20 % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a 50 -70 % above average prescription figure during spring time (february to may) and a 25 to 40 % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. 25, 79104 freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα12/13 and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα12/13 family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st-2 cells and in primary osteoblasts from rat calvaria. st-2 cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st-2 cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c57bl/6 and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c57bl/6 or balb/c were subjected to high (25cmh 2o) or low pressure (8cmh2o) ventilation for 240 minutes. after 180 minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another 60 minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c57bl/6 mice showed higher tnf, il-1β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl-1, cxcl-2) and il-6. kinase activities (jnk, akt, erk1/2, p38 map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by 50% and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c57bl/6 and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m1 macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m2 macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh 2) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh2 nanoparticles for up to 6 days. analysis of cell viability revealed that ps-nh2 but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh2 trigger apoptosis in macrophages. we further polarized macrophages to either m1 or m2 using ifn-γ and lps or il-4, respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m1 and m2 macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp1313. loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. 20, 89081 ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh 2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, thp-1 monocytic leukemia cells, and by pmadifferentiated thp-1 cells via different mechanisms. in buffer, macrophages and thp-1 rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd64 receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp-1 cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp-1 cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh 2 were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp1313. specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh2) of ~100 nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp3 inflammasome activation and the subsequent release of il-1β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp3 protein resulting in a conformational change of the pyrin domain of the nlrp3 protein as predicted by molecular modeling. txnip interaction with nlrp3 led to assembly of the nlrp3 inflammasome complex, to caspase-1 activation, and release of il-1β. using an in vitro knockdown approach, we showed that ps-nh 2 induced activation exclusively of nlrp3, whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase-1 activation and the subsequent release of il-1β caused by ps-nh2 nanoparticles. these data reveal a novel mechanism of the nlrp3 activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp1313. the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a2 and s100a10. addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within 30 min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h 2dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase 3 activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p65/rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph 7.4, helenalin interacts with rela (k d = 4.8 µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic50 of 5.0 µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine 38 by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg35, lys37, gly44 and ile118 of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of 24 µm, though no significant interaction was observed at ph 7.4. thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of 60 nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of 20 nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp1313. prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [4] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [2, 3] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [1] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [5] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v79. induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with 4',6-diamidino-2-phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a2 (pga2), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the 15dpgj2 and pga2 -induced significant decrease in the tgsh level in v79 may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd86. determination of the concentration required to cause a half-maximal increase in cd86-expression (ec 50) allowed a quantitative evaluation. the irritative potential of the substances was assessed by 7-aad (7-amino-actinomycin d)-staining. the concentration required to devitalize 50 % of the examined cells compared to a zero control was termed ec50%. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec50-values ranging from 16.67 µmol/l (pentylparaben) to 325.36 µmol/l (methylparaben). due to a pronounced cytotoxicity (ec50% = 70.94 µmol/l), we could not estimate an ec50-value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin 4 by protein kinase dyrk1a at serine 107 affects protein stability soppa u. 1 septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the 13 family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin 4 (sept4) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept4 by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase 1a (dyrk1a). dyrk1a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept4 and overexpression in hela cells we identified serine 107 as the major phosphorylation site of dyrk1a and generated a phosphospecific antibody. transient coexpression of sept4 and dyrk1a in hela cells increased phosphorylation of serine 107 by 50% in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk1a mutants k188r and d287n did not increase serine 107 phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk1a inhibitor harmine reduced phosphorylation of exogenous sept4 at serine 107 about 25% in hela cells. furthermore, down regulation of dyrk1a by rna interference lead to decreased phosphorylated serine 107. these results indicate that endogenous dyrk1a contributes to sept4 phosphorylation in hela cells. finally we analyzed protein stability of wild type sept4 compared to the phosphorylation resistant s107a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept4 s107a mutant is more stable than wild type sept4. in summary, our results suggest phosphorylation at serine 107 by dyrk1a as a novel mechanism to regulate sept4 stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, 89069 ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee 3, 53175 bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. 2002) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. 2003) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca 2+ influx. in the present study we analyzed whether kca channels of sk4 and bk type play a role in sustaining ca 2+ oscillations at g1-to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk4 modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca 2+ ]i oscillations and peak amplitude were determined using ca 2+ indicator fura-2am. results: sk4, but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk4 inhibition by tram-34 dose-dependently (0,1 to 10 µm) inhibits the growth of primary mammary tumor cells probably by a g1 cell cycle arrest. ca 2+ oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk4 channels. -dependent cell cycle progression is dependent on sk4 activity. blocking sk4 disrupts a feed-forward loop that coordinates ca 2+ influx via trp or crac channels in tumor cells. the consequences of sk4 inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of 5-hydroxymethyl-5-methylpyrroline n-oxide stolze k. 1 esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap 5,5-dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a 4-carboxybutyltriphenylphosphonium-substituent to the spin trap 5-hydroxymethyl-5-methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. 9, 97078 würzburg, germany type 2 diabetes mellitus (dm2) is a growing health problem affecting more than 150 million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm2 leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o 2-) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm2 complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc1 is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc1 for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn 2+ quenching and qpcr analysis were applied. furthermore, the effect of trpc1 knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc1 was not able to function as a homomeric channel. instead, trpc1 subunits formed functional receptor-operated heteromeric channel complexes with trpc3, 4, 5, 6, and 7. heteromers containing trpc1 subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc1 further reduced calcium permeability. in gnrh neurons endogenously expressing trpc1, 2, 5, and 6, downregulation of trpc1 by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc1 was not involved in store-operated cation influx in gnrh neurons. moreover, trpc1 suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc1 and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin-3 (trpm3) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm3 is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm3-like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm3 -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm3 is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm3 blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic50 values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm3 blockers. furthermore, we could show that these blockers are effective on endogenous trpm3 in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm3 activity in vivo. in sensory neurons, trpm3 may exert similar functions as trpv1. thus, trpm3 blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec50) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): 1 /25× ec50, 1 /10× ec50 , 1 /3× ec50, and 1× ec50. each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative γh2ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec50 values of substances were found (mmol/l;mean +/-sem): tmp(eo)9ta a, b 0.087 ± 0.011; 1,6-hddma b, c 4.500 ± 0.700; etma a, c ; 12.000 ± 1.100; a significantly (p < 0.05) differently to 1,6-hddma, b significantly (p < 0.05) differently to etma, c significantly (p < 0.05) differently to tmp(eo)9ta. after six hours of exposure with tmp(eo)9ta at 0.00348 mm there were induced 0.55 γ-h2ax foci-formations in hgfs, at 0.00870 mm 0.67 foci, at 0.02900 mm 0.86 foci and at 0.08700 mm 0.97 foci. after exposure with 1,6-hddma at 0.180 mm there were induced 0.45 γ-h2ax foci, at 0.450 mm 0.64 foci, at 1.500 mm 0.94 foci and at 4.500 mm 1.32 foci. after exposure with etma at 0.48 mm there were induced 0.43 γ-h2ax foci, at 1.2 mm 0.50 foci, at 4.0 mm 0.61 foci and at 12 mm 0.71 foci. the negative controls dmso and medium cultures displayed 0.31 -0.34 γ-h2ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: 1,6-hddma > tmp(eo)9ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. 1 , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and 2-hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative analysis of the γ-h2ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec 50 (mmol/l) of triethylenglykol dimethacrylat (tegdma) and 2-hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < 0.05) higher dsbs compared to hema (1.91 ± 0.04 vs 1.66 ± 0.02). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed (2.02 ± 0.06), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed (1.89 ± 0.07), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed (1.64 ± 0.04), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed (0.76 ± 0.02), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys 101 was replaced by ala (c101a) resulting in destabilization of enos has been generated (enos-c101a). transgenic mice carrying c101a were generated on a c57bl/6 background using the endotheliumspecific tie-2 promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c101a (enos-ko/enos-c101a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c57bl/6, enos-ko, and enos-ko/ enos-c101a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c101a-tg in aorta (37.1±8.4%, n=9), skeletal muscle (45.4±5.3%, n=10) and myocardium (17.4±4.9%, n=7) and revealed an increased phosphorylation of enos on ser1176/79 (470±47%) as compared to c57bl/6 (p<0.05, n=8). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c101a-tg (p<0.05, n=5-8), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c101a as a source of elevated radical generation. endothelium-specific introduction of enos-c101a at ~ 35% of c57bl/6 level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc)2 indicated that endothelium-dependent relaxation in enos-ko/enos-c101a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p<0.05, n=4-7). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n=4-7, p<0.05). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c101a-tg was substantially lower then in enos-ko and tended to be similar to c57bl/6. conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. 1 , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle-6tn and 16hbe14o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk1/2 were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn3o4 or mno2 with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. 1 numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g12/13, or gq/11 g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/11 signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga11 selectively in nociceptors to investigate the contribution of the entire gq/11signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/11 in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/11 deficient mice, suggesting a novel function for gq/11 in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/11, whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/11 branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany ap duration and ca 2+ cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within 6 hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for 1 second (5hz; 10hz; 5hz + s2-stimulus after 50-80ms) followed by a 4s rest period. the resting-membrane-potential (rmp) was observed over 30 cycles. we observed rmp-fluctuations of different length (amplitude <5 mv; % of 13 observed cardiomyocytes, mean events/cell; <1s: 100%, 17.1; <3s: 92%, 11.5; >3s: 69%, 5.8), spontaneous depolarizations (>5 mv; 92%, 5.1) and spontaneous aps (62%, 1.9). intracellular ca 2+ transients and sarcomere shortening were measured after loading cardiomyocytes with indo-1/am. after preconditioning (10 min/1 hz) cells were measured under basal and continuous isoprenaline (10 -6 m) stimulation (iso). a 4 min pacing period was followed by a 1 min interval of no pacing. pacing frequency was reduced after each cycle (1 hz, 0.5 hz, 0.25 arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca 2+ overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord1, bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi500 and cgi6000 with apparent cgmp affinities of 500 nm and 6000 nm, respectively. one mouse line expresses cgi6000 driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm22alpha promoter directs cgi500 expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase 3, 8 and 9, activated caspase 3, and aif). exposure of hepatocytes to α-ama at concentrations of 0,2 µm, 0,5 µm and 1 µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of 30 µm and 1mm, silibinin at 50 µm and 100 µm and a combination of both (30 µm benzylpenicillin and 50 µm silibinin, 1mm benzylpenicillin and 100 µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept 50 µm silibinin at 0,2 µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations (0,5 µm and 1 µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy 926) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh 4), we analyzed the amount of bh4 in ea.hy 926 by hplc. a concentration-dependent reduction of bh4 and also bh2 (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh4 can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch1, acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh4 involving dihydrofolate reductase, dhfr). treatment of ea.hy 926 cells with dex decreased mrna and protein expression of both gch1 and dhfr. because bh4 is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh4 which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap-1 and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh10tert foreskin fibroblasts (behaving like primary cells) and sv40-immortalized gm637 fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap-1 binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression (30-90 min after exposure) was not translated into the protein, the second wave of transcription (4-24h after uvc exposure) resulted in c-fos protein expression, 18-48h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p38k and erks) were immediately induced upon uvc exposure and stayed active for at least 24h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed 24-40h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch 665/2-1). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p450 (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp2c19 alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel (75mg qd), albeit carriage of cyp2c19 loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp2c19 genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type 2 (octn2/slc22a5) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc22a5 gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn2 expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn2 expression in monocytes and thp-1 cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp-1 and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn2 expression was characterized on monocytes and thp-1 cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn2 specific signals as well as a localization in the plasma membrane. following thp-1 cells were activated using lps (10ng/ml) for up to 6h, thereby indicating the expected cytokine response as demonstrated by increased tnfα (24fold induction) and il-1β (37fold induction) mrna levels. in addition, octn2 expression was analyzed identifying an initial reduction of around 60% compared to untreated cells. in parallel activated thp-1 cells were coincubated with increasing concentrations of the octn2 substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by 64% in the presence of 50mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp-1 cells represent a useful model to study octn2 expression and function. in addition, we demonstrate immunosuppressive effects of octn2 substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than 3000 years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg-01 cells to ricinoleic acid caused an increase in [ca 2+ ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca 2+ ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e2 receptors ep3 and ep4 as mediators of ricinoleic acid-induced effects. to test if ep3 and ep4 receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep3-deficient (ep3 -/-) or ep4-deficient mice (ep4 -/-). while ep4 -/mice responded similarly to the wt mice, ep3 -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep3 receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep3 receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep3, consistent with a preferential expression of the ep3 receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep3 receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep3 receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected 5 drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in 2011. in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but 17 additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an 8-times (cipralex ® : 5 → 40) and 5-times (onbrez ® : 2 → 10), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : 34 of 40; onbrez ® : 10 of 10). an additional amount of 22 conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports (21 of 227). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type 1 and type 2 patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc1). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in 24h as measured by an ha-binding protein linked assay (starved, 0g/l glucose: 100±4.7%; 1g/l: 185.7±44.8%; 4g/l: 362.6±58.3%; full medium 100±15.2%; 155.3±32.3%; 422.7±32.0%). surprisingly, total ha concentrations were about 2.2-2.8 fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with 10 mg/l insulin for 24h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, 1g/l glucose: 100±6.5% vs 67.83±4.4%; 4g/l: 100±2.2% vs 71.8±6.6%). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc1 cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut1, glut4) were analyzed by qrt-pcr. the relative abundance was 24.32±3.49 in favor of glut1 indicating the presence of insulin-independent glucose transport. conclusion: in osc1 the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type 2, where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type 2 and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße 56, 72074 tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb1-mutated tumours. larch-derived diterpenes are potent and selective trpc6 blockers urban n. 1 , kübler w. the transient receptor potential channel trpc6 is a poorly ca 2+ -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc6 expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc6 activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc6 and trpc3. indeed, turpentines and resins, but not coniphere oils blocked trpc6 and trpc3 in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc6-prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc6-selective block. larixol acetates displayed an ic50 towards the dag-or receptor-stimulated trpc6 activity of about 0.3-1 µm, but did not strongly inhibit a number of other trp channels, including trpv1, trpm2, trpm3, trpm8, or trpa1. selectivity for trpc6 compared to its closest relative, trpc3, was about 30-fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc6. electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc6 -/mice. we conclude that trpc6 blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still 2d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt4 cells were cultured in mccoy's 5a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) 50 mg of protein sample was mixed with 5% of coomassie blue g-250 (cbb g-250) and loaded in each lane of 4-15% polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a 12% sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina3n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. 1, 2 , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina3n, a serine protease inhibitor, which is homologous to human a1-antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina3n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina3n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina3n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina3n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina3n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany introduction. activated β1-adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk1/2 at threonine 188 (erk thr188phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr188 -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β1-receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ 3 h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk2 showed a significant increase in [ 3 h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr188 -phosphorylation deficient mutants (erk2 t188a and erk2 t188s ) or pretreatment with the erk inhibitor, pd98059, significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for 14 days to wild-type mice and transgenic mice overexpressing either wild-type erk2 t188t or erk2 t188s . echocardiography and histological analyses revealed that erk t188s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk1) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk2 t188a or erk2 t188s are retained in the cytosol and prevented elk1-phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr188 -phosphorylation. conclusion. taken together, gβγ-subunits participate in β1-adrenergic receptor mediated hypertrophy by enhancing erk thr188 -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. 1 , burke m. introduction: alpha-toxin, a 34 kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation (1). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets (2) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp1, pp2 or su6654 (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr-416-phospho-src from calbiochem and cell signaling, respectively (3). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner (0.18 -3.0 µg/ml of toxin). pre-incubation with 3 src inhibitors (pp1, pp2 or su6654) reduced α-toxin-induced platelet aggregation by about 50%. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr-416 followed by a fast and complete dephosphorylation within 10 min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr-416 phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin (1µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr-416 typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m2 and m4 prefer coupling to gi proteins, whereas the odd-numbered receptors m1, m3 and m5 prefer coupling to gq proteins. with respect to ligand binding and m2 receptor activation, the conserved epitope trp 7.35 at the beginning of tm7 displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m2 receptor [1] . in the inactive m2 receptor, it provides subtype-independent baseline affinity for allosteric antagonists [1] . in the active receptor, m2 trp 7.35 affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [2] . to study the role of trp 7.35 for m3 receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm3 wild-type-cells and hm3 trp 7.35→ala-cells, amounting to 0.07 and 0.11x10 6 receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m3 trp 7.35→ala relative to m3 wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m2 and the corresponding mutant. in the case of pilocarpine, replacement of trp 7.35 by alanine in m3 did not reduce intrinsic efficacy. this finding is in contrast to m2, where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m3 trp 7.35→ala mutant relative to m3 wild-type. this finding is also in contrast to m2, at which pilocarpine's potency was not sensitive to the trp 7.35→ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp 7.35→ala mutation between the m3 and the m2 receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human 3dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these 3d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such 3dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of 20 substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in 3t3 and a549 cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the 3d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca 2+ handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. 1 background: in atrial myocytes ca 2+ entry through l-type ca 2+ channels (ica,l) triggers a larger ca 2+ release (ca 2+ transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo-3) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, 1µm) and the nonselective phosphodiasterase (pde) inhibitor 3-isobutyl-1-methylxanthine (ibmx, 10µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l (3.3±0.3pa/pf, n=12/6 [myocytes/patients] vs 6.2±0.8 pa/pf, n=15/10, p<0.01) and cat (182.2±26.8nm vs 307.5±30.9nm, p<0.01) were lower than in ctl myocytes, whereas diastolic [ca 2+ ]i levels were unchanged (caf, 312.3±56.7nm; ctl, 305.3±43.6nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to 10.4±2.0pa/pf (n=7/4) in caf and to 14.3±1.7pa/pf (n=9/7) in ctl. the corresponding cats increased to 493.0±132.3nm in caf and to 545.0±79.0nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca 2+ ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, +202.3±47.3% vs ctl, +98.4±16.1%, p<0.05) and cat (caf, +215.4±57.8% vs ctl, +101.9±20.3%, p=0.06) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, 9.9±1.5pmol/mg, n=6 vs ctl, 5.0±0.6pmol/mg, n=7, p<0.05), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. 1 g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o 6 -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o 2 -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ 0.05 mm 2 /s, g-proteins ~ 0.1mm 2 /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g2. molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac2 and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s13 and n62) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz3/bcl6 or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac2-mediated phospholipase c-β 2 (plcβ2) and plcγ2 activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ2, plcβ2, and plcδ1, but that it had little or no effect on the activity of plcγ1 and plcε. the amino acid residues s13 and n62, likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac2 caused inhibitory effects on rac2-mediated plcβ2 and plcγ2 stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ2 distinct from those which are necessary for rac2 interaction, as the split pleckstrin homology domain of plcγ2, which is essential for its interaction with activated rac2, is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ2 and plcγ2, both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ2 activation. effect of rac1 inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse 1, 40225 düsseldorf, germany background: the small gtpase rac1 is a well characterized member of the rashomologous (rho) family. rac1 is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap1). furthermore, rac1 can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac1 is still unclear. here, we address the question how rac1 influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac1, human hepatoma cells were pretreated with the rac1-inhibitor eht 1864 before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac1 inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac1 inhibitor showed a higher viability, less phosphorylation of h2ax (s139) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac1 resulted in a reduced phosphorylation of topo iiα (s1106) and an increased interaction of topo iiα with hsp90 in doxorubicin treated cells. the data indicate that inhibition of rac1 protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg2 cdna-clone sequences were submitted to the ncbi genebank (eu570105, gq141082 and gq241418). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst 33342 was measured and the selectivity was determined by the bcrp inhibitor ko143. inhibition studies using hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. 52, 67663 kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [1] . as an α,b-unsaturated aldehyde, ac forms 1,4-michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-(3-hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-(2-carbamoylethyl)-cysteine (aama), and (n-acetyl-s-(2-hydroxy-2-carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [2] . nevertheless, in a pilot study in humans urinary 3-hpma excretion of 14 non-smokers was reported to be about three fold higher, as compared to aama [3] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips (175 g), equivalent to an uptake of 44 µg aa (absolute amount), together with an as yet unknown amount of acrolein [4] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to 24 h after test meal uptake. the results demonstrated kinetics of 3-hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of 3-hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [1] stevens, j.f. and maier, c.s. (2008) molecular nutrition & food research 52; 7 [2] osorio, v. m. and de lourdes cardeal, z., (2011) journal of chromatography a 1218; 3332 [3] schettgen, t., musiol, a., and kraus, t., (2008) rapid communications in mass spectrometry 22; 2629 [4] ewert, a., granvogl, m., and schieberle, p., (2011) lebensmittelchemikertag 2011 450 protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str.31, 78457 konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase 1 (parp-1) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to 2 fold in unstimulated [1] and 1.5 fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine 3':5' monophosphate (camp) and guanosine 3':5' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic 3':5' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [1] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [2] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr1. we chose this gene because it is regulated by numerous stimuli including camp [3] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr1 gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr1. esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am (3, 10, 33, 100 µm) over 15 to 240 min 24 h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr1 expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb 153, sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr1 expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr1 expression in hela cells. methyl-cpg-binding protein 2 (mecp2) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp2 is diminished in murine and human heart failure. prevention of mecp2 downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp2 gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp2 on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp2 of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp2. in order to investigate the cardiac function of mecp2, two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp2 in cardiac myocytes under control of the tetracycline-system (mecp2-tg) and mice with targeted ablation of mecp2 in myocytes (mecp2 mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp2 did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp2 expression could be completely prevented by the mecp2 transgene. cardiac contractility and relaxation were significantly decreased in mecp2-tg animals. upon electron microscopical investigation, transgenic mecp2 expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp2 caused a rightward shift in the size distribution of mitochondria as compared with mecp2-tg hearts. epigenetic processes, including the recognition of dna methylation by mecp2, may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp2 expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren2 rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass 9, 97078 würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren2 rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of 13 and 32 weeks. untreated ren2 rats exhibit increased blood pressure from the age of 8 weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren2 animals, a significant higher superoxide production could be observed in kidneys already in 13 week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h2ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren2 rats compared to control rats. as another organ affected by hypertension the heart of the ren2 animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d541a pore mutation leads to complete inactivation of trpv6 channels in epididymis weißgerber p. 1 , kriebs u. replacement of aspartate residue 541 by alanine (d541a) in the pore of trpv6 channels in mice disrupts ca 2+ absorption by the epididymal epithelium resulting in abnormally high ca 2+ concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv6 d541a proteins (sci signal 4, ra27, 2011) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv6 d541a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv6 -/and trpv6 d541a/d541a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv6 mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv6 -/and trpv6 d541a/d541a males. the profound reduction in ca 2+ uptake by the epididymal epithelium was identical in trpv6 -/and trpv6 d541a/d541a males. this direct comparison of these parameters indicate that deleting trpv6 does not further aggravate the phenotype observed in trpv6 d541a/d541a mice, and -in our opinion -allows the conclusion that the d541a pore mutant of the trpv6 protein leads to complete inactivation of the trpv6 channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n996i) on herg channel function in hek293 cells sellmaier v. 1 , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome 2 (lqt2) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh2 or kv11.1). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as 2 isoforms (1a and 1b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing 2 of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg1 gene (n996i) in a patient with lqt. both herg1a and herg1b subunits were cloned from a human heart cdna library and the specific n996i mutation introduced by site directed mutagenesis. hek 293 cells were transiently transfected with equal amounts of mutated herg1a and wild type (wt) herg1b cdnas and the resulting potassium current compared to herg1a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at -27 mv for wt and -29 mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = 62 ms and tslow = 480 ms versus tfast = 90 ms and tfast = 620 ms in wt channels, determined from the tail current at -40 mv after a 5-second pulse to + 50 mv. the half-maximal steady-state inactivation of the tail current was shifted by 20 mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc2 is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc2 polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc2 haplotypes by the influence of micrornas. methods: abcc2 cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 3'utr sequence of abcc2 whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc2 were identified form an mirna array after rifampicin stimulation of hepg2 cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 3'utr vs. mod. 3'utr) concerning the -24c/1249g/3972c (cgc) abcc2 wt in hepg2 cells. using the fl vector construct, the expression level of cac haplotype protein was increased (136,15% ± 20%). transfection assays with the mir-397, which was identified as a candidate mirna targeting abcc2 mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir-379 was able to down regulate the abcc2 protein expression. there was no significance in downregulation for one abcc2 haplotype, respectively (modified 3'utr: cgc 22-34%; cac 20-40%, fl 3'utr: cgc 20-25%; cac 17-44% down regulation compared to mir-negative control). discussion: differences of abcc2 protein expression level are due to the epigenetic regulation of abcc2 haplotypes. to further characterize the effect of mir-379 on tcg, tgt and cgt abcc2 haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg 14, 07743 jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk886 effectively reduced ltb 4 pleural levels in female but not in male rats treated with carrageenan, and mk886 increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk886. in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, 21:102, 2009 ): male wistar rats were exposed to the test items for 6 h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of 10%, the instilled dose corresponded to the aerosol concentration of 10 mg/m 3 used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of 60-120 nm. in the last 2 years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd 427) with 65 zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v79 cells, both showing negative results whereas the mouse lymphoma mutation test / l5178y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in 5-days, 14-days and 90-days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of 1.5 mg/m3 in the 90-day study. in a prenatal developmental toxicity study according to oecd tg 414, with repeated inhalation exposure to female wistar rats from gestation day 6 through 19, maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. (1996) . the initial report used a database of 613 organic substances compiled from publicly available sources. in total, 2941 noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per 3 november 2011, containing data for more than 3900 registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to 6 were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the 5 th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than 1000 substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. 1 , schumann b. b-and l-moc can be cultivated up to 70 days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h322 to tpm (30 mg/l) reduced viability to 95% or 83% after 24 or 72h, respectively. in a549 viability was 67% after 72h with tpm (30 mg/l). the same dose of tpm lead to a decrease in viability to 82% (24h) or 25% (72h) in nhbec and to 74% (24h) or 28% (72h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm (5 mg/l). in h322 the induction was 1.6 and 1.2 fold after 24 or 72h, respectively. while in a549 it was 1.3 (24h) and 1.4 fold (72h). in nhec and plc tpm (5 mg/l) did not have a significant effect on gsh-levels after 72h. in n-moc tpm (5 mg/l) also did not modulate gsh after 24h, but it diminished gsh-level by 0.5 fold upon prolonged contact (72h). in b-moc gsh concentrations also decreased to 0.6 or 0.8 fold the level of controls after 24 or 72h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. 1 , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by 20 % (wt 0,25 ± 0,01 mg/g vs. tg 0,30 ± 0,02 mg/g, p<0.05, n=12). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, 100 µl 1 mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet (1%) was performed. after 28 days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration (16 ± 0.5 ms vs. 14 ± 0.7 ms, p<0.05) and qtc interval (61 ± 2 ms vs. 50 ± 3 ms, p<0.05) in tg (n=7) compared to wt (n=5) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße 9, 97078 würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca 2+ -atpase serca2a, thereby decelerating cytosolic ca 2+ clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca 2+ transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l37a, i40a and c41f) or favor pentamer formation (i45a, v49a). transfected hek293 cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with 0.25µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with 2.5µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature (14°c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek293 cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. 1, 2 , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a 5-fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. 1 camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r2) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s49 wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s49 kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n 6 -mb-camp and into 2'-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) 1 . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s49 wt und s49 kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s49 wt nor in s49 kincells. interestingly, we observed apoptosis in s49 wt and s49 kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. (1) wolter s, golombek m, seifert r (2011) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s49 cells : induction of apoptosis in s49 wt and in s49 cells lacking the catalytic subunit of pka (s49 kin-) with a) db-cnmps and b) cnmp-ams for 72h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. 67, 55131 mainz, germany nebivolol is a third generation β1 receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. 48 spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment (5 mg/kg/day for 10 days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam-1, vcam-1) and pro-inflammatory cytokines (e.g. il-6). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde4 inhibitors using two novel pde4 reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg 18a, 42096 wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde4 reporter cell lines. plasmid constructs encoding human pde4b1 or pde4d3 were stably co-transfected with the beta1-adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde4 and non-pde4 inhibitors, we could show that our novel pde4b1 and pde4d3 reporter cell lines specifically monitor pde4 inhibition with high sensitivity. pde4-selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta1-adrenoceptor reporter cell line with no recombinant pde4 expression, pde4 inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde4 inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde4d3 from cell lysates. two different groups of pde4 inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d159153, showed high cellular activity and much better inhibition of full-length versus truncated pde4d3. the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde4d3. the results imply that these novel pde4 reporter cell lines are well-suited for the characterization of the cellular activity of pde4 inhibitors and may also support a better understanding of the complex pde4 pharmacology. plexin-b2 is required for kidney regeneration after acute renal failure xia j. 1 , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b2 belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b2 is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b2 during kidney regeneration we generated mice lacking plexin-b2 specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b2 conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within 3 weeks after injury, plexin-b2 knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b2 is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b2 might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p22phox) and several cytosolic regulatory components including p47phox, p67phox, p40phox and the small gtpase rac1. we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox2 and nox4 in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac1 membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin 1 (sirt1). we then wanted to find out whether the effect of resveratrol on rac1 was mediated by sirt1. sirt1 is a histone/protein deacetylase. in vitro incubation of rac1 with sirt1 led to a reduction of lysine acetylation. deacetylation of rac1 on lysine 166 could be identified by mass spectrometry analyses. the lysine 166 lies within the p67phox-binding region of rac1. consistently, in vitro incubation of rac1 with sirt1 markedly reduced its binding activity to p67phox. in conclusion, we provide evidence that rac1 is a direct target molecule of sirt1. sirt1 deacetylates rac1 on lysine 166 and thereby inhibits its interaction with p67phox. this is a novel mechanism of nadph oxidase inhibition by sirt1/resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany dexrazoxane (drz, icrf-187) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top2). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top2b, the dominant cardiac top2 isoform. this was investigated using top2b mutants resistant to drz, which were expressed in cells depleted of wild-type top2 isoforms. top2b-mediated double-strand dna breaks were assessed as γ-h2ax. the levesl of dsb generated by the top2b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top2b. preincubation with drz depleted wild-type top2b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top2b depletion nor the reduction of dsb by drz was seen in drz-resistant top2b mutants. furthermore, the cardially ineffective drz analog icrf-161, capable of iron chelation but not of top2 binding, affected neither the stability of top2b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top2b rather than by iron chelation. they also suggest a cardioprotective function of top2b, which is currently under investigation using cardiomyocyte-specific top2b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw3 were cultivated for 3, 6, and 9 days in dmem supplemented with three different concentrations of amikacin, streptomycin (30 mg/l, 100 mg/l, 300 mg/l), gentamicin or tobramycin (10 mg/l, 30 mg/l, 100 mg/l). we determined the expression of two junctional proteins (connexin 43, ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested (30 mg/l) connexin 43 and n-cadherin were reduced to 55±19% and 92±10% of the controls (n=6). no change was recognized for vimentin (102±16%). effects obtained with streptomycin were less pronounced for these these proteins (68±16%, 96±7%, and 102±13%, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of 10 mg/l (connexin 43: 88±15% and 81±15%; n-cadherin: 95±18% and 103±11%; vimentin: 81±12% and 102±19%) and 30 mg/l (connexin-43: 84±19% and 78±18%; n-cadherin: 98±19% and 103±13%; vimentin: 102±8% and 115±15%). after incubation for 6 and 9 days the effects occurred in the same range. the substances showed no influence on the viability of serw3 sertoli-cells up to 300 mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin 43 and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of 59 amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ 50% inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s700e) in bk-strex, resulted in a ~50% reduction of basal channel activity, whereas the s700a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c12:13a) or by pharmacological inhibition of palmitoyl transferases with 2-bromopalmitate (2-bp). both, mutation and pretreatment with 2-bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ 50% inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s700a channel mutant pretreated with 2-bp. in a clonal rat somatomammotroph pituitary cell line (gh3b6), in which pcr products without (zero) and with the 174 bp strex exon could be identified, the pkc activator pma blocked channel activity by ~25 %. this inhibition was increased to over 50% when gh3b6 cells were pretreated with 2-bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser700, probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met-5a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. 1 , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. 03x0109a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv40transformed, non-malignant pleural mesothelial cell line met-5a was chosen as the main in vitro model in this project. in the present study part, met-5a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos (2, 10, and 20 µg/cm 2 ) was demonstrated in met-5a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg1modified comet assay. thus, met-5a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met-5a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than 10% cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met-5a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm7 channels zierler s. 1 transient receptor potential melastatin 7 (trpm7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm7. waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg 2+ ) concentration. mutating a mg 2+ -sensitive site on the trpm7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg 2+ . waixenicin a failed to inhibit the closely homologous trpm6 channel and did not significantly affect trpm2, trpm4, and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm7 ion channels. consistent with trpm7 inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg 2+ -dependent block of trpm7, waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg 2+ . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße 4-6, 30449 hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first 5 litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total 16 water pipe strands of 11 domestic installation systems were examined. they comprised 379 single water samples and 5306 single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc4 protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. 1, 66421 homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical 4 (mtrpc4) were identified. the trpc4 protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc4 gene exist, trpc4a (974 aa) and trpc4b (890 aa), trpc4b lacks aa 781 to 864 of the trpc4a variant. both trpc4 variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc4-interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc4a (aa 1 to 324; aa 622 to 974). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc4 in hek 293 cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc4. in addition, changes of cytosolic calcium were monitored and trpc4 currents were recorded in hek 293 cells expressing the candidate cdnas and stably expressing the trpc4a or trpc4b and the muscarinic receptor type 2 cdnas. the tarbp2 protein, one of the candidates shown to interact with trpc4, changed calcium influx when coexpressed with trpc4. in order to identify the domains of trpc4 responsible for its interaction with the tarbp2 protein, six trpc4-gst-fusion proteins covering the carboxyl terminal 353 aa of trpc4a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc4 could be identified to interact with tarbp2. one of these domains is well conserved within the trpc5 protein, corresponding to the result, that trpc5 and tarbp2 effectively co-immunoprecipitate, too. the tarbp2 protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer1, eif2c2/ago2 and tarbp2 (chendrimada et al. 2005) . by its interaction it may link trpc4 to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c57bl/6-mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600ng/kg · min during 28 days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type 1 (at1) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at1 receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at1 receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at1 receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine 3´, 5´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek 293 cells that stably express hcn1, hcn2, hcn3 or hcn4, respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn2 and hcn4 channels, ccmp shifted the membrane potential for half maximal activation (v0.5) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec50 for ccmp was 30 µm which is about 5 times higher than the ec50 of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only 65 % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of 1 mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v0.5 of ih by about +5 mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about 20 % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a 3d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. 1 the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [1] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [2, 3] , outperformed the current standard for the therapy of actinic keratosis, 5-fluoruracil, when tested in the tumour cell line scc25, in normal human keratinocytes and fibroblasts [4] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the 3d construct of scc developed by höller and co-workers [5] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc12 seeding to grow an aklike construct with scc12 cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki-67, marker for scc: cytokeratin-10, axl, marker for invasion: mmp2, marker for apoptosis: caspase-7, nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin-18 and its caspase-induced cleavage product into the culture medium following drug exposure for up to 7 days. efficacy of a 0.05% oxbu solution proved close to or even better when compared to both a 0.1% 5fluorouracil and 0.025% aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [4] . -therefore, these 3d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other 3d models of skin diseases currently gaining increased interest as test platforms. ima910, a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd8+ and cd4+ t-cell responses associated with improved survival walter s. 1 , kuttruff s. ima910 is a novel peptide-based vaccine consisting of 10 hla-a*02 and 3 hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima910 was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima910 vaccinations in combination with low-dose gm-csf (cohort 1; n=66) or ima910 / gm-csf plus topically applied imiquimod (cohort 2; n=26). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd8 + t-cell responses and by ics assay for cd4 + tcell responses. as immune status biomarkers, 6 phenotypically defined myeloid derived suppressor cell populations (mdsc1-6) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima910 overall was immunogenic in 73/81 (90%) evaluable patients, with 43% and 65% of patients mounting multiple cd8 + and cd4 + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd8 + cell responses as detected by ics (p=0.016). multiple cd8 + and multiple cd4 + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd8 + and multiple cd4 + responses. these patients had significantly higher dcr at 6 months (p=0.002), improved ttp (p=0.006) and improved pfs (p=0.009) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p=0.088, hazard ratio 0.53). in the study population, levels of 5 different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc4 and mdsc5 high frequencies were associated with shorter os (p=0.007 and p=0.019, respectively). to summarize, both hla-a*02 and hla-dr restricted peptides in ima910 were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima910. acrylamide (aa), a genotoxic carcinogen (iarc class 2a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp450 2e1 into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n7 of guanine (n7-ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-(2-carbamoylethyl)-cysteine (aama), and n-acetyl-s-(2-hydroxy-2carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to 10 mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n7-ga-gua dna adducts in liver, kidney and lung was measured 16 h after application, a time point where cmax of n7-ga-gua was reached. the untreated control group was found to excrete about 0.8 nmol (aama plus gama) in the urine (16 h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of 0.1 µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n7-ga-gua adduct levels in any organ tested (limit of detection, lod, 0.2 adducts/10 8 nucleotides). at the tenfold higher dose (1 µg/kg bw), adducts were found in kidney (about 1 adduct/10 8 nucleotides) and lung (< 1 adduct/10 8 nucleotides), but not in liver. at 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg aa/kg bw (about 1-2 adducts/10 8 nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure (0.1-10 µg/kg bw ) leads to n7-ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. 27, 01307 dresden health insurance costs in germany have grown by 3% p.a. over the last ten years and amount to approx. 280 bn eur in 2009. while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from 2007 to 2011. the data of a private health insurance company with more than 600.000 customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. 1, 2 , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically 2 d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than 60 million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for 1) replacement of tests on juveniles and (sub-) adult rodents, 2) stages with impossibility of sensation of pain in the individuals, 3) highly sensitive prospects of modes of action of chemicals, which 4) might show consequences in the next generations. 3 channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav 2.3 e/r-type voltage-gated calcium channel transcriptional upregulation of cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav3.2 t-type calcium channels histidine residues in the is3-is4 loop are critical for nickel-sensitive inhibition of the cav2.3 calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct2 (slc22a2) functional characterization of the human organic cation transporter 2 variant p.270ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp-2 tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs5 domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc-631570 (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago2 for microrna processing and gene silencing index a 009, 019, 035 011, 014 003, 044 objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr3c2), rs2070951 modulating blood pressure, renin, and aldosterone levels, and rs5534, which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels >140 mmol/l were defined as 'high'. after application of 5 µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤10%), or 'strong' (>10%). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n=12), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p=0.06). both nr3c2 variants were associated with a change >10% in endothelial stiffness after amiloride treatment. the rs2070951 c allele was significantly associated with a strong amiloride response (p=0.024), while the rs5534 a allele only showed a trend towards stronger amiloride effects (p=0.06). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr3c2 variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. 55, 45122 essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb1 (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p450 (cyp) enzymes, including cyp2e1. cytotoxicity of acetaminophen (aap) is based on its cyp2e1-catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp2e1-overexpressing ctnnb1mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb1-mutated tumors. administration of a single dose of aap (300 mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about 90% necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb1-mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp2e1-overexpressing hepatoma. release of 5,6-epoxyeicosatrienoic acid (5,6-eet) upon neuronal activity induces trpa1-dependent mechanical pain hypersensitivity sisignano m. 1 , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp450) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of 5,6-eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception 5,6-eet-levels increased in the drg and it is released from activated sensory neurons in vitro. 5,6-eet potently induced a calcium flux [10 nm] in cultured drg-neurons which was completely abolished when trpa1 was deleted or inhibited. in spinal cord slices 5,6-eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, 5,6-eet-induced enhancement of sepsc frequency was abolished in trpa1 null mice, suggesting that 5,6-eet pre-synaptically facilitates spinal cord synaptic transmission via trpa1. finally, intrathecal injection of 5,6-eet caused mechanical hyperagesia in wild type but not trpa1 null mice. we conclude that 5,6-eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa1 at central afferent terminals in the spinal cord. sisnaiske j. 1 , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep2010/002811). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy5y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca2+, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox4 by histone deacetylases siuda d. 1, 2 , zechner u. 3 , prawitt d. 4 nox4 is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox4-mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox4 transcription by histone deacetylase (hdac). in cultured human ea.hy 926 endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox4 mrna expression. a similar down-regulation of nox4 mrna expression can be achieved with sirna-mediated knockdown of hdac3. hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox4 promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox4 promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox4 transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox4 mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox4 promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox4 promoter is significantly inhibited as well, which may explain the reduction in nox4 transcription. in conclusion, hdac inhibition decreases nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox4 promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v79 cells solecki g. m. 1 key: cord-004534-jqm1hxps authors: nan title: abstract date: 2009-06-09 journal: eur biophys j doi: 10.1007/s00249-009-0478-1 sha: doc_id: 4534 cord_uid: jqm1hxps nan standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organisation of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. we have combined two existing methodologies to tackle these challenges: tandem affinity purification (tap) and nanoflow esi-ms. we use methods designed to maintain non-covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. structural models will be presented for three oligomeric protein complexes of unknown structure: the yeast exosome and the human u1snrnp and eif3 complexes. these models will then be examined within the context of their function. recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross-section. using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross-sections. very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase . this enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. in vivo molecular sensing: fluorescence beyond labeling f. beltram scuola normale superiore, i-56126 pisa, italy fluorescent molecules are powerful reporter tools that have much extended the impact of optical microscopy, particularly thanks to the flexibility of genetically encoded tags. detection can now target single molecules even in the complex environment of intact live cells offering unprecedented insight on biological processes in real time in live cells and tissues. fluorescent labels, however, can do more that this. our increased ability to tailor molecule and, more in general, nanosystem properties allows us to design, produce and exploit intelligent tags that can actually analyze the cellular environment. today multifunctional nanosystems can be produced that provide a signal dependent on the value of a specific biochemical parameter. importantly these nanosystems can target specific subcellular domains and have the ability to be used also in the case of live organisms. recent results will be discussed that highlight the impact of nanobiotechnology in this context with a particular emphasis given to methods suitable for in vivo studies that can be transferred to the biomedical world. far-field optical nanoscopy s. w. hell max planck institute, göttingen, germany the resolution of a far-field optical microscope is usually limited to d = λ/ (2 n sin α) > 200 nm, with n sin α denoting the numerical aperture of the lens and λ the wavelength of light. we will discuss lens-based fluorescence microscopy concepts that feature a resolving power on the nanoscale. all these concepts share a common basis: exploiting selected (pairs of) states and transitions of the fluorescent marker to neutralize the limiting role of diffraction. specifically, the fluorophore is switched on and off, that is, between a bright and a dark state, to detect the emission of adjacent features sequentially in time. the first viable concept of this kind was stimulated emission depletion (sted) microscopy in which the fluorescence ability of the dye is switched off by stimulated emission. in the sted microscope, the extent of the region in which the molecule is able to fluoresce follows d ≈ λ/ 2 n sin α 1 + i/i s , meaning that fluorophores that are further away than d can be separated. i is the intensity that drives a fluorophore from the bright fluorescent state to the dark ground state by stimulated emission. i s depends (inversely) on the lifetime of the states. for i/i s → ∞, it follows that d → 0, meaning that the resolution can be molecular). altogether, far-field optical 'nanoscopy' is a fascinating development in optics with high relevance to the many areas of sciences, in particular the life sciences. since it has already been a key to answering important questions in biology, and owing to its simplicity and commercial availability, we expect far-field fluorescence 'nanoscopes' to enter most cell biology and many nanoscience laboratories in the near future. grabbing the cat by the tail: discrete steps by a dna packaging motor and the inter-subunit coordination in a ring-atpase c. bustamante, j. moffitt university of california, berkeley, california, u.s.a. as part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid. bacteriophage ϕ29 packages its 6.6 mm long double-stranded dna into a 42 nm dia. x 54 nm high capsid using a multimeric ring motor that belongs to the asce (additional strand, conserved e) superfamily of atpases. a number of fundamental questions remain as to the coordination of the various subunits in these multimeric rings. the portal motor in bacteriophage phi29 is ideal to investigate these questions and is a remarkable machine that must overcome entropic, electrostatic, and dna bending energies to package its genome to near-crystalline density inside the capsid. using optical tweezers, we find that this motor can work against loads of up to ∼55 piconewtons on average, making it one of the strongest molecular motors ever reported. we establish the force-velocity relationship of the motor. interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to dna compression attaining the value of ∼6 megapascals at the end of the packaging. we show that the chemical energy of atp is converted into mechanical work during phosphate release. using ultra-high resolution optical tweezers, we determined the step size of the motor and established the coordination of the polymerases around the ring. we propose a comprehensive model of the operation of this motor. watching proteins function in real time via timeresolved x-ray diffraction and solution scattering p. a. anfinrud laboratory of chemical physics/niddk, nih, bethesda, maryland, u.s.a. to generate a deeper understanding into the relations between protein structure, dynamics, and function, we have developed x-ray methods capable of probing changes in protein structure on time scales as short as 150 ps. this infrastructure was first developed on the id09b time-resolved x-ray beamline at the european synchrotron and radiation facility, and more recently at the id14b biocars beamline at the advanced photon source. in studies of ligand-binding heme proteins, a picosecond laser pulse first photolyzes co from the heme, then a suitably delayed picosecond x-ray pulse passes through the protein and the scattered x-rays are imaged on a 2d detector. when the sample is a protein crystal, this "pump-probe" approach recovers time-resolved diffraction "snapshots" whose corresponding electron density maps can be stitched together into movies that unveil the correlated protein motions that accompany and/or mediate ligand migration within the hydrophobic interior of the protein. when the sample is a protein solution, we recover timeresolved small-and wide-angle x-ray scattering patterns that are sensitive to changes in the size, shape, and structure of the protein. scattering studies of proteins in solution unveil structural dynamics without the constraints imposed by crystal contacts; thus, these scattering "fingerprints" complement results obtained from diffraction studies. this research was supported in part by the intramural research program of the nih, niddk dc-sign is a trans-membrane protein expressed on antigen presenting cells and recognizes pathogens like hiv-1, hepatitis c virus and ebola. by electron microscopy and near-field optical nanoscopy, we demonstrated that at the plasma membrane dc-sign is organized in well-defined nanodomains of 100-180 nm in diameter. intensity-size correlation analysis revealed remarkable heterogeneity in the nanodomains molecular packing density. we constructed and characterized several dc-sign mutated forms lacking specific molecular domains. by immunogold labeling and spatial point pattern analysis, we show that the extracellular neck domain is essential for dc-sign nanoclustering. finally, we present a model that describes the probability of a cell to have a certain number of receptors joining the contact site in the initial encounter with an external object. monte carlo simulations subsequently define the parameters that are determinant in the object-cell encounter. our results show that receptor nanoclustering is of particular importance for binding objects of sizes comparable to the nanocluster size, indicating that the nanoscale spatial organization of dc-sign is optimized for binding to virus-sized objects. imaging of mobile stable lipid rafts in the live cell plasma membrane m. brameshuber 1 , j. weghuber 1 , v. ruprecht 1 , h. stockinger 2 , g. j. schuetz 1 1 johannes kepler university linz, austria, 2 medical university of vienna, austria the organization of the cellular plasma membrane at a nanoscopic length scale is believed to affect the association of distinct sets of membrane proteins for the regulation of multiple signaling pathways. based on in vitro results, conflicting models have been proposed which postulate the existence of stable or highly dynamic platforms of membrane lipids and proteins. here we directly imaged and further characterized lipid rafts in the plasma membrane of living cho cells by single molecule tirf microscopy. using a novel recording scheme for "thinning out clusters while conserving stoichiometry of labeling" 1 , molecular homo-association of gpi-anchored mgfp was detected at 37 • c and ascribed to specific enrichment in lipid platforms. the mobile mgfp-gpi homo-associates were found to be stable on a seconds timescale and dissolved after cholesterol depletion. having confirmed the association of mgfp-gpi to stable membrane rafts, we attempted to use an externally applied marker to test this hypothesis. we used bodipy-gm1, a probe that was recently reported to be enriched in the liquid-ordered phase of plasma membrane vesicles. when applied to cho cells at different surface staining, we found that also bodipy-gm1 homo-associated in a cholesterol-dependent manner, thus providing further evidence for the existence of membrane rafts. [1] appl phys lett 87, 263903 (2005) . synapsin knock-out mice as an in vitro model of human epilepsy studied with multi-electrode arrays d. f. boido, p. farisello, p. baldelli, f. benfenati department of neuroscience and brain technologies, italian institute of technology, genova, italy mutant mice lacking synapsins (syn), a family of synaptic vesicles (sv) proteins implicated in the regulation of neurotransmitter release and synapse formation, are epileptic. the attacks appear after the third month of age and severity increases with age. several mutations of syn genes have been found in families of patients with epilepsy. we used micro-electrode arrays (meas) to study spontaneous and chemically evoked epileptiform activities in cortico-hippocampal brain slices obtained from wild-type (wt) and synko mice. 6-months old synko mice show sporadic ictal (ic) events in the entorhinal cortex. a potassium channel blocker, 4aminopyridine (4ap), elicits ic and inter-ictal (i-ic) events in both wt and synko slices. in the hippocampus of young synko (15-days old) mice, 4ap induces i-ic events at higher frequencies than in wt mice. also the frequency of ic events, mainly observed in the cortex, is higher in synko. the analysis of adult (1-year old) mice, revealed a clear age-related aggravation, which paralleled the increase in the severity of the epileptic phenotype observed in vivo. many slices from adult synko mice showed an ic event, while wt slices were refractory at this age to experience ic activity. synko mice are useful to study how neuronal network hyperexcitabilty due to mutations in sv proteins leads to the development of epileptiform activity. meas proved themselves to be useful tools to characterize the epileptic signals foci and patterns of propagation. high electron mobility transistor (hemt) structures were used to bridge the gap between the analysis of biological reactions and biophysical characterization. the combination of nanotechnological measurement approaches with biological reactions provides new possibilities for living cell examinations after exposure to ionizing radiation and basically during the irradiation experiments itself. in this transdisciplinary approach experimental data and handling of biological material enables the identification and specification of systems properties of biological responses to ionizing radiation at different hierarchical levels. gan/algan-heterostructures form a hemt with a gate very sensitive to ph-value changes and potential changes in general. to record cell membrane potentials and ion fluxes during and after irradiation experiments living cells are cultivated on the functionalised biocompatible chip surface. here, we present results of x-ray stimulated cell responses grown on gan-chip surfaces. we recorded transistor signal changes of 0.13 µa within 60 s caused by an irradiated cell monolayer. to measure cell potentials, not only after irradiation experiments but also during the irradiation itself expands the examination restrictions in an enormous way. measuring diffusion by spatial-cross-correlation e. gratton, m. a. digman laboratory for fluorescence dynamics, university of california, irvine, u.s.a. fluorescence correlation spectroscopy (fcs) has emerged as a very powerful method to study the motions of proteins both in the interior and exterior of the cell. it provides information at the single molecular level by averaging the behavior of many molecules thus achieving very good statistics. single particle tracking (spt) is also a highly sensitive technique to measure particle movement. however, the fcs method suffers in spatial resolution while the spt technique only allows for the tracking of isolated molecules. here we propose a change of paradigm in which using spatial pair cross-correlation functions we can overcome this limitation. our method measures the time a particle takes to go from one location to another by correlating the intensity fluctuations at specific points on a grid independently on how many particles are in the imaging field. therefore we can trace the average path of the particles. for example, our method could be used to detect when a protein passes the nuclear barrier and the location of the passage. this information cannot be obtained with the frap (fluorescence recovery after photobleaching) technique or the image correlation spectroscopy method. the interaction of the bax c-terminal domain with membranes j. c. gomez-fernandez, s. sanchez-bautista, a. perez-lara, s. corbalan-garcía departamento de bioquimica y biologia molecular. universidad de murcia, murcia, spain the c-terminal domain of the pro-apoptotic protein bax (bax-c) acts as a membrane anchor during the translocation to the membrane of this protein leading to programmed cell death. we have used static and mas-nmr techniques to show that the interaction of bax-c with membranes is modulated by the presence of a negatively charged phosphatidylglycerol. the width of the resonance peaks were considerably more increased by bax-c, in the presence of phosphatidylglycerol. bax-c substantially decreased the t 1 relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol but they were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. 13 c-mas-nmr showed that t 1 values were decreased when bax-c was incorporated and, when phosphatidylglycerol was also present, the decrease in t 1 affected considerably more to some carbons in the polar region. these results indicate that bax-c interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of bax-c with the membrane determines the membrane disposition of this domain. fluorescence spectroscopy showed that the trp residues of bax-c were located in a microenvironment more hydrophobic when phosphatidylglycerol was present. h. g. franquelim 1 , l. m. s. loura 2 , n. santos 1 , m. castanho 1 1 instituto de medicina molecular, univ. lisboa, portugal, 2 faculdade de farmácia, univ. coimbra, portugal since the efficacy of hiv fusion inhibitors was previously reported to be related to an ability to interact with membranes, we studied the interaction of the hiv fusion inhibitor sifuvirtide, a 36 aa negatively charged peptide, with lipid vesicles. since this peptide has aromatic residues, fluorescence spectroscopy techniques were used with no need for attached probes. results showed no significant interaction with both zwitterionic fluid phase and cholesterol-enriched membranes; however extensive partition to fluid phase cationic membranes were observed. in the dppc gel phase, however, an adsorption at the surface of these membranes was detected by using a differential quenching approach with lipophilic probes, as well as by fret. moreover, the interaction with gel phase membranes seems to be specific towards pc vesicles, since no significant interaction was retrieved for membranes composed by shingomyelin and ceramide. besides fluorescence, atomic force microscopy and zeta-potential were used to further investigate this issue. our results show a selectivity and specificity of the peptide towards rigid domains, where most of the receptors are found, and help explain the importance of the interaction with membranes in the improved efficacy of sifuvirtide compared to other fusion inhibitors, by providing a local increased concentration of the peptide near the fusion site on both cellular and viral membranes. the study of molecular dynamics at the single-molecule level with fluorescence far-field optics offers new detailed insights into scientific problems, especially in living cells. unfortunately, the resolution of common far-field techniques is limited to about 200nm in the lateral direction by diffraction. in recent years, several concepts such as stimulated emission depletion microscopy (sted) have been successfully applied to overcome the diffraction barrier by exploiting the photophysical properties of fluorescent labels. we present the combination of high resolution sted microscopy with different fluorescence fluctuation techniques providing the unique ability to study molecular dynamics with high spatial (<40nm) and temporal resolution (<1ms) in living cells. using fluorescence correlation spectroscopy (fcs) and general single-molecule analysis, we were able to explore single-molecule dynamics in up to 70-fold reduced focal volumes on two-dimensional samples such as lipid membranes with excellent signal-to-noise ratios. special attention is drawn to inhomogeneous lipid diffusion on the plasma membrane of living cells 1 . by extending the available spatial scale of standard single-molecule fluorescence far-field spectroscopy techniques, our experiments outline a new way of approaching scientific problems. modulation of the properties of membrane microdomains as a control mechanism in cellular physiology p. o'shea cell biophysics group, institute of biophysics, imaging & optical science, school of biology, university of nottingham, nottingham ng7 2rd, u.k. this presentation will outline the molecular-physical rationale of how membrane microdomains may modulate the behaviour of membrane receptor systems as a controlling mechanism in cell signaling. a number of external factors that modulate these properties will be indicated that have a bearing on controlling cellular behaviour. some of key questions will be considered such as the factors that control the assembly and disassembly of microdomains, the size and numberdensity of the microdomains and the lifetime that they exist within the membrane. the technical challenges that these questions identify will also be outlined with some possible solutions. throughout this presentation, correlations will be made between theory and experiment as well as between model membrane systems and real cellular systems. structural and dynamic properties of caveolin-1 and -2 fragments at the membrane interface c. le lan 1 , j. gallay 2 , m. vincent 2 , j.-m. neumann 1 , b. de foresta 1 , n. jamin 1 1 cea, ibitecs, sb2sm, & ura cnrs 2096, gif-sur-yvette, france, 2 ibbmc, université paris-sud, umr8619-cnrs, ifr115, orsay, france caveolins are major protein components of caveolae, microdomains of the plasma membrane involved in a large number of biological functions, including signal transduction, cholesterol homeostasis and transport. the consensus topological model of caveolin-1 includes a small central intramembrane region (102-134) flanked by two cytosolic amphiphilic domains (82-101 and 135-150) which probably constitute in-plane membrane anchors. we investigated the interaction of the cav-1(94-102) juxta-membrane segment with various membrane mimics, using fluorescence, cd and nmr. this segment partitioned better in dpc and in dm/anionic lipids micelles than in dm micelles and this partitioning was coupled with the formation of an amphipathic α-helix. this amphipathic helix was located in an average shallow position, in the polar head group region of the dpc micelle, as shown by fluorescence data and intermolecular noes, with the aromatic doublet w98-f99 probably pointing towards the inside of the micelle on average. the peptide encompassing the homologous sequence of cav-2 was also localized to the dpc micelle polar head group region, in which it adopted a more stable helical conformation than cav1(94-102) . these data brings experimental support for the role of this segment as an interfacial membrane anchor. resveratrol (trans-3,4',5-trihydroxystilbene) , a phytoalexin present in grapes and its analogue piceatannol (trans-3,4,3',5'-tetrahydroxystilbene) are biologically active compounds and possess potential chemopreventive and anticancer properties. the activity of resveratrol and piceatannol can be mediated by membrane effects since structure of lipid membrane domains may play an important role in cell signalling pathways. drugs interactions with dmpc bilayers was investigated using a combination of esr spectroscopy and differential scanning calorimetry. spin probes used in epr experiment were located in different part of lipid bilayer. study was performed at temperatures below and above phase transition temperature (t m ). epr spectra were simulated and displayed with ghost condensation method. the values of ϑ and ϕ (the main and asymmetry cone angles of wobbling spin probe, respectively) were taken for free rotational space parameter (ω) calculation. the decrease of ω values was observed in the presence of both compounds and the effect was more pronounced in lipid gel phase. order parameter and correlation time were also determined and presented in form of ghost patterns. using this approach the differential influence of studied compounds on membrane heterogenity was revealed. separating hydrostatic pressure from cellular strain: development of an in vitro model system r. sulley 1 , j. whatmore 1 , c. p. winlove 2 , a. shore 1 , j. tooke 1 , r. ellis 2 , k. gooding 1 1 peninsula medical school, 2 school of physics, university of exeter, uk endothelial cells (ecs) line blood vessels & are constantly subjected to haemodynamic and mechanical stresses and strains. these stimuli are known to influence ecs, modifying their morphology, intracellular signalling & gene expression. most reported systems exposing ec to mechanical forces in vitro alter pressure & strain simultaneously, making it impossible to distinguish the two potentially independent stimuli. this distinction is particularly relevant when examining the interaction of haemodynamic forces on microvascular ecs, which are exposed to low hydrostatic pressure but significant strains. this research aims to create an in vitro system that can independently examine the effects of pressure and strain, over a range experienced by ecs in the microvasculature. human ecs are seeded (12x10ˆ4cells/cmˆ2) on to the inner surface of compliant 4mm diameter tubing. which is mounted on a perfusion rig inside a sealed, fluid-filled chamber. a continuous sinusoidal cyclical strain of 5-10% is created by a pump attached to the external chamber. lumenal pressure is generated using two hydrostatic pressure heads. validation experiments show that pressure & substrate strain can be independently varied & controlled over a physiological range. the system is now being used to investigate the effects of pathophysiological haemodynamic abnormalities on ec function. membrane potential dynamics of living cells in response to femtosecond laser irradiation n. i. smith 1 , j. ando 2 , k. fujita 2 , s. kawata 2 1 photonics advanced research center, osaka university, suita, osaka 565-0871, japan, 2 dept of applied physics, osaka university, suita, osaka 565-0871, japan the ultrashort pulsed near-infrared femtosecond laser has had a large impact in biomedical research fields and in microscopy, where it has enabled new imaging methodologies. at high intensities, the focused beam of a femtosecond laser has been used to irradiate specific locations inside a cell, often beneath the cell membrane, exploiting the inherent penetration and localized absorption that comes from the multiphoton absorption physics. this has been applied in photobleaching, photouncaging, laser surgery and other experiments where the light is used not merely for observation but is instead an integral tool to interact with the dynamics of cells, to probe and perturb the cell condition. in this talk i will discuss biological and mechanical effects that can be generated by short exposures to femtosecond laser irradiation, such as calcium waves, membrane hyperpolarization, and cell contraction. this talk will concentrate on the changes in membrane potential that can occur when the cell is subjected to focused femtosecond laser beams. both depolarization and hyperpolarization of the membrane potential could be evoked, depending on the laser parameters and on the position of the laser focus. these results have implications for the use of laser beams in microscopy, optical gene transfection, and laser nanosurgery. in a recent study we showed that the melting behavior of supported lipid bilayers (slbs) on mica can be influenced by the solution ionic strength and by the slb preparation temperature [1] . by changing these parameters we could control the coupling between the two bilayer leaflets obtaining a coupled or decoupled melting behavior. thus, we could provide evidence that the slb model system is also suited for the study of lipid/protein interactions which had been questioned in the past. then we investigated the mutual interactions between the membrane lipids (pope:popg 3:1) and the kcsa potassium ion channel by studying kcsa proteins reconstituted in slbs. in particular, we studied the melting behavior of the slb and the ion channel distribution relative to the different membrane phases by temperature controlled atomic force microscopy (afm). by decreasing the temperature we found that the proteins underwent diffusion so to be excluded from the growing solid ordered regions. further, the ion channels tended to accumulate at the domain boundaries or they aggregated in the liquid disordered phase. both effects have been suggested to affect protein function. when we started from a low temperature at which the membrane was mainly in the solid ordered phase the membrane melting processes started in the vicinity of the included ion channels. we report fluorescence recovery after photobleaching (frap) measurements performed at variable spot radius for t7-egfp-hmop receptors on sh-sy5y neuroblastoma cells in the presence of ligands. two different agonists, damgo and morphine, caused markedly different changes to receptor diffusion as compared to the basal state. like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joint semi-permeable domains, but with smaller domain size and diffusion coefficient. this effect was inhibited by pertussis toxin, suggesting that this dynamic behaviour is associated with early steps of signaling. in the presence of damgo, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. hypertonic sucrose buffer suppressed this effect which we attribute to receptor entry into clathrin-coated pits. it is likely that the observation of distinct receptor dynamics in the presence of damgo and morphine involves the agonist-selective phosphorylation of the receptor. the alteration of the physiological transcriptional program is one of the constant features of cancer cells. however, the characterization of the chromatin changes at single-gene level requires going beyond the diffraction limit affecting conventional fluorescence microscopy. the ability of molecular biology techniques to obtain a detailed view of the chromatin status at a sub-promoter resolution has to pay instead the price of averaging over a cell population. we report here the application of an approach based on high-resolution cytometry, chromatin immuno-precipitation and transcriptional profiling (dna microarray) for the characterization of the transcriptional and chromatin changes induced by the oncogenic transcription factor pml/rarα. the presentation will focus on the imaging protocol employed to observe the effects on the chromatin status and the extent of the deregulation induced on transcriptional activity in acute promyelocytic leukemia cells. this multiple-approach examination provides a further step towards the comprehension of the hierarchy of chromatin modifications leading to the establishment of a malignant transcriptional program. dna is rigid negatively charged polymer and in solution exists in extended conformation. i n vivo, volume occupied by dna must be reduced to fit to tiny space of cell nucleus. to condense dna, dna-dna electrostatic repulsion must be cut off that is achieved by interaction with cationic ligands. in binding to dna, oligocations compete with salt cations (k + , na + , mg 2+ ). description of salt dependence of oligocation-induced dna condensation is still lacking. we studied dna condensation by model oligocations, ε-oligo(l-lysines), with variation of charge from +3 to +31. combination of light scattering, uv-monitored precipitation assay and isothermal titration calorimetry allowed covering wide range of dna (c dna ) and salt (c kcl ) concentrations. salt dependence of dna condensation efficiency of the ligand, ec 50 (ligand concentration at the transition midpoint) displays two regimes: salt-independent at low c kcl and salt-dependent at higher c kcl (steep increase of ec 50 with c kcl ). simple formula describing ec 50 as function of ligand charge, c dna and dissociation constant of ligand-dna complex (k d ), was proposed. in the salt-independent regime ec 50 is defined by c dna . salt-dependence of ec 50 is rooted in the variation of k d with c kcl earlier described in ligand-dna binding studies. importance of our findings for description of chromatin is discussed. interaction between proteins from linker region of nucleosome in presence/absence of dna in solution i. b. kipenko 1 , e. v. chikhirzhina 2 , a. m. polyanichko 1 1 faculty of physics, saint-petersburg state university, russia, 2 laboratory of cell biochemistry, institute of cytology, ras, saint-petersburg, russia interactions in the linker region of the nucleosome play a key role in the structural organization of the chromatin. the most fascinating and least understood is the interplay between non-histone chromatin protein hmgb1 and a linker histone h1. it is known that both h1 and hmgb1 bind the linker region of the dna in vivo. however it is still a matter of debate as to whether these proteins assist each other or compete upon binding. the main attention in this work is paid to the investigation of the interactions between the hmgb1 and h1 proteins in physiological environment. using circular dichroism (cd) spectroscopy we have studied the interactions between hmgb1 and h1 at various hmgb1/h1 ratios (r). it has been shown that there is a cddetectable interaction between the proteins h1 and hmgb1 at r < 1. we have demonstrated that the interaction between these proteins results in changes of their secondary structure. cd indicates that the structural impact of the unordered fragments decreases while the net α-helicity of the proteins increases upon the interaction. we have also shown that large higher order structures are formed in solution. in this work we have also discussed the dna-binding properties of the hmgb1 and h1 proteins. the work was supported by a rfbr grant (09-08-01119) and the government of staint-petersburg. t. u. rodionova 1 , a. m. polyanichko 1 , v. i. vorob'ev 2 1 faculty of physics, saint-petersburg state university, russia, 2 institute of cytology of the russian academy of sciences, saint-petersburg, russia hmgb1 is a nonhistone chromosomal protein. data regarding the structure of the hmgb-proteins obtained so far are rather different. thermodynamic experiments reveal predominant α-helical structure of the proteins only at temperatures below +5 • c, i.e. under physiological conditions they are mainly disordered. despite a lot of experimental data biological role of hmgb1 still remains unclear. it is believed that the proteins perform structural functions in chromatin and participate in various regulatory processes in cell. using circular dichroism spectroscopy and dna melting analysis we have shown that hmgb1 changes its structure upon binding to dna. it was shown that at room temperature only about 25% of amino acid residues form α-helices, while in dna-hmgb1complex the degree of the α-helicity of the protein increases to approximately 50%. based on the data obtained we estimate the size of hmgb1 binding site as 80-100 b.p. we have also demonstrated that despite of strong dna-bending properties of hmgb1 its binding to dna results in increase of the double helix termostability. the authors are grateful for the financial support from the russian foundation for basic research (grants 09-08-01119, 07-04-01072) and the government of saint-petersburg. structural organization of supramolecular complexes of dna with chromosomal proteins hmgb1 and h1 a. m. polyanichko 1 , h. wieser 2 1 faculty of physics, saint-petersburg state university, russia, 2 dept. of chemistry, university of calgary, canada a combination of uv and ir absorption and circular dichroism spectroscopy together with atomic force microscopy was applied to investigate the structure and formation of large supramolecular dna-protein complexes. this combination of techniques was used to overcome limitations of uv-cd spectroscopy due to considerable light scattering in such solutions. based on the analysis of ftir and uv circular dichroism spectra and afm data the interaction of dna with highmobility group non-histone chromatin protein hmgb1 and linker histone h1 was studied. it is believed, that hmgb-domain proteins perform both structural and regulatory functions in chromatin. however, the particular mechanisms of it functioning remain unclear/ our data show that histone h1 facilitated binding of hmgb1 to dna by interacting with the sugar-phosphate backbone and binding of asp\glu amino acid residues of hmgb1. acting together, hmgb1 and h1 stimulated the assemblage of supramolecular dna-protein structures. the organization of the ternary complexes is modulated by the interactions between hmgb1 and h1 molecules. the dna-proteins interactions in the presence of metal ions were different, causing prominent dna compaction and formation of large intermolecular complexes. the work was supported by rfbr (grant 09-08-01119). biophysical properties and mechanisms of phage dna ejection t. mdzinarashvili 1 , m. khvedelidze 2 , a. ivanova 1 , t. partskhaladze 1 , n. shengelia 1 1 i. javakhishvili tbilisi state university, tbilisi, georgia, 2 institute of molecular biology and biophysics, tbilisi, georgia to determine the requirements for phage adsorption on bacterial cell and for the realizing resources of following dna ejection thermodynamic and hydrodynamic methods were employed. the temperature, bacterial membrane fragments and receptors had been chosen as such external factors. the phages with short and long tail, both contractile and noncontractile have been studied. our viscometric studies of the phage dna ejection induced by receptor by the example of t5 phage and its receptor fhua have shown that the minimum protein-to-phage ratio necessary for complete dna release is 300 to 1. the viscometric study of ddvi phage dna ejection induced by membrane fragments obtained from its host cells has shown that the environmental conditions play significant role in ejection process. both methods show that the thermally induced phage dna ejection for all investigated by us phages have shown that this process is nonenthalpic. finally from our experimental results we conclude that the start of the dna ejection process from the phage particle occurs without additional energy from either a physical or chemical source. we thank gnsf for the financial support. nitroxides induce apoptosis through caspase-3 activation and collapse of mitochondrial potential k. matczak 1 , a. koceva-chyla 1 , k. gwozdzinski 2 , z. jozwiak 1 1 department of thermobiology, 2 department of molecular biophysics, university of lodz, lodz, poland nitroxides are new class of antioxidants that have been proved to show high reactivity toward free radicals. they act as superoxide dismutase mimics dismutating superoxide anions, but can also exert pro-oxidative properties. in view of their possible dual activity nitroxides could be of great importance in medicine. we have investigated pro-apoptotic activity of pyrroline and pyrrolidine nitroxides pirolid (pd) and pirolin (pl) in human breast cancer cells. in cancer, it is the failure of malignant cells to undergo apoptosis that is crucial. using microplate fluorescence methods, we estimated kinetics of changes in mitochondrial transmembrane potential and caspase-3 activity in breast cancer cells mcf-7 treated with pirolin or pirolid. these features are connected with induction of apoptosis in some type of cancer cells. we observed steady-state increase in caspase-3 activity up to 12 h of postincubation that was followed by a decrease in the enzyme activity at 24 h. caspase-3 activation was considerably greater in cells treated with pirolid. both nitroxides also caused notable decrease in mitochondrial transmembrane potential, which suggest that they can induce apoptosis in breast cancer cells through mitochondrial pathway. o. chernyavskiy 1 , l. vannucci 2 , p. bianchini 3 , f. difato 4 , l. kubínová 1 1 dept. of biomathematics, inst. of physiology, as cr, prague, czech republic, 2 dept. of immunology, inst. of microbiology, as cr, prague, czech republic, 3 lambs, dept. of physics, university of genoa, italy, 4 dept. of neuroscience and brain technologies, the italian institute of technology, genoa, italy the second harmonic generation (shg) imaging along with confocal laser scanning microscopy in reflectance mode can be applied to imaging unstained tissues in vivo, so it can be considered as a fast and non-invasive tool for in vivo studies. murine b16f10 melanoma cells after subcutaneous inoculation in syngeneic mice were let to develop into tumor up to 15-20 mm in diameter. microscopic images were taken before and after microwave hyperthermia treatment (mwht). the microscopic images were acquired by 1-photon imaging in reflectance mode, shg imaging and 2-photon imaging of tissue autofluorescence. the evaluation of changes in the images after mwht of the tumor demonstrated changes in the architecture and organization in both the tumor capsule and tumor mass. the presented study was supported by the academy of sciences of the czech republic (grant iaa500200510, institutional research concepts no.av0z50200510, av0z50110509), and ministry of education, youth and sports of the czech republic (research program lc06063). intracellular delivery and fate of peptide-capped gold nanoparticles: towards cellular biosensors y. cesbron 1 , v. sée 1 , p. free 1 , p. nativo 1 , d. g. spiller 1 , m. r. h. white 1 , m. brust 1 , b. lounis 2 , r. lévy 1 1 liverpool institute for nanoscale science, engineering and technology, liverpool, uk, 2 université bordeaux i / cnrs, bordeaux, france gold nanoparticles (nps) have extraordinary optical properties that make them very attractive single molecule labels. although understanding their dynamic interactions with biomolecules, living cells and organisms is a prerequisite for their use as in situ sensors or actuators. while recent research has provided indications on the effect of size, shape, and surface properties of nps on their internalization by living cells, the biochemical fate of nps after internalization has been essentially unknown. here we show that peptide-capped gold nps enter mammalian cells by endocytosis. we demonstrate that the peptide layer is subsequently degraded within the endosomal compartments through peptide cleavage by the ubiquitous endosomal protease cathepsin l. preservation of the peptide layer integrity and cytosolic delivery of nps can be achieved by a combination of cathepsin inhibition and endosome disruption. this is demonstrated using a combination of distance-dependant fluorescence unquenching and photothermal heterodyne imaging. these results prove the potential of peptide-capped gold nps as cellular biosensors. current efforts focus on in-vivo labeling of nps, nanoparticle-based real-time sensing of enzyme activity in living cells, and the development of photothermal microscopy for single nanoparticle imaging in living cells. towards intravital two photon microscopy study of lymphocytes mobility in lymphonodes m. caccia 1 , l. sironi 1 , m. collini 1 , i. zanoni 2 , t. gorletta 2 , m. di gioia 2 , g. francesca 2 1 dipartimento di fisica, università di milano bicocca, italy, 2 dipartimento di biotecnologie e bioscienze, università di milano bicocca, italy during the last 30 years the edge between optical fluorescence based microscopy and the world of bio-medical research has become thinner and thinner and two photon laser scanning microscopy (tplsm) is one of the most powerful tool for immunological and medical research. one of the most limiting step in intravital microscopy is the preparation of the animal model and the number of animals to be sacrificed to get good statistics. alternative routes must be searched. we employ tplsm to explanted lymphnodes. two photon excitation and a non descanned detection mode allow to increase, respectively, the excitation and detection efficiency while the explanted organs are kept very close to the condition they experience in live animals by means of an home-made temperature controlled box surrounding the microscope and a system for the flux of physiological fluids. experiments performed on explanted lymphonodes, kept under constant flux of co 2 -o 2 saturated buffers and at 36 o c, agree with literature for what concern t-cell homing and motility. this seems to confirm that t-cells behavior in explanted organs mantained in physiological conditions is very similar to that observed in live animals. we then believe that our tplsm microscope would allow to study cell behaviors in in-vivo with high efficiency and a little technical effort. a generalized quantitative frap method with no restriction on the size of the photobleached area heterochromatin protein 1 in dna damage response -recruitment or dissociation from repair sites? j. w. dobrucki division of cell biophysics, faculty of biochem. biophys. and biotechnol., jagiellonian university, kraków, poland we studied recruitment of dna repair proteins to damage sites in live cells, by microscopy approaches, using a new method of inflicting local, sublethal damage in nuclei of live cells. oxidative damage, which was inflicted by exciting dna-intercalated ethidium with focused green light, triggered recruitment of base excision repair enzymes. surprisingly, an epigenetic regulator, heterochromatin protein 1 (hp1) (zarebski et al., 2009 ) was recruited to damage as well. hp1 is a constitutive component of heterochromatin, and plays an important role in transcriptional repression and regulation of euchromatic genes, however it was not known to be required for repair of oxidative damage. the finding of hp1 recruitment is particularly puzzling, since in another study hp1 was shown to dissociate from chromatin as a result of dna damage (ayoub et al. 2008) . technical aspects of live cell imaging that may explain these contradictory results will be discussed. in this presentation, we report a method for determining both the presence and the stoichiometry of protein complexes at pixel resolution and apply it to disassembling focal adhesions. the method is derived from fluorescence fluctuation methods that have single molecule sensitivity and is based on our previously described n&b (number and brightness) method that measures the number and brightness (aggregation state) of fluorescent molecules in every pixel of a confocal microscope image. the new method exploits the correlation of fluorescence amplitude fluctuations for two colors and detects the presence of molecular complexes and their stoichiometry. while the original n&b method was developed for one color, i.e., a single molecular species, the new method, ccn&b, extends the analysis to two colors and introduces the concept of cross-variance. this method is similar in concept to the two-color pch analysis. however, the covariancebased ccn&b method also generates pixel resolution maps of protein complexes and can be used on commercial confocal microscopes. the method is highly sensitive and has relatively high temporal resolution. we have applied this method to adhesion complexes in cells. in addition of their structural role, to link the extracellular substratum to actin filaments, they also serve as signaling centers that regulate many cellular processes including their own assembly and turnover, migration, gene expression, apoptosis, and proliferation. spatio-temporal analysis of membrane lipid remodeling during phagocytosis s. de keijzer 1 , d. kilic 1 , c. g. figdor 1 , s. grinstein 2 , a. cambi 1 1 department of tumor immunology, radboud university of nijmegen, nijmegen, the netherlands, 2 department of biochemistry, university of toronto, toronto, canada the constant threat posed by pathogens and cell debris is tackled by phagocytosis, the process through which cells engulf and destroy dangerous material. the signaling, targeting and trafficking during phagocytosis is dependent on cytoskeleton rearrangements and membrane remodeling. it is becoming increasingly evident that lipids play an important role and can affect the phagocytic response. they assemble microdomains which can act as signaling platforms and confer charge and curvature to the membrane surface promoting electrostatic attraction and retention of proteins. little is known about the mechanism(s) regulating membrane lipid remodeling during phagocytosis. here we used fluorescently labeled biosensors based on k-ras and h-ras proteins to obtain spatio-temporal information on phagosomal membrane lipid remodeling during fcreceptormediated phagocytosis. the results show that cell activation by cytokines modulates the kinetics of anionic lipids thus affecting the membrane charge and the recruitment of cytosolic proteins to the phagosomal membrane. our data emphasize the fundamental role of lipids in the generation and transduction of signals in phagocytosis, and we believe this can be extrapolated to many important processes in a cell. fluorescence correlation spectroscopy (fcs) is a useful technique for characterizing the mobility and concentration of fluorescent molecules both in vitro and in vivo. we utilize two-photon fcs to characterize the concentration and mobility of fluorescent molecules within living cells of bacillus subtilis. autocorrelation functions were measured in bacteria expressing green fluorescent protein (gfp) under the lac promoter in both nutrient rich and nutrient poor culture medium. although considerable heterogeneity was evident from cell to cell, on average, both intracellular concentration and mobility were found to be dependent upon culture medium. we also investigated bacteria expressing gfp under control of native promoters for involved in the regulation of the carbon metabolic cycle in bacillus subtilis. the gfp concentration, which should be related to promoter activity, was investigated for single cells and cell populations under different metabolic conditions. some photobleaching was observed during the course of the measurements as a decrease in the average fluorescence intensity. this is due to the small size of the bacteria (∼1 fl) and low basal expression levels of gfp (∼100 nm) in the absence of iptg. methods to take this into account during data analysis are discussed. a. fassio 1 , s. congia 2 , p. baldelli 2 , f. benfenati 2 1 dimes, university of genoa, genoa, italy, 2 dnbt , iit, genoa, italy several mutations have been discovered in the synapsin i (syn) gene in families with epilepsy, but the mechanism inducing the epileptic phenotype is unknown. syn is a protein associated with synaptic vesicles (svs) that control sv trafficking and neurotransmitter release. the syn mutations subject of this study are a non sense (ns-1) and of two missense (ms-2, ms-3) . syn knockout (ko) hippocampal neurons were transfected with the either wild type (wt) or mutated syns. all syns presented a common punctate pattern of expression at the level of axonal arborizations but, while ns-1 syn targeted to synapses as wt-syn, ms-2 and ms-3 syns reached the presynaptic terminal less efficiently. we next set up a live cell imaging experiment using synaptophysin-phluorin and evaluated the effects of ns-1, ms-2 and ms-3 syns on the size of sv pools. restoring wt-syn in syn i ko terminals led to a increase of all sv pools. restoring ms-2 and ms-3 syns resulted in a phenotype not significatively different from syn i ko background whereas restoring ns-1 syn caused a decrease of all sv pools as compared either with wt-syn or syn i ko background. these data suggest an alteration in the subcellular distribution and function of syn in patients carrying ms-2 e ms-3 mutation and a more severe effect on synaptic activity in patient carrying ns-1 mutation. imaging dynamics of dc-sign at the plasma membrane of hiv-1-stimulated dendritic cells o understanding how viruses interact with host receptors is essential for developing new antiviral strategies. dendritic cells (dcs) can efficiently capture and take up hiv-1 through multiple attachment factors, such as the c-type lectin dc-sign. however, the initial interactions between hiv-1 and this receptor on dcs are not fully understood yet. in this work, we have used single-molecule epi-tirf microscopy in combination with fluorescently labelled hiv-1 virus like particles (vlps) and dc-sign-specific antibodies to image the dynamic interaction between hiv-1 and dc-sign nanoclusters at the plasma membrane of dcs. by tracking individual trajectories of dc-sign on the cell surface of living dcs we have found heterogeneity in the modes of motion of dc-sign: some clusters are immobile whereas others move very quickly, with a few ones showing a directed motion on the cell membrane. to investigate how such motion might be correlated to dc-sign function as virus attachment factor, we have developed glass platforms functionalized with hiv-1 vlps to locally stimulate living dcs. these virus platforms have allowed us to measure dc-sign diffusion rates and motion modes over the cell surface of stimulated dcs and represent a powerful tool for studying the dynamic interactions between hiv-1 and the dc membrane. a. esposito 1 , t. tiffert 2 , j. mauritz 1 , s. schlachter 1 , j. n. skepper 2 , v. l. lew 2 , c. f. kaminski 1 1 dept. of chemical engineering and biotechnology, univ. of cambridge, u.k., 2 dept. of physiology, development and neuroscience, univ. of cambridge, u.k. plasmodium falciparum (pf ) causes the most lethal form of malaria in humans. early research exposed two paradoxes: 1) during its intraerythrocytic cycle pf permeabilizes the host cell so much that a comparably permeabilized healthy red blood cell (rbc) would lyse prematurely, and 2) pf digests far more hemoglobin than needed for its metabolism. a model of the homeostasis of a pf infected rbc suggested a common explanation of both puzzles: excess hemoglobin digestion is required to reduce the colloidosmotic pressure within the host cell thus ensuring its osmotic stability to the end of the pf asexual cycle. we investigated these predictions with direct measurements of [hb] and volumes of parasitized rbcs. reliable volume and morphological data was obtained by confocal microscopy and quantitative surface reconstruction. furthermore, we developed a new fret-based method to measure hemoglobin molecular crowding by exploiting the reduction in fluorescence lifetime of a donor fluorophore loaded in the rbc cytosol. fret imaging techniques are powerful tools for probing the biophysics of living cells. these tools provided a first validation of the colloidosmotic hypothesis and a deeper understanding of the homeostasis of the intraerythrocytic stage of pf. hiv-1 assembly and release occur at the plasma membrane of infected cells and are driven by the gag polyprotein. using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated assembly of fluorescently labeled hiv-1 at the plasma membrane of living cells with high time resolution. gag assembled into discrete clusters corresponding to single virions. after their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1-2 hours. using a photoconvertible fluorescent protein, we determined that assembly was nucleated by membrane bound gag molecules, while both membrane-bound and cytosol derived gag polyproteins contributed to the growing bud. assembly kinetics were rapid and three phases are observed. in phase i, the number of gag molecules at a budding site increases following a saturating exponential with a rate constant of ∼ 5 x 10 −3 s −1 . hence, gag assembly is complete in ∼ 200 s. in phase ii, a plateau in fluorescence intensity is observed with no exchange of gag protein. the fluorescence intensity decays in phase iii. this decay, in some cases, corresponds to the release of a virion. the time scale from the onset of assembly to release of extracellular particles was measured to be ∼1,500+/-700 s. fast 3d chromatin dynamics studied in living yeasts using a novel lab on chip technology h. hajjoul 1 , m. dilhan 1 , i. lassadi 2 , k. bystricky 2 , a. bancaud 1 1 laas-cnrs, université de toulouse, france, 2 lbme, cnrs umr 5099, toulouse, france we present a novel lab-on-chip technology for 3d particle tracking yeast abased on v-shaped mirrors, which are used to observe fluorescent specimens from multiple vantage points, providing stereo-images that can be recombined for 3d reconstruction. our technology is based on v-shaped mirrors, which are fabricated by wet etching of silicon wafers, and used as optical and fluidic components. after rigorous optical optimization of the device in vitro, the lab-on-chip is applied to study chromatin dynamics in vivo using budding yeasts as a model system. yeasts cells are visualized using gfp fused to the associated repressor protein. we confirm earlier observations that telomeric sequences, i.e. located close to chromosome ends, accumulate at the nuclear periphery, whereas genes found midway along chromosome arms are mostly present in the nuclear lumen. the dynamics of these sequences is followed in 3d with an unpreceded temporal resolution of 10 ms, showing that chromosome dynamics is non linear in the short time regime and switches to a linear regime at larger timescales. notably, this behavior is reminiscent of universal responses observed in polymer solutions, and is related to the confinement and the structure of chromosomes. this technique shows a great potential for studying dynamic processes in small living organisms. retrovirus induced remodeling of the host cell actin architecture m. gladnikoff, i. rousso department of structural biology, weizmann institute of science, rehovot, israel retrovirus budding is a key step in the virus replication cycle. yet, despite substantial progress in the structural and biochemical characterization of retroviral budding, the underlying physical mechanism remains poorly understood, primarily due to technical limitations preventing visualization of bud formation in real time. using atomic force-, fluorescence-and transmission electron microscopy we find that both hiv and moloney murine leukemia virus (mlv) remodel the actin cytoskeleton of their host cells and utilize the forces it generates to drive their assembly and budding. highly dynamic actin-filamentous structures which varied in size over the duration of budding appeared to emanate from the assembled virion. these actin structures assemble simultaneously or immediately after the beginning of budding, and disappear as soon as the nascent virus is released from the cell membrane. analysis of sections of cryo-preserved virus infected cells by tem reveals similar actin filaments structures emerging from every nascent virus. substitution of the nucleocapsid domain implicated in actin binding by a leucine-zipper domain resulted in budding of virus-like particles that was not accompanied by remodeling of the cell's cytoskeleton. notably, budding of viruses carrying the modified nucleocapsid domains was an order of magnitude slower than that of the wild type. the results of this study show that retroviruses utilize the cell cytoskeleton to expedite their assembly and budding. t. gensch institute of structural biology and biophysics (isb-1) cellular biophysics, research centre jülich, germany the determination of ion concentrations in cells -in particular in neurons -is very important for understanding cell function and life. ca 2+ is an ubiquitous messenger in almost all cell types, cl − has several roles, e.g. plays an important role in some neuronal signaling pathways including olfaction, nociception and vision. fluorescence lifetime imaging (flim) is of advantage over intensity based fluorescence microscopy, when comparisons between micro-domains of one cell or between different cells of one cell type are performed. several (organic chromophores and fluorescent protein based) ca 2+ -and cl − -sensors have been tested in culture cells with respect to their applicability in flim studies. the ca 2+ -and cl − concentration in rodent olfactory sensory neurons, dorsal root ganglion neurons and neurons of the retina is investigated by time-resolved flim with two-photon excitation. viscosity is one of the main factors which influence diffusion in condensed media. in a cell viscosity can play a role in several diffusion mediated processes, such as drug delivery and signalling. previously, alterations in viscosity in cells and organs have been linked to malfunction; however, mapping viscosity on a single-cell scale remains a challenge. we have imaged viscosity inside individual cells using novel fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent. 1, 2 this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to 100× higher than that of water. 1 we have also shown that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt). 2 we have demonstrated the effect of such viscosity increase on intracellular reactions by directly monitoring the rates of formation and decay of a short lived toxic intermediate, crucial in pdt, called singlet molecular oxygen, in light perturbed cells. characean algae, close relatives of higher plants, represent a convenient model for studying the effect of propagating electrical signals, action potentials (ap) on photosynthesis. illuminated chara corallina cells produce coordinated spatial patterns of chlorophyll fluorescence (chl fl) and extracellular ph. photosynthesis is higher in the cell regions adjacent to acid zones compared to alkaline zones. under physiological conditions, the electrically induced ap differentially and reversibly suppresses photosynthesis in the alkaline and acid regions. in this work we examined the effects of an artificial psi acceptor, methyl viologen (mv), on chl fl with imaging-pam technique. mv is a divalent cation and poorly permeates through biological membranes. the presence in the medium of mv had no effect on fl as well as on p700 + absorbance signals until the application of a single excitatory stimulus. once an ap was generated in the presence of mv, it induced irreversible inhibition of native (nadpdependent) electron flow and a strong non-photochemical fl quenching all over the cell. this indicates that ap redirects electron flow from the main pathway to the artificial acceptor. we concluded that ap generation opens access for permeation of mv from the medium to the chloroplast stroma across two membrane barriers, plasmalemma and chloroplast inner membrane. we suggested that mv might enter chara cell via plasmalemma ca 2+ -channels activated during ap. evaluation of cell damage after photodynamic and sonodynamic treatment h. kolarova, k. tomankova, s. binder, p. kolar, r. bajgar, m. strnad department of medical biophysics, faculty of medicine and dentistry, palacky university, hnevotinska 3, 775 15 olomouc, czech republic photodynamic therapy (pdt) and sonodynamic therapy (sdt) is a new, combined therapy for treating cancer. the basis of the therapy is to administer a small amount of photosensitizer and sonosensitizer, which are selectively taken up by cancer cells, and then expose the body to light and ultrasound to activate these sensitizers. when the sensitizers absorb light of an appropriate wavelength, it may cause their excitation with subsequent energy transfer to oxygen; the oxygen then becomes highly reactive in cancer cells and produces reactive oxygen species (ros). the resulting damage to organelles within malignant cells leads to tumor ablation. sdt uses an agent that is sensitive to ultrasound, allowing deeper penetration and destruction of abnormal cells. changes in human melanoma cells were evaluated using fluorescence microscope and atomic force microscopy.we focused on obtaining pictures of the topography and pictures involving elastic properties of cell surface. the production of ros was investigated with the molecular probe cm-h2dcfda, and morphological changes in cells were evaluated using fluorescence microscope. the quantitative ros production changes in relation to phthalocyanine concentration, irradiation doses and ultrasound intensity were measured by a fluororeader. activity correlation imaging: visualizing function and structure of neuronal populations s. junek 1 , t.-w. chen 1 , m. alevra 1 , d. schild 2 1 department of neurophysiology and cellular biophysics, university of göttingen, germany, 2 dfg research center for molecular physiology of the brain (cmpb), university of göttingen, germany understanding a neuronal network relies on knowledge about both the function and the structure of its neurons. while he simultaneous observation of hundreds of neurons is possible by densely staining brain tissue with functional dyes, the low contrast of these stainings does not allow the identification of neuronal processes from the raw fluorescent images. however, as neurons are known to exhibit complex temporal patterns of activity, fluctuations in signal intensity over time could be exploited to generate contrast in densely stained tissue. we demonstrate that the uniform calcium signals within individual neurons of the olfactory bulb can be exploited to visualize the morphology and projection patterns of these cells in tissue slices. as different neurons exhibit distinct time patterns, it is possible to generate a highcontrast multi-color visualization of the network's active neurons, solely based on the specificity of temporal fluctuations of a single-color calcium dye. it is thus possible to use other spectral channels for additional labelling, for example using cell-type or protein specific markers. the ability to map function and structure of neuronal populations online opens up a number of intriguing applications, such as selecting cells or cell pairs with certain functional or projection profiles for targeted recordings, ablation or stimulation. -live cell imaging investigation of the dynamics of redox elements in live cells by using fluorescence ratio microscopy g. maulucci 1 , g. pani 2 , v. labate 2 , m. mele 2 , e. panieri 2 , m. papi 1 , g. arcovito 1 , t. galeotti 2 , m. de spirito 1 1 istituto di fisica, università cattolica s. cuore, roma, italy, 2 istituto di patologia generale, università cattolica s. cuore, roma, italy oxidative stress or signaling events can affect cellular redox environment, which act simultaneously as regulator and indicator of key cellular functions in both physiological and pathological settings. by using a redox-sensitive protein (rxyfp), employed ratiometrically, it is possible to generate high resolution redox maps of cells. the spatial distributions of oxidized and reduced elements have been discriminated in human embryonic kidney cells by the deconvolution of the histograms of redox maps. by transfecting cell with glutaredoxin v (grx-v), a significant shift towards more reduced state with respect to that recovered from non-transfected cells is observed. despite such large differences, a common behaviour in the spatial distribution of reduced and oxidized couples can still be observed: oxidized population shows a pronounced localization on the cell borders, near the plasma membrane, while reduced population appears as a collection of well separated spots homogeneuosly distributed thoroughly the inner part of the cell with a mean dimension of 2 um. furthermore we observe that the role of grx-v consists in causing a shift towards reduced values of the highly reduced region, while leaving unaltered the redox-balance of the intracellular side of the plasma membrane. fluorescence resonant energy transfer (fret) is a popular approach to studying molecular interactions. fluorescence lifetime imaging (flim) provides a robust read-out of fret but has largely been restricted to fixed cells owing to relatively long acquisition times. we present a highspeed wide-field multidimensional fluorescence microscope for flim fret in live cells on second timescales for high content analysis and studying fast dynamics. the system uses a nipkow disc for optical sectioning and a time-gated image intensifier for wide-field flim. a spectrally selected supercontinuum excitation source facilitates versatile realtime flim of live cells transfected with fluorescent proteins. for cell signalling studies we have developed a multiplexed fret approach. ras activation at the plasma membrane is demonstrated using flim to read out tagrfp-raf rbd interaction with mplum-h-ras. simultaneously, a spectral ratiometric read-out is used for a second fret (cfp/yfp cameleon) probe to monitor the downstream calcium flux. to further develop multiplexed fret as a tool for imaging multiple components of cell signalling networks, we are working to include polarisation-resolved imaging for steady-state and time resolved fluorescence anisotropy measurements. monitoring gene expression via novel nucleic acid and delivery methods k. lymperopoulos 1 , c. spassova 1 , a. seefeld 1 , h. s. parekh 2 , d. p. herten 1 1 bioquant institute,heidelberg university, germany, 2 school of pharmacy, university of queensland, australia application of single-molecule and high-resolution fluorescence methods to monitor gene expression in living cells increase the demand on novel probes and delivery methods.they require fluorophores with high photostability and quantum yield and highly-efficient delivery methods that ensure the minimum interference with cell processes such as metabolism and signal transduction. we used a novel class of dendrimers with varying generations and branching factors and different number of positive charges due to different moieties and functional groups.these different properties were tested for their efficiency to transfect eukaryotic cells with fluorescent oligonucleotides (odns).different parameters (temperature,concentration of dendrimers, ratio of dendrimers and odns) were evaluated and optimised.we utilised these established optimal conditions to deliver a modified concept of smartprobes to mammalian cells targeting mrnas involved in signal pathways.we tested different mrna targets and we optimised the fluorescence signal by varying a range of parameters,namely the fluorescent label and the intrinsic properties of the smartprobe (length of the loop and stem, conformation and number of guanosines).in the near future,we plan to use these probes for monitoring gene expression levels using diffusion imaging microscopy. we present a novel near-field scanning microwave microscope (smm) capable of providing surface impedance measurements of samples with nanometric resolution. the instrument is the integration of a microwave vector network analyzer (vna) and a scanning probe microscope (afm/stm). a key point is that our software, controlling and synchronizing both the instruments, creates simultaneously images of the sample at several frequency points. this can be used to extract several features of the sample depending on the frequency. moreover, close frequencies show the same features, added to random noise. exploiting this redundancy of information, we have achieved remarkable results. we have been working on the optimization of this system for biological applications, to detect functional characteristics of cells generating a variation of their dielectric properties. this instrument offers the possibility of performing local impedance measurements on a single live cell and, if correctly calibrated, it provides also quantitative information (e.g. absolute measurements of membrane permittivity). the system was demonstrated to work on saccharomyces cerevisiae. a better model for a full test of the potentialities of the new technique is given by excitable cells, characterized by a greater variability of dielectric properties. a challenging objective could be directly imaging ion channels. hiv-1 to efficiently complete a replication cycle has to integrate its genome into the host cellular dna.after hiv-1 enters target cells,neosynthesized viral dna forms along with other proteins the pre-integration complex (pic).pics are then transported into the nucleus where integration,catalyzed by the viral integrase,takes place.hiv-1 viral particles engineered to incorporate integrase fused to egfp have proven effective to study pics within nuclei of infected cells.in this study we report the live imaging analysis of nuclear pic dynamics obtained by time-lapse microscopy.intranuclear trajectories of in-egfp-labeled pic were collected in three dimensions and examined by both mean squared displacement (msd) and cage diameter (cd) analysis.in cd the maximum distances measured between two positions occupied by a pic in a time window of 2 minutes were calculated while in our msd analysis 5-minute long trajectory segments were considered.remarkably,msd revealed the presence of an underlying active transport mechanism.to test the possible role of actin filaments,pic nuclear trafficking was analyzed in cells treated with latrunculin b (actin polymerization inhibitor).preliminary results suggest that the disruption of actin function impairs the active nuclear movement of pics. second harmonic generation microscopy reveals sarcomere contractile dynamics of cardiomyocytes n. prent 1 , c. a. greenhalgh 1 , r. o. cisek 1 , j. aus der au 2 , s. elmore 3 , j. h. van beek 3 , j. squier 2 , v. barzda 1 1 department of physics and institute for optical sciences, university of toronto, toronto, canada, 2 department of physics, colorado school of mines, golden, usa, 3 department of molecular cell physiology, vrije universiteit, amsterdam, netherlands cardiomyocytes, like other striated muscles, exhibit strong inherent second-order nonlinear optical properties that make them exemplar for live cell dynamic studies with second harmonic generation (shg) microscopy. laser scanning shg microscopy with an incident wavelength around 1µm, enables fast imaging for extended periods of time with negligible tissue damage. strong shg signal originates from the anisotropic (a-) bands which are comprised of regularly arranged myosin molecules, while actin molecules, the main constituent of the isotropic (i-) bands, produce negligible shg. consequently, the alternating bands along the myofibril are clearly visualized, therefore, enabling the determination of individual sarcomere lengths and the study of sarcomere length dynamics during macro-scale contractions. shg intensity is shown to positively correlate to sarcomere length, which leads to the development of real-time inherent force sensors for in vivo myocytes. rich sarcomere dynamics can be observed during myocyte contraction, which can be used for medical diagnostic purpose of muscular degenerative diseases. imaging cellular communication in insulin secretion ex vivo and in vivo d. w. piston vanderbilt university, nashville, tennessee, u.s.a. the islet of langerhans is the functional unit responsible for glucose-stimulated insulin secretion (gsis), and thus plays a key role in blood glucose homeostasis. the importance of the islet is demonstrated by the proven ability of islet transplants to reverse type i diabetes pathologies in human patients. we are interested in understanding the multicellular mechanisms of islet function, and their role in the regulation of blood glucose under normal and pathological conditions. in many ways, the islet appears to function as a syncytium, which exhibits synchronous behavior of membrane action potentials, ca 2+ oscillations, and pulsatile insulin secretion across all β-cells in the islet. in other ways, the islet works as individual cells, especially in the regulation of gene transcription. using our unique quantitative optical imaging methods and novel microfluidic devices, the dynamics of these molecular mechanisms can be followed quantitatively in living cells within intact islets. these investigations utilize transgenic and tissue-specific knock-out mouse models with demonstrated phenotypes, as well as traditional biochemical and molecular biological approaches. do retinal rod outer segments carry out oxydative phosphorylation? i. panfoli 1 , p. bianchini 2 , d. calzia 1 , s. ravera 1 , a. morelli 1 , a. diaspro 3 1 biology dept., university of genova, italy, 2 lambs-microscobio res.centre, university of genova, italy, 3 neuroscience and brain technology dept., i.i.t. genova, italy visual transduction in vertebrate retinal rod outer segments (os) is very energy demanding. however, atp supply in os, that are devoid of mitochondria, is still puzzling. by a proteomic analysis we identified in purified bovine os disks, proteins involved in vision, as well as the respiratory chain complexes i to iv and f 1 f o -atp synthase, whose activity was comparable to that of mitochondria and sensitive to specific inhibitors. rhodamine 123 fluorescence quenching experiments showed the presence of a proton potential difference across disks. disks consumed oxygen. confocal laser scanning and transmission electron microscopy showed that cytochrome c oxydase and atp synthase are localized on disks. mitochondrial vital dyes stained os ex vivo, and disks. rhodopsin and mitotracker fluorescence co-localized in os. data, suggestive of an aerobic metabolism in os, point to the existence of "mitochondrial inner membrane-like membranes" ectopically producing atp through oxidative phosphorylation, with respect to mitochondria. new scenarios open on the patho-physiology of many retinal diseases associated to mitochondrial dysfunction. -live cell imaging -abstracts 123 unmixing using lifetime-excitation multidimensional confocal fluorescence microscopy data s. schlachter 1 , s. schwedler 2 , a. esposito 1 , a. d. elder 1 , g. s. kaminski schierle 1 , c. f. kaminski 1 1 cambridge university, u.k., 2 universität bielefeld, germany fluorescence imaging provides a powerful tool to probe biological systems, enabling the high resolution investigation of the localization, interaction and biochemical modification of biomolecules. multidimensional imaging, in particular, is a growing application of confocal and other forms of fluorescence microscopy. it seeks to measure not only fluorescence intensity in three spatial dimensions, but also other features of fluorescence emission, such as lifetime and fluorescence spectra. although multi-dimensional fluorescence microscopy has been demonstrated before, little attention has so far been paid to the problem of data interpretation and representation when dealing with such large datasets. we present here an instrument capable of recording fluorescence lifetime and excitation data by combining tcspc for lifetime determination and a supercontinuum excitation source for extracting excitation spectra. these technologies permit the acquisition of 2d and 3d images with lifetime and excitation spectrum information at every pixel. we demonstrate a method whereby the multidimensional datasets acquired with this instrument are processed to yield biologically relevant parameters, (e.g. unmix fluorophores in a multiply labelled sample). our method relies on a global analysis approach and uses ab plots for displaying multidimensional data. we demonstrate the instrument & processing method on dye and multiply labelled biological samples. advanced neuroimaging with diffractive optical elements p. saggau baylor college of medicine, houston, texas, usa studying neural systems is complicated by the large number and small size of its cellular elements. the traditional way of exploring neuronal function by electrical monitoring with micropipettes is increasingly replaced by molecular imaging. fluorescent molecules allow optical monitoring of neuronal signaling with cellular and often subcellular resolution. in addition to monitoring activity, analyzing the functional properties of individual neurons or neuronal populations requires their controlled activation. optical approaches are well-suited, including molecular photolysis of inert precursors and light activation of engineered proteins that control ionic membrane currents. to fully utilize optical techniques for exploring neural systems requires more than adequate spatial resolution to distinguish neuronal elements and sufficient temporal resolution to induce and/or monitor neuronal signaling. because of the non-linear and non-stationary nature of the studied system it is necessary to access many neuronal sites simultaneously. in modern imaging, wide-field illumination and imageformation are often replaced by patterned excitation and nonimaging photon collection. in many instants, diffractive illumination schemes can substitute for conventional reflective or refractive designs. in particular, the availability of programmable diffractive optical elements has made this an attractive alternative, since they permit highly versatile microscope designs and support a significant increase in the overall spatio-temporal resolution. i will present an advanced imaging approach using diffractive optical elements to analyze structure and function of live neurons in brain slices and intact cortex. efficient evaluation of fret in image cytometry with acceptor photobleaching and ratiometric methods j. roszik, d. lisboa, j. szöllősi, g. vereb department of biophysics and cell biology, university of debrecen, debrecen, hungary fluorescence resonance energy transfer (fret) is a powerful technique that can be applied to study nanoscale intra-and intermolecular events and interactions of molecules in situ in biological systems. a robust, easy to use, self-controlled fret method, independent of donor and acceptor concentration and stochiometry, is acceptor photobleaching fret, which requires only simple image mathematics. another approach with more complicated calculations is the intensitybased ratiometric method, which is not based on destroying the acceptor fluorophores, making it applicable for following molecular interactions in live cells. as the need for using fret in image cytometry for evaluating molecular interactions increases, we have undertaken to develop softwares for these methods involving the calculation and usage of all correction factors needed to obtain reliable energy transfer efficiencies. correction possibilities of the acceptor photobleaching method include unwanted photobleaching of the donor, fluorescent photoproduct of the acceptor after photobleaching, cross-talk of unbleached acceptor into the donor channel and partial photobleaching of the acceptor. in the case of intensity-based ratiometric fret, we can correct for all channel cross-talks and calibrate the fret efficiency calculations. both programs provide registration and semiautomatic processing, and they are freely available. the pendrin (slc26a4) gene is responsible, when mutated, for the pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid disfunctions. pendrin is an anion exchanger. the way it works and its role in different tissues, owing to the lack of known isoforms, is matter of wide research and debate. we focused on a still unexplored pendrin function, that is most important in the inner ear: cellular volume control and cl − fluxes regulation. we used hek cells over-expressing wild type pendrin or a mutated isoform together with the eyfp. we challenged cells with hypo-osmolar solutions and followed their volume variations in time. taking advantage of the confocal optical sectioning we independently measured cell volume and fluorescence intensity. in this way, given the dependence of eyfp fluorescence from [cl − ] and ph (measured with snarf5) we could estimate at the same time cl − fluxes, volume and ph variations. the contemporary measurements of the three variables, not yet reported in living cells, allowed to assess the role of pendrin in volume regulation and evidenced its participation to cl − fluxes as compared to the mutated isoform or controls. particle tracking inside the cell largely benefits of the ability to spatially and temporally mark specific structures to follow their "signalling" over a "dark" background as made possible since the advent of the photo-activatable markers. in terms of spatial confinement of the photo-activation process, the use of multiphoton excitation provides several favourable aspects compared to single photon confocal microscopy in photomarking biological structures to be tracked: the confined excitation volumes, of the order of magnitude of subfemtoliter, due to the non-linear requirements provide a unique control of the excitation and consequently photoactivation in the 3d space. in this context photoactivation experiments can be used to assess quantitative information about the binding kinetics of a macromolecule expressed in different cellular compartments. in this work we extended to photoactivation procedures and models originally developed for the quantitative analysis of frap experiments and we evaluated, for different proteins of medical interest (rac-pagfp), the diffusive behaviour in the cytoplasm and the binding kinetics at the large endosomes. the results are compared with standard photobleaching experiments, in order to evidence the gained sensitivity obtained with photo-activatable proteins. motile plaques involved in stress fiber assembly revealed by high-speed spm for living cells k. tamura, t. mizutani, h. haga, k. kawabata division of biological sciences, graduate school of science, hokkaido university, kita-ku, sapporo, japan stress fibers, which are contractile actin cables aligned in a highly-ordered manner, are important for cell migration, mechanical support of plasma membranes and extracellular matrix organization. although stress fibers are dynamically disassembled and assembled again in cells, it is poorly understood how actin filaments are organized into cables with highly-ordered alignment for stress fiber assembly. for investigation of actin cytoskeletal dynamics during actin cable formation, we performed time-lapse observation of actin cytoskeleton in lamella of living fibroblasts by using scanning probe microscopy (spm). high-speed spm observation revealed that motile plaques defined front-side ends of new actin cables and that preexisting mesh-form actin networks were remodeled into new actin cables. directional order analysis of movement of plaques and pharmacological experiments clarified that plaques were driven by myosin-ii-based retrograde actin flow, indicating that plaques are associated with actin cytoskeleton. immunofluorescence experiments showed that plaques were localized on foci of vinculin, a component of cell-substratum adhesions, suggesting that plaques can bind to extracellular substrata via vinculin. based on these results, we propose a model for actin cable formation that motile plaques initiate remodeling of preexisting actin networks into actin cables aligned in a direction of actin flow by associating with extracellular substrata. abscisic acid (aba) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. recently, aba was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells and by human and murine pancreatic β cells. aba autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin (ptx)-sensitive receptor/g-protein complex, cyclic amp, cd38-produced cyclic adp-ribose and intracellular calcium. here, the aba receptor on human granulocytes and on rat insulinoma cells is identified as the lanthionine synthetase c-like protein lancl2. co-expression of lancl2 and cd38 in the human hela cell line reproduces the aba-signaling pathway. the ptx-sensitive g protein coupled to lancl2 is identified as g i by transfection of cd38 + /lancl2 + hela with a chimeric g protein (gα q/i ). identification of the mammalian aba receptor will enable the screening of synthetic aba antagonists as prospective new anti-inflammatory and anti-diabetic agents. investigation of post-thaw damage of s.cerevisiae yeasts using fluorescent dyes 2-dab and 3-dab t. s. dyubko 1 , i. a. buriak 2 , v. d. zinchenko 2 , i. f. kovalenko 2 1 state scientific institution "institute for single crystals", national acadey of sciences of ukraine, kharkov, ukraine, 2 institute for problems of cryobiology and cryomedicine, national academy of sciences of ukraine, kharkov, ukraine we investigate applicability of the fluorescent probes 2-dab and 3-dab available from seta biomedicals (www.setabiomedicals.com) to study the post-thaw damage of saccharomyces cerevisiae yeast cells. the leaving cells stained with these dyes have bright fluorescence in the yellow and red region, respectively. however, the freezing followed by post-thawing causes cell damage, which results in a change of fluorescence intensity. in the native living cells the dyes are preferably localized in the cell membranes and organelles which are highly fluorescent. partially damaged cells have even brighter fluorescence as compared to the living cells. however, their cell ultra-structure is not well-distinguished in the fluorescence mode. ultimately destroyed cells are almost non-fluorescent: the cell membranes are not visible in the fluorescent mode and their organelles are only weakly fluorescent. the number of intensively fluorescing cells with partially destroyed membranes, which were obtained by freezing to -20 • c, is lower compared to those frozen at -196 • c. 2-dab and 3-dab enable not only to distinguish damaged and undamaged cells, but also allow quantitative estimation of the extent of damage in membranes by cryogenic effects. the thylakoid membrane is a structured network in higher plants which is organised into stacked granal thylakoids that are interconnected by single 'stromal' lamellae. we have studied the mobility of a resident protein of the stromal lamellae, hcf106, part of the tat protein translocase. hcf106-green fluorescent protein fusion (gfp) was targeted into thylakoids and studied using photobleaching approaches. we show that small regions fail to recover significant levels of hcf106-gfp fluorescence within 3 minutes after photobleaching. autofluorescence from the photosystem ii light-harvesting complex (lhcii) in granal stacks likewise fails to recover over this time scale. although the thylakoid membrane is a single continuous entity, these data show that both hcf106-gfp and lhcii are constrained within this network. since the hcf106 homologue, tatb, is highly mobile in the escherichia coli plasma membrane, we believe that the stromal lamellae take the form of distinct domains that are effectively constrained by boundaries within the thylakoid network. cytomechanical modifications induced by drugloaded carriers uptake by breast cancer cells the novel opportunities offered by the nanotechnologies have attracted great interest in the development of novel biomaterials for targeted drug delivery in cancer research. the desired features of pharmaceutical drug delivery for intravenous administration are their small size, biodegradability, high drug content, prolonged circulation in the blood and the ability to target required areas. in this work we have compared efficacy of different type of carriers having complementary properties for pharmaceutical delivery in cancer therapy. drug nano-colloids encapsulated by combination of layer by layer (lbl) techniques and ultrasonication, phytochemical encapsulated artificial oleosomes and drug-loading clay/carbon nanotubes have been used for uptake into breast cancer cells. analysis of viscoelastic response of neoplastic cells induced by cargo-carriers uptake has been carried out by a combination of high resolution optical and scanning force microscopy techniques. furthermore, the effects of drug-reservoir carriers on the cytoskeleton (re)organisation of neoplastic cells were further investigated by confocal microscopy using different fluorescent probes. mapping diffusion by raster image correlation spectroscopy (rics) with analog detection time lapse and flow cytometry data integrated in a common cell cycle proliferation model p. ubezio, v. colombo, m. lupi, f. falcetta istituto di ricerche farmacologiche "mario negri", milano, italy we present a cell cycle simulation tool, connecting the basic proliferation process to the cell population data obtained by time lapse and flow cytometry. the computer program is a general framework within which cell cycle progression can be interactively modeled at the desired level of complexity, including g1/s/g2m cell cycle phases with variable duration, g0 phase, cell subpopulations belonging to different generations or differentiation stages, distinct block and cell death parameters for each phase. in this way, we achieved a detailed rendering of cell proliferation of normal or tumor cell populations, additionally including cytostatic and cytotoxic effects of treatments. the program gives as output simulated measures, reproducing those obtained by absolute cell counting, growth inhibition tests, flow cytometric dna histograms and cell cycle percentages, pulse continuous-labeling studies, time-lapse intermitotic times and generation-wise analyses. each technique provides a piece of information related to the underlying proliferation process, catching only some of the phenomena in play, and the measures often cannot be univocally interpreted. we used the cell cycle simulator to fit together time lapse and flow cytometry time courses measures, to achieve a detailed reconstruction of the cell cycle proliferation of an ovarian cancer cell line in vitro after x-ray exposure. -live cell imaging detection of functional modes in protein dynamics j. s. hub 1 , b. l. de groot 2 1 deptartment of cell & molecular biology, uppsala university, sweden, 2 computational biomolecular dynamics group, max-planck-institute for biophysical chemistry, göttingen, germany proteins frequently accomplish their biological function by collective atomic motions. yet the identification of a collective motions related to a specific protein function from, e.g. a molecular dynamics trajectory, is often non-trivial. here, we propose a novel technique termed`functional mode analysis' that aims to detect the collective motion that is directly related to a particular protein function. based on an ensemble of structures, together with an arbitrary`functional quantity' that quantifies the functional state of the protein, the method detects the collective motion that is maximally correlated to the functional quantity. the functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. two different correlation measures are applied. first, the pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. the new method is illustrated using various biomolecules, including a polyalanine-helix, t4 lysozyme, trp-cage, and leucine-binding protein. logic estimation of the optimum source neutron energy for bnct of brain tumors boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron-10. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding conditions on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating 65 different source neutron energies, between 0.01 ev and 13.2 mev. mcnp4b monte carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about 30 kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. the results were discussed. v. l. cruz, j. ramos, j. martinez-salazar instituto de estructura de la materia. csic cannabinoid receptors are an important class of g protein coupled receptors. in particular cb1 and cb2 have received considerable interest because they mediate a variety of physiological responses in the central nervous and immune systems. their tertiary structure is still unknown. experimental information suggests the presence of both, an active and an inactive conformation. cb1 and cb2 share a common structural framework consisting in a seven transmembrane α-helix bundle connected by three extracellular and three intracellular loops. however, the knowledge of structural differences between both receptors may serve to design new ligands to activate/deactivate selectively only one of those receptors. in the present research, we report a multi-nanosecond molecular dynamics simulation of both receptors in solution, starting from a structure obtained by homology modelling using the x-ray determined bovine rhodopsin protein. we look for differences in the behaviour of cb1 and cb2 during the simulation process to shed light about those structural features which can be important for ligand selectivity. in this sense, we observed that cb1 tend to present a more flexible and opened helix bundle than cb2. thus, it is expected than the former receptors would present less steric hindrance for ligand binding. we expect these results will be useful to design more selective ligands. self-assembly and equilibration of bola-lipids membranes studied by molecular dynamics m. bulacu, s. j. marrink groningen university, the netherlands bola-lipids consist of two monopolar, twin-tailed lipids that are held together by chemical linkage between one or both ends of the tails from one lipid and the corresponding ends from the other one. membranes formed by these lipids or by their mixtures with monopolar lipids are known to have additional mechanical stability while retaining membrane fluidity. this is traditionally attributed to the fact that, in the membrane phase, the bola-lipids have predominantly spanning configuration (the two polar heads are positioned at opposite membrane-water interfaces) in detriment to looping configuration (both head groups are located in the same membrane-water interface). we perform molecular dynamics simulations, using the coarse grained martini forcefield. we start with self-assembly simulations of bola-lipids followed by bilayer equilibration. an artificial pore is created in the membrane that significantly increases the flip-flop mobility of the lipids and hasten equilibration. the membrane properties are characterized (area per lipid, thickness, order parameter, pressure profile) with emphasis on the spanning/looping ratio. our study can help designing new artificial membranes, with higher stability under extreme conditions. -multiscale simulation evidence for proton shuffling in a thioredoxinlike protein during catalysis d. narzi 1 , s. w. siu 1 , c. u. stirnimann 3 , j. p. grimshaw 2 , r. glockshuber 2 , g. capitani 4 , r. a. böckmann 1 1 theoretical and computational membrane biology, center for bioinformatics, saarland university, germany, 2 institute of molecular biology and biophysics, eth zürich, switzerland, 3 structural and computational unit, embl, heidelberg, germany, 4 paul scherrer institute, villigen psi, switzerland proteins of the thioredoxin (trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. all members of the trx family with thioldisulfide oxidoreductase activity contain the characteristic cys-x-x-cys motif in their active site. here, using poisson-boltzmann-based protonation-state calculations based on 100-ns molecular dynamics simulations, we investigated the catalytic mechanism of dsbl, the most oxidizing protein known to date. we observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. these results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. a consistent catalytic mechanism for dsbl, probably conferrable to other proteins of the same class, is presented. our results suggest a functional role of hydration entropy of active-site groups . the role of water in zn(ii)-abeta(1-16) complexes beta-amyloid (aβ) peptides are the main component of amyloid fibrils detected in the brain of alzheimer patients. fibrils display an abnormal content of cu and zn ions whose binding to aβ-peptides has been recently studied by x-ray absorption spectroscopy [1] and interpreted in terms of ab initio simulations. in order to perform such simulations it is of the utmost importance to find a compromise between the need of having a realistic description of the actual physical system and the difficulty of dealing with too many atoms and electrons. what is usually done is removing solvent (water molecules), thus studying the system in the so called "gasphase". in this work we investigate the relevance of water in the zn-aβ 1−16 coordination mode. relying on a combination of classical [2] and quantum chemistry [3] methods we find a significant difference in the zn coordination geometry depending on whether water is present or not. this information is exploited in building a full model system for subsequent car-parrinello simulations where two aβ peptides in water are in interaction in the presence of zn. [ although experiments which can directly probe the mechanical properties of proteins have only been performed recently, it is clear that the complex folds of polypeptide backbones lead to very heterogeneous mechanical behavior. this heterogeneity is likely to play an important role in protein function and interaction and it would be useful to be able to predict what mechanical (and dynamical) properties will result from a given structure. we have been using coarse-grain elastic network models to investigate this question and have found that these models are able to link specific mechanical properties to a number of functional features including enzymatic active sites, folding nuclei and changes in behavior due to point mutations. these models are also adapted to looking at complex formation between proteins and how specific recognition is achieved, while being fast enough to be applied to very large numbers of interactions. refinement of protein model structures using biasing potential replica exchange simulations s. kannan, m. zacharias school of engineering and science, jacobs university bremen, d-28759 bremen, germany comparative protein modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. frequently, the quality of the target-template sequence alignment is non-uniform along the sequence: parts can be modeled with a high confidence, whereas other parts differ strongly from the template. in principle, molecular dynamics (md) simulations can be used to refine protein model structures but it is limited by the currently accessible simulation time scales. we have used a recently developed biasing potential replica exchange (bp-rex) md method (kannan, s. zacharias, m. proteins 2007, 66, 697-70) to refine homology modeled protein structure at atomic resolution including explicit solvent. in standard rex-md simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. in a bp-rexmd simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. the method requires much fewer replicas for efficient sampling compared with standard temperature rexmd. bp-rexmd simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional md simulations. m. s. p. sansom, p. j. stansfeld, c. l. wee, k. balali-mood department of biochemistry, university of oxford, u.k. coarse-grained molecular dynamics simulations may be used to probe the interactions of membrane proteins with bilayers and their component lipids on an extended (∼1 µs) timescale (1 ) . conversion to atomistic resolution allows more detailed protein/lipid interactions to be examined. this multiscale approach will be examined via three examples: (i) interactions of a small ion channel toxin (vstx1) we present a new competitive approach for the treatment of biomolecular flexibility to provide an alternative to the limitations of current methodologies such as molecular dynamics and normal mode analysis. this method, called static mode method, is based on the "induced-fit" concept and is aimed at mapping the intrinsic deformations of a biomolecule subject to any external excitations: direct mono or multi-site contact, electrical etc... the algorithm allows obtaining a set of deformations, each one corresponding to a specific interaction on a specific molecular site, in terms of force constants contained in the energy model. such a process can be used to explore the properties of single molecular intrinsic flexibility, as well as to predict molecular docking or molecule/surface interactions. from a modelling point of view, the interaction problem can be expressed in terms of reactive sites between the interacting entities, the molecular deformations being extracted from the pre-calculated static modes of each separated ones. the first applications of our method have focused on the intrinsic flexibility of biomolecules like nucleic acids and proteins. more recently, this new methodology allowed us to investigate the folding of the region 1-16 of the amyloid β-peptide, via the docking of a zinc ion on the reactive sites of the molecule. conformational study on a myelin basic protein fragment: molecular dynamics simulations in membrane e. polverini 1 , g. harauz 2 1 dipartimento di fisica, università di parma, parma, italy, 2 department of molecular and cellular biology, university of guelph, guelph, ontario, canada myelin basic protein (mbp) is a multifunctional protein of the central nervous system whose principal role is in maintaining the compactness and integrity of the myelin sheath, the multilamellar membrane wrapped around nerve axons. however, mbp also interacts with other proteins such as cytoskeletal and signalling proteins, adapting its structure to the different roles. mbp is a candidate autoantigen in the human demyelinating disease multiple sclerosis. this study investigated at atomic detail the conformation of a highly conserved central fragment of mbp, constisting of two consecutive regions with different relevant functionalities. the first one is associated with the membrane and comprises the primary immunodominant epitope in multiple sclerosis; the second one was predicted to be a ligand for sh3-domains of signalling proteins. molecular dynamics simulations were perfomed in the presence of dodecylphosphocholine micelle, starting from a structure extrapolated from experimental data (harauz and libich, curr. protein pept. sci., 2009). the results confirm the experimetal hypothesis, showing, in the micelle, a stable alpha-helix anchored to the membrane for the first region and, for the proline-rich second one, a poly-proline type ii helix pointing outwards, ready to interact with the signalling proteins. multiresolution modelling of drug and hormone permeability through a lipid bilayer traditional atomic-level (al) modelling of biomembranes is time-consuming, and hence limited in the range of systems and phenomena that can be simulated. to alleviate this problem, we designed a coarse-grain (cg) representation where each lipid molecule, in reality consisting of more than 100 atoms, is modelled with only 10 cg sites. our cg technique proves two orders of magnitude less demanding of computational resources than traditional al methodology. a unique feature of our approach is that the cg potentials are directly compatible with standard al models, thus facilitating the simulation of multiresolution systems, where the "chemically-sensitive" components (e.g., the solutes in membrane permeation studies) are modelled atomistically, while the surrounding environment is coarse-grained. in this contribution, we present a summary of our multiscale methodology, together with its application to the permeation of large molecules -drugs and steroid hormones. the calculated permeabilities are compared to the available experimental measurements and al simulation data. molecularlevel insights regarding the permeation mechanism are obtained and rationalised. -multiscale simulation the processes of life involve a variety of events that occur on different scales, ranging from a fewå/ps of the triggering steps of the biochemical reactions, up to their macroscopic effects in cells and organs. intermediate steps involve the structural rearrangement of the bio-molecules (∼nm/10-100ns), their aggregation, folding (∼10nm-µm/ µs-ms), internal cell diffusion and dynamics (µm-mm/ms-hours), evidently requiring a multi-scale modeling approach. here the multi-scale approaches are first briefly illustrated with a particular attention to the issue of matching the different resolutions, which is essential to achieve a coherent descripition. the focus is then fixed on the coarse grained (cg) models, typically spanning the nm-µm and µs-ms scale, and in particular on their minimalist -simplest and computationally cheapest -versions [1, 2] . a parameterization strategy that combines accuracy and predictive power within these models is presented here and applications are shown to relevant cases including the proteins involved in the hiv replication, the green fluorescent proteins and examples of macromolecular complexes. [1] controlled degradation of collagen is an important process in tissue remodeling and wound healing. collagenase cleaves fibrillar collagens about three quarters of the distance from the amino-terminus. even though the determination of the cleavage site and the collagenase structure took place decades ago, the mode of action of collagenase on collagen is not clear. to understand the mechanism of collagenase activity on collagen, the structure, stability and dynamics of collagen, its conformation around the cleavage site and the possibilities of conformational rearrangements between the two domains in collagenase was explored using md simulation. the results of principal component (pca) and normal mode (nm) analysis of the collagen and collagenase suggests that the c-terminal domain of collagenase recognizes the collagen, and then the n-terminal catalytic domain undergoes rearrangement on the substrate (with the help of linker regions). the sda software for carrying out brownian dynamics simulations of protein-protein diffusional association with the help of biochemical constraints is being used to predict the mode of interaction of collagen and collagenase. different conformations obtained from pca and nm analysis are being used for the docking. the predicted structures of the complex will help us to understand how collagenase recognizes and binds collagen specifically. acknowledgment: daad, germany, csir-srf india, and the klaus tschira foundation. membrane aoration by antimicrobial peptides combining atomistic and coarse-grained descriptions d. sengupta, a. j. rzepiela, n. goga, s. j. marrink university of groningen, the netherlands antimicrobial peptides (amps) comprise a large family of peptides that include small cationic peptides, such as magainins, which permeabilize lipid membranes. previous atomistic level simulations of magainin-h2 peptides show that they act by forming toroidal transmembrane pores. however, due to the atomistic level of description, these simulations were necessarily limited to small system sizes and sub-microsecond time scales. here, we study the long-time relaxation properties of these pores by evolving the systems using a coarse-grain (cg) description. the disordered nature and the topology of the atomistic pores are maintained at the cg level. the peptides sample different orientations but at any given time, only a few peptides insert into the pore. key states observed at the cg level are subsequently back-transformed to the atomistic level using a resolutionexchange protocol. the configurations sampled at the cg level are stable in the atomistic simulation. the effect of helicity on pore stability is investigated at the cg level and we find that partial helicity is required to form stable pores. we also show that the current cg scheme can be used to study spontaneous poration by magainin-h2 peptides. over-all, our simulations provide a multi-scale view of a fundamental biophysical membrane process involving a complex interplay between peptides and lipids. the varkud satellite (vs) ribozyme is the largest of the nucleolytic ribozymes, and the only one for which there is no crystal structure. we have determined the overall architecture of the complete vs ribozyme using small-angle x-ray scattering in solution. the substrate stem-loop docks into the tertiary fold of the ribozyme, allowing an intimate loop-loop interaction to occur. this brings two key nucleobases a756 and g638 into close proximity of the scissile phosphate, and we believe that these nucleobases are involved in general acid-base catalysis of the phosphoryl transfer reactions. this is supported by functional group substitution, and the ph dependence of the reaction rate for the natural and modified rna. although possessing totally different folds, the functional elements of the vs and hairpin ribozymes are topologically and mechanistically very similar if not identical. this has probably arisen by convergent evolution. other nucleolytic ribozymes have diversified to employ hydrated metal ions and even small molecules to participate in genera acid-base catalysis. by contrast, the larger ribozymes seem to have adopted a different, metalloenzyme, catalytic strategy. why these different groups have evolved different catalytic chemistries is an interesting challenge to our understanding of biocatalysis. comparison of dna and sirna binding and nuclease protection by non-viral vectors for gene delivery j. lam, k. witt, a. j. mason, l. kudsiova, m. j. lawrence pharmaceutical science division, king's college london, uk the biggest obstacle for the success of gene therapy is delivery. with the discovery of rnai, the use of sirna to regulate gene expression as potential therapy has attracted much attention recently. in contrast to dna, sirna delivery does not require nuclear entry. with one less barrier to overcome, the delivery of sirna seems to be easier. it is frequently assumed that comparable delivery strategy could be used for both dna and sirna. in fact the two types of nucleic acids are fundamentally different with distinct properties, which impact their delivery strategies. in the present study it was found that most non-viral delivery vectors including polymers, peptides and lipids were generally more efficient in binding with dna than sirna as shown in gel retardation assay. the inferior sirna binding is possibly due to the rigid structure of sirna, resulting in weaker electrostatic interaction with the cationic vectors. surprisingly, it was observed that all the vectors studied offered better nuclease protection for sirna than dna despite poorer sirna binding. either the nuclease protection for sirna is easier to achieve due to its small size, or the gel retardation assay did not truly reflect the binding efficiency as the weaker sirna complexes may dissociate during electrophoresis. not only the delivery strategy, the results between dna and sirna study must also be carefully adapted and interpreted. single molecule studies of spliceosomal rnas u2 and u6 z. guo, d. rueda department of chemistry, wayne state university, detroit, michigan, u.s.a. splicing is an essential step in the maturation reaction of mrna, in which intervening introns from exons. the spliceosome is a dynamic assembly of 5 small nuclear rnas and >150 proteins that catalyzes splicing. u2 and u6 are the only 2 snrnas strictly required for splicing. major conformational changes are expected to take place during spliceosomal assembly and catalysis. we have developed a single-molecule fluorescence assay to study the structural dynamics of a protein-free u2-u6 complex from yeast. our previous data have revealed a 2-step large amplitude conformational change of the u2-u6 complex. the 1 st step is a mg 2+ -induced conformational change where helix iii and the u6-isl are in close proximity in low mg 2+ and separated in high mg 2+ . the 2 nd step corresponds to the formation of the highly conserved helix ib. we now examine the role of the highly conserved bases in the folding dynamics of the u2-u6 complex. the data show that u80 and the acagag loop play an important role in stabilizing the interaction between helix iii and the u6-isl. we hypothesize that u80 flips out of the u6-isl and interacts with the acagag loop to bring them in close proximity. our results raise the interesting possibility that this interaction plays an important role in bringing the 5' splice site and the branched a into close proximity of u80, which may be critical for catalysis. a structure-based approach for targeting hiv-1 genomic rna dimerization initiation site s. freisz, s. bernacchi, p. dumas, e. ennifar ibmc -cnrs, strasbourg, france all retroviral genomes consist in two homologous single stranded rnas. hiv-1 dimerization initiation site (dis) is a conserved hairpin in the 5' non-coding region of the genomic rna and essential for viral infectivity. the dis initiates genome dimerization by forming a kissing-loop complex, further stabilized into an extended duplex upon interaction with the ncp7 nucleocapsid protein. x-ray structures of the dis kissing-loop and extended duplex revealed similarities with the bacterial 16s ribosomal rna a-site, which is the target of aminoglycoside antibiotics. as a result, aminoglycosides also bind the hiv-1 dis as shown by our x-ray structures of the dis kissingloop bound to aminoglycosides. using fluorescence, uvspectroscopy and microcalorimetry, we further characterized hiv-1 dis/aminoglycosides interactions. we found that the affinity of aminoglycosides for the dis was higher than for their natural target, the 16s a site. they strongly stabilize the dis kissing-loop, blocking its conversion into the duplex form. finally, we also solved x-ray structures of the dis duplex bound to aminoglycosides, revealing an important conformational change following drug binding. these structures show that it is possible to target the hiv-1 dis dimer before and after the ncp7-assisted rna maturation with the same molecule. altogether, our studies create the basis for a rationally driven design of novel potential drugs targeting the hiv-1 genome. the core protein of hepatitis c virus is a multifunctional protein of 179 aa, consisting of a hydrophilic n-terminal domain with three basic domains (bd1-bd3) responsible for the interactions with rna and a hydrophobic c-terminal domain. the n-terminal domain exhibits nucleic acid chaperone properties similar to those of the nucleocapsid protein from hiv. here, we characterized the mechanism of the chaperone properties of a peptide e corresponding to the bd2 and bd3 clusters of the n-terminal domain. to this end, we monitored the promotion by this peptide of the annealing of dtar, the dna analogue of the transactivation response element to its complementary sequence, ctar dna, taken as models. the annealing involves two second-order kinetic components that are activated by at least three orders of magnitude by peptide e. this activation was correlated with the ability of peptide e to destabilize the lower half of dtar stem. using, ctar and dtar mutants, the two kinetic components were assigned to two pathways which are connected with the fast annealing of the terminal bases of ctar to dtar and slow extended duplex formation, limited kinetically by the nucleation of central segments of ctar and dtar stems. structure and conformational dynamics of a unique dead box helicase m. rudolph 1 , m. linden 2 , r. hartmann 3 , d. klostermeier 2 1 hoffmann-la roche, basel, switzerland, 2 biozentrum, univ. of basel, switzerland, 3 univ. of marburg, germany dead box helicases couple atp hydrolysis to rna structural rearrangements. t. thermophilus hera consists of a helicase core and a c-terminal extension (cte) with a putative rnase p motif. crystal structures show that the cte is bipartite, forming a highly flexible dimerization motif with a novel fold and an additional rna-binding module. we provide a first glimpse on the orientation of an rna-binding domain outside the helicase core. in a structure-based model for the complete hera dimer, the rna-binding sites of the helicase cores face each other, allowing for inter-subunit communication. the plasticity of the dimerization motif allows for drastic changes in the juxtaposition of the helicase cores within the dimer. in single molecule fret experiments we identified fragments of the 23s rrna and rnase p rna as substrates for hera. both substrates switch the hera core to the closed conformation and stimulate the intrinsic atpase activity. rna binding is mediated by the cte, but does not require the putative rnase p motif. atp-dependent unwinding of a short helix in 23s rrna suggests a specific role for hera in ribosome assembly, in analogy to the e. coli and b. subtilis helicases dbpa and yxin. in addition, the specificity of hera for rnase p rna may be required for rnase p rna folding or rnase p assembly. simultaneous action of two hera subunits on the same rna molecule may be important for efficient rna remodeling in vivo. structural basis for the encapsidation process of turnip yellow mosaic virus m. petersen 1 , j. hansen 1 , s. s. wijmenga 2 1 nucleic acid center, department of physics and chemistry, university of southern denmark, odense, denmark, 2 physical chemistry/biophysical chemistry, radboud university, nijmegen, the netherlands formation of hairpins with internal loops with c c and c a mismatches in the 5' untranslated region is a common characteristic among the plant viruses belonging to the tymovirus genus. turnip yellow mosaic virus possesses two such hairpins, hp1 and hp2. hp1, and in particular the presence of the c c and c a mismatches in its internal loop, is important for initiation of the encapsidation of the viral genome. the encapsidation occurs under acidic conditions at the neck of invaginations of the chloroplast membrane. we have now determined the 3d structures of revertants involved in the evolutionary pathway using nmr spectroscopy. these structures reveal how the grooves in the hairpin become increasingly positively charged in successive generations of evolution. the similarity between the ccca mutant and the wild-type hairpin (hp1) is striking and explains why this mutant gives rise to a viable virus. in addition, a characteristic tilt of the backbone is observed upon occurrence of a protonated c c base pair. both the positively charged major groove and the kink in the rna backbone appear to be crucial for interactions with the viral capsid. at neutral ph, the structure of hp1 resembles the watson-crick base paired mutant which explains why encapsidation only occurs under acidic conditions. a. percot 1 , j. vergne 2 , m.-c. maurel 2 , s. lecomte 3 1 ladir, umr7075, 94320 thiais, france, 2 lbeam, upmc, 75005 paris, france, 3 cbmn, umr5248, 33607 pessac, france the existence of "rna world" as an early step in the history of life increases the interest for the characterization of these biomolecules. the studied hairpin ribozyme is a self-cleaving/ligating motif found in the minus strand of the satellite rna associated with tobacco ringspot virus. surface-enhanced raman spectroscopy (sers) was successfully used to detect sub-picomole quantities of nucleic acids. sers takes advantage of the strongly increased raman signals generated by local field enhancement near metallic (typically au and ag) nanostructures. sers spectra of dna or rna are strongly dominated by stockes modes of adenine. through an interaction of adenyl residues with silver colloid, adenyl raman signal is 10 6 times increased compared to raman scattering. in controlled conditions, sers signal is proportional to the amount of free residues adsorbed on the metal surface. upon rna cleavage, residues are unpaired and free to interact with metal. in the present study, we proposed to follow the cleavage reaction of hairpin ribozyme (hpr85) using the sers signal of the liberate adenyl residues. as the sers signal is proportional to the adenyl residues, reactivity of hr85 was monitored by measuring the raman intensity of the fragment liberated during the cleavage of hairpin. the results obtained were compared with the electrophoresis method performed on the same sample and similar results were obtained. -rna world eur biophys j (2009) the development of arrays for biomolecular recognition for a broad range of applications in biomedical diagnostics is receiving a constantly increasing attention. the design of efficient protein biochips, however, requires the optimization of protein immobilization protocols for improving the device sensitivity. moreover, innovative platforms for in-vitro detection in highly diluted, small volume samples need to be developed, for which standard micro-fabrication techniques are not suitable. therefore, the development of nano-scale platforms for protein and antibody detection is essential. we report here on a novel approach for the fabrication of multiple protein nanosensors using atomic force microscopy based nanografting and dna-directed immobilization (ddi), which takes advantage of the specific watson-crick hybridization of oligonucleotide-modified proteins to surfacebound complementary oligomers. using nanografting, single-stranded dna nano-structures with well defined local environments were fabricated on a flat surface. dna-protein conjugates were then anchored on the engineered binding sites by ddi in a single chemical operation and detected by the corresponding topographic height increase of the relevant patches. immunological assay were used to demonstrate the biochemical functionality of the immobilized proteins, proving the specificity of biomolecular recognition of our nanodevices, in the micro-molar to pico-molar concentration range. dna accessible surface area and indirect protein-dna recognition: study by bioinformatical approach o. p. boryskina 1 , m. y. tkachenko 1 , a. v. shestopalova 1 , m. y. tolstorukov 2 1 institute of radiophysics and electronics nas of ukraine, 2 harvard-partners center for genetics and genomics, usa revealing the mechanisms of protein-dna recognition is essential for understanding the regulation of many cellular processes. there is growing evidence that recognition through sequence-specific contacts (direct readout) can be enhanced by recognition via dna sequence-dependent deformability (indirect readout). the role of changes in dna accessible surface area (asa) in distorted dna configurations in complexes with proteins is not fully understood yet, even though such changes and related changes in polarity of dna surface are among key factors of indirect readout. to fill this gap we have developed a publicly-available internet database of protein-dna complexes, which integrates the data on dna conformational parameters with information on asa of dna atoms in the minor and major grooves, protna-asa. the database has been used to analyze the effect of changes in dna backbone configuration on asa of dna atoms in major and minor grooves. we observe that sugar puckering and conformation of torsion angle γ affect the accessibility of dna atoms in both grooves to a noticeable extent. we also report sequence specific preferences of the nucleotides for structural domains of γ. these results can shed new light on the mechanisms of indirect protein-dna recognition. a. arakelyan biology dpt., yerevan state university, yerevan, armenia the influence of ligand, irreversibly binding with dna, on the isotherm of adsorption of ligand binding with dna reversibly has been modeled theoretically. the isotherm of adsorption of etbr on dna in presence of cis-ddpt has been considered at low concentrations of cis-ddpt and etbr.. the isotherm of adsorption of etbr on dna has linear form in scatchard coordinates at low degrees of occupation. the comparison with experimental isotherm permits to estimate the parameters of etbr binding with dna. with taking into account the pseudo-ring structures formation with partially molten regions we consider two types of binding regions, linear and ring at low degrees of occupation. the isotherm of adsorption of etbr on dna and also variation of the number of bounded etbr molecules are calculated with taking into account these two types of binding regions. it was shown that at low concentrations cis-ddp, bounding with dna, changes the isotherm of ligands adsorption. the linear in scathcard coordinates isotherm of adsorption transforms into non-linear isotherm. the degree of transformation depends on the fraction of dna in the ring regions, on the ratio between number of etbr binding cites in the ring and linear regions, and also on the binding constants for these regions. it was shown that low concentrations of cis-ddp affect the dependence of dispersion on the concentration of ligands, changing both the magnitude of maximum and its position. tunable nanoconfinement structures for dna manipulation e. angeli, l. repetto, g. firpo, c. boragno, u. valbusa nanomed labs, advanced biotechnology center and physics department, university of genoa, italy nanostructures, such as nanochannels [1] or nanoslits [2] , have been successfully used to confine and stretch dna molecules, offering interesting opportunities of investigation on conformational changes induced by confinement, physical and biological properties, etc. the integration of these nanostructures on lab-on-chip systems has shown their great potential for applications such as dna sieving or single molecule manipulation [3] , [4] . arrays of nanochannels fabricated, using a focused ion beam (fib) on a silicon master, are replicated using elastomeric materials, such as poly(dimethylsiloxane) (pdms), and soft-lithography techniques. the cross-section of these flexible polymeric nanoconfinement structures can be reversibly and dynamically tuned, in order to vary biomolecules transport characteristics and confinement conditions of trapped dna molecules. moreover, these nanochannels, with tunable cross-section, are used to study and exploit the sieving mechanism of "entropic recoil" [5] for the separation of long dna chains. [1] mannion jt, et al., (2006 ) biophys. j., 90, 12:4538-4545. [2] jo k, et al., (2007 ) pnas, 104, 8: 2673 -2678 . [3] fu j, et al., (2007 ) nat. nanotechnol., 2: 121-128 [4] huh d, et al., (2007 nat. mater., 6: 424-428. the nucleocapsid protein ncp7 of hiv-1 is characterized by two conserved zinc fingers and plays crucial roles in the virus, through its binding to nucleic acids. ncp7 is required for efficient proviral dna synthesis, by promoting the initiation of reverse transcription and the two obligatory strand transfers. using fluorescence techniques as well as fcs and nmr, we investigated the chaperone properties of ncp7 on the primer binding sites (pbs) and transactivation response elements (tar) sequences involved in the two obligatory strand transfers. we showed that ncp7 binds mainly to the (-)pbs loop, which results in an extension of the loop and a destabilization of the upper base pair of the stem. these structural changes chaperone a kissing complex with the complementary (+)pbs loop and its further conversion into an extended duplex. in contrast, the ncp7-promoted annealing of ctar-tar results from the destabilization of the bottom of ctar stem, which favors the invasion of the tar stem. by developing new fluorescence methodologies to site-specifically characterize these interactions, we further showed that ncp7 slows down the ps to ns conformational fluctuations of its nucleic acid targets and freezes the local mobility of the bases contacted by the zinc fingers. exploring dna orientation in flow e. l. gilroy, m. r. hicks, a. rodger department of chemistry, university of warwick, uk dna is one of the most important biomolecules. in order to undertake its biological role the dna needs to fold and unfold for which its structure and flexibility are crucially important. we have studied the characteristics of dna in flow to probe its structure. flow aligned linear dichroism (ld) is a technique that uses light polarised parallel and perpendicular to an orientated sample. it can be used to measure how aligned a sample is, and the orientation of any interacting molecules. to create this alignment, the sample is placed between two concentric cylinders where one is spun to create a shear flow. the longer and more rigid the molecule is the better the orientation and the signal. as the sample is in flow there are other factors than need to be considered when using ld. these include viscosity and temperature. there are many methods to measure the viscosity of a sample of dna at varying temperatures. these include viscometer, rheometer and dynamic light scattering. the effects temperature has on viscosity and the sample itself need also to be considered. the findings of all these studies have been reported and show the significance that viscosity and temperature have on dna ld measurements. possible applications of using ld to study dna have also been discussed, showing the importance of the use of ld and in the results shown. a.-m. florescu, m. joyeux laboratoire de spectrométrie physique, université joseph fourier grenoble 1, france we present a dynamical model for simulating non-specific dna protein interactions, which is based on the "beadspring" model for dna with elastic, bending and debye-hückel electrostatic interactions, and where the protein interacts with the dna chain through electrostatic and excluded-volume forces. we study the properties of this model using a brownian dynamics algorithm that takes hydrodynamic interactions into account and obtain results that partially agree with experiments and predictions of kinetic models. for example, we show that the protein samples dna by a combination of three-dimensional diffusion in the solvent and one-dimensional sliding along the dna chain. this model evidences the presence, in a certain range of values of the effective protein charge, of facilitated diffusion, i.e. a combination of the two types of diffusion that leads to faster than 3-dimensional diffusion sampling of dna. moreover, the analysis of single sliding events shows that the number of base pairs visited during sliding is comparable to those deduced from single molecule experiments. in contrast to kinetic models, which predict an increase of the number of different base pairs visited by the protein proportional to the square root of time, our model however suggests that this number increases linearly with time until it reaches a value that is close to the total number of dna base pairs in the cell (published in j. chem. phys. 130, 015103 (2009)). use of md simulation to identify the critical radiation-induced lesions of a dna binding protein n. chalabi, s. goffinont, n. garnier, d. genest, 45071 orléans cedex 2, france a key step in the regulation of gene expression, dna structuring and dna repair is the binding of some proteins to specific dna sequences. we have shown previously that ionizing radiation destabilizes such dna-protein complexes mainly through damage to the protein. for the typical lactose operator -repressor system we have shown by fluorescence measurement and mass spectrometry that upon irradiation, all the four tyrosine residues of the dna binding domain (called headpiece) are oxidized into 3,4dihydroxyphenylalanine (dopa). a circular dichroism study revealed a global conformational change and the destabilization of the headpiece. to decide which lesion is critical for the induction of these effects, a molecular dynamics simulation study was undertaken in parallel with a site-directed mutagenesis one. each tyrosine residue of the headpiece was replaced by another amino-acid that mimics the damaged tyrosine. the most common amino-acid used in site-directed mutagenesis being alanine, we have replaced one tyrosine (7, 12, 17 or 47) in the nmr-based structure of the headpiece from pdb databank (1lqc) by an alanine. the conformational stability of each tyr→ala mutant has been studied by molecular dynamics simulation (md) and compared to that of the native sequence and of the different tyr→dopa mutants. the mammalian high mobility group protein at-hook 2 (hmga2) is a nuclear protein associated with mensenchymal cell development and differentiation. disruption of its normal expression pattern is directly linked to oncogenesis and obesity. our laboratory has utilized a variety of biochemical and biophysical methods to investigate the molecular mechanisms of hmga2 recognizing at-rich dna. using a pcr-based selex procedure, we discovered that hmga2 is a sequence-specific dna-binding protein and recognizes the following two sequences: 5'-atattcgcgawwatt-3' and 5'atattgcgcawwatt-3', where w is a or t. using a double-label emsa assay, we found that hmga2 binds to at-rich dna as a monomer. using isothermal-titrationcalorimetry, we demonstrated that hmga2 binds to at-rich dna with very high binding affinity whereby the binding of hmga2 to a-tracts is entropy-driven and to alternate at sequences is enthalpy-driven. this is a typical example of enthalpy-entropy compensation in which the hydration difference between hmga2-dna complexes is a main reason for the compensation. interestingly, the binding of hmga2 to different at-rich sequences is accompanied with a large negative heat capacity change indicating an important role of solvent displacement and charge-charge interaction in the linked folding/binding processes. partition of gibb´s free energy of drug-dna complexation we report a computation methodology, which leads to the ability to partition the gibb's free energy for the complexation reaction of aromatic drug molecules with dna. using this approach it is now possible to calculate the absolute values of the energy contributions of various physical factors to the dna binding process, whose summation gives a value that is reasonably close to the experimentallymeasured gibb's free energy of binding. application of the methodology to binding of various aromatic drugs with dna provides an answer to the question 'what forces are the main contributors to the stabilization of aromatic ligand-dna complexes' ? we report the effects of 22 khz ultrasound irradiation of double-stranded dna solutions under conditions of transient cavitation. a new method was developed for studying these effects which is based on combination of two procedures: ultrasound irradiation of the solutions of double-stranded dna fragments and subsequent analysis of the high resolution denaturing gel electrophoresis data. statistical treatment of the data on the mobility of 3'-end-labelled restriction fragments with known sequence allowed us to discover the phenomenon of sequence specific ultrasonic cleavage of dna. our analysis results in the following conclusions: 1) all steps with 5'-ward cytosine [5'-d(cpn)-3'] have significantly higher cleavage rates than others; the intensity of cleavage diminishes in the order cpg > cpa ≈ cpt > cpc; 2) the cleavage rates of all 16 steps depend on the type of flanking base pairs; 3) the cleavage rates of the complementary base pair steps are not identical. thus, subtle sequence specific conformational and physical-chemical variations modulate the reaction of sugar-phosphate single bonds on the ultrasound exposure. a theory of the mechanisms on the simultaneous binding of two aromatic drugs to dna it has long been recognized that certain combinations of dna-binding aromatic drugs, x +y, lead to synergistic biological effects. considering x as a basic ligand and y as an added ligand, the change of the integral biological response of x in the presence of y has been interpreted in terms of two mechanisms: the interceptor and protector action of y on x. this mechanisms have been characterized by two criteria, r d and a d , reflecting the removal of x from dna by y (biophys. chem., 2008, vol.132, pages 148-158 ) . in this work we develop the theory of the interceptor-protector action of a mixture of two biologically-active dna-binding aromatic drugs. the theory is based on solution of a general system of mass balance equations in the three-component system x -y -dna with respect to the two factors, r d and a d . the outcome is a set of expressions enabling estimation of the change in biological response of x on addition of y as a function of equilibrium parameters under different restrictions. the results are in good agreement with known in vitro data on caffeine+antibiotic action in leukemia cell lines. the influence of mn 2+ ions on the structure of natural calf thymus dna was studied by raman spectroscopy. measurements were done at room temperature and ph 6.2 ± 0.2, in the presence of the physiological concentration of 150 mm na + ions, and in the presence of mn 2+ concentrations that varied between 0 and 600 mm. no condensation of dna was observed. dna backbone conformational changes were not detected in the whole concentration range of mn 2+ ions as judging from the raman spectra. no evidence for dna melting was identified. binding of manganese(ii) ions to the charged phosphate groups of dna, stabilizing the double helical structure, is indicated in the spectra. as judged from the marker band of dc near 785 cm −1 , altered nucleoside conformations in dc residues are supposed to occur, in the mn 2+ concentration range of 400-600 mm. binding of divalent ions to n7 of guanine and, possibly, in a lesser extent to adenine was observed as judging from the raman marker bands at 1336, 1376, 1490 and 1578 cm −1 . toward rapid dna sequencing -ab initio study of nucleotide sandwiched between au(111) plates c. morari, d. bogdan, i. turcu national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania recently a new technique for dna sequencing based on measurement of transversal conductive properties of a single strained dna molecule has been proposed. such a method would allow single-base resolution by measuring the electrical current perpendicular to the dna backbone. until now, it is still not clear if the electrical signals obtained for the four nucleotide can be clearly distinguished by a hypothetical experimental setup. several factors -like the influence of the pentose group or the presence of water -may influence quite strongly the electrical signatures of the four bases. therefore, in order to obtain a working device, the understanding and detailed description of the conduction mechanisms through dna bases connected to a metallic electrode is essential. the goal of theoretical studies in this field is to describe the electric signatures of dna bases from a theoretical point of view. our study is focused on the detailed description of the electronic structure of dna's base pairs "squeezed" between two au plates. while such a geometrical model closely mimic the sequencing devices proposed in the literature, it allows us to compute meaningful physical properties such as density of states, charge transfer and orbital localizationby using "ab initio" methods. the results allow us to give qualitative prediction over the potential use of such a device in the dna's sequencing technology. multinuclear platinum complexes represent a new class of anticancer agents, distinct in dna binding and antitumor activity from their mononuclear counterparts. bbr3464 as a first representative of this class has undergone phase ii clinical trials for treatment of ovarian and lung cancers. the structure of this trinuclear pt drug consists of two trans-ptcl(nh 3 ) 2 units bridged by a transthe main lesions formed by multinuclear pt complexes in dna are long-range intra-and interstrand cross-links (cls) bridging two guanines separated by up to four base pairs. since interstrand cls can prevent dna strand separation interfering with critical cellular events they represent a serious obstacle in cell survival. in order to contribute to understanding the biological effects of dna interstrand cls of bbr3464, we analyzed the effect of the single, site-specific 1,4-interstrand cl formed by this metallodrug between two guanine bases on opposite strands in the 3'-3' and 5'-5' direction on the thermal stability and energetics of short dna duplexes. the results demonstrate that 1,4-interstrand cls of bbr3464 in both 3'-3' and 5'-5' directions exist as two distinct conformers that are not interconvertible and affect thermodynamic stability of the dna differently. side-by-side and end-to-end attraction of doublestranded dna c. maffeo 1 , b. luan 2 , a. aksimentiev 1 1 university of illinois at urbana-champaign, urbana, usa, 2 ibm watson research center, yorktown heights, usa genomic dna is densely packed inside cell nuclei and viral capsids. such close packing suggests that electrostatic repulsion between negatively charged dna in the condensed states is balanced by counterion-induced attraction. several theoretical models have been proposed to explain dna attraction, however, specific microscopic mechanisms are not known. here, we report all-atom molecular dynamics simulations of the effective force between double-stranded dna in side-by-side and end-to-end orientations. in the side-by-side orientation, the dna molecules were found to form a bond state in the presence of magnesium ions. in the bond state, the dna molecules contact each other via their negatively charged phosphate groups, bridged by magnesium ions. the maximum attractive force in the side-by-side orientation is about 40 pn per dna turn. in the end-to-end orientation, a strong ( > 60 pn) attractive force was observed at short (< 0.8 nm) end-to-end distances regardless of the electrolyte concentration. the presence of a phosphate group at the 5'ends of the fragments was found to direct dna end-to-end self-assembly and produce bound states resembling a continuous dna molecule. the computed potentials of the mean force suggest that the end-to-end attraction, rather than being mediated by counterions, is likely caused by hydrophobic and van der waals interactions between terminal nucleobases of the fragments. doxorubicin (dox) is an anticancer antibiotics with a four-membered ring system containing an anthraquinone chromophore, and an aminoglycoside. it has good anticancer activity against a wide spectrum of tumors and is one of the most extensively used antitumor chemotherapeutic compounds currently in clinical use. interestingly, conversion of dox to 2pyrrolinodoxorubicin analog (p-dox) exhibits 500-1000 times higher toxic effects in human breast cancer cell line (nagy, a. et al., pnas, 93 (1996 ) 2464 . molecular mechanisms responsible for this enormously enhanced cytotoxicity have not been entirely clarified. there is good evidence that a key component of the mechanism of action of dox is its intercalation into dna and the formation of dox-dna adducts. therefore, we have examined in detail, using the methods of molecular biophysics, dna interactions of p-dox in cell-free media and compared these results with the same studies focused on the parental dox. we find distinct differences between dna interactions of dox and p-dox and suggest that these differences are responsible at least in part for different biological effects of these two anticancer drugs. design of a microfluidic devices for the detection of oligonucléotides by sers designing fast and efficient analytical tools allowing the detection of free dna or rna at very low concentration within small volumes, without specific molecular labeling, remains a major issue of significance to perform diagnostic or to detect pathogen agents. our strategy is to use surface-enhanced raman scattering (sers) to probe selectively the spectral signature of each base in polynucleotides. sers takes advantage of the strongly increased raman signals of species when adsorbed on adapted silver colloids. we already demonstrated that adenyl raman signal of pa in presence of silver colloids is 10 6 times enhanced compared to bulk signal. we plan to use nanoliter droplets of uniformed size that form spontaneously in microchannels when two immiscible fluid streams merge. these tiny droplets are almost ideal reactors as they create homogeneous control condition. we will design an optimized channels network platform that result in droplets production hosting both nucleotides and silver colloids: internal fluids recirculation provide fast and efficient mixing, favoring base adsorption on silver nanoparticles. sers will be used to determine the chemical composition of the droplets. nicks represent the most common damage in dna which occurs naturally in living cells. structural properties of nicked dna fragments have been an object of numerous studies due to its special role in reparation processes. here we report experimental results covering ultrasound irradiation of nicked dna solutions. several single-stranded nicks were produced into one strand of dsdna fragments by the nicking enzyme bst9i. we have quantitatively estimated the ultrasonic cleavage rates in nicked dna fragments with known sequences using the polyacrylamide gel electrophoresis. computer analysis of the cleavage pattern in the 3'-end labeled and primarily intact strand reveal cleavage enhancement in the regions of about 10 b. p. up and down the nicks which were initially produced into complementary strand. the intensity of cleavage near the nicks is (in average) about 20 times higher than cleavage in the same sites of the intact dsdna fragments. at the same time, the cleavage rates in positions beyond the regions of the nick markedly grow weak even comparing to the sequence-specific cleavage of intact double-stranded dna fragments. thus, the presence of the nick serves as an expressive structural indignation, which exceeds modulation of the structure caused by the base-pair sequence and is capable of absorbing mechanical stresses applied to the nearby sites of the molecule. comparing the native and an irradiated lactose repressor-operateur complex by md simulation g. naudin, n. garnier, d. genest, rue charles sadron, 45071 orléans cedex 02, france the function of the e. coli lactose operon requires the binding of a protein, the tetrameric repressor, to a specific dna sequence, the operator. the formation of this complex involves the interaction of at least one protein dimer with the operator sequence. this occurs via the dna-binding domains (called headpieces) of the two constitutive monomers. we have previously shown that upon irradiation with gamma rays the complex is destabilised mainly because the repressor losses its dna binding ability. radiation-induced lesions were identified that may be responsible for this deleterious effect: all tyrosine residues of the headpieces are oxidized into 3,4-dihydroxyphenylalanine (dopa). in order to unravel the mechanisms leading to the observed destabilization of the operator-repressor complex, we compare by md simulations two complexes: 1-the native complex formed by a dimer of two headpieces and a fragment of dna with the operator sequence and 2-the damaged complex in which all tyrosines are replaced by dopa. analysis of these trajectories shows a loss of stability and binding energy as well as changes in the structure of the damaged complex with respect to the native one. by comparing precisely the evolution of the two complexes we can explain how the oxidation of the tyrosine residues of the headpieces into dopa may trigger the destabilization of the complex. local conformation of confined dna studied using emission polarization anisotropy in nanochannels with dimensions smaller than the dna radius of gyration, dna will extend along the channel. we investigate long dna confined in nanochannels with dimensions down to 50*50 nm, using fluorescence microscopy. studies of the statics and dynamics of the dna extension or position in such confinements as a function of e.g. dna contour length, degree and shape of confinement as well as ionic strength has yielded new insight into the physical properties of dna with relevance for applications in genomics and fundamental understanding of dna packaging in e.g. viruses. our work extends the field by not only studying the location of the emitting dyes along a confined dna molecule but also monitoring the polarization of the emission. we use intercalating dyes whose emission is polarized perpendicular to the dna extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched dna, information on the local spatial distribution of the dna backbone can be obtained. we will discuss results in shallow (60 nm) and deep (180 nm) channels and describe how the technique can be used to investigate non uniform stretching of a single dna molecule. raman spectroscopy of dna modified by new antitumor nonclassical platinum complexes o. vrana, v. kohoutkova, v. brabec institute of biophysics as cr, královopolská 135, cz-61265 brno, czech republic platinum anticancer agents (cisplatin, carboplatin, oxaliplatin) are in widespread clinical use especially against testicular, ovarian and head and neck carcinomas. there is a large body of experimental evidence that dna is the critical target for the cytostatic activity of cisplatin. platinum complexes form several types of adducts, which occur in dna with a different frequency and differently distort the conformation of dna. clinically ineffective trans isomer of cisplatin (transplatin) also covalently binds to dna bases. the trans geometry in dichloridoplatinum(ii) complexes was activated by various ways. the replacement of at least one amine ligand by planar amine ligand represents example of such activation. raman spectroscopy is powerful technique for examining both structural and thermodynamic properties of nucleic acids in solution. interactions of cis-and trans-pt(ii) complexes having nonplanar heterocyclic amine ligand such as piperidine, piperazine and 4-picoline with dna have been investigated by laser raman spectroscopy. raman difference spectra reveal that the pt(ii) complexes induce great structural changes in b-dna and indicate disordering of b-dna backbone, reduction in base stacking and base pairing and specific metal interaction with acceptor sites on purine residues. acknowledgement: this work was supported by grant as cr, iqs500040581. a. v. vargiu 1 , a. magistrato 2 , p. carloni 3 , p. ruggerone 1 1 cnr-infm-slacs and physics dept., university of cagliari, cagliari, italy, 2 cnr-infm-democritos and sissa/isas, trieste, italy, 3 iit and sissa/isas, trieste, italy the minor groove of dna is the target of several anticancer agents, which interfere with replication and translocation processes, leading to cell death. the molecular recognition event is a key step to achieve detailed knowledge of the interactions behind selectivity and affinity of a ligand towards a particular nucleic acids sequence. recognition is a multiroute process which can involve many steps before the formation of the most stable adduct. in particular, many studies have pointed out the importance of events like sliding along the groove and dissociation (which is a relevant step in the translocation among different sequences) for the affinity and the specificity of minor groove binders. despite this, no studies on the dynamics of this event were reported in the literature. in this contribution i present our recent work on the subject. umbrella sampling and metadynamics were used in the framework of classical md to characterize mechanisms andfree energy profiles of molecular recognition routes by the antitumoral agents anthramycin, duocarmycin and distamycin. our results are in very good agreement with the available experimental data, and provide insights on the influence of factors like size, charge and flexibility on the molecular recognition process. amplification of oligonucleotide sequence recognition using bioresponsive hydrogels s. tierney, b. t. stokke biophysics and medical technology, dept. of physics, the norwegian university of science and technology, ntnu, no-7491 trondheim, norway we describe development and characterization of oligonucleotide functionalized hydrogels for amplifying the molecular recognition signal occurring on hybridization between dioligonucleotides. the 50 µm radius hemispherical hydrogels were integrated on a high resolution interferometric fiberoptic readout platform supporting determination changes in the optical length of the hydrogel with 2 nanometer resolution. the hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network as network junctions in addition to the covalent crosslinks or oligonucleotides grafted to the network chains. the probe oligonucleotide destabilizing the junction point by displacement hybridization yielded an optical signal about five times as large as for binding within the hydrogel design with a comblike grafted oligonucleotide. the optical signal was found to be dependent on the concentration of the probe, the sequence and matching length between the probe and sensing oligonucleotide. concentration sensitivity applied as specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. the current design support detection in excess of 1x10 12 sequences. amplification of the molecular recognition employing the developed oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequence designed as aptamers for determination of other types of molecules are discussed. tracing t-cells by paramagentic nanoparticles in the brain of the rat model of als d. bataveljic 1 , g. vanhoute 2 , g. bacic 3 , p. andjus 1 1 inst. for physiology and biochemistry, univ. of belgrade, serbia, 2 bio imaging lab, univ. of antwerpen, belgium, 3 school of physical chemistry, univ. of belgrade, serbia amyotrophic lateral sclerosis (als) is a devastating neurological disorder affecting upper and lower motoneurons. since immune disbalance is known to be an important manifestation of the disease we were particularly interested in following the labeled immune cells in the familial als rat model, hsod-1 g93a . a t2-or t1-weighted mri protocol was used with a mini surface coil placed over the skull of the anesthetized animal in a 1.5 t wide bore magnet. in order to compare this approach to standard high field small animal imaging a 7.0 t mri system was also used. there was a congruence of images of lesions in the brainstem at the two field strengths. it was confirmed with gd-dtpa contrast that the blood brain barrier (bbb) is compromised at the interbrain level. in order to study immune cell infiltration rats were i.v. injected with magnetically labeled antibodies against helper cd4+ or cytolytic cd8+ killer t cells. by combined t1, t2 and t2* weighted imaging cd4+ lymphocyte infiltration was observed in the brainstem-midbrain region while the cd8+ cells were more confined to the brainstem region. comparison of mri of labelled cd4+ vs. cd8+ lymphocytes reveals the relevant cellular inflammatory mechanism in als. the appearance of the mri signal from the latter t cell type points to the mechanism of bbb disruption as suggested from a recent study on the role of cd8+ t cells in a model of multiple sclerosis. emodin uptake study in u-87mg cells using optically trapped surface-enhanced raman scattering probes s. balint 1 , s. rao 1 , p. miskovsky 2 , d. petrov 1 1 icfo -the institute of photonic sciences, barcelona, spain, 2 department of biophysics, university of p.j.šafárik, košice, slovakia emodin (1,3,8-trihydroxy-6-methylantraquinone) is a photosensitizing pigment present in plants of herbal laxatives. emodin inhibits nuclear transcription factor-κb activation and induces free radical production in human mononuclear cells resulting in its antiviral and anti-cancer activity. the uptake and distribution of emodin inside the u-87 mg cell line is studied by combining optical tweezers and surfaceenhanced raman spectroscopy (sers). sers greatly enhances the spectrum of an otherwise weakly scattering material which is achieved by attaching nano-sized silver colloids to micron-sized dielectric beads. the distribution of emodin in the cell is studied by simultaneously trapping and exciting the sers bead and scanning it across the membrane while recording the emitted light. secondly, the beads are statically placed inside the cell and excited at certain intervals in order to track the migration of emodin through the membrane. the results give new insight in to the metabolic pathways of emodin and demonstrate a new imaging and detection technique that is fast and less invasive than current standards. acknowledgement: miin fis2008-00114 (spain), fundació cellex barcelona, nadacia spp (slovakia), apvv-0449-07 (slovakia) amphotericin b (amb) is a polyene antibiotic that has been widely used for treatment of systemic fungal infections. the main mechanism of biological mode of action of amb is considered to be associated with formation of ionic membrane pores or channels in the lipid membranes. the aim of this work was to study the influence of the k + and na + ions on the aggregation process of amb in aqueous medium. the analysis of electronic absorption and fluorescence spectra of amb shows that the increasing k + concentration have influence on the level of aggregation of the drug much more than the same amount of na + ions. this effect is especially noticed at neutral ph values. the rls technique was used to study aggregation of amb in solution, in the environment of the k + and na + ions. the application of this technique makes it possible to study the electronically coupled chromophores, especially molecular aggregates. the results of the atr-ftir and raman spectroscopic studies also support this conclusion. these results provide a better understanding of the interaction between k + and na + ions and antibiotic which has not been previously considered to be significant for biological action of amb. g-quadruplexes: combining theory with experimental spectroscopic methods guanine-rich oligonucleotides can form unique tetrameric structures with four coplanar guanine bases, known as gquadruplex motifs. the g-quadruplexes have been found in vivo in the terminal parts of telomeres and other genomic regions. ligands, specifically binding to the g-quadruplex regions inhibit telomerase activity and thus can play an important role in the cancer therapy. most recently, the potential use of these structures has been tested in biosensors and nanotechnology industry. in the present work we use the infrared spectroscopy, including the relatively novel technique of the vibrational circular dichroism (vcd), in a combination with molecular dynamics and quantum chemistry computational methods to investigate the structure and spectroscopic response of the quadruplexes formed by the d(g) 8 and selfassociated dgmp. we obtained a good agreement between the computed and experimental spectra, confirming that the proposed geometrical models are realistic. the vcd technique appears especially convenient for the studies as it can detect the liquid-crystalline phases of the g-quadruplexes by an anomalous enhancement of the signal. j. bogdanovic pristov, a. mitrovic, k. radotic, i. spasojevic institute for multidisciplinary research, belgrade, serbia fructose, due to its high antioxidative capacity, represents a significant component of non-enzymatic defense of some plants against cold-provoked oxidative stress. in the present study, we have investigated role of fructose in seasonal adaptation of picea omorika (pančić) purkinye to cold. this endemic coniferous species is exposed to subfreezing temperatures that range from -10 to -30 • c during the autumn/winter and high temperatures exceeding 30 • c during the summer. characteristic epr signal of free or weakly bound mn 2+ was used as an indicator of oxidative status of needles, since coldrelated oxidative damage leads to mn 2+ release from photosystem ii. it was observed that prooxidative conditions developed in the autumn, at the beginning of cold season, which corresponded to significant increase of fructose level. total sod, as well as mnsod activity also rose significantly higher in the autumn. observed activation of antioxidative system (non-enzymatic and enzymatic) led to adaptation of needles to cold, as oxidative status during winter was decreased and similar to the status of needles in cold-free seasons. calyx of held: sted nanoscopy of a glutamatergic terminal p. bingen 1 , t. m. staudt 2 , c. kempf 3 , h. horstmann 3 , j. engelhardt 1 , t. kuner 3 , s. w. hell 2 1 german cancer research center / bioquant, 69120 heidelberg, germany, 2 max planck institute for biophysical chemistry, 37077 göttingen, germany, 3 institute for anatomy and cell biology, university of heidelberg, 69120 heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of <50 nm inside 3 µm thick tissue sections. three-dimensional shim and 2pem to study collagen arrangement and crimping pattern p. bianchini 1 , m. franchi 3 , l. leonardi 3 , a. diaspro 2 1 lambs-ifom microscobio research centre and department of physics, university of genova, genova, italy., 2 italian institute of technology (iit), genova, italy, 3 department of human anatomical sciences and physiopathology of the locomotorapparatus, university of bologna, italy ligaments have been generally described as multifascicular structures with collagen fibre bundles cross-connecting to each other or running straight and parallel with crimps. a different collagen array and crimping pattern in different ligaments may reflect a different mechanical role. aim of this study was to relate the 3d collagen arrangement and crimping pattern by backward and forward second harmonic imaging microscopy (shim) and 2-photon excitation microscopy (2pem). shim on a laser-scanning system is a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of tissue architecture. although it is a coherent process the multiple scattering through the tissue give us the capability to acquire signal in both backward and forward direction [1] . shim and 2pem were combined in a dual-mode nonlinear microscopy to find out collagen fibre arrangement and crimping pattern. both polarization dependence and differences between forward and backward signals allowed to yield information on local structure [2] . currently used tcspc flim systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. the systems are, however, restricted to count rates on the order of a few mhz. in the majority of applications, such as fret or autofluorescence, the photostability of the samples limits the count rate to much lower values. the limitation of the count rate is therefore no problem. however, if flim is used for ion concentration measurements or imaging of chlorophyll in plants the available count rates can exceed the counting capability of a single tcspc channel. we therefore developed a flim system that uses eight fully parallel tcspc channels. by using a polychromator for spectral dispersion and a multichannel pmt for detection we obtain multi-spectral flim data at a rate of several frames per second. we will demonstrate the application of the system to dyamic changes of the fluorescence lifetime of chlorophyll in living plants. -imaging and spectroscopy scattering effects on non linear imaging of thick biological samples f. cella 1 , z. lavagnino 1 , a. diaspro 2 1 lambs-ifom, microscobio research center, university of genoa, italy, 2 iit, italian institute of technology, genoa, italy non linear optical scanning microscopy became a useful tool for living tissue imaging. biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. while performing imaging of biological scattering tissues in two photon excitation (2pe) regime, the localization of the maximum 2pe intensity was found to shift closer to the surface 1 and the imaging depth limit appears strongly limited by near surface fluorescence 2 . in this work we computed illumination and photobleaching intensity distribution 3 in order to characterize the effects induced by scattering. simulations of 2pe illumination and photobleaching intensity profiles have been performed for different scattering coefficients and at different focus depth. furthermore imaging of fluorescent immobile sample (polyelectrolyte gel) allowed to perform an experimental test on thick turbid media. results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface. [1]ying et al., appl. opt. 38, (1999) . [2] theer p. and denk w, j. opt. soc. am. a. (2006) [3]mazza d. et al., appl. opt. 46 (2007) . biospectroscopic probes for real time measurement of hydrogen-deuterium exchange p. carmona 1 , m. molina 2 1 instituto de estructura de la materia (csic), madrid, spain, 2 escuela universitaria deóptica, madrid, spain isotopic exchange has long been used for the analysis of biomolecular structure and dynamics. hydrogen-deuterium exchange rates depend on ph, temperature and biomolecular environment. this is due to hydrogen bonding, low solvent accessibility, and steric blocking. time resolved measurement of hydrogen-deuterium exchange for subsequent 2d correlation spectroscopic analysis can, then, be very useful to obtain structural information from the said viewpoint. we have developed a microdialysis quartz cell for use in conjunction with raman spectroscopy to investigate hydrogen-isotope exchange reactions of biomolecules. the system requires only 40 µl volumes of the initial substrate and perturbing effluent solutions. we have obtained a d 2 o efflux rate of k d = 0.38 ± 0.008 min −1 with the greatest mwco (18 kda) used here, which involves that an exchange rate of 2.5 min −1 is the limiting rate that could be resolved with the said cell system. analogous results have been obtained using an infrared biospectroscopic microdialysis probe. the use of the method described here has the advantage of avoiding sample dilution (and subsequent signal loss) involved in the known stop flow methods. acknowledgements: the authors gratefully acknowledge financial support from the spanish ministerio de ciencia e innovación (project ctq2006-04161/bqu). polarized transient absorption to resolve electron transfer between tryptophans in dna photolyase photoactivation of dna photolyase comprises electron transfer through the chain fadh • -w382-w359-w306. photo-excited fadh • abstracts an electron from the tryptophan residue w382 in ∼30 ps (monitored by transient absorption spectroscopy). the subsequent electron transfer steps (from w359 to w382 •+ and from w306 to w359 •+ ) are difficult to resolve experimentally, because electron transfer between chemically identical species does not give rise to net absorption changes. to overcome this difficulty, we make use of the fact that polarized excitation (pulse laser) induces a preferential axis (that of the excited flavin transition) in the system (photoselection), and that w359 and w306 form different angles with that axis (known from the crystal structure). thus, polarized detection should allow distinguishing between them. using polarized "classical" transient absorption on a nanosecond time scale and the pump-probe technique on a picosecond scale, we demonstrate the feasibility of the method and provide evidence that electron transfer from w306 to w359 •+ is faster than the 30 ps time constant of the initial electron transfer from w382 to excited fadh • . synchrotron based fourier transform infrared (sr-ftir) microspectroscopy was applied to investigate apoptotic death of u-87 mg cells induced by the photosensitizer hypericin (hyp), in using different transport systems (hyp alone vs. hyp/ldl complexes) and incubation protocols. the differences between ir spectra of non-treated and hyp treated cells are mainly manifested in the positions of amide i and amide ii vibrational bands of proteins. these vibrational shifts are attributed to the protein structure changes from dominantly alfa-helix, in the non-treated cells, to beta-sheets and random coil structures, which prevail 4h and 24h after photodynamic treatment, respectively. the observed conformational changes of proteins can be explained as the consequences of the processes leading to apoptosis as was verified by flow cytometry experiments. the results confirm suggestion that ir spectroscopy can be successfully applied for the detection of early apoptotic processes. a. k. de, d. goswami indian institute of technology kanpur, india molecular fluorescence has been an indispensable tool in modern day optical imaging. one of the state-of the-art challenges in fluorescence microscopy is having better depth resolution as embodied by the confocal and multi-photon laser-scanning microscopic techniques. however, each technique bears its own limitation in having sufficient out-of-focus signal for the former while the low non-linear photon absorption cross-section for the latter. for confocal microscopy using one-photon excitation, we have shown how the clever choice of pulsed illumination instead of continuous-wave excitation leads to a gigantic enhancement in fluorescence that also has immediate applications in microscopy. moreover, single-photon illumination with ultrafast pulses leads to a novel way of achieving axial resolution along with numerous other advantageous applications e.g. reduced photo-bleaching of the chromophore. on the other hand we have thrown new insight demonstrating that the use of mode-locked laser pulses in multi-photon microscopy induces severe solvent-induced photo-thermal damage and prescribed methods to get rid of it. besides, the use of pulse pair excitation in multi-photon microscopy leads to probe and control the dynamics of fluorophores which has crucial role in selective excitation of fluorophores from quantum control perspectives. all these cutting edge research works will be presented in addition to our recent work on application of laser pulse shaping in multi-photon microscopy. interkingdom signalling in pseudomonas aeruginosa b. davis 1 , r. jenson 2 , p. williams 2 , p. o'shea 1 1 institute of biophysics, imaging and optical science, university of nottingham, u.k., 2 institute of infection, immunity and inflamation, university of nottingham, u.k. quorum sensing is the process through which some bacterial species coordinate cell-cell communication. pseudomonas aeruginosa; the pathogen responsible for over 90% of chronic lung infections and the leading cause of mortality in cystic fibrosis patients, expresses two major classes of quorum sensing molecules characterized as n-acyl homoserine lactones and 2-alkyl-4-quinolones. these compounds, in addition to their signalling roles have also been found to possess virulent properties, not only against competing species of bacteria such as staphylococcus aureus but also eukaryotic cells such as t-lymphocytes. the process of bacterial quorum signalling molecules influencing eukaryotic cell activity is termed 'interkingdom signalling'. to date it has been suggested that the quorum sensing molecules outlined above act on eukaryotic cells through interactions with an as yet unidentified plasma membrane or cytosolic receptors. this project is directed towards developing an understanding of how these compounds elicit eukaryotic response through a combination of membrane based interactions at physiologically relevant concentrations. particular emphasis is placed on studies of ligand binding with membrane microdomains in and the consequent downstream signalling are also considered. this work is significant as it will not only lead to a better understanding of pseudomonas infection, but may also lead to the discovery of new classes of agents for the treatment of infective diseases. a xas study of the sulphur environment in human neuromelanin and its synthetic analogues neuromelanin is a complex molecule accumulating in the catecholaminergic neurons that undergo a degenerative process in parkinson's disease. it was shown to play an either protective or toxic role depending on whether it is present in the intraneuronal or extraneuronal milieu. in the present study x-ray absorption spectroscopy is employed to investigate the sulphur binding mode in natural human neuromelanin, synthetic neuromelanins and in certain structurally known model compounds, namely cysteine and trichochrome c. based on comparative fits of human and synthetic neuromelanin spectra in terms of those of model compounds, the occurrence of both cysteine-and trichochrome-like sulphur coordination modes is recognized and the relative abundance of these two types of structural arrangement is determined. data on the amount of cysteine-and trichochrome-like sulphur measured in this way indicate that among the synthetic neuromelanins those produced by enzymatic oxidation are the most similar ones to natural neuromelanin. c. cremer kirchhoff-institute for physics, university of heidelberg, germany here we report on "spectral precision distance/position determination microscopy (spdm) with physically modifiable fluorochromes (spdm phymod ) to analyse the spatial distribution of single nuclear proteins and dna sequences at the macromolecular optical resolution level. like other methods of "spectrally assigned localization microscopy" (salm), spdm phymod is based on labelling 'point like' objects (single molecules) with different spectral signatures, spectrally selective registration and high precision localization monitoring by far field fluorescence microscopy. the intranuclear spatial location of single molecules was determined up to a density up to ca. 1000 molecules/µm 2 of the same type, and distances down to 15 -30 nm were nanoscopically resolved. quantum dots (qds) are semiconductor nanoparticles with increasing application as fluorescent markers in biology.we investigated structure of the cell walls of different species complexed with cdse qds using fluorescence microscopy, fluorescence spectroscopy and ftir techniques. in the experiments we used the cell walls isolated from three distinct plant species: arabidopsis thaliana, acer sp. and picea omorika. we studied both unlabeled and cdse-labeled cell walls. fluorescence spectroscopy and microscopy were used for detection of qds alone or complexed to the cell walls. emission spectra were deconvolved using the nelder-mead algorithm in matlab 6.5. we calculated approximate probability distribution (apd) for positions of spectral component maxima. there was certain difference between unlabeled cell walls and those complexed with qds. the ftir spectra also show some difference between the complexed and pure cell walls. the results show that structure was changed, but not significantly in reaction with cdse qds. these results are promising in context of use of qds as labels in cell wall studies. the characterization of the complex of cell wall structure with qds is a part of the study of nanoparticles application in investigations of plant materials. modulating the response of single neurons and neuronal networks with biophysical stimuli f. difato, a. maccione, l. berdondini, f. benfenati, a. blau italian institute of technology, department of neuroscience and brain technologies, genoa, italy during differentiation, cell processes create connections with other cells to form tissue capable of performing complex tasks. biophysical constraints provide necessary inputs for cellular organization in living organism 1 . to better understand how biophysical conditions influence tissue development, it is necessary to bridge the gap between experiments on single cells and complex tissues 2,3 . to achieve this goal we pair optical tweezers with electrophysiology measurements 4 . by adopting neuronal networks as a biological model, neuronal signal transmission can be recorded either by patchclamp electrophysiology or microelectrode arrays (meas). dissociated neurons will be cultured on meas to record neuronal network activity at different sites of the network while applying spatio-temporally defined biophysical stimuli to individual neurons. a. diaspro 1 , k. cortese 2 , p. bianchini 2 , c. gagliani 2 , c. tacchetti 2 1 iit -italian institute of technology, morego, genova, italy, 2 microscobio, university of genoa, italy correlative light/electron microscopy (clem) is becoming increasingly frequent in molecular and cellular biophysics. we successfully applied the method to analyze the 3d structure of rough and smooth russell bodies used as model systems. the major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibilitˆto perform 3d correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of the high data analysis capability of flm with the high precisionaccuracy of transmission electron microscopy in a clem hybrid morphometry analysis. we have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3d reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach. the relevance of the presented approach lies in two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques. multidrug resistance is a well known phenomenon which limits effectiveness in treating malignancy with chemotherapy by modifying the internalization and/or externalization flow of the drugs through the cancerous cells. combined chemotherapies, such as mvac, are therefore currently used in bladder cancer treatment. however, about 30% of patients do not respond this chemotherapy because of inherent or acquired drug resistance. we developed a non invasive predicative test on urinary cells to estimate the chemotherapy effectiveness before treatment, based on the fluorescence emission of mvac. we first studied the mvac photophysical properties in solution and using five cell lines: a drug sensitive cancer cell line mgh-u1s, its multidrug resistant subline mgh-u1r, a not tumorigenic cell line sv-huc-1, its tumorigenic counterpart mc-sv-huc t-2 and a cell line from transitional cell carcinoma t24. the results revealed a penetration and localization of the drug depending of the cell line type, allowing us to find a specific fluorescence signature for the identification of mvac resistant cells. similar data have been obtained for cytospined fixed culture cells and patients urinary cells. -imaging and spectroscopy -abstracts multilayered photoresist system as a ghost model for biological samples in confocal microscopy. l. ferrari, f. cilloco, f. r. bertani, s. selci istituto dei sistemi complessi cnr rome we have realized a bilayer photoresist system as a model to perform optical characterization in the vis-ir range of multilayered complex biological specimen. our goal is to obtain structural and spectroscopic reference parameters from the identification of reflectance pattern features within a novel project granted by miur (skintarget, ideas firb). our model is composed by a 1300 nm thick layer (shipley photoresist 1813) with a refractive index n= 1.6-1.8, wavelength dependent, and a 600 nm tick hsq (hydrogen silsesquioxane) layer with a refractive index n around 1.38, both spin-coated over a glass substrate. we present the analysis of reflectance pattern that can be obtained by a confocal laser reflective system as compared to the expected values as coming from analytical calculations, using a matrix approach, or a microscopic electromagnetic finite element analysis. interfacial roughness and consequent optical scattering are analyzed using a neural network approach. translational biophysics: multimode imaging for preclinical and clinical applications d. l. farkas cedars-sinai medical center, los angeles, usa our focus is where light and patient meet, and improvements yielding better outcomes. surgery is moving towards minimally invasive intervention, where biophotonics represents a major area of hope and growth. the translation of useful laboratory-derived knowledge into clinical practice has been hampered by the difficulty of detecting, characterizing and monitoring molecules and cells in the human body, especially dynamically. advanced bi ophotonic imaging is best suited for studying such entities, but has been lagging in clinical acceptance, in spite of major advances, and a clear need for the kind of resolution (spatial, temporal, spectral) and specificity that it alone can offer. biophysics-based new strategies are needed to address this challenge. the development of biophysical methods for translational medicine will be reviewed, with emphasis on our recent advances. our approach is a multimode one -combining methods to achieve early, quantitative detection of abnormalities. with imaging fulfilling its dual role of better describing anatomy and physiology, intrasurgical histopathologyequivalent molecular and cellular imaging is achievable in vivo, as is a closer spatio-temporal connection between imaging and intervention. some application areas to be covered: cancer (early detection by spectral reflectance/autofluorescence; progression quantitation by oct; nano-and targeted chemotherapy assessment in vivo); neurobiology (imaging fast calcium transients and alzheimer's plaques); hyperspectral mie scattering imaging for in situ displasia; design and use of an advanced multimode imaging endoscope with in vivo delineation of hirschsprung's disease for better intervention; monitoring of stem cell fate in vivo. immobilization of liposomes in a sol-gel matrix: a fluorescence confocal microscopy study r. esquembre 1 , s. n. pinto 2 , j. a. poveda 1 , m. prieto 2 , c. r. mateo 1 1 ibmc, universidad miguel hernández, elche, spain, 2 cqfm, instituto superior técnico, lisbon, portugal immobilization of liposomes shows interesting applications in protein biology, membrane biophysics and biosensor technology. previous fluorescence spectroscopy works revealed that the entrappement of zwitterionic phospholipid liposomes in a silica sol-gel matrix alters the thermodynamic properties as well as the fluidity of the lipid bilayer. interactions between the polar head of phospholipids and the porous surface of the host matrix could be responsible of such behaviour. in order to get more insight into this possibility we have immobilized, for the first time, giant unilamelar vesicles (guvs) and the shape and size of these structures as well as the possible existence of lipid domains have been visualized through fluorescence confocal microscopy. this technique allows for direct observation of the effect of encapsulation on an individual liposome, in contrast to the averaged information given by macroscopic spectroscopic techniques. liposomes composed of pure popc or dopc as well as mixtures dopc/dppc were labelled with the fluorescent probes bodipy, rhd-pe and rhd-dope. preliminary results shows that only the smallest guvs (6-10µm) survive to the encapsulation process but often with a slight loss of its sphericity, probably due to pressures suffered during the matrix gelation. however no change in the gel/fluid phase proportion has been observed for immobilized dopc/dppc guvs, regarding to solution. solvent fluctuations play a key role in controlling protein motions and functions. here, we have studied how the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (por) couple with solvent dynamics (g. durin et al, biophysical j. (2009 ) 96, 1902 . to simultaneously monitor the catalytic cycle of the enzyme and the solvent dynamics, we designed temperature-dependent uv-visible microspectrophotometry experiments, using flash-cooled nano-droplets of por. the temperature-dependant formation of the first two intermediates in the por reaction were measured, together with the solvent glass transition temperature (t g ) and the build-up of crystalline ice. we find that formation of the first intermediate occurs below t g and is not affected by solvent dynamics, whereas formation of the second intermediate occurs above t g and is influenced by solvent dynamics. these results suggest that internal protein motions drive the first step of the por reaction whereas solvent slaved motions control the second step. we propose that the concept of solvent slaving applies to complex enzymes such as por. amphotericin b (amb) is one of the main polyene antibiotics widely used to treat deep-seated fungal infections. the mechanism of biological action of amb is most probably directly related to the ability of the drug to form hydrophilic pores in the membrane core, thus affecting physiological transport of ions. the effects of amb-cu 2+ complexation are demonstrated by the electronic absorption and fluorescence spectra. the absorption spectra of amb in water (ph=7) after the injection of water solution of copper(ii)sulfate display a complex structure with hypsochromic-and bathochromic-shifted bands indicative of formation of molecular aggregates of the drug. formation of the electronically coupled chromophores of amb, especially aggregates, was analyzed at different cu 2+ concentrations by the rls (resonance light scattering) technique. intensity of the fluorescence emission spectrum (characteristic of the dimeric form of amb) decreases after the amb-cu 2+ complex formation. this effect of the formation of the amb aggregated structures by amb-cu 2+ are different from the spontaneous molecular aggregation process, as deduced from the spectroscopic analysis. morfo-functional asymmetry of the olfactory receptors of the honeybee apis mellifera l e. frasnelli 1 , g. anfora 2 , f. trona 2 , f. tessarolo 3 , r. antolini 3 , g. vallortigara 1 1 cimec, centre for mind/brain sciences, university of trento, italy, 2 iasma research and innovation center, fondazione e. mach, s. michele all´adige (tn), italy, 3 biophysics and biosignals lab., dept. of physics, university of trento, italy lateralization, i.e. the different functional specialisation of the left and right side of the brain, has been documented in many vertebrate and, recently, invertebrates species. in the honeybee apis mellifera l. olfactory memory seems to involve at first the use of the right antenna. the present study investigated physiological and anatomical differences between left and right antennae of honeybees. electroantennographic responses (eag) were recorded from the left and right antennae of 16 honeybees to linalool, a floral volatile compound, and isoamyl acetate, an alarm pheromone, at 5 doses (from 0,25 to 2500 µg). the number of sensilla on the left and right antennae was recorded by scanning electron microscopy (sem). each antenna segment, from 14 insects, was observed from 4 different viewpoints in order to image the whole antenna surface and compute the number of sensilla. the tested compounds induced higher eag responses on the right than on the left antenna at every dose. sem showed that the placoidea olfactory sensilla were slightly more abundant on the right antenna surface than on the left one. results suggest an asymmetry in the peripheral odour perception mechanism in the honeybee a. mellifera. super-resolution imaging of dna through single molecule switching of intercalating cyanine dyes c. flors, c. n. j. ravarani, d. t. f. dryden school of chemistry, university of edinburgh, u.k. a growing trend in far-field super resolution fluorescence microscopy involves the replacement of photoactivatable fluorophores by common dyes such as cy, atto or alexa [1] . it has been shown that these dyes can blink in useful timescales for single-molecule based imaging by adding suitable buffers. this strategy greatly simplifies the sample preparation and imaging scheme, enabling its application to a wider range of biological systems. we have explored if a similar approach might be useful to study dna topology using intercalating cyanine dyes such as yoyo-1. there are two main advantages of this approach: i) dna labelling with intercalating dyes is straightforward, and ii) the free dye in solution is essentially non-fluorescent, greatly reducing the fluorescence background. we show that yoyo-1 can blink in the absence of oxygen and in the presence of cysteamine, which allows its application to nanoscale imaging. we exemplify its use by imaging λ-dna and a puc19 plasmid. we also explore the compatibility of several intercalating dyes with biological systems such as enzymes or cells. our results suggest that dna intercalating dyes are a promising option for fluorescence super-resolution studies of dna topology. seeing more in total internal reflection fluorescence (tirf) microscopy r. fiolka, a. stemmer nanotechnology group, eth zürich, zürich, switzerland total internal reflection fluorescence (tirf) microscopy is an effective widefield imaging tool that selectively excites a very thin sample layer within the evanescent excitation field at the glass-water interface. lateral resolution of standard tirf microscopy is limited to approx. 240nm using green emission, which can be insufficient for a large class of biological investigations. additionally, the evanescent excitation field is prone to light scattering, creating out of focus blur in the final image. we present several techniques that address these mentioned shortcomings of standard tirf microscopy. using evanescent standing waves, the lateral resolution in tirf microscopy can be extended by a factor of 2.5, reaching 90nm. we further show techniques to reduce the blur induced by light scattering of the evanescent field. finally, we demonstrate optical sub-sectioning capabilities in tirf microscopy by acquiring several images with different penetration depth of the evanescent field and applying suitable post processing algorithms. thereby the obtainable z-resolution exceeds the classical limit of widefield microscopy, and object structures lying within the evanescent field can be reconstructed. the natural photosentizer hypericine (hyp) exhibits potent properties for tumor diagnosis and photodynamic therapy. evidences of hyp release from ldls prior to passive diffusion within cells are addressed in this study. fluorescent properties of hyp have been used for dynamic studies of its interaction with low-density lipoproteins (ldls) and u87 glioma cells. subsequent non-specific staining of intracellular membranes compartment were observed by mean of colocalization fluorescent imaging studies. it was shown, that monomers of hyp are only redistributive forms. increasing of hyp concentration leads to the formation of non-fluorescent aggregates within ldls as well as within the u87 cells, and can preclude its photosensitizing activities. in all experiments, hydrophobic character of the molecules appears as the driving force of its redistribution process. acknowledgment: this work was supported by the slovak res. and dev. agency contracts no. lpp-0337-06. we also kindly thank the synchrotron soleil for using the detection system of the disco beamline. ledgf/p75 switches from a dynamic to a tight chromatin interaction upon binding to hiv-1 integrase j. hendrix 1 , z. debyser 2 , y. engelborghs 1 1 laboratory of biomolecular dynamics, katholieke universiteit leuven, belgium, 2 laboratory of molecular virology and gene therapy, katholieke universiteit leuven, belgium human transcriptional co-activator ledgf/p75 is hijacked by hiv-1 integrase (in) during the replication of hiv. little is still known about the molecular complex of these two proteins in the living cell. in this work we first studied the cellular chromatin interaction of egfp-tagged ledgf/p75 with tunable-focus fluorescence correlation spectroscopy (tf-fcs) and show that ledgf/p75 is in equilibrium between a free brownian motion and a very slow movement on the chromatin. being dependent on the size of the laser focus, this slow movement represents a continuous associationdissociation-reassociation process that is governed by diffusion. concentration-dependent continuous photobleaching measurements (cp) furthermore revealed the existence of high-affinity chromatin binding sites. next, we co-expressed mrfp-tagged in and confirmed its intracellular interaction with ledgf/p75 by fluorescence cross-correlation spectroscopy (fccs). interestingly, cp and fluorescence recovery after photobleaching (frap) indicated that the affinity of this complex for chromatin is exceptionally high. by twophoton fluorescence lifetime imaging (2p-flim) we verified if the cellular stoichiometry was altered when the proteins were expressed together. we believe that this work is useful for the understanding and targeting of hiv-replication. biophysical identification of orf10 from clavulanic acid biosynthesis cluster as a cyp450 l. s. goto 2 , c. o. hokka 1 , j. f. lima 2 , o. r. nascimento 2 , a. p. u. araújo 2 1 grupo de engenharia bioquímica, ufscar, br, 2 grupo de biofísica molecular "sérgio mascarenhas", usp, br streptomyces clavuligerus produces the clinically important β-lactamase inhibitor clavulanic acid. biosynthesis related genes reside in three gene clusters, one of these, named clavulanic acid gene cluster, includes most of the known clavulanic acid biosynthetic enzymes. the penultimate step along clavulanic acid biosynthesis remains unclear. required transformation involves at least two events: oxidative deamination and double epimerization of (3s , 5s )-clavaminic acid into (3r, 5r)-clavaldehyde. downstream the known part of clavulanic acid cluster lays orf10 , a putative gene encoding a tentative cytochrome p450-like protein which knockout has been proven deleterious to clavulanic acid biosynthesis. should such protein exist, it would be candidate to fulfill the clavulanic acid pathway missing step. in this work, orf10 encoded protein is characterized aiming to place it as a real p450. for this task, molecular cloning and recombinant expression of orf10 were accomplished. purified protein was submitted to spectroscopic measurements such as circular dichroism and electron paramagnetic resonance which indicate p450 features, including catalytic relevant heme iron redox states and homolytic peroxide scission mechanisms. further, peroxide reaction adducts were characterized by spin trapping. this work is supported by fapesp. muscle structure and gabaergic innervations in the limbs of barnacle cyprid l. gallus 1 , s. ferrando 1 , c. gambardella 1 , a. diaspro 2 , p. bianchini 2 , p. ramoino 3 , v. piazza 4 , g. tagliafierro 1 1 libiom, dibio, università di genova, italy, 2 ifom-lambs/microscobio, difi, università di genova, italy, 3 dipteris, università di genova, italy, 4 ismar cnr, venezia, veneto, italy balanus amphitrite is a sessile crustacean that settles at the larval stage of cyprid. in this stage we studied the gabaergic innervation of limb striated muscular fibers, by immunohistochemistry. the second harmonic generation (shg) and 2-photon excitation (2pe) microscopy were used to set out the muscle structure and its relationship with nerve terminal. sections were observed at a multimodal nonlinear microscope composed by the leica clsm. the laser system used is a ti:sapphire chameleon-ultra (coherent inc, santa clara, ca, usa), tunable between 690 nm and 1040 nm and characterized by a pulse width of 140 fs delivered at a repetition rate of 90 mhz by means of a homebuilt set-up (bianchini p. and diaspro a. j biophotonics 2008 1:443-50). the z-stacks were performed in order to obtain the 3-dimensional distribution of the muscular fibers. in the posterior ganglion gad immunoreactive (ir) motor neurons were arranged in 12 clusters near the emergence of the limb nerves. gaba and gababr1 ir neuromuscular junctions (nj) were localized in the limb muscle fibers; vgat ir cells surrounded each limb muscles. these results suggest that gaba plays a key role in the regulation of limbs movement. the shg was very useful to outline the relationship between nerve terminals and limbs muscle fibers. giant unilamellar vesicles (guvs) are very useful model membrane systems to study many aspects of lipid-lipid and lipid protein interactions, particularly employing fluorescence microscopy related techniques (bagatolli 2006) . the use of this model system can be particularly useful to study aspects related with the lateral structure of bacterial membranes using the aforementioned approach (i.e. fluorescence microscopy). bacterial cells have a size close to the resolution limit of optical microscopy and details about the organization of their membranes are not easy to achieve using such technique. recently a new electroformation method to prepare guvs composed of compositionally complex mixtures under physiological conditions was developed in our laboratory (montes 2007). in the present work we further extended this electroformation method to prepare guvs composed of bacterial lipid extracts and lipopolysaccharide (lps). in our experiments we used e.coli lipid extract to prepare small vesicles containing various lps species, from smooth strains and rough mutants. suvs were used as starting point to electroform guvs using various types of buffers with high ionic strength. the successfully obtained bacterial-guvs were used to study the interaction of these membranes with known lps-binging proteins (lung surfactant protein d, sp-d) and peptides (polymyxin b). our results remark the usefulness of this particular bilayer models to perform studies mimicking bacterial membranes. mechanical properties of polymeric membranes probed by afm m. kocun 1 , i. mey 1 , w. müller 2 , m. maskos 2 , a. janshoff 1 1 georg-august universität, institute for physical chemistry, göttingen, germany, 2 johannes gutenberg universität, institute for physical chemistry, mainz, germany many biological cell functions are dependent on the mechanical properties of the membrane. polymeric membranes that mimic native cell membranes are valuable research tools which can be used to better understand the physics of biological membranes. we have investigated free standing artificial membranes prepared from polybutadiene-b-polyethylene oxide (pb-b-peo). the membranes were prepared from vesicles ruptured on porous silicon substrates. these polymeric membranes were studied by confocal laser scanning microscopy (clsm) and atomic force microscopy (afm). force indentation curves were performed on the membranes and theoretical models were used to extract elasticity constants from the results. the study of polymeric membranes can give insight to the function of biological membranes, furthermore polymeric membranes can be used to create new hybrid systems by incorporating biological (lipids, proteins), artificial (polymers, dyes) and inorganic (nanoparticles) components. site-directed spin labeling study of the lightharvesting complex cp29 the topology of the long n-terminal domain of the photosynthetic light-harvesting complex cp29 was studied using electron spin resonance (esr). cp29 is a minor antenna complex of the photosystem ii (psii), a multisubunit protein complex. wild-type cp29 protein containing a single cysteine at position 108 and nine single cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. in all cases the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. the spin label esr spectra were analyzed in terms of a multi-component spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. these results permit to trace the structural organization of the long n-terminal domain of cp29. we took the crystal structure of light-harvesting complex ii (lhcii), major antenna complex of psii available in pdb as a starting point and constructed a model for cp29 based on esr data. present opportunities and future developments in soft x-ray transmission and emission microscopy b. kaulich, a. gianoncelli, v. babin, m. kiskinova elettra -sincrotrone trieste, s.s. 14 km 163.5 in area science park, i-34012 trieste-basovizza, italy soft x-ray transmission and emission imaging and spectromicroscopy are bridging the gap to other microscopy techniques in terms of lateral resolution, penetration depth and chemical sensitivity. the novel soft x-ray spectromicroscopy approach of the twinmic instrument at the elettra synchrotron radiation facility is combining several imaging modes for morphology characterization, such as scanning, projection and full-field imaging with several contrast techniques including brightfield, darkfield, differential phase and interference contrast at sub-100 nm lateral resolution. complementary chemical information is provided by chemical imaging and micro-spectroscopy using the photon-in/photon-out xray absorption and x-ray fluorescence. unique for twinmic is the low-energy x-ray fluorescence setup operated at 280 -2200 ev photon energies, which allows simultaneous analysis of the morphology, and the distribution of light elements on cellular and sub-cellular level. in the presentation the principles of the methods used in the twinmic instruments the performance and potentials of the instrument will be demonstrated by several examples of applications in the field of human, animal and plant biology, biophysics and chemistry, physiology and genetics. the potential impact of microscopy techniques using free-electron x-ray lasers on biophysics will be illustrated by the diproi project at fermi@elettra. -imaging and spectroscopy fluorescence anisotropy and afm used as tools to characterized porin´s reconstituion in luv´s s. c. lopes, i. sousa, p. eaton, p. gameiro requimte, faculdade de ciências, universidade do porto, rua do campo alegre, 4169-007 porto, portugal a major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that assure, not only, a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. for this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. in this study we report the reconstitution of ompf in preformed dmpc and e. coli liposomes using two different techniques for detergent removal: (1) exclusion chromatography and (2) incubation with detergent adsorbing beads. the incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine ompf effect on the structural order of membrane lipids. these results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. moreover more anisotropy and atomic force microscopy studies will allow a better characterization of bacterial model system membranes. the wavelength dependence of the luminescence for different sized aunp by 2-photon clsm k. li, m. schneider pharmaceutical nanotechnology, saarland university, campus a4 1, d-66123 saarbrücken, germany gold nanoparticles (aunp) with different sizes can exhibit original luminescent properties if excited with pulsed nearinfrared laser light which makes them a suitable object to be detected in biological tissues. the main obstacle is to distinguish between the object of interest (emitted light) and the autofluorescence from the sample which limits the scope of application of aunp. therefore, our aim is to characterize the luminescent properties of such nanoparticles regarding their excitation and emission. in this study, the excitation and emission spectra of aunp with different sizes below 50 nm in an excitation range of 720 nm to 900 nm were investigated. our study shows the emission spectra curves of aunp are broadband spectrum and vary with the changing of excitation wavelength from 720 nm to 900 nm. the results also suggest the minimum laser power necessary to trap the aunp depends on particle size and excitation light wavelength and a maximum power above which the particles are destroyed. in all the experiment above, to avoid the simultaneous effects from slide and cover slip is a must. rocking, tumbling, and sliding: real-time nanomotion of a membrane-bound virus p. kukura 1 , h. ewers 2 , c. mueller 1 , a. renn 1 , a. helenius 2 , v. sandoghdar 1 1 laboratory of physical chemistry, eth zurich, ch-8093 zurich, switzerland, 2 institute of biochemistry, eth zurich, ch-8093 zurich, switzerland the interaction of a virus with its receptors in the plasma membrane is decisive for its infection of cells. optical studies have revealed that after binding, virus particles move laterally on the membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings. here, we examine a model system, in which single simian viruses (sv40) interact with their gm1 ganglioside receptors in supported lipid bilayers. we employed scattering interferometry and single molecule fluorescence localization to visualize the vectorial rotational motion of virions. at low receptor concentration, we observed sliding and tumbling of single virions during rapid lateral diffusion. in contrast, at increased receptor concentration the virions repeatedly underwent periods of standstill, reminiscent of their behavior prior to endocytosis. by an unprecedented combination of millisecond time and nanometer spatial resolutions, we revealed that during these immobile periods, the virions rock back and forth among nanoscopic spots separated by 9 nm. our insights, together with the structure of the viral capsid, suggest aggregation of receptors in nanodomains and recurrent swap of binding between receptor molecules and neighboring viral protein pentamers. herein, we have developed different membrane probes binding spontaneously to the outer plasma membrane leaflet and showing sensitivity to membrane properties such as surface charge and phase state. the first two types of probes were based on 3-hydroxyflavone fluorophore. one of them showed a high sensitivity to the surface charge and to the phase state of lipid bilayers, while the other was only sensitive to the surface charge. the third type, which was based on nile red fluorophore, was sensitive only to the phase state. surprisingly, the probes that were sensitive only to the surface charge did not respond to apoptosis, while the other two types of probes showed significant spectroscopic response to it. moreover, the latter exhibited response to cholesterol depletion, which was similar to that observed on apoptosis. thus, according to our data, the intact living cells present a remarkable fraction of the cholesterol-rich domains, while apoptotic loss of the transmembrane asymmetry decreases it dramatically. these probes represent a new tool for quantification of surface charge and cholesterol-rich domains in cell membranes. -imaging and spectroscopy due to its porosity and unique optical properties porous silicon (psi) is an attractive template to develop biomaterials and biosensors. porous silicon microcavity (psimc) structures were prepared then functionalized for covalent protein attachment of glucose oxidase (gox) or solubilized bacteriorhodopsin (br). functionalization and protein infiltration was monitored by specular reflectometry sensitive to change in refractive index, when a molecule is attached to the large internal surface of psi. protein infiltration into the porous scaffold was confirmed by edx spectroscopy and the structures were imaged by biphoton microscopy. second harmonic generation and enhanced two-photon excited fluorescence emission from porous silicon was observed when resonantly exciting the structures. in addition, when the microcavities were infiltrated with gox or br, the proteins acted as a very efficient internal two-photon-excited fluorescence emitter, hence protein infiltration enabled the in-depth visualization of the porous structure by taking advantage of the optical sectioning capacity inherent to the non linear optical microscopy technique. patterning of bio-molecules: methods for characterization of neuron-substrate interfaces we investigated how to use and improve micro printing techniques to obtain molecules patterns on cell culture substrates. with micro contact printing (µcp), we generated geometrically defined depositions of poly-d-lysine (pdl) using poly-dimethylsiloxane stamps and optimized neuronal culturing conditions. rat and mice hippocampal neurons grown on those patterns showed to be alive and functional. we then applied µcp to study axonal development by combining cell culture assays with atomic force microscopy (afm) to investigate in more detail the molecule deposition on the surface and to measure morphological changes in the growth cone (gc) during the early phases of neurite development. we found distinct shapes of the gcs depending on whether they were growing on l1 adhesion molecule patterned by indirect-µcp or on pdl coated surfaces. we also attempted to transfer such patterns on multi electrode arrays in order to constrain neuronal cell bodies on the electrode area and to improve electrophysiological recordings from neuronal networks. other patterning techniques were therefore explored using a nano-drop printing system. patterned surfaces were analyzed with afm and scanning electron microscopy to combine different approaches aimed to the improvement and characterization of the printing techniques. insights on the mechanism of electron transfer in complex i a. l. maniero 1 , c. bergamini 2 , m. bortolus 1 , r. fato 2 , s. leoni 2 , g. lenaz 2 1 università degli studi di padova, padova, italy, 2 università degli studi di bologna, bologna, italy complex i (nadh dehydrogenase) plays a central role in cellular energy production, transferring two electrons from nadh through a series of iron-sulfur clusters (fes) to ubiquinone (coenzyme q); the electron transfer is coupled to the translocation of protons across the membrane. the fes center n2 is the last acceptor in the electron-transfer chain, but the mechanism through which the enzyme couples the 1e − reduction of the fes centers to the 2e − reduction of ubiquinone (q→sq→qh 2 ) is unclear [1] . in our experiments, submitochondrial particles were treated with different inhibitors, in the absence or in the presence of different quinone analogues, and nadh addition initiated the electron transfer. we assess by epr (electron paramagnetic resonance) spectroscopy the relative abundance of the reduced n2 center and of the semiquinone radical, and coupled epr data to enzymatic activity assays and to fluorescence measurements on the effect of inhibitors on reactive oxygen species (ros) production. we identify two different classes of inhibitors showing different effects on ros production. moreover the redox state of the complex has shown to depend both on the inhibitors and on the quinone analogues. a possible mechanism of the electron transfer, that can explain the experimental findings, will be presented. conventional fluorescence microscopy suffers from a resolution limit imposed by the diffraction of light. stimulated emission depletion (sted) microscopy overcomes this resolution barrier without being limited by the wavelength by taking advantage of the photophysics of the observed sample into the image formation process, and has proven to be a powerful approach for exploring relevant biological issues. the outstanding resolution of sted microscopy is achieved by drastically minimizing the spatial extent of the focal region from which fluorescent molecules can emit signal in the sample. so far, mainly complex and relatively expensive lasers systems providing pulsed beams have been used to inhibit the fluorescence in the outer area of the focal region. here we report on the development of a new setup based on a fast beam scanning confocal microscope using compact turn-key and inexpensive continuous wave lasers. the great potential of this simple configuration is demonstrated for a selection of commonly used fluorescent markers. characterization of hepatitis b antigen particles by atomic force microscopy each year, over one million people die from hepatitis b virusrelated chronic liver disease, including cirrhosis and hepatocellular carcinoma. the major surface antigen of hepatitis b virus (hbsag) is a cysteine-rich, lipid-bound protein with 226 amino acids. recombinant hbsag produced in yeast can self-assemble into 22-nm immunogenic spherical particles that are used in licensed hepatitis b vaccines (protein/lipid ratio is 60/40 in mass). hbsag size and shape have been mainly investigated by transmission electron microscopy after negative staining of the particles. however, under these conditions, no details of the particle surface can be obtained because of the shadowing effect due to the uranium salts. here we describe new structural insights of hbsag particles using atomic force microscopy (afm) performed under physiological conditions. we applied atomic force microscopy to define structural details of the surface organization with a resolution in the nanometer range. as expected, the diameter of hbsag particles is 26,8 ± 2,5 nm in average. the surface of these particles clearly shows the presence of protuberances that most probably correspond to proteins. indeed, reduction and alkylation induces the disappearance of the protuberances. the number of the protuberances estimated from afm micrographs is about 70 per spherical particles. a biophysical study of equine herpesvirus-1 entry into cells g. mckenzie 1 , p. o'shea 2 , j. kydd 1 , c. rauch 1 1 school of veterinary medicine and science, university of nottingham, uk, 2 institute of biophysics, imaging and optical science, university of nottingham, uk equine herpesvirus-1 (ehv-1) can cause respiratory disease in young horses, varying degrees of paralysis and abortion during the later stages of pregnancy. furthermore, there has been a recent increase in the number of outbreaks involving paralysis of thoroughbred horses in training. virus disseminates rapidly after initial infection via cell associated viraemia. controlling this may prove crucial in combating the pathogenicity of the disease. we have investigated the cellular interactions of ehv-1. initially, binding of ehv-1 to fluorescein phosphatidylethanolamine (fpe)-labelled phospholipid vesicles of various compositions was examined. a variety of microscopy techniques were then employed to study the events surrounding the binding of virus to equine peripheral blood mononuclear cell (pbmc) membranes. confocal microscopy images have highlighted possible colocalisation of ehv-1 with membrane 'rafts'. total internal reflection fluorescent microscopy was then used to identify viral binding at the membrane with high contrast images, able to observe single virus particles. establishing the viral entry pathway into pbmcs would allow drugs that target this process to be employed, reducing clinical viraemia. the interpretation of in-vivo binding rate measurements allows inferring the molecular interactions that regulate cellular functions. fluorescence recovery after photobleaching (frap) is a widely used tool to quantify binding reactions in vivo. however the lack of "golden standards" for these measurements requires the measured binding rates to be validated with other techniques. we present a new fluorescence correlation spectroscopy (fcs) method to measure the in vivo bound fractions and residence times for molecules that interact with an immobile substrate. we apply this method to measure binding of mutants of the transcription factor vbp (vitellogenin binding protein) to the dna. comparison of fcs with frap results in comparable estimates of the measured diffusion constants and bound fractions. however, fcs provides an estimate of the vbp residence time at the dna, while frap does not. this limitation in the analysis of vbp is due to the larger photobleaching volume used in frap, if compared to the observation volume of an fcs experiment. in sum, we present a method to measure binding rates with fcs. substantial agreement of this method with frap is shown. however, further validation on tightly bound molecules will be necessary to assess if frap and fcs agree in the measurement of residence times. -imaging and spectroscopy we have investigated the changes in the mechanical properties of the zona pellucida (zp), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. the response of the zp to mechanical stress has been recovered according to a modified hertz model. zp of immature oocyte's shows a pure elastic behaviour. mature and fertilized oocyte's zps evidence, instead, a transition from a purely elastic behaviour, which occurs when low stress forces are applied, towards a plastic behaviour has been observed. the high critical force necessary to induce deformations, that well supports the non-covalent long interactions lifetimes of polymers, increase after the cortical reaction. afm images show that oocytes' zp surface appear to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles more wrinkled surface characterize marure oocytes with respecto to immature and fertilized oocytes from a mechanical point of view, the transition of the mature zp membrane toward fertilized zp, through the hardening process, consists in the recovery of the elasticity of the immature zp, while maintaining a plastic transition that, however, occurs with a much higher force with respect to that required in mature zp. dynamical behavior of ribose and deoxyribose supercooled water solutions s. e. pagnotta 1 , s. cerveny 1 , a. alegria 2 , j. colmenero 3 1 centro de fisica de materiales, centro mixto csic-upv/ehu, san sebastián, spain, 2 departamento de fisica de materiales, universidad del pais vasco (upv/ehu), facultad de quimica, san sebastián, spain, 3 donostia international physics center, san sebastián, spain ribose and 2'-deoxyribose are probably the most widespread monosaccharides in nature. they can be extensively found in ribonucleic acid (rna) and 2'-deoxyribonucleic acid (dna), respectively, where they form, together with a nitrogenous base and a phosphate group, a peculiar building-block structure called nucleotide. in the present work, the relaxation dynamic of ribose and deoxy-ribose water solutions at different concentrations has been studied by broadband dielectric spectroscopy and differential scanning calorimetry in the temperature range of 150-250k. two relaxation processes are observed for all the hydration levels; the slower (process i) is related to the relaxation of the whole solution whereas the faster one (process ii) is associated with the reorientation of water molecules in the mixture. as for other polymeric water solutions, dielectric data for process ii indicate the existence of a critical water concentration above which water mobility is less restricted. moreover, according with these results, atr-ftir measurements of the same sugar solutions showed an increment in the intensity of the oh stratching sub-band close to 3200 cm −1 as water content increases. the regulation of the formation of cytoskeletal protein complexes by actin-binding proteins m. nyitrai, a. vig, t. kupi, z. ujfalusi, s. barkó, g. hild university of pécs, faculty of medicine, department of biophysics, pécs, hungary in living cells various groups of proteins are associated to supramolecular actin filament structures, often in a nucleation factor dependent manner. for example, actin structures associated with formins can bind tropomyosin and profilin, while those polymerised by the nucleation of the arp2/3 complex bind cofilin and myosin i. the molecular mechanisms underlying the regulation of the formation of these protein complexes is still ambiguous. we have shown recently that formins can bind the actin filaments and change their conformational state. subsequent binding of other actin-binding proteins, such as tropomyosin and myosin, can reverse these changes. it appears that the reversal effect assumes that the actin-binding protein binds the filaments in a well-defined and specific binding site. the altered conformational state of the actin filaments observed after the binding of these proteins provides a possible explanation for the modified affinity of the filaments for other-actin binding proteins. based on the results available so far we assume that the affinities are modified differently by different nucleation factors, and the conformational changes introduced to actin by actin nucleation factors can serve as the molecular bases for the regulation of the formation of actin based intracellular protein complexes. experiments are currently in progress to test and further corroborate the existence of such regulatory mechanisms in living cells. correlation between sub-cellular distribution of photoactive drug hypericin (hyp), determined by applied delivery systems (hyp vs hyp/ldl), and mode of the cell death is addressed in this study. co-localization of hyp with mitochondria, lysosomes and golgi apparatus in u-87 mg glioma cells was determined by confocal laser scanning microscopy in using organelle specific fluorescent dyes as well as by time resolved fret experiments. flow cytometry experiments were realized to study a photodynamic effect of hyp (598 nm/4 jcm −1 ) on cells. significant differences in the proportional representation of live, apoptotic and/or necrotic cells were observed for different types of delivery systems of hyp 24 hours after hyp (5x10 −7 m) photoactivation. conclusions: i) sub-cellular distribution of hyp depends on using delivery systems, ii) the mode of cell death depends more on concentration of hyp inside cells, than on different type of delivery systems (for non selective wide-field cell illumination), iii) fluorescence lifetime is sensitive parameter to study sub-cellular distribution of hyp. novel time-resolved spectroscopic methods have been used to investigate the interactions between a fluorescently-labelled mutant of the peptide melittin and supported lipid bilayers, formed by self-assembly at a silica-water interface via vesicle deposition. time-resolved evanescent wave-induced fluorescence spectroscopy (trewifs) is a surface-selective technique in which the evanescent field from a pulsed laser source is used to photoexcite fluorescent species at an interface. the resulting fluorescence decay kinetics, measured using time-correlated single-photon counting, report on the micro-domains experienced by those fluorescent species at an interface. extending trewifs to time-resolved evanescent wave-induced fluorescence anisotropy measurements (ew-trams) provides dynamic rotational information of a fluorescent species, reporting on its mobility at an interface. presented here are trewifs and ew-trams data obtained from the fluorescence of an alexa 430-labelled melittin mutant interacting with a dipalmitoylphosphocholine bilayer at room temperature, physiological ph and ionic strength. the results provide new insights into the conformation, location and motion of cytolytic peptides interacting with cell membranes. optical tweezers are used to controllably apply forces to red blood cells and the resulting chemical and structural changes are monitored using raman spectroscopy. the forces are applied in vivo and mimic that which the cell undergoes mechanically as it passes through vessels and smaller capillaries. the first result presented will be spectroscopic evidence of a transition between the oxygenation and deoxygenation states of hemoglobin that is caused by the stretching of the red blood cell. the transition is due to mechanically induced enhancements of hemoglobin-membrane and hemoglobin neighbor-neighbor interactions. the latter, lesser known effect is further studied by modeling the electrostatic binding of two of the protein structures using molecular dynamics methods. secondly, polarized raman spectroscopy is utilized to study the packing and ordering of the hemoglobin proteins in the red blood cell as it is stretched. depolarization ratios for a number of heme group modes change, indicating that the applied force additionally packs and orders the proteins inside the cell which further demonstrates the role of cell deformation in the oxygen transport kinetics. acknowledgements: this work was supported by miin fis2008-00114 (spain) and fundació cellex barcelona fret microscopy is widely used to study protein-protein interactions in living or fixed specimens. currently, most commonly used visible fluorescent proteins for live-cell fret studies are the cerulean and venus variants of the cyan and yellow fluorescent proteins. even though this fret pair appears to be ideal for monitoring protein-protein interactions, the most commonly used fixed laser wavelengths do not excite cerulean at peak absorption. recently, we characterized an ideal donor, the monomeric teal fluorescent protein (mtfp), which is excitable using the commonly available 457 (8) opt. june/july, 2008). we used teal as a donor for various red fluorescent proteins as acceptors including tdtomato, mko2, morange2, mtagrfp, mkate. we have employed a "fret standard" genetic construct to minimize variability in separation distances and positioning of the fused donor and acceptor fps. using spectral fret imaging and fluorescence lifetime measurements in living cells expressing the fused proteins, we have characterized both sensitized acceptor emission and the change in the donor lifetime distribution as a result of quenching for each of the fused fp pairings. our results indicate that some red fps are better acceptors than others in terms of quenching the teal donor and sensitizing the emission of the acceptor indicating a fret event. s. m. perepelytsya, s. n. volkov bogolyubov institute for theoretical physics, nasu, 14-b metrologichna str., kiev, 03680, ukraine stability of the dna double helix is determined by na + counterions, neutralizing the negatively charged phosphate groups of the macromolecule backbone. but under spatial conditions they may be replaced by much heavier ions, for example cs + . to determine the influence of heavy counterions on internal dynamics of the double helix the conformational vibrations of na-and cs-dna are studied. for this purpose the model of conformational vibrations of dna with counterions is used [perepelytsya s.m., volkov s.n., eur.phys.j. e. 24, 261, 2007] . as the result the frequencies and amplitudes of vibrations for b -dna with na + and cs + counerions are calculated. the frequencies of internal modes of the double helix are about 110, 79, 58 and 15 cm −1 . the frequencies of ion-phosphate modes are about 180 and 110 cm −1 for na-and cs-dna respectively. the calculated amplitudes of vibrations show that light counterions not disturb the dna internal dynamics, but heavy counterions make move all structure elements of the double helix. using the valence-optic approach the intensities of the dna vibrations in raman spectra are calculated. the calculations show that the ion-phosphate mode in cs-dna spectra is prominent, in contrast to na-dna spectra, where it has very low intensity. obtained results describe the intensity increase of the band 100 cm −1 in cs-dna spectra that was observed in [bulavin l.a., et al., arxiv:0805.0696v1]. s. soria 1 , f. quercioli 3 , r. mercatelli 3 , f. bianco 2 , i. cacciari 2 , g. righini 2 1 centro studi e ricerche "e. fermi", p. del viminale 2, 00184 rome, italy, 2 ifac-cnr, istituto di fisica applicata "n. carrara", via madonna del piano 10, 50019 sesto fiorentino (fi), italy, 3 isc-cnr, istituto dei sistemi complessi, via madonna del piano 10, 50019 sesto fiorentino (fi), italy we report on the application of a simple white light source based on the supercontinuum generation from commercial photonic crystal fibres to confocal fluorescence microscopy and fluorescence lifetime imaging (flim) microscopy. the coherent white light can be tuned by varying the wavelength and intensity of the pump, a ti:sapphire laser. there are several advantages jn the use of sc sources: spatially coherent white radiation, tuning ranges of approximately 400 nm, high brightness, a robust compact system (potentially all-fibre) and relatively low cost. being pulsed, sc sources are suitable for flim. we have used this system for measuring foerster resonance energy transfer (fret) in order to study interactions between ions channels and proteins of membrane within lives cells resolving the quaternary structure of plague f1 capsular antigen a. soliakov 1 , j. r. harris 1 , m. r. hicks 2 , a. rodger 2 , r. woody 3 , a. watkinson 4 , j. h. lakey 1 1 cell and molecular biosciences inst., newcastle univ., uk, 2 chemistry dept., univ. of warwick, coventry, uk, 3 biochemistry and molecular biology dept., colorado state univ., usa, 4 pharmathene uk, billingham, cleveland, uk most gram-negative pathogens express multi-subunit fibres on their surfaces that mediate host cell attachment, biofilm formation, invasion of host defenses and protection against phagocytosis. here we have studied capsular antigen fraction 1 (or caf1), secreted through the conserved chaperone/usher pathway by the plague agent yersinia pestis [1] . caf1 is highly immunogenic and is used in a recombinant subunit vaccine against plague. recent immunological studies indicated vaccines containing polymeric caf1 have higher protective efficacy than vaccines containing its monomeric variant. this difference in protective efficacy was attributed to the quaternary structure. however the quaternary structure of caf1 has not been characterized [2] . in our study we have used transmission electron microscopy of negatively stained specimen and linear dichroism spectroscopy to determine the quaternary structure of recombinant caf1. whereas electron microscopy revealed morphology of caf1 fibres bound to the surface of a carbon coated grid, linear dichroism gave the orientation of subunits in flow aligned caf1 fibres in solution. our results show recombinant caf1 comprises extended linear fibres and circularized fibres, both of which have high degree of conformational freedom. despite the vast application of lambert-beer law in biology, this empirical law cannot accurately describe the light absorption process of molecules with a long-lived excited state. this family of molecules includes most fluorescent molecules, which are very important in biology. lambert-beer law is in fact only valid at very low intensity of incident light and very low concentration of chromophore. if the system doesn't meet these conditions, it falls into a nonlinear regime when it is affected by a phenomenon which we call "dynamic photobleaching": the depletion of chromophores from the first layers, due to their transition to the excited state, leads to a sub-exponential propagation of light in the medium [1] . this phenomenon leads to the necessity of a new formula for the light absorption dynamics which depends on the lifetime of the excited state of the chromophore. the predictions of the theory were successful in describing the absorption dynamics of azobenzene [2] , but now they have been tested also on a biologically relevant molecule like chlorophyll. the results indicate that the absorbance is affected by the intensity of the incident light, and it is therefore a non reliable way of determining, for example, the concentration of the molecule. annular pupil filter to improve spatial highfrequency signal to noise ratio in linear and non linear microscopy e. ronzitti 2 , v. caorsi 1 , a. diaspro 3 1 lambs, microscobio, department of physics, university of genoa, genoa, italy, 2 semm, ifom-ieo, university of milan, milan, italy, 3 neuroscience and brain technologies department, iit, genoa, italy shot-noise significantly affects and deteriorates the imaging capabilities of typical two-photon excitation and confocal laser scanning microscope, especially in biological applications where the detected signal can be remarkable slight [1] . in particular, shot-noise substantially influences the spatial high-frequency range inducing a remarkable reduction of the optical transfer bandwidth. the insertion of an annular filter on the microscope objective lens in the illumination light pathway is here proposed to retrieve the high frequencies information loss [2] .the electromagnetic interference effect induced by the filter insertion, gives a redistribution of the optical transfer function. in particular, the microscope frequency response in filter scheme exhibits an enhancement of signal to noise ratio at the high frequencies able to recover the high frequencies hampered by shot-noise [3] . the goal of biomarker studies is to develop simple noninvasive tests that identify disease states. focus is beginning to shift from identification of individual biomarkers to identification of biomarker panels comprising multiple targets of different molecular species. there are, however, no current technologies available that allow for a comprehensive and simultaneous analysis of the expression levels of multiple cellular components, i.e. proteins and rna. surface plasmon resonance (spr) polaritons are surface electromagnetic waves that propagate in a direction parallel to the interface between a metal surface and an external medium e.g., liquid. these oscillations are very sensitive to any change of this boundary, a phenomenon that has been exploited to facilitate label-free, real-time detection of biological interactions, e.g. protein binding interactions. we are utilising the power of spr to develop technologies that facilitate diagnostic procedures for complex diseases such as alzheimer's disease and chronic obstructive pulmonary disease, through identification and detection of patterns of biomolecules indicative of disease. our approach will facilitate better disease characterisation, improve early detection strategies and aid drug discovery. surface generated fluorescence detection by supercritical angle confocal microscopy d. verdes, m. rabe, s. seeger institute of physical chemistry, university of zürich, winterthurerstrasse 190, zürich, switzerland a two channel confocal microscope for surface and simultaneously in solution fluorescence detection is presented. the microscope's core element is a parabolic mirror objective that collects the fluorescence above the critical angles for total internal reflection (tirf) of the water/glass interface. an aspheric lens incorporated into the parabolic mirror is used for diffraction limited focusing and collecting the fluorescence at low angles with respect to the optical axis. this low angles excitation approach is technically straightforward and gives an advantage over high numerical objectives that require very high angles for tirf illumination. by separating the collection of the fluorescence into supercritical and subcritical angles, two detection volumes highly differing in their axial resolution are generated at the interface. the surface selectivity of the detection volume is obtained on the basis of the dipole emission profile near a dielectric interface. its angular distribution is highly anisotropic and consists of a superposition of traveling and evanescent waves, which both are detected using the parabolic mirror objective. unlike with objective tirf microscopy, the parabolic mirror objective achieves easily diffraction limited excitation/detection volume at the water/glass interface. the objective optical performance is shown by measuring the actin cytoskeleton of cultured cells, fret energy transfer within adsorbed clustered proteins and single molecules detection. differential polarization laser scanning microscopy. anisotropy in biological samples g. steinbach 1 , i. pomozi 2 , g. garab 1 1 biological research center, hungarian academy of sciences, szeged, hungary, 2 pi vision bt., budapest, hungary differential polarization (dp) spectroscopy provides unique information on the anisotropic organization of biological samples [tinoco et al. 1987 ann rev biophys biophys chem 16:319]. however, anisotropy is often a microscopic property. the dp-lsm, constructed in our laboratory enables us to image the main dp quantities: linear and circular dichroisms (ld&cd), also in fluorescence detection ( . further applications include: periodic structure of isolated amyloids, anisotropy variations in cell walls related to drought resistance, and strong anisotropy of 'artificial chlorosomes', nanorods of synthetic porphyrins. dp-lsm might thus represent a novel tool in the better understanding of highly organized molecular macroassemblies. t. m. staudt 2 , p. bingen 1 , c. kempf 3 , h. horstmann 3 , j. engelhardt 1 , t. kuner 3 , s. w. hell 2 1 german cancer research center/ bioquant, 69120 heidelberg, germany, 2 max planck institute for biophysical chemistry, 37077 göttingen, germany, 3 institute for anatomy and cell biology, university of heidelberg, 69120 heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of <50 nm inside 3 µm thick tissue sections. -imaging and spectroscopy r. worch, t. weidemann, p. schwille biotec, biophysics group, technische universität dresden, germany interleukin-4 (il-4), a small soluble protein, is a principal regulatory cytokine, playing an important role during the maturation and clonal expansion of antigen specific b-cells in mammals. at the plasma membrane, il-4 is recognized by a receptor that consists of two single spanning transmembrane proteins: a high affinity il-4r alpha chain, and a low affinity il-2r gamma chain. it is still controversial by which molecular mechanism the signaling complex is fully activated: dimerization of chains, large conformational change upon il-4 binding or a combination of both. moreover, the influence of the lipid environment in which the activation takes place is poorly characterized. to address these issues we aim to reconstitute the receptor component in artificial membrane systems to study the various mutual interactions by means of fluorescence-based techniques, mainly fluorescence correlation spectroscopy. due to reduced background in a chemically defined system this may provide details not yet accessible in the living cell. k. wicker, s. sindbert, r. heintzmann randall division of cell and molecular biophysics, king's college london, se1 1ul london, u.k. fluorescence confocal microscopy, an indispensable tool of modern biology, allows the imaging of live fluorescent specimen with high lateral as well as axial resolution. through the introduction of a sufficiently small confocal pinhole, the lateral resolution can be enhanced compared to that of a wide field microscope. however, this gain in resolution comes at the cost of a decrease in detection efficiency, as light blocked by the pinhole is lost. we present a method for improving the lateral resolution (extending work of sandeau et al. [1] ) and detection efficiency of scanning microscopes by adding an interferometer with image inversion in one of its arms to the detection pathway [2] . this surpasses the lateral resolution achievable in a conventional confocal microscope (closed pinhole) while increasing the detection efficiency substantially. point spread function measurements for a uz-interferometer (uzi) are shown. the light in this setup follows threedimensional u-and z-shaped paths and relies on reflections off planar surfaces only in order to achieve image inversion. we achieved an interference contrast of 91% for white light, and excellent agreement with theoretical predictions. g. vicidomini, a. schönle, j. keller, r. schmidt, c. von middendorff, s. w. hell max planck institute for biophysical chemistry, department of nanobiophotonics, göttingen, germany fluorescence far-field microscopy is a indispensable tool in modern life science. however, the resolution of its standard variants is limited by diffraction to ∼200nm in the focal plane and ∼600nm along the optical axis. to overcome this limit, a new class of super-resolution microscopy techniques has been designed. one example is 4pi microscopy. to obtain isotropic resolution, 4pi uses interference of the wave fronts from two opposing lenses. the point spread function (psf) of 4pi system is characterized by multiple maxima/sidelobes, which replicate the object in the image. therefore image restoration is mandatory to render unambiguous imaging. the situation is further complicated because the positions and the relative heights of the multiple maxima/sidelobes of the psf depend on the phase difference (pd) between the two wave fronts at the focus. if the refractive index of the sample varies in space, this pd becomes a function of position and 4pi image formation process looses its shift invariance. the pd function (pdf) may not even be known a-priori and must then be estimated from the image, leading to a blind image restoration problem. here, we propose a maximum a-posteriori based method to solve the problem. we either assumed a mathematical model for the pdf that depends on a small number of parameters or allowed for an arbitrary pdf but introduced a smoothing constraint. designer multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes [1] [2] [3] . here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid dna to escape from endosomes with extremely high efficiency [4] . endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid dna throughout the cytoplasm and in the nucleus [4] . fluorescence spectroscopy and stopped-flow technique were utilized for the study of the kinetics of incorporation of hypericin (hyp), a natural photosensitizing pigment, into lowdensity lipoproteins (ldl). triphasic kinetics of hyp association with ldl was observed when solutions of hyp and ldl were mixed together. the most rapid phase of hyp incorporation is completed within tens of msec, while the slowest one lasts 10-20 min. the most of hyp molecules are incorporated into ldl in the slowest phase. the kinetics of the incorporation of hyp into ldl particles pre-loaded with hyp were also investigated. the observed decrease of the lifetime and total intensity of hyp fluorescence with the increase of the incubation time of hyp with hyp/ldl complex is a sign of the formation of aggregates and the dynamic quenching of singlet excitation state of hyp inside ldl. to study the kinetics of a transfer of hyp molecules between ldl particles, the time evolution of the stopped-flow and time-resolved fluorescence experiments were investigated after the mixing of the complex hyp/ldl=200:1 with appropriate amounts of free ldl. for each final hyp/ldl ratio the increase of the lifetime and total intensity of hyp fluorescence was observed. the half-time of this process is similar to that one of the slowest phase of hyp incorporation into free ldl. a. bosca 1 , r. magrassi 2 , g. firpo 2 , l. repetto 2 , c. boragno 2 , u. valbusa 2 1 italian institute of technology, genova, italy, 2 nanomed labs (difi-cba), genova, italy the technique of choice to measure the electrophysiological activity of neuronal cells is the so called patch-clamp method because of its precision and sensitivity; this procedure could play a major role also in the investigation of the behavior of a biological neuronal network and so represents an important tool for understanding its functionality and for screening the effects of drugs and compounds on it. the final aim of this project is the development of a planar patch clamp device suited to measure simultaneously the electrical activity of cultured neurons associated in a network. this device will be made of polymeric disposable material and will include a microfluidic perfusion system. in order to create a smooth micro sized pore in a thin polymeric membrane we exploited the prototyping capabilities of focused ion beam etching and we extended the air moulding technique by combining it with soft moulding, obtaining micro structured substrates with the requested features. we also subjected our substrates to a chemical treatment capable of rendering its surface stable and hydrophilic and we verified that it makes them suitable for neuron culturing. use of lipoamino acids for nasal delivery of therapeutic proteins c. bijani 1 , s. sice 1 , j. elezgaray 2 , c. degert 1 , o. broussaud 1 , e. j. dufourc 2 1 physica pharma, 33600 pessac, france, 2 umr 5248 cbmn, cnrs-université bordeaux1, iecb, 33607 pessac, france most of the therapeutic proteins are clinically administered through an intravenous injection several times a day / week. because the repeated injections are not convenient and cause pain in patients, an alternative route of administration is desirable such as oral or nasal. unfortunately, proteins are easily degraded by proteolytic enzymes in the gastrointestinal tract and, therefore, have a low bioavailability when administered via oral route. physica pharma has gained experience in forming sprayable solutions combining lipoamino acids (laa) and small therapeutic organic compounds with the aim to improve their intranasal absorption. in the present work we investigated the ability of laa to complex large molecule such as human erythropoietin, human growth hormone and salmon calcitonin in order to form easily sprayable colloids. these three proteins are used respectively to treat anaemia, growth problem and hypercalcemy. circular dichroism and dynamic light scattering were used to further characterize the laa-protein colloids. a specific molar ratio of laa versus protein was found for wich the proteins keep their secondary structure and have an overall isotropic size slightly increased. molecular dynamics show that proteins are indeed coated with laa. such a complex is shown to pass very easily through a culture of nasal cells growth at confluence. in this study we describe two systems based on soft matter designed for the drug delivery and for the replacement of synovial fluid in osteoarticular pathologies. (i) a new class of temperature sensitive hydrogels pva/poly(ma m nipaam n ) shaped as microspheres obtained with a water-in-water emulsion photocrosslinking reaction. microparticles of pva/poly(ma m nipaam n ) with m:n theoretical molar ratios equal to 1:0; 1:4, 1:8 have been studied in terms of average size and responsiveness to temperature characterized by confocal laser scanning microscopy (clsm), dynamic light scattering (dls) and differential scanning microcalorimetry (dsc). (ii) a physical network based on hyaluronic acid with a small extent (degree of substitution: 1%) of hydrophobic moiety grafted on the backbone, hyadd4, has been characterized in order to account for the influence of thermal treatment on the stability of the hydrogel. dynamic light scattering (dls) and small angle neutron scattering (sans) have been used for dynamic-structural characterization of hyadd4 hydrogels. diffusion of macromolecular probes has been studied by fluorescence recovery after photobleaching (frap) to study the mesoscopic texture of the hydrogel and molecular dynamic (md) simulations were used to approach the time evolution of the physical junction points and of chains clusters. multi-scale estimation of water soluble diffusivity in polysaccharide gels g. eisele 1 , s. bertini 1 , d. paganini 1 , l. piazza 2 1 ronzoni institute, milan, italy, 2 distam, university of milan, italy diffusion properties in gels play important role in food and biotechnological applications. an attractive goal is to design gels in such a way that the active molecules are delivered by the material according to specific release sequences. the transport of macromolecules within polymeric gels depends: on the obstruction effects of the surrounding gel strands, on the molecular interactions between the gel and the solute, on the interactions between the solute molecules themselves and the interactions between the solute and the solvent. physical polysaccharide gels were evaluated in this work to probe diffusion over both microscopic and macroscopic distance scales. physical gels from agar and starch were investigated by high and low resolution nmr techniques in order to characterize their structures. obstruction effects of the surrounding gel strands were considered by studying diffusion of glucose. local diffusion, due to brownian motion, was quantified by low resolution nmr spectroscopy. the fickian diffusion coefficient was measured by modelling experimental concentration-distance curves obtained by means of a two-compartment diffusion-cell. diffusion coefficients depend on the viscoelastic properties of the gel matrix and on water-polysaccharide interactions. j. l. cuellar, g. koehler, m. fischlechner, e. donath medizinische institut für physik und biophysik, leipzig, germany rather than being just pathogens, from the view point of biotechnology and materials science viruses can be regarded as nanocontainers in which nucleic acids have been enclosed in a protective self assembled protein cage. in some viruses an additional lipid-protein envelope wraps the capsid shell. the capsid is constituted of several copies of one single protein subunit or just a few, arranged in a regular fashion showing icosahedral symmetry. the mechanical properties of the cage are important for understanding the infection mechanism involving the release of the encaged genome. by means of atomic force microscopy, it becomes possible to probe such material properties by nanoindentations of single viral particles. it is very interesting to learn how strong or brittle a virus can be. here we have studied the mechanical properties of empty rubella virus particles (rlps) due response of external applied forces. we found that rlps are extremely soft comparable to that of some rubber materials. a peculiarity of the rubella virus is that the capsid is considerably smaller than the surrounding shell filling only a fraction of the lumen provided by the envelope. the envelope in rubella has a distinguishable response on the material properties of the virus. deformation and fracture of the capsid requires comparatively larger forces. our results indicate that the ph is a major factor influencing on rubella particle material properties. this can be related to the infection mechanism. pentavalent antimony (pa) (glucantime, sanofi-aventis or glaxo) are the mainstream agents of choice for leishmaniasis treatment. in therapeutic doses the pa treatment has cardiac side effects like electrocardiographic (ecg) alterations, that include qt segment prolongation, t wave flattening or inversion, inversion of st segment, p, r and t waves amplitude reductions, torsade de pointes arrhythmias and sudden death by cardiac arrest. the objective of this study was to characterize the arrhythmogenic potential of pa. we used 30 guinea-pig to assess the chronic effects of pa therapeutic dose on corrected qt interval, qt dispersion, ventricular action potential (ap) amplitude and duration at 30% and 90% of maximal repolarization and survival rate. guinea-pig received daily 30mg/kg pa or saline for 15 days. eight lead ecg were recorded before and in the last treatment day. at the end the animals were killed and the left ventricle papillary muscles excised for ap recording with the intracellular microelectrode technique. our results of chronic pa treatment showed significant increase of qrs complex duration, qt interval duration, qt dispersion and incidence of t wave flattening or inversion and arrhythmias. the ap analysis demonstrated prolongation at 90% duration. the treatment was lethal in 30% of the animals. we concluded that pa is a proarrhythmic drug that upon chronic use may causes arrhythmias and mortality by disturbances in the ventricular repolarization process. m. m. khvedelidze 1 , t. j. mdzinarashvili 2 , t. partskhaladze 1 , n. nafee 3 , m. schneider 4 1 ivane javakhishvili tbilisi state university, tbilisi, georgia, 2 institute of molecular biology and biological physics, tbilisi, georgia, 3 biopharmaceutics and pharmaceutical technology, saarbrücken, germany, 4 pharmaceutical nanotechnology, saarbrücken, germany we have studied the thermodynamical properties of chitosan-coated nanoparticles (cnp) and non-coated nanoparticles (np) and have gained some insights about plga nanoparticles' properties using supersensitive differential microcalorimetry. the experiments show that in a wide ph interval the changes in transition temperature did not take place. it was shown that such nanoparticles could be used in acidic surrounding for drug transfer. stability and their other properties are less depended on either the particles were in bidistilled or deionized water, or the suspension of particles were located in buffer. to determine the interaction of plga nanoparticles with dna. in the case of dna presence in cnp solution the calorimetric experiments show that the heat absorption peak is constricted, what biophysically means that interaction between them takes place. for more exact determination the contribution of cnp in spectrum, we have compared the spectra of pure dna with the spectrum of the same concentration dna plus cnp. the optimal ratio for dna loading onto the cnp was found to be 7:1. protein-based "epoxy-like" physical hydrogels for stem cell transplantation s. c. heilshorn, c. wong po foo, j. s. lee materials science and engineering, stanford univ., u.s.a. stem cell transplantation has emerged as a promising therapy for multiple injuries and diseases; however, cell survival after transplantation is often poor and unpredictable. we hypothesize that co-injection of stem cells encapsulated within an optimized physical hydrogel will enhance viability. whereas, current physical hydrogels require a shift in environmental conditions (e.g., ph, temperature) to initiate the sol-gel phase transition during encapsulation, our newly designed molecular-recognition gels do not require environmental triggers. instead, these "physical epoxy-like" gels consist of two components that undergo a sol-gel transition upon mixing due to specific hydrogen bonding. the gel viscoelasticity is predictably tuned through precise variation in the molecular-level design of the two components, created using recombinant protein techniques. the design of the two components is based on simple polymer physics considerations and utilizes bio-mimimcry. adult neural stem cells or mesenchymal stem cells are encapsulated within these gels with high viability at constant physiological conditions. the gels promote the growth and differentiation of neural progenitors into neuronal phenotypes, which adopt a 3d-branched morphology. the gels are further optimized for use in the central nervous system by tethering neuroprotective peptides to the gel through molecular-recognition sites. these peptides are released-on-demand by cells through the action of proteolytic enzymes. we consider the integration of the portal protein of the bacteriophage virus φ29 into lipid bilayers of giant unilamellar vesicles (guvs) membranes with the aim of constructing a functional cargo-device able to transport dna and later translocate it outwards. our nano-engineering plan consists of growing guvs from bilayer membranes built up from proteoliposomes previously prepared by extrusion. we have designed two alternative chemical routes for integrating the portal protein in the lipid bilayer, the first considering the native protein and the second a mutant modified with a hydrophobic belt made of histidine-tags. in our contribution we will present details on the different nano-engineering strategies and experimental evidence about the integration of the portal protein in the membrane with an orientation adequate to allow for functional dna translocation. p.-h. guelluy 1 , m.-p. fontaine-aupart 2 , m. hoebeke 1 1 biomedical spectroscopy, ulg, belgium, 2 laboratory of molecular photophysics, univ. of paris-sud, france ppme is a second generation photosensitizer (ps), and a promising candidate for photodynamic therapy (pdt) treatment. we have previously demonstrated that ppme can be easily and efficiently encapsulated in dmpc liposomes, used as ps-vector. we therefore compared the photophysical and photochemical properties of free and encapsulated ppme incubated with human carcinoma cells. absorption and fluorescence microspectroscopy as well as flim analysis allow evaluating the aggregation state of ppme inside the different cellular organelles and the extracellular medium. confocal microscopy established undoubtedly the colocalization of ppme, by robust probabilistic exclusion method, within mitochondrion (central siege of apoptosis). after ps activation (4h and 24h), the balance apoptosis-necrosis was double-estimated by facs device and fluorescence confocal microscopy. quantification of hydroxyl radical was purchased by spin trapping-esr spectroscopy and quenching technique. all these techniques required peculiar settings because of the fluorescence activity of ppme. these results allow to ascertain that the vectorization of ppme affords a better cellular penetration and a monomeric state of ppme. the presence of ppme inside mitochondrion orientated the cellular death in apoptosis 4h following ps-activation. but necrosis is the major actor 24h after treatment. active substrates to study mechanotransduction j. le digabel, p. hersen, b. ladoux laboratoire matière et systèmes complexes, université paris diderot -cnrs, paris, france cellular processes imply an important coordination of interactions with the extracellular medium. accumulating evidences demonstrate that cell functions can be modulated by physical factors such as the mechanical forces acting on the cells and the extracellular matrix, as well as the topography or rigidity of the matrix. these extracellular signals can be sensed by mechanosensors on the cell surface or in the cell interior to induce various cell responses. we have developed an original approach based on micro-fabricated substrates of polydimethylsiloxane (pdms) to study cell migration. we used a closely spaced array of flexible micropillars (diameter∼1?m) to map the forces exerted by cells on their substrates. in this case, the micropillars act as passive force sensors. here, we propose to analyze the cell response to an external applied stress by a well-controlled actuation of the substrate. to do so, we used magnetic pillars. such substrates allow us to modify dynamic adhesion conditions of cells and to better understand the coupling phenomena between mechanical sensing and biochemical activity of a living cell. using polyacrylamide hydrogels doped with ferromagnetic iron oxide particles or ferrofluids, we can make magnetic pillars with diameters of 5 to 10 microns while a magnetic field can be locally applied with a magnetic needle. such a technique will be helpful to study the mechanical response of cells to an external force or to local changes in their microenvironment. glucose scavenging activities of pamam dendrimers m. labieniec 1 , t. gabryelak 1 , c. watala 2 1 university of lodz, deptartment of general biophisics, lodz, poland, 2 glycation is a spontaneous, non-enzymatic modification of biomacromolecules with hexoses, mainly glucose. in terms of its pathophysiological relevance, it targets predominantly proteins, but also nucleotides and phospholipids, and is of major importance to both physiology (ageing) and pathology (diseases with a metabolic background, like diabetes). earlier we demonstrated in vivo that the administration of pamam g4 to diabetic rats resulted in a significantly reduced blood glucose, as well as the early (hba 1c ) and late (ages) protein glycation products. in this study we investigated the ability of pamam g2 (16 surface nh 2 groups) and g4 (64 nh 2 ) to inhibit glycation of proteins in plasma, and a model protein -bsa. pamam g2 and g4 competed chemically with protein nh 2 for the binding of glucose, and hampered protein glycation. in a high-glucose medium they underwent an excessive glycation themselves. this modification was more effective in pamam g2, in which surface nh 2 were more mobile and accessible. pamam modification with glucose rendered these dendrimers less polycationic in aqueous solutions. pamams neither affected bsa conformation nor formed stable complexes with a protein. we conclude that pamams are very effective glucose scavengers. thus, even less toxic pamams of lower generations, like g2, may appear useful in further medical applications as the agents attenuating the detrimental effects of sever hyperglycaemia on biomacromolecules. selective drug delivery and novel drug approaches by polyelectrolytes s. krol cbm-cluster in biomedicine, area science park, s.s. 14, 163.5 km; trieste, italy the use of polyelectrolytes in the past was mainly related to targeted drug delivery and nanoparticle preparations for medical applications. but rarely the polyelectrolytes were investigated for the own features as a drug. in our present work we use a special physical feature of cancer cell membranes as a target for a specific polycation. we found that the polycation is selectively up-taken by cancer cells (leukemia, hepatocarcinoma, cancer stem cells) while normal cells remain unaffected. another interesting application of polyelectrolytes are in form of a multilayer coating on the surface of nanogold particles. for this topic, two new approaches were developed for a drug delivery through the blood brain barrier. in the first case, high amounts of creatine were bound to the gold particles and delivered as protective agents for ischemic stroke (viota et al., j colloid interface sci. 2009, 332 (1):215-23). in the second case coated multifunctional gold particles were prepared as a drug for neurodegenerative disease on the basis of protein aggregates. the use of gold as core for coated nanoparticles offers the possibility to study our systems as "theranostics", system which are modified to recognize selectively diseased cells and carry the moieties to treat or destroy the malfunctioning cells. possible treatments can be local hyperthermal therapy by using the particles as amplifier and enhancer or photodynamic therapy with gold as a drug. m. koneracka 1 , v. zavisova 1 , m. muckova 2 , p. kopcansky 1 , a. jurikova 1 , n. tomasovicova 1 , g. lancz 1 , m. timko 1 , m. fabian 3 1 institute of experimental physics, slovak academy of sciences, košice, slovakia, 2 hameln rds a.s., modra, slovakia, 3 institute of geotechnics, slovak academy of sciences, košice, slovakia the aim of this study was to develop biodegradable and biocompatible paclitaxel loaded magnetic plga nanospheres (nps) suitable for biomedical applications. biodegradable poly(d,l-lactic-co-glycolic acid) (plga) was used as a capsulation material and the magnetic fluid was used as a magnetic carrier. incorporation of magnetic particles and drug in the plga polymer matrix was confirmed by infrared spectroscopy (ftir) and differential scanning calorimetry (dsc). the release of the drug from the prepared nps to the surroundings under the different conditions was studied also. the prepared magnetic plga nps with encapsulated paclitaxel (spherical shape, size 200 -250 nm) have good stability in the presence of high nacl concentration at 25 • c, the toxicity of prepared samples declared 3 times higher value of lethal dose ld 50 in comparison with pure paclitaxel (ld 50 = 33mg/kg) and showed the significant response to external magnetic field which is useful from the point of view to achieve pharmaceutically acceptable drug delivery systems for tumour treatment. success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety. many cationic polymers have been used to condense dna by electrostatic interaction into small particles (polyplex), for protecting the dna from degradation and enhancing uptake via endocytosis. polyethylenimine (pei) appears to be one of the most advanced delivery system that can condense dna efficiently forming pei-dna polyplex complex. the physicochemical properties of different molecular weights of pei, such as condensation ability, buffer capacity, time kinetics, ftir and surface charges of the pei-dna complexes may be important factors to obtain a higher transfection efficiency of the polycation vectors. our intent in this study was to characterize pei-dna complexes to see whether these physicochemical properties have any influence on their disposition characteristics and cellular uptake process. we found that pei-dna complexes, obtained by the 25 k pei at n/p ratio > 4, were more stable in the presence of tissue culture medium & serum, and did not dissociate in nacl easily. key words: polyethylenimine, polyplex, polycations, dna, transfection, gene delivery. effect of nanopatterned substrate on neuronal growth cones activity in the last20years an increasing interest has been address to explore, at the level of the single cell, the physical interaction between neurons and micro-and nano-patterned surfaces which mimic the biological environment and can induce specific biological behavior. a consistent number of substrates have been tested (nano-grooves, pillars, gap nanowires) but the effect of nano-topographical features at subcellular level e.g. branching and pathfinding of growth cones(gc)is still unexplored. using nanoimprinting lithography we fabricated gratings on glass with grooves of variable pitch, depth and width all in the nm range. embryonic stem cell derived neurons were seeded on nanostructured and on flat control glasses. we investigate gc morphology with nm resolution by afm. a significant effect on sub-cellular architecture was observed.on nanopatterned substrates72% of gc were branched with a large number of long and thin filopodia(average length and height4.3µm and 75nm)while on control only34%of gc were branched with an higher percentage of long and thick filopodia (average length and height 6,5µm and 300nm). on the contrary we did not observe a significant influence of the nanopatterning on the alignment and elongation of neurites. in both cases the distribution of angles between axon and filopodia showed a preferential direction at30 • . in conclusion, the tested nanopatterns do not influence the neurite directions but do enhance the gc morphology and explorative activities. growth enhancement and adhesion control of pc12 on micropatterned ns-tio 2 thin film c. lenardi 1 , a. v. singh 2 , p. milani 3 1 c.i.ma.i.na., dip. di scienze molecolari applicate ai biosistemi, università di milano, italy, 2 c.i.ma.i.na., semm, european school of molecular medicine, fondazione ifom, milano, italy, 3 c.i.ma.i.na., dip. di fisica, università di milano, italy cluster assembled nanostructured titanium dioxide (ns-tio 2 ) has been explored as novel substrate for in vitro cell culture. in this work, we report micropatterned ns-tio 2 thin film as putative microdevice for neuron culture and growth. in additions, we show a simple scheme of molecular patterning of bovine serum albumin (bsa) as cell anti-adherent substrate complementary to ns-tio 2 micropattern which favors a selective spatially confined adhesion of neurons. bsa was drop coated and physisorbed over glass coverslip covered with a thin conducting layer of indium tin oxide (ito) and then pmma was spin coated over it using standard protocols. later, using combinations of e-beam lithography and pulsed microplasma cluster source (pmcs), a thin layer of ns-tio 2 was deposited over micropatterned pmma. further, lift off process enabled us to generate complementary micropatterns of hydrophobic bsa (cell repellent) and hydrophilic ns-tio 2 (cell adhesive). cell culture studies have confirmed that pc12 cells like to grow on ns-tio 2 substrate and not on bsa layer. the technique offers a novel approach for neuronal cell assay applications. gene delivery with chitosan: influence of chain length on intracellular trafficking and dissociation s. lélu 1 , c. d. l. davies 1 , n. reitan 1 , s. p. strand 2 1 department of physics, ntnu, trondheim, norway, 2 department of biotechnology, ntnu, trondheim, norway chitosan, a cationic polysaccharide presenting low cytotoxicity, is a promising nonviral gene delivery vector. chitosan complexes dna into nanoparticles, and the complexation and cell transfection efficacy are function of the chitosan/dna ratio and of the intrinsic properties of chitosan, i.e. degree of polymerization dpn, charge density, and molecular structure. nanoparticles formed by shorter chitosan (dpn31-41) mediated higher transgene expression than nanoparticles based on longer chitosans. the purpose of this work is to relate the dpn of linear chitosan with the cellular uptake, intracellular trafficking and dissociation of chitosan-pdna complexes, measured by confocal laser scanning microscopy and fluorescence correlation spectroscopy (fcs). cells were incubated with oligomers (dpn 31 and 88) either free or complexed with plasmid-dna. both chitosan oligomers seem to penetrate cell nucleus in association with or free from pdna. 24 hours after incubation, accumulation of oligomers in cell nucleus is similar for free and complexed dpn31, whereas it increases for complexed dpn88 compared to free, indicating a possible delayed dissociation of the complexes based on dpn88 in the nucleus and suggesting a dissociation of pdna-dpn31 in cytoplasm. these data are consistent with previous studies which suggested that longer chitosan chains led to tighter complexes, and hence to a delayed dissociation process and lower transfection efficiencies compared to shorter chitosans. material surface properties greatly influence dna purification and pcr yield in microsystems c. potrich 1 , l. lunelli 1 , l. marocchi 1 , l. pasquardini 1 , g. guella 2 , d. vozzi 3 , l. vanzetti 1 , p. gasparini 3 , c. pederzolli 1 1 fbk, trento, italy, 2 univ.trento, italy, 3 univ.trieste, italy modern microchip platforms integrate dna purification, target amplification by pcr and dna detection in a single device. combination of these processes minimizes sample loss and contamination problems as well as reduces analysis time and costs. different strategies are available to perform dna extraction on a chip. here we exploited amino-coated materials as a tool for specific binding of dna through the electrostatic interaction between amine groups and nucleic acids. we analyzed the ability of different treated substrates to selectively absorb/desorb the genomic dna with the aim to purify dna from unwanted components. amino-coated substrates were characterized by afm, xps, fluorescence microscopy and absorption spectroscopy to define the surface chemical and morphological properties. the distribution of the dna adsorbed on materials was homogeneous and the eluted dna was tested for pcr. same materials were analyzed for their compatibility with pcr and the use of different enzymes and reagents or proper surface treatments was employed. we established the best conditions for dna amplification in silicon/pyrex microdevices depending on the type and the fabrication method used and on the quality of reagents more than on the passivation treatment or increment in standard taq polymerase concentration. nanoporous alumina fabricated using unconventional acids for enhanced biomolecular physisorption n. patra, m. salerno, r. losso, r. cingolani italian institute of technology, genova, italy in the last decades the available porecell sizes of porous alumina have been extended, but operating conditions to obtain some pore diameters still have to be optimized, particularly below the 50 nm limit. in this range the use of biocompatible porous alumina thin films could find applications in biosensors, after functionalization by appropriate physisorbed biomolecules. while pore ordering and film growth rate are mainly influenced by the electrical parameters of voltage and current, respectively, the typical pore size primarily depends on the electrolyte. in the search for alternative anodization conditions, we have investigated several acids that have never been used before, namely gallic, lactic, propionic, and glycolic acid. the anodizations were carried out in galvanostatic conditions at fixed concentration and durations, by varying the current. atomic force microscopy was used to test the oxidized surface morphology. in particular, lactic and propionic acids demonstrated feasible. lactic acid gave best results for ∼30 ma/cm 2 current density, corresponding to roughly constant ∼15 v potential, with resulting pore diameter in the 4060 nm range, whereas propionic acid performed best for ∼5 ma/cm 2 , corresponding to ∼43 v, resulting in 7090 nm pore diameter. both kind of films looked lightgray, different from the yellowish oxalic acid porous alumina, which can be a hint of lower ion contamination. chitosan-arabic gum nanoparticles as potential vehicles for peptide delivery l. moutinho, s. rocha, m. coelho, m. c. pereira lepae, chemical engineering department, faculty of engineering, university of porto, portugal chitosan (cs) -arabic gum (ga) nanoparticles were produced by an ion-ion interaction process, using different weight ratios of polysaccharides. according to zeta potential (zp) measurements, particles are ionically stable. zp values ranged from 30 to 70 mv for cs:ga ratios of 1:3.3 and 1:0.8, respectively. cs:ga 1:5 yielded uncharged nanoparticles and aggregates, showing that, at this ratio, the negative charges of ga neutralize the positive charges of cs. the particle diameters ranged from 300 to 500 nm, as measured by dynamic light scattering (dls), and displayed a slight tendency for decreasing as the ga concentration increased. transmission electron microscopy (tem) images showed numerous spherical nanoparticles. peptides were encapsulated within the nanoparticles, by mixing them with chitosan solution prior to adding ga. this system can protect short peptides from rapid metabolization, prolonging their blood half-life. physical characterization of nanocarriers for drug delivery s. motta 1 , y. gerelli 2 , g. sandri 3 , e. ricci 3 , p. brocca 1 1 dept. of chem, biochem and biotech for medicine -university of milan, italy, 2 dept. of physics -university of parma, italy, 3 dept. of pharmaceutical chem -university of pavia, italy nanoparticles used as nanocarriers for pharmaceuticals can improve solubility and bioavailability of problematic drugs and protect labile or toxic molecules. fundamental parameters like drug encapsulation efficiency and release kinetics are tuned by the physico-chemical features of the drug/carrier complex. a challenging aspect of the pharmaceuticallyoriented issues resides in the relatively low number of molecules allowed to build up nanosystems with different properties and morphology, according to the specific drug and to the intended therapy. we have studied: 1) soybeanlecithine/chitosan nanoparticles for progesterone and tamoxifen delivery; 2) solid-lipid nanoparticles (compritol 888 ato, poloxamer 188, tween 80 and chitosan) for cyclosporine-a delivery via ophthalmic formulation. both void carriers and drug-loaded nanoparticles have been studied, to understand how the structure of the carriers can be modified by the active molecules. several non-invasive physical techniques have been used to achieve a detailed knowledge of the systems: a) dynamic light scattering for the dimensional distribution of the nanocarriers, b) zeta potential to determine surface charge, c) cryo-tem for morphological analysis d) x-ray scattering (in small angle configuration) and dsc to access information on the inner structure. . a. schollier 1 , a. halperin 3 , m. sferrazza 2 , g. fragneto 1 1 institut laue-langevin, grenoble, france, 2 universite libre de bruxelles, belgium, 3 cnrs-ujf grenoble, france protein adsorption on surfaces is responsible of several unwanted effects in technological and pharmaceutical applications: fouling of contact lenses, clotting in blood containing devices, inflammation around artificial organs for instance. those phenomena can be repressed with certain types of polymer brushes, in particular peg (polyethylene glycol) brushes, and a better understanding of the mechanism of adsorption could lead to improvements in the design of biomaterials. we have developed a theory describing this mechanism and carried out measurements to confirm the theoretical predictions [langmuir 2007, 23, 10603] . three cases are predicted: primary adsorption at the grafting surface, secondary adsorption at the outer edge of the brush, and ternary adsorption within the brush itself. we prepared samples with different grafting densities and different degrees of polymerization and used different protein size and concentrations. neutron reflectivity experiments, able to determine the structure and composition of material interfaces with a fraction of nanometer resolution, were carried out and, by using deuterated proteins (anti-freeze protein, dihydrofolate reductase and myoglobin) on different peg compositions and grafting densities, it was possible to locate the proteins in the brush and to distinguish between the different kinds of adsorption : primary adsorption is dominant for short brushes and ternary adsorption for long brushes. effect of powder air polishing on nanocomposite dental materials measured by atomic force microscopy m. salerno 1 , g. derchi 2 , a. m. genovesi 2 , l. giacomelli 2 1 italian institute of technology, genova, italy, 2 istituto stomatologico tirreno, lido di camaiore, italy in dentistry, airpolishing with glycine and bicarbonate powders is widely used to remove accumulated plaque. however, the resulting teeth and gingival surface roughness, which is a risk factor for further plaque accumulation, has to be considered. in this in vitro study the effect of the above mentioned technique on the surface of a novel nanocomposite dental material is evaluated by means of atomic force microscopy (afm). square specimens (0.5×0.5 cm 2 size) were airpolished either with glycine or bicarbonate, using different application times (2, 8, 30 sec) and distances (2, 4, 6 mm) . four specimens were evaluated for each timedistance combination, and checked versus untreated specimens (controls). the specimens were imaged with tappingmode afm at two different scan sizes (3×3 and 30×30 µm 2 ) in two different regions for each sample. the surface roughness was measured as the rms of the feature heights. for the 2 sec2 mm group airpolishing resulted in an increased surface roughness as compared to the controls; however, glycine was associated with a lower roughness than bicarbonate (glycine: 238±91 nm; bicarbonate: 436±75 nm; controls: 14±14 nm; p<0.0001 for treated specimens vs controls, p<0.001 for glycine vs bicarbonate, anova test). similar results were obtained for all the other timedistance combinations. the work comprises the design of polysaccharide based nanoparticles for drug delivery. chitosan/arabic gum and arabic gum/maltodextrin nanoparticles were prepared by polyelectrolyte complexation and spray drying [1] , respectively. dynamic light scattering characterization established that arabic gum/maltodextrin nanoparticles are more polydisperse (diameters ranging from 8 to 400 nm) than chitosan/arabic gum particles (diameters of approximately 500 nm). scanning electron microscopy measurements demonstrated that the particles are spherical and have a smooth surface. the systems are highly stable to forces up to 80 nn as observed by atomic force microscopy. due to their nature they are hydrophilic and biodegradable. the nanoparticles were used to entrap short peptide sequences and antioxidants and were proved to be efficient in maintaining the biological activity of the molecules. kyotorphin (ktp) was found in 1979 in bovine brain. despite revealing remarkable analgesic properties, analgesia was only induced after central delivery. this limited ability to cross the blood-brain barrier (bbb) combined with the unknown mechanism of action largely confines its pharmacological use. to surpass these problems, we designed new ktp derivatives: ktp-rc and ktp-rc-lipogen. biophysical studies were carried out using fluorescence methodologies to characterize the peptides' interaction with biomembrane model systems. partition coefficient quantification showed a clear preference of the derivatives towards fluid zwitterionic and anionic membranes. moreover, a relationship between anionic lipid percentage and in-depth insertion in membrane was established. additionally, studies with fluorescent probe di-8-anepps revealed different membrane-interaction profiles of morphine and ktp-derivatives, suggesting distinct actions between them. the analgesic efficacy of the compounds was studied in vivo after systemic administration in models of acute and tonic pain. unlike ktp, both ktp-rc and ktp-rc-lipogen displayed high efficacy from doses as low as 1.67 and 0.85 mg/100g, respectively, indicating bbb crossing. the observed correlation between higher partition/insertion in the membrane and enhanced analgesic action proved the biophysical rationale to be a powerful strategy for early screening in cns drug development. metabolic syndrome (ms) is now regarded as a major risk factor for cardiovascular disease. as prooxidant/antioxidant balance is disturbed in the course of this disorder, it is possible that melatonin may exhibit protective effect against oxidative damage of blood cells. ms was diagnosed according to international diabetes federation definition (2005) . the investigated group consisted of patients before and after the melatonin supplementation (5 mg per day for two months) and was compared to control group of healthy individuals on normal diet. our experiments show that erythrocytes from patient group exhibit significantly higher tbars and total cholesterol levels whereas the protein thiol concentration, na + k + atpase and glutathione peroxidase activities were decreased in comparison to those of healthy volunteers. after two month melatonin supplementation, tbars and cholesterol concentrations significantly decreased, whereas the na + k + atpase, catalase and glutathione peroxidase activities increased. the glutathione concentration was also higher. these results show that melatonin supplementation has a protective effect on erythrocytes of ms patients. action of ferritin, a nanoparticle model, on ros formation and glutamate uptake in synaptosomes t. waseem, a. alekseenko, s. fedorovich institute of biophysics and cell engineering, minsk, republic of belarus nanoparticles are currently used in medicine as agents for targeted drug delivery and imaging. however it has been demonstrated that nanoparticles induce neurodegeneration in vivo and kill neurons in vitro. the cellular and molecular bases of this phenomenon are still unclear. we have used the protein ferritin as a nanoparticle model. ferritin contains iron particles (fe 3+ ) with size 7 nm and a protein shell. we investigated how ferritin influences uptake and release of [ 14 c]glutamate and free radical formation as monitored by fluorescent dye dcfda in rat brain synaptosomes. we found that even a high concentration of ferritin (800 µg/ml) did not induce spontaneous [ 14 c]glutamate release. in contrast the same concentration of this protein inhibited [ 14 c]glutamate uptake two fold. furthermore ferritin induced intrasynaptosomal ros (reactive oxygen species) formation in a dose-dependent manner. this process was insensitive to 30 µm dpi, an inhibitor of nadph oxidase and to 10 µm cccp, a mitochondrial uncoupler. these results indicate that iron-based nanoparticles can cause ros synthesis and decrease glutamate uptake, potentially leading to neurodegeneration. functionalized carbon nanotubes 1 (cf-cnts) have a promising future as vectors for drug delivery 2 and for neuronal cell growth and communications 3 . it has been demonstrated that cnts can be employed in drug delivery systems. the toxicological properties of cnts result strictly correlated with nanotube solubility 4 . in this study we focused our attention on multi-walled cnts (mwcnts) because they are easy to manipulate and we covalently functionalize them in different ways to increase their solubility.. we grew different derivatives of a dendrimer on mwcnts surface with positive charges at the periphery, in order to study the different solubilisation properties and correlate them to the biological activity in gene therapy 5 . slowdown of 1-40 β-peptide aggregation by addition of two synthetic biocompatible polymers p. sciortino 1 , r. carrotta 1 , g. cavallaro 2 , d. bulone 1 , p. l. san biagio 1 1 ibf-cnr, palermo, italy, 2 ctf dept., university of palermo, palermo, italy fibril deposit formation of amyloid β-protein (aβ) in the brain is a hallmark of alzheimer disease (ad). fibrils formation is triggered by molecular conformational changes and protein-protein interactions involving partially unfolded regions of different aβ peptide molecules. increasing evidence suggests that toxicity is linked to diffusible aβ oligomers, which have been found in soluble brain extracts of ad patients, rather than to the insoluble fibres. new therapeutical approach, based on searching molecules capable of regulating the peptide aggregation, is currently developing. here, we study the effects on the aggregation of aβ 1-40 peptide of two different synthetic polymers with structure similar to that of a protein (α,β-polyasparthylhydrazide -pahy and α,β-polyasparthylhydrazide-polyethyleneglycol -pahy-peg). static and dynamic light scattering measurements showed that the aggregation kinetic is slowed down by the presence of both polymers. optical microscopy revealed the presence of aggregates of different dimension in all samples. transmission electron microscopy allowed to establish that all aggregates are made of fibers, as confirmed by fluorescence spectroscopy measurements on thioflavine binding. a. smorodchenko 1 , d. sittner 1 , a. rupprecht 1 , j. goyn 1 , a. seiler 2 , a. u. bräuer 1 , e. e. pohl 1 1 institute of cell biology and neurobiology, charité-universitaetsmedizin, berlin, germany, 2 german federal institute for risk assessment, zebet, berlin, germany ucp4 is a member of the mitochondrial anion transporter family and one of the three ucps (ucp2, ucp4 and ucp5) associated with the nervous system. in our previous work we have shown that ucp4 appears in brain at embryonic day 14 (e14). we hypothesized that the ucp4 expression can be related to neuronal differentiation. to prove this idea we now investigated protein and mrna levels in two different systems: (i) mouse embryonic stem cells of line d3 (mesc) and (ii) brains from pre-and postnatal mice. ucp4 was not present in undifferentiated mesc. during differentiation of mesc in neurons the expression of neuronal marker map2 and ucp4 started at the same time -as early as on day 7 in culture -and were increasing simultaneously. (ii) the comparative analysis of gene transcripts prepared from whole embryos, brains and different brain regions (neocortex, hippocampus, cerebellum) demonstrated that levels of ucp4 mrna were increasing from e8 till e19, reached an expression peak between e19 and postnatal day 0 (p0) and remained constant in adult animals. in contrast, expression of ucp5 was increasing permanently until birth, whereas ucp2 expression was invariant in time. our results suggest that ucp4 contributes to specific neuronal functions. the plant hormone abscisic acid stimulates the proliferation of human hemopoietic progenitors through the second messenger cyclic adp-ribose s. scarfì 1 , c. fresia 2 , c. ferraris 1 , s. bruzzone 2 , f. fruscione 1 , c. usai 3 , m. magnone 2 , m. podestà 4 , l. sturla 2 , l. guida 2 , g. damonte 2 , a. salis 2 , a. de flora 2 , e. zocchi 2 1 advanced biotechnology center, genova, italy, 2 dept of experimental medicine, biochemistry section, and cebr, university of genova, italy;, 3 institute of biophysics, cnr, genova, italy, 4 stem cell center, s. martino hospital, genova, italy abscisic acid (aba) is a hormone involved in pivotal physiological functions in higher plants. recently, aba was demonstrated to be produced by human granulocytes, β pancreatic cells and mesenchymal stem cells (msc) and to stimulate cell-specific functions. here we show that aba expands human hemopoietic progenitors in vitro, through a cadpr-mediated increase of the intracellular calcium concentration. incubation of cd34 + cells with micromolar aba also induces transcriptional effects, which include nf-κb nuclear translocation and transcription of cytokines encoding genes. stimulated human msc produce and release aba at concentrations sufficient to exert growth-stimulatory effects on co-cultured cd34 + cells, as demonstrated by the inhibition of colony growth in the presence of an anti-aba monoclonal antibody. these results provide a remarkable example of conservation of a stress-hormone from plants to humans and identify aba as a new hemopoietic growth factor involved in the cross-talk between hp and msc. t. golovanova, g. b. belostotskaya sechenov institute of evolutionary physiology and biochemistry, russian academy of sciences, saint petersburg, russia a subpopulation of small cells (volume 141±6 µm 3 ) have been found in the neonatal cardiac myocytes culture whereas the same parameter of the remaining cells was 819±68 µm 3 on the 1 st day. in contrast to main part of hypetrofied cardiomyocytes, the small cells were able to proliferate, form colonies and differentiate spontaneously into cardiac myocytes in the culture. on the 8 th day there were slight, slow (2-3 beats/min), arrhythmic contractions in the centre of colonies. pulsing cells were not united by common contraction and had individual beating profile. on the 11-12 th days, the colonies displayed comprehensive contractile activity with the pulsation rate 25-28 beats/min and reached 46 beats/min on the 23 d -25 th day and 58 beats/min on the 26-30 th days in the culture. it has been shown that receptors of the surface membrane and sarcoplasmic reticulum of colony cells gradually mature. by estimating the ca 2+ transition under the specific agonists action: acetylcholine, kcl and caffeine, we have detected the activity of basic structural and functional elements of excitation-contraction coupling in contractile cells inside colonies. the small cells ability to proliferate and differentiate under the influence of near mature cardiomyocytes allows us to put forward a hypothesis, that they belong to a category of resident stem cells. changes in the gating properties of na + channels were studied in es-derived neural stem (ns) cells during in vitro neuronal differentiation with the use of the whole-cell and cell-attached variants of patch-clamp technique. ns cells represent a novel stem cell population remaining stable and highly neurogenic over multiple passages.voltage-clamp recordings during neuronal differentiation of ns cells indicated significant changes in the key properties of na + currents. a voltage-gated and tetrodotoxine-sensitive na + current,absent under self-renewal conditions, was first recorded following application of differentiative agents. current density increased with time of exposure to differentiating conditions. whole-cell and single channel analysis revealed that the observed increase in current density was due at least in part to changes in steady-state activation and inactivation properties. namely, half activation potential shifted from -34 mv to -45 mv, while half-inactivation potential shifted from -94 mv to -78 mv. furthermore, a contribution to the increase in na + current density could also be given by an enhancement in channel expression, as suggested by an augmentation in the number of single channels per patch area, with increasing neuronal differentiation. interestingly, those changes in na + channel activity well correlate with the capability of ns cells to generate action potentials during in vitro neuronal differentiation. a. viale, g. bonizzi, a. cicalese, f. de franco, c. pasi, s. pece, a. orleth, p. di fiore, p. pelicci european institute of oncology, milan, italy we are characterizing the biological differences between normal and transformed scs. scs are defined by their ability to generate more scs, termed self-renewal, and to produce cells that differentiate (asymmetric cell division). scs, however, possess the ability to expand in number (i.e. during development, in adulthood after injury/disease); this increase is not accounted by asymmetric divisions, in which only one daughter cell maintains sc identity. recent findings in invertebrates indicate that scs can also generate daughter cells destined to acquire the same fate (symmetric cell division). on the other hand, sc quiescence is critical to maintain tissue homeostasis after injury. here we show increased symmetric divisions of cscs in breast tumors (due to inactivation of the p53 tumor suppressor) and dependency of leukemia development on quiescent leukemia scs (due to transcriptional up-regulation of the cell cycle inhibitor p21 by leukemia-associated fusion proteins). we suggest that sc asymmetric divisions function as a mechanism of tumor suppression, that sc quiescence is critical to the maintenance of the transformed clone and that symmetric divisions of scs permit their geometric expansion. a. uccelli department of neurosciences ophthalmology and genetics, genoa, italy stem cells are considered as a possible source of cells for tissue repair. in this scenario damaged tissues will be reconstructed by newly formed cells with the result of a recovery of lost functions. thus, the rationale for utilizing stem cells for the treatment of neurological diseases such as multiple sclerosis (ms) stemmed from the idea that they differentiate in neural cells regenerating the damaged tissues at the basis of irreversibile disability. however, while multipotential embryonic stem cells may provide in the future an optimal source of cells competent for myelin and axons repair in ms, their use is still far from being exploitable in a clinical setting. moreover, it is widely accepted that adult stem cells may in vitro transdifferentiate in neural cells but recent experimental data have challenged the biological importance of this event in vivo. nevertheless, several experimental studies have provided new evidences supporting the use of adult stem cells derived form different human tissues for the treatment of ms. in the cases of mesenchymal stem cells and neural stem cells current experimental data support an unexpected therapeutic plasticity mediated by diverse paracrine mechanisms including neuroprotection, induction of local neurogenesis and modulation of the immune response. therefore, current experimental and clinical studies support the use of stem cells for the treatment of neurological diseases such as ms. unravelling the structure of the water splitting site of photosynthesis and implications for mechanism of catalysis j. barber imperial college london, u.k. photosystem ii (psii) is a multi-subunit membrane protein complex which catalyses the oxidation of water to molecular oxygen and reducing equivalents. the reaction occurs at a catalytic centre composed of 4 mn ions and a ca ion, is thermodynamically demanding and generates highly oxidised species. unavoidable side reactions cause detrimental effects on the protein environment leading to the rapid turnover of the reaction centre d1 protein. to understand the mechanisms of water oxidation and d1 turnover structural information is required. initially the positioning of various protein subunits and their transmembrane helices were determined by electron microscopy. more recently a refined structure of the cyanobacterial psii unit has been elucidated by x-ray crystallography giving details of specific environments of the redox active cofactors. the implications of these structural studies will be discussed in relation to the unique facets of psii function, particularly the water splitting reaction. importantly this new knowledge is providing a blue print for the design of photochemical catalysts which can mimic the photosynthetic water splitting reaction and thus give hope that new technologies will emerge to provide humankind with a sustainable energy supply. photochemically controlled molecular devices and machines v. balzani department of chemistry "g. ciamician", university of bologna, italy the macroscopic concepts of a device and a machine can be extended to the molecular level. molecular-level devices and machines operate via electronic and/or nuclear rearrangements and, like macroscopic devices and machines, they need energy to operate and signals to communicate with the operator. the extension of the concepts of a device and a machine to the molecular level is of interest not only for basic research, but also for the growth of nanoscience and the development of nanotechnology. if a molecular device or machine has to work by inputs of chemical energy, it will need addition of fresh reactants ("fuel") at any step of its working cycle, with the concomitant formation of waste products that compromise the operation. currently there is an increasing interest in the use of light to power molecular devices and machines. the lecture will illustrate examples of recent achievements [1, 2, 3] , which include molecular wires, switches, plug-socket systems, extension cables, antennas, and light powered nanomotors. biophysics laboratory, institute of botany, azerbaijan national academy of sciences, baku, azerbaijan it is well known that fast component of induction curves of millisecond delayed chlorophyll fluorescence (ms-df) originates via radiative recombination of reaction center with product on the donor side of psii. in view of our previous data (j. photochemistry and photobiology b: biology 86, 2007) it was shown that partners for radiative recombination of p 680 q a − localized as a hole on the donor side of psii. depending on ph this hole might be on the camn 4 -cluster or on y z and their recombination with p 680 q a − can be monitored by the ms-df fast component. for analysis of site of damages, photoinhibition of psii particles from spinach at different ph was monitored by ms-df. during photoinhibition of psii (4000 µmol photons m −2 s −1 ) the fast component of ms-df was shown to be more stable at ph 5.5 and ratio of the fast component to the steady-state level of ms-df was approximately constant, while at ph 8.5 the fast component essentially decreased, the steady-state level increased and ratio of these components rapidly went down. it is possible concluded that strong light damaged of recombination reaction with in the presence ppbq -artificial electron acceptor -the protection of psii from photoinhibition has been observed only at acidic condition. stress factors such as heavy metals, strong light and low temperature were investigated by measurement of transient pictures of ms-df in intact plants, isolated chloroplasts and pure psii particles. the heavy metals action on psii was investigated on wheat seedlings. the targets for toxic action of al, mn and co ions were found to be a q a -q b acceptor side of psii. the damage site for cd 2+ may be partners for recombination with p680 + -depend on of medium ph -either y z or camn 4 -cluster. investigated ions (mn 2+ , al 3+ , cd 2+ and co 2+ ) have lead to reduction of chlorophyllprotein complexes pigment fund and cd 2+ and co 2+ destroyed also an apoproteins, especially of psii. the photoinhibition of psii was investigated in intact leaves of barley and maize seedlings at low and normal temperature. a very sharp reduction of intensity of ms-df second fast component, possibly y z * p680ÿq a − radiative recombination of maize seedlings was observed even after short illumination by strong light at 4 • c. ph dependence photoinhibition of chloroplasts and pure psii particles have shown that strong light damaged of camn 4 -cluster in great degree than y z . hydrogenases are considered as potential energy sources. in particular, the ability of the green alga c . reinhardtii to reduce protons to hydrogen gas upon illumination by means of a [fefe]-hydrogenase is a phenomenon of great scientific interest, as it would need only light and water to generate energy. however, the catalytic activity is strongly inhibited by the o 2 produced during photosynthesis; furthermore, the protein is expressed at very low levels, only in conditions of strict anaerobiosis. this mutually exclusive nature of o 2 and h 2 photoproduction represents a crucial problem in the development of h 2 bio-production. the study of the structurefunction relationship of [fefe] hydrogenases, which would help to clarify the molecular mechanisms underlying both h 2 production and o 2 sensitivity, requires the characterization of purified native and modified proteins, which can be obtained by site-directed mutagenesis. we expressed the algal hydrogenase in the cyanobacterium synechocystis sp. pcc 6803, which holds a bidirectional [nife]-hydrogenase with a well known maturation system (1) . we obtained two constructs to stably transform synechocystis, enabling it to express the c. reinhardtii hydrogenase in an active form. this suggests that the [nife]-hydrogenase maturation pathway is able to drive the biosynthesis of functional [fefe] enzymes. these data open new perspectives about the indispensable presence of hyde, hydf and hydg auxiliary proteins (2, 3) to obtain a correctly folded [fefe]-hydrogenase. the a2 low energy monomeric chlorophyll of cp29 to investigate the role of low energy spectral forms in energy transfer among chlorophyll protein complexes, we characterized one of the redmost chlorophylls of psii antenna complexes, the a2 of cp29. pigment stoichiometry of the wild type (cp29wt) and mutant lacking the a2 binding residue (cp29a2) reconstituted complexes confirmed the loss of a chlorophyll a. to exclude the possibility of a marked excitonic interaction of the a2 with other chlorophylls, we quantified the decrease in dipole strength after mutagenesis as a function of the frequency and performed a second derivative analysis of the difference absorption spectrum cp29wt minus cp29a2. these data, along with circular dichroism spectra of both complexes, support the idea that the a2 is a monomer in cp29, in disagreement with recent suggestion of involvement in an excitonic dimer (mozzo et al., bba 2008) . chla a2 reorganization energy and inhomogeneous broadening were then determined through a thermal broadening analysis in the 80-285 k temperature range. these parameters allowed us to calculate the förster overlap integral of the homogeneously broadened a2 bandshape, a fundamental factor for energy transfer rate estimations between chromophores. a comparison with foi of chlorophyll in solution suggests that chla a2 may connect cp29 complex to psii core favoring energy transfer from cp29 towards other inner antenna low energy chlorophylls. in oxygenic photosynthetic organisms, reaction centre complexes catalyze electron transport from water to co2 using light energy absorbed by tetrapyrrole pigments bound to so called "antenna proteins". a major challenge in performing this type of photosynthesis consists on the difficulty of operating "one electron" transport through a multi-step pathway in the chloroplast environment which is the source of oxygen of biosphere. in fact, synthesis of ros, mainly singlet oxygen and superoxide anion and consequent photoinhibition is an intrinsically unavoidable consequence of photosynthesis that must be prevented and/or controlled in order to avoid photodestruction and death. mechanisms involved include chlorophyll (chl) triplet quenching, ros scavenging and controlled heat dissipation of excess chl singlet excited states. the latter process has been studied for over 40 years with little success in elucidating its mechanism. reverse genomics and ultrafast-spectroscopy led to the proposal that the transient formation of carotenoid radical cations, followed by charge recombination might be the underlying mechanism of energy dissipation while three proteins belonging to the lhc superfamily could be the site hosting the reaction. probing the dark cycle to reveal regulatory networks controlling photosynthetic efficiency the food, fibre and fuel needs of an ever-increasing population are one of the challenges facing current society. higher plant photosynthetic efficiency is ∼1% whereas the theoretical maximum is thought to be ∼6-8% giving potential for great improvements. it is not however clear where in the photosynthetic process the additional losses are occurring. we are adopting a systems biology approach to reveal the regulatory networks that control photosynthetic efficiency, the challenge being to make quantitative measurements of the input parameters to models of the network of photosynthetic reactions, and also to identify missing physical parameters and processes. the aim being to understand how they interact with other key physiological pathways and what impact they hold over photosynthetic efficiency. we report initial results in this poster from experiments using conventional and novel proteomic methods. e.g. an optical technique based on a non-linear 2dir method is being developed for proteomic and metabolomic analysis. unlike many conventional proteomic methods, which provide information on the relative levels of various proteins and metabolites, the viability of the 2dir method as a quantitative, sensitive and high throughput proteomics platform has recently been demonstrated. in collaboration with klug this project will employ 2dir as a both a proteomic and metabolomic tool. t. morosinotto 1 , p. arnoux 2 , g. saga 1 , g. m. giacometti 1 , r. bassi 3 , d. pignol 2 1 dip. di biologia, padova, italy, 2 cea, cadarache, france, 3 dip. di biotecnologie, verona, italy violaxanthin de-epoxidase (vde) is the enzyme responsible for zeaxanthin (z) production. the synthesis of this carotenoid in plants exposed to high light conditions is an important photoprotection mechanism, enhancing excess energy dissipation and reactive oxygen species scavenging. the inactive enzyme is normally soluble but, upon activation by low ph, it binds to the thylakoids membrane, where its substrate is found. we present here the first structural data on this enzyme obtained at both acidic and neutral ph. at neutral ph, vde is monomeric with its active site occluded in the lipocalin barrel. upon acidification, the barrel opens up resulting in a functional dimerization of the enzyme. the channel linking the two active sites of the dimer can harbour the entire carotenoid substrate and thus allow the parallel de-epoxidation of the two violaxanthin β-ionone rings, making vde an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate. structural data opened the possibility to investigate deeper this enzyme and further work allowed the identification of its active site, the protein domains responsible of its membrane association as well as key residues involved in the ph dependent conformational change. hydrogen is considered the fuel of the future if produced from sun light-driven water splitting. a hydrogen economy based on genetically modified organisms, immobilized enzymes or biomimetic synthetic catalysts requires a profound knowledge of the structure and function of the respective enzymes in nature. 1 light-induced water splitting is performed by a tetranuclear manganese cluster located in photosystem (ps) ii of oxygenic photosynthesis. the protons generated by ps ii can be converted to molecular hydrogen by the enzyme hydrogenase, which is for example found in green algae and cyanobacteria. [nife]-and [fefe]-hydrogenases are the two main classes of this enzyme. they contain bridged binuclear transition metal cores, which are tuned by a special ligand environment to efficiently convert protons to hydrogen -or vice versa -via a heterolytic mechanism. 2 rhodobacter sphaeroides strain a-10 isolated from mineral springs in armenia produces h 2 with high rate in anaerobic conditions under light. in our previous work the inhibition of h 2 production by n,n' -dicyclohexylcarbodiimide (dccd), the f 0 f 1 -atpase inhibitor, was shown, and dccd-inhibited atpase activity was determined. therefore it is possible to admit a role of this atpase in h 2 production by r. sphaeroides; otherwise dccd inhibition of hydrogenase is not ruled out. in order to examine the mediatory role of proton-motive force (pmf) or proton atpase in this process transmembrane electrical potential (∆ψ) and ∆ph are determined and the atpase activity is studied in r. sphaeroides grown under light. pmf was determined under anaerobic conditions in the dark. at ph 7.5 the ∆ψ was of -94 mv and the reversed ∆ph was +30 mv, resulting in the pmf of -64 mv. but ∆ψ was not affected by dccd. moreover, adenine nucleotide phosphates (anp) content is essential for cell functioning and may result with atpase activity. the percentage of atp was calculated from the total quantity of anp in whole cells, it was 46.6 %. a relatively high concentration of adp (∼35 %) and amp (∼18 %) and accordingly low energetic charge were noted; these might be indicative for the atpase activity, a further study is required. relationship between h 2 production and atpase activity by rh. sphaeroides is suggested; possible mechanisms are discussed. identification of the sites of chlorophyll triplet quenching in relation to the structure of lhc-ii from higher plants. evidence the chl a molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric lhc-ii are identified by time-resolved and pulse epr techniques. the concept of spin conservation during triplet-triplet energy transfer is exploited. the sites with the highest probability to form triplet states, which are quenched by the central luteins, are chl603 and chl612. unquenched chl triplet states are also produced by photo-excitation in the lhc-ii complex. putative sites of these triplet states are chl614, chl611, chla604 and chl613, since they do not contribute to the formation of the observed carotenoid triplet states. fluorescence lifetime spectrum of the plant photosystem ii core complex the photosystem ii kinetic model (diffusion or trap-limited) is still much debated. there is discussion about whether energy transfer from the core antenna (cp47 and cp43) to the reaction center complex (d1-d2-cyt b559) is rate-limiting (transfer to trap-limited). this study investigates this problem in isolated core particles by exploiting the different optical properties of the core antenna and the reaction center complex near 680 nm, due to p680 and an isoenergetic pheophytin. this was used as a marker feature for the reaction center complex. if the transfer to the trap-limited model were correct, assuming excited-state thermalization, the specific reaction center fluorescence decay lifetime should be shorter near 680 nm, where there is reaction center complex specificity, than at the other emission wavelengths. such a selective reaction center feature was not observed in fluorescence decay measurements. at the experimental resolution used here, we conclude that the traplimited energy transfer to the reaction center could, at the most, be 20% limiting. thus, the transfer to the trap-limited model is not supported. photosynthetic apparatus response under heat stress n. pshybytko 1 , i. divak 1 , l. kabashnikova 1 , e. lysenko 2 1 institute of biophysics and cell engineering, national academy of sciences of belarus, belarus, 2 institute of plant physiology, russian academy of sciences, russia the mechanisms of psii thermoinactivation and adaptation under high temperature impact and participation of hydrogen peroxide at these processes were studied. the twice suppression of oxygen evolving activity of thylakoids with simultaneous decrease in d1 protein content and the release of extrinsic 33 kda polypeptide from woc after 15-20 min heating at 40 o c were registered. using inhibitor analysis it was shown that thermoinduced degradation of d1 protein after 20 min heating occurred by proteases. the participation of ftsh protease in thermoinduced d1 protein degradation was observed. level of transcription of psba gene in chloroplast was raised after 20 min heating and was decreased through 1 h. the content of hydrogen peroxide was increased three times after 20 min of heating and was decreased to normal level through 1 h and was raised after 2.5 h again. it is interesting that level of peroxidation lipids products was increased after 2.5 h heating only. received data indicated that hydrogen peroxide is signal molecular at the photosynthetic apparatus under heat stress. during heating the inactivation of woc and d1 protein is occurred. as result the h 2 o 2 is generated. hydrogen peroxide as signal molecule activates transcription of psba. turnover of psii is occurred. more long heating induces degradation of proteins and lipids and h 2 o 2 represents as the destructive agent. a. nurisso 1 , m. a. morando 2 , f. j. cañada 2 , j. j. barbero 2 , a. imberty 1 1 cermav-cnrs, grenoble, france, 2 centro de investigaciones biológicas, madrid, spain the mechanism of symbiosis between legume plants and rhizobial bacteria is a relevant topic of interest since it is at the basis of the nitrogen fixation process. this mechanism is strictly related to the production of lipochitooligosaccharides, nod factors (nf), which allow the bacterial invasion into the legume host roots. in medicago truncatula, the recognition of nf from the symbiont sinorhizobium meliloti requires the nfp gene, able to encode a lysm-motif receptor-like kinase. the extracellular part of this protein is characterized by three lysm motifs: only one of them seems to be involved in the nf recognition. herein, we report an in silico investigation of different structural aspects of this process. first of all, the conformational behaviors of a new generation of nf analogues were elucidated by mds simulation. then, a homology model of the lysm2 domain from m. truncatula was proposed and compared with a model of lysm domain from pisum sativum: docking calculations of natural nf identified a common binding site in which the carbohydrate portion is the main responsible of the binding. both studies provide support for the idea that the carbohydrate part of nf plays a key role in the interaction with lysm domains, while the lipid moiety modulates ligand specificity probably interacting with a potential second receptor. interaction between frutalin with biomembrane models and its correlation with cell adhesion property frutalin is a homotetrameric lectin d-galactose (d-gal) binding that activates natural killer cells in vitro and leukocyte migration in vivo, being a potent lymphocyte stimulator. in this study we investigated the interaction of frutalin with different phospho/glyco-lipids using langmuir monolayers as biomembrane mimetic system. the results attest the specificity of the protein for the carbohydrate d-gal when attached to the biomembrane model. the adsorption kinetics for frutalin to the mixed monolayers containing glycolipids showed that the interaction depends on the presence of charged groups and on the position of the d-gal on the polar head of the glycolipid. using brewster angle microscopy (bam), we investigated the morphology of the interface for the binary mixtures containing galcer, where small domains were formed at high lipid packing, suggesting that frutalin can induce the formation of likelipid rafts domains in vitro. the results obtained with the membrane models were associated with those from fibroblast adhesion induction. the cell adhesion promoted by frutalin is in accordance with the results observed in langmuir monolayers, which probes the specificity of the interaction between the lectin and d-gal on cell-membrane surfaces. based on these results, frutalin can be considered as a promise biotechnological tool to actuate in tissue engineering regeneration. supported by fapesp, cnpq and capes aggregation and glycation processes of proteins are of peculiar interest for several scientific fields. serum albumins are widely studied proteins for their ability to self-assemble in aggregates and also to undergo to non enzymatic glycosylation in cases of diabetes. in this work we report a study on thermal aggregation of glycated bovine serum albumin (bsa) prepared with different concentrations of glucose at ph 7.4. increasing concentration of sugar modulates the effect of different glycation levels on the protein aggregation. fluorescence spectroscopy, ftir absorption, static and dynamic light scattering are used to follow the time evolution of the aggregation process and of protein conformational changes. conformational changes of secondary and tertiary structures are measured by ftir absorption; the kinetics of amide i, amide ii and amide ii' bands are monitored. the kinetics of tryptophans fluorescence give complementary information on the tertiary structure changes and on the polarity modification of the fluorophores environment. the aggregates growth is studied by dynamic light scattering measurements and rayleigh scattering peak. the results show that the partial unfolding of the protein is not affected by the glycation, while the presence of increasing amounts of glycated molecules progressively inhibits the aggregates formation. solvent occupancy analysis in ligand structure prediction: the case of galectin-1 s. di lella, m. a. martí, d. a. estrin inquimae-conicet, universidad de buenos aires, argentina formation of protein ligand complexes is a fundamental phenomenum in biochemistry. during the process, significant solvent reorganization is produced along the contact surface. using md simulations in explicit solvent combined with statistical mechanics analysis, thermodynamic properties of water molecules around proteins can be computed and analyzed in a comparative view. based on this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin-1 (hgal-1). specifically, we defined water sites (ws) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. we then succesfully extended the analysis of the role of the surface associated water molecules in the ligand binding and recognition process to many other carbohydrate binding proteins. our results show that the probability of finding water molecules inside the ws, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an oxhydril group of the ligand in the protein-ligand complex. this information can be used to predict possible complex structures when unavailable, and suggest addition of ohcontaining functional groups to displace water from high p(v) ws to enhance drug, specially glycomimetic-drugs, protein affinity and/or specificity. glyconanoparticles: nano-biomaterials for application in biotechnology and biomedicine s. penadés laboratory of glyconanotecnology, cicbiomagune -ciber-bbn, san sebastian, spain to study and intervene in carbohydrate interactions our laboratory has developed a chemical strategy (glyconanotechnology) to produce sugar functionalized gold nanoclusters with multivalent carbohydrate display (glyconanoparticles, gnps). by combining these tools with biophysical and analytic surface techniques (itc, afm, tem, spr) we have demonstrated and evaluated ca 2+ -mediated carbohydratecarbohydrate interactions involved in cell adhesion processes. the gnp system complements other currently available multivalent systems incorporating carbohydrates and presents some advantages as: 1) exceptionally small core size; 2) high stability in water and physiological buffers; and 3) multivalency and multifunctionality with control over ligands number. specific gnps have been applied to the inhibition of melanoma metastasis in mice and as inhibitors of hiv-trans infection. magnetic gnps have also been used in cellular labelling and imaging as probes for mri. in this lecture, the glyconanotechnology strategy will be presented and some application will be highlighted. -glycobiophysics expanded ataxin-1 induces membrane mechanical instability: evidences from model systems and cells c. canale 1 , s. averaimo 2 , d. pesci 3 , d. paulis 2 , v. fortunati 3 , a. gliozzi 4 , m. mazzanti 2 , c. jodice 3 , a. relini 4 1 iit, genoa, italy, 2 university of milan, italy, 3 university of tor vergata, rome, italy, 4 university of genoa, italy spinocerebellar ataxia type 1 is a neurodegenerative disorder involving the expansion of a polyglutamine stretch beyond a threshold in the protein ataxin-1, with intracellular deposition of amyloid aggregates. ataxin-1 variants with polyglutamine stretches of pathological length are not associated to disease when the stretch is interrupted by histidines. mounting evidence suggests that ataxin-1 aggregates interact with the nuclear membrane. to get insight into the mechanisms leading to neurological disorders, we studied model lipid membranes containing an expanded pathological form of ataxin-1 or expanded normal forms interrupted by histidines. electrical measurements on planar bilayers showed the occurrence of current steps, much larger for the pathological form, indicating the formation of pore-like structures. atomic force microscopy measurements showed that the longer the polyglutamine tract, the smaller was the force required to penetrate the bilayer with the afm tip. the smallest penetration force, and then the strongest membrane destabilization, was observed for membranes containing the expanded pathological form. experiments on cos1 cells provided evidence that pathological protein aggregates damage the nuclear membrane eventually causing cell autophagy. modification by dopamine adducts links αsynuclein to oxidative stress in parkinson disease m. bisaglia 1 , e. greggio 2 , l. tosatto 1 , f. munari 1 , i. tessari 1 , p. polverino de laureto 1 , s. mammi 1 , m. r. cookson 2 , l. bubacco 1 1 university of padova, italy, 2 nih, bethesda, md, usa oxidative stress has been proposed to be involved in the pathogenesis of parkinson disease (pd). a plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (da) and produce various toxic molecules, i.e., free radicals and quinone species. α-synuclein (αsyn), a small protein found in lewy bodies characteristic of pd, is also thought to be involved in the pathogenesis of pd. to investigate the possibility of a synergistic role of oxidative stress and αsyn in pd, we analyzed the modulation of da toxicity by αsyn overexpression in dopaminergic human neuroblastoma cells. our results indicate that the increased expression of αsyn enhances the cellular toxicity induced by the accumulation of intracellular da. we then correlated our results with the structural modifications induced by the oxidation products of da on αsyn by studying two potential pathways for the oxidative chemistry associated with da. the first one is the auto-oxidation reaction which leads to the concomitant formation of radical and quinone species. the second is the enzymatic oxidation, mediated by tyrosinase, which leads only to the accumulation of quinones. our data suggest a link between da and αsyn in the progression of pd and provide novel insights on both the mechanisms involved in the oxidative chemistry and the aggregation properties of αsyn. dynamic light scattering aggregation studies of aβ (1-40) peptide s. bertini, r. beretta, d. gaudesi, a. naggi, g. torri ronzoni institute, milan, italy heparan sulfate (hs) proteoglycans play a role in formation of amyloid plaques by facilitating formation of amyloid aggregates. heparins and low-molecular weight heparins which mimic hs sequences could interact with amyloid peptides putatively involved in neurodegenerative processes. the aim of this work is to study the influence of chain length and structure of heparin oligosaccharides on the aggregation of amyloid. to this purpose, the interaction with amyloid peptide aβ 1−40 with a number of heparin/heparin oligosaccharides had been investigated using dynamic light scattering (dls) which permits to determine the size and the stability of molecular aggregates in solution. as indexes of aggregation in solution, two parameters were chosen, i.e., the area under the autocorrelation function (af) and the average hydrodynamic ratio (rh). we evaluated the aggregation kinetic of different preparations of aβ and the ph of solubilization. a clear indication was obtained for the trend of aggregation of the peptide aβ 1−40 alone and in the presence of oligosaccharides. the size of the aggregate in the presence of the tetrasaccharide is definitely lower than for the peptide alone. the size of the aggregates increases with increasing size of the oligosaccharides. in fact, the octasaccharide promotes further aggregation. it is crucial for pharmaceutical protein formulations to have low aggregate content. using the monoclonal igg1 antibody rituximab as a model system, we have studied the mechanisms by which antibodies aggregate at physiological ph when incubated at 52-65 • c. light scattering showed two coupled stages: an initial fast stage followed by several hours of exponential growth of the scattered intensity. data analysis showed the fast formation of a large species, which subsequently increased in size. the aggregate number density had a maximum suggesting that aggregates increase in size by coagulation. the analysis also predicted the actual underlying increase in aggregate mass to be linear and reach saturation. this was confirmed by size-exclusion chromatography of incubated samples. in an arrhenius plot the activation energy of the first stage was similar to the unfolding energy of the ch2 domain, suggesting a pivotal role of this domain in the aggregation process. cd and fluorescence showed only minor structural changes in the temperature interval studied. we conclude that coagulation is the main mechanism driving rituximab aggregation and that aggregation is due to small structural changes. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils -abstracts 123 solid-state nmr structural studies of alzheimer's disease amyloid β(1-42) in lipid bilayers j. d. gehman 1 , a. k. mehta 2 , f. separovic 1 1 school of chemistry, bio21 institute, university of melbourne, 3010 vic, australia, 2 department of chemistry, emory university, 1515 dickey dr., 30322 atlanta, ga, usa amyloid-β peptides are believed to cause loss of nerve cell function in individuals suffering from alzheimer's disease, where evidence suggests that interaction with the cell membrane correlates strongly with cytotoxicity. previous studies report a range of different but plausible structures, which depend on the molecular environment of the peptide. this sensitivity to sample preparation suggests that the structure relevant to disease is found in lipid bilayer membranes. while model membrane vesicles are too large to be studied by conventional solution nmr, solid-state nmr is one of the few technologies available to study such systems. we present recent measurements which suggest a novel peptide structure in a lipid bilayer environment: (i) rotational-echo double-resonance (redor) distance measurements between selectively enriched 13 c-carbonyl and 15 n-amide positions constrain dihedral angles of intervening residues and suggest that at least part of the aβ(1-42) peptide folds into a β-sheet like conformation, in contrast to the helical and coiled structures in previous reports; and (ii) double quantum filtered draws measurements indicate that the extended strand does not assemble into an in-register parallel sheet as reported for amyloid fibrils. a mutation in app gene with dominant-negative effect on amyloidogenesis: a light scattering study e. del favero 1 , g. di fede 2 , f. tagliavini 2 , m. salmona 3 , l. cantù 1 1 university of milan, segrate, italy, 2 "carlo besta" national neurological institute, milan, italy, 3 istituto di ricerche farmacologiche "mario negri", milan, italy we studied the effect of a single-point mutation (a673v) on the aggregative properties of alzheimer's peptides aβ1−40. by laser light scattering it was possible to follow the early stages of aggregation of aβ1−40 wt and aβ1−40 mut (within 48 hours after sample preparation), when intermediates in the aggregation pathway, rather than the mature insoluble fibrils, are formed. results show that both the kinetics and the extent of aggregation are strongly enhanced by the mutation. on the reverse, coincubation of mutated and wild-type peptides slows down the aggregation process and results in aβ aggregates that are much more unstable against dilution. it is evident that the interaction between mutant and wild-type aβ1−40 interferes with nucleation or nucleation-dependent polymerization, hindering amyloidogenesis. these results account for the highly amyloidogenic effect of the mutation in vitro, if alone, reversely reduced by the mutated-wild type mix. also a clinical case exists that constitutes the in-vivo evidence of these two opposing effects. this finding may offer grounds for the development of therapeutic strategies, based on modified aβ peptides or peptido-mimetic compounds, for the potential treatment of alzheimer's disease. the collagen i is the major structural protein of the body and it is found organized in a hierarchical manner in the extra-cellular matrix of several tissues (bone, tendon, cornea and sclera, etc..). it is responsible for their specific architecture. it is already known that collagen i is capable of forming cholesteric liquid crystalline phases that once stabilized by a ph increase, lead to collagen matrices that mimic the organization of the organic matrix found in bone [1] . in the present work, we analyze physico-chemical ways to modulate long range organizations obtained in this liquid state. we study the effect of collagen concentration, ph (2.6 / 3.6) and acids (hydrochloride acid / acetic acid) over the liquid crystalline order. in a original approach, correlation of collagen endofluorescence and shg signals has enabled us to quantify cholesteric pitch and phase transition as a function of collagen concentration within a gradient. cholesteric pitch have proved to be very responsive to physico-chemical conditions. indeed, the results allowed us to quantify a i / cholesteric transition as a subsequent transition from cholesteric to a phase hexagonal or colummar. prion diseases are deadly neurodegenerative diseases affecting human and mammalian species. according to the 'protein-only' hypothesis, the key event in the pathogenesis is the conversion of the α-helix-rich monomer (prp c ) into a polymeric β-sheet-rich pathogenic conformer (prp sc ). we have used a combination of biophysical techniques with molecular dynamics simulations (md) to elucidate the molecular mechanisms of prp c unfolding and polymerization. under well established conditions, three β-sheet-rich soluble oligomers were generated from the partial unfolding of the monomer, which were found to form in parallel. to obtain a deeper insight into the molecular events, doublecystein mutants were designed, thus 'locking' different regions of prp. single mutations were also performed, which affected dramatically and selectively the prp oligomerization pathway. furthermore, we have now identified the minimal region that leads to the same oligomerization profile as the full-length prp, namely, h2h3. the existence of at least three distinct oligomerization pathways and the effect of single mutations reveal the conformational diversity of prp and a possible relationship with prion strain phenomena. the identification of domains involved in the conversion process may lead to a better understanding of the effect of mutations or gene polymorphism on the evolution of prion pathology. investigating toxic protein nanoclusters and their interactions with living cells s. nag, b. sahoo, a. bandyopadhyay, c. muralidharan, r. abhyankar, s. gurav, s. maiti tata institute of fundamental research, homi bhabha road, colaba, mumbai 400005, india amyloid protein aggregation is responsible for many neurodegenerative diseases, such as alzheimer's and parkinson's. it appears that quasi-stable small soluble aggregates are the key to amyloid toxicity. though amyloid aggregation appears to be a nucleation mediated process, simple nucleation theory is unable to explain the stability of these intermediate species. we investigate this issue with fluorescence correlation spectroscopy in solution and on cell membranes. our initial data, varying the ph and the ionic strength of the solution, show that a charged colloid model can explain the overall stability of these species. in addition, this understanding suggests possible ways of modulating the stability of these aggregates in solution. some of these strategies successfully decimate the stable aggregate population, and also reduce the toxicity of amyloid beta to cultured neurons. afm studies of artificial amyloid fibrils and filaments p. mesquida 1 , a. kurtossy 1 , c. macphee 2 1 department of mechanical engineering, king's college london, london, u.k., 2 school of physics, university of edinburgh, edinburgh, u.k. amyloid fibrils are beta-sheet-rich superstructures of peptides or proteins. although these aggregates have first been found in connection with protein-misfolding diseases, such as alzheimer's or parkinson's disease, there is evidence that the ability to form fibrils is a generic property of any polypeptide rather than a result of specific, disease-related amino-acid sequences. fibrils can easily be formed in vitro from nondisease-related proteins and even from synthetic peptides. these artificial fibrils can thus serve as model systems to investigate the biophysical properties of amyloid fibrils. the system presented here is built from the artificial peptide ttr 105−115 , which contains 11 amino-acids and which forms well-defined nanorods of ca 10 nm diameter. we present atomic force microscopy (afm) results of their inner morphology by chemically dissecting the rods into their constituent filaments without completely breaking up the aggregates. in contrast to the stiff rods, their constituent filaments of ca 1-2 nm diameter were much more flexible. this shows that the particular way filaments are arranged around each other can massively change the mechanical rigidity of the resulting structures in amyloid fibrils, which could be one cause of the different strains in amyloid diseases. the formation of insulin fibrils is characterized by an initial apparent lag-phase, related to the formation of oligomers, protofibrils and aggregation nuclei. afterwards, the aggregation proceeds via fibril elongation, thickening and/or flocculation, and eventual gelation. here, we focus on the formation of such a gel, made of insulin amyloid fibrils, upon incubation at high temperature and low ph. by light scattering and rheological techniques, we monitor the development of the structural, dynamical and mechanical properties of fibrillar aggregates, up to the dynamic arrest of the sample and to the appearance of a non-ergodic behaviour, which marks the onset of gelation. our experiments were able to reveal the structural details hidden in the apparent lag-phase, displaying the slow fibril nucleation and elongation. this initial stage is followed by the known exponential growth of structures of different sizes. these two kinetic stages of structural growth are mirrored by the kinetics of the viscoelastic properties and, in particular, by the growth of the elastic modulus. our results show that the appearance of a noteworthy elastic network, is associated with the initial fibril nucleation and elongation more than with the formation of large structures, which causes the eventual gelation. capturing the initial events of in-cubo crystallization of membrane proteins c. v. kulkarni 3 , o. ces 1 , s. iwata 2 , r. h. templer 1 1 chemical biology centre and department of chemistry, imperial college london, united kingdom, 2 division of molecular biosciences, imperial college london, united kingdom, 3 institute für chemie, heinrichstrasse-28, university of graz, austria lipid based methods for the crystallization of membrane proteins including in-cubo crystallization are becoming popular in recent times. however, a complete understanding of the basic principles behind these methodologies is still elusive. the crystallization of membrane proteins in the lipid cubic phases involves following major steps: removal of membrane proteins from the native membrane using detergentlike molecules, followed by their insertion and equilibration into a lipid bilayer. the crystallization itself commences with precipitant induced osmotic dehydration which in turn stimulates the nucleation that subsequently leads to a crystal growth. using time-resolved x-ray scattering (saxs) and ultraviolet spectroscopy we have been able to gain new insights into the mechanism behind protein insertion into these complex three dimensional lipid structures including information on the timescales of protein folding relative to crystal growth and the effect of protein insertion on the morphology of the surrounding lipid matrix. several proteins can form amyloid fibrils under given environmental or thermodynamic conditions that affect their native conformation. lysozyme forms amyloid fibrils upon incubation of solutions at acid ph and at about 65 • c for a few days. differential scanning calorimetry experiments confirm that lysozyme is mainly unfolded above 60 • c at ph 2. we monitored the growth of aggregate size by dynamic light scattering for some days at different temperatures. our results show that the fibrillogenesis is characterized by an initial apparent lag phase and a subsequent growth with quadratic dependence upon time of the scattering intensity. this behaviour recalls a simple kinetic model of nucleation and elongation, with nuclei in equilibrium with monomers. at the end of the incubation at high temperature, we collected atomic force microscopy images which show fibrils with a diameter of a few tens of nanometers and a length of a few microns, characterized by a periodicity along the elongation axis. interestingly, the fibrils morphology exhibits no branching or thickening. this is consistent with the non-exponential growth observed in light scattering experiments. in order to elicit the role of repulsive electrostatic interaction in protein unfolding and self-assembly we extended our study at different ph. in this work we used afm to follow the amyloidogenesis pathway of transthyretin (ttr) to form protofilaments. single-molecule force spectroscopy (smfs) of native ttr and protofilaments were also compared in order to evaluate dynamic and structural differences. we observed that this pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers. although implicated in cytoxicity, the role of such oligomers within the amyloidogenesis pathway is poorly understood. we show that the annular species display a tendency to stack, forming tubular-like structures that precede the formation of protofilaments. the protofilament height and pitch resemble those of ttr amyloid reported in previous structural studies. upon solvent exchange, we also observed protofilament disassembly that revealed structures reminiscent of the initial ttr annular oligomers. smfs of protofilaments showed a time-dependent increase in the length of the manipulated structures, suggesting that associations between monomers stabilize with time. force spectra of native ttr and protofilaments contained transitions spaced by ∼4 nm, indicative of sequential unfolding of individual β-strands. based on these results a model of ttr protofilament assembly is proposed. ability to undergo amyloid aggregation is affected by protein size c. parrini 1 , h. ramshini 2 , f. chiti 2 , a. relini 1 1 physics department, university of genoa, italy, 2 biochemical sciences department, university of florence, italy short polypeptide chains, typically between 28 and 253 residues, are generally involved with the conversion from the native state into insoluble fibrillar aggregates. the low prevalence of large proteins is disproportionate with their high occurrence in the human proteome. in order to explore the propensity of large proteins to form amyloid-like fibrils, the 486-residue hexokinase-b from saccharomyces cerevisiae (yhkb) has been induced to aggregate under two separate conditions, at low ph in the presence of salts and at ph 5.5 in the presence of trifluoroethanol. such conditions are among the most promising to form amyloid-like fibrils by normally globular proteins. under both conditions yhkb aggregates very rapidly into species with significant β-sheet structure, as detected by circular dichroism, and a weak thioflavin t and congo red binding. atomic force microscopy revealed globular aggregates eventually clustering into large amorphous aggregates at low ph, while in the presence of trifluoroethanol ribbon-like structures with distinct morphology from typical amyloid fibrils were observed. they had irregular width, no twist, and were connected by thinner fibril segments perpendicular to them. in general, yhkb aggregates displayed an unusual softness, as they were very easily perturbed by the afm tip. these results suggest that inability to form amyloid fibrils may prevent large proteins from being associated with protein deposition diseases. the initial stage of proteins aggregation leading to amyloid fibrils: a saxs study m. g. ortore 1 , f. spinozzi 1 , f. carsughi 1 , t. narayanan 2 , g. irace 2 , s. vilasi 3 , p. mariani 3 1 dip. saifet, univ. pol., ancona, italy, 2 esrf, grenoble, france, 3 dip. biochim. biofis., ii univ., napoli, italy under some conditions, a protein converts from its soluble form into highly ordered aggregates called amyloid fibrils, which are associated with many human diseases. in order to tackle the prevention and treatment of these diseases, we need to understand the mechanism of the pathological aggregation of proteins. in vitro amyloid fibril formation is preceded by the formation of metastable non fibrillar forms, which are responsible for cytotoxicity underlying neurodegeneration. the molecular mechanisms leading proteins into prefibrillar aggregates are still unclear. we present a saxs study performed at id02 beamline of esrf on the apomyoglobin mutant w7fw14f, which at physiological ph, firstly aggregates in prefibrillar forms that are cytotoxic and then forms amyloid fibrils. the first stages of w7fw14f oligomerization are induced by a ph jump. data show that big changes in w7fw14f in solution happen in less than 100 ms. singular value decomposition (svd) of the data yields a set of functions, from which all the scattering curves can be reproduced. the major result of our study is the determination of the presence of different oligomers in each step of the process. hence, time-resolved saxs experiments together with the estimation of different oligomers via svd method, can be a new and useful approach to investigate the first stages of amyloidogenesis. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils study of structure/toxicity relationship of amyloids by infrared spectroscopy h. p. ta amyloid diseases (alzheimer, parkinson, type ii diabetes, prion) correlate with protein aggregation. all the proteins associated with these pathologies aggregate into amyloid fibrils with a common ß-cross structure. according to many in vitro studies, the toxicity of various amyloids seems to be linked to some intermediates formed during the aggregation pathway and to their interaction with membranes. our aim is to understand the link between structure and toxicity of amyloids by studying their interaction with model membranes. for this, we use as an amyloid model, the prion-forming domain pfd218-289 (wild type wt) of het-s, a prion protein of the fungal p. anserina. when expressed in yeast, wt is not toxic whereas one of these mutants, m8 is toxic. in vitro, m8 forms very unusual short amyloid fibers contrary to wt which polymerizes as long fibers. furthermore, atr spectra have shown that m8 is essentially assembled into mixed parallel and anti-parallel ß-sheets whereas wt displays a predominant parallel organization. we are currently studying the interaction between wt or m8 with lipid langmuir film by polarized modulation-infrared reflexion absorption spectroscopy (pm-irras). this technique allows determining the secondary structure of proteins and their orientation in the membrane. the study of interactions with different charged phospholipids is following. a. stirpe, m. pantusa, r. bartucci, l. sportelli, r. guzzi dipartimento di fisica, laboratorio di biofisica molecolare & udr cnism, università della calabria, rende (cs), italy an increasing number of experimental studies is demonstrating that the propensity of forming amyloid fibrils is not limited to proteins related to neurodegenerative diseases but it is a more general phenomenon. we present data on human serum albumin (hsa) aggregation induced by a combination of thermal and metal ions effects investigated by optical density, fluorescence and electron paramagnetic resonance (epr). the turbidity experiments as a function of temperature show that the hsa aggregation starts at about 70 • c which is higher than the denaturation temperature (∼ 60 • c). in the presence of copper and zinc metal ions the onset temperature for protein aggregation is markedly reduced as the metal:protein molar ratio is increased from 1:1 to 10:1. moreover, the copper is more effective in inducing protein aggregation compared to zinc ion. the hsa aggregation analyzed by tht fluorescence at different incubation temperatures lacks a lag phase and the kinetic traces can be fitted by double exponential functions. the tht fluorescence increase evidences the formation of protein aggregates with fibrillar features. epr experiments for the cu(ii)-hsa complex show the binding of cu(ii) in the protein native state in a square planar coordination with 4 equatorial n atoms, and is not influenced by the heat treatment of the protein. the overall results suggest that hsa aggregation is compatible with a downhill process that does not require the formation of an aggregation nucleus. protein condensation diseases -a colloid physicists viewpoint p. schurtenberger adolphe merkle institute, university of fribourg, ch-1723 marly 1, switzerland a broad class of diseases, such as cataract, alzheimer's disease and sickle-cell disease, involve protein association phenomena as an essential aspect. the basic element common to all members of this class of molecular condensation diseases is the subtle interplay between protein interactions that produces condensation into dense, frequently insoluble mesoscopic phases. among this class of diseases, cataract is particularly important as the world's leading cause of blindness. this disease is most often the consequence of an uncontrolled aggregation (or phase separation) of the proteins in the eye lens that results in a loss of its transparency. the high concentration protein mixtures present in the eye lens are normally stable and produce a high refractive index that aids the eye in adaptive focusing of light. moreover, the proteins themselves exhibit a rich variety of repulsive interactions, attractive interactions, sizes, phase transitions and self-association. these mixtures also exhibit the pathological aggregation and opacification of the cataract disease that has inspired their study. in my presentation i will illustrate how we can use a combination of small-angle neutron and xray scattering experiments combined with molecular dynamics computer simulations to identify, measure and model the molecular interactions and emergent optical and viscoelastic properties and the phase behavior of the relevant, complex cytoplasmic mixtures. fluorescence microscopy studies of iapp fibrillation at model and cellular membranes d. c. radovan 1 , n. opitz 2 , r. winter 1 1 department of physical chemistry i -biophysical chemistry, tu dortmund university, dortmund, germany, 2 max-planck institute for molecular physiology, dortmund, germany type 2 diabetes mellitus (t2dm) is characterized by islet amyloid deposition and beta cell death, the main culprit being a small 37 a.a. peptide hormone, islet amyloid polypeptide (iapp), which forms fibrils under pathological conditions. we studied the interaction of iapp with giant (guvs) and large (luvs) unilamellar vesicles as well as with ins-1e cells. by using confocal / two-photon excitation fluorescence microscopy and guvs, we tested the influence of charge (dopc:dopg 7:3) and model raft systems, displaying liquid-ordered (l o ) / liquid-disordered (l d ) phase coexistence (such as dopc:dppc:cholesterol 1:2:1), respectively, on the kinetics of iapp fibril formation. a preferential partitioning into the l d phase was observed and fibrils grew along with lipid uptake. fluorescence spectroscopy leakage experiments with carboxyfluorescein-filled luvs and the corresponding tht kinetics of iapp fibril formation were carried out as well. moreover, using the wst-1 reduction assay and fluorescence microscopy, we could show that the red wine compound resveratrol is a potent inhibitor of iapp fibrillation and its cellular toxicity on ins-1e cells, these findings highlighting the potential role of resveratrol in future clinical applications, i.e., in the treatment of t2dm. a remarkably high viscosity has been induced in aqueous solutions of lysozyme by the addition of certain structurally related organic solvents, such as tetramethylurea (tmu), dimethylsulfoxide (dmso), dimethylformamide (dmf), and hexamethylphosphortriamide. dmso-induced gelation is observed in samples fulfilling the two following requirements: (1.) lysozyme concentration in excess of 5 mm, and (2.) volume fractions of dmso exceeding 0.7. based on spectroscopic data, the whole process was characterized as consisting of two mutually independent stages. the first involves an extensive transition of the polypeptide backbone, from a predominantly helical to increased random coiled and beta-sheet structures, with the occurrence of non-orthodox protein secondary structures at regions above the solvent critical point. the second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. here we present a set of scattering and microrheology experiments that investigate both the structural and dynamic properties of the gels under various experimental conditions. studying alpha-synuclein aggregation by fluorescence polarization based kinetics l. tosatto, f. munari, i. tessari, g. de franceschi, m. pivato, p. polverino de laureto, m. bisaglia, l. bubacco università degli studi di padova, padova, italy alpha-synuclein (syn) is linked to parkinson's disease (pd) by two evidences: the accumulation of amyloid fibrils of the protein and the autosomal dominant forms of the disease (a53t, a30p and e46k mutants). protein oligomers seem to be the most toxic species, causing dopaminergic neuronal death, probably disrupting cell membrane (volles & lansbury, 2002) . the aggregation of syn follows a nucleation dependent mechanism, i. e., aggregation is favoured only after the formation of an oligomer composed of a critical number of monomers (wood et al., 1999) . as the constitution of the nucleus is a rare event, early aggregation stages are difficult to study. to explore the early stages of the aggregation process a fluorescence polarization (fp) based method (luk et al., 2007) was applied to study syn oligomerization. fp depends on the size of the fluorescent molecule, therefore it is suitable for the detection of oligomers formation. we measured aggregation kinetic properties under several different conditions. a 100 to 1 mixture of syn and oregon green labelled syn was used to analyze the aggregation behaviour of wild type (wt) and pd mutants. the wt protein shows the fastest aggregation rate. this aggregation process is slowed down by the presence of the chaperone 14-3-3eta in wt syn samples. finally, dimers formed by a disulfide bond at the n-terminus or c-terminus of syn, when tested for their aggregation behaviour, show a different propensity to aggregate. a. pardini 1 , r. bizzarri 2 , a. diaspro 3 , p. bianchini 3 , c. usai 1 , p. ramoino 3 , g. checcucci 1 , g. colombetti 1 1 istituto di biofisica, cnr, italy, 2 a) nest, sns, iit udr, pisa, italy; b) nest, cnr-infm, pisa, italy, 3 univiversity of genova, genova, italy the colored ciliates blepharisma japonicum and fabrea salina show photomotile responses triggered by endogenous pigments of the hypericin family. b. japonicum exists in two forms: the wild one contains red blepharismin; the other one, generated by irradiating the cells with dim visible light, blue blepharismin. b. japonicum shows step-up photophobic response, whereas f. salina, that contains fabrein, shows step-down photophobic responses and positive phototaxis. we showed by confocal microscopy that the pigments are localized not only in pigment granules, but also in the cilia. this fact implies that the structure of the pigment in the cilia differs from that in the cell body, (pigment granules are too big to fit in the axoneme) and suggests that ciliary pigments might play a decisive role in photoreception. fluorescence lifetime imaging microscopy (flim) shows that there is a spatial distribution of lifetimes, which are shorter for the pigment in the cilia. this might indicate a functional role for these pigments. furthermore, lifetimes for f. salina are always longer than those of blepharisma. in order to further characterize the structure of ciliary pigments by means of spectroscopic methods, we have also performed spatially resolved static and time-resolved fluorescence anisotropy measurements. biosensing applications of micro/nano structured silicon several topics related to porous silicon (ps) biosensing properties were carefully considered in this study. ps allows the increasing of the immobilised biomolecule number on its surface and the creation of stable covalent bonds due to its controllable chemistry. beside this, ps is suitable for electrical (conductance, impedance), electrochemical and optical amplification of the detected signal. the experimental results on the fabrication of the ps microstructures such as: (i) protein immobilization and detection using microarray technique; (ii) dna biomolecule detection by impedance or by fluorescence spectroscopy; (iii) very sensitive sers biosensors (raman signal of 11-mercaptoundecanoic acid); (iv) sensitive element for neurons in nutmix culture are in detail presented. various characterisation techniques have been used, optical and scanning electron microscopy (sem), x-ray diffraction, raman, laser fluorescence and impedance spectroscopy for investigation molecule attachment on the au/ps structures. we have demonstrated that different morphologies of ps as-prepared or coated with gold nanoparticles have an important role in biomolecule/cell detection, due to its large internal surface combined with specific optical properties, being in the same time sensing element/support for immobilization of sensing biomolecules as well as transducer for biochemical interactions. heterotrimeric g-proteins interact with their g-protein coupled receptors (gpcrs) via key binding elements comprising the c-terminal segment of the α-subunit and the two lipid anchors at the α− and γ−subunit. direct information about diffusion and interaction of gpcrs and their g-proteins is mandatory, as these properties will affect the timing of events in the complex signal transduction cascade. in the case of the photoreceptor rhodopsin, receptor packing in the membrane and the related diffusion coefficients are discussed controversially (1, 2). by using single particle tracking we show that the encounters of rhodopsin with the fluorescently labeled cterminus of the α-subunit as well as with the holo-g-protein transducin change upon rhodopsin light-activation. our results indicate confined areas of interaction for the c-terminal segment of the α-subunit with inactive rhodopsin disk membranes and less restricted diffusion of the receptor-bound cterminal segment after light-activation. this suggests dynamic short-range order in rhodopsin packing and specific structures for efficient interaction (3) in vivo, skeletal muscle actively shortens and also resists lengthening, for example when landing after a jump. we study the energy during shortening and during lengthening by measuring the rate of pi release which results from atp hydrolysis. we show here the rate of pi releases in an in vitro protocol that involves muscle stretched followed by muscle shortening. it is hypothesised that the magnitude of deceleration of pi release in the stretch phase is less than the acceleration during the release due the energy input of the motor that is used to apply the length changes. therefore the end point of pi release is similar to isometric conditions. pi release responses to a ramped stretch (5%) followed 100 ms later by a symmetrical release at low velocities (0.5 l 0 /s −1 ) were measured in permeabilised fibre bundles of rabbit psoas at 20 • c. laser diffraction and high speed video were also recorded to confirm length change. we show that the rate of pi release drops during the stretch phase, returns to isometric levels during the length hold phase, and finally accelerates during the ramped release. tracking of sarcomere length change using video analysis demonstrated that laser diffraction is unreliable at times during stretches due to lack of uniformity of the sarcomere spacing. microbial rhodopsins: receptors, channels, and pumps from a single design j. l. spudich, e. negri-spudich center for membrane biology, university of texas, houston, texas, u.s.a. the microbial rhodopsin family is comprised of ∼5000 homologous proteins containing 7 transmembrane helices forming a pocket for the chromophore retinal. most are lightdriven ion pumps ("transport rhodopsins") and others are photosensory receptors ("sensory rhodopsins"). phylogenetic analysis indicates frequent lateral gene transfer of proton pumps among prokaryotic and unicellular eukaryotic species, followed by coupling of the pump's mechanism to the cell's existing signal transduction machinery to create photosensors. this evolutionary path is strongly supported by our studies of sensory rhodopsins in various organisms which demonstrate remarkably diverse signaling mechanisms with diverse transducer partners. the best studied are the phototaxis receptors in haloarchaeal prokaryotes (sri and srii) and in eukaryotic algae (channelrhodopsins). sri and srii transmit signals by protein-protein interaction to control a phosphorylation cascade that modulates motility. channelrhodopsins are light-gated cation channels that depolarize the membrane mediating calcium ion influx into the flagellar axoneme. crystallography and molecular biophysics have begun to clarify how modifications of the same architecture enable the rhodopsins to carry out their distinctly different molecular functions. interconversions of their functions by mutation reveal the elegant simplicity by which evolution uses existing genes to create proteins with novel functions. gold colloids-fluorophore complexes for protein detection assay l. sironi 1 , s. freddi 1 , l. d'alfonso 1 , m. collini 1 , m. caccia 1 , g. tallarida 2 , s. caprioli 2 , g. chirico 1 1 dipartimento di fisica, università di milano-bicocca, italy, 2 laboratorio nazionale mdm, agrate brianza (mi), italy noble metal nanoparticles (np) are endowed with peculiar optical properties related to the surface plasmon resonances (spr). the interaction of surface plasmons of gold nanoparticles with fluorophores a few nanometers away from the surface modifies their brightness and excited-state lifetime, and this effect can be exploited to obtain nanodevices for proteinprotein recognition. we studied different types of constructs based on gold nps on which derivatives of fluorescein were bound. the interaction of this fluorophore with the gold surface plasmon resonances, mainly occurring through quenching, affects its excited-state lifetime, that is measured by fluorescence burst analysis in standard solutions. the binding of proteins to the gold nps through antigen-antibody recognition further modifies the dye excited-state lifetime. this change can therefore be used to measure the protein concentration. streptavidin-functionalized gold nps of size 5-10 nm are used to bind biotin-fluorescein and biotin-antibodies for specific proteins. we have first tested the constructs for bovine serum albumine (bsa) detection. the data reported here indicate that one can measure the concentration of bsa in solution with an apparent limit of detection of 5±2 pm. we have then extended the study to the antitumor protein p53 -p53 antibody interaction in standard solution and directly in cellular extracts. calcium transport and phototransduction in isolated rod outer segments g. rispoli dip. biologia ed evoluzione, università di ferrara, italy ca 2+ concentration in photoreceptor rod outer segment (os) strongly affects the generator potential kinetics and light adaptation. light stimuli may produce voltage changes exceeding 40 mv: since the os ca 2+ extrusion is entirely controlled by the na + :ca 2+ ,k + exchanger (nckx), it is important to assess how the nckx ion transport is affected by voltage and intracellular factors. the nkcx regulation was investigated in whole-cell recorded os, using ionic conditions that activated maximally forward and reverse exchange. in all species examined of amphibia and reptilia, the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. since hyperpolarization increases ca 2+ extrusion rate, the recovery of the dark level of ca 2+ (and of the generator potential) after light stimuli results accelerated. mg-atp doubled the size of forward and reverse exchange current without modifying their voltage dependence, indicating that mg-atp regulates the number of active exchanger sites and/or the nckx turnover number. ca 2+ jumps achieved via photolysis of caged-ca 2+ produced current transients, possibly originating from electrogenic partial reactions. no monovalent cation substituted for na + at the nckx binding sites, but rb + substituted for k + , while sr 2+ , ba 2+ , mg 2+ substituted for ca 2+ with an apparent permeability ratio of 0.78, 0.20, <0.05:1, respectively. a. pfeifer 1 , k. zikihara 2 , s. tokutomi 2 , j. heberle 1 , t. kottke 1 1 bielefeld university, bielefeld, germany, 2 osaka prefecture university, sakai, japan the blue light receptor phototropin regulates the growth of plants towards the light. it contains two light-, oxygen-, or voltage-sensitive (lov) domains and a kinase domain. lov domains bind noncovalently flavin mononucleotide (fmn) as chromophore. upon illumination, the triplet excited state of flavin reacts within few microseconds with a nearby cysteine under formation of a photoadduct, which represents the signaling state. in response to adduct formation, a jα helix adjacent to the lov2 domain dissociates and allows for autophosphorylation by the kinase domain. the mechanism of the photoreaction and the signaling pathway from fmn to the jα helix are still unclear. we have investigated the lov2 domain of arabidopsis phototropin 2 by microsecond ft-infrared spectroscopy. the difference spectrum recorded at 2 µs provides evidence that the flavin is unprotonated in the triplet excited state. therefore, a previously proposed ionic mechanism of bond formation is disfavored. changes in secondary structure were detected concomitant with adduct formation that relax with a time constant of 120 µs. this early adduct intermediate has not been previously characterized. the final adduct state is formed in milliseconds by further alterations in secondary structure. these findings raise the question of whether the early or late adduct intermediate propagate the signal to the jα helix. -photosensory biophysics the psychostimulant amphetamine increased no generation measured by epr as well as amino acid release in the rat brain nitric oxide (no) is a novel messenger that modulates many functions of the nervous system. the involvement of no in brain damage was shown mainly by indirect evidence and the data are controversial. the short half-life of no makes its direct detection difficult. we measured no generation using epr spectroscopy based on determination of the amount of paramagnetic mononitrosyl-iron complexes. the aim was to elucidate whether psychostimulant drug amphetamine (amph) modulates formation of no and lipid peroxidation (lpo) products as well as the neurotransmitter release in rat brain. the output of glutamate, aspartate, gaba and acetylcholine (ach) was monitored in striatum by microdialysis with hplc detection. amph produced 2fold elevation of no generation and lpo formation in brain areas. while amph increased the aspartate, gaba and ach release, the glutamate output was not affected. pretreatment with the neuronal nos inhibitor was highly effective in abating the rise of no and neurotransmitter levels but failed to influence the lpo intensity elicited by amph. the findings suggest that activation of no synthesis is a potent factor in the amph-induced neurotransmitter release. light scattering study of dna over a wide chain length range: comparison with wormlike model p. baeri, m. zimbone dipartimento di fisica e astronomia, università di catania, italy this work reports light scattering measurements on dna in aqueous solutions (100 mm nacl ,1mm edta and 10 mm tris-hcl buffer, ph 7.8) over a wide range of molecular weights (10 2 -10 5 base pairs) and shows that, in the above standard solvent, shorter chains ( < 10 4 base pairs) behave as a "wormlike chain" and their diffusion coefficient as obtained by dynamic light scattering measurements, confirm the prediction of standard wormlike model, whilst longer chains ( >10 4 base pairs) behave in a different manner. dynamic and static light scattering and sem analysis indicate that dna molecules 10 5 base pairs long, condense into compact structures in our solvent condition. calculations done using a wormlike model are also presented and discussed in comparison both to our experimental data and to other data reported in the literature. a. asandei, t. luchian 'alexandru i. cuza'university, faculty of physics, laboratory of biophysics and medical physics, blvd. carol i no. 11, iasi, romania amphotericin b (amb) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. in this study we investigated the influence of ph upon the selectivity and the transport properties of amb channels inserted in reconstituted, ergosterolcontaining zwitterionic lipid membranes. our electrophysiology experiments carried out on single and multiple amb channels prove that at ph=2.8 these channels are anion selective, whereas at neutral and alkaline ph's (ph=7 and ph=11) they become cation selective. we attribute this to the ph-dependent ionization state of the carboxyl and amino groups present at the mouth of amb molecules. surprisingly, our data reveal that the single-molecule ionic conductance of amb channels varies in a non-monotonic fashion with ph changes, which we attribute to the ph-dependent variation of the surface and dipole membrane potential. we demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low ph values, amb channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition. we report a detection method for the redox state of proteins which combines fret-based fluorescence/confocal microscopy on dye-labeled protein with cyclic voltammetry. by using this combined method, electron transfer properties can be revealed from protein to electrode or from redox enzyme to substrate. we applied the fluorescent detection to azurin, a blue copper protein from the bacterium ps. aeruginosa, fluorescently labeled on the n-terminus for monitoring the redox state of the protein. the dye fluorescence is quenched by energy transfer to the copper in oxidized, but not in reduced azurin. fluorescence results demonstrated that cy5-labeled wtazurin switched in fluorescence intensity by up to 85% by varying the applied potential. labeled zinc-azurin was used as a control sample and did not show any fluorescence switching. for single molecule studies wt-azurin was labeled with atto 655 dye and a mixed sam was used, with 8-hydroxy-1octanethiol as a blocking agent to prevent non-specific binding of protein on the surface, and 1,10 decanedithiol for the specific covalent binding to the protein. preliminary data show that single-molecule fluorescence switching with the potential is indeed possible. -single molecule biophysics is there a specific erythrocyte membrane receptor for fibrinogen? an atomic force microscopy approach f. a. carvalho 1 , s. connell 2 , r. a. ariëns 2 , n. c. santos 1 1 instituto de medicina molecular, univ. lisbon, portugal, 2 university of leeds, u.k. fibrinogen (fg) contributes to erythrocyte (rbc) hyperaggregation by an increase in. rbc-fb protein binding considered to be non-specific. glycoprotein α iib β 3 is a specific integrin receptor for fibrinogen on platelets. we showed that there is a single molecule interaction between fg and an unknown receptor on rbc membrane, with a lower affinity when compared with platelet binding. we evaluated if rbc-fg binding is through an integrin-like receptor or not. interactions between fg and platelet/rbc receptors were studied by force spectroscopy. force curves were performed between fg-functionalized atomic force microscope tips and rbc or platelets. to evaluate if the fg-rbc binding is calciumdependent, similar studies were performed in the presence of ca 2+ or edta. we also carried out studies in the presence of a α iib β 3 inhibitor and of methyl-ß-cyclodextrin (to disrupt lipid rafts by cholesterol depletion). rbc-fg single forces were of 300-380 pn and of 400-480 pn for platelet-fg binding in presence of calcium. a significant decrease of the platelets-fg force-rupture was obtained in the presence of edta or α iib β 3 inhibitor. significant lower fg-rbc force value was obtained in the presence of mβcd, but not in the presence of edta. conclusion: fg-rbc binding seems not to be calcium-dependent but the existence of cholesterol on rbc membrane is important. nanoscopic and spectroscopic investigation of p53-based complexes at single molecule level a. r. bizzarri biophysics and nanoscience centre, cnism, facoltà di scienze, università della tuscia, largo dell'università -01100 viterbo, italy p53 is a transcription factor that plays a widely recognized role in preventing cancer development in response to dna damage. the tumor suppressor activity of p53 involves the formation of several complexes whose detection and study, at single molecule level, could be extremely relevant to understand, in detail, the mechanisms governing the cancer defense processes, as well as to develop ultrasensitive biosensors. p53-based complexes, are investigated at molecular level, by combining atomic force microscopy (afm), atomic force spectroscopy (afs) and surface enhanced raman spectroscopy (sers) with the support of computational docking. our attention is mainly devoted to study complexes between p53 and the electron transfer azurin which has been demonstrated to interact with p53, by promoting its stabilization. a possible competition between azurin and the cellular oncogene mdm2 is also investigated. unwinding the dna helix in force clamp condition p. bianco 1 , l. bongini 2 , m. dolfi 1 , l. vincenzo 1 1 physiolab, dbe, university of florence, italy, 2 university of florence and centro interdipartimentale studio dinamiche complesse, firenze, italy we use a dual-laser optical-tweezers (dlot, smith et al., science, 1996) to define the highly cooperative conformational transition in the molecule of dna, where the natural b-dna has converted into a new overstretched conformation called s-dna (bensimon et al., phys. rev. lett., 1995; cluzel et al., science, 1996) . single molecules of double stranded λ-phage dna (in a solution with 150 mm naci, 10 mm tris-hcl, 1 mm edta, ph 8.0. and 27 • c) are stretched either in length clamp or in force clamp mode. when the dna molecule is stretched in length clamp mode with a ramp lengthening, it shows the previously described highly cooperative overstretching transition at ∼60 pn, attributed to unwinding from the b-form to the 1.7 times longer s-form. stretching the molecule in force clamp mode with a staircase of force steps at 5 s intervals (step size 1-2 pn, rise time 1-2 ms) shows, for any given clamped force f in the region of the overstretching transition, different amounts of dna elongation (∆l) with exponential time courses. the analysis of the elongation rates allows to recover all the necessary parameters for an effective two-state model able to reproduce the out-of-equilibrium properties of the system. the results imply an unwinding cooperativity of 27 bps. this value is significantly lower than that obtained assuming force independent rate constants. supported by miur, ente cassa di risparmio di firenze and itb-cnr (milano). c. m. becker 1 , a. benedix 1 , b. l. de groot 2 , a. cafisch 3 , r. a. böckmann 1 1 saarland university, saarbrücken, germany, 2 mpi for biophysical chemistry, göttingen, germany, 3 university of zürich, zürich, switzerland modifying the stability or the binding behavior of the involved proteins by mutation can influence the activity of cellular processes. for an efficient identification of possible mutation-sites a fast calculation of the free energy of proteins is crucial. here we developed a fast and reliable method (cc/pbsa) [1] for the prediction of the change in stability of proteins and binding affinity of protein-protein complexes upon mutation. the energy function of cc/pbsa is based on gas phase energies, solvation free energies and entropic contributions. the protein flexibility is taken into account by generating random conformations based on geometrical constraints only applying the concoord [2] program. we applied cc/pbsa on the tem1-blip complex, which is important in bacterial antibiotic resistance. the results of single-and double-point alanine scanning are used to detect hot spots, cooperative effects, and the corresponding energy distribution. cc/pbsa is freely accessible on our web-server: http://ccpbsa.bioinformatik.uni-saarland.de the human recombinase hrad51 is a key protein for the maintenance of genome integrity and for cancer development. this protein plays a central role is the dna strand exchange occurring during homologous recombination. here we report the polymerization and depolymerization of hrad51 on duplex dna observed with a new generation of magnetic tweezers, allowing the measurement of dna twist with a resolution of 5 • in real time. at odds with earlier claims, we show that, after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition of single proteins, involving dna twisting steps of 65±5 • . simple numerical simulations support that this mechanism is an efficient way to minimize nucleoprotein filament defects. this behavior, consisting of different stoichiometry for nucleation and growth phases, may be instrumental in vivo. fast growth would permit efficient continuation of strand exchange by rad51 alone while the limited nucleation would require additional proteins such as rad52, thus keeping this initiation step under the strict control of regulatory pathways. besides, our results combined with earlier structural information, suggest that dna is somewhat less extended (4.5 versus 5.1å per bp) and more untwisted (18.2 versus 15 • per bp) by hrad51 than by reca, and confirm a stoichiometry of 3-4 bp per protein in the hrad51-dsdna nucleoprotein filament. biofunctional micropatterned surfaces to study the spatio-temporal organisation of lfa-1 r. diez-ahedo 1 , d. normanno 1 , c. g. figdor 2 , a. cambi 2 , m. f. garcia-parajo 1 1 bionanophotonics, ciber-bbn and ibec, barcelona, spain, 2 tumor immunology, nijmegen center for molecular life sciences, the netherlands lymphocyte function associated antigen-1 (lfa-1) adhesion depends on receptor occupancy and lateral organization on the cell membrane. however, the signals and mechanisms which dynamically reorganize lfa-1 into high avidity clusters are still a subject of many studies. to obtain deeper insight on the mechanisms that control and regulate lfa-1 clustering, patterned surfaces of immobilized lfa-1 ligand areas were fabricated using microcontact printing. the diffusion of lfa-1 expressed by monocytes stretched over patterned surfaces was followed in time using single molecule tirf microscopy. single lfa-1 nanocluster trajectories on individual cells showed an increase of immobile lfa-1 fraction and a slow-down of diffusing lfa-1 on the ligand areas compared to the non-ligand areas. moreover, single-cluster intensity analysis indicated a reorganization of lfa-1 nanoclusters in microclusters upon ligand binding. finally, single particle motion analysis of lfa-1 trajectories in close neighborhood to the ligand areas showed no assisted diffusion of lfa-1 towards the adhesive regions, consistent with random ligand-encountering and binding. we are currently investigating the effect of cell membrane organizers to regulate the spatio-temporal organization of lfa-1. r. diez-ahedo et al, small, in press. optical and electrophysiological detection of single phages across a lipid membrane n. chiaruttini 1 , p. boulanger 2 , m. de frutos 3 , l. letellier 2 , u. bockelmann 1 , v. viasnoff 1 1 nanobiophysique, espci paristech, cnrs, paris, france, 2 ibbmc, université paris xi, cnrs, orsay, france, 3 lps, université paris xi, cnrs, orsay, france we present an investigation study of the ejection of single t5 bacteriophages. in vivo studies of dna ejections from the bacteriophage capsid show that the t5 genome is introduced in the bacterial host in two steps. first 8% of the genome is ejected then after a pause of a few minutes the rest is internalized. bulk in vitro studies showed that various mutants of t5 eject their genome in solution following a single or a multistep process. by immobilizing single bacteriophages on a surface and following their ejection by fluorescence microscopy we showed that in all cases the ejection occurs in one step, but some mutants seem to have a subpopulation for which the triggering signal of the ejection is transmitted more slowly to the capsid entrance. we then reconstituted the phage receptor fhua into giant liposomes and followed the ejection of the dna into the liposome by fluorescence. finally we incorporated fhua in a suspended bilayer and followed the infection of the phages through the bilayer both by fluorescence labeling and electrophysiological measurements. we will discuss the influence of the cross membrane potential on the ejection speed of the dna. helixlike pili is a prerequisite of uropathogenic e. coli to adhere to host and withstand urine flow the gram-negative uropathogenic escherichia coli (upec) bacteria, invades the urinary tract region and cause in some cases severe infections, pyelonephritis, if they can withstand the rinsing action of urine and ascend to the kidney, via the bladder and ureters. to mediate adhesion, upec express quaternary surface organelles that are assembled from ∼10 3 identical subunits into a helix-like coil, with a single adhesin located at the tip. it is believed that the single adhesin mediate attachment to host cells while the helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow. to unravel the biomechanical properties of such quaternary structures, in particular in terms of their force-elongation and kinetic behavior, force-measuring optical tweezers (fmot) have been used. a plethora of different types of pili have been identified in the literature and we show, using fmot, that those dissimilarities might reflect the host environment. for example, we have found differences among pili expressed at diverse environment inside the urinary tract, which imply that pili presumably have evolved to resist specific forces under in vivo conditions. it is thus worth striving for understanding bacterial adhesion in order to figure out alternative to the over-abundance of antibiotics worldwide. single molecules studies have probed protein conformational fluctuations and monitored the oscillating activity of enzymes that remained masked in ensemble measurements. various statistical models have attempted to connect the conformational motions to the fluctuating activity of enzymes and suggested that they adopt inter-converting conformations each one of them exhibiting different catalytic activity. the main drawback of these studies is the non specific interactions that may bias the observed catalytic behavior. we have developed new innovating tools to study the behavior of single enzymes. as a first step we utilized liposomes as scaffold to confine enzymes while keeping them under physiological conditions. keeping them in their native conformation allowed us to monitor the inherent properties of their behavior. as a second step we developed a new model that accurately connects enzyme activity behavior to conformational motions. using this approach we accurately predicted the behavior of single lipases in a highly controlled environment. we modulated the enzyme's conformational mobility and activity by systematically varying its accessibility to liposomes. as we anticipated, that resulted in altering the time the enzyme spends on active conformations.our results provide new insights on interpreting the behavior of enzymes. simulations of single-molecule fret experiments on ribozymes m. hajdziona, a. molski adam mickiewicz university laboratory for dynamics of physicochemical processes, poznan, poland rnazymes are important biological molecules and that can catalyze the cleavage of their own nucleotide chains. this precess is called self splicing [1] . there are several methods to to study kinetic properties of ribozymes at the singlemolecule level. in this work we focus on fret (förster resonance energy transfer) of single, immobilized molecules. single-molecule fluorescence spectroscopy gives an insight into the behavior of individual molecules rather than the average behavior of an ensemble of molecules, which makes it possible to observe the kinetic heterogeneity. we simulated fret trajectories for single immobilized ribozymes. in our computer simulations we consider two-and three-state kinetic models motivated by actual experiments, published in [2] and [3] . we fitted the histograms of on and off times to recover the kinetic parameters of the models. we investigated the bias and standard deviation of the recovered parameters when one or two thresholds are applied to fret trajectories between adjacent states. we found that two thresholds give better parameter estimates than one-threshold analysis. references frap probes the average dynamical properties of a population of fluorescently labeled molecules whereas spt obtains these properties from observations of the trajectories of individual molecules tagged with a colloidal particle (the "bead"). when results of frap are compared with those of spt, frap yields significantly higher diffusion constants than spt. to understand the origin of this difference, we have developed and tested a model system to evaluate the influence of the bead on the dynamics of a diffusing molecule. we use a dsdna tether to attach a bead to a supported lipid bilayer (slb). the dna tether is modified at one end by cholesterol for anchoring to and diffusion in the membrane, and at the other end by biotin for tagging with a bead. with this system, we can vary several parameters: distance between slb and bead, size and nature of the bead, lipid composition of the slb, external force. we can also model the brownian motion of the bead and the hydrodynamic flow around it. we will present results using φ = 200 nm neutravidin-coated latex particles attached to an eggpc slb via a 15 bp to 90 bp dna tether. elucidation of the mechanism of the lambda bacteriophage epigenetic switch l. finzi physics department, emory university, 400 dowman dr, atlanta, ga 30322, usa the lambda bacteriophage epigenetic switch determines the growth lifestyle of the virus after infection of its host (e. coli ). it is now clear that the switch consists of a ∼2.3 kbplong dna loop mediated by the lambda repressor protein. using tethered particle microscopy (tpm), magnetic tweezers and afm, our laboratory has novel, direct evidence of loop formation and breakdown by the repressor, the first characterization of the thermodynamics and kinetics of the looping reaction and its dependence on repressor non-specific binding and dna supercoiling. these in vitro data provide insight into the different possible nucleoprotein complexes and into the lambda repressor-mediated looping mechanism which leads to predictions for that in vivo. the significance of this work consists not only of the new insight into a paradigmatic epigenetic switch that governs lysogeny vs. lysis, but also the detailed mechanics of regulatory dna loops mediated by proteins bound to multipartite operators and capable of different levels of oligomerization. mechanical force at the molecular level is involved in the action of many enzymes. for example, the phi29 dna polymerase mechanically unwinds the dna helix as it moves processively along the dna replicating one strand of the dna molecule. using optical tweezers we have developed a single molecule mechanical assay to elucidate the physical mechanism of dna unwinding by the phi29 dna polymerase as the protein replicates the dna. a single dna hairpin is hold between an optical trap and a mobile surface. as a single polymerase works on the dna hairpin, its replication and unwinding reactions can be measured in real time by measuring the change in extension in the dna polymer, revealing the fluctuations of its rate in response to the dna sequence. moreover, by gradually pulling on the opposite strands of the dna hairpin we can promote the controlled mechanical unwinding of the dna helix and determine the effect of increasing unwinding forces on the polymerization and unwinding rates. the effect of force and dna sequence on the activities of the wild type and an unwinding-deficient polymerase mutant will allow us to determine the inner workings of this molecular motor. single centrosome manipulation reveals its electric charge and associated dynamic structure we demonstrate laser manipulation of individual early drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively-charged organelle with a very low isoelectric region (3.1 ± 0.1). from this single-organelle electrophoresis, we infer an effective charge smaller or of the order of 10 3 electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. by investigating the centrosome hydrodynamic behavior, we show that its charge has a remarkable influence over its own structure. specifically, we find that the electric field which drains to the centrosome expands its structure to a physiological size a 60% larger than previous electron microscopy determinations, a self-effect which modulates its structural behavior via environmental ph. this methodology further proves useful to study the action of different environmental conditions, such as the presence of ca 2+ , over the thermally-induced dynamic structure of the centrosome. magnetic contrast neutron reflectivity delivers significant improvements in resolution for membrane structure analysis neutron reflection (nr), with its high resolution and use of contrast variation, is a unique tool to gain information on the orientation and structure of lipid bilayers in the z-axis perpendicular to the surface. to improve the use of nr we have used a highly oriented and stable layer of membrane proteins and lipids made possible by self-assembly upon a gold surface. this also provides a model of the bacterial outer membrane. as the dimensions of the layer can be predicted with accuracy, the system provides a molecular ruler for improvements in methodology and the complexity of the layer structure adds significantly to the modelling challenge. early improvements in resolution were obtained through sample preparation and gold smoothness. further improvement however required clearer discrimination between similar models. this was achieved using the new approach of magnetic contrast variation which uses a magnetic layer to provide two different scattering length densities for oppositely polarised neutrons. during this data collection the sample is unchanged. we show that this approach delivers significant improvements in data analysis and resolution of the protein, lipid and solvent structures. the method will provide a new level of understanding of membrane structure and dynamics. single molecule force spectroscopy and recognition imaging p. hinterdorfer institute for biophysics, johannes kepler university of linz, altenbergerstr. 69, a-4040 linz, austria in single molecule force spectroscopy, interaction forces of ligand-containing tips with receptors on probe surfaces are quantified. here he attachment of human rhino virus 2 (hrv2) to the cell surface, the first step in infection, was characterized. sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra. this suggests that multiple receptors bound to the virus in a timely manner. unbinding forces required to detach the virus from the cell membrane increased within a time frame of several 100 ms. the number of receptors involved in virus binding was determined and estimates for on-rate, off-rate, and equilibrium binding constant were obtained. furthermore, we show that accurate free energy values of membrane protein unfolding can be obtained from single molecule force measurements. by applying a statistical theorem developed by jarzynski, we derived equilibrium unfolding free energies of from unfolding force data acquired at different force loading-rates and temperatures. finally, we present a method for the localization of specific binding sites and epitopes with nm positional accuracy. a magnetically driven afm tip containing a ligand covalently bound via a tether molecule is oscillated at a few nm amplitude, during scanning along the surface. in this way, topography and recognition images on membranes and cell surfaces were obtained simultaneously. mapping of the forces acting on biomolecules in cell membranes has spurred the development of effective labels, e.g. organic fluorophores and nanoparticles, to track trajectories of single biomolecules [1] . standard methods use particular statistical observables, namely the mean square displacement (msd), to extract cues on the underlying dynamics. yet, msd is not an appropriate tool to access force fields and becomes easily a biased estimator in the presence of positioning noise. here, we introduce general inference methods [2] to fully exploit information hidden in the experimental trajectories, providing sharp estimates for the forces and the diffusion coefficients within membrane microdomains. rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. the inference method is then applied to infer forces and potentials acting on the receptor of the ε-toxin labelled by lanthanide-ion nanoparticles. results show a constant diffusivity inside a complex force field confining the receptor inside a specific domain. our scheme is applicable to any labelled biomolecule, and results presented here show its general relevance to the issue of membrane compartmentation and protein motion. large scale domain motions are structural rearrangements often adopted by enzymes to achieve their full functionality. understanding the mechanism responsible for such movements by means of computational approaches is of great interest especially in rational drug design, since induced fit or population shift effects are closely related aspects of the problem. unfortunately, the simulation time required to sample these events by conventional techniques such as plain molecular dynamics, is unfeasible. here, the domain motion required by adenosine kinase (ak) to achieve its (pre-)catalytic conformation was studied using well-tempered metadynamics [barducci, a. et al. phys rev lett. (2008) , 100: 020603] with path collective variables (pcvs) [branduardi, d. et al. j chem phys. (2007) , 126: 054103]. first, a low energy path for the apo form of the enzyme was obtained, and the potential of mean force along the pcvs was reconstructed. then, the large scale movement was simulated in the presence of two inhibitors known to bind to the enzyme in different conformational states. the adopted approach was proven to be successful both to understand the mechanistic features of the ak domain motion and to provide a picture of the population shift effects upon ligand binding. force measurement study of the interaction between 23s rrna and the ribosomal protein l20 single molecule force measurements enable to probe the complex structure and folding dynamics of rna molecules and furthermore to investigate the numerous interactions with proteins that affect rna folding in vivo. we use a double optical tweezers setup to study a region of the ribosomal rna 23s from e.coli and its interaction with the essential ribosomal protein l20. the apparatus permits us to probe the dynamics of the rna structure with milisecond time-resolution. first, we pull the rna and show that it mechanically unfolds in several reproducible steps. we use these results in combination with structure prediction tools to evaluate the possible structures of the rna fragment in vitro. the most probable structure exhibits a unique binding site for l20. then, force measurements are done on the rna in presence of l20. we show that the protein specifically binds the rna and stabilizes it. the binding site is recognized with a resolution of a few bases. finally, two bases are mutated on the rna fragment. in presence of these mutations, in vivo and in vitro binding of l20 to 23s rna is abolished. the single molecule approach gives an explanation of this result: the force measurements show that the mutated rna folds differently from the natural one and that it does not bind l20. control of the translocation of single dna molecules through alpha-hemolysin nanopores g. maglia, h. bayley department of chemistry, university of oxford, ox1 3ta, oxford, uk the analysis of single nucleic acid molecules by electrophoretic threading through nanopores is under intense investigation as a rapid, low cost platform for dna sequencing. biological nanopores such as staphylococcal alpha-hemolysin (hl) have an added advantage over solid-state nanopores because they can be modified by genetic engineering with atomistic precision. although we showed that all four dna bases can be identified in an immobilized ssdna molecule (1), the translocation of free dna is too quick to observe single bases. here we show that by a small increase of the net internal charge of the hl nanopore (e.g. by introducing a ring of 7 arginine residues), we have augmented the frequency of dna translocation events through the pore and dramatically lowered the voltage threshold required for dna translocation (2) . by further increasing the net positive charge of the transmembrane barrel region of the pore (e.g. by introducing 49 extra positive charges) we have also reduced the speed at which dna translocate the pore by more than two orders of magnitude. these experiments provide a means of controlling dna translocation with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. the atomic force microscope (afm) is a tool for imaging, measuring and manipulating matter at the nanoscale. single-molecule pulling experiments give information on the thermodynamics and kinetics of biomolecules. the purpose if this work was to develop software to simulate single-molecule pulling experiments and to analyze singlemolecule pulling data. the long term objective is to asses the accuracy and precision of the parameters recovered from single-molecule pulling data.we carried out simulations for two different models [1, 2, 3] , using a wide range of loading rates. from the simulated force-extension curves we extracted the kinetic parameters and compared them with the values used for simulations. the kinetic parameters were the intrinsic rate coefficient (k 0 kramers rate), the location of transition state (x ) and the free energy of activation (∆g). we have found that the loading rates have a small effect on the recovery of the free energy of activation, but have a significant effect on the recovery of the kramers rate. we report the tracking of single myosin v molecules in their natural environment, the cell. myosin v molecules, labeled with quantum dots, are introduced into the cytoplasm of living hela cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. we perform intracellular measurements of key parameters of this molecular transporter: velocity, processivity, step size and dwell time. our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells. a mathematical model of neurotransmission at the input stage of the cerebellum t. nieus 1 , s. solinas 3 , l. mapelli 2 , e. d'angelo 3 1 dept. neuroscience and brain technologies, iit, italian institute of technology, genova, italy, 2 dept. of biomolecular sciences and biotechnology, milan, italy, 3 dept. physiological and pharmacological sciences and cnism, university of pavia, italy the granule cell (gc) of the cerebellum has some peculiar properties compared to other cells of the vertebrate brain. the gc has a small soma (diam=6 microm) and just a few (2 to 6) excitatory and inhibitory inputs (e&i). the e&i gc synaptic inputs are formed inside the cerebellar glomerulus, which favors neurotransmitter diffusion between neighboring sites (1) protracting postsynaptic receptor activation and (2) causing cross-tall between e&i synapses through presynaptic receptors. neurotransmitter release probability (p) can be regulated by long-term plasticity (ltp and ltd at e synapses) and by gaba b and mglu presynaptic receptors (both at e&i synapses). the p change in turn modifies shortterm plasticity, affecting the first response in a train much more than the followings. to gain insight into the role that p changes might have in computations performed at the cerebellum input stage, we have built detailed biophysical models of the e&i synapses. the model has allowed to investigate glomerular processing of high frequency inputs reaching the cerebellum. optimal synaptic transmission through the mfs inputs resulted when gos inhibited synchronously the gcs. the role of feed-forward inhibition onto synaptic transmission is under investigation. unzipping dna with a nanopore j. muzard, n. chiaruttini, u. bockelmann, v. viasnoff cnrs/espci nanobiophysique, 10 rue vauquelin, 75005 paris, france the use of proteinaceous or artificial nanometer size pores has become a promising approach for sensing biomolecules at the single molecule level. it was shown that alphahemolysin, a toxin from staphylococcus aureus, can be employed to sense the translocation of dna strands through a lipid bilayer. the pore dimension allows the electrophoretically driven passage of single stranded dna whereas double stranded structures need to open prior their translocation. we study the unzipping process of the double stranded part of dna both experimentally and theoretically. we show that in a first approximation the duplex opens progressively in a sequence dependent manner. the experimental results can be accounted for by modeling the unzipping process as a free diffusion of the unzipping fork in the energy landscape defined by the sequence of the basepaires. we then discuss the effect of the pore/dna interaction on the translocation process. we further characterize the effects of the pore geometry, showing that the pre-confinement of the dna in the pore vestibule is essential to the unzipping process. we eventually discuss the possibility to use this nanopore approach to sequentially probe rna secondary structures. we report on the interaction forces in the range 100 --400 pn determined between pairs of the lectin soybean agglutinin (sba) and a modified porcine submaxillary mucin (tn-psm) using a single-molecule approach. lectins are carbohydrate-binding proteins with biological activities related to i.e. cellular recognition, adhesion, growth and metastasis. here, dynamic force spectroscopy is used to investigate pairs of sba and tn-psm with the aim to understand the mechanisms of binding and cross-linking of multivalent lectins. the unbinding force increased from 103 pn to 402 pn with increasing force loading rate and the lifetime of the complex in the absence of applied force was 1.3 -1.9 s. published kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range 3.3 x 10 −3 -2.5 x 10 −3 s. the long lifetime of the sba -tnpsm complex is compatible with a previously proposed "bind and jump" mechanism. this mechanism has also been suggested for lectins binding to multivalent carbohydrates and globular glycoprotein. the bind and jump mechanism is also similar to that observed for binding of proteins to dna, and suggest a common conserved binding mechanism of ligands to the two biopolymers and possibly between ligands and all biopolymers, as recently suggested. the rate of topoisomerase ii activity correlates with persistence length of dna q. shao 1 , l. finzi 1 , d. dunlap 2 1 dept. of physics, emory university, atlanta, georgia, u.s.a., 2 dept. of cell biology, emory univ. med. school, atlanta, georgia, u.s.a. type ii topoisomerases catalyze the transection of one double helical segment by another to modify the topological state of dna. reversible 5' linkages to the phosphate-sugar backbones are established on each strand of the "gate" segment to create an opening through which the enzyme drives the "transfer" segment. a recent crystal structure shows that the "gate" segment bends approximately 150 • upon binding to the enzyme. bending a stiff polymer like dna requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. using modified deoxyribonucleotides in pcr reactions, more rigid dna fragments have been produced and used as substrates for topoisomerase ii-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. before relaxation the persistence lengths were measured by fitting force extension data with the worm-like chain model. topoisomerase ii was found to release mechanically introduced supercoils more slowly in stiffer dna suggesting that dna bending might be a rate limiting step in topoisomerase ii activity. h. seidel 1 , t. klose 2 , h. lilie 2 , c. g. hübner 1 1 institute of physics, ratzeburger allee 160, 23538 lübeck, germany, 2 institute of biochemistry and biotechnology, kurt-mothes-str. 3, 06120 halle, germany one essential element of a virus is its protein shell, the viral capsid, which encloses the viral genome. the murine polyomavirus is a non-enveloped dna tumor virus with an icosahedral t=7d structure. besides the knowledge of the structure, it is of utter importance to understand the process of viral assembly [1] . the assembly reaction of polyoma vp1 does not show the typical sigmoidal kinetics in light scattering experiments. the apparent kinetics is of fourth order, which appears rather unrealistic. in order to gain knowledge on capsid composition during assembly beyond ensemble average, we apply methods of single molecule fluorescence, namely fluorescence correlation spectroscopy (fcs), fluorescence-intensity-distribution-analysis (fida), and single-particle-imaging (spi). the molecular brightness and the diffusion time reported by fcs are in agreement with the results from light scattering. fida as well as spi, moreover, point to a polymerization of subunits to complete capsids along the assembly pathway without pronounced intermediates. mixing experiments show that already early in the course of the assembly reaction no exchange of pentamers between capsids occurs, and that the effect of breathing [2] can be excluded for polyoma vp1. genetic information coded in dna provides complete instructions to cells regarding metabolism and proper functioning. a number of endogenous and exogenous sources can damage the genomic dna. unrepaired dna damage can give rise to mutations and may cause cell death. cells have evolved different mechanisms to repair damaged dna and to protect genetic integrity. uracil in dna occurs as a result of incorporation of dump in the place of dtmp during replication and deamination of cytocine, resulting in u:a and u:g base pairs, respectively. uracil-dna glycosylase (ung) is a dna repair protein that searches and removes uracil from dna. ung removes mismatched base by flipping it out from the base stack into the active site. the exact mechanism by which ung searches dna for uracil is unknown. therefore, in our study we use atomic force microscopy (afm) to investigate the interaction between single ung molecules with dna carrying the u:a or u:g mismatches. furthermore, we study the complexation of dna and ung protein. afm images of ung bound to dna reveal the structural changes at the level of single complex, e.g. dna kinking that may occur upon binding of ung to dna. imaging of dnaprotein complexes can provide a a new level of understanding of ung-dna interaction. atomic force microscopy (afm) has been a useful device to visualize cellular and molecular structures at a single-molecule resolution. especially, afm imaging under the trec t m (topography and recognition) mode (recognition imaging) can map a specific protein of interest within an afm image. in this study, we employed an antibody-coupled afm cantilever in recognition imaging, and dissected the structural/functional domains of α actinin-4, an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction. a use of monoclonal anti-α actinin-4 antibody enabled us to map its epitope in the amino-terminal actin-binding domain within a single molecule afm image of α actinin-4. counting fluorescent molecules by photonantibunching h. ta, m. schwering, d. p. herten bioquant, heidelberg university, germany information on the stoichiometry of labelled biomolecules is highly desirable. a method called photo-antibunching has successfully been used to determine the number of fluorescence emitters in multichromophoric systems. a statistical model to estimate the number of fluorescent molecules in a confocal observation volume is established based on photonantibunching in time-correlated single-photon counting (tcspc) scheme with 4 avalanche photon diodes (apds) for detection of individual photons. numerical simulations based on realistic experimental conditions show that the model is able to estimate the number of molecules with moderate errors. experiments on immobilized double-strand dna oligonucleotides with photon stabilizing agents show promising results even when the number of molecules is ∼ 20. the proposed method allows us to monitor labelled single molecules and in the near future we plan to implement it in complex biological systems (cell extracts/live cells). single molecule afm force spectroscopy and steered molecular dynamics of contactin-4 protein j. w. strzelecki, k. mikulska, a. balter, w. nowak institute of physics, nicolaus copernicus university, grudziadzka 5, 87-100 torun, poland contactin-4 (cntn4) is an axonal cell adhesion protein that contains six igc2 and four fniii domains and is responsible for creating neural outgrowths. recent research shows that mutation of cntn4 gene may be responsible for autism, while its absence in transgenic mice results in lack of smell sense. we use a homemade afm single molecule puller and steered molecular dynamics simulation to test its elastic properties through force spectroscopy. stretching experiments show unfolding of fniii domains while igc2 domains stay coiled as they are stabilized by disulfide bonds. unfolding of contactin-4 molecule appears to play role of buffer that helps to protect neural contacts from damage when neural cells are subjected to shock or tumor. single molecule studies of the thermophilic bacillus ps3 f 1 -atpase have revealed kinetic and structural information that cannot be discerned using other methods, including the presence of 40 and 80 degree physical substeps (yasuda et al. 2001 ) and the order and kinetics of chemical substeps. we are interested in using single molecule techniques to observe the effects of mgi mutations on enzyme kinetics and torque production in f 1 from the yeast saccharomyces cerevisiae. using a high speed imaging camera, we have captured the rotation of wild-type and mutant forms of yeast f 1 -atpase. rotation data for the wild-type and preliminary data for some mgi strains will be presented. we show for the first time that at saturating atp, wild-type yeast f 1 rotates approximately four times faster than the thermophilic f 1 . kinetic and substepping behaviour in yeast appears to be similar to that observed in bacterial f 1 . -single molecule biophysics biophysical characterization of a dna gquadruplex formed in the promoter of human mef2d gene w. zhou 1 , l. ying 2 1 molecular medicine, national heart and lung institute, imperial college london, london, sw7 2az, uk, 2 chemical biology center, imperial college london, sw7 2az, uk g-quadruplex is believed to be involved in many crucial biological processes, such as the gene regulation and the maintenance of human telomere. mef2d, a member of mef2 (myocyte enhancer factor-2) family of transcription factors, regulates the response of heart to cardiac stress signals. we found that a g-rich sequence starting at -145bp of mef2d promoter can form a very stable intramolecular g-quadruplex. here we present a detailed biophysical characterization of this quadruplex. we also found that this quadruplex is more stable than its duplex form under physiological conditions by cd melting and single molecular fret. we observed subpopulations in smfret measurements possibly due to different conformations of the quadruplex. we characterized its unfolding process by monitoring the change in fret when it hybridizes to its c-rich complimentary strand. finally, we proposed several possible structures of this quadruplex based on the smfret and fluorescence dms footprinting results. this research is supported by national heart and lung institute foundation. by stretching a polymer in solution using single molecule techniques it is possible to infer about its physical properties. afm stretching experiments allow for a full characterization of the elasto-mechanical properties of the sample under study. in the presented work, single molecule afm force spectroscopy experiments were used to determine mechanical properties of a peptide obtained from exon 28 (ex28) of the human elastin gene. elastin is a protein with important mechanical properties and, in particular, it shows quasi ideal elastic behavior associated to the presence of many hydrophobic unstructured domains (such as ex28) into the protein structure. ex28 coded polymer was used as a starting point to obtain bio-materials with specialized elastomechanical functions. in particular, a mutated polypeptide based on the ex28 sequence was synthesisized with the aim of obtaining a new polymer with the same mechanical and physical properties of the native molecule but with increased aggregation properties, induced by a cross-linking reaction. afm stretching experiments were used to verify the mechanical properties of the engineered proteins at a single molecule level. the obtained results allowed not only to address this question, but also to give some insight into the first aggregation steps of the polymer towards the formation of reticulated structures. luminescent lanthanide-ion doped nanoparticles (nps) are attractive single-biomolecule labels. they are synthesized directly in water, are highly photostable, and display narrow emission without intermittency. we coupled y 0.6 eu 0.4 vo 4 nps to ε-toxins produced by clostridium perfringens, which bind to a specific receptor on mdck cells. single-molecule tracking experiments using these labels produce long (5 min) uninterrupted trajectories with temporal resolution down to 20 ms or localization precision down to 15 nm. we found that the toxin receptor exhibits confined motion in cell membrane microdomains. to analyze the receptor trajectories, we used a novel approach based on an inference method [1] . this method fully exploits the information of the ensemble of the trajectory, in contrast to the usual mean square displacement analysis. applying both techniques to recorded trajectories, we highlight the difference in extracted parameters. from the shape of the confining potential, obtained by mapping the forces inside the domain, we can deduce information about the mechanism of confinement. in combination with experiments on cholesterol depletion and cytoskeleton destruction, this technique will shed light into the nature of the membrane micropatterning. background: implantable cardioverter defibrillators (icds) are save-life device for patients at risk of sudden cardiac death, and help cardiac patients to avoid slow beat-rate by means of anti-bradichardia pacing (abp) feature. however, recent literature evidenced the presence of ventricular tachyarrhythmias (vts) immediately following abp, leading to the hypothesis that icd devices might be pro-arrhythmic. aim: study differences between pacing-associated tachyarrhythmias (pat) and other spontaneous vts. signals and methods: 165 spontaneous vt episodes are retrieved from 37 patients with icd. vts are examined and pat episodes, classified. characterization of vts is based on the analysis of 20 seconds immediately preceding vt-onset quantified by: heart cycle (hc-prevt), prevalence of premature ventricular contraction (pvcprev) and prevalence of paced beats (pprev). significant differences are evaluated by student-t or chi-square tests with p<0.05. results: 20 vt episodes from 8 patients are pat episodes (12%). cardiac activity preceding pats vs. non-pats differs: pat episodes have higher hc-prevt (p<0.01), greater pprev (p<0.001), and greater pvcprev (p<0.001). analysis also shows that pat episode often occur when the paced beat immediately follows a premature contraction. conclusion: the study indicates that new icd generations should avoid abp after premature ventricular contraction. towards mechanistical understanding of adsorption: combining technologies in situ and in real time p. h. bjöörn q-sense ab, västra frolunda, sweden a growing number of researchers in different surface related fields present evidence from more than one analytical technique when detailing their findings. thus a logic and useful development is to combine technologies for simultaneous measurements on a single sensor surface. to develop a biosensor platform such as an assay fast and accurately, mechanistical understanding of why the platform works is essential. quartz crystal microbalance-with dissipation (qcm-d) technology and instrumentation provides an open platform and enable easy and precise quantification of mass, thickness and viscoelastic properties of surface bound molecules. these parameters provide both a reference tool in assay development, but also detecting and identifying your target molecules as the combination of mass and material property info in many cases provide a unique response for a specific molecule. by combining qcm-d with other technologies, a range of useful info is put within easy reach of the researcher. recent advances will be presented where simultaneous real time and in situ measurements using qcm-d together with electrochemistry, ellipsometry and fluorescence microscopy enables manipulation of films as well as quantification of the variation of water content as a result of conformational changes in immobilized molecules. examples will include new data from the formation of protein films, polyelectrolyte multilayers and polymer brushes. intrinsic gravity versus metabolically inert infrastructure and basal metabolic rate in living mass i. r. bhattacharjee 1 , r. kashyap 2 , b. shaptadvipa 2 1 assam agricultural university, jorhat-785013, india, 2 international institute of intrinsic gravitation biology (i3gb), jorhat-785013, india 'self gravitation bio' is an emerging concept in biophysics. intrinsic or 'self' gravitational force might exert when mass increases beyond critical level in biological particle pyramid. (http://en.wikipedia.org/wiki/biomechanics of intrinsic gravity) 'metabolically inert infrastructure' (mii) consists of total body mass (body water, dissolved substances, mineral and organic deposits) and serves as storage of nutrients, transport and distribution of these materials. to act independently as living body, mii is suggested also to provide structural support to the organism with density-gradient buoyant force against intrinsic and extrinsic gravitational attraction for the biological mass. it is shown that 'amniotic fluid', 'isotonic saline to ailing patient', 'growth factors', 'cultural medium' and other 'medium matrices' act as counter-gravity mechanism for micro to macro living organisms under center-of-biomass reference frame. controversy over relationship between bmr (basal metabolic rate), rmr (resting metabolic rate), pal (physical activity level), on one hand, and mass of the living organism, on the other, (as described in rubner's 2/3 law, kleiber's 3/4 law that continued to be contested by many) can be favorably resolved substituting the concept of self gravitation bio, considering 'mass' as synonym of gravitational force under center-of-biomass reference frame. bioenergetics would be an unequal but opposite entity of self-gravity reinforced by extrinsic gravity. platelet arrest on von willebrand factor (vwf) occurs transiently via the platelet receptor gpib. depending on the combination of shear stress and surface density of vwf binding sites the platelets either only adhere, pull tethers or generate microparticles. these processes are influenced by the properties of the platelet membrane and the cytoskeleton. under shear flow conditions these platelet characteristics were examined with reflection interference contrast microscopy and a viscosimeter. in addition we quantified platelet adhesion energy and tether pulling forces. disrupting platelet f-actin had several effects. the tethers were shorter, the membrane contact area was larger, the receptor αiibβ3 density of the microparticles was depleted and no platelet spreading occurred. breaking up the microtubular system had different implications. the number of severed tethers tripled, the contact area was smaller, microparticle formation was increased and the area of spread platelets was reduced. however changes in the membrane elasticity had no effect on the platelets. our results suggest that platelet attachment and adhesion is not only determined by the platelet adhesion receptors and their cytoplasmic linker proteins, but that cytoskeletal structures have also a crucial influence on how platelets interact with thrombogenic surfaces. gene expression is orchestrated by a host of regulatory proteins that coordinate the transcription of dna to rna. regulatory proteins function by locating specific binding sequences of dna and binding to these sequences to form the transcription initiation complex. in many instances, these regulatory proteins only have several hundred copies that must efficiently locate target sequences on the genome-length dna strand. the non-specific binding of regulatory proteins to random sequences of dna is believed to permit the protein to slide along the dna in a stochastic manner. periodically, a thermal kick or an interaction with another bound protein will disengage the regulatory protein from the dna surface, leading to three-dimensional diffusion. eventually, the protein will reattach to the dna at some new location that is dictated by both the diffusivity of the protein and the dna configuration. cycling through these random events constitutes a search strategy for the target site. we build a reaction-diffusion theory of this search process in order to predict the optimal strategy for target site localization. the statistical behavior of the dna strand acts as a necessary input into the theory, and we consider several governing behaviors for the dna strand. we explore the impact of dna configuration and protein occlusion on target site localization in order to predict how protein expression will vary under different experimental conditions. a. di garbo 1 , s. alloisio 2 , s. chillemi 1 , m. nobile 2 1 istituto di biofisica cnr, via g. moruzzi 1, 56124 pisa, italy, 2 istituto di biofisica cnr, via de marini 6, 16149 genova, italy in the nervous system of vertebrates there are more glial cells than neurons: from 10 to 50 times, depending on the animal type. glial cells are not able to generate action potentials but, nevertheless they play an important role for the functioning of the different brain's area. the astrocytes are the more abundant cells of the macroglia and through their processes they modulate synaptic activity. in this contribution a biophysical neural network model consisting of a pyramidal neuron, an interneuron and the astrocyte is studied. the corresponding dynamical properties are mainly investigated by using numerical simulations. the results show that the presence of the atp and of the interneuron strongly impacts the neural activity. moreover, it is shown that the fluxes of calcium through the cellular membrane strongly affect the modulation of the neural activity arising from the astrocyte. microscopic origin of the very-low energy vibrational dynamics in proteins g. d'angelo 1 , v. conti nibali 1 , c. crupi 1 , a. paciaroni 2 , u. wanderlingh 1 1 department of physics, faculty of science, messina university, italy, 2 department of physics, faculty of science, perugia university, italy important functions of biological processes require large atomic rearrangements and conformational fluctuations. for proteins, atoms are mostly displaced along the soft directions identified by the delocalized, low frequency vibrations. the study of very low energy vibrational spectrum of proteins is therefore of particular interest. as a step toward understanding their functional role, we have investigated the low frequency vibrational motions (0.03÷10 mev) in different proteins by performing inelastic neutron scattering and low temperature (1.5÷30 k) specific heat measurements. for the first time for biological systems, the well-known boson peak found in neutron scattering spectra at ∼ 3 mev is put in relationship with a clear anomaly of the measured specific heat located at around 7 k. this departure from the debye-like behavior further expands the analogy of proteins with glassy systems. quite interestingly, in the very low sub-mev energy range and below ∼3k, we observe an additional anomalous behaviour, which could be ascribed to the existence of twolevel-systems states. increasing the hydration degree, the low energy vibrational region is drastically altered, revealing that the addition or removal of the hydrogen bond network around the protein deeply modifies the interatomic forces, affecting the character of the vibrational modes. a. cupane 1 , m. levantino 1 , m. cammarata 2 , g. schirò 1 1 department of physical and astronomical sciences, university of palermo, via archirafi 36, i 90123 palermo, italy, 2 european synchrotron radiation facility, grenoble, france our efforts in recent years have been to study in great detail the way hemoglobin works in confined geometries [1] [2] [3] . to this aim we have contributed to the development of a new experimental technique, time-resolved wide-angle x-ray scattering (tr-waxs), that enables one to watch proteins at work in solution [4, 5] . a very recent and challenging application of this technique is the study of hemoglobin functioning inside intact red blood cells (rbc). our preliminary results show that, by using laser pulses of about 150 ns, it is possible to photolyse hemoglobin inside rbcs obtaining about 40% -60% photolysis. good structural signals from hemoglobin are obtained, with limited radiation damage to the cells: this opens the possibility of studying the conformational transitions of hemoglobin in its "natural" environment. preliminary results concerning the timing of the r->t quaternary transition inside the rbc and comparison with the behaviour in dilute solution will be discussed. physical review letters and physical review e invite submission of your best work in biological physics. submissions must present significant new results in physics; topics may range from the microscopic to the macroscopic. we will provide information on the different types of articles published in the journals, on authors and referees, and on the review process. for publication in physical review letters, the work should be important and of broad interest. for rapid communications in physical review e, the work should be important for the field. biological physics papers published in physical review are indexed in medline. in an effort to bring important research to the attention of a wider community, the physical review journals have recently begun highlighting important work with viewpoints in the online publication physics. all physical review journals may be accessed through the website http://publish.aps.org/. modeling of fibrin gels using confocal microscopy, light scattering and turbidimetry f. ferri 1 , d. magatti 1 , b. cardinali 2 , a. profumo 2 , m. rocco 2 1 dipartimento di fisica e matematica, università dell'insubria, como, italy, 2 istituto nazionale per la ricerca sul cancro (ist), genova, italy fibrin gels are biological networks playing a fundamental role in blood coagulation. confocal microscopy of fibrin gels shows a collection of straight fibers, not uniformly distributed in space, connected together at low-order (3) (4) branching points. although each fiber is quite straight (mass fractal dimension d m =1), for the overall system d m >1. based on the confocal images, we generated threedimensional (3d) synthetic gels made of cylindrical sticks of diameter d, joined together at randomly distributed nodal points. the resulting 3d network is no more random but ordered on length scales of the order the average fiber length, and exhibits a fractal morphology with d m ∼1. 2-1.7 . the gel structure is analyzed by means of its 3d correlation function g 3d (r)=<n(x) n(x+r)> x , where n(x) is the gel local density. since the gel is isotropic, g 3d depends on the modulus r=|r| and can be obtained by averaging 2d correlation functions evaluated at different heights of the gel volume. from this analysis we recover the fiber diameter d (fwhm of g 3d ), the fractal dimension d m (power-law decay of g 3d ) and the gel mesh-size ξ (minimum in g 3d ). furthermore, the 3d-fourier transform of g 3d (r) gives the gel power spectrum i(q), which compares quite well with elastic light and multi-wavelength turbidimetry data taken on real gels. a model coupling vibrational and rotational motion for the dna molecule e. drigo filho, j. r. ruggiero, r. a. d. s. silva unesp -sao paulo state university, brazil a largely used mechanical model for vibrational motion of dna has been proposed by peyrard and bishop (pb). in this model, dna is represented by a pair of harmonic chains coupled by a nonlinear potential. the most frequently used nonlinear potential for this purpose is the morse potential. some extensions of the original model are proposed considering, for example, the helicoidal structure of dna. an important objective for this kind of model is to understand the phenomenon of thermal denaturation and, through this, get some knowledge about other processes such as the genetic transcription and drug intercalation. it is possible to obtain from this type of model interesting properties, such as the average stretching between base pairs as a function of the temperature using the transfer integral operator. dynamical properties of this model were also explored and several phenomenological quantities are studied, such as energy localization. in this work, we extend the original pb model by introducing rotational motions for the nucleotides. in this way, both the vibrational and rotational motion for each nucleotide are considered. the stretch of the base pairs is given by the morse potential in the same way as in the original pb model. however, the coupling between the two kinds of motion, rotation and vibration, is obtained through a nonlinear combination of them in the morse potential. a. dobovišek 1 , m. brumen 2 , p.županović 3 , d. juretić 3 1 university of maribor, faculty of natural sciences and mathematics, maribor, slovenia, 2 jožef stefan institute, ljubljana, slovenia, 3 faculty of science, university of split, split, croatia mepp is a physical principle, widely used for quantitative explanation of non equilibrium phenomena in physics, chemistry and biology [1] [2] [3] . here, we applied mepp to study two and three state reversible michaelis-menten kinetic schemes of enzymatic reactions. by applying constraints as a diffusional limit for kinetic constants, constant free energy differences between enzymatic states and constant thermodynamic force, we calculated shannon information entropy and entropy production of the entire reaction system as a function of forward rate constants. we found maxima in the net steady-state metabolic flux, total entropy production and shannon entropy for equal values of forward rate constants. moreover, for these values an analytical expression derived gives a relation between substrate and product concentrations at which enzymes operate in the optimal way. in conclusion, we demonstrated that mepp is an appropriate selection principle for evolutionary optimization of enzymes. attractive interaction between like-charged lipid surfaces mediated by spherical nanoparticles with spatially distributed charge is theoretically described by using functional density theory and mc simulations. the spatial distribution of charge within a single nanoparticle is considered by two effective charges at a finite distance. the minimization of the free energy is performed to obtain the equilibrium configuration of the system. both, the rigorous solution of the variational problem and the mc simulations show that orientational ordering of nanoparticles subject to the gradient of the electric field gives rise to an attractive interaction between charged lipid surfaces for high enough charge densities of the interacting surfaces and large enough separations between charges within the nanoparticle. the attraction is explained by orientational ordering of dimeric charges in the electric field which lowers free energy. viral genomes encode for a series of membrane proteins which are embedded or attached to the lipid membrane of the host, or penetrate them during the very first step of viral invasion. we are focussing especially to those of the former which are known to assemble and from channels or pores for small ions or substrates. with these channel forming proteins the virus manipulates the host cell interior for the benefit of its replication. strategies are developed to answer the question about the assembly process of these proteins based on the 'two stage model' and the substrate flux. only few experimental data are available to answer these questions, consequently we stress computational methods to derive the adequate answers. the methods applied are ab inito molecular dynamics (md) simulations based on density functional theory (dft) and conventional md simulations. potential routes for assembly of the three transmembrane domains of 3a protein from sars-cov will be outlined and compared with computational data from other viral membrane proteins. with a novel pore lining motif suggested for 3a, the dynamics of ions at selected positions within the putative pore will be assessed. probing the molecular mechanism of antibiotics diffusion through the ompf channel e. hajjar 1 , a. kumar 1 , m. winterhalter 2 , p. ruggerone 1 , m. ceccarelli 1 1 department of physics, university of cagliari, italy, 2 jacobs university, bremen, germany in gram-negative bacteria, the outer membrane porin-f (ompf) is the preferred entry point of antibiotics. bacteria can resist to antibiotics by altering the expression and the structures of ompf. a key feature in the structure of ompf is the presence of a constriction zone, characterized by both spatial and electrostatics restrictions. to study the process of antibiotics translocation at a molecular scale, we performed molecular dynamic simulations accelerated with the metadynamics algorithm. we studied the diffusion of antibiotics with different structural and chemical properties through ompf wild-type and variants. the free energy surface suggests faster translocation for the cephalosporins compared to the penicillins antibiotics, and also for ompf mutants compared to the wild type. further, the conservation of favored orientation and affinity sites of antibiotics inside the ompf channel reveal which specific interactions govern translocation. the calculated energy barriers and rate determining interactions for translocations compared well with the electrophysiology measurements and liposome swelling assays from our collaborators. this study demonstrates how theory and experiments can be combined to reveal the structural determinants and mechanism of ompf permeation. this will benefit to the design of antibiotics with improved transport properties. m. g. gauthier, j. bechhoefer dept. of physics, simon fraser univ., burnaby, bc, canada dna replication requires two distinct processes: the initiation of pre-licensed replication origins and the propagation of replication forks away from the fired origins. experiments indicate that these origins are triggered over the whole genome at a rate i(t) (the number of initiations per unreplicated length per time) that increases throughout most of the synthesis (s) phase, before rapidly decreasing to zero at the end of the replication process. we propose a simple model for the control of dna replication in which the rate of initiation of replication origins is controlled by the interaction with a population of ratelimiting proteins. we find the time set by reaction-diffusion kinetics for such proteins to find, bind to, and trigger a potential origin. the replication itself is modeled using a formalism resembling that used to study the kinetics of first-order phase transitions. analyzing data from xenopus frog embryos, we find that the initiation rate is reaction limited until nearly the end of replication, when it becomes diffusion limited. initiation of origins and hence i(t) is suppressed when the diffusionlimited search time dominates. we find that, in order to fit the experimental data, the interaction between dna and the rate-limiting protein must be subdiffusive. we also find that using a constant nuclear import of the limiting proteins leads to a more accurate description of the experimental data. the microsoft research -university of trento centre for computational and sisytems biology, trento, italy we propose a new method for inferring the structure of a biochemical network from the time-series of the reactant species. it consists of two parts: the first is the quantification of the correlation between the time-series profiles. correlation in time series data can be used to reveal dependencies between variables and to infer the graph of connectivity among species. the second part consists in the elimination from the connectivity graph of the relationships that have a non-null correlation coefficient, but that are not biochemically plausible. the cutting of false correlations from the graph is performed through the estimation of the parameters ("calibration") of the network. to calibrate the network for detecting null dynamics correlations, we developed the software tool kinfer (knowledge inference). based on a new probabilistic model of the variations in reaction concentrations, kinfer infers the values of the kinetic rate constants, their confidence regions and the level of noise in the input data by maximizing the likelihood to obtain the observed variations given the network model. the a priori knowledge required as input is minimal, as it consists only in the time-series of reactant concentrations. the minimal a priori knowledge and the probabilistic formulation of the calibration method make the accuracy of the predictions strong against experimental, biological and stochastic noise, and allows to use it to cut the null-dynamics edges of the connectivity graph. a. kuzmanic, b. zagrovic mediterranean institute for life sciences, split, croatia root-mean-square deviation (rmsd) is a measure used to give information on the global structure of macromolecules. for example, pairwise rmsd (prmsd) is used to assess similarity of the lowest energy nmr structures or for clustering large ensembles of structures. on the other hand, to obtain information on the local structure of a macromolecule and its dynamics, root-mean-square fluctuation (rmsf) is often used. rmsf can be calculated from md simulations, but also from experimental x-ray b-factors. since prmsd and rmsf report on different features, it is interesting to ask what the relationship between them is. first, we provide a mathematical derivation showing that, given a set of conservative assumptions, the <prmsd> rms is directly related to the <rmsf> rms and, consequently, experimental b-factors. second, we demonstrate this on structures taken from distributed-computing md simulations of the native and unfolded state of villin headpiece. both our analytical and computational results suggest a strong correlation: <rmsf> rms = [(s-1)/2s] 1/2 <prmsd> rms , where s is the number of compared structures. furthermore, if <prmsd> rms is defined as a generalized radius of gyration in the space of 3d structures and using rmsd as a measure of distance, the following identity holds: <rmsf> rms = <prmsd> rms. our results provide a basis for determining the level of structural diversity of molecular ensembles, as captured by <prmsd> rms , directly from experiment. charge migration along dna helices may be biologically important because extended electronic states could play a role in the processes of sensing of dna damage and/or dna repair via long-range charge transfer. we measured conductivity and other physical characteristics of several models of natural and diversely damaged molecules of dna. dna polymers were mimicked by various sequences of 32nucleotide-long double helices with fully watson-crick (wc) paired bases, with several central bases mismatched, and also with chemical modifications that included removal of bases from a few central nucleotides (abasic dna), and neutralization of phosphate charges by their derivatization. the model dnas were investigated by scanning tunneling microscopy, time-resolved thz spectroscopy, raman spectroscopy, circular dichroism, and modeled by molecular dynamics simulations. dna has the highest conductivity in its biologically most relevant double helical form with wc base pairs and negatively charged phosphates equilibrated with counterions. mismatches and all chemical modifications always lower the conductivity. the mechanism of charge transfer is consistent with electron or hole hopping between parallel stacked bases. these observations and data showing that the natural dna has also the most regular double helical form suggest that the continuous base stacking is critical for charge transfer. to control the passage across the bacterial cell wall nature created a large number of "nano"-channels which may act as selective gates for water soluble molecules. here we focus on porins from e. coli which control permeation through interactions with the channel surface. comparing single channel temperature dependent conductance measurements with all atom modeling allow conclusions on the mode of permeation. for example, surprisingly modeling ompf-conductance revealed not only a good agreement with the experiment over a broad range of temperature but also the selectivity for ions. the primary task of porins is to provide facilitated diffusion. we investigated facilitated diffusion of maltooligosaccharide or antibiotics. time resolved conductance measurements allows conclusion on the flux and molecular modeling identifies the limiting interactions with the surface, reveal potential barriers and pathways. exploiting the selectivity of natural or bioengineered channels has promising applications for detecting molecules, characterizing molecular interaction, sequencing dna, protein folding etc. traditionally, studies of diffusion-controlled reaction of biological macromolecules have been made in diluted solutions. however, the high concentration of macromolecules in intracellular environments results into non-specific interactions (macromolecular crowding), which have a great importance on the kinetics and thermodynamics of possible reactions that occur in these systems. in the literature there are studies concerning monte carlo (mc) simulations, giving results that are satisfactory agreement with experimental data, showing, for example, that the protein diffusion in cell cytoplasm is reduced considerably. in addition, there are mc studies about enzymatic reaction, which predict a temporal dependency of the velocity constant in macromolecular crowding. in this work, we try to compare the predicted behavior by mc simulation with the results obtained from the study of the diffusion and reaction of a model protein (alpha-chymotrypsin) using spectroscopic techniques in highly confined media in order to study experimentally the temporal dependence of its diffusion and reaction coefficients. a. paciaroni 1 , a. orecchini 1 , c. petrillo 1 , a. de francesco 2 , f. sacchetti 1 1 university of perugia, italy, 2 cnr-infm, genova, italy the single-particle and collective dynamical properties of protein hydration water have been studied by neutron scattering experiments in a wide temperature and hydration range. an unprecedented accuracy has been achieved thanks to the availability of a large amount of fully deuterated protein powder and the use of the high-flux spectrometers in5 and brisp. the protein under investigation was the maltose binding protein (mbp), which is a well-known and widely studied model of biosensor systems. we found that the low-temperature single particle dynamics of mbp hydration water shows clear features that can be traced back to amorphous systems. more in detail, its vibrational density of states is simply described as the superposition of the contributions of low-density and high-density amorphous ice. the quasielastic signal, which appears at the higher temperatures, can be excellently described with a fractional power law which may put in relationship with the peculiarities of fractal systems. quite strikingly, there is a strong similarity, on both the qualitative and quantitative point of view, with the behaviour of hydrated proteins. the collective dynamics of protein hydration water is characterised by the presence of two modes, whose dispersion curves are reminiscent of those of bulk water. however, the relevant damping factors suggest a strong similarity with glassy systems. m. malferrari 1 , f. francia 1 , s. sacquin-mora 2 , g. venturoli 1 1 università di bologna, bologna, italy, 2 cnrs upr 9080, paris, france the coupling between electron transfer and protein dynamics has been compared in reaction centers (rc) from the wild type (wt) and the carotenoid-less mutant r26, by combining brownian dynamics simulations and the kinetic analysis of charge recombination. upon incorporation of the rc into a progressively dehydrated trehalose matrix the electron transfer between the primary photoreduced quinone and the photoxidized donor accelerates progressively and becomes broadly distributed. this behaviour reflects the hindrance of protein relaxation following charge separation and the inhibition of interconversion between conformational substates. in extensively dehydrated matrices the recombination kinetics is two-times faster and three-times more distributed in the wt rc, indicating a larger inhibition of the internal protein dynamics. in line with this findings brownian dynamics simulations reveal a larger rigidity of the carotenoid-containing structure, in which a cluster of residues close to the quinone acceptors is stiffened as compared to the r26 rc. the in silico and experimental results indicate that the introduction of an internal void in the rc structure has long-range effects on the protein dynamics and that the coupling between the glassy matrix and the rc interior depends markedly on the local mechanical properties of the protein. n. maghelli 1 , v. krstic 2 , n. pavin 2 , f. julicher 2 , i. tolic-norrelykke 1 1 mpi-cbg, dresden, germany, 2 mpi-pks, dresden, germany in the fission yeast schizosaccharomyces pombe, the nucleus is positioned at the cell center. since the nucleus determines the cell division site, keeping the nucleus at the center is crucial for ensuring symmetrical cell division (1 ) . microtubules push against the cell ends and exert force on the nucleus (2 ), but how the cell regulates these forces in order to center the nucleus remains unknown. here we tackle this problem by using a combination of live cell imaging, cell manipulations by optical tweezers, and a theoretical model. we show that microtubule pushing forces can center the nucleus because of a larger number of contacts between the microtubules and the proximal cell end than the distal one. moreover, kinesin-8 motors (klp5/6) increase the rate of microtubule catastrophe (transition from growth to shrinkage) in a microtubule length-and contact-dependent manner. thus, the motor behavior results in a longer contact between a microtubule and the proximal than the distal cell end. taken together, our experimental and theoretical results provide a novel centering mechanism, where kinesin-8 motors increase the efficiency of nuclear centering. electropermeabilization is a commonly used physical method which can induce a transient permeabilization of the cell membrane allowing the entry of therapeutic molecules into the cell and is thus of great interest in the fields of cancer treatment and gene therapy [1] . however, very little is known about the mechanisms occurring at molecular level. there is clearly some microscopic reorganization of the membrane which is responsible for this change in its transverse transport properties. rather than studying the change of these transport properties, we adopt a simple strategy based on the use of giant unilamellar vesicles and videomicroscopy, as described below. we apply a series of permeabilizing electric pulses to the liposomes, and we observe a size decrease down to a critical radius beyond which their size no longer changes. this decrease in size points to the fact that during the physical processes leading to electropermeabilization, lipids are lost from the vesicles. our results published in [2] suggest different possible modes for lipid loss, which can be small vesicles, pores, or tubules formation. imaging of brain activation using core techniques as fmri, pet and synchrotron radiation in parallel c. poitry-yamate, g. margaritondo, r. gruetter ecole polytechnique fédérale de lausanne, switzerland functional magnetic resonance imaging (fmri), magnetic resonance spectroscopy (mrs), positron emission tomography (pet) and synchrotron x-ray emission imaging form a highly complementary set of imaging core technologies for studying brain function and energy metabolism. owing to differences in the information each conveys and the temporal and spatial scales on which they measure labeled substances, a single working hypothesis can be tested from different angles to provide a cross-validated, consistent and coherent explanation. the cibm is a unique research facility in europe for advancing our understanding of biomedical processes in health and disease. the housing of a 7-tesla human magnet, 9.4 and 14.1-tesla animal magnets, an animal pet imaging facility and fully-equipped neurochemistry and rf laboratories has enabled us to develop and perform well-targeted experiments around one research theme. in parallel, a longterm collaborative project using synchrotron x-ray transmission and emission imaging at elettra enables us to combine these core technologies towards understanding brain function in vitro and in vivo, from tissue to cells. a brief presentation of these methods will be followed by their application in studying the visual system from man to mouse. p. picone 1 , r. carrotta 2 , d. giacomazza 2 , m. di carlo 3 1 dip. chimica e tecnologie farmaceutiche, università di palermo, italia, 2 ibf -cnr, palermo, italia, 3 ibim -cnr, palermo, italia diabetes and alzheimer's disease are connected in a way that still is not completely known. diabetes has been implicated as a risk factor for developing alzheimer's disease. some diabetes drugs appear to decrease the cognitive decline associated with alzheimer's disease. it has been recently demonstrated that extracellular injection of insulin is able to protect neurons against a-beta amyloid cell death. one of the proposed theories to explain such an effect is that the hematic glucose levels affect the metabolism of the hippocampus, a part of the brain (associated with memory, emotion and motor skills), which is strongly damaged in alzheimer's patients. the aim of this study is to investigate the effect of insulin on the a-beta induced degeneration and oxidative stress on the neuroblastoma lan5 cell line. in particular, the present study looks into the role of insulin in the inhibition of abeta specific degenerative apoptotic pathways. preliminary results indicate that insulin dissolved in culture medium in its hexameric form (as tested by absolute scale light scattering) is able to reduce neurodegeneration induced by a-beta amyloid in a dose dependent manner. the link between diabetes and alzheimer's disease may provide new targets for future alzheimer's treatments. moreover, due to the increased incidence of diabetes in western countries, a deeper understanding of such a link is relevant in order to control the escalation in the number of people dealing with dementia. a. pelizzola dipartimento di fisica, politecnico di torino, torino, italy many features of protein folding have been shown to be described by an ising-like model (one-dimensional, with longrange, multispin interactions) whose equilibrium thermodynamics is exactly solvable [1] [2] [3] . we have generalized such a model to the problem of mechanical unfolding. the equilibrium thermodynamics is still exactly solvable, and the characteristic kinetic responses found in force ramp and force clamp experiments are well reproduced [4, 5] . unfolding and refolding pathways and intermediates can also be studied, again with good agreement with experiments [6]. applications to various proteins and rna fragments will be discussed. [ the key role of water in living systems has been widely studied in literature, along with its anomalies, consequence of the extensive three-dimensional hydrogen bonding of water molecules. moreover protein-water interactions take place at protein surface where cell water has been recognized to behave differently from bulky water. the two-states theory of water assumes that water is a mixture of microdomains of different structure and density, the low-density water (ldw) and the high-density water (hdw) domains, and ions partition selectively into ldw or hdw domains. the idea developed in this work was to explore the ordered water structure by measuring delayed luminescence (dl) from salt aqueous solutions in which water structuring is anticipated. it appeared that dl signal from salt solutions is significantly relevant when prevalence of ldw domains is foreseen, with a decay time probability distribution function characterized by a broad maximum in the microsecond range. the obtained results support the ability of dl to reveal the different properties of ldw and hdw domains induced by salt molecules. moreover, the results reveal the existence of clusters, whose characteristics strongly depend on the specific ion effects, of surprisingly long lifetimes not observed till now. this could give new insight into biological water properties. self assembly of patchy particles and dnafunctionalized dendrimers f. sciortino dipartimento di fisica e infm-cnr-soft università 'la sapienza', roma, italy i will report numerical results on the phase behavior of very simple models of patchy particles with the aim of understanding the interplay between phase separation and selfassembly and how the fraction of surface allowing for attractive interactions controls the collective behavior of the system. the case of janus particles, particles characterized by a surface divided evenly into two areas of different chemical composition, will be discussed. i will also discuss the self-assembly of a simple model for four single strands of dna tethered to a central core, and show that the model exhibits a rich phase diagram that includes at least four thermodynamically distinct amorphous phases (polyamorphism) in a one-component system. the dense phases consist of a hierarchy of interpenetrating networks, reminiscent of a woven cloth. peptide dimer motifs in the phospholipid environment -structure, interaction and molecular design p. e. schneggenburger 1 , a. beerlink 2 , t. salditt 2 , u. diederichsen 1 1 iobc, universität göttingen, germany., 2 irp, universität göttingen, germany. based on recently reported homodimeric peptide pores with a d,l-alternating configuration a novel double helical hairpin-motif of a membrane active gramicidin a analog was designed. [1, 2] the cd spectroscopic analyses of the peptide-lipid complexes revealed the structural preservation and elucidated the importance of a zwitterionic interaction of the peptide termini. [3] the peptide design was enhanced regarding the versatile functionalization with analytical probes as well as molecular recognition moieties like peptide nucleic acids (pnas) to observe the effects of aggregation and specific organization within model lipid membranes even at high peptide-to-lipid ratios. [3] x-ray reflectivity on lipid bilayer stacks in combination with heavy atom labeling and spectroscopic studies of vesicle systems provides information about the peptide structure and interaction in the native fluid state of the membrane system. [2, 3] for this, the fmoc-diiodo-allylglycine building block was created to serve as a novel iodine label pinpointing at a certain position with respect to the membrane normal. we study thermal undulations of giant unilamellar vesicles (guvs) of lipids by flickering spectroscopy. getting values for the mechanical parameters of lipid bilayers requires the experimental fluctuation spectra to be scrutinized in view of the classical helfrich's theory. pure bending modes are revealed unable in predicting the large fluctuations systematically found at high wavevectors. hybrid curvature-dilational modes have been invoked as a more efficient mode of motion in producing high curvatures. a bimodal spectrum of the thermal undulations has been theoretically developed for the shell-like topology. from this new description, two important consequences emerge a priori, the dependence of the fluctuation dynamics on either vesicle size and on bending/compression parameters. for popc and dopc vesicles containing cholesterol the experimental fluctuation spectra are well described by the new spectrum. reconciliation between experiments and theory is achieved when this bimodal spectrum is considered. the new theory opens enormous possibilities for better exploring membrane mechanics in guv models. under normal conditions, platelets circulate in the vascular system having very low interaction with each other and with other cells. the platelets become activated when the biological system is disturbed, for instance by vascular damage in which blood gets in contact with collagen. upon activation, different types of receptors/molecules are exposed on the cell membrane to support adhesion, spreading and aggregation of the platelets onto the damaged vessel. the measurement of altered platelet function is particularly important in cardiovascular diseases such as thrombosis. we are investigating biosensor technologies for the detection of functional properties of platelets. an important study is the specific and non-specific stimulation of platelets in a biosensor cartridge. we will present experimental results on biosensor platelet activation using the platelet-specific membrane markers p-selectin and gp1b. multi-joint analysis of locomotion in the first neonatal rats flown in space d. sulica, j. vinersan "carol davila" university of medicine and pharmacy, bucharest, romania the first mammalian neonatal animals in space were the rats flown on the space shuttle endeavor during a 9-day mission, sts-72. the development of locomotion in weightlessness was evaluated using two litters of neonatal rats, launched at postnatal days 7 and 15. age-and cage-matched animals were used as ground controls. free walking was videotaped from the landing day. although preliminary analysis of walking showed differences in both hindlimb and forelimb joint angles and a hyperextension of the hindlimbs was apparent, the numerical values reached the significance level only for the ankle angle measured at specific moments of the step cycle: foot contact, maximum loading with weight, foot lift and maximum flexion during swing. we report here on the behavior of all the joints during the whole step cycle, by computing the integral of these angles over the step cycle. the results were affected by the differences in the walking speed (the young animals walked faster than the controls), so we scaled the integrals by the step cycle duration. we found that, besides the ankle, the knee was also more extended throughout the whole step cycle in both groups of animals. moreover, all the joints (including the toe and the hip) were affected in the same way (hyperextended), since the differences were still significant when we added together these angles. the animals recovered slowly, with significant differences remaining after 14 days of readaptation. the effect of dextran 70 concentration on red blood cell deposit formation j. strzelecka, b. grzegorzewski department of biophysics, collegium medicum in bydgoszcz, nicolaus copernicus university 85-067 bydgoszcz, poland red blood cell (rbc) deposit formation was examined by means of an optical method. blood was obtained from healthy donors and measurements were performed at initial hematocrit 40%. the intensity of scattered light was measured during sedimentation of rbcs suspended in saline -dextran 70 solutions at different polymer concentrations (2 -6 g/dl). the changes in the intensity of the scattered light manifest rbc aggregate formation, their sedimentation and the process of deposit formation. the deposit formation curve was determined. it is shown that the concentration of dextran affects the deposit formation. an empirical model has been used to describe the experimental data. the parameters of the deposit formation curve as a function of dextran concentration are analyzed. water is essential to life and a major scientific interest lies in a detailed understanding of how it interacts with biological macromolecules in cells. we studied water dynamics in whole cells with neutron scattering [1, 2, 3] . the cellular environment is extremely crowded with macromolecules and water molecules are permanently in close contact to biological interfaces. we measured water dynamics in e. coli and human red blood cells with neutron scattering [2, 3] . the data revealed two populations of water in the cells: a major fraction which has dynamical properties similar to those of bulk water (relaxation times ∼ps) and a minor fraction in the order of ∼10% which is interpreted as bound hydration water with significantly slower dynamics (relaxation times >49 ps). in this contribution we report on investigation of model membrane dynamics by means of quasi elastic and inelastic incoherent neutron scattering and on the effect of membrane inserted pore forming peptide gramicidin. model membrane are realized by highly oriented, hydrated phospholipid bilayer stacks of dmpc (1,2-dimyristoyl-snglycero-3-phoshatidylcholine) hydrated with d2o in excess of solvent condition. the bilayer were supported on mica substrates and prepared at different concentrations of gramicidin, a 15-residue oligopeptide showing antimicrobial activity by forming pores on the membrane surfaces which allow water and small ions to permeate across the membrane. incoherent qens and ins spectra, measured on in5 and in13 spectrometer at ill, allows to obtaining information on the mean dynamics of the hydrogen atoms in the system. moreover, by proper orientations of the membrane plane respect to the scattering wave vector q, we were able to derive information on in plane and out of plane motions of the phospholipids. the using of media products for the creating attracted bioenergetic brain rhythmus advertisement v. i. vlastopulo, v. g. nikolajev str. gen. petrova 92 , app. 44, 65065 odessa, ukraine the technical efficiency of bioenergetical influence of advertisement is present with assistance of consciousness of memory at revision and hearing of advertisement on tv, radio stations, mobile phones, at supermarkets and other places. in a basis of useful model it is put a task to improve the method of creating the attracted advertisement, in which the creation of bioenergetical influence by the oscillation of not less 2 electromagnetic fields or video-images is introduced with their creating as the base on the spatial or flat structure formative macro matrix or matrixes with repeatable structure with the brain α-rhythm frequency of extreme attention and δ-rhythm frequency of meditation. the point of the patent on the device is in the bioenergetical influence increases in addition to the information influence by advertisement of pictures and audio oscillations the bioenergetical influence increases at the consciousness contribution of human memory at the moment of watching or hearing of television, radio stations, working in the internet, music in the supermarkets, banks, clubs, metro and other places. cell adhesion and motility are processes involved in fundamental biological phenomena. they imply multimolecular scaffolds as anchorage points and actin cytoskeleton filaments to build internal stress and eventually crawl onto the substrate. these processes, very dynamic by nature are out of equilibrium. we study cell adhesion on micro-patterned substrates where the introduction of a finite distance between the possible anchorage points of the cell modifies drastically the organization of the cytoskeleton and the anchorage point's distribution. because statistical quantification shows that some shapes are more likely than other, we believe they represent particular organizations of the system which should minimize the energy dissipation. we checked this hypothesis by using the cellular potts model. shapes obtained by simulation are in excellent qualitative agreement with experimental shapes. they depend on phenomenological parameters such as interaction between cells and the extra cellular matrix, a line tension and an elastic modulus. the aim of this work is to link model parameters to physico-chemical properties of cells and to establish phenomenological relations between relevant biochemical regulators controlling the cytoskeleton organization. c. canale, d. ferrera, f. benfenati, l. gasparini the italian institute of technology, 16163 genova, italy alzheimer disease (ad) is characterized by cerebral extracellular deposits of β-amyloid (aβ) fibrils. aβ aggregation is a multi-step process involving the formation of various conformational species including soluble intermediate species (i.e. aβ oligomers), protofibrils and fibrils. such aggregates may have various effects on neuronal and glial function and differentially contribute to ad neurodegeneration. aim of this study was to investigate the structural properties of distinct aβ aggregated species and dissect out their effects on neuronal viability. recombinant aβ42 and aβ40 peptides were aggregated in vitro in conditions differing by ionic strength, temperature and ph and were analyzed by gel electrophoresis, thioflavin t binding assay and atomic force microscopy (afm). afm analysis was performed using both hydrophilic and hydrophobic substrates, to analyze the full spectrum of structural species. stable low molecular weight oligomers were obtained when aβ42 was incubated at 37 • c for 5 days in low salt concentration buffer. doughnut-shaped conformational species were detected by afm alongside globular aggregates (1.6-5.2 nm height range). acidic ph promoted aggregation of aβ42 into thioflavin-positive fibrils and protofibrils. protofibrils appeared as beaded chains having a mean height of 3.65±1.49 nm. effects of aβ on viability of mouse hypppocampal neurons were assessed and correlated with their conformational features. a. bellova 1 , e. bystrenova 2 , m. koneracka 1 , p. kopcansky 1 , f. valle 2 , j. bagelova 1 , f. biscarini 2 , z. gazova 1 1 1 institute of experimental physics, slovak academy of sciences, kosice, slovakia, 2 2 ismn cnr, bologna, italy peptide amyloid aggregation is a hallmark of amyloid diseases including azheimer's disease or type ii diabetes. recent works have addressed the potential of nanoparticles to affect amyloid aggregation. the experimental data are very controversial suggesting that particle characteristics markedly influence the final effect of nanoparticles on the amyloid aggregation (initiation, acceleration or inhibition of amyloid aggregation). we investigate the ability of electrostatically stabilized magnetic nanoparticles of fe 3 o 4 to affect the amyloid aggregation of lysozyme, as a prototype amyloidogenic protein. we have used a combination of spectroscopic (tht fluorescence) and local microscopic techniques (afm). we found, that the ability of magnetic nanoparticles to inhibit formation of amyloid aggregates or destroy pre-formed amyloids exhibit concentrationdependence. the values of inhibition ic50 and depolymerization dc50 were determined suggesting that nanoparticles interfere with lysozyme aggregation at stoichiometric concentrations. the observed features make magnetic nanoparticles of potential interest as a therapeutical agent against amyloid diseases. (this work was supported by project of esf 26220120021 and by slovak academy of sciences in frame of cex nanofluid, vega grants 7055, 0056 and 0038 and eu-strp 0032652 biodot.). a. bellova 1 , l. balogova 2 , b. chelli 3 , e. bystrenova 3 , f. valle 3 , j. imrich 2 , p. kristian 2 , l. drajna 2 , j. bagelova 1 , f. biscarini 3 , z. gazova 1 1 institute of experimental physics, sas, kosice, slovakia, 2 faculty of sciences, p. j. safarik university, kosice, slovakia, 3 ismn cnr, bologna, italy numerous diseases have been linked to a common pathogenic process called amyloidosis, whereby proteins or peptide clump together to form amyloid aggregates in the body. an attractive strategy to develop therapies for these diseases seems to be reduction of polypeptide aggregation. we have tested several acridine derivatives characterized by various glycosyl groups for their potential to affect the lysozyme amyloid aggregation in vitro. the ability of glycosyl acridines to interfere with lysozyme aggregation was investigated by tht assay. we found that structure of acridine side chain is factor affecting their anti-aggregation activity significantly. for the most effective compounds the values of ic50 and dc50 were obtained. the reduction of protein aggregation was confirmed by afm. to investigate influence of the glycosyl acridines on the cell processes we examined effect of compounds on cell viability. we performed glycosyl acridines characterization by high anti-aggregation activity and low toxicity suggesting their possible application for therapeutical purpose. (this work was supported by project of esf 26220120021 and by slovak academy of sciences in frame of cex nanofluid and vega grants 7055, 0056 and 0038 and eu-strp 0032652 biodot.). a. j. beevers, a. m. dixon department of chemistry, university of warwick, uk due to the immense medical importance of proteins which span the membrane of cells, detailed molecular structural information of these systems is essential. practical difficulties in employing high-resolution structural elucidation techniques have resulted in a relative paucity of fully resolved membrane protein structures. therefore a variety of lower-resolution techniques are used to determine structural information of the transmembrane (tm) domains of proteins. one example of such a membrane protein is erbb-2, a receptor tyrosine kinase responsible for triggering cell division and which is prone to a mutation in its transmembrane domain resulting in permanent activation and oncogenic effects. we have predicted an interface for the mutated tm domain dimer using site-specific infrared spectroscopy containing a repeating sequence of ile, val and leu 1 . applying the in vivo toxcat assay to the tm domain sequence and to specific mutants of it, confirms this proposed interface whilst another proposed interface is discounted. current studies are focussing on the effect of the tm mutation to the activation of the erbb-2 receptor and to any possible change in this interface. bacteriophages are complex molecular assemblies which multiplication relies on bacteria infection. the process starts with the binding of the phage on its specific host receptor and the injection of its genome into the host cytoplasm. our work aims to determine the physical mechanisms and forces driving the dna transfer from the phage capsid. the in vitro dna ejection has been analyzed by using light scattering and gel electrophoresis measurements for three phages (t5, spp1 and lambda) belonging to the same family (syphoviridae). our results reveal two forces contributing to drive the dna transfer: the first one is originated from the pressurization due to the strong confinement of dna into the capsid; the second one comes from a pulling mechanism originated by the presence of condensed dna outside the capsid. these two contributions were characterized in in vitro conditions but they likely play a role in the in vivo transfer. the ejection kinetics was also analysed and the characteristic time of the mechanism was studied as a function of the temperature. it appears to follow an arrhenius law, allowing the determination of the activation energy that governs the transfer. the energy values are close for the different phages, suggesting that the mechanism regulating the ejection is common for a given phage family. below these general features, our studies also reveal differences between the three phages. the effect of &beta-amyloid peptide on polymer cushioned membranes s. dante 1 , r. steitz 2 , t. hauss 2 , c. canale 1 , n. a. dencher 3 1 istituto italiano di tecnologia, genova, italy, 2 bensc, helmholtz center berlin, germany, 3 tu-darmstadt germany beta-amyloid (aβ) is a peptide implicated in the neurodegenerative process characteristic of the alzheimer's disease (ad). to clarify its mechanism of action it is crucial to elucidate the interaction of aβ with the neural membrane. in previous work we demonstrated the capability of aβ to penetrate and perturb stacked lipid bilayers. in this study we considered polymer cushioned lipid bilayers as a model for neural membranes. the polymer cushion is aimed to preserve the membrane natural fluidity; it is obtained depositing charged polyelectrolites layer-by-layer; the lipid membrane is built on the top of the polymer film by fusion of unilamellar vesicles. the floating membranes were kept always in contact to the subphase. the kinetics of adsorption of the lipid double layer at the polymer/water interface was monitored by neutron reflectivity; different experimental conditions to obtain the best surface coverage were exploited. after administration of aβ to the subphase the lipid membrane still adhered to the polymer cushion, but its structure was modified by the interaction with aβ. neutron reflectivity showed a change of the scattering density profile in the direction perpendicular to the membrane plane, suggesting a penetration of aβ inside the double layer. a change in the surface morphology was detected by afm imaging; afm film-rupture experiments showed that aβ weakens the lipid packing. x. cheng 1 , r. pacheco-gomez 2 , a. rodger 1 , h. matthew 1 , d. i. roper 1 1 university of warwick, u.k., 2 university of birmingham, u.k. ftsz, the ancestral homolog of eukaryotic tubulins, is a gt-pase that assembles into a cytokinetic ring structure (z ring) essential for cell division in prokaryotic cells. the z ring also recruits other proteins (e.g. zapa, ygfe, zipa) to the division site, where they participate in formation of the septum that separates the two daughter cells. we have studied ftsz polymerization and its dynamic behaviour in real time by right angle light scattering. similar to tubulin, ftsz polymerizes into dynamic protofilaments in the presence of gtp; polymer assembly is accompanied by gtp hydrolysis. the kinetics of inorganic phosphate (p i ) released from the gtp hydrolysis have been studied as well, employing a fast and sensitive colourimetric assay. at ph 6.5, approximate 90% of the p i was released into the media within 20 minutes of gtp addition. the effects of gtp, ph, k + , and mg 2+ were studied in both cases, and the results were used to build up a model for the mechanism of fibre assembly and disassembly. ygfe, a ftsz accessory protein, is identified as a functional zapa orthologue. finally, we have studied the ygfe bundling to ftsz polymers. it strongly promotes ftsz bundling and is an inhibitor of the gtpase activity. many genomes of viruses encode small membrane spanning proteins which are proposed to modify membrane permeability for ions and small molecules. these channel or pore forming proteins are getting into the focus for antiviral therapy since they are essential for some of the viruses. one of the general themes of the mechanism of function of the proteins is to self-assemble to form the functional form. we present a study on the open reading frame (orf) 8a membrane protein encoded in structural region of human severe acute respiratory syndrome coronavirus (sars-covs). the full length orf8a protein is 39 residues long and contains a single transmembrane (tm) domain. full length protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers forms cation-selective ion channels. the bilayer recordings show cation selection channel activity with a major conductance level of around 8.5 ps also at elevated temperatures (38.5 • c). in silico studies with a 22 amino acid tm domain are done to assess conformational space of the monomeric orf8a helix. with this monomeric helix homooligomeric helical bundle models are built and embedded in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycerol-3phosphatidylcholine (popc) bilayer. results of both experimental channel recordings and computational modeling show sars orf8a to act as a channel forming protein. -biomolecular self-assembly microtubules are involved in many vital processes. their rigid structure can resist high forces while their intrinsic ability to switch stochastically between growth and shrinkage phases allows them dynamically to reorganise. in cells, a sizeable network of microtubule binding proteins control and regulate microtubule dynamics. alp14 and dis1 are members of the dis1/ xmap215 family that are major players in s.pombe. the deletion phenotypes of alp14 and dis1 are similar, but nonetheless distinct. both are involved in the formation of spindles but alp14 is also involved in the maintenance of cytosolic microtubules in interphase. the restrictive temperatures of alp14-deletion and dis1-deletion mutants are different. alp14 interacts with alp7, a potential member of the tacc protein family. i am working to reconstitute alp14/dis1-dependent microtubule dynamics in vitro, using purified s. pombe tubulin. both alp14 and dis1 express well in insect cells and can be readily purified. preliminary data show that both proteins bind tubulin at low salt concentrations and that both influence the dynamics of pig brain microtubules. my goal is a complete functional analysis of alp14 and dis1, individually and in combination, to test candidate molecular mechanisms for alp14/dis1-catalysis of s. pombe microtubule dynamics. the syphoviridae coliphage t5 is a well-suited model to study the assembly of large viral capsids. biochemical and biophysical approaches were used to reconstitute in vitro the assembly pathway of its capsid. the t5 structure was recently solved from cryo-em and image reconstruction. its icosahedral capsid (t = 13) is built from the major head protein (pb8, 775 copies) forming both the pentons and hexons and from the portal protein (pb7, 12 copies) located at one vertex. its assembly proceeds by steps. pb8 and pb7 first assemble into a precursor structure called prohead i, which is converted to prohead ii by proteolysis of pb8 and pb7 by a head maturation protease. packaging of the 121 kbp dsdna is then driven through the portal pore by a molecular motor, the terminase. this promotes expansion of prohead ii leading to the mature capsid. the different assembly steps and the conformational changes accompanying capsid maturation were characterized using proheads i either self-assembled from the overproduced and purified capsid proteins or isolated from a phage mutant. these precursor capsid structures were analysed by small angle x-ray scattering. the 3d structure of prohead ii and of the expanded capsid were solved from cryo-em. our data show that the assembly process of a large icosahedral capsid can be efficiently reconstituted in vitro. amyloid beta peptide fibril formation modulated by phospholipid membranes e. hellstrand 1 , e. sparr 2 , s. linse 1 1 lund univ., department of biophysical chemistry, sweden, 2 lund univ., department of physical chemistry, sweden disease-causing amyloid fibril formation can be modulated by many factors including interactions with biological lipid membranes. an increasing amount of evidence suggests that the process of fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. alzheimer's is probably the most well-known amyloid disease and the associated amyloid beta peptide originates from the membrane incorporated amyloid precursor protein (app). we use recombinant abeta m1-40 and abeta m1-42 produced in escherichia coli, which allows us to perform large scale kinetics assays with good statistics where the amyloid formation process is followed in means of thioflavin t fluorescence. the lipid membranes are introduced in the system as large unilamellar vesicles composed of dopc, dppc and sphingomyelin, with and without incorporation of cholesterol. we find that the phase behanviour of the membrane in the vesicles has a large effect on the lag time of the amyloid formation process for both abeta m1-40 and m1-42. all membranes increase the lagtime to some degree but dppc has the largest effect. by comparing different phases we can conclude that the translational diffusion in the membrane seems to be more important than the acyl chain ordering. furthermore, electrostatics, concentration dependence and membrane addition at different time points in the amyloid formation process have been investigated. equilibrium/non-equilibrium transitions in macromolecule interactions p. dumas 1 , g. gibrat 2 , s. bernacchi 1 , e. ennifar 1 1 ibmc-cnrs, strasbourg, france, 2 llb (cea/cnrs), saclay, france usually, so called 'relaxation phenomena' occur on a fast time-scale and 'p-jump' or 't-jump' techniques are required to follow such events lasting (much) less than 100 ms. we report that, during stability studies of proteins or nucleic acids, such relaxation events can be observed on astonishing long time-scales. we first performed 'melting studies' with nucleic-acid duplexes by using linear variations of temperature (t) with time (t). we observed that even very low rates dt/dt could lead to a frozen state for temperature values below a sharp temperature range, and relaxation to equilibrium beyond that range. this allows defining a 'relaxation temperature' t r separating the two regimes. numerical simulations very accurately described the related hysteresis phenomenon observed upon a heating-cooling cycle, which is the hallmark of departure from equilibrium. analogous observations were made with protein oligomers submitted to either a variable pressure, or variable concentration in denaturant. importantly, a single theoretical frame predicts that the critical relaxation value x r (x standing indifferently for temperature, pressure or denaturant concentration) depends on ln(dx/dt). one may ask whether some thermosensor rnas known for switching on or off genetic expression by 'feeling' a temperature variation, might also 'feel' dt/dt. if true, the exact switching temperature would depend on dt/dt and faster temperature changes would increase t r . -biomolecular self-assembly the major component of amyloid plaques in the gerstmann-sträussler-scheinker disease is a prion peptide fragment from 81-82 to 144-153 residues. here, we present a structural study of prp82-146 in form of oligomers and fibrils by fourier transform infrared spectroscopy (ftir) and atomic force microscopy (afm). after incubation at 37 • c, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands and by a wide distribution of oligomer volumes. after a lag phase, a conformational rearrangement of oligomers into fibrils, with a parallel orientation of the cross β-sheet structures, was observed. by afm, different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly. in addition, we also studied thermal and random aggregation. the prp82-146 peptide was found to undergo several aggregation pathways, whose end products display different structural properties and intermolecular interactions. these findings underline the high plasticity of the prion peptide, a peculiar feature of prion proteins to overcome species barriers (natalello et al. j.mol.biol. 2008;381:1349-1361). the role of proline isomerisation in the aggregation process and fibril formation of alpha-synuclein j. meuvis 1 , m. gerard 2 , v. baekelandt 3 , y. engelborghs 1 1 lab.of biomolecular dynamics, ku leuven, belgium, 2 lab.of biochemistry, campus kortrijk, belgium, 3 lab.for neurobiology & gene therapy, ku leuven, belgium alpha-synuclein (α-syn) plays a central role in parkinson's disease. the aggregation of this protein, which contains five proline residues (p108,p117,p120,p128,p138), is accelerated in vitro by fk506 binding proteins (fkbps), a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (ppiase). fkbps catalyze the cis-trans conformational change of proline, often a rate limiting step in protein folding. to elucidate the role of the proline residues in aggregation, we constructed a mutant p(108,117,120,128,138)a α-syn . the kinetics of the aggregation of the mutant were studied with turbidity and thioflavin t fluorescence (tht). turbidity measurements show the formation of early, tht negative aggregates which is as fast for both wt and mutant. fibril formation however is faster for the proline-deficient mutant. we also studied the effect of fkbp12 on the aggregation of the mutant. although wt α-syn early aggregate formation is accelerated by the addition of 1 µm fkbp12, this effect disappears in the mutant. addition of (1pm-1µm) fkbp12 accelerates the fiber formation of wt α-syn, which is abolished in the mutant. we can conclude that α-syn fiber formation is accelerated for the proline-deficient mutant, which suggests a role for the proline residues in fiber formation. furthermore all accelerating effects of fkbp12 are abolished in the mutant which suggests that the ppiase activity of fkbp12 is responsible for the accelerating effect on the aggregation of wt α-syn. materials used as gene delivery vehicles must be able to condense dna into small sizes to facilitate transport and crossing various barriers. one of the polycations investigated for dna compaction is chitosan, which has the advantage of being safe and biodegradable. as a step towards reducing the aggregation behaviour of dna-chitosan complexes, chitosans were modified by grafting peg-chains on the backbone. it is known that the transfection efficacy depends on the chitosan chain length. additionally, the degree of pegylation might influence the condensation process. here a systematic biophysical study of pegylated chitosans and how the interplay between chitosan chain length and degree of pegylation affect the compaction of dna in terms of particle size and structure, stability in pbs and when exposed to serum, and transfection efficacy is presented. three different chain lengths of chitosans are employed, and for each sample three pegylation degrees are investigated and the properties of the dna-pegchitosan complexes compared to complexes formed when employing the original, chitosan for dna compaction. it is found pegylation of chitosans can be used to increase both the stability of the dna-chitosan complexes when exposed to serum as well as increase their transfection efficacy in hek293 cells. max-planck institute for polymer research, mainz, germany model membranes mimic the essential function of a natural membrane. however, the complexity is reduced in order to allow the study of fundamental processes. tethered membranes consist in principle of a lipid bilayer that is covalently linked to a solid support through a spacer group. this architecture allows the characterization of the membrane itself as well as of incorporated membrane proteins using surface analytical techniques. we have established a versatile system of various anchor lipids, which allow membrane formation on different surfaces. the architectures have been characterized by surface plasmon techniques, neutron reflectivity and electrochemical methods. the membranes are electrically insulating and allow for the functional incorporation of ion channel proteins. polymerizable lipids allow to pattern the membrane and to study lateral diffusion processes. furthermore, the membranes can be used as a sensing platform, where embedded membrane proteins act as actual sensing units. a. perico, s. pietronave, l. arcesi, c. d'arrigo consiglio nazionale delle ricerche (cnr), institute for macromolecular studies (ismac) the electrostatic free energy (fe) of two parallel rigid likecharged polyelectrolytes (pes) is given as a function of the separation distance. 1 for high linear charge density, z , the fe shows a minimum due to the increasing of the counterion condensation and condensation volume as the two pes approach. the interaction fe is governed by a critical linear charge density, z c , inversely proportional to the counterion valence. for highly charged pes (z > z c , like dna), the pes attract the stronger the smaller is the counterion valence, because the fe is dominated by the entropic term due to condensation of counterions in a volume displaying a maximum at short distances. for weakly charged pes (z < z c /2 ) the pes remain undercritical in the whole separation range and therefore repel. for moderately charged pes (z c /2 < z < z c ), the infinitely separated pes are undercritical but become supercritical as they approach a critical distance and charge condensation and condensation volume expansion start: in these circumstances the pes may attract if the counterion valence is sufficiently large. in the case of many dna rods, hexagonal clusters may be formed. upon interaction with hydrophobic surfaces, proteins show a tendency to expose regions that are normally buried in the hydrophobic core. unfolding is generally perceived as an undesired process in studies aimed to anchor functional proteins at surfaces. upon an upset of perspective the fine control of the unfolding/re-assembly process could be regarded as a strategy to build up molecular nanostructures for the development of organic-inorganic assemblies. we show that molecular layers patterned at the nanoscale, with longrange order properties extending over the microscopic scale, can be obtained upon adsorption of proteins onto the hydrophobic and ordered surface of pyrolytic graphite. upon adsorption, proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. the first layer, in contact with the substrate, and the second molecular layer show corduroy-like nanopatterns of different periodicity, with a relative orientation between the first and second layer patterns of 30 • . surface-induced protein unfolding and polypeptide chain reassembly according to a layered ordered structure is a rather general phenomenon since it is observed for different proteins irrespectively of their specific structural properties. the possibility of using these ordered molecular structures as templates for the subsequent patterned deposition of supramolecular aggregates will be discussed. understanding protein-protein interactions and assemblies to control the hierarchical building of well-ordered supramolecular structures is highly relevant to new tailormade biomaterials. we previously evidenced that contrary to native calcium-loaded α-lactalbumin (holo α-la), calcium-depleted form (apo α-la) has the ability to selfassemble with lysozyme (lys) to form different supramolecular structures in temperature-dependent manner. in the present work, the events occurring at molecular scale were explored using fluorescence techniques. fluorescence anisotropy and fluorescence lifetime measurements provide a powerful and sensitive mean to measure intermolecular interactions. we showed that lys interacts with both apo α-la and holo α-la to form oligomers, assumed to be heterodimers, at 10 • c and 45 • c. the dissociation constants for dimerization, found to be in the µm range, were sensitive to the ionic strength. correlation time calculations suggest that formed heterodimers holo α-la/lys and apo α−la/lys differed in their shape and/or conformation. such conformation differences could explain why holo α-la/lys complexes are trapped as heterodimers while the apo α-la/lys complexes have the ability to further self-assemble into previously reported various supramolecular structures. polyglutamine aggregation and neurodegeneration g. nicastro, l. masino, a. pastore national institute for medical research, the ridgeway, nw71aa london, u.k. polyglutamine (polyq) diseases are rare but dominant misfolding diseases linked to neurodegeneration. they are caused by the expansion of cag codon repeats, which encode polyq tracts in the corresponding gene products. aggregation of polyq proteins is thought to be triggered by polyq expansion but be strongly modulated by the protein context. in the attempt of understanding the molecular bases of polyq diseases, we are studying the structures, interactomes and aggregation properties of selected polyq proteins. here, we present recent work on ataxin-3, taken as a representative example of the whole family. ataxin-3 is a ubiquitin specific cysteine protease, involved in the ubiquitinproteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. the enzymatic site resides in the n-terminal josephin domain of ataxin-3. we have characterized, using different biophysical techniques, the structure in solution and the aggregation properties of josephin both in isolation and in a ubiquitin complex. we demonstrate that interaction with ubiquitin strongly modulates the aggregation properties of ataxin-3 and suggest the importance of protein-protein interactions in preventing aggregation. our study also provides new insights into the molecular mechanisms which determine ataxin-3 specificity for poly-ubiquitin chains of the correct length and cross-linking. förster resonant energy transfer (fret) from an optically excited to a non-excited molecule has been widely used to probe molecular interactions in living cells. changes in the molecular makeup of a cellular region occurring during the acquisition of fluorescence images place tight constraints on the fret technology and data analysis, which could not be addressed satisfactorily until recently. we will describe a method for imaging protein complex distributions in living cells with sub-cellular spatial resolution, which relies on a spectrally resolved two-photon microscope (raicu et al, 2009, nature photon. 3: 107-113) and a simple theory of fret in oligomeric complexes (v. raicu, 2007, j. biol. phys. 33:109-127) . then, we will overview recent results on the determination of the supra-molecular structure and distributions in living cells of oligomeric complexes of some g protein-coupled receptors. observing protein aggregates on surfaces m. rabe, d. verdes, s. seeger institute of physical chemistry, university of zurich, switzerland protein aggregation is an important topic of current protein research as it is associated with several human diseases including alzheimer's disease, parkinson's disease, and type ii diabetes. although protein aggregation mechanisms and conditions have been comprehensively investigated, studies on the formation and the fate of protein aggregates in contact with solid interfaces are scarce. we have comprehensively investigated the structure of protein assemblies that form spontaneously upon protein adsorption on solid interfaces using a surface sensitive fluorescence imaging technique based on super critical angle fluorescence (saf) detection. combining this technique with fret we not only succeeded to detect protein aggregates deposited on surfaces but also to characterize their behavior in real time, i.e., their emergence, growth, or spreading. the model protein bsa, for instance, was found to exhibit a certain tendency for aggregation in the buffer solution. these protein clusters can deposit onto solid surfaces and spread resulting in a large, flat structure after some time. 1 a different possibility how protein aggregates emerge on surfaces consists of a direct deposition of protein monomers to pre existing aggregates. such a growth of protein aggregates on the surface has been observed in a model system using the protein α-synuclein, which is tightly associated with the parkinson's syndrome. we present the results of a spectroscopic ellipsometry (se) study of the adsorption process of yeast cytochrome c (ycc) on gold and graphite substrates, according to methods already applied to study the growth dynamics of organosulphur sams on gold [1] . se investigation was carried out both in situ, at room temperature during protein deposition, and ex situ. on gold, se data demonstrate the formation of an about 3-4 nm thick layer, consistent with the formation of a dense monolayer of ycc molecules, confirmed by afm inspection. both in situ and ex situ measurements were characterized by well defined spectral features related to the soret band. analysis of the fine position of this feature allowed to obtain information on the oxidation state of the iron ion of the heme group. se data suggest that proteins have preserved their native structure. a completely different adsorption mechanism was observed on highly oriented pyrolytic graphite (hopg) [2] . ex-situ se data on ycc/hopg, supported by afm observations, indicate the formation of an ultrathin molecular layer (∼0.7nm) related to complete protein unfolding at the hydrophobic surface. the role of phospholipid anisotropy in the stability of inverted hexagonal phase was considered. the equilibrium configuration of the system was determined by the minimum of the free energy involving the contribution of the isotropic and deviatoric bending and the interstitial energy of phospholipid monolayers. the shape and local interactions of a single lipid molecule were taken into account. the minimization with respect to the configuration of the lipid layers was performed by the monte carlo simulated annealing method. at high enough temperature the lipid molecules attain a shape exhibiting higher intrinsic mean and deviatoric curvatures which fits better into the inverted hexagonal phase than into the lamellar phase. for the mathematical model the advanced geometry with non-spherical cross-section of inverted hexagonal phase was calculated, resulting in lower energy in non-spherical cross-section. theoretical results are in a good agreement with the small angle x-ray scattering experimental data. for a long while the conventional view has been that alzheimer disease is brought about by the beta-amyloid fibrils found in the senile plaques, but more recently it has been suggested that the main neurotoxic species would be the soluble oligomeric species, apparently prone to interact with cell structures and macromolecules potentially inducing neuronal dysfunction. peptide-peptide interactions resulting in self-assembly phenomena of beta-amyloid yielding fibrils can be modulated and influenced by small organic molecules that might also be effective therapeutic tools to ideally target both oligomeric and fibrillar species. in this perspective, polycyclic aromatic molecules are of special interest because they might disrupt the molecular architectures precursors of beta-amyloid fibrils by means of weak, non-covalent aromatic interactions, like stacking interactions. we have performed an in vitro spectroscopic study (light scattering, circular dichroism, ftir and fluorescence) of the effects on beta-amyloid fibrillogenesis of the natural pigment hypericin extracted from hypericum perforatum. our results show that, thanks to its structural characteristics and peculiar spectroscopic features, hypericin can be easily used to in vitro monitor the appearance of initial aggregation states of beta-amyloid peptides and, more importantly, that hypericin can interfere with the early stages of polymerization process, playing the role of an aggregation inhibitor. peptaibols are peculiar peptides produced by fungi associated to plants. they are composed by 4 to 20 amminoacidic residues and exhibit antibiotic and antifungal properties. due to their amphypatic nature, they can form ion channels in biological membranes. by making use of experimental models of biological membranes (biomimetic membranes) currently employed in the laboratory of bioelectrochemistry, and models of plant membranes (corn seed root), that are used in the international laboratory of plant neurobiology (linv), we characterized synthetic peptides such as trichogin gaiv and its shorter homologues (4 and 8 residues). we studied these peptaibols in a dioleoylphosphatidylcholine monolayer supported by hg using different electrochemical techniques (ac,vc,eis). the experimental technique employed in the linv (clark microelectrode coupled to mife system) allows to measure oxygen flux in the solution contacting plant cell membranes, after treatment with different peptide concentrations. preliminary results might indicate that short peptides can influence the whole metabolism of the plant and can therefore be used as "elicitors" in order to induce an acquired systemic resistance. supported biomimetic membranes (sbm) were developed for protein-membrane interactions studies. phospholipid vesicles were chemically linked onto amine grafted gold or glass surfaces; after an osmotic choc and liposomes fusion a continuous membrane bilayer was formed. the anchoring phospholipid molecule (dspe-peg-nhs) incorporated into the vesicles allowed the formation of a water-filled compartment between the surface and the bilayer. this first sbm model was used to monitor the membranes binding properties (dependent of calcium) of the adenylate cyclase toxin (cyaa) from bordetella pertussis. the sbm model was improved in order to study the translocation of the catalytic domain of cyaa across the bilayer. naturally, the cyaa catalytic domain, when it reaches the target cell cytosol, associates with intracellular calmodulin (cam) an activator of the adenylate cyclase activity of cyaa. to mimic this biological phenomenon, cam was first immobilized on the surface (gold or glass) and in a second step membrane construction was performed over the cam layer. the formation of the biomimetic membrane onto the cam layer was monitored by spr while membrane fluidity and continuity were analysed by fluorescence. our results demonstrated the potentialities of sbm for the study of protein insertion into and translocation across biological membranes. a multi-resolution approach to the structure and function of integrin αiibβ3 m. rocco 1 , c. rosano 1 , j. w. weisel 2 , d. horita 3 , r. r. hantgan 3 1 istituto nazionale per la ricerca sul cancro (ist), genova, italy, 2 university of pennsylvania, philadelphia, pa, usa, 3 wake forest university, winston-salem, nc, usa. integrins are heterodimeric transmembrane receptors involved in mechanical anchoring and two-way signaling. each α and β subunit has a modular structure with a large extracellular portion, a single transmembrane region, and a cytoplasmic domain. integrins activation mechanism is regulated by controversial conformational changes: while crystallography revealed similar bent shapes for resting and primed extracellular region constructs, ligand binding-induced large structural rearrangements in smaller constructs suggested extension, "opening" and tails separation. in a multiresolution approach, we used experimental and computed hydrodynamics to discriminate among αiibβ3 integrin models built on x-ray, nmr, and em data. in contrast with xray data and 3d em maps, an extension is needed to match the hydrodynamics of full-length, solubilized αiibβ3; an electron tomography-based model fares better. consistent with that, and with our averaged 2d em images, a conformational change in the head region (β3 hybrid domain swingout) coupled to a simple transmembrane helices shift matches priming agents-induced frictional changes in full-length αiibβ3. our multi-resolution study thus suggests that in integrins extension and immediate tail separation are uncoupled from head domain rearrangements following activation. -biomolecular self-assembly stabilizing effects of α s1 -casein, a natively unfolded protein, on the aggregation of biomolecules a. trapani, r. carrotta, p. l. san biagio, d. bulone ibf-cnr palermo, italy α s1 -casein is one of the four types of caseins, a group of calcium phosphate-binding proteins that, in the form of micellar aggregates, makes up the largest protein component of bovine milk. the structure of α s1 -casein is that of a triblock polymer with a hydrophilic tract interposed between two hydrophobic blocks. due to the lack of a compact folded conformation, this protein can be classified as one of the intrinsically disordered (or natively unfolded) proteins. this class of proteins is known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces that partially unfolded molecules may expose to the solvent. here we present results on α s1 -casein effects on the thermally induced aggregation of gluthathione stransferase, a ligand-binding anzyme, and 1-40 β-amyloid peptide involved in alzheimer's disease. by means of light scattering and circular dichroism experiments, we attempt to reveal the molecular details of α s1 -biomolecules interaction. bacterial protein self-assembly on surfaces of well-defined chemistry s-layers are one of the most common cell envelope components of prokaryotic organisms and represent the simplest biological membrane developed during evolution. these (glyco)proteins, which can self-assemble into 2-d crystalline nanostructures on lipid films, liposomes, and polymers, play already an important role in nanobiotechnology. in this work, we present new findings concerning the recrystallization of bacterial proteins on substrates with defined chemistry. manipulation of the protein-sample interaction was carried out by changing the relative height of oh and ch 3 terminated moieties in self-assembled monolayers (sams). we have found that differences in chain length lead to: i) protein bilayer-protein monolayer transition, ii) preferential protein side adsorption, and iii) increase of the crystal lattice parameters. further manipulation of the protein-sample interaction was achieved by using silane chemistry. we will show that sample hidrophobicity speeds up recrystallization kinetics and reduces the crystalline domain size (and layer compliance). the protein-adsorbed mass per unit area on these substrates is reported for the first time. self-assembly of phenylalanine oligopeptides: insights from experimental and computational studies p. tamamis 1 , l. adler-abramovich 2 , m. reches 2 , k. marshall 3 , p. sikorski 3 , l. serpell 3 , e. gazit 2 , g. archontis 1 1 dpt. of physics, univ. of cyprus, cyprus, 2 dpt. of molecular microbiology and biotechnology, tel aviv univ., israel, 3 dpt. of biochemistry, univ. of sussex, u.k. the diphenylalanine peptide (ff) forms well ordered nanotubes and its derivatives form nano-assemblies of various morphologies with promising material applications. we demonstrate for the first time by electron and laser microscopy and ftir spectroscopy that the related, triphenylalanine peptide (fff) assembles into rather planar nanostructures, rich in β-sheet. in addition, we conduct 0.4-µs replica exchange m.d. simulations of aqueous ff and fff solutions in implicit solvent. the peptides coalesce into aggregates and participate frequently in open or ring-like linear networks, as well as elementary and network-containing structures with β-sheet characteristics. polar and nonpolar interactions, as well as the surrounding aggregate medium contribute to the network stabilities. within a network, consecutive peptides are linked by head-to-tail interactions; the aromatic sidechains of neighbor peptides assume approximately "t-shaped" orientations. these features are observed in ff crystals and could characterize early formations, or stabilize the mature nanostructures. the fff aggregates acquire higher stability and peptide-network propensity compared to the ff aggregates due to energetic contributions 1,2 . chlorophyll biosynthesis is light-dependent in angiosperms because the reduction of protochlorophyllide (pchlide) into chlorophyllide is driven by a photoenzyme, nadph:pchlide oxidoreductase (por). the unique properties of por are due to its ability to assemble into dimers and oligomers within the prolamellar body (plb) membranes of etioplasts studied mainly in leaves of dark-grown seedlings under laboratory conditions. we extended these studies to plant organs developed in the nature: cabbage heads, leaf primordia inside buds, pericarp-covered regions of sunflower cotyledons, potato tubers and seedlings germinating under the soil. in electron microscopic and fluorescence spectroscopic studies we found in many of these organs poorly developed plbs in which por was mainly in monomer state. as a consequence, the chlorophyll accumulation was slow and photo-oxidation processes occurred at illumination. in vitro we artificially induced the aggregation of por monomers into oligomers in glycerol and sucrose containing buffers. this resulted in the increase of the photoreduction rate at the expense of photo-oxidation. these results underline the importance of the self-assembly of por and the plbs in chloroplast development and chlorophyll synthesis in nature. v. vetri 1 , g. ossato 2 , v. militello 1 , m. a. digman 2 , m. leone 1 , e. gratton 2 1 dsfa, university of palermo, palermo, italy, 2 lfd, university of california, irvine, ca, usa we report an experimental study on concanavalina (cona) aggregation in live cells. in vitro, close to physiological temperature, cona readily forms fibrils involving secondary structure changes leading to β-aggregate structures. the effect of cona on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. in particular, we monitored protein aggregation in live cells by means n&b analysis, cross-n&b and rics. n&b showed the aggregation kinetic and the progressive formation of cona oligomers at cell surface. this suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. in parallel, the morphology of the cells changes indicating the progressive cell compaction and death. aggregation and binding of small aggregates to the cell surface were assessed by rics: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. oligomers formation may stimulate non-specific cellular responses due to the exposure of reactive regions of protein structure and of progressive formation of cross−β structures. moreover, aggregates stoichiometry was measured during the kinetic by cross-variance n&b. the two conductive pathways of p2x7 purinergic receptor: different modulation and selectivity r. barbieri 1 , s. alloisio 1 , a. di garbo 2 , m. nobile 1 1 institute of biophysics, cnr, via de marini 6, 16149 genoa, italy, 2 institute of biophysics, cnr, via g. moruzzi 1, 56124 pisa, italy the p2x 7 purinoceptor (p2x 7 r) is an atp-gated cation channel that is able to activate a cell permeabilizing pore. p2x 7 r cytosolic c-terminal tail is thought to modulate this function. this study was aimed to characterise the biophysical properties of p2x 7 r compared to those of the variant lacking the entire c-terminus tail (trp2x 7 r) by measuring whole-cell currents and intracellular ca 2+ variations. a mathematical model is used to describe the experimental results. in p2x 7 r expressing hek-293 cells, the potent agonist 3'-o-(4-benzoyl)benzoyl adenosine 5'-triphosphate (bzatp) -evoked ionic currents depending on concentration and frequency of agonist applications. the currents were strongly inhibited by extracellular mg 2+ in a noncompetitive way. by contrast, in trp2x 7 r cells, only high bzatp concentrations elicited small currents not affected by mg 2+ . interestingly, bzatp-induced ca 2+ influx was present both in p2x 7 r and in trp2x 7 r cells, albeit in the latter the intracellular ca 2+ elevation was smaller. importantly, in trp2x 7 r the intracellular ca 2+ rise maintained a competitive mechanism of mg 2+ inhibition similar to that observed in p2x 7 r. the experimental data and the modelling findings support the tenet of a functional structure of p2x 7 r possessing two distinct conductive pathways. the review of our data on the effects of physical and chemical weak signals on physicochemical properties of water, cell volume, activity and the number of membrane proteins (receptors, ionic channels and enzymes, na + /k + pump and na + /ca 2+ exchanger), intracellular signal systems in norm and pathology (cancer and nerve disorders) would be presented. light microscopic, cell voltage-and patch-clamp, isotope, standard biochemical and genetic engineering methods were used. weak signal-induced effects on cell functional activity (intracellular enzymes activity, the number of functionally active membrane proteins) are realized by changing the physicochemical properties of extra-and intracellular aqua medium. the latter induces the modulation of na + /k + pump-induced cell hydration, which serves as a primary mechanism through which the autoregulation of pump and regulation of membrane excitability and chemosensitivity are realized. by genetic engineering method in oocytes it was shown that the correlation between na + /k + pump and na + /ca 2+ exchanger, which is realized through intracellular messenger systems, plays a crucial role in weak signals transduction in cells and determination of aging-induced increase of cell pathology. listeriolysin pore forming ability in planar lipid membranes at different ph listeriolysin o (llo) is a cholesterol-dependent cytolysin secreted by the intracellular pathogen listeria monocytogenes. its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. llo exhibits optimal cytolytic activity at low ph but it is still able to bind membranes at physiological or even slightly basic ph values in a cholesterol-dependent fashion. high cholesterol concentrations in the membrane restore the low activity of llo at high ph values. based on this broad ph activity we investigated the electrophysiological properties of pores formed by llo at room temperature and at different phs using planar lipid bilayer technique. llo is able to form pores both at ph 5.5 and 7.5 with a similar permeabilizing ability and similar heterogeneous conductances in the range of picosiemes to nanosiemens. cholesterol content directly correlates with llo activity but it does not change the pore characteristics. collectively, our results demonstrate that llo activity at physiological ph cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for listeria pathogenesis. s. aimon, g. toombes, p. bassereau institut curie, paris, france the physics of membrane/channel and channel/channel interactions is difficult to investigate in cells where it is nearly impossible to modify relevant parameters to deduce physics laws. to overcome these difficulties we built a model system in which voltage gated ion channels were reconstituted in giant unilamellar vesicles (guvs) for the first time. as a first step, we successfully expressed kvap (a voltage gated potassium channel) in e-coli. the channel was purified, fluorescently labelled and reconstituted in small liposomes. its functionality was checked with electrophysiology via fusion of these liposomes into black lipid membranes (blm). as a second step, guvs were formed from these small proteoliposomes using electroformation in a buffer containing 100 mm kcl salt. the proper incorporation of proteins into guvs was controlled using confocal microscopy. functional proteins were detected using the patch clamp technique. with our protocol, we are thus able to prepare guvs containing functional voltage-gated ion channels. one goal is now to study the effect of channel activity on its spatial distribution in these guvs. ryr activation in cultured shr cardiomyocytes at the end of the prehypertensive period the rate of [ca 2+ ] i elevation after the ryanodine receptor (ryr) activation by 4-chloro-m-cresol (4-cmc) and l-type ca 2+ channels (dhpr) activation by bay k8644 was studied in cultured (5 days) cardiomyocytes of spontaneously hypertensive (shr) and normotensive rats (wky, wistar) during 6 weeks of postnatal development. the differences in dh-prs and ryrs activities began to be evident after 3 weeks age when cicr formation has finished and became more expressed at the end of prehypertensive period (6 weeks). in response to 4-cmc (2 mm), a drastic increase in the rate of [ca 2+ ] i accumulation (2.85±0.8 times) in shr myocytes was registered after 20 days age versus a decrease in the rates of ca 2+ efflux from the sarcoplasmic reticulum of wistar and wky rat cardiomyocytes. bayk (80 µm) also induced more sharp [ca 2+ ] i elevation in shr myocytes (3.35±0.25 times) as compared with wistar (2.32±0.16 times) and wky (1.22±0.09 times) ones of the same age. our results argue that in shr and wky cardiomyocytes, as opposed to normotensive wistar rats, gradual growth of dhpr activity is observed, which follows in parallel with cicr formation in the excitation-contraction coupling during early postnatal ontogenesis, and drastic activation of ryr2 in shr myocytes after the process termination. cytochrome ba 3 from thermus thermophilus belongs to the large family of structurally related heme-copper oxidases. it accepts electrons from cytochrome c 552 at the p-side of the membrane and uses them to reduce oxygen to water. the energy released in this reaction is used for proton pumping across the membrane to form of an electrochemical proton gradient, used by the cell for formation of atp. in this work we followed the kinetics of single-electron injection into the oxidized nonrelaxed state (o h →e h ) of cytochrome ba 3 by time-resolved optical spectroscopy. two main phases of electron transfer were resolved. the first (τ∼17 µs) includes oxidation of cu a and simultaneous reduction of both low and high spin hemes. the second (τ∼420 µs) reflects reoxidation of both hemes by cu b . this is in significant contrast to the o h →e h transition of aa 3 -type oxidases, where the fastest phase is due to transient reduction of the low-spin heme a only. on the other hand, the single-electron reduction of the relaxed o state in ba 3 oxidase consisted of only rapid electron transfer from cu a to heme b, which is similar to that in aa 3 oxidase. this indicates a functional difference between the relaxed o and the pulsed o h states of cytochrome ba 3 . as opposed to the phospholamban pentamer, sarcolipin forms anion-selective channels in biomembranes l. becucci 1 , c. b. karim 3 , d. d. thomas 3 , g. veglia 2 , r. guidelli 1 1 chemistry department, florence university, 50019 florence,italy, 2 chemistry department, university of minnesota, minneapolis, mn 55455, 3 3department of biochemistry, molecular biology, and biophysics, university of minnesota, minneapolis, mn 55455 sarcolipin (sln) and the phospholamban pentamer (pln) are two membrane proteins that inhibit ca-atpase of the sarcoplasmic reticulum at low concentrations. in contrast to pln, sln stimulates maximal ca 2+ uptake rates. sln and pln were incorporated in a bilayer lipid membrane (tblm) tethered to a mercury electrode through a hydrophilic spacer. electrochemical impedance spectroscopy measurements show that sln forms channels permeable to chloride ion, weakly permeable to phosphate ion and impermeable to inorganic cations such as na + and k + . a relationship between this property of sln and its regulatory function on ca-atpase of sarcoplasmic reticulum is proposed. atp increases the permeability of a tblm incorporating sln to phosphate ion by associating to sln with an association constant of 0.1 µm. an explanation for this behavior is provided. sln can be identified with the " p i transporter" described by a.g. lee et al. conversely, both electrochemical impedance spectroscopy measurements and molecular dynamics simulations provide strong evidence that the pore of the pentameric form of pln does not act as a chloride channel. "social" domain organization and dynamics of nicotinic acetylcholine receptor at the cell membrane f. j. barrantes unesco chair biophys. & mol. neurobiology. univ. nac. sur, 8000 b. blanca, argentina a combination of experimental techniques (patch-clamp, confocal frap and fcs, single-particle tracking, highresolution fluorescence microscopy) has been used to analyze the supramolecular organization of the acetylcholine receptor (achr), the dynamics of the receptor at the cell surface, and the kinetics of receptor internalization. changes in cholesterol (chol) content affected muscle and neuronal-type achr organization and dynamics at the cell surface. chol depletion produced gain-of-function of single-channel dwell time. submicron-sized (∼250 nm) domains, stable over a period of hours at the cell membrane, could be resolved into achr "nano-clusters" with a peak size distribution of ∼55 nm by sted microscopy. chol depletion reduced the number of nanoclusters, increasing their size, and changed their supramolecular "social" organization on larger scales (0.5-3.5 microns). frap, fcs and spt experiments provided information on the dynamics of achr nanoclusters, disclosing the dependence of their mobility on chol content and cortical cytoskeleton. chol content at the plasmalemma may thus modulate cell-surface organization and dynamics of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute achr losses from the cell surface. m. czaplinska, k. gwozdzinski, a. koceva-chyla division of research of structure of biopolymers university of lodz, lodz, poland doxorubicin (dox) and paclitaxel (ptx) are anticancer drugs commonly used in chemotherapy of breast cancer therapy, however, the use of these drugs is limited by the risk of developing heart failure. generation of reactive oxygen species contributes to the cardiotoxicity of doxorubicin. nitroxides are low molecular weight, stable free radicals reacting with ros and they present antioxidant properties. the aim of this study was to analyze the effects of pirolid (pd) on the oxidative stress induced by dox and taxane in mcf-7 breast cancer cell line. results from mtt test revealed that ptx is more cytotoxic towards mcf-7 cells than dox. the ic 50 was 0.3µm and 3µm, respectively. pd alone does not influence cell viability. pd in combination with both drugs did not change viability of cells. both drugs increased the level of carbonyl groups in cells. the highest level of peroxide was observed in cells incubated with dox (approx. 3-fold). nitroxide alone did not influence the level of peroxide in the whole range of concentrations. combination of pd with dox and ptx reduced the level of carbonyls depending on its concentration. pd did not affected on the level of peroxide in cells suspension. dox and ptx increased (2-fold) the level of carbonyls. pd decreased the level of peroxides in cells treated with dox and ptx. the lowest concentration of peroxide was observed at 50µm of pd. these results show that pd protect mcf-7 cells against oxidative stress induced by drugs. open channel structure of mscl from fret microscopy and simulation mechanosensitive channels open in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing and osmoregulation. here, we have determined the likely structure of the open state of the mechanosensitive channel of large conductance from e. coli (mscl) in a natural environment using a combination of patch-clamp studies, fret spectroscopy, epr data, molecular and brownian dynamics simulation. structural rearrangements of the protein are measured while controlling the state of the pore by modifying lipid bilayer morphology. fret efficiency changes can be related to distance changes using a monte carlo analysis program in conjunction with detailed orientational analysis. these measurements are used as restraints in all atom molecular simulations in order to determine the likely structure of the open state, whose probable conductance is derived from brownian dynamics simulations. transition to the open state occurs via large rearrangements throughout the protein that create a wide pore nearly 30 a in diameter. both transmembrane helices are found to line part of the pore. the n terminal helix is found to lie along the face of the membrane where it can act to sense membrane tension and directly transfer this to the pore lining helices. the method of coupling spectroscopic data with simulations is likely to be of great value for studying conformational changes in a range of membrane proteins. putative potassium channels in synechocystis sp. pcc 6803 v. checchetto, m. zanetti, g. m. giacometti, i. szabò, e. bergantino department of biology, university of padova, italy we are interested in the identification and characterization of potassium channels in the cyanobacterium synechocystis sp. pcc 6803, an organism which is considered the ancestor of plant chloroplasts. a bioinformatic screening of synechocystis proteome identified, among others, two proteins on which we focused our attention. the first one (syncak) displays sequence homology to mthk, a ca 2+ -dependent potassium channel from m . thermoautotrophicum. the second one (synk) is predicted to contain six transmembrane regions and the typical selectivity filter of all potassium channels. our goal is to understand their roles in the physiology of cyanobacteria. we cloned their coding sequences in fusion with gfp and the hybrid proteins were expressed in chinese hamster ovary cells. we evaluated the presence of both proteins in plasma membrane by fluorescence microscopy and then we proceeded to their functional characterization using patch clamp technique.this analysis will allow us to gain information about channel activity, regulation and pharmacology. we also plan to evaluate the importance of syncak and synk channels in photosynthesis. to test the hypothesis that they could be involved in regulating this process, we will produce deletion and site-specific mutants in synechocystis. finally we would also identify the homologous of these channels in the higher plant a. thaliana and obtain some information about their localization and function. j. braunagel max-planck institute for polymer research, ackermannweg 10, 55128 mainz, germany the cyclododecadepsipeptide valinomycin is composed of two amino acids (l-valine and dvaline) and two hydroxyl acids (d-α-hydroxy-isovaleric acid and l-lactic acid). they form a 36membered ring of alternating amino and hydroxyl acids. in the cyclic structure, the polar groups are oriented towards the central cavity, whereas the rest of the molecule is relatively nonpolar. this enables the complexation of ions and valinomycin acts as a selective ion transporter (k + ) in lipid membranes. when one of the amino acids is exchanged, e.g one l-valine by an l-lysine, selected functionality can be engineered into the depsipeptide while maintaining its ion conducting properties. we induced several modifications into valinomycin, i.e. a biotin binding unit or a ferrocene group to induce an electrochemical active center. the ion conducting properties of the modified ion carriers have been probed in planar lipid bilayers as well as in solid supported membranes. the role of the membrane dipole potential (ϕ d ) is of a particular interest due to a powerful impact of this potential on the membrane permeability and lipid-protein interactions. channel forming activity of gramicidin a, alamethicin, syringomycin e, hpa3 peptide, and ompf porin are influenced by ϕ d . we have studied the effect of the membrane dipole modifier, phloretin, on the properties of single channels formed by a wild-type alpha-hemolysin in planar lipid bilayers. the single channel of a ∼1000 ps conductance exhibits transitions into a number of low-conductance states as the transmembrane voltage exceeds ∼130 mv (regardless of the voltage polarity). the phloretin addition to the bathing solutions (20 µm) (after the hemolysin channel was formed in the membrane) shifts dramatically the channel voltagedependence. transitions to the low-conductance states are observed at ∼30 mv. the effect of the phloretin addition was not observed in the case when it was introduced into the bathing solution before alpha-toxin. since phloretin reduces ϕ d , the data may report on the influence of the electric potential profile on the energy of the low-conductance state of the alpha-hemolysin channel. the alternative explanation of this effect consists in a specific interaction between the phloretin and toxin channel. the work is supported in part by rfbr (# 09-04-00883), ss (# 1135.2008.4) , and the program of the ras «molecular and cell biology». atypical mechanism of conduction in potassium channels c. domene 1 , s. furini 1 1 physical and theoretical chemistry laboratory, department of chemistry, university of oxford, oxford, u.k., 2 department of electronics, computer science and systems, university of bologna, bologna, italy potassium channels can conduct k + ions with rates of up to ∼ 10 8 ions per second at physiological conditions, and they are selective to these species by a factor of 10 4 over na + ions. ion conduction has been proposed to involve transitions between two main states, with two or three k + ions occupying the selectivity filter separated by an intervening water molecule. the largest free energy barrier of such a process was reported to be of the order of 2-3kcal mol −1 . here, we present an alternative mechanism for conduction of k + in k + channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of k + -water-k + . we exploited the au-deposited self-assembled monolayers of the type: [−s−(ch 2 ) n −ch 3 ] (where n = 4, 9 and 15) with hydrophobically adsorbed redox protein -azurin to verify intrinsic electron transfer mechanisms according to the charge-transfer theory. the enthalpies and volumes of activation were determined through the variation of temperature (2-50 o c) and pressure (5-150 mpa) and experimental values were compared with those expected on the theoretical grounds. for the case of n = 4 the activation enthalpy definitely contains a large contribution originated from frictional-like dynamics of protein, and the activation volume has a small positive value. for n = 15 the value of activation enthalpy directly matches 1/4 of that for the reorganization energy, and the activation volume attains substantially negative value. for n = 9 we observed the intermediary performance. the whole kinetic pattern is consistent with the smooth changeover between adiabatic and nonadiabatic mechanisms of electron transfer. two gating modalities in the pore of the miniature k + channel kcv kcv is a viral protein that forms functional k + channel in heterologous systems. because of its miniature size (94 amino acids) we use kcv as a model system to study and manipulate basic properties of the k + channel pore. by analysing single-channel recordings we highlighted two voltage-dependent modalities of gating in kcv: a slow and a fast gating. the presence of a slow gating is revealed by the very low (in the order of 1-3%) mean open probability. slow gating is not related to the presence of a bundle crossing, as shown by accessibility of the cavity to mts reagents. channel opening might involve the transient formation of salt bridges between residues at the n and c termini of the channel, as suggested by mutational experiments inspired by molecular dynamics simulations of kcv. fast gating, analyzed by beta distributions, is responsible for the negative slope conductance in the single-channel i/v curve at extreme potentials and can be explained by depletion-aggravated instability of the filter region. aca8 is a type 2b caatpase with a regulatory n-terminus whose autoinhibitory action can be suppressed by binding of calmodulin (cam). aca8 n-terminus is able to bind a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. to define the role of this interaction in autoinhibition we have analysed a number of single point mutants produced by mutagenesis of aca8 e252-n345 sequence. mutation to ala of any of 6 acidic residues (e252, d273, d291, d303, e302, d332) originates an enzyme with normal activity in the presence of cam, but less camstimulated. these results highlight the relevance of a negative charge of the surface area of the small cytoplasmic loop in aca8 autoinhibition. the most deregulated mutant is d291a aca8, which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the d291a mutant is stronger than that of d291n aca8 suggesting a more direct involvement of this residue in autoinhibition. of the other mutants (i284a, n286a, p289a, p322a, v344a, n345a), only p322a aca8 has a basal activity higher than that of the wt. these results provide the first evidence that the small cytoplasmic loop of a type 2b caatpase plays a role in the attainment of the autoinhibited state. complex i, the first member of the respiratory chain, serves as a proton pump catalyzing transfer of two electrons from nadh to ubiquinone coupled with the translocation of four protons across the membrane. so far the mechanism of energy transduction by complex i is unknown. the nadh-binding cavity of complex i has a very prominent feature -the presence of two invariant amino acid residues, glutamate and tyrosine, that are exposed to the solvent and located in the vicinity of the fmn, the primary electron acceptor in the enzyme. it was suggested that they might be involved in the binding of nadh through interaction with its nicotinamide moiety. in this work we assessed the function of corresponding glu95 from the nuof subunit of e. coli complex i by mutation for glutamine. we showed that the negative charge of glutamate in the catalytic site is needed for the electrostatic repulsion of negatively charged phosphates of nucleotides. this process facilitates release of the product nad + and, as a result, accelerates turnover of complex i. we also found that glutamate, as one of the four negatively charged amino acid residues surrounding the isoalloxazine ring of the fmn at a distance of 4-6å, has a share of 40 mv of the overall 130 mv depression of the midpoint potential of this redox cofactor. l. erokhova 1 , p. kügler 2 , p. pohl 1 1 institute of biophysics, johannes kepler university, linz, austria, 2 ricam, austrian academy of sciences, linz, austria the mechanism of water transport through epithelia is still under debate. in the present work we tested the hypothesis of isosmolal water transport using mdck cells stably expressing the human sodium-glucose cotransporter (hsglt1). tagging hsglt1 with egfp enabled determination of its abundance in the plasma membrane by fluorescence correlation spectroscopy (fcs). by monitoring tiny shifts in the concentration of water-soluble dyes in the vicinity of epithelia, fcs also allowed assessment of the water fluxes through confluent cell monolayers grown on permeable supports. fitting the set of differential equations for the osmotic drift and for the back diffusion to the experimentally determined dye distribution permitted calculation of water flow both in the presence and in the absence of an osmotic gradient. from the calorimetric measurements of glucose transported across the cell monolayer, the water:glucose stoichiometry was derived. dividing the increment in osmotic water flux due to hsglt1 expression by the number of hsglt1 copies in the plasma membrane resulted in a single transporter water permeability p f of 4.6 x 10 −14 cm 3 /sec. thus, p f is close to the single channel water permeability of aquaporin-1. consequently, even small osmolyte concentration differences between the cytoplasm and the basolateral buffer solution are sufficient to drive a substantial water flux. m. emre, s. kavak cukurova university school of medicine, department of biophysics, balcali-adana, turkey experimental studies have shown that the at1-receptor antagonists telmisartan (tel) has a ppar-activating property, but there does not appear to be a class effect. to test telmisartan's importance, we investigated its effect on electrical activities (ea) in diabetic (d) rats. the purpose of this study was to investigate the effects of the tel on ea of diabetic papillary muscle (dpm) with stz-induced. in this study, we used four groups: (1) nondiabetic control (ndc) group (c), (2) tel-treated ndc group (c+tel), (3) diabetic group (d), and (4) tel-treated d group (d+tel). diabetes was induced by a single i.v injection of stz. in the study, membrane potential (mp) and action potential (ap) recorded after the establishment of diabetes. 1) mp was decreased significantly in both tel-treated c and d rats (from -71,4±0,7 to -64,7±0,8 mv and from -69,9±0,6 to -63,8±0,7 mv). 2) ap unchanged in d group, whereas c+tel and d+tel groups showed increase in ap compared with c and d groups. 3) repolarization time was prolonged in diabetic rats. 4) in c+tel and d+tel groups depolarization rate values increased significantly. on the other hand, in d group repolarization rate values descreased increased significantly compared to baseline values in tel solution. as a result, our data suggest that the beneficial effects of tel-treatment on the ea of the dpm appear to be due to the diminished k + currents. cytochrome c oxidase is a terminal complex (cco, complex iv) of a respiratory chain that is located in an internal membrane of mitochondria or plasma membrane of bacteria. cco is an electron transfer enzyme that reduces o 2 and uses the redox energy of the o 2 reduction for the proton translocation across the membrane. the electron-and proton-transfer generate a transmembrane electrochemical gradient (∆µh + ) that is used for atp synthesis and all other kinds of work for the cell needs. the proton translocation mechanism of cco requires 'channels' for the h + uptake and expulsion within the enzyme. the proton transfer occurs on a time-scale of micro-to-milliseconds. in order to study the proton transfer in cco, a flow-flash approach based on a time-resolved ftir spectroscopy was developed and applied. our ftir flow-flash approach (the measurement of the reaction of cco with o 2 ) allows to reach a time resolution up to tens milliseconds. with this approach and site-specific mutants of cco where the catalysis is slowed down, separate steps of the proton transfer were studied. the results showed that a unique cross-linked tyr-280 (in a combination with a time-resolved visible spectroscopy and electrometry) serves as a proton donor for the dioxygen bond cleavage during the o 2 reduction by cco. furthermore, the protolytic transitions of glu-278 -a key amino acid in the proton transfer mechanism in cco -were shown for the first time. role of calcium ions in nickel potentiation of nmda currents p. gavazzo, i. zanardi, p. guida, c. marchetti biophysics institute ,cnr, genova, italy nmda receptors are glutamate-gated channels distributed throughout the brain in the excitatory synapses and are critical for the nervous system function. they are assembled from two types of subunits, the essential nr1 and at least one nr2(a,b,c,d). nickel (ni) modulates the current flowing through nmda receptors in a different way depending on the nr2 subunit present. we have recently identified several domains of the channel involved in ni interaction, but many aspects of this modulation remain elusive. in this work we intended to determine the role of calcium (ca) ions in the potentiation induced by ni on the current through nr1/nr2b recombinant nmda receptors. when ni was applied in the presence of the physiological concentration of ca (1.8 mm) , a voltage-independent potentiation of the current was observed with a kp of 2.5 µm. this effect was progressively reduced by decreasing ca concentrations and it was no more detectable with 0.18 mm ca or in the presence of barium (ba, 0.3 mm). in this last case the effect of ni on nr1/nr2b receptors was mainly inhibitory (ki(-60mv)=156µm). therefore a physiological concentration of ca is necessary to induce ni amplification of the current. many data in the literature indicate a correlation between ca ion entrance through the channel, nmda current facilitation and cytoskeleton; however, in our experiments, the application of the actin perturbing agent cytochalasin-d did not produce major modifications in ni effect. heteromerization properties of voltage dependent potassium channels f. gambale 1 , l. pedemonte 1 , a. naso 1 , i. testa 2 , c. usai 1 , a. diaspro 2 , c. picco 1 1 istituto di biofisica, cnr, genova, italy, 2 lambs-microscobio, department of physics, genova, italy voltage-gated potassium channels are either homomeric or heteromeric tetramers composed of four α-subunits. in order to bring a contribution to the comprehension of channel heteromerization we have been investigating the properties of two plant voltage-gated k + -channels by using electrophysiological and fluorescence techniques. experiments were focussed on kdc1 and kdc2, coexpressed in xenopus laevis oocytes. kdc1, the first potassium channel cloned from daucus carota, belongs to the subfamily of α-modulatory silent channels as it doesn't form functional homomeric channels by itself. on the contrary kdc1 forms functional heteromeric channels when coexpressed with homologous subunits. kdc2, the last k + channel cloned from d. carota, belongs to the kat1 family and shares an overall identity of 63% with kat1. to correlate kdc1 functional properties with its localization in oocytes, kdc1 and/or kdc2 subunits were labelled with gfp and their properties investigated by confocal microscopy and voltage-clamp. we found that the kdc1-egfp fusion protein is not targeted to the plasma membrane unless it is coexpressed with kdc2. moreover electrophysiological experiments demonstrated that the heteromeric kdc1-kdc2 channel has altered selectivity and activation properties with respect to homomeric kdc2 channel. circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome (ards) [1] . we present a microfluidic investigation of the roles of actin organization and myosin ii activity during the different stages of leukocyte trafficking through narrow capillaries using specific drugs. the deformation rate during entry reveals that cell stiffness depends strongly on f-actin organization and hardly on myosin ii activity, supporting microfilament role in leukocyte sequestration. in the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. conversely, membrane unfolding was independent of leukocyte stiffness. the surface area of sequestered leukocytes increased by up to 160% in absence of myosin ii activity, showing the major role of molecular motors on microvilli wrinkling and zipping. finally, cell shape relaxation was largely independent of both actin organization and myosin ii activity, whereas a deformed state was required for normal trafficking through capillary segments [2] . [1] g.s. worthen et al., science, 245, 183-186 (1989) excitation-contraction coupling in skeletal and cardiac muscle is tightly regulated by the calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor (ryr). we could previously show that suramin is a potent activator of the ryr via the calmodulin binding site. calmodulin shows dualistic action, i.e. activation or inhibition of the ryanodine receptor, depending on the absence or presence of ca 2+ . screening of suramin analogues identified nf676 as a use-dependent inhibitor of the skeletal muscle ryr (ryr1). here we show that nf676 inhibits high affinity [ 3 h]ryanodine binding and single channel recordings of the purified ryr1. nf676 induced a reduction of open probabilities in a concentration dependent manner, with no effect on current amplitude and unitary conductance. importantly, nf676 triggers flickering episodes of channel openings and closings before the ryr1 is frozen in a complete non-conducting state, which is fully reactivated by the ryr agonist atp. moreover, zwitterionic behaviour of nf676 facilitates plasma membrane permeation, which prevented caffeine induced ca 2+ transients in skeletal muscle cells and cardiomyocytes. conversely, ip 3 mediated ca 2+ signals were not altered by nf676. this work was supported by herzfelder'sche familienstiftung and fwf . beyond steady-state protein dynamics t. hauß 1 , j. pieper 3 , a. buchsteiner 1 , r. e. lechner 2 , n. a. dencher 2 1 helmholtz-zentrum berlin für materialien und energie, berlin, germany, 2 technische universität darmstadt, germany, 3 technische universität berlin, germany to study protein dynamics beyond steady-state experiments we have developed a novel laser-pump:neutron-probe experiment which allows us to monitor temporal changes in protein dynamics during a working cycle of a protein. protein dynamics has been extensively studied, but so far, the correlation of internal protein dynamics with the function of proteins was investigated only indirectly in steady-state experiments by variation of external parameters by variation of external parameters like temperature or hydration. the method comprises of an in-situ optical activation of a protein and a time-dependent sampling of the dymamic response using quasi-elastic neutron scattering. with the membrane protein bacteriorhodopsin, a light driven proton pump, we can demonstrate for the first time temporary alterations in the protein dynamics after triggering the working cycle. this observation is a direct proof for the functional significance of protein structural flexibility, in connection with the largescale conformational changes in the protein structure occurring during the operation of a "molecular machine". the slow vacuolar (sv) channels are ubiquitous in all tissues of higher plants. the sv channel is a non-selective cation channel permeable to both monovalent and divalent cations. sv currents recorded in a typical patch-clamp experiment require unphysiologically high cytosolic and low vacuolar calcium concentrations for full activation. we aim at looking for endogenous plant substances which might be able to modify or shift the voltage activation threshold of this channel towards more physiological conditions. flavonoid naringenin [nar] is present in all plant species where it plays a central role in the flavonoid biosynthetic pathway. nar is stored in the vacuoles in glycosylated form called naringin. when nar was added to cytosolic bath solution, we recorded a dose-dependent reversible decrease in sv channel activity. when we investigated the effect of nar on the voltage dependence of the channel, we observed that the activation threshold of the sv channel is shifted towards more positive voltages. our group has evidences that approximately 10% of the total sv current at high (e.g.> 50 mv) positive voltages is mediated by calcium. therefore, in order to verify whether nar affects both potassium and calcium conductance, we performed experiments by combining the patch clamp technique with fluorescence measurements using the fluorophore fura-2: both sv currents and calcium signals were abolished by 1 mm [nar]. determination of calcium currents in cation channels using a novel fluorescence/patch-clamp approach p. v. k. gutla, a. gradogna, a. carpaneto istituto di biofisica, consiglio nazionale delle ricerche, via de marini 6, 16149 genova, italy the patch-clamp technique combined with fura-2 fluorescence detection is suitable to investigate calcium fluxes. we used the excised patch configuration and focused the photomultiplier to the tip of the recording pipette where the fluorescent dye was present (fluoresence combined with excised patch = flep). this configuration has several advantages, i.e. absence of delay in loading the fluorophore, of interference by endogenous calcium buffers and of photobleaching. here we present an application for the determination of fractional calcium currents (pf) in a plant non-selective cation channel, showing that pf can be modulated by cytosolic calcium and potassium. flep is very efficient for measuring small calcium currents (<1 pa) of sufficiently long duration; fluorescence signals are amplified by integration in time, as the calcium/fura-2 complex accumulates at the tip of the recording pipette and diffuses slowly. we propose this technique not only for the study of calcium transport pathways, but also for other transporters of divalent cations as nickel and manganese known to quench fluorescence thus reducing both 340 and 380 nm components. moreover, using the appropriate fluorophore the technique may be extended to further ion species, e.g. bcecf for investigating proton transport pathways. ref: we introduced an original method for the monitoring of the changes in the electrostatic surface potential, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. this approach opens new way to understanding the dynamic processes within the proteins. our experiments revealed that the conformation of the na + /k + -atpase large cytoplasmic loop (c45) in the presence of the atp (without magnesium) substantially differed from the conformation in the presence of mg 2+ or mgatp or in the absence of any ligand not only in the sense of geometry but also in the sense of the electrostatic surface potential. moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. this property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain. influx of antibiotics into the periplasm of gram-negative bacteria is facilitated by porins that form channels in the outer membrane. we propose that certain natural antibiotics have been optimized by co-evolution to take advantage of the charge distribution in non-specific porins to achieve binding and thereby facilitating their uptake in bacteria. we investigate the permeation pathways of antibiotics into bacteria by reconstitution of a single porin into an artificial lipid bilayer and measuring the binding of antibiotic molecules through the time-resolved modulation of a small ion current. we have been able to characterize facilitated translocation of several antibiotics through escherichia coli and enterobacter aerogenes porins. noise analysis of ion currents through a porin in the presence of effective antibiotics revealed binding kinetics at a single molecule level. we report for the first time temperature dependent antibiotic translocation that revealed complete energy profile. combining these results with microbiological assays and molecular dynamics simulations, we conclude the molecular mechanism of antibiotic permeation. our approach may contribute to the rational design of new antibiotics against clinical bacterial strains for the most efficient delivery to target sites. mitochondria regulate ca 2+ influx and determine patterns of er ca 2+ refilling in acinar cel o. kopach, i. kruglikov, t. pivneva, n. voitenko, n. fedirko bogomoletz institute of physiology, kiev, ukraine store-operated ca 2+ entry (soce) is mediated by activation of soc-channels of plasma membrane following the emptying of endoplasmic reticulum (er) ca 2+ stores. the soce is required for calcium signaling, secretion of neurotransmitters and proteins, but the mechanisms of natural soce regulation are not well understood. we utilized several imaging methods to measure ca 2+ signals in cytoplasm ( ] mit transients, observed both in egta-and bapta-buffering inside solutions. these data suggest that ca 2+ sequestration by mit is associated with the formation of microdomains and prevents ca 2+ -dependent socc inactivation. we also found that inhibition of mit under prolonged cell stimulation resulted in complete inhibition of soce as well as decrease and deceleration of er refilling. thus, mit regulate calcium recycling and maintain the soce controlling dynamic interplay between soce and sustained er refilling under prolonged stimulation. bioelectrochemical devices composed of au electrodes coated by self-assembled monolayers (sams) of different composition and thickness are ideal systems to probe et patterns and mechanisms for redox proteins. representative proteins, cytochrome c and azurin were studied by using the combinations of four different strategies including the variation of sam thickness ([−s−(ch 2 ) n −ω], with n running over the range 3 to 20, throughout), solution viscosity (varied by adding of the viscose additive -glucose), temperature (0 to 50 o c) and hydrostatic pressure (up to 150 mpa), aiming the identification of different intrinsic et patterns and interplay between them in the framework of generalized charge-transfer theory. we demonstrated the full adiabatic (frictional) control for the case of thinner sams, the intermediate (mixed) regime, and the complete changeover to the nonadiabatic mechanism (long-range tunneling) for the case of thick sams owing to the variation of electronic coupling, in a nice agreement with theoretical predictions. h. nury 1 , f. manon 1 , b. arnou 2 , m. le maire 2 , e. pebay-peyroula 1 , c. ebel 1 1 institut de biologie structurale (ibs) cea cnrs ujf grenoble france, 2 cea ibitec cnrs ura2096 univ. paris-sud gif-sur-yvette france adp/atp carriers (aacs) are major and essential constituents of the inner mitochondrial membrane. they drive the import of adp and the export of newly synthesized atp. they were described as functional dimers from the 1980s until the structures of the aac shed doubt on this consensus. we aimed to ascertain the published biophysical data claiming that aacs are dimers and to characterize the oligomeric state of the protein before crystallization. analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine aac is a monomer in 3-laurylamido-n,n 2 dimethylpropylaminoxide (lapao), whereas in triton x-100 and reduced triton x-100, higher molecular mass species can also be identified. neutron scattering data for monomeric bovine aac in lapao does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. we discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of aacs within the mitochondrial membrane. local anaesthetic binding to shaker channels: role of aromatic residues j. nilsson, h. ullman, k. sahlholm, p. arhem the nobel institute for neurophysiology, department of neuroscience, karolinska institutet, se-171 77 stockholm, sweden local anaesthetic, antiepileptic and antiarrhythmic drugs acting on nav and herg channels have been assumed to bind to aromatic residues in the internal vestibule; to f1764 and y1771 in nav (ragsdale et al., 1996 and to y652 and f656 in herg (mitcheson et al., 2000) . despite a lack of such residues in kv channels, local anaesthetics, antiepileptic and antiarrhythmics bind to kv channels with a considerable affinity. to explore the role of aromatic residues for the binding we investigated the effect of bupivacaine, benzocaine, phenytoin and quinidine on shaker channels mutated to residues corresponding to the most c-terminal of the two aromatic residues in the s6 segment of the nav and the herg channels, (v473y and p474f respectively). the channels were expressed in xenopus oocytes and the currents measured with the two-electrode voltage-clamp technique. the results suggest that aromatic residues do not increase the binding affinity of the studied compounds to kv channels. rather, the affinity decreases (as reflected in typical k d values for bupivacaine on v473f, p474f and wildtype channels, being 550, 740 and 300 µm, respectively). thus, aromatic residues seem not to be necessary for high-affinity binding of the studied compounds to kv channels. how this relates to their suggested roles in the nav and herg channels, remains to be evaluated. (molina et al, 2006) . the occurrence of a similar behavior in other channels points out to clustering and coupled gating as a potentially important drug target to modulate channel activity. we have identified molecular determinants involved in single and coupled channel gating in kcsa. first, we detected that clustering and coupled gating of kcsa is modulated by anionic lipid. also, a model for the interaction between two kcsa open channels was built. the docking predicts intermolecular sites which includes the non-annular lipid binding site. this explains how an excess of anionic lipid disrupts interactions between channels, destabilizing clustering and coupled gating in kcsa. in addition, the docking model reveals molecular determinants involved in single and coupled channel gating. this interaction involves w87, which affects the neighbouring channel through specific interactions in the extracellular mouth stabilizing the selectivity filter in an open conformation. the coupled gating is also explained since this mechanism affects the opposite channel in a mutual manner. finally, mutants kcsa e71a and kcsa w87a disrupt the coupled gating of kcsa, thus, supporting the model. we think that this coupled gating phenomenon could correspond to the second gate previously detected by fluorescence methods (blunck, et al, 2006) . supported by grants from the spanish bfu2008-00602/bmc and csd2008-00005. a. kumar, e. hajjar, p. ruggerone, m. ceccarelli university of cagliari, monserrato, italy the striking presence of outer membrane (om) in gramnegative bacteria of e.coli represents a strong barrier for any molecule to penetrate inside bacteria. in particular for β-lactam antibiotics, which have their target located inside the bacteria; the first step towards reaching inner part of bacterium is the cellular uptake. ubiquitous presence of porins (such as for instance ompf, ompc) in the om, function as a channel facilitating the transport of molecules (such as, for instance antibiotics) across the om. bacteria can exhibit resistant towards antibiotics by: (i) decreasing their uptake by under-expressing the porins or/and (ii) production of inactivating enzymes such as ß-lactamases. to combat the latter mechanism ß-lactamase inhibitors (such as, sulbactam for instance) are prescribed together with the antibiotics. like the antibiotics, the inhibitors must penetrate the om, the main path being through porins. it is thus evident the biological relevance of investigating the mechanisms by which porins can regulate entry/exit across the om. to achieve this goal, molecular dynamics simulations were performed to explore the structure and dynamics of pores formed by ompf and ompc porin. from the analysis of data obtained from our simulations, we identified the key residues buried behind the l3 loop, which may play be crucial for porins to exert their biological role. as a case study, we report results about the diffusion of sulbactam through the two porins. the pentadecapeptide gramicidin forms a cation-specific ion channel in membrane environment. the two main conformations are the head-to-head helical dimer (hd) known as the channel conformation and the intertwined double helical form (dh) often referred to as non-channel conformation. in this comparative study [1] , the energetics of single potassium ion permeation by means of the potential of mean force (pmf) for both gramicidin conformations embedded in a dmpc bilayer has been addressed by molecular dynamics simulations. a significantly decreased free energy barrier by ∼25 kj/mol for potassium ion passage through dh as compared to hd is reported. favorable electrostatic side chain-cation interactions in hd are overcompensated by phospholipid-cation interactions in dh. the latter are coupled to an increased accessibility of the channel entrance in dh due to distributed tryptophans along the channel axis. this result underscores the importance of the lipid environment of this channel not only for the equilibrium between the different conformations but also for their function as cation channels. y. sawada 1 , m. murase 2 , m. sokabe 1 1 dept. physiol. nagoya univ. grad. sch. med., nagoya, japan, 2 icorp/sorst cell mechanosensing, jst, nagoya, japan the bacterial mechanosensitive channel of large conductance mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer α-helices, and its 3d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. although several models for the opening process have been proposed with molecular dynamics (md) simulations, as they do not include mscllipid interactions, it remains unclear which amino acids sense membrane tension and how the sensed force induces channel opening. we performed md simulations for the mechano-gating of mscl embedded in the lipid bilayer. upon tension in the bilayer, phe78 in the outer helix was dragged by lipids, leading to a tilting of the helices. among amino acids in the outer helix facing the bilayer, phe78 at the water-lipid interface showed the strongest interaction with lipids, thus may work as a major tension sensor. neighboring inner helices cross each other in the inner leaflet, forming the most constricted part of the pore. as tension increases, the crossings move toward the cytoplasm associated with an expansion of the constricted part. during the movement, a hydrophobic water block environment around the constricted part was broken followed by water penetration and permeation. the k + channel kcsa is an integral membrane protein from s.lividans, used as a model system for studies on ion channels and oligomeric membrane proteins. its atomic structure has been solved by x-ray diffraction, which shows an assembly of four identical subunits around a central aqueous pore, including the so-called selectivity filter. this channel is able to permeate k + at high flux rate and it is blocked by na + (physiological blocker). fluorescence, circular dichroism and fourier transform infrared experiments carried out in our laboratory demonstrated that k + and na + are able to bind to kcsa in a competitive manner. this binding is indeed associated with channel conformational changes, which seem to be related to the permeation and blockade processes. to further investigate this phenomenon we carried out a detailed study of chemical and thermal denaturation of wild-type and mutant kcsa channels. these two types of experiments were in agreement and indicate that both cations are able to stabilize the channel through conformational changes, being k + the more efficient one, even more when lipids are present. particularly, mutant channels with a structurally altered selectivity filter show that ion and lipid-induced global conformational changes are intimately associated to the conformation of this selectivity filter (conductive and non-conductive forms). modulation of the voltage-gated sodium channel nav 1.5 by rcssii, a toxin from the scorpion centruroides suffusus c. picco 1 , g. corzo 2 , l. d. possani 2 , g. prestipino 1 1 institute of biophysics, cnr, genova, italy, 2 instituto de biotecnologia, unam, cuernavaca, mexico the main cardiac voltage-gating sodium channel, na v 1.5, generates the fast depolarization of the cardiac action potential and plays a key role in cardiac conduction. its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene scn5a, which encodes na v 1.5, that are linked to various cardiac deseases. subsequently, studies of this channel localization have led to its identification in immature and denervated skeletal muscle and in the brain neurons. in our effort to identify high affinity ligands for this channel, we have investigated the effects of the recombinant cssii (rc-ssii), a four disulfide-bridged scorpion toxin isolated from the venom of the scorpion centruroides suffusus. human cardiac sodium channel α subunit scn5a was expressed in cho cells and macroscopic na + currents were recorded with patchclamp technique in whole cell configuration. the electrophysiological experiments have highlighted a strong affinity for the channel at low nanomolar concentration. compared with control conditions, rcssii toxin affects in reversible way the kinetics of activation and inactivation and marked decrease the peak na + influx. the extrusion mechanism of substrates in rnd family efflux pumps: a molecular dynamics study a. v the rnd transporters of the acrab-tolc (e.coli) and mexab-oprm (p.aeruginosa) systems are able to export structurally and chemically different substrates outside bacteria through the membrane, being responsible of multidrug resistance. on the basis of crystallographic information, an extrusion process conceived as a three-cyclic peristaltic pumping has been proposed, but further microscopically well-funded investigations are needed to understand the mechanism. using different computational methods like adaptive bias force (abf) and targeted molecular dynamics (tmd), we have investigated the mechanism of substrate uptake and pumping at a molecular level. with the first method we have investigated the passage of antibiotics from the periplasm into the internal pore of the pump, while tmd has been used to assess the effect of conformational changes on the extrusion of drugs (which have been located into one of the proposed binding pockets). comparison between the active pumps acrb and mexb (which show different resistance patterns despite their homology) provide insights into the microscopic details of their functioning. in arabidopsis thaliana there are twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iglrs). each protein displays a pore-forming loop, flanked by two conserved helices (plus a third c-terminal helix), a glutamate-binding domain and an n-terminal region. through pharmacological and/or genetic approaches, many physiological functions have been attributed to plant glurs, such as the regulation of cytosolic calcium, photomorphogenesis, water balance and carbon/nitrogen sensing and assimilation. according to the endosymbiotic theory, the cyanobacteria are considered to represent the precursors of the present chloroplasts. the first prokaryotic glutamate receptor (glur0) was identified in the cyanobacterium synechocystis. the putative products of the atglr3.4 and atglr3.5 genes display a possible targeting sequence for chloroplast location and show a high degree of homology with glur0. using specific antibodies and confocal microscopy, we have localized the two members of the atglr subgroup 3, glr3. 4 and glr3.5 (splicing variant) , to the chloroplast in arabidopsis and to the inner envelope membrane in spinach. electrophysiological experiments indicate the presence of an activity which is compatible with that of glutamate receptors. furthermore, oxygen evolution measurements suggest that chloroplast-located glutamate receptors may play a role in the regulation of photosynthesis. under extreme conditions many cells control their volume responding to osmotic challenges by unloading or loading solutes to recover their original volume. a faster volume regulatory role triggered by membrane tension has been disclosed for aquaporins in kidney proximal tubule cells, where aqp1 is the main water channel, using isolated brush border membranes. in conventional osmotic studies in animal cells it is common to disregard internal hydrostatic pressures because they are insignificant compared to osmotic forces. however, by using low osmolarity buffers in small radii vesicles, we detected a rise on the internal pressure that creates surface tension and causes membrane stress, with a negative outcome on aquaporin water permeability. these findings suggested a mechanism for volume regulation in kidney proximal tubule epithelia where massive solute and fluid transport occurs. to further explore aquaporin regulation by membrane tension, yeast cells were used as a model that could bare surface tension some orders of magnitude higher than animal cells due to the existence of a cell wall. the effect of increasing levels of membrane tension on yeast water channel activity was evaluated. an impairment of aquaporin activity correlated with the increase of membrane tension corroborates the volume regulatory role of aquaporin in different cells. deuterium isotope effects on fast gating of the chloride channel clc-0 g. zifarelli, a. r. murgia, p. soliani, p. michael istituto di biofisica, cnr, via de marini, 6, i-16149 genova, italy gating of the torpedo cl − channel clc-0 is modulated by intracellular and extracellular ph, but the mechanism responsible for this regulation has remained so far elusive. using inside-out patch clamp measurements we studied the dependence of the fast gate on ph int and [cl − ] int . only the closing rate, but not the opening rate showed a strong dependence on these intracellular factors. using mutagenesis we excluded several candidate residues as mediators of the ph int dependence. we propose a model in which a proton generated by the dissociation of an intrapore water molecule protonates e166 leading to channel opening. deuterium isotope effects confirm that proton transfer is rate limiting for gate opening and that channel closure depends mostly on [oh − ]. the model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on ph int and [cl − ] int . deletion of the c-terminus destabilizes phosphorylated na/k pump state containing 3na ions n. vedovato, d. c. gadsby the rockefeller university, new york, u.s.a. the na/k pump's extended c-terminus (compared to the serca ca pump's) links 3 transmembrane helices, and its truncation lowers cytoplasmic na affinity for forming the occluded e1p(na 3 ) state. here we test the effects of c-terminal truncations on interactions with external na. we deleted the last 2 (yy) or 5 (kesyy) residues in xenopus α1 β3 pumps made ouabain resistant by mutations q120r-n131d (rd) or c113y (c-y), and then used two-microelectrode voltageclamp recording in xenopus oocytes to measure pump currents as 10 mm ouabain-sensitive currents while endogenous na/k pumps were silenced with 1 µm ouabain. inhibition by external na of steady outward pump current ([k] o =15 mm) at large negative voltages was somewhat weaker in both rd and c-y pumps than in wt pumps, but was severely impaired in all c-terminal truncated pumps. consistent with this, the voltage dependence of transient charge movements under na/na exchange conditions ([k] o =0 mm) was strongly shifted to more negative potentials in the truncated pumps relative to the parent rd or c-y pumps, shifts comparable to those seen in wt pumps on decreasing [na] o several-fold. together, the results suggest that these c-terminal deletions lower the apparent affinity for external na ions to bind and become occluded in the na/k pump. the c-terminus therefore provides contacts important for stabilizing the occluded e1p(na 3 ) conformation, regardless of the route of na ion entry into the binding pocket. muscle contraction is driven by molecular motors that adapt their energy utilization according to the demands made on them. we test the hypothesis that rate constants controlling the biochemical steps involved in atp hydrolysis by myosin atpase are affected by the force of the muscle. here we use fluorescence lifetime imaging microscopy (flim) of a fluorescently labelled atp analogue to investigate changes in the environment of the myosin atpase, caused by different loads applied to skeletal muscle. single muscle fibres were subjected to cycles of stretches and releases in the presence of rigor solution and 10 µm of coumarin-labelled atp. flim acquisition was synchronised with stretch/release cycles and force measurements, which allow us to investigate the effect of strain on the lifetime of the labelled atp bound to the actomyosin complex. characterization of the fluorescence decay by a bi-exponential function resolved the time constant of two populations, namely, free fluorophore (τ 1 = 0.47 ± 0.03 ns; mean ± s.d.) and fluorescent nucleotide bound to the actomyosin complex (τ 2 = 2.21 ± 0.06 ns at low strain). these experiments showed that while the time constant of the free fluorophore did not change with force, the time constant of the fluorescent nucleotide bound to actomyosin showed a linear dependence with the force applied to the muscle of 0.43 ± 0.05 ps/kpa. neck linker docking coordinates the kinetics of kinesin´s heads i. derenyi, a. czovek, g. j. szollosi department of biological physics, eotvos university, pazmany p. stny. 1a, h-1117 budapest, hungary conventional kinesin is a two-headed motor protein, which is able to walk along microtubules processively by hydrolyzing atp. its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. considering the two neck linkers as entropic springs that are allowed to dock to their head domains and incorporating only the few most relevant kinetic and structural properties of the individual heads, we have developed the first detailed, thermodynamically consistent model of kinesin that can (i) explain the cooperation of the heads during walking and (ii) reproduce much of the available experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck linker length and composition, etc.). besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion. the positive role of noise on the transport efficiency of na, k atpase c.-h. chang 1 , t. y. tsong 2 1 institute of physics, national chiao tung university & physics division, national center for theoretical sciences, hsinchu, taiwan, 2 institute of physics, academy of sciences, taipei 115, taiwan na, k atpase is a molecular motor which is able to transport ions through cell membranes, even against the transmembrane ion concentration gradient. while in vivo this nanoscale soft machine consumes atp, it may be driven by external fluctuating electric fields, no matter they are periodic or random. theoretically, the motor conformations can be described by a conformation vector v(t) governed by a multi-dimensional kinetic equation. given an oscillating electric field with a slight fluctuation, the boltzmann distributions of these conformations will change with time. the instantaneous transported ion flux is a functional of the quasi-cyclic trajectory v(t) of this non-autonomous dynamical system. various interesting dynamical properties of this ion pump, including stochastic resonance, can be studied theoretically, some of which have good agreement with recent experimental findings. in situ measurements of the molecular motor of muscle with nanometer-microsecond resolution in a contracting muscle, arrays of the dimeric motor protein myosin ii pull the actin filament towards the centre of the sarcomere during cyclical atp driven interactions. when the external load is smaller than the array force, the sarcomere works as a motor, converting metabolic energy into mechanical work; when the external load is larger than the array force, the sarcomere acts as a brake resisting the load with reduced metabolic cost. to investigate the molecular basis of the work production and the braking action of muscle, we use sarcomere-level mechanics and x-ray interferometry in intact single cells from frog skeletal muscle. during isometric contraction, each motor bears a force of about 6 pn. during shortening against high and moderate loads, the number of myosin motors attached to actin reduces in proportion to the external load while the force per attached motor is maintained similar to the isometric value (piazzesi et al., cell 131, 784-95, 2007) . rapid stretches of 1-5 nm between each overlapping set of myosin and actin filaments in a muscle sarcomere cause the stiffness of the array of myosin motors to increase up to twice the isometric value within 2 ms (brunello et al., pnas usa 104, 20114-19, 2007) , indicating that the high resistance of active muscle to stretch is due to recruitment of the second motor domain of the myosin molecules with the first domain already attached to actin. supported by miur and ente crf (italy), nih (usa), mrc (uk), embl, esrf. kinesin-1 is a molecular motor that moves cellular cargo along microtubules. its functional mechanism is well understood for individual motors. however, the way that many kinesin-1 motor proteins bound to the same cargo move together is not. we addressed the structural basis for this phenomenon using video microscopy of single microtubulebound full-length motors and various spectroscopy methods were employed to study synthetic peptides derived from hinge-1 region. these peptides show an unexpected profile of secondary structure forming propensities. video microscopy of single microtubule-bound full-length motors reveal the sporadic occurrence of high compliance states alternating with longer-lived, low compliance states. the deletion of hinge-1abolishes transitions to the high compliance state. from the results we hypothesize that strain accumulated during multiple kinesin motility populates the high compliance state by unfolding helical secondary structure in the central hinge 1 domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple motor situations. titin is a giant protein of vertebrate skeletal and cardiac muscles. cardiac titin is expressed in two main isoforms: short n2b (∼3000 kda) and long n2ba (∼3400 kda). we have studied changes of titin isoform composition in myocardium of hibernating ground squirrels and spontaneously hypertensive rats (shr). using electrophoresis we have revealed considerable decrease (by 2-3 times) in the content of titin relative to myosin heavy chains in shr heart as compared with that for normotensive rats. surprisingly that the data of qrt-pcr showed the increase in mrna content of n2ba and n2b-isoforms in hypertrophic heart more than 2 times in comparison with norm. we suppose that such a result is an effect of depressed translation of mrnatitin in pathology. we have observed the decrease (by 1,5 times) of total titin amount in heart of hibernating animals in comparison with that for summer active animals. however n2ba/n2b ratio in the heart upon hibernation was increased by 2 times. similar trend was not revealed for the mrna level of corresponding isoforms, although we have showed the decrease of mrna of both titin isoforms in heart of hibernating ground squirrels as compared to their content of summer animals. the decrease in total mrna level may be explained by repressed transcription or mrna degradation in the cell during hibernation. these discrepancies in protein and mrna levels may be considered as the posttranscriptional regulation of titin isoforms expression. actomyosin cross-bridges formed when the globular heads of myosins bind to actin filaments are the molecular engines that drive muscle contraction, fuelled by atp hydrolysis. critical to this process is the change in shape of the cross-bridge and the change in the interactions with actin, in response to force applied to the muscle, and to the status of the nucleotide in the binding pocket. although molecular detail is known from x-ray crystallography and biochemistry, understanding of the interplay between cross-bridge shape and chemical state requires studies in muscle fibres generating force. we use fluorescence life time imaging microscopy (flim) as a probe of the cross-bridge environment.with a fluorescent analogue of atp 1 , fluorescence life-time (flt) changes when the crossbridge binds to actin. now, we show preliminary experiments on the effect of force on flt. the essential light chain of myosin (elc) is a ∼20 kda peptide that wraps around a 9nmlong α-helix of the myosin cross-bridge known as the lever arm which tilts during force generation. using a recombinant elc, labelled with a fluorophore at a strategic cys, we replace the native elc and introduce the fluorescent elc in muscle fibres. preliminary experiments demonstrate that the elc fluorophore also is sensitive to force applied to the muscle fibre. in addition, förster resonance energy transfer occurs between the nucleotide and elc fluorophores, opening the way for studying structural changes in cross-bridges during force generation by fret. muscle contraction: pitfalls in the determination of the contractile response e. grazi dipartimento di biochimica e biologia molecolare, università di ferrara, ferrara, italy the contractile response of an active muscle depends on the load. the load is a force /cross-section. there are three fundamental dimensions: the mass, m; the space, l; and the time, t. from these three dimensions are built up all the physical dimensions. as an example the acceleration, a, is given by, a=l.t −2 . once the direction and versus are settled the modulus fully defines the physical effect of the acceleration. what about the force? the force, f, is given by, f=m.a. at variance with the acceleration, once the direction and versus are settled, the modulus does not define the physical and the biological effects of the force: the same force is generated by an infinite number of mass-acceleration couples that display different physical and biological effects. the same occurs with the load. thus defining the load that opposes the contractile force does not define the contractile system. in the studies on muscle contraction the acceleration of the load is not considered nor it is provided a way to extract this information. thus these systems are poorly defined from the physical as well as from the biological point of view. models of muscle contraction that consider explicitly both the mass and the acceleration of the load show that, at the same load, the decrease of the acceleration of the load significantly delays the pre-steady state of the contraction and decreases the stiffness of the active fibre. r. shahapure 1 , f. difato 1 , a. laio 1 , d. cojoc 3 , e. ferrari 3 , j. laishram 1 , g. bisson 1 , v. torre 2 1 int. school for advanced studies, trieste, italy, 2 iit-sissa unit, trieste, italy, 3 lab. nazionale tasc, trieste, italy polymerization of actin filaments is the main source of motility in lamellipodia and is controlled by many regulatory proteins. the underlying molecular mechanisms are only partially understood and now a determination of the dynamical properties of force generation is needed. using optical tweezers we measured with millisecond temporal resolution and pn sensitivity the force-velocity (fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. when force and velocity are averaged over 3-5 s, fv relationships can be flat. on a finer time scale, random occurrence of fast growths and sub-second retractions become predominant. maximal power dissipated by lamellipodia over a silica bead with a diameter of 1 µm is 10 −16 w. due to the presence of adhesion forces, beads in close contact with a lamellipodium can seal on its membrane reducing the amplitude of brownian fluctuations often by more than 10 times. under these conditions, when lamellipodia grow and push the beads, discrete jumps varying from about 5 to 50 nm are detected. when lamellipodia retract, pulling the beads, no discrete events are observed. our results on the dynamical properties of force generation are: a) force generation is a probabilistic process; b) underlying biological events have a bandwidth up to at least 10 hz; c) fast growths of lamellipodia leading edge alternate with local retractions; d) force generation is produced in discrete steps with varying amplitude up to 0.2 pn. pushing on microtubules: dominant spindle centering mechanism in c. elegans embryo? j. pecreaux, s. redemann, a. a. hyman, j. howard mpi-cbg, pfotenhauerstr 108, 01307 dresden, germany asymmetric cell division, where the content of the two daughter cells -as well as their sizes -differ, is found in many organisms. strikingly, the spindle, first centered, starts to be displaced out of the center only in late metaphase. in c elegans embryo, the spindle rocks and is posteriorly displaced during anaphase by force generators asymmetrically localized on cell cortex. prior to anaphase onset, the spindle is usually assumed to be centered by the same pulling force. it thus requires the force generators to be carefully repressed to distribute forces symmetrically. on live embryos, we measured positional fluctuations of centrosomes during metaphase with 20 nm accuracy. fourier analysis shows an extremely accurate centering respect to the number of force generators and microtubules. furthermore, spectrum is close to a lorentzian, modeled by a spring and a dashpot, suggesting a spindle centering more likely by pushing on microtubules than pulling. deviation at high frequencies indicates a subdominant pulling force. rnai of gpr-1/2, known to control force generation, increases slightly centering accuracy; this result supports the hypothesis of an independent centering mechanism. conversely, zyg-9(rnai), a microtubule growing factor, decreases centering accuracy, modeled spring stiffness and damping modulus. conclusion: first, the spindle centering mechanism is independent of cortical pulling force generator. second, microtubules pushing is likely to center the spindle. mechanical forces are important in the regulation of cellular adhesion and migration. the focal adhesion kinase (fak) has been suggested to transduce cellular forces and govern cell migration. to obtain more insight in the functioning of fak, a fret-based optical biosensor for fak was designed to relate integrin-mediated conformational changes in its ferm domain to focal adhesion behavior during cell spreading and migration in living cells. imaging of the kinetics of ferm-based fak conformational changes in spreading cells revealed two consecutive stages of focal adhesion activation. heterogeneous ferm conformational responses were observed in individual focal adhesions of adherent motile cells, with the active ferm conformation being enriched in growing and sliding fas, but not in stable and shrinking focal adhesions. inhibition of the cellular actomyosin system revealed the involvement of rho-rock rather than mlckinduced tension signaling in the modulation of the ferm response. our results place the ferm conformational change of fak at the interface between integrin and force sensing. the time course of inorganic phosphate release in permeabilized cardiac trabeculae of the rat c. mansfield, t. west, m. a. ferenczi imperial college london, u.k. the rate of p i release was determined in permeabilized rat trabeculae. contraction was elicited at 20 • c by laser-flash photolysis of npe-caged atp, and time-resolved p i release was monitored using mdcc-pbp, a coumarin-labelled phosphate binding protein, which increases its fluorescence intensity five-fold upon p i binding. the atpase rate during the first turnover of the total crossbridges (assuming 100 µm myosin heads) was 23s −1 . the rate decreased to a steady state of 4s −1 after the eighth turnover (0.5-0.6s after activation). this steady state rate is comparable to published values of 3 -10s −1 , made ∼15s after activation using an nadh-linked enzyme assay of adp release. the advantage of using mdcc-pbp is that the control of mechanochemical coupling can be examined from the onset of force production and as it progresses toward the steady state. force production and p i release were simulated using a seven step scheme. force was attributed to the states in the sequence a.m.adp.p i ↔ a.m.adp ↔ a.m.adp, with strain sensitivity incorporated into the isomerisation of a.m.adp. the a.m.adp.p i and a.m.adp states populated rapidly as force was increasing. in contrast, the a.m.adp state accumulated slowly after the force plateau was reached and became the dominant force bearing state at the time of the eighth crossbridge turnover. experiments are on-going to examine how the distribution of a.m states changes in response to rapid length-changes. pulling as a factor in forming the heterophasic structure of immunoglobulin proteins structure of proteins of immunoglobulin superfamily: human igg3 kuc and muscle protein titin, has been investigated by methods of electron microscopy and diffraction with the use synchrotron radiation. super elasticity of titin, the protein of immunoglobulin superfamily, is a key parameter that determines the mechanical properties of muscle. however, the structural-physical mechanism of titin elasticity under tension remains poorly understood. here both tension transduction and high elasticity of titin are explained in terms of crystalline polymer physics. x-ray data suggest a model of titin as a nanoscale, morphological, aperiodical array of rigid ig-and fn3-type domains covalently-connected by conformationally variable short loops. the line group symmetry of the model can be defined as s m with axial translation τ ∞ . homologous domains would have similar stability, but the structure of different domains on stretching is subject to different forces because they have different orientations relative to the axis of the molecule. under the force influence the structure of any domain can become either rigid or flexible depending on its orientation in the titin strand. pulling geometry forms an active axial structure from latent isotropic random coil structure of titin strand. we are suddenly faced with nanophase-separated morphology of igg3 kuc. study was supported by rfbr grant 07-02-01281. strain response of myosin essential light chain in permeabilized skeletal muscle fibres d. s. ushakov, d. ibanez-garcia, t. g. west, p. m. w. french, m. a. ferenczi imperial college london, uk we applied fluorescence lifetime imaging microscopy (flim) to investigate the relation between conformation of myosin head and mechanical force in skeletal muscle fibres. recombinant myosin essential light chain (elc) was expressed in e.coli and labelled at cys-178 with coumarin. the labelled elc was exchanged with native elc in single permeabilized rabbit m.psoas fibres. fluorescence lifetime was measured using leica sp5 upright confocal microscope equipped with becker & hickl time-correlated single photon counting module and 63x 1.2 na leica planapo dipping objective, with the two-photon fluorescence excitation at 856 nm by sapphire pulsed laser. after acquiring flim images of muscle fibres in relaxed state, the solution was changed to ca-free rigor. further images were acquired in rigor with or without 0.5-1% stretch applied by a motor. both single and double exponential fluorescence decay analysis showed that the lifetime in rigor was lower compared to relaxed (about 80 ps difference for single exponential fit) and to rigor fibres under strain (about 40 ps). these data suggest a change in the microenvironment of coumarin induced by nucleotide binding and strain. this change is likely to be due to interaction between c-terminal domain of elc and the n-terminal domain of myosin heavy chain related to the lever arm re-orientation process. supported by bbsrc. alpha-synuclein and its a30p mutant affects the actin cytoskeleton structure and dynamics v. sousa 1 , s. bellani 1 , g. ronzitti 2 , f. valtorta 1 , j. meldolesi 1 , e. chieregatti 2 1 department of neuroscience, hsr, milano, italy, 2 department of neuroscience, iit, genova, italy alpha-synuclein (syn) is a soluble protein abundant in the brain, primarily enriched at pre-synapses. syn overexpression and the expression of its a30p mutant participate in the pathogenesis of parkinson's disease. many roles have been proposed for syn, including the regulation of synaptic vesicle pools and of neurotransmitter release. the actin cytoskeleton regulates many aspects of synaptic function and its dysregulation may be a cause of neurodegeneration. working both in cell-free and in vivo conditions we demonstrate that syn and the a30p mutant have different effects on the actin cytoskeleton dynamics. our results show that syn binds actin, and decreases actin polymerization rate probably by monomer sequestration. on the contrary, a30p accelerates actin polymerization in vitro and disrupts the cytoskeleton of intact cells. in particular, during dynamic cytoskeleton remodeling, a30p induces the assembly of discrete actin-rich foci. actin trapping and the impairment of filaments reassembly lead to inhibition of cell movement and of the re-establishment of cell-cell contacts. in a30p expressing cells cytoskeleton-based processes, such as cell migration and the exo/endocytic traffic are inhibited. elucidating the dynamics of syn interaction with actin may contribute to the understanding of its role in neuronal physiology as well as in neurodegeneration. on the physics of muscle contraction m. l. shur ural state university, yekaterinburg, russian federation whichever energy source is chosen as an engine, its force will decrease with increasing velocity. this is connected with a limited power of any engine. thus, we state that hill's formula is a mere sequence of the law of energy conservation. to derive a mathematical dependence "force-velocity", all the means of consumption of fuel energy should be determined -in our case, the energy of the atp hydrolyze. moreover, the conformation energy of the crossbridges attached serves as the force source as well. we state that part of the energy release transforms into the energy of oscillations of myosin proteins; the other part goes into thermal energy of the sarcoplasmic solution. interaction of the oscillating myosin system with the sarcoplasmic solution controls the process of force generation by a muscle. it is just this interaction that leads to the temperature dependence of force. the presentation is devoted to constructing a theory based on these simple considerations. a discussion and constructive critic is especially wanted. -biological motility and molecular motors meiotic nuclear oscillations in the fission yeast schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. we report a mechanism of these oscillations based on collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. by combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. the redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, due to the intrinsic motor properties, generate regular large-scale movements in the cell (1 ) m. versaevel, s. gabriele, p. damman university mons-hainaut, 7000 mons, belgium the remodeling of blood vessels in response to changes in blood flow is mainly realized by endothelial cells (ecs) that convert mechanical stimuli from flowing blood into changes in cell signaling through a process called mechanotransduction. many of the biological responses to external forces originate at two types of microscale structures: focal adhesions linking cells to their extracellular matrix and adherens junctions that link adjacent cells. this study aims to elucidate the role of the cytoskeleton, cell-matrix and cell-cell junctions in transducing fluid shear stress into intracellular signals in ecs. by using microcontact printing of proteins, we design substrates with defined adhesive islands in order to control shapes of living cells. this confinement of ecs allows to study the organization and the contractile activity of the cytoskeleton in order to redistribute their intracellular forces in response to externally applied forces. we design microfluidic channels with sizes and geometries close to small blood vessels to apply a physiological range of shear stresses on ecs. our results indicate that cells deposited on a precisely defined adhesive area inside microchannels and subjected to shear stress reorganize their cytoskeleton, their focal adhesions and adherens junctions in response to blood flow. drugs interfering with the cytoskeleton are used to underline the role of its different components in the cellular adaptation to the mechanical environment. s. mahdavi 1 , b. ranjbar 1 , s. gharibzadeh 2 , m. toosi 2 , m. javan 1 1 tarbiat modares university, tehran, iran, 2 amirkabir university of technology, tehran, iran multiple sclerosis (ms) is the main known pathology of myelinating cells. an autoimmune reaction occurs against myelin sheets of neurons, so action potential (ap) propagation along the affected nerve fibers has been destroyed and it causes various disorders. here, we propose a novel strategy for ms symptoms treatment. we modeled neuron by orcad software and simulated the action potential propagation along the axon in normal condition. our model simulated normal neuronal behavior. then we destroyed the myelin sheet as it occurs in ms and observed destroyed ap propagation as it was reported in ms disease. we investigated the effect of changes in the voltage-gated sodium channel (vgsc) threshold on the efficiency of ap propagation. the results demonstrated that reduction of vgsc threshold improves the propagation of ap by increasing the amount of sodium flux during ap propagation. although, some researches have proposed vgsc blocker as ms symptom treatment, our result suggests that the increase of sodium current produced by reduction of vgsc threshold, improves ap propagation and probably cure some ms symptoms. so, we suggest that vgsc gating modifiers can be considered as novel strategy for ms treatment. surely, this results needs to be confirmed by experimental studies. a. gradogna, e. babini, a. picollo, m. pusch istituto di biofisica, consiglio nazionale delle ricerche, via de marini 6, 16149 genova, italy clc-ka and clc-kb are highly homologous cl − channels expressed in the kidney and the inner ear where they mediate transepithelial chloride transport. both channels heteromerize with the beta subunit barttin. mutations in clc-kb and barttin genes lead to bartter's syndrome. we analyzed the modulatory effect of extracellular ca 2+ and h + on clc-k channels using the xenopus oocyte expression system. clc-ka currents increased with increasing [ca 2+ ] ext without full saturation for [ca 2+ ] ext up to 50 mm. however, in the virtual absence of ca 2+ , clc-ka currents are about 18 % of currents measured in 10 mm [ca 2+ ] ext , demonstrating that ca 2+ is not strictly essential for opening. vice versa, clc-ka was blocked by increasing the [h] + ext with an almost complete block at ph 6. among various reaction models tested, the model that best fitted all state-steady data predicts an allosteric regulation of channel opening by separate binding sites for ca 2+ and h + . moreover, the best fit suggests that one ca 2+ and two h + bind to the channel. kinetic analysis of current responses upon [ca 2+ ] ext and ph jumps confirmed the allosteric character of modulation. in support of the presence of two separate binding sites we identified several mutations that selectively altered ca 2+ or h + sensitivity. our data represent a first step towards a molecular picture of ca 2+ and proton regulation of clc-k channels and suggest that it is of physiological relevance. the extracellular matrix molecule hyaluronic acid modulates l-type voltage-dependent ca2+ channels e. dvoretskova 1 , g. kochlamazashvili 2 , o. bukalo 2 , c. henneberger 3 , d. rusakov 3 , m. schachner 2 , a. dityatev 1 1 italian institute of technology, genova, italy, 2 centre for molecular neurobiology, hamburg, germany, 3 institute of neurology, university college london, london, uk we studied the effects of hyaluronic acid (ha), a major extracellular matrix molecule, on activity of l-vdccs in a heterologous expression system and in hippocampal slices. we recorded currents mediated by a major neuronal subtype of l-vdccs (ca v 1.2c, β 1 b, and α 2 δ 1 ) expressed in cho cells. a five-minute application of 0.1 mg/ml ha potentiated l-vdcc currents at -20, -10, 0 and +10 mv by approximately 50%. analysis of boltzmann curves showed that ha increased maximal conductance rather than other parameters (v 0.5 or k ). treatment with hyaluronidase removed endogenous ha in murine hippocampal slices and specifically impaired long-term potentiation (ltp) induced at ca3-ca1 synapses by repetitive theta-burst stimulation. blockade of l-vdccs reduced ltp in control slices to the levels seen after hyaluronidase treatment. a potentiation of l-vdccs with bay k 8644 fully restored ltp after hyaluronidase treatment. removal of ha reduced ca 2+ transients elicited by backpropagating action potentials in individual dendritic shafts and spines of ca1 pyramidal cells, whereas pretreatment with nifedipine fully occluded this effect. thus, ha potentiates postsynaptic l-vdccs and by this way influences use-dependent synaptic plasticity. whole-cell patch clamp recordings from a variety of human cancer cells showed that functional voltage-gated sodium channel (vgsc) expression occurred specifically with strongly metastatic cells. in addition, where studied, this was accompanied by down-regulation of outward (mainly potassium) currents. this has led to the celex ("cellular excitability") hypothesis of cancer according to which metastatic cell membranes are excitable and this promotes their hyperactivity. importantly, the vgsc genes expressed are embryonic splice variants, which are normally developmentally regulated, hence the phenomenon is 'oncofetal'. in breast cancer, where the predominant vgsc is nav1.5 the neonatal and adult forms are significantly different and this is reflected in channel activity whereby the neonatal vgsc has much slower inactivation kinetics. the double-charge change at position 211 is critical for this difference. the slow kinetics results in much greater influx of na + into cells and one consequence of this is activation of protein kinase a. this is a tonic effect and, under steady-state resting conditions, it results in a positive feedback effect promoting post-translational trafficking of vgsc protein to plasma membrane. the unique amino acid sequence of the spliced region has enabled the production of a polyclonal blocking antibody specific to neonatal nav1.5. it is concluded that vgscs represent novel biophysical targets for clinical management of metastatic disease. m. pusch cnr, istituto di biofisica, genoa, italy clc proteins form an evolutionary conserved gene-family that comprises 9 members in mammals. four of the human clcs are passive plasma membrane cl − ion channels. the other 5 clcs are expressed in intracellular organelles. clc-4 and clc-5, mutations of which lead to dent's disease, are secondary active cl − /h + antiporters, similar to the bacterial clc-ec1, and with identical 2 cl − : 1 h + stoichiometry. cl − and h + transport activity of the exchanger clcs depends on two glut residues. mutating a 'gating glutamate' (e211 in clc-5) converts the exchanger into anion conductances. neutralizing the 'proton glutamate' (e268), but not its replacement by some other titratable groups, abolishes cl − and h + transport. noise analysis indicated that clc-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 ps, indicating a very large transport turnover. no 3 − uncouples h + transport but mutating the highly conserved s168 to p, as found in the plant no 3 − / h + antiporter atclca, led to coupled no 3 − : h + exchange. clc proteins are a fascinating example of how a very similar protein architecture can be used to provide either a passive electrodiffusive permeation pathway or a strictly coupled secondary active ion transporter. (supported by telethon italy -grant ggp08064). the role of ion dynamics in zebrafish fin regeneration the specific and directional ion transport across cell membranes or tissue layers results in differential accumulation of ions and endogenous electric currents. these phenomena have been shown to be important for vertebrate organs regeneration. however, the specific ion nature of such electric currents remains unknown, as well as the role of cellular ion dynamics during regeneration and the molecular signalling pathway that transduces electric cues into cellular responses. we use zebrafish caudal fin as an adult regeneration model to unveil the specific ion composition of the currents associated with wound healing and regeneration, using a non-invasive ion-specific scanning microprobe setup. our data suggests a role for potassium (k + ), calcium (ca 2+ ) and protons (h + ) at different stages of the regeneration process. k + and ca 2+ extracellular effluxes have both been detected during the wound healing stage. h + efflux is triggered during wound healing and is maintained throughout regeneration. we are validating these data with genetic and pharmacological approaches, as well as advanced ion imaging. overall, our results suggest ion-driven mechanisms underlie adult tissue regeneration and its comprehension may open way for new therapeutic strategies, both in regenerative and developmental medicine and in cancer therapy. cardiac effects of anabolic steroids: an electrophysiological approach anabolic androgenic steroids (aas) have been used by athletes and non athletes for almost five decades in order to improve performance. however, the illicit abuse of high-doses of aas has been attributed as a main cause of several cardiovascular disorders such as arterial hypertension, lipid profile abnormalities, heart failure, hypertrophic cardiomyopathy, arrhythmia and sudden death. the aim of this study was to investigate qt interval and transient outward potassium current (i to ) changes in rats treated with nandrolone decanoate (deca). male wistar rats received weekly 10 mg/kg of deca (n=10) or vehicle (control, n=10). electrocardiogram was recorded weekly, and qt interval was measured. after 8 weeks hearts were excised and single myocytes were isolated from the ventricles of animals. i to was recorded by means of the whole cell patch clamp technique. qt interval was larger in deca group from 4 th to 8 th week (p <0.01). analysis of i to showed a decreased current density (p <0.01) in ventricular cardiomyocytes of deca group, compared to control group. in conclusion, our results show that alterations on ventricular repolarizaton may constitute an early consequence of the chronic administration of high doses of anabolic steroids in rats, and demonstrated qt prolongation and i to density reduction, which may constitute an important marker of arrhythmia vulnerability and sudden death. -ion channels in channelopathies and cancer denitrifying bacteria control no and no 2 cytosolic levels by regulating the expression of denitrification gene clusters via redox signalling of specific transcriptional factors that may act as no sensors in vivo. a protein belonging to the subclass dnr (dissimilative nitrate respiration regulator) from pseudomonas aeruginosa has been recently suggested to be a heme containing protein. very recently the three dimensional structure of the apo-form of dnr (in the absence of heme) has been determined by x-ray crystallography, whereas the holo-form (in the presence of heme) has not yet been crystallized. we have investigated the heme local structure in solution of ferric, ferrous, co bound and no bound holo-dnr by x-ray aborption spectroscopy (xas) and we added a kinetic study of the co bound form by means of a flash photolysis setup using uv-visible absorption as a spectroscopic probe. the combination of fe k-edge xanes fingerprints and kinetic study reveal a heme pocket able to bind exogenous ligands like no and co with increased plasticity, thus supporting its role as the cofactor involved in no sensing activity. molecular examination of motifs that lead to the formation of s-nitrosylated proteins i. alicea, e. r. schreiter university of puerto rico rio piedras, san juan, puerto rico physiologically, a wide range of proteins experience structural and functional modifications after the addition of a nitric oxide (no) moiety to cysteine thiol. this posttranslational modification, known as s-nitrosylation, regulates a large number of cellular processes like vasodilatation, cell signaling and others, and the products of s-nitrosylation can be involved during the development of different human diseases. however, little is known about the mechanism by which different proteins specifically bind the no moiety to their cysteine. here we show a bio-statistical analysis of some properties of cysteine that are s-nitrosylated in different proteins, including the pk, electrostatic environment, solvent accessibility of the target cysteine and identity of surrounding amino acids. we also chose model proteins (human thioredoxin and s100 protein) to make specific targeted amino acid substitutions around selected cysteines to alter the properties described above. the reactivity and stability of these mutant proteins towards s-nitrosylation will be examined. sampling the flexibility of ppar-γ s. aci-sèche 1 , n. garnier 1 , d. genest 1 , s. bourg 2 , c. marot 3 , l. morin-allory 3 , m. genest 1 1 upr cnrs 4301, orléans, france, 2 fr pcv cnrs 2708, orléans, france, 3 umr cnrs 6005, université d´orléans, france a promising approach to consider the flexibility of proteins in docking studies consists in performing multiple rigid docking on a representative set of the receptor conformations. molecular dynamic (md) simulation is one of the best adapted methods for structural sampling, but exploring the conformational diversity of a protein is computationally expensive. we present a protocol for generating a wide range of conformational states of a receptor using restrained md and a partitioning protocol to select a few representative conformations of the binding site from this md. a way to speed up efficiently md calculation is using an implicit model to represent the solute-solvent interactions. we explore a protocol using a distance-dependant permittivity function to represent solvent effect and an ensemble of controlled restraints applied on a subset of specific atoms in order to prevent artefactual structural distortions, but preserving receptor's flexibility. ten 100ns simulations have been performed using different sets of parameters and compared to a reference 30ns simulation with explicit solvent. to select a representative set of conformations, partitioning (k -means algorithm) was applied on the ensemble of simulated conformations. this methodology was applied to the ligand binding domain of peroxysome proliferator-activated receptor-γ. 4 department of biomedical sciences, university of antwerp, antwerp, belgium the complex system of cavities identified in human neuroglobin (ngb) has been postulated to be of functional significance to the putative no dioxygenase activity of the protein. the interconnected hydrophobic cavities may support this catalytic activity by acting as reservoir for reactants and providing preferential pathways assisting product removal from the active site. we thus decided to investigate co rebinding kinetics to ngb embedded in silica gels to expose ligand migration processes in the geminate phase. encapsulation of the co complexes of reduced neuroglobin, leads to a slight increase in geminate recombination after nanosecond laser photolysis. increasing the viscosity of the medium, by soaking the gels in glycerol, completely inhibits escape of the photodissociated ligand to the solvent, and highlights a complex, multiphasic kinetic pattern. this finding can be rationalized by assuming the existence of a discrete set of temporary docking sites, capable of trapping the photodissociated ligand for very long times, up to a few ms after photolysis. g. bartolommei, f. tadini-buoninsegni, m. r. moncelli bioelectrolab -department of chemistry, university of florence, italy ion pumps are integral membrane proteins devoted to ion transport through a lipid membrane phase. the ion pumps ca-atpase and na,k-atpase are prominent members of the p-type atpases family. due to fundamental physiological roles of these proteins, they are very promising drug targets. bioelectrolab has a wide expertise in the study of ion transport by these proteins [1] . our attention has been recently focused on the interaction of these enzymes with molecules of potential pharmacological interest. frequently, drugs exert an inhibitory action on the transport activity of an ion pump, usually confining it in an inactive conformation. molecules like thapsigargin and cyclopiazonic acid belong to high (nanom) affinity inhibitors of the ca-atpase, whereas clotrimazole and curcumin are medium (microm) affinity inhibitors of both ca-atpase and na,k-atpase. for each of these compounds a mechanism of action is proposed. moreover, recent results concerning ca-atpase inhibition by clotrimazole analogues will be shown: the relevance of this type of molecules is due to their potential employment as an alternative to traditional drugs against malaria parasite. financial support of ente cassa di risparmio di firenze and of miur (prin project) is gratefully acknowledged. [1] tadini-buoninsegni f., bartolommei g., moncelli m.r., fendler k. 2008. arch. biochem. biophys. 476:75-86 (review). s. asthana 1 , s. shukla 1 , g. giliberti 1 , f. luliano 1 , m. ceccarelli 2 , r. loddu 1 , p. ruggerone 2 , p. la colla 1 1 department of biomedical science and technology, università di cagliari, cagliari, italy, 2 department of physics, università di cagliari, cagliari, italy studies of protein-inhibitors interactions are helpful to elucidate the mode of action of ligands and thereby providing clues for rational drug design. bvdv, is an important target of drug discovery activities largely because it is essential for viral replication. in bvdv rdrp no specific nni binding site has been reported till now. experimental results have shown that different class of inhibitors (benzimidazole, imidazoquinolines and pyridoxyquinolines), have resistant mutations located in the finger domain of the rdrp. all the reported mutations are spatially very close to each other. thereby, indicating that binding sites of nni's may lie in the finger domain for these different class of inhibitors.herein, we have utilized docking procedure to investigate binding sites, binding modes as well as binding affinity of different class of inhibitors.we then used all atom molecular dynamics (md) simulations to investigate the stabilizing interaction between inhibitor-receptor pairs. our md results are in good agreement with experimental data and provide deep insights into the dynamical features of the high affinity inhibitorreceptor binding. thus identifying the binding modes of our inhibitors and mechanism leading to inactivity of the enzyme can help us to build a microscopically well-funded picture of the functioning of these enzymes. we present an investigation of the molecular basis of ligand binding and reactivity of heme proteins using computer simulation. a combination of classical molecular dynamics and hybrid quantum-classical (qm-mm) calculations are applied to explore distal and proximal effects on diatomic ligand binding to the heme. trends in binding energies and in the kinetic constants are illustrated through a number of selected examples. an investigation of the interplay between ligand migration and protein dynamics obtained through classical molecular dynamics techniques in combination with advanced sampling tools is also presented to yield information about free energy profiles and possible secondary docking sites. results for truncated n hemoglobin of mycobacterium tuberculosis, presented as an illustrative example, suggest that the truncated hemoglobin n has evolved a dual-path mechanism for selective/distinct migration of o 2 and no to the heme, to achieve efficient no detoxification. finally, we present also an analysis of the molecular basis of hexacoordination in human neuroglobin, which suggest that the flexibility of the cd plays a key role in determining the ligand binding properties. thermodynamic bases of nucleoplasmin-histone complexes recognition by the nuclear transport machinery i. arregi, j. falces, s. bañuelos, m. a. urbaneja, s. g. taneva unidad de biofísica (csic/upv-ehu), departamento de bioquímica y biología molecular, universidad del país vasco, spain the nuclear transport of the chromatin remodeling (nucleoplasmin) and chromatin building (histones) proteins is mediated by importins. nucleoplasmin (np) contains a classical bipartite nuclear localization signal (nls) that is recognized by importin α, while histones present multiple sequence elements (nls-like motifs) for nuclear targeting. besides, ternary importin/np/histone complexes might represent a putative coimport pathway for nuclear import of linker (h5), nucleosomal core (h2ah2b) histones and their chaperone protein np, enhancing the histone import efficiency. to better understand np and histone recognition by the transport machinery we studied the thermodynamics of complex formation of importin α (a truncated form) and importin β with histones and np, and with np/histone binary complexes by means of isothermal titration calorimetry. data show that importins interact with the two histone types and np, and that importin and histones can simultaneously bind to np. analysis of the binding energetics reveals an enthalpy driven formation of high affinity binary and ternary complexes. we demonstrate that different amount of importin molecules can be loaded on np/binary complexes dependent on the histone type, linker or core, and the amount of the bound histones. g. breuzard, i. di maïo, p. barbier, d. allegro, c. brault, v. peyrot cro2 inserm u911, ufr de pharmacie, marseille, france since a decade, our laboratory has already studied the interaction of tau variant with tubulin (tub) or microtubules (mts) and phosphorylation process on this interaction. indeed, fret assay was a powerful tool to achieve binding parameters between tau and tub in vitro: 1/ with mts stabilized by fluorescein-coupled taxol (flutax-2) as donor and rhodamine-labelled tau (rho-tau) as acceptor, and 2/ in living cells by confocal laser scanning microscopy (clsm) with tau/α-tub fused to egfp/mcherry, respectively. results revealed 47 ± 3 % energy transfer efficiency from flutax-2 to rho-tau and a donor-to-acceptor distance of 54 ± 1å. by titration, the dissociation constant of tau was determined to 1.0 ± 0.5 µm. a cleavage procedure of αβ-tub was performed to determine the influence of the c-term tails of αβ-tub on the tau-mt interaction. no difference in distances and binding parameters was observed. clsm images displayed a heightened concentration of fluorescent tau in patches along mts. fret experiments revealed in particular higher efficiencies between gfp-tau to mcherry-α tub proteins in these locations. overall, our results suggested no involvement of the hypervariable and highly acidic c-term tails of tub in mt/tau binding. a molecular model is proposed in which flutax-2 is directly accessible to tau molecules. besides, the modified distribution of fluorescent tau could be implicated in local change of mechanical properties of mts. a. boreham 1 , k. winkler 1 , c. gebhard 2 , k. rueck-braun 2 , p. henklein 3 , e. michalsky 3 , r. preissner 3 , r. misselwitz 3 , a. ziegler 3 , u. alexiev 1 1 freie universität berlin, berlin, germany, 2 technische universität berlin, berlin, germany, 3 charité-universitätsmedizin berlin, berlin, germany peptide presentation by major histocompatibility complex (mhc) molecules is crucial for immune responses. photocontrol of peptide dynamics by means of photo-switchable peptide analogs will provide insights in peptide dynamics and its dependence on mhc polymorphism (1) (2) (3) . we have designed a hemithioindigo (mhti) photo-switch bearing peptide using the viral epitope rrrwrrltv (plmp2) as a template. incorporation of mhti in the peptide backbone should result only in a minor change of the overall peptide structure given the relaxed conformation of the zisomer. indeed, the human mhc molecule hla-b*2705 can tolerate nonpeptidic elements in plmp2, as evidenced by normal peptide binding. using the autofluorescent properties of mhti we determined the stability of the hla-b*2705/mhti-plmp2 complex. photoswitching from z →e results in a decrease of hla-complex stability. based on computer modeling this decrease is due to a reduced interaction of the peptide c-terminus with hla-b*2705. truncated hemoglobins (trhbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. three phylogenetic groups (n, o, and p) have been identified in trhbs. the crystal structure of truncated hemoglobin o of b.subtilis, does not show an evident tunnel/cavity system connecting the protein active site with the solvent, a fact that cannot be easily rationalized considering the very high oxygen association rate. moreover, resonant raman results of the co bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. for these reasons we performed classical molecular dynamics simulations of the oxy, carboxy and deoxy protein, and computed the free energy profiles associated with ligand migration to the active site. our results suggest that there is a key residue, glne11, that may present an alternate conformation in which a wide ligand migration tunnel is formed, consistently with the kinetic data. the results for the co and o 2 bound protein show also that glne11 is directly involved in the stabilization of the coordinated ligand, playing a similar role as tyrb10, and trpg8 in other trhbs. our results not only reconcile the structural data with the kinetic information, but also provide additional insight about the general behaviour of trhbs. patterned functionalization of surfaces for guided transport on molecular motors tracks m. bhagawati 1 , s. ghosh 2 , t. surrey 1 , j. piehler 2 1 department of biophysics, university of osnabrueck, osnabrueck, germany, 2 european molecular biology laboratory, cell biology and biophysics unit, heidelberg, germany chelator head groups with multiple nitrilotriacetic acid (nta) moieties have been very well characterized and successfully utilized as high affinity adapters for functional immobilization of oligohistidine tagged proteins to surfaces. we have recently established a generic method for patterning nta functionalized surfaces by selective photodestruction via a light induced fenton reaction. efficiency of different transition metal ions for catalyzing this reaction was tested. functionality of the patterned protein was confirmed using the interaction between interferonα2 and its receptor. implementation of this technique in a confocal laser scanning microscope allowed us to control surface density of binding sites, providing the possibility to vary the surface concentration of immobilized proteins in a spatially resolved manner. we also applied this approach for exploring guided transport of microtubules by kinesin selectively immobilized onto trisnta patterns. rheumatoid arthritis (ra) is an autoimmune disorder, leading to pathological damage at the level of joints, associated with the hla class ii allele hla-dr4. although etiology of ra is unknown, type ii collagen (cii) is a potential antigen candidate and it is believed that t cell responses in collagendependent ra are directed towards the immunodominant pathogenic epitope cii(261-273). despite recent advances in characterization of class ii major histocompatibility complex (mhc) and t-cell receptor (tcr) contacts in this epitope, the atomic details of tcr-cii(261-273)-mhc complex are not known. here, homology modeling and molecular docking studies have been used to derive a three-dimensional model of tcr β chains, obtained from a dr4+ subject, in complex with cii(261-273)/hla-dr4. the best complex from docking was further refined using molecular dynamics simulations for 20 ns. the proposed model represents a reasonable structural basis for understanding cii(261-273)-mhcii complex recognition by tcr and for rational design of inhibitors targeting tcr-pmhc interface. probing bio-molecular bonds with magnetic force for biosensor applications a. m. de jong 1 , a. jacob 1 , x. j. janssen 1 , j. m. van noorloos 1 , l. j. van ijzendoorn 1 , m. w. prins 2 1 eindhoven university of technology, eindhoven, the netherlands, 2 philips research laboratories, eindhoven, the netherlands we investigate new technologies to be applied in next generation biosensors, which not only measure biomarker concentrations but also probe bio-molecular interactions. the concept is based on the response of ligand-receptor pairs to an applied force or torque [1, 2] . we use functionalized magnetic beads and magnetic fields to apply translational and rotational forces on the molecular bonds. in a model experiment, polystyrene surfaces were coated with anti-biotin and beads were coated with biotin. after incubation, a constant magnetic force was applied to the beads and the number of bound beads was measured as a function of time. this was repeated for a range of forces. the dissociation rate (k o f f ) is determined for each force and k o f f at zero force is extracted from these data. rotational forces were exerted on protein-g coated beads bound to igg antibodies immobilized on a surface. rotating fields revealed an oscillating behavior, which can be understood from the balance between the applied magnetic torque and the torque due to the deformation of the biological bond. studying interactions between cop1 regulatory protein and hy5 transcription factor d. s. dalafave the college of new jersey, ewing, nj 18940, usa this work addresses important questions of protein-ligand interactions and selective protein recognition. proteinligand bindings are crucial for many cellular processes. dependable methods for predicting binding sites would lead to a better understanding of proteins' selective recognition and would, in turn, help research on fighting diseases. presented here is a study of interactions between the wd domain found in a regulatory protein cop1 and the motif v-p-e/d-φ-g (φ=hydrophobic residue) found in a transcription factor hy5. cop1 and hy5 proteins have opposing roles in developmental regulation. cop1 can repress hy5 by directly binding with it. the repression involves specific interactions between the wd domain and the motif v-p-e/d-φ-g. previous experimental research showed that mutations in the motif's v-p pair resulted in a large decrease in hy5 repression. to study effects of similar mutations, residues in the motif v-p-e/d-φ-g were systematically substituted with other residues. interactions between the mutated motif and the wd domain were studied. distributions of binding sites, bond lengths, local shape complementarity, and interaction potentials were modeled for each residue substitution. the study identified binding sites critical for the cop1-hy5 binding. some hy5 residues in the vicinity of the motif were also found to be important in the binding. the significance of the results for understanding selective protein recognition is discussed. determination of protein-ligand binding thermodynamics by thermal shift assay p. cimmperman, a. zubriene, l. baranauskiene, e. kazlauskas, j. matuliene, d. matulis laboratory of biothermodynamics and drug design, institute of biotechnology, vilnius, lithuania thermal shift assay determines the effect of ligand binding on the protein thermal denaturation equilibrium. several models have been derived to describe the general cases of protein-ligand binding. first, the model describing protein stabilization and destabilization by ligand binding to the native and unfolded states. second, the split of protein melting transitions by tightly binding ligands is presented when the transition of free protein and ligand-bound protein occur separately. mathematical equations describing the protein unfolding and ligand binding thermodynamic parameters at various temperatures are presented. the protein melting temperature shift can be determined by various techniques such as differential scanning calorimetry, circular dichroism, and intrinsic or extrinsic fluorescence. the advantages of fluorescent techniques are presented. the melting temperature shift caused by ligand binding is dependent on the thermodynamic parameters of protein unfolding and ligand binding, including enthalpy, entropy, and heat capacity, thus allowing determination of binding thermodynamics. application of the models in the design of hsp90 chaperone and carbonic anhydrase inhibitors is discussed. comparison with isothermal titration calorimetry data is presented. recent reports have shown that the bacterial redox protein azurin can enter into cancer cells and induce apoptosis by stabilizing p53. the formation of a complex between the two proteins has been demonstrated, but little is known about binding features. for the first time, we show here that azurin binds to the n-terminal region of p53 with a dissociation constant in the 5-10 µm range. trp phosphorescence lifetime measurements revealed conformational changes of azurin induced by the interaction with p53(1-63). acrylamide quenching of trp phosphorescence also indicated a significant increase of the overall flexibility of azurin upon binding to p53 . no change of the fluorescence emission of p53(1-63) was detected in the presence of azurin. the latter finding suggests that w23 of p53 is not directly involved in domain binding to azurin, indicating that the binding site is distinct from that of mdm2. the present results may assist the design of novel cancer treatments based on p53 stabilization by azurin. a. eleta 1 , r. georgieva 2 , h. bäumler 2 , j. l. toca-herrera 1 1 cic biomagune, 20009 donostia-san sebastián, spain, 2 charité-universitätsmedizin berlin, berlin, germany human serum albumin (hsa) is the most abundant nonglycosilated plasma protein in the human body. this multifunctional protein has ligand-binding and transport properties, antioxidant functions and enzymatic activity [1] . bilirubin interacts with albumin in order to be transported from the blood to the liver where it is secreted. the interaction occurs specifically in hsa i-domain of its three domains [2] . in our work, we investigate the interaction between hsa and bilirubin by quartz crystal microbalance with dissipation (qcm-d) and atomic force microscopy (afm) [1, 3] . albumin was adsorbed on negatively charged silicon oxide. however, hsa was removed after rinsing with pbs. hsa adsorbed on positively charged polyelectrolyte multilayers leads to a stable layer of surface mass density of 272 ng/cm 2 . cross linked albumin with glutaraldehyde after its adsorption on nh 2 -terminated thiols is also stable reaching surface mass density of 296 ng/cm 2 . a preliminary bilirubin adsorption results on cross linked hsa substrate show that 200 ng/cm 2 is immobilized. taking into account that hsa-bilirubin stoichiometry is 1:1, the outcome demonstrates that the 70% of hsa i-domains remain active. antimicrobial peptides (amps) are short positively charged polypeptides. they are important due to their potential to provide an alternative to conventional therapy against bacterial infections. rbpi 21 is a 21 kda peptide based on the nterminal region of the neutrophil bactericidal/permeabilityincreasing protein (bpi). it was shown that this amp possesses bactericidal effects on gram-negative bacteria and higher affinity for lipopolysaccharide (lps), neutralizing its effect. the peptide use against meningitis, is in phase iii clinical trials. here, we demonstrate that rbpi 21 promotes aggregation of negatively charged large unilamellar vesicles (luv) and lps aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (popc) luv the size remains unchanged. the aggregation increases with peptide concentration until peptide promotes massive aggregation followed by sample flocculation/precipitation. with the rbpi 21 -lipid interaction there is a progressive change in the zeta-potential of the luv systems and lps aggregates. luv systems composed of phosphatidylglycerol (popg) and popc:popg mixtures have higher zeta-potential variations than popc luv. for lps aggregates, rbpi 21 neutralizes the surface charge and at higher peptide concentrations overcompensates it. results demonstrate that the interaction of the peptide rbpi 21 with lps aggregates and luv systems has electrostatic and hydrophobic contributions. serratia marcescens heme acquisition system: heme transport and protein:protein interactions m. delepierre unité de rmn des biomolécules cnrs ura 2185, institut pasteur, paris, france heme transport systems in bacteria are required and might be potential target for antibacterial drugs. the heme acquisition system, has, exists in pathogenic as well as in opportunistic bacteria but only for the latter one extensive studies have been conducted, constituting as such a model system. the outer membrane receptor hasr, the central component of this system, functions in synergy with a secreted high affinity heme binding protein, the hemophore hasa. hasa extracts heme from host hemoproteins and returns it to hasr. then, the energy given by a protein complex of the inner membrane is used to allow heme entrance across the bacterial membrane and to eject the empty hemophore from the receptor. reconstitution of this heme acquisition system in e coli, overexpression and purification of its various components have allowed us to obtain sufficient amount of protein to perform nmr and biophysical studies to analyse at the molecular level the different steps of heme acquisition by hasr. protein-protein interactions (ppi) are the central pillar supporting most of biological functional activity on the molecular level. a binding event between two proteins typically consists of two stages: 1) diffusional search of the binding partners for each other, and 2) specific recognition of the compatible binding surfaces followed by the formation of the complex. we focus here on the non-specific component of ppi, which refers to all physico-chemical properties of the binding partners (such as size, charge, isoelectric point, hydrophilicity etc.) that are independent of the exact details of their binding sites, but which could in turn affect their localization or diffusional search for one another. it is known that proteins co-localize due to segregation into different cellular compartments, sequestration via anchor and scaffold proteins or even chemical modifications. we suggest that the non-specific component of ppi determines in part the co-localization and clustering of the binding partners, which then directly in a non-specific fashion influences their interactions. we examine the possibility that such signature might be encoded within the experimental 3d structures of a large set of known mutually interacting proteins. we provide preliminary evidence that this indeed may be the case, and corroborate our findings by using different statistical tests to compare those features of the known interacting partners, and ascertain correlations and commonalities between them. a link between hinge-bending domain motions and the temperature dependence of catalysis in ipmdh i. hajdú, a. szilágyi, j. kardos, p. závodszky institute of enzymology, hung acad sci, budapest, hungary enzyme function depends on specific conformational motions. since conformational flexibility strongly depends on temperature, temperature dependent enzyme kinetic studies with measurements related to dynamics can give us some insight at atomic level into these functionally relevant motions. the catalytic efficiency (k cat /k m ) of 3-isopropylmalate dehydrogenase for its substrate (ipm) has unusual temperature dependence, showing a local minimum at 35 • c. in search of an explanation, we measured the individual constants k cat and k m,ipm as a function of temperature, and found that the van't hoff plot of k m,ipm shows a sigmoid-like transition in the 20-40 • c temperature range. by means of various measurements including h-d exchange and fret, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperature above 30 • c. the thermodynamic parameters of ligand binding determined by itc as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. because the binding of ipm is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with ipm binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency. a simulation approach to multiple sclerosis: study of a peptide with a pharmaceutical potential c. guardiani 1 , s. marsili 2 , p. procacci 2 , r. livi 3 1 centro dinamiche complesse, università di firenze, italy, 2 dipartimento di chimica, università di firenze, italy, 3 dipartimento di fisica, università di firenze, italy multiple sclerosis (ms) is an autoimmune disease of the central nervous system, leading to premature death. one of the potential targets of the autoimmune reaction is the myelin protein mog that has been crystallized in complex with the 8-18c5 ms-autoantibody. the analysis of contacts and buried surface area combined with an alanine scanning computation reveals the key role of mog fragment 101-108 for the interaction with 8-18c5. a docking simulation shows that the 101-108 fragment, excised from mog, and kept in crystal-like conformation, is still capable of fitting into the binding pocket of the antibody. we then studied, through replica exchange molecular dynamics simulations, the structural equilibrium distribution of the free peptide and of a number of analogs stabilized by a disulfide bond. we found that the free peptide yields a significant fraction of crystal-like conformations and the proportion of native-like structures is further increased by the disulfide bridge. when we tried to dock the centroids of the most populated clusters to 8-18c5, we discovered the existence of a docking funnel whose bottom is populated by stable complexes where the peptide occupies the same spatial region as in the crystal. we therefore conclude that the mog 101-108 fragment may be used to develop a diagnostic assay or a drug for ms. the escherichia coli membrane insertase yidc reversibly binds its substrate pf3 coat protein u. gerken, s. winterfeld, a. kuhn institute of microbiology and molecular biology, university of hohenheim, germany the membrane insertase yidc of e. coli belongs to the oxa1 family of mitochondria and plays an essential role in facilitating the insertion and assembly of membrane proteins. we have previously shown with detergent-solubilized (c 12 pc) yidc, labelled with ans, and pf3 coat that the initial step of the membrane insertion process, the binding of the substrate pf3 coat to yidc, is reversible [biochemistry 47, 6052-6058 (2008) ]. the dissociation constant k d for that particular system is about 1 µm. in order to obtain data for the native system we used in this study membrane-reconstituted (dopc and dope/dopg) yidc. the effect of the initial binding was examined in vitro by fluorescence quenching of the 11 tryptophan (trp) residues of yidc which are highly sensitive fluorescent probes for changes of the tertiary structure. quenching of the trp fluorescence after titration with a trp-free pf3 mutant indicates a change in the yidcs tertiary structure upon binding to its substrate. the binding data show a k d value in the range of 0.5-1.8 µm. the influence of different environments (lipid membranes, ddm micelles) on the secondary structure of yidc as well as on the yidc large periplasmic domain p1 was investigated by circular dichroism (cd). the cd data show that the secondary structure of yidc changes upon reconstitution into a membranes when compared to the detergent solubilized state. particularly, the p1 domain is considerably affected by the detergent c 12 pc. b. karasulu, b. erman, o. keskin koc university, istanbul, turkey histone proteins are fundamental to the cells since they are involved in cell regulatory processes, such as chromatin regulation, gene silencing and transcription, cell cycle control, and epigenetics, which are controlled via post-transcriptional modifications of the histone protein tails. these modifications are categorized under four main groups: methylation, acetylation, ubiquitination, and phosphorylation. among them methylation has been very recently proven to be reversible with the discovery of histone demethylase proteins and this modification type has been extensively studied, because the abnormal methylation rates cause the excess proliferation of the cell, which, in turn, triggers the cancer. therefore, understanding of the details of reaction mechanisms of histone methylation/demethylation dynamics provide the required knowledge basis for preventing the abnormal methylation rates by designing proper inhibitor (drug) molecules. in this study, we display possible reaction mechanisms (such as amine oxidation via lsd1) for the demethylation of specific histone tail proteins. we try to explain the role/importance of residues that take place in or near to the reaction pocket for the demethylation reaction. we also carry out md simulations and reaction path (free energy profile) analysis using free energy perturbation (fep) method for qm/mm hybrid systems in order to compare different possible reaction pathways. the sonic hedgehog (shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors patched (ptc) and smoothened (smo) , which lead to birth defects, cancer or neurodegenerative diseases. however, little is known about ptc, the receptor of the shh protein, and the way ptc regulates smo, the receptor responsible for the transduction of the signal. to develop structure-function studies of these receptors, we expressed human ptc (hptc) in the yeast saccharomyces cerevisiae. we demonstrated that hptc expressed in a yeast membrane fraction is able to interact with its purified ligand shh, indicating that hptc is produced in yeast in its native conformational state. using surface plasmon resonance technology, we showed that fluorinated surfactants preserve the ability of hptc to interact with its ligand after purification. this is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor ptc. this work will allow the scale-up of hptc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by shh signalling dysfunction. dissecting the colicin translocon c. l. johnson 1 , a. solovyova 1 , p. callow 2 , s. a. holt 3 , l. a. clifton 3 , k. weiss 4 , j. h. lakey 1 1 inst. for cell and molecular biosciences, newcastle univ., uk, 2 inst. laue langevin, grenoble, france, 3 isis, rutherford appleton lab., didcot, uk, 4 oak ridge national lab., centre for structural molecular biology, oak ridge, usa pore-forming colicin n hijacks e. coli outer membrane protein ompf and exploits it as both a receptor and translocator to cross the outer membrane [1] . it is currently a matter of debate if the translocation route is through the ompf lumen or the interface between ompf and the lipid bilayer. recent electron microscopy data from our laboratory suggests the latter route for translocation [2] . the colicin n/ompf complex in detergent has been studied by sans to examine the translocation pathway undertaken by colicin n. by using a combination of deuterated ompf and hydrogenated colicin we have been able to derive a low resolution structure of individual proteins in the binary complex. low resolution structural studies supplemented by targeted mutagenesis and screening techniques including itc, potassium efflux assays and auc have allowed us further our understanding of colicin n translocation. dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly49a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined. here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. effect of osmolytes on the dhfr activity, structure and dynamics b. legrand 1 , s. renaud 2 , m. collen 1 , c. tascon 3 , s. bonnassie 2 , e. gautier 1 , j. mellet 1 , c. blanco 2 , e. le rumeur 1 , j.-f. hubert 1 , a. bondon 1 1 rmn-ilp, 2 duals, 3 cbp, umr cnrs 6026, univ. de rennes1, france osmolytes are small molecules accumulated by a wide variety of organisms in response to hyperosmotic stress. they contribute to save the cellular integrity and to stabilize the macromolecules from environmental stress. dihydrofolate reductase activity is inhibited by several osmolytes. we studied the impact of osmolytes on the dhfr structure and dynamics by various techniques. we observe that substrate (dhf) and cofactor (nadph) diffusions are quite different in glycerol and betaine despite similar viscosities. we demonstrate that the overall structure is maintained at high osmolyte concentrations while no direct interactions can be detected with the enzyme. the k o f f of substrate analogues decreases with increasing the osmolyte concentrations. the enzyme dynamics, in various media, has been compared with the dhfr behaviour in water described in the literature. the osmolyte impact appears only partly conditioned by its viscogenic properties which reduce the molecules diffusion and the k o f f of the product controlled by the m20 loop of the dhfr. we suggest that the osmolytes decrease m20 loop mobility. comparing the results obtained with different osmolytes, we offer a better understanding of the osmolyte nature dependence of the dhfr inhibition. estrogen receptor (er) is a well characterized member of the nuclear receptor superfamily that modulates the expression of estrogen-responsive target genes in response to estradiol and other natural and synthetic chemicals mimicking the estradiol structure. in human, two ers, erα and erβ, lied on two distinct chromosomes, are known. er exhibits several functional domains: two conserved domains, a short domain c involved in dna-binding and a large domain e/f responsible for ligand-binding and hormone-dependent transcription activation, are linked by a hinge domain d; a poorly conserved a/b domain, at the n terminus, mediating interactions with the general transcription machinery, is involved in hormone-independent transcription activation. upon estrogen binding, ers can specifically bind to a dna fragment, called estrogen response element ere, and activate the transcription. the optimal ere sequence consists of two six base-pair half-sites, aggtca, organized as inverted repeats with a three base-pair spacing. in this study, we have investigated, by fluorescence methods, the effect of kcl concentrations, on the protein conformational flexibility and the thermodynamic stability of hers -eres complexes. we show, here, that electrostatic interactions, inside hers, contribute to its conformational flexibility and its thermal stability. moreover, the specific interaction between hers and eres is poorly sensitive to changes in ionic strength, in opposite to unspecific complexes. kinetic and structural explanation for the low enantioselectivity of human 3-phosphoglycerate kinase p. lallemand 1 , j. rouhana 1 , l. chaloin 1 , b. roy 2 , s. arold 3 , t. barman 1 , c. lionne 1 1 cpbs umr 5236, 4 bd henri iv cs69033, 34965 montpellier cedex 2, france, 2 ibmm umr 5247, 3 cbs umr 5048 l-nucleosides comprise a new class of antiviral and anticancer agents that are converted to pharmacologically active nucleoside triphosphates in vivo. the last step of the cascade may be catalyzed by 3-phosphoglycerate kinase (pgk), an enzyme that has low specificity for nucleoside diphosphate: ndp + 1,3-bisphosphoglycerate ↔ ntp + 3-phosphoglycerate. here we compare the kinetics of formation of the complexes of human pgk with different d-and their mirror images, l-nucleoside diphosphates, and the effect of 3-phosphoglycerate thereon. two types of experiment were carried out: equilibrium experiments allow the estimation of dissociation constants, and stopped-flow experiments the transient kinetics of the interactions. in addition, by the rapid-quench-flow technique, we compare the kinetics of the phospho-transfer and product release steps with each d-or l-nucleotide. crystallographic and molecular modelling studies allow defining the structural reasons for the low enantioselectivity of pgk. the aim of this basic work on the mechanism of action of human pgk with non-natural nucleotides is to obtain information for the optimization of therapeutic nucleoside analogues that are poorly phosphorylated by pgk. anrs is gratefully acknowledged for financial support. a new itc assay for measuring high-and lowaffinity protein-ligand interactions g. krainer 1 , j. fanghänel 2 , s. keller 1 1 leibniz institute of molecular pharmacology (fmp), berlin, germany, 2 bayer schering pharma ag, berlin, germany isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions [1] . however, reliable determination of the dissociation constant (kd) is typically limited to the range 100 µm > kd >1 nm. nevertheless, interactions characterised by a higher or lower kd can be assessed indirectly, provided that a suitable competitive ligand is available whose kd falls within the directly accessible window [2] . unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of high-and low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-affinity (kd <1 nm) and low-affinity (kd >100 µm) protein-ligand interactions. a molecular dynamics study of the cftr nucleotide binding domains interaction v. martorana 1 , r. noto 1 , o. moran 2 1 cnr-istituto di biofisica, palermo, italy, 2 cnr-istituto di biofisica, genova, italy the cystic fibrosis transmembrane conductance regulator (cftr), the dysfunctional protein in cystic fibrosis, contains two transmembrane domains, two nucleotide-binding domains (nbds), and a regulatory domain. opening of the pore have been linked to the atp-driven tight dimerisation of nbd. we have studied the nbd1-nbd2 interactions on wild type and cystic fibrosis-related mutations by steered molecular dynamics simulations (smd). a fully solvated dimer, including the two bound atps, was separated by pulling one monomer with an external, increasing force. interestingly, the force needed to break the mutated (g511d) dimer is significantly smaller than in the wild type case. the effect of a cftr potentiator, the genistein, has also been tested by repeating the smd simulations with the small molecule docked at the interface between the two nbd domains. to test the validity of our results we have repeated the separation process for different simulation lengths and force strengths. the amount of distortion on the pulled nbd domain has also been studied. this work is partially supported by the italian cystic fibrosis foundation (prog ffc #2/2008), with the contribution of "mille bambini a via margutta"onlus"blunotte", "lega italiana fc toscana" stability and aggregation studies of human septin 3 (sept3) j. n. a. macêdo, r. c. garratt, a. p. u. araújo instituto de física de são carlos, usp, são carlos, brazil the septins are a conserved family of nucleotide binding proteins firstly identified in saccharomyces cerevisae as proteins required for the completion of the cell cycle. septins are implicated in several cellular functions such as cytokinesis, vesicle trafficking, exocytosis, cytoeskeletal dynamics, cell polarity and sperm motility. furthermore, they are associated with alzheimer's and parkinson's disease. sept3 was identified in rat brain being highly enriched in presynaptic nerve terminals. it colocalizes with synaptophysin and dynamin i and is associated with synaptic vesicle. in this work, human sept3 without its n-terminal domain (sept3gc) was expressed in e. coli and purified by affinity and size exclusion cromatographies. structural stability studies were performed with recombinant sept3gc using circular dichroism spectroscopy (cd), right-angle light scattering, and fluorescence spectroscopy. the sept3gc cd spectrum showed a conformational transition from a predominantly α-helical starting structure to one dominated by β-sheet, just above physiological temperatures. the formation of irreversible aggregates, detected by light scattering, and their ability to bind thioflavin-t suggested that sept3 forms amyloidlike structures, as has been previously observed for human septins 2 and 4. our data suggest that amyloid formation by isolated septins in vitro may be a general phenomenon whose physiological relevance needs to be further investigated. pln149 is an antimicrobial peptide produced from lactobacillus plantarum nric149. analog pln149 (pln149a) and a ser-derived (pln149s, tyr to ser replacement) were synthesized on solid phase and their interactions with biomembrane model systems and inhibitory property on s.aureus and p.aeruginosa were investigated by cd, leakage assays, fluorescence spectroscopy, and differential scanning calorimetry. both peptides share the same unordered structure-like cd spectrum in aqueous solution, but a helicoidal induction in the presence of negative vesicles were observed, however pln149s showed lower helical content than pln149a. the ser-derived peptide presented 30% decrease of its leakage activity in different liposome compositions, a threefold increase in the dissociation constant from the liposomes than pln149a, and close to 40% reduction for the inhibitory activity against p. aeruginosa growth. we can conclude that besides the electrostatic contacts between the amphipathic &alpha-helix, formed by the cationic residues from pln149 and negatively charged phospholipids, the pln149-membrane binding is a process driven for the hydrophobic interactions from non-polar residues. in this case, there was a significant contribution of the tyr residue, which must be allocated in a lipid interface, described as the preferential position to this residue in membrane proteins. supported: fapesp label free detection using deep-uv laser-based fluorescence lifetime imaging microscopy q. li, s. seeger physikalisch-chemisches institut, universität zürich, winterthurerstrasse 190, ch-8057 zürich, switzerland label free detection based on native fluorescence excited at uv region shows great potential for the life sciences. it offers simple, low-cost and fast method for sensitive detection of important biological analytes without modification. in this contribution we present a deep uv fluorescence lifetime imaging microscopy system (duv-flim) based on a picosecond deep uv laser using time-correlated single-photon counting method. the described setup is well-suited for biological applications for ultrasensitive detection. we have showed single uv dye (bmq) and single protein (ecβ gal ) molecules detection using duv-flim. further, the label free detection of protein interaction between ecβ gal and monoclonal anti-ecβ gal has been demonstrated by means of steady-state and time-resolved fluorescence spectroscopy. we achieved detection sensitivity for the ecβ gal/ anti-ecβ gal pair down to the nanomolar concentration range. we also extended this method to study the interaction of therapeutic drug porphyrin with bsa protein. fluorescence resonance energy transfer between protein and alexa fluor 350 has been investigated using duv-flim. the intrinsic fluorescence and fluorescence lifetime changes of donor biotin β-galactosidase have been measured. energy transfer efficiency and donor acceptor distance have been obtained. fluorescence images of acceptor af 350 due to fret have been observed when excited at 266 nm. the monomeric heme-containing indoleamine 2,3dioxygenase (ido) catalyses the oxidative cleavage of the indole moiety of l-tryptophan (l-trp). enhanced l-trp degradation contributes to various physiological disorders including depression or failure of the immuno-regulating system. the regulation of ido by inhibitors is extensively studied. however, the catalytic mechanism of ido on the molecular level is still unknown. in addition to l-trp, a wide range of substrates are degraded including d-tryptophan, melatonine or tryptamine. in contrast, indole or histidine do not function as substrates for ido. to understand the determinants for substrate specificity, we investigate the interaction of the heme iron, the heme-bound ligand and the substrate. we use (time-resolved) uv/visible and fourier transform infrared (ftir) spectroscopy over a wide temperature range (4 -300 k) to monitor the binding of diatomic ligands to the heme iron and the binding of different substrates to coligated ido. changes in the spectra upon addition of l-trp are analyzed and compared to those induced by other substrates or inhibitors. archaeal protoglobin 3d-structure: novel ligand diffusion paths and heme-reactivity modulation protoglobin (pgb) from methanosarcina acetivorans c2a, a strictly anaerobic methanogenic archaea, is the latest entry in the hemoglobin superfamily. our previous crystallographic studies on pgb have shown that protoglobinspecific loops and a n-terminal extension completely bury the heme within the protein matrix (1) . access of o 2 , co, and no to the heme is granted by protoglobin-specific apolar tunnels reaching the heme distal site from locations at the b/g and b/e helix interfaces. here we report structural and kinetic data on pgb mutants engineered to probe the protein structural and kinetic properties. six crystal structures (pgb mutants: ∆20 (missing 20 nter residues), y(b10)61→a, y(b10)61→w, f(b12)63→w, f(g7)145→w, i(g11)149→f) show that the mutations engineered essentially restrict access to ligand tunnel 1. an accurate molecular level description of the signaling mechanism in ca 2+ transducers necessitates the knowledge of the kinetics and energetics of conformational changes associated with ca 2+ binding to various calcium binding proteins. with this in mind, we have developed an approach that combines the laser-induced photolysis of photolabile "caged" ca 2+ compound, dm-nitrophen, with the photothermal beam deflection (pbd) technique to determine thermodynamic profiles associated with the ligand binding to calcium chelator, edta, and ca 2+ sensor, calmodulin. this approach allows us to monitor time profiles of volume and enthalpy changes on the microsecond to millisecond timescale. the initial pbd study of ca 2+ photo-relase from ca 2+ loaded dm-nitrophen reveals the presence of two phases. the first step takes place within first 20 µs upon and is associated with a volume decrease of -7 ml mol −1 and enthalpy change of 66 kcal mol −1 . on the longer timescale (τ = 200 µs), the second event with a positive volume change of 7 ml mol −1 and enthalpy change of 8 kcal mol −1 was detected. on the other hand, ca 2+ photorelease in the presence of calmodulin is accompanied with an additional phase with a distinct lifetime and volume and enthalpy changes that reflects the metal binding to calmodulin and concomitant structural changes. antimicrobial peptides: linking partition, activity and high membrane-bound concentrations antimicrobial peptides (amps) have been intensively studied at micro and macroscopic levels for over twenty years. knowledge from these two domains has, however, contributed little to a comprehensive understanding of amp action; rather, in vivo amp performance has been only remotely correlated to biophysical properties. we focus on the assessment of peptide accumulation on bacterial membranes as an example of this separation: amp-bilayer interactions have been subject to extensive biophysical characterization, but conversion of that information into educated estimates of in vivo membrane-bound amp concentrations is lacking. this has led to the overlooking of important factors for activity. using simple partition models we were able to analyze available information on amp activity and interaction with membranes to show that unexpectedly high membranebound peptide concentrations are likely in vivo and may, in some cases, be required for triggering bacterial death. e. e. schäfer 1 , s. schuy 2 , a. janshoff 1 1 georg august-university göttingen, germany, 2 johannes-gutenberg-university mainz, germany retrovirus entry into cells occurs through fusion of the lipid bilayers that surround the virus and the lipid bilayer of the host cell. fusion proteins, present on the surface of the virus membrane play an essential role in the early stage of virus entry. understanding of the molecular mechanism is important for the design and function of modern fusion inhibitors. in this project we analyze the fusion of active conformation of the envelope gp41 from the human and simian immunodeficiency viruses (hiv and siv). during the infection process gp41 undergoes a sequence of conformational changes where the n -terminal nhr develop a trimer pre-hairpin intermediate. afterwards the three chr fold back towards the central nhr and a six helix bundle is formed. this rearrangement forces the viral and the host membrane into close contact and fusion pores may induce membrane fusion. this decisive molecular step in retroviral fusion has been modeled by rational design of lipopeptide assemblies that mimic a coiled-coil structure serving as a receptor for potential antagonists. for this purpose, sslbs were functionalized in an in situ coupling reaction with peptides originating from the nhr (n-peptides) of siv and hiv to monitor the interactions with the specific chr peptides (c34 and t20). binding of antagonists to surface confined coiled-coil structures has been quantified by ellipsometry, quartz crystal microbalance and was visualized by atomic force microscopy. a. rupprecht 1 , e. sokolenko 2 , e. e. pohl 1 1 institute of cell biology and neurobiology, charité -universitätsmedizin, berlin, germany, 2 frumkin institute of physical chemistry and electrochemistry russian academy of sciences, moscow, russia the production of reactive oxygen species (ros) in mitochondria is very sensitive to the proton motive force and can be decreased by mild uncoupling, mediated e.g. by uncoupling proteins (ucp) 1 . in contrast, the activation of uncoupling proteins by ros as a negative feedback loop is a highly controversial hypothesis. ucp activation in mitochondria by 4-hydroxy-2-nonenal (hne, aldehydic product of lipid peroxidation) was first demonstrated by echtay et al. 2 . here we investigate the ability of hne to activate and/or to regulate the expression of ucp in two different systems: (i) in lipid membranes reconstituted with recombinant ucp and (ii) in primary neuronal cells. total bilayer conductance was enhanced in the presence of hne, but this effect was independent on ucp1 and ucp2. the results concerning the hne-mediated ucp expression after induction of ros-production and/or after exogenous addition of hne are discussed for three brain-associated proteins (ucp2, ucp4, ucp5) in view of their possible functions. 1. beck, v et al. 2007 . faseb j. 21:1137 -1144 . 2. echtay, k. s. et al. 2003 . t. rudack, j. schlitter, k. gerwert ruhr university, department of biophysics, nd 04 north, 44780 bochum, germany the gtpase ras p21 which is linked to the membrane via a lipid anchor, is a crucial switch in the cellular signal transduction processes that control cell growth and proliferation. docking simulations can be seriously hampered by the great difficulty to accurately estimate the ligand-protein binding affinity constant k, which is usually derived via the computation of the binding free energy ∆g. unfortunately, due to the involved logarithmic relationship, errors of less than 1.5 kcal/mol in the computation of ∆g result in about one order of magnitude inaccuracy on k. this can hinder computational methods from discriminating micromolar from nanomolar compounds. to improve the reliability of docking prediction, we make use of enhanced sampling methods, ranging from steered molecular dynamics 1 to metadynamics 2 . we also test several descriptors, such as the recently developed path collective variables 3 , to identify the most suitable reaction coordinates accounting for binding and unbinding processes. using these approaches we investigate ligand docking and undocking and we attempt to describe at an atomistic level the kinetics of binding, which we intend to exploit for drug design purposes. here, an example of application in drug design is reported. 1. isralewitz, b. et al.; j. mol. graph. model. 2001, 19 , 13-25. 2. laio, a. and parrinello, m.; pnas 2002, 99 , 12562-6. 3. branduardi, d.et al.; j. chem. phys. 2007, 126 , 054103. prediction of protein-protein complex structures using wang-landau simulations a. solernou, barcelona supercomputing center, jordi girona 29, barcelona, spain protein-protein interactions are essential in the majority of life processes, so they have keen interest in several knowledge areas. however, experimental data on protein complexes is being produced at a quite low pace in comparison to that of the individual components. thus, in recent years attention has focused into computational approaches to the proteinprotein docking problem. a variety of docking protocols have been recently reported, sharing usually the following strategy: fast rigid-body search of the interacting subunits, followed by scoring and refinement of the interfaces. although this kind of strategy has proven some good results in the capri blind test it has two main limitations. on one hand one should include full flexibility on the protein structures. on the other, the evaluation should be made with free energy calculations instead of using a scoring function. in this work we propose to search the native complex structure in the minimum of the free energy landscape. we use the coarse grained potential unres to get the potential energy of the possible conformations. they are generated changing the dihedral angles, the side chain rotamers, and lastly the mutual orientation using a new sampling protocol we have developed (rotation-based uniform sampling; rotbus). finally, the free energy calculations are performed using an omp parallelization of the wang-landau algorithm. s. shukla 1 , s. asthana 1 , g. giliberti 1 , f. luliano 1 , m. ceccarelli 2 , r. loddu 1 , p. ruggerone 2 , p. la colla 1 1 department of biomedical science and technology, università di cagliari, cagliari, italy, 2 department of physics, università di cagliari, cagliari, italy the virus encoded rdrp has emerged as a prime target in the search for specific hcv and other flaviviridae antivirals. recently, the determination of the hcv rdrp structure in complex with certain benzimidazoles has been reported, these nni's bind to the surface of the thumb domain, thereby disrupting its interaction with the finger domain, which is necessary for catalytic activity. on the other hand, we have found that, in bvdv, the mutations conferring resistance to same class of inhibitors lie in the finger domain of rdrp, indicating that the mode of inhibition of benzimidazole class of compounds is different in both hcv and bvdv rdrp. herein, we have applied docking approaches (binding orientation), molecular dynamics (md) simulations combined with metadynamics to elucidate the microscopic mechanism of the interactions between the ligand and the receptor in order to identify features barely seen in experiment. the recently designed algorithm overcomes the time scale problem by accelerating properly defined reaction coordinates. it mimics the real dynamics of a ligand staying or leaving the receptor and in doing so reconstructs the free energy surface, which in turn gives an idea of the residence time of the inhibitor in the cavity.finally we identified the binding modes and different mechanism of inhibition of benzimidazole class of compounds in rdrp of two closely related rna viruses. . c. seifert 1 , w. stacklies 2 , f. graeter 2 1 protein mechanics and evolution, bioquant, inf 267 bq0031, 69120 heidelberg, germany, 2 ag graeter, picb, 320 yueyang lu, shanghai 200031, china we use a new method that detects force distribution in proteins. based on molecular dynamic simulations, changes in inter-atomic forces are calculated, here caused by different ligands. these changes will then be analyzed to detect a signal transfer through a protein initiated by the binding of a ligand to the protein. chaperones are ubiquitous proteins, which help other proteins to fold into a native conformation. they are able to refold misfolded proteins with a great variety of mechanisms. in this project, our group focuses on molecular dynamic simulations of htpg, an e. coli homolog of the human hsp90 (heat shock protein 90kda). the full structure of htpg was published by shiau et al. in 2006, but the mechanism of how htpg performs its function is still not understood. the folding mechanism is an atp driven reaction cycle, in which the functional entity is a homodimer of htpg. we separate the cycle in three main states: apo, adp-and atpbound state. conventional molecular dynamics simulations are used to build a stable simulation system and provide structure trajectories and primary information about the behavior of htpg in its different states of the folding process. experiments indicate that the molecular movement is atp driven. we use force distribution analysis to elucidate how atp effects htpg conformation and dynamics. the small size of myoglobin makes it the preferred candidate to investigate the structure-function paradigm. in its interior five docking sites have been identified and for long time these xenon cavities have been classified as packing defects. recently, it was shown that they might be involved in ligands migration path. however, some questions regarding its role as oxygen carrier no scavenger remain yet open as well as the microscopic mechanism regulating these biological functions. in this work we made use of standard md simulations of solvated myoglobin to characterize internal cavities. our principal results is that we have found several secondary cavities showing volume and occurrence at least comparable to that of xenon cavities. in order to analyze and rationalize internal cavities we applied special cluster-analysis: we classified all cavities with respect to the position, size and occurrence ascribing them to different clusters. this analysis highlights possible intrinsic migration paths for small ligands within the protein matrix controlled by spontaneous fluctuations of the protein itself. moreover, we identified some key residues playing a fundamental role in controlling internal pathways. our suggestion that the secondary cavities constitute the preferred path for ligand escape is also supported by explicit metadynamics simulations of ligand escape. a. varga 1 , p. lallemand 2 , j. szabó 1 , p. závodszky 1 , m. vas 1 , t. barman 2 , l. chaloin 2 , c. lionne 2 1 institute of enzymology, brc, hungarian academy of sciences, budapest, hungary, 2 cnrs-université montpellier 1 -université montpellier 2, institut de biologie, umr5236, montpellier, france 3-phosphoglycerate kinase (pgk) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. pgk is a key enzyme in glycolysis; it catalyses the reversible reaction 1,3-bisphosphoglycerate + adp ↔ 3-phosphoglycerate + atp. here we explored the catalytic role in human pgk of the highly conserved lys 215 that has been proposed to be essential for pgk function, by a transient and equilibrium kinetic study with the active site mutant k215a. by the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type pgk and mutant k215a. by the use of a chemical sampling method (rapid-quench-flow) under multi and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild type pgk follows product formation, whereas with the mutant it is the phospho-transfer step itself that is rate limiting. these data are supported by saxs measurements which showed no direct role of lys 215 in the domain closure of the enzyme i.e. the isomerisation step of the reaction. the results are explained by the structural data of the enzyme. characterization of different recombinant nrp1 proteins and interactions with heparin k. a. uniewicz, y. ahmed, d. g. fernig school of biological sciences and centre for glycobiology, biosciences building university of liverpool, l69 7zb liverpool, u.k. neuropilin-1 (nrp1) is a mammalian membrane glycoprotein involved in tip sprouting processes like angiogenesis and neurogenesis. it has been shown that the interaction of nrp1with heparin/heparan sulfate is implicated in its enhancement of growth factor signalling, however the mechanism is not yet known. commercially available extracellular domain of nrp1 is available either as a truncated his-tagged human protein (hnrp1) or as the rat protein fused to histagged human fc and expressed as a dimer (fcrnrp1). biochemical properties such as kinetics of heparin binding and structural requirements for sugar binding together with biochemical tests of protein properties were performed in order to characterise these commercial proteins. fc rnrp1 shown high affinity to heparin (kd=2.5 nm) and required a minimum of dp10 to effectively compete with fc rnrp1 binding to immobilised heparin. competition experiments with various modified sugars show that interaction of fc rnrp1 with heparin is highly ionic and dependent on the position of sulfate groups along the heparin chain. the hnrp1 did not bind to heparin immobilised via nhs-biotin, though it did bind to some heparin affinity chromatography matrices. organic compounds of tin are among wide spread environmental pollutants. due to physical and/or chemical actions, poly-substituted alkyltins speciate into less substituted, more toxic species. tetra-or tri-alkyltins show a marked delay in their toxic action with respect to the corresponding di-and mono-alkylated analogs. it has been hypothesized that the delayed toxicity may results from the progressive dealkylation of alkyltins in more toxic di-and mono-derivatives, which bind and inhibit essential enzymes. it has been proposed that alkyltins preferentially target enzyme sulphydryl groups. previously, we showed that a nine amino acid linear peptide (i 1 lgcwcylr 9 ) containing a cxc motif is able to bind and dealkylate tri-substituted alkyltin compounds into the corresponding dialkyl derivatives. here, we investigated the time dependence of the degradation of the most common alkyltin derivatives by this peptide. we monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position 5 of the peptide. we found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudo-enzymatic reaction mechanism. the end-point of the reactions was the formation of a covalent complex between the disubstituted alkyltin and the peptide. these data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds. parkinson's disease (pd) is a multifactorial neurodegenerative condition characterized by the progressive loss of dopaminergic neurons in the substantia nigra and by the presence of intracellular inclusions, composed predominantly of fibrillar alpha-synuclein (as). post-mortem studies indicate the presence of oxidative damage in the nigral neurons. dopamine oxidation, which leads to the formation of highly reactive quinones (daq), may account for the specificity neurodegeneration observed in pd. daq have many potential protein targets for chemical modifications. among them, we focused our attention on dj-1, of which mutated forms have been found in familial cases of pd. a possible function of dj-1 is its redox-dependent chaperone activity that could prevent as aggregation and fibril formation. in the present work, we analyzed the structural and functional modification induced on dj-1 by daq. 15 n-hsqc spectra of dj-1 were recorded in the presence of different amounts of daq and chemical shift perturbations were used to identify the dj-1 residues target of daq and their relative reactivity toward daq. aggregation assays were also performed to evaluate the functional effects of the daq modifications on the chaperone activity of dj-1. rabies virus (rabv) infects neurons exclusively and causes lethal encephalitis. pathogenic rabv strains favor neuronal survival, whereas non-pathogen strains lead to neuronal apoptosis. the use of recombinant rabv showed: 1/ the g protein determined the induction of the survival or death phenotypes; 2/ the last 4 cooh amino acids of the g protein cytoplasmic domain (cytog) are critical. these residues form a binding site for pdz domain (pdz-bs). one of the 6 amino acid differences between survival and death gproteins are located in this pdz-bs. results of two-hybrid experiments showed that cytog survival interacted pdz domain of ser/thr kinases (mast), while cytog death interacted also with 3 additional pdz containing host proteins. to understand the fine structural basis for the specificity of the pdz-cytog complexes, we determined the 3d structure and the dynamics of the mast-cytog complexes by nmr. the structures, as well as the affinities and constant kinetics, of the mast2 pdz complexes with both cytog are similar. we conclude that difference by one aa in the pdz-bs of the two strains cannot drastically modify the interaction with mast2-pdz, in agreement with the two-hybrid data. preliminary results suggest that the interaction of cytog death with one additional cellular partner blurs the pro-survival signals engaging the infected cells through apoptotic trails. functional protein immobilization on glass-type surfaces s. waichman, m. bhagawati, y. podoplelova, j. piehler institute of biology, department of biophysics,university of osnabrück , barbara st.11 osnabrück, germany the immobilization of membrane proteins onto solid supports enables protein interactions and conformational changes to be probed by spectroscopic techniques under highly defined conditions. here, we present a novel method for covalent protein immobilization on glass-type surfaces using a bottom-up approach. in this approach the 4'phosphopantetheinyl group of coenzyme a (coa) was transferred to the acyl carrier protein-derived ybbr tag of the target protein by means of the phosphopantetheinyl transferase sfp. the glass-support was rendered biocompatible by coating it with an ultrathin layer of peg (polyethylene glycol), followed by functionalization with coa through maleimide chemistry. immobilization of ybbr-tagged proteins in presence of sfp was followed in real time by label-free detection using reflectance interference spectroscopy. the immobilization procedure was thus systematically optimized by means of binding specificity, enzyme activity and functionality of the immobilized protein. this approach was employed for immobilizing the type i interferon ifnα2 in order to probe ligand recognition by ifnar1 and ifnar2 and ligand-induced ternary complex formation. the versatility of this technique was further enhanced by its combination with photopatterning methods. this immobilization technique can provide a beneficial tool for bioanalytical and biophysical applications at the single molecule level. r. vijayan, p. c. biggin department of biochemistry, university of oxford, south parks road, oxford, ox1 3qu, uk ionotropic glutamate receptors mediate excitatory synaptic transmission in the brain and are heavily implicated in memory and learning as well as in numerous neuropathological conditions. one family of iglurs, the kainate receptors, show unusual sensitivity to changes in external ion species resulting in an apparent requirement for both sodium and chloride ions for activation. our recent work revealed the location and selectivity of the cation binding sites. despite this progress, it is still unclear how the cation binding sites confer sodium selectivity and how apparent affinity for chloride is influenced by the presence of cations. we have attempted to address these questions by performing extensive free energy calculations using all-atom molecular dynamics simulations. the rank order of cation binding obtained from relative binding free energy calculations is in agreement with experimental measurements of apparent affinity. these calculations also reveal that the pair of cation binding sites in the dimer interface act independently. binding free energy calculations performed using a reduced model of the binding site show that cation selectivity can been attributed to both the rigidity and high charge density of the binding sites. finally, a potential of mean force derived from umbrella sampling simulations indicate that the presence of cations stabilize the anion binding site considerably. the effect of toxins on the inorganic phosphate release during the actin filament formation a. vig, t. kupi, g. hild, m. nyitrai university of pécs, faculty of medicine, department of biophysics, szigeti str. 12, 7624 pécs, hungary actin can be found in monomeric (g-actin) and filamentous (f-actin) form in eukaryote cells under physiological circumstances. the first step of actin polymerisation is the formation of actin nuclei by atp-binding actin monomers. the next step in is the elongation when monomers are associated to the previously formed nuclei or to the ends of the growing filaments. after the association of monomers the bound atp is hydrolysed to adp.p i , and with first order kinetics the release of inorganic phosphate occures. the rate constant of the release is 0,006 s −1 , which is a slower process than the hydrolysis itself (0,02 s −1 ).phalloidin, a cyclic peptide from amanita phalloides can bind to the filaments and stabilizes their structure. jasplakinolide is another cyclic peptide from marine sponge (jaspis johnstoni) which binds actin filaments. the aim of this study was to investigate whether the binding of toxins to the newly created filaments has an effect on the kinetics of the inorganic phosphate release or not. we used absorption photometry measurements to measure the rate of phosphate release. phalloidin decreased the rate of the release substantially. although the effect of jasplakinolide was weaker, the results showed that the binding of these toxins to the actin can modify the rate of the release of inorganic phosphate from the filaments. these observations are in agreement with the molecular mechanisms by which these toxins stabilise the actin filamens. fluorescent proteins (fps) are invaluable fluorescent markers in cell biology. however, their use is often limited by photobleaching of the chromophore, notably in single-molecule, time-resolved or super-resolution imaging studies. we will present the crystallographic studies at near atomic resolution of a photo-activatable fluorescent protein irisfp that has been observed in a transient radical state en route to photobleaching. we took advantage of x-rays to populate the radical, which, under illumination with visible light, presumably forms with low probability from the triplet state. the combined x-ray diffraction and in crystallo spectroscopic data (from uv-vis and raman spectroscopies) reveal that radical formation in irisfp involves strong but reversible distortion of the chromophore, suggesting a transient loss of pi-conjugation. these results will help unravel the mechanisms of blinking and photobleaching in fps, which is of importance to rationally design variants of higher photostability. optimizing photoactivatable fluorescent proteins for live-cell imaging recently, novel fluorescent proteins (fps) have been reported which perform spectral changes in response to irradiation with light of a particular wavelength. reversibly photoactivatable proteins switch between a bright and a dim fluorescent state. this process is accompanied by photoisomerization of the protein chromophore. irreversible photoactivation results from photochemical processes within the protein, e.g., photolysis of an amino acid side chain, or an extension of the alternating π-electron system of the chromophore by a β-elimination reaction. fps have became valuable tools in live-cell imaging because they allow intracellular protein labeling by using them as fusion tag, and photoactivatable fps are powerful tools for application in novel subdiffraction imaging techniques. however, the available fps still offer potential for improvement in various ways. they frequently show a tendency to aggregate or oligomerize, incomplete chromophore maturation, fluctuating emission and low photostability. here, we will present our recent progress towards engineering the 'perfect' fp. simultaneous intracellular chloride and ph measurements using gfp-based sensor in ldcv d. arosio 3 , f. ricci 1 , l. marchetti 2 , l. albertazzi 1 , f. beltram 2 1 italian institute of technology, udr pisa, italy, 2 nest, scuola normale superiore, pisa, italy, 3 nest, infm cnr, pisa, italy chloride ion participates in many physiological functions including control of neuronal resting potential, charge balance during endosome acidification, and regulation of cell volume. as a consequence dysfunctions in regulating membrane chloride permeability lead to severe diseases including motor disorders, cystic fibrosis and epilepsy. at present processes regulating intracellular chloride ion concentrations are still widely unexplored mainly as a consequence of limiting methods to quantify chloride fluxes in living cells. in the present work a highly specific, genetically encoded sensor is developed for detecting simultaneously intracellular ph and chloride concentration. the sensor is obtained by fusion of a red fluorescent protein (dsred-monomer), insensitive to chloride and ph, to a gfp variant containing a specific chloride-binding site (gfp-chl). dsred-gfp-chl binds the chloride ion following a fluorescence static quenching mechanism, which allows measurements of intracellular ph in a chlorideindependent manner. the sensor has been successfully tested in different living cells, in a ph range 5-8 and chloride concentration up to 200 mm. for the first time, to the best of our knowledge, it allowed to measure the chloride concentration of dense core vesicles in the secretory pathway. applicability to high-throughput screening, range of validity and accuracy of time-lapse maps will be discussed. we aim to understand the basis of the photophysical changes in fluorescent proteins (fp) induced by reactive oxygen species (ros). indeed, ros might be involved in photochemistry of fp, leading to their photobleaching or photoconversion. in addition, fp may be used to investigate cellular events like phagocytosis or mitochondrial activity, where ros are naturally produced. in the latter cases, an accurate analysis of fp's fluorescence signals requires the full knowledge of reactions between ros and fp. in the future, this work may help in developing either photoresistant or photoswitchable fp and improving their use for imaging under oxidative stress conditions. using γ-radiolysis as a quantitative source of ros, we investigated the reactions of oh and o 2 − radicals on the cyan fluorescent protein (cfp) and the modifications of the cfp's photophysical properties by oh radicals were explored in detail (submitted). in order to address the corresponding chemical changes in the protein, we devised a mild proteolysis protocol that for the first time offers a peptide mass fingerprint almost covering the cfp sequence (alvarez et al. biochemistry 2009). then, we achieved the meticulous characterization of the cfp oxidation products by mass spectrometry and proposed a mechanism to account for their formation by pulse radiolysis. since the cloning of the green fluorescent protein from aequoria victoria, numerous screens have been performed to improve the brightness of this protein, its spectral variants and fluorescent proteins from other species. the improvement is evaluated by comparing fluorescence intensity of individual bacteria or colonies. in this way also expression level, folding and maturation efficiency, and thickness of the bacterial colony contribute. here we report a screening method that, in addition to fluorescence intensity, quantifies the excited state lifetime of a fluorescent protein, which is independent of intensity or expression level, and provides a direct measure for the quantum efficiency of the fluorescent protein. the novel approach was used to screen a library of cyan fluorescent protein (cfp) variants randomly mutated at position 65, 148 and 224, yielding an improved bright cyan fluorescent protein named, mposeidon, with a markedly increased fluorescence lifetime and quantum yield, increased photostability and improved fluorescence intensity in vivo. it is shown that mposeidon is the brightest cyan fluorescent protein in mammalian cells. in addition, several lifetime variants were identified that can be used for lifetime unmixing. it is demonstrated that three cfp variants can be separated and their distribution quantified in a single detection channel. a. r. faro 1 , p. carpentier 2 , d. bourgeois 3 , e. rosny 4 1 irtsv, cea, ufj, grenoble, france, 2 ibs, cea, cnrs, ufj, grenoble, france, 3 ibs, cea, cnrs, ufj, grenoble, france, 4 ibs, cnrs, ufj, grenoble, france enhanced yellow fluorescent protein (eyfp) is extensively used as a fluorescent marker. like other photo activatable fluorescence proteins (pafp), it exhibits photo-switching properties. however, the mechanism by which fluorescence can be swiched on or off upon light irradiation is not fully understood at the molecular level. the bright to dark conversion involves a protonation step and structural rearrangements of the chromophore, but it is not clear which of these two steps is the triggering event. to answer this question, we carried out photo-switching experiments at cryotemperatures. our data suggest that a photo-induced protonation step (probably in the triplet state) is the primary event in the bright to dark conversion. our results may bear relevance to other pafps, such as iris, eos, dronpa, padron or kaede. how misfolding and aggregation of proteins constitutes a toxic insult to neurons remains largely unknown. in order to obtain insight into the molecular biology of neurodegenerative disease, we have developed a number of gfp-based biosensors for the detection and quantification of cellular clearing mechanisms for aggregated proteins. the high load on these protein quality control mechanisms, and their failure to meet normal physiological demand ultimately results in a "de-compensation" condition from which the nerve cell cannot recover. our fret/flim-based bioassays visualize protein ubiquitination and degradation; proteasomal activity; foldase activity using a folding-impaired gfp mutant which gains fluorescence conditional on the upregulation of chaperone activity; chaperone binding to unfolded proteins; and autophagosome formation/lysosomal integrity via the targeted and sensitive fret-based measurement of ph changes. these sensors are employed in cellular model systems for parkinson's and alzheimer's disease, and amyotrophic lateral sclerosis (als) to delineate the molecular pathway of cellular demise, and to gain a mechanistic understanding of the toxicity of protein aggregates and the basis for the vulnerability of neurons. millisecond photo-switching dynamics of e222q gfp mutants for sensor and imaging applications m. collini 1 , v. quercioli 1 , l. d'alfonso 1 , g. baldini 1 , b. campanini 2 , s. bettati 2 , g. chirico 1 1 dipartimento di fisica, università di milano bicocca, italy, 2 dipartimento di biochimica e biologia molecolare, università di parma, parma, italy e222q mutants of the green fluorescent protein are known to possess photo-chromic properties: the anionic emission, primed by a pump 488 nm laser beam, can be switched between two levels of different brightness by irradiation with a blue, ∼ = 400 nm, probe laser light. we have studied here the amplitude and the dynamics of the brightness enhancement of the e222q mutant of gfpmut2. the fluorescence emission increases almost threefold, under saturating probe laser excitation, for pump excitation intensity in the linear regime. two characteristic activation times, estimated by means of modulated two colour fluorescence correlation spectroscopy, are detected in the 1-30 ms range, independent of solution temperature and viscosity. the brightness enhancement factor and the characteristic activation times depend markedly on the solution ph. these results indicate that this mutant can be used as a high sensitive intracellular marker for local proton concentration and for modulated excitation imaging. the advent of fluorescent proteins (fp) gave researchers the opportunity to study proteins in situ. fluorescence resonance energy transfer (fret) benefited from this. cell fixation is a commonly used approach when working with microscopy. however, we have found that fret efficiency (e) in cells transfected with cerulean and venus chimeras could not be reproducibly measured after fixation. to evaluate this problem in detail, we measured e of 3 cerulean-venus standard constructs by acceptor photobleaching fret, intensity-based ratiometric fret and flim-fret. the constructs were produced as standards (biophys j, 2006, 91, 99) with 43, 38 and 31% e values, comprising donor-acceptor separations of 5, 17 and 32 amino acids, respectively. transient transfection of the fusion plasmids was performed into hela cells and e was measured in live and pfa or methanol fixed cells. literature e values were reproduced when measuring live cells. conversely, cell fixation caused a deviation of e values. methanol fixed cells showed e between 1-5% for all the constructs. the effect of pfa fixation on both fluorescence intensity and fret varied vastly among independent experiments regardless of the measurement modality. thus, fixation should be avoided due to the effects it has on fp's fluorescence and consequently on fret efficiency. to explain the improvement in the fluorescence properties of cerulean when compared to ecfp, we have determined the x-ray crystallographic structures of these two proteins at physiological ph, and performed molecular dynamics simulations. both proteins exhibit a structural heterogeneity in the nterminal half of their seventh strand, which forms a specific set of van der waals interactions with the chromophore. the critical h148d mutation present in cerulean induces a modification of these interactions, and allows the chromophore to be more planar and better packed, albeit only intermittently. as a consequence, the probability of non-radiative decay is significantly decreased. our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins, and provide insights which should allow the design of mutants with enhanced optical properties. the fluorescence lifetime of green fluorescent protein g. jung biophysical chemistry, saarland university, 66123 saarbruecken, germany biotechnological design of the green fluorescent protein (gfp) and the discovery of other proteins boosted the development of the life sciences in the past decade. tracking protein movements and high resolution microscopy are only a few recent applications which were realized by fluorescent protein technology. among these examples, the switching between two chromophore state is exploited. our aim is to establish fluorescent proteins for bioanalytical fluorescence lifetime measurements. despite the progress in other fields, quantification with gfp still imposes practical problems [1] . in the past, we observed that the uv-light driven decarboxylation of the nearby aminoacids glu222 distorts the fluorescence lifetime of gfp [2] . we found out that this chemical reaction also occurs under excitation of the anionic chromophore state with a high quantum yield [3] . recently, we could show by time-resolved spectroscopy that, indeed, the more susceptible state for this kind of photoconversion is the anionic chromophore state [4] . suppression of this reaction therefore enables the design of autofluorescent proteins which can be used e.g. for the quantification of ions and which are beneficial as donors in fret applications. the fluorescent properties of tryptophan residues (w) in proteins are highly dependent on their immediate protein environment. however, the multi exponential decay of single w proteins is not completely understood. the most cited hypothesis contributes a multi exponential decay to the existence of several micro conformations (rotamers) of the w residue within the protein matrix. to determine rotamers we apply a method based on dead-end elimination (dee) and molecular dynamics simulations (md). low energy rotamers are calculated by dee while dynamics and further refinement is accomplished using md. the method was applied on several test cases including the protein mutant bc-csp l66e from bacillus caldolyticus, which contains a solvent exposed w residue. as resolved by x-ray crystallography, this w residue occupies two conformations. using dee and md we were able to retrieve the w conformations found in the x-ray structure. the results demonstrate the ability of the method to obtain valuable w rotamers, both for solvent shielded as exposed residues. the determined conformations were compatible with the findings based on a method using replica exchange simulations. k. nienhaus 1 , h. nar 2 , r. heilker 2 , j. wiedenmann 3 , g. u. nienhaus 4 1 inst. of biophysics, universität ulm, ulm, germany, 2 dept. of lead discovery, boehringer ingelheim, biberach/riss, germany, 3 national oceanography center, university of southampton, southampton, uk, 4 inst. of applied physics, universität karlsruhe (th), karlsruhe, germany eqfp611 is a red fluorescent protein (rfp) with the chromophore in a co-planar trans orientation, whereas the cis isomer is preferred by most other rfps. by using x-ray crystallography, we determined the structures of the dimeric variants d1eqfp611 and d2eqfp611 at high resolution (up to 1.1å). for d1eqfp611, we had previously seen a redshifted species upon irradiation with 532-nm light. concomitant changes in the raman spectrum were interpreted as evidence of a trans-cis isomerization of the chromophore. here we have combined x-ray crystallography and site-directed mutagenesis to assess whether we can create a stable fluorescent, red-shifted eqfp611 variant with a cis chromophore. in a first step, we introduced the n143s substitution. this variant, d2rfp630, is highly fluorescent, with the absorption (emission) maximum red-shifted by 24 (19) nm. with an additional s158c mutation, the chromophore is found completely in the cis form. the variant, rfp639, is highly fluorescent, with excitation and emission maxima at 588 and 639 nm. still further red shifts appear to be in reach. k. nienhaus 1 , v. adam 2 , d. bourgeois 2 , g. u. nienhaus 3 1 of biophysics, universität ulm, ulm, germany, 2 esrf, grenoble, france, 3 institute of applied physics, universität karlsruhe (th), karlsruhe, germany dendra2 is an engineered, monomeric gfp-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green-to a red-emitting state upon exposure to purple-blue light. this process occurs in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized π-electron system. we have measured the x-ray structure of green dendra2 and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. the structure, which is very similar to those reported for the closely related proteins eosfp and kaede, revealed a local structural change next to the chromophore, involving mainly arg66 and a water molecule. we propose that this structural change explains the blue shift of the absorption and emission bands, as well as the markedly higher pks of the hydroxyphenyl moiety of the chromophore. the 20-fold enhancement of the neutral species in dendra2 at physiological ph accounts for the observed higher photoconversion yield of this protein in comparison to eosfp. photochromic green fluorescent protein mutants: chromophore states unveiled by raman spectroscopy s. luin, v. voliani, g. lanza, r. bizzarri, r. nifosì, p. amat, v. tozzini, m. serresi, f. beltram nest, scuola normale superiore, cnr-infm and italian institute of technology, pisa, italy the most widespread genetically-encodable fluorescent markers used for studies in living cells and tissues belong to the green fluorescent protein (gfp) family. reversibly switchable fluorescent proteins (rsfps) were developed that can undergo repeated transitions between different states, e.g. bright and dark forms. this property makes rsfps particularly attractive as active labels in biological systems for selective photolabeling applications or subdiffraction imaging. we shall present pre-resonant raman results unveiling the photophysical mechanism underlying the observed photochromic behavior. the variation of spectral properties before and after photoconversion of chemically-synthesized isolated chromophores under different protonation and/or isomerization have been analyzed, and compared to results obtained for the case of complete folded proteins comprising the same chromophores. experimental results have been analyzed within a time-dependent density functional theory, allowing us to assign all relevant vibrational modes. these results make it possible to discriminate between the effect of cis-trans isomerization and of diverse protonation states of the chromophore in the photoproducts of these proteins. s. luin et al, j. am. chem. soc. 131, 96-103 (2009 ). r. bizzarri et al., anal. bioanal. chem. 393, 1107 -1122 . a combined study of the interaction of outer membrane proteins with cephalosporin antibiotics m. lovelle, i. barroso, m. j. feio, p. gameiro requimte , fac. ciências, universidade do porto, portugal gram-negative bacteria characteristically are protected by an outer membrane that serves as a selective permeation barrier. most of the β-lactam antibiotics appear to penetrate the outer membrane through these non-specific channels, and it becomes important to understand the possible interactions between β-lactams and the porin. fluorescence techniques have been largely used to characterize both the conformation and the dynamic behavior of large biological structures such as membranes and proteins. the fluorescence of the tryptophan residues is quenched in the presence of the different cephalosporin antibiotics. this reaction between the excited state of the fluorophores and the drug can be described as a formation of a non-fluorescent complex. the dependence of the fluorescence intensity upon quencher concentration for static quenching is proportional to the binding constant for complex formation. since β-lactam susceptibility is closely related to the presence of these non-specific porins, minimum inhibitory concentration (mic) by micro-broth dilution in microplate were used to assess the bactericidal activity of cephalosporin antibiotics upon on escherichia coli strain bl21(de3 ) and a series of bl21(de3) mutated in different outer membrane proteins. this combined study of the interactions at single molecular level and at in vivo level provides new insights for a better understanding of the antibiotic translocation. when used in combination with e.g. a cyan fluorescent protein, keima offers the unique opportunity to perform dual color fluorescence cross-correlation spectroscopy using a single laser line to excite both fluorophores. the molecular determinants of the large stokes shift of mkeima have been characterized structurally by combining x-ray crystallography with in crystallo uv-visible absorption, fluorescence and raman spectroscopy. our results reveal a ph-dependant "reverse chromophore protonation" of mkeima, driven by the key residue asp157. moreover, the chromophore protonation state is shown to be coupled with different chromophore conformations, cis conformation at ph 3.8, and mostly trans conformation at ph 8.0. these results will help unravel the mechanisms of fluorescence in fps, which is of importance to rationally design and develop new fluorescent markers. recently reversibly switchable fluorescent proteins (rsfps) have become a new branch of the green fluorescent protein (gfp) like family. the rsfps may be reversibly switched between a fluorescent and a non-fluorescent state by irradiation with light of distinct wavelengths. the key process of this switching behaviour is a light induced change of the chromophore between a cis-and a trans-isomeric state. the proteins are becoming increasingly important for diverse applications like protein tracking, sub-diffraction resolution microscopy and data storage. based on the switching mechanism, we created novel rsfps with unique and improved characteristics. padron and bs-dronpa are two of these new switchable proteins. padron features a reversed switching mechanism as compared to all other green rsfps known to date while bsdronpa exhibits a very broad absorption spectrum extending into the uv. utilizing the characteristics of both proteins, we performed multi label single detection colour microscopy as well as dual colour sub-diffraction microscopy, the latter with a resolution of 20 nm. further, we recently introduced the first monomeric rsfp exhibiting red fluorescence: using a semi rational mutagenesis approach on the non-switchable mcherry, we generated the switchable monomeric protein rscherryrev. the use of this protein in single molecule switching microscopy allowed us to record time-lapse live-cell images of the endoplasmic reticulum with sub-diffraction resolution. a spectroscopic approach to the study of chromophoric dissolved organic matter (cdom) in the sea c. santinelli, r. lavezza, l. nannicini, a. seritti cnr-ibf, via moruzzi 1, 56124 pisa, italy dissolved organic matter (dom) in the ocean represents the largest reservoir of reactive carbon on the earth. it is produce at each level of the marine food web and it represents the food for heterotrophic bacteria, which can use the different pools of dom (labile, semi-labile, refractory) with a large range of turn-over times (from minutes to millennia). dom plays a central role in the global carbon cycle and it has a high ecological significance. chromophoric dom (cdom) is the fraction of dom capable of adsorbing light at uv and visible spectral regions. it determines the underwater light availability in open and coastal regions with important implication on primary production and biological activity. it is photodegraded to co 2 , co, with a significant impact on the role of the ocean as source and/or sink of atmospheric co 2 and to highly reactive compounds, dangerous for marine organisms. in its pool "humic-like" and "protein-like" fluorophores have been individuated. cdom optical properties (absorption and fluorescence) together with doc data collected in some key regions of the mediterranean sea will be presented and discussed, in order to (i) investigate the powerful of cdom optical properties to get information on cdom "quality" (i) asses the role of dom in carbon export at depth, (iii) underline the main unresolved question on dom and cdom dynamics in the ocean, with particular emphasis to their biological lability. mechanism and applications of photo-and redox-switchable fluorescent proteins s. j. remington, j. n. henderson, x. shu, j. lohman institute of molecular biology, university of oregon, u.s.a. photoswitchable fluorescent proteins have significant advantages over conventional fluorescent labels, and in a revolutionary application, now allow cell biologists to exceed the diffraction limit in light microscopy by a factor of ten. atomic resolution crystal structures and time resolved spectroscopy of both reversible (mtfp0.7) and irreversible (pa-gfp) photoswitchable fluorescent proteins in the light and dark states explain the long term stability of either state, as well as how illumination at the appropriate wavelength causes the molecules to switch between states. the photoswitching mechanisms will be discussed in terms of photochemistry, light induced chromophore isomerization and excited state proton transfer (espt). mutagenesis of espt pathways provide insight into the nature of the ratedetermining steps in proton transfer and lead to practical applications, such as redox-sensitive gfp biosensors. k. i. willig, b. hein, s. w. hell max-planck-institute for biophysical chemistry, goettingen, germany stimulated emission depletion (sted) nanoscopy is a light microscopic technique offering a resolution far beyond the diffraction limit: the excitation beam is overlapped with a doughnut-shaped, red-shifted sted beam, which switches off the ability of the molecules to fluoresce in the outer region of the excitation spot. scanning the nanosized focal spot through the sample renders sub-diffraction images with a sub-second frame rate. we used the yellow fluorescent protein citrine to image individual structural elements of living mammalian cells: vimentin and the tubular network, structures of the cytoskeleton, were recorded with a lateral (x,y) resolution < 50 nm inside the living cell, corresponding to a 4-fold improvement over that of a confocal microscope. also, time lapse sted imaging of dendritic spines of yfppositive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. as an alternative to fluorescent proteins we used a genetically encoded protein tag which can be labelled with modified organic dyes within living samples. thus nanoscale imaging of structures in the interior of living cells greatly expands the scope of light microscopy in cell biology. pulling membrane tubes from solid-supported lipid bilayers with atomic force microscopy j. w. armond 1 , j. v. macpherson 2 , m. s. turner 1 1 department of physics, university of warwick, u.k., 2 department of chemistry, university of warwick, u.k. information on the elastic and dynamic properties of membranes is essential for a thorough understanding of biological processes such as exocytosis, endocytosis, cell division, and pore formation. we use an atomic force microscope to pull on solid-supported lipid bilayers and observe that an energy barrier must be overcome prior to the formation of membrane tubes. we have modified elastic models of lipid bilayer vesicles to describe the free energy of a planar lipid bilayer in adhesive contact with a surface. from this model we are able to extract force-distance curves for the formation of a membrane tether, including the associated energy barrier. the experimental data can thus be understood in a quantitative fashion. this work enables the atomic force microscope as a quantitative instrument for measuring membrane pulling mechanics, and in future work will allow two-dimensional mapping of elastic properties under tension. from pores to micelles -a peptide-membrane interaction study t. l. andresen, j. r. henriksen technical university of denmark, dtu nanotech, frederiksborgvej 399, b124, 4000 roskilde, denmark. email: thomas.andresen@nanotech.dtu.dk. research in the partitioning of peptides into lipid membranes has been intense for several years. along with scattering techniques and nmr, isothermal titration calorimetry (itc) has proven to be a powerful tool for thermodynamic characterization of peptide-membrane interactions. we have studied two antimicrobial peptides, mastoparan-x and melittin, and found that these peptides induce a range of structural transitions of popc and popc/popg membrane systems at different peptide-lipid ratios. we have found that itc can be used to elucidate the threshold where transitions occur, including the threshold for pore formation and micellation. this has been achieved using a thermodynamic model based on gouy-chapman theory, which provides the partitioning constant of the peptide-membrane interaction and thereby the concentration of peptide on the membrane surface. the structural changes have furthermore been visualized by cryo-tem. we have further investigated the pore formation in detail and found that the thermodynamic parameters of pore formation can be fully characterized using a system specific temperature where the enthalpy of peptide partitioning becomes zero. this allows for an exclusive study of the pore formation process. lactoferrin is a glycoprotein with two globular lobes, with of two domains each. since its discovery, the research on antimicrobial properties has been extended to peptides derived from this protein. the largest family studied so far is known as lactoferricin b, obtained from the protein by digestion with pepsin. more recently, a new family of antimicrobial peptides derived from lactoferrin was discovered by bolcher et al and named lactoferrampin (lfampin). the original sequence of lfampin contained residues 268-284 from the n1 domain of lactoferrin. we studied 3 peptides derived from lfampin obtained by extension and/or truncation at the c-or n-terminal sides, keeping the essential characteristics, in order to unravel the main structural features responsible for antimicrobial action. the peptides were tested against model membranes. the ability to adopt helical conformation was followed by cd, the perturbation of the membrane phase transition by dsc, the energetics of interaction by isothermal titration calorimetry (itc), the partition of the peptide to the membrane by trfs and the importance of charge effects assessed by zeta potential measurements. the results are discussed and compared to the antimicrobial and hemolytic activities obtained by microbiology techniques. defensins are small cysteine-rich cationic proteins or peptides found in both vertebrates and invertebrates. they can be active against bacteria, fungi and many enveloped and non-enveloped viruses; thus, being also classified as antimicrobial peptides (amps). in the present work a comparative study between psd1 (a plant defensin with antifungic properties obtained from the garden pea pisum sativum) and hnp1 (a human neutrophils defensin) was conducted, in order to shed some light on the mechanism of action at the molecular level of these defensins. large unillamelar vesicles with different lipid compositions were used for this purpose as biomembranes model systems; namely, popc/cholesterol (characteristic of the outer leaflet of vertebrates cell membranes) and popc/ergosterol (fungal) mixtures. changes on the intrinsic fluorescence of the tryptophan residues present in these peptides upon membrane binding/insertion were followed by fluorescence spectroscopy. experimental results show the affinity of both defensins for mammalian and fungal sterols. the partitioning behavior of psd1 showed a high selectivity for ergosterol rich membranes, while hnp1 has preference for cholesterol rich membranes. preliminary characterization of atomistic computer models of galactolipid bilayers k. baczynski, m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university, krakow, poland the main lipid component of thylakoid membranes are galactolipids, which constitute more than 75% of its lipid composition. the most common types of galactolipids found in thylakoid are monogalactosyldiacylglycerol(mgdg), whose headgroup consists of a single molecule of beta-d-galactose and digalactosyldiacylglycerol(dgdg), whose headgroup consists of two galactose molecules: alpha-d-galactose and beta-d-galactose linked by o-glycosidic bond majorty of thylakoid galactolipid have gamma-linolenic acid both in the sn-1 and sn-2 position. atomistic computer models of mgdg and dgdg molecules have been constructed and parametrized in the opls-aa force field. the molecules were used to construct three bilayer systems for molecular dynamics (md) simulations:(1) composed entirely of dgdg molecules,(2) composed entirely of mgdg molecules,(3) composed of a mixture of dgdg and mgdg molecules the ratio 1:2. the systems have been md simulated using the gromacs 3.3.1 package. the generated trajectories were analysed to determine a number of structural parameters among them: membrane thickness, average area per lipid, electron density profile accross the bilayer, order parameters, organisation of the bilayer/water interface as well as several dynamic parametrers like diffusion coefficients, lifetimes of conformational states, lifetimes od lipid lipid interactions. single mixed-lipid guv method reveals interaction of viper venom with lipid membranes n. m. ayvazyan, n. a. zaqaryan, n. a. ghazaryan dpt. biophysics, yerevan state university, armenia studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including bilayer lipid membranes (blms), small and large unilamellar vesicles. giant unilamellar vesicles (guvs) with a mean diameter of 30 µm have a minimum curvature and mimic cell membranes in this respect. guvs are ideal for studying lipid/lipid and lipid/protein interactions using microscopy techniques with membrane fluorescence probes. guvs were formed from the total lipid fraction from bovine brain by the electroformation method, developed by angelova and dimitrov (1987) . vipera lebetina obtusa venom was added to the sample chamber before the vesicles were formed. the membrane fluorescence probes, ans and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. ans and pyrene allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. the presence of viper venom in guvs media reveals a noticable decreasing of membrane fluidity compare the control, while the binding of fluorophores with guvs modified by venom lead to appearance of channel activity. it was recognized early that the vipers' venom components preferred an organized lipid substrate near the lipid's phase transition and were particularly active against micellar lipids. these studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom. the attenuated total reflection fourier transform infrared (atr-ftir) spectroscopy is ideal for highly absorbing samples such as water suspensions or even bulk water due to the small light penetration depth. it is also suited for experiments on lipid membranes in excess water. for example, we have studied the hydrogen bonding (h-bonding) between cholesterol (ch) and phosphatidylcholine (pc) or sphingomyelin (sm), which could be important for the stabilization of the cholesterol-rich lipid domains. the atr-ftir method enabled comparison of the carbonyl band shape for pc/ch samples in excess h 2 o or d 2 o, and has confirmed similar behavior for both [1] . secondly, we were able to analyze the amide ii band for sm/ch samples in excess h 2 o. the observations confirm the presence of h-bonds between ch and n-h group in sm [2] . another example is the study of the interlamellar water structure, which could influence the water-mediated phenomena in membranes. h-bonds in interlamellar water in partially hydrated lipid multibilayers are stronger with respect to bulk water. in contrast, we show by atr-ftir spectroscopy that the h-bonds are weaker in multibilayers in excess water [3] . the bactericidal activity of antimicrobial peptides is linked to their ability to perturb the permeability of bacterial cells. they often show α-helical conformation, and many peptides have a kink in the middle of this structure, caused by pro or gly. in order to understand the role of the kink we designed various analogues of p5, in which the central pro residue was moved from its central position, or removed altogether (in analogue p5f). displacement of the pro residue caused a decrease of the antimicrobial activity, and an increase in the toxicity against erythrocytes, with the less active and more toxic peptide being p5f. fluorescence studies suggest that both p5 and p5f bind on the membrane surface. fluorescence experiments show that the activities of the two analogues correlate with their affinity for different kinds of lipid bilayers: the kinked p5 peptide has a dramatically higher affinity for negatively charged vesicles (mimicking the composition of bacterial membranes) than for neutral liposomes (similar to mammalian cells), while analogue p5f exhibits comparable affinities for both membranes. therefore, our data suggest that the central pro-induced kink is involved in selectivity, inhibiting peptide binding to the membranes of eukaryotic cells. d. behn 1 , h. hoffmeister 2 , r. witzgall 2 , c. steinem 1 1 institute for organic and biomolecular chemistry, university of göttingen, germany, 2 institute for molecular and cellular anatomy, university of regensburg, germany polycystin-2, encoded by pkd2, is an integral membrane protein with a size of 110 kda and 968 amino acids. the protein, which is known to be a ca 2+ permeable, non selective cation channel, interacts with several proteins such as polycystin-1, pigea14 or pacs-1/-2 etc. the interaction takes place through a proposed coiled-coil domain of polycystin-2 located at the c-terminus of the protein.if mutation of either pkd2 or pkd1 (gene product of polycystin-1) occurs, the interaction between the proteins is disturbed leading to increased formation of renal cysts. this autosomal polycystic kidney disease (adpkd) is one of the most common genetic diseases causing renal failure due to the enormous cyst formation. in this study, the interaction of the c-terminus of polycystin-2 with its postulated specific interaction partners has been investigated in terms of its biological relevance. binding affinities as well as kinetic parameters of the interaction were determined. in particular, the interaction of c-polycystin-1 and pigea14 immobilized on solid supported membranes with c-polycystin-2 has been quantified by means of the quartz crystal microbalance (qcm) method. a dissociation constant of about 100 nm was obtained. the results are compared with those obtained by surface plasmon resonance (spr) technique using a different immobilization strategy. correlation of the lateral structure of lipid bilayers and monolayers using two photon excitation fluorescence microscopy l. a. bagatolli membrane biophysics and biophotonics group/memphys -center for biomembrane physics, bmb, university of southern denmark most of the reported fluorescence microscopy applications on langmuir lipid films are focused in obtaining fluorescence intensity images using particular fluorescence probes. in this type of experiments the probes are generally utilized to obtain "contrast" between membrane regions (lipid domains) displaying dissimilar physical properties. the obtained information largely depends on the partition of the fluorescent probes for the lipid domains and the obtained fluorescence intensity pictures are not providing any information about the local physical properties of the lipid film. local physical properties of these distinct regions can be determined by exploring fluorescent probe related parameters such lifetime, emission shift, polarization. however these fluorescent probe's properties are almost unexplored in this type of experiments. this presentation will focus in describing a new experimental setup that includes a specially designed film balance on top of a custom built multiphoton excitation fluorescence microscope. this setup allows obtaining laur-dan gp images (1) many studies on phase separations of lipids in bilayers and their leaflet asymmetry make use of fluorescence techniques. we used a dithionite assay, steady-state fluorescence anisotropy and fluorescence resonance energy transfer (fret) for characterization. dithionite assay was performed to measure the fluorophore distribution in the inner and outer leaflets of symmetric membranes. for low concentrations, the fluorophore is distributed almost homogeneously, whereas for concentrations > 0.5 mol % it accumulates in the outer leaflet. we discuss these effects taking into account the presence of multilamellar liposomes and dynamic effects produced by higher local bending elasticities. the results point out possible artifacts in the use of fluorophores to characterize bilayers under the assumption of their homogeneous distribution. dithionite relative bilayer permeability is discussed. the results won by fret and steady-state fluorescence anisotropy regarding lipid phase transitions are in good coincidence to each other and to results reported in the literature. the photosensitizing properties of three chlorins are compared in solution and when incorporated in dioleoylphosphatidylcholine vesicules. in solution, they possess a similar efficacy to generate singlet oxygen and a similar ability to induce the peroxidation. however, the role of the photosensitizer localization within the lipidic bilayer is strongly highlighted, when chlorins are incorporated in liposomes, both by the changes in order of peroxidation efficacy but by the measurements of the photoinduced permeation of the liposomes. the results are discussed in relation with the technology of photochemical internalization, pci. then, using giant unilamellar vesicles, we asymmetrically oxidize the membranes. we observed different shape transitions, such as budding, typical of membrane curvature modifications. the asymmetry of the shape transitions are in accordance to a lowered effective spontaneous curvature of the leaflet targeted. this effect is interpreted as a decreased preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation. permeabilization of guv by photooxidation is interpret as the opening of a pore above a critical membrane tension due to the budding of vesicles. the effective spontaneous curvature of photosensitized vesicle at lysis was estimated. additionally photooxidation was shown to be fusogenic. influence of polyphenol extracts from fruits on biological and model membranes d. bonarska-kujawa, h. pruchnik, j. sarapuk, j. oszmiański, h. kleszczyńska univ. of environmental and life sciences, wroc law, poland biological activity of polyphenol extracts from apple, strawberry and chokeberry was studied. polyphenols were shown to be good antioxidants and to act as antyhemolytic agents. both the activities are the result of polyphenols incorporation into biological membranes. to check potential changes they induce in membranes some experiments were performed with the use of erythrocytes, and lipid membranes. it was found that the extracts studied induced shape transitions of erythrocytes. they were classified according to the bessis-brecher scale. strawberry extract induced mainly discocytes and discoechinocytes. populations of discocytes, echinocytes and some discoechinocytes were found on applying apple extract, while chokeberry induced mainly the formation of echinocytes and spheroechinocytes. the results evidence that the polyphenols incorporate into the external monolayer of lipid bilayer of the erythrocyte membrane. the results of the fluorimetric experiments showed that all the extracts changed fluidity in the hydrophilic part of rbc membrane. the changes observed were biggest for chokeberry extract and smallest for strawberry one. incorporation was also followed by changes in electrical resistance of black lipid membranes and in shifting the temperatures of main transition (t m ) and pretransition (t p ) in liposomes. this work was sponsored by ministry of science and education, scientific project no. n n305 337034. a. boll 1 , n. czudnochowski 2 , m. geyer 2 , c. steinem 1 1 institue of organic and biomolecular chemistry, university of göttingen, germany, 2 mpi for molecular physiology, dortmund, germany hiv-1 tat belongs to the accessory proteins of hiv and has regulatory functions. tat is concentrated in the nucleus and nucleolus of infected cells. the protein is composed of 86 amino acids with a molecular weight of 11 kda. tat is a transcriptional activator protein, which stimulates rna polymerase ii-mediated transcription elongation. therefore, tat interacts with cyclin t1 and binds to the tar rna element. tat has different domains. with respect to the interaction with lipid membranes, the most important structural motif is its basic region, including 6 arginine and 2 lysine residues. the peptide derived from this basic region belongs to the cell-penetrating peptides and is able to translocate through lipid membranes. the major aim of this study is to investigate the influence of full length hiv-1 tat on artificial lipid membranes. as a starting point solid-supported membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (popc) immobilized on silicon dioxide were used. the influence of the lipid head groups on the interaction with tat was analysed by using membranes composed of the negatively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (pops) in a mixture with popc. fluorescently labelled tat was used to localise the protein in the membrane. the impact of tat on lipid membranes was investigated by fluorescence and atomic force microscopy, showing that it is capable of perturbing lipid membranes. g. bocchinfuso 1 , a. palleschi 1 , b. orioni 1 , g. grande 1 , f. formaggio 2 , c. toniolo 2 , y. park 3 , k. s. hahm 3 , l. stella 1 1 univ. of rome tor vergata, dept. of chemistry, italy, 2 dept. of chemistry, univ. of padova and cnr, italy, 3 chosun univ., rcpm, south korea. most antimicrobial peptides exert their activity by perturbing the permeability of bacterial membranes, but the molecular details of this process are still debated. here, we compare fluorescence experiments and molecular dynamics simulations regarding the interaction of two antimicrobial peptides (pmap-23 and trichogin ga iv) with lipid membranes, showing that their mechanisms of bilayer perturbation are significantly different. pmap-23, a cationic peptide member of the cathelicidin family, associates to the membrane close to its surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. on the other hand, trichogin ga iv, a neutral peptide member of the peptaibol family, undergoes a transition from a surface-bound state to an inserted orientation. in the first arrangement it does not cause any strong membrane perturbation, while in the second orientation it may span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. lipopolysaccharide (lps) is the main component of the outer membrane of gram negative bacteria. lps is also known as endotoxin because of its potency to induce sepsis, a serious source of mortality in many clinical cases. among lps-neutralizing agents, polymyxin b (pxb) is considered as the "gold standard" due to its high efficiency of binding and detoxification of endotoxin. in this work, we have further explored the interaction of pxb to lps from the rough mutant of salmonella minnesota (re-lps) both in the gel and in the liquid crystalline phase, using isothermal titration calorimetry (itc) and fluorescence based techniques. the effect of pxb binding on lps-membrane integrity was determined with a fluorescence quenching assay treating vesicles of lps labeled with nbd-pe with dithionite. thermodynamic parameters associated with the binding process as well as binding stoichiometry were obtained from itc experiments. finally, itc was conducted with enterococcus faecalis and salmonella typhimurium, as representative examples of a gram negative and a gram positive bacterium respectively. pressure jumps -an accessible trigger for biomolecular transformations high pressure can be used to induce many structural changes in biological systems: from triggering phase changes in model membranes to causing protein unfolding, in fact any change involving a volume reduction is promoted by pressure. as well as being broadly applicable, pressure changes can be applied very quickly both up and down in contrast to other structure change triggers such as temperature jumps. fast pressure jumps allow the trigger to be decoupled from structural changes, so with fast structure probe techniques such as time resolved x-ray diffraction, the out-of-equilibrium evolution of these systems can be monitored. despite great advantages, high pressure remains under-utilised primarily due to its technical difficulty. in response to this technology vacuum a high pressure user facility based around a pressure jump cell for small and wide angle x-ray diffraction has been commissioned at beamline i22, diamond light source, uk and will be freely available to the user community. the cell is highly robust requiring virtually no user intervention during an experiment and the pressure system is computer controlled with a graphical user interface and is integrated with the beamline. pressures between 0 and 0.5 gpa are accessible and jumps can be carried out in approximately 5 ms at temperatures from -10 to 120 • c. sample changing has been made simple and fast with a dedicated sample loading port and modular sample holders allow optimisation for a broad range of samples. the transformation of vesicle and lateral distribution of mobile membrane inclusions b. bozic institute of biophysics, faculty of medicine, university of ljubljana, lipičeva 2, si-1000 ljubljana, slovenia membrane inclusions such as membrane embedded peptides or proteins exhibit curvature dependent interaction with the surrounding lipid matrix due to the mismatch between their intrinsic curvature and the local membrane curvature. this interaction causes an inhomogeneous lateral distribution of the inclusions and a corresponding adjustment of the vesicle shape. by taking into consideration that the membrane free energy includes elastic energy of the lipid bilayer and a contribution due to an inclusion-membrane interaction, the configurations of lipid vesicles with mobile inclusions have been studied theoretically. the variational problem to calculate equilibrium vesicle shapes is solved by applying a ritz method based on an expansion in spherical harmonics. in general, vesicle shapes adjust to the presence of inclusions by increasing regions with favorable curvature and decreasing regions of unfavorable curvature in such a way that the lateral distribution of inclusions becomes inhomogeneous. if the number of inclusions or the inclusion-membrane interaction exceeds a certain value, the prolate shapes become globally stable. investigating the structure of pores formed by antimicrobial peptides using epr spectroscopy m. bortolus 1 , k.-s. hahm 2 , a. l. maniero 1 1 universita' di padova, padova, italy, 2 chosun university, kwangju, south korea spin label electron paramagnetic resonance (epr) is a spectroscopical technique effective to study the molecular mobility of membrane components and the membranepeptide interactions, as the timescale of epr is optimally matched to the rotational motions of lipids in membranes. we applied epr to study the pore-forming mechanism of two antimicrobial peptides (amp) that create pores of different dimensions when interacting with liposomes. hp (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) is derived from the n-terminus of helicobacter pylori ribosomal protein l1, and hpa3 is an hp (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) analogue where gln and asp at positions 17 and 19 were substituted by trp. we studied the interaction of the two amp with zwitterionic and negatively charged vesicles, doped with phospholipids spin labelled in the lipid head or at the c5 or c10 positions; the different phospholipids allow us to study the peptide-membrane interaction at different depths relative to the membrane surface. we studied the interaction of the amp with vesicles following the influence of amp on label mobility as a function of temperature and membrane depth. we also prepared spin-labelled dmpc/dhpc bicelles, doped with lanthanide ions (dy 3+ /tm 3+ ) that allow us to macroscopically orient the system using the magnetic field of the epr spectrometer. we studied the interaction of amp with the oriented bicellar system monitoring the effect of amp on the order parameter of the phase. a. chattopadhyay centre for cellular and molecular biology, hyderabad, india we addressed the organization and dynamics of the human serotonin 1a receptor fused to enhanced yellow fluorescent protein (serotonin 1a r-eyfp) expressed in cho cells. serotonin 1a receptors are prototypical members of the gprotein coupled receptor superfamily and represent a prime target for therapeutic actions of several anxiolytic and antidepressant drugs. interestingly, we observed significant retention in fluorescence of serotonin 1a receptors upon triton x-100 treatment of intact cells at low temperature demonstrating their detergent insolubility. we analyzed the role of cholesterol in the plasma membrane organization of the serotonin 1a receptor by fluorescence recovery after photobleaching (frap) measurements with varying bleach spot sizes. our results show that lateral diffusion parameters of serotonin 1a receptors are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin 1a receptors in the plasma membrane. interestingly, results from frap measurements performed under conditions of mild cytoskeletal destabilization suggest that receptor signaling is correlated with receptor mobility, in agreement with the 'mobile receptor hypothesis'. our current work is focused on exploring the oligomerization of the receptor using photobleaching anisotropy measurements and indicates the presence of constitutive oligomers of the serotonin 1a receptor in live cells. expression and reconstitution of connexin43 in pore-suspending membranes c. carnarius 1 , s. kaufmann 2 , m. tanaka 2 , c. steinem 1 1 institute of organic and biomolecular chemistry, university of göttingen, tammannstrasse 2, 37077 göttingen, germany, 2 institute of physical chemistry, university of heidelberg, im neuenheimer feld 253, heidelberg, germany the intercellular communication and electronic coupling between adjacent cells of vertebrates are mediated by gap junctions. these proteins are composed of two connexon hemichannels, whereas each connexon consists of six connexin subunits. each subunit is characterized by two conserved motives: two extracellular loops and four transmembrane α-helices. in this study, we focused on cx43. to obtain large amounts of this protein, we expressed cx43 and cx43+gfp in a rather new expression system: dictyostelium discoideum. in contrast to human tissue cultures, the system allows for high cell densities up to 30 million cells per ml and the cells can be cultivated by fermentation. cx43+gfp was successfully visualized in d. discoideum by confocal laser scanning microscopy, where it was preferentially found in the plasma membrane.after the cells were harvested, plasma membranes were prepared and both proteins (cx43 and cx43+gfp) were verified by western blot analysis. the proteins were solubilized by addition of 5% n-octyl-β-d-glucopyranoside and purified by ion metal chelate affinity chromatography. the activities of both proteins were confirmed by a cytochrome c assay. after the purification of both proteins, they were reconstituted in µm-sized pore-suspending membranes. in the near future, we plan to determine the mobility of cx43 and cx43+gfp in these membranes by fluorescence recovery after photobleaching. o. cañadas, c. casals dpt. biochemistry and molecular biology i, and ciber enfermedades respiratorias, complutense university of madrid, madrid, spain inhaled bacterial lipopolysaccharide (lps) may incorporate into the lung surfactant monolayer. in this study, the effect of smooth lps (s-lps) on the surface activity of lung surfactant was evaluated. to that end we investigated the behavior of dppc films containing s-lps, with and without surfactant protein a (sp-a) in the subphase, using epifluorescence microscopy combined with a surface balance. our data show that s-lps injected into the subphase incorporated into dppc films forming mixed dppc/s-lps monolayers. cospread s-lps fluidized the dppc monolayer as demonstrated by epifluorescence images and changes in the compressibility modulus of the monolayer as a function of s-lps molar fraction (x s−lps ). the presence of low amounts of s-lps in the monolayer promoted early collapse, preventing high surface pressures to be reached. moreover, s-lps hampered the re-spreading of dppc molecules during dynamic compression at s-lps concentrations as small as x s−lps = 0.02. such inhibitory effects could not be relieved by repeated compression-expansion cycles or by adding surfactant protein a. however, sp-a facilitated the squeeze-out of s-lps from dppc/s-lps mixed monolayers, suggesting that sp-a is an s-lps scavenger. cholesterol displaces ceramide from its tight packing with sphingomyelin in the absence of ld phase j. v. busto, j. sot, j. requejo-isidro, f. m. goñi, a. alonso unidad de biofísica (csic-upv/ehu) and departamento de bioquímica, u. país vasco (upv/ehu), spain a set of biophysical approaches have been applied to study the phase behaviour of palmitoylsphingomyelin (psm)/cholesterol (chol) model membranes upon palmitoylceramide (pcer) addition. fluorescence spectroscopy of di-4-aneppdhq-stained psm/chol vesicles reveals no segregation of large liquid-ordered (l o ) microdomains. in contrast, the formation of disperse, compositionally homogeneous l o psm/chol (3:1) nanodomains over a psm gel (l β ) phase is proposed. dsc measurements show that pcer addition to vesicles with coexisting l o and l β phases (low [chol] ) induces the formation of large gel-like psm/pcer microdomains, coexisting with a l o psm/chol phase. the ∆h for the psm/pcer phase at high psm/(chol+pcer) ratio is close to that of the binary mixture in the absence of chol, supporting immiscibility, but no displacement, between chol and pcer-rich phases. on the contrary, both confocal microscopy of guvs and the dsc data coincide in showing that a rise in pcer increases the gel-like phase to a lower extent than in the pure psm/pcer mixture, revealing some cholinduced restriction. in the presence of a pure l o psm/chol phase (high [chol] ), pcer addition is unable to induce the formation of large psm/pcer microdomains. the present data support the role of chol as the key determinant in controlling its own displacement from l o domains by ceramide. hydroquinones modifying lipid membrane morphology c. di vitta 1 , l. marzorati 1 , v. rebbin 2 , s. s. funari 2 1 iqusp, university of são paulo, são paulo, brasil, 2 hasylab, hamburg, germany quinones are important molecules in cells. flavina, ubiquinone and coenzyme q, coq, are associated with electron transfer. coq, having a hydrophobic tail, is soluble in the internal lipid membrane of mitochondria. their reduction leads to the equivalent hydroquinones. we synthesized novel alkylthioquinones aths, to investigate their interaction with phospholipids. one or more long hydrophobic chains attached to the quinone ring alter their hydrophobicity and electron availability. we aimed at their influence on the structure of membranes. different phs highlights the charge/polarity effect on the interaction and on the morphology of the bilayer. for that, pope and popc, lipids with different charges, but identical chains were selected. x-rays diffraction on pope/2,6bath, 100/1 at different temperatures and ph, showed cubic phases coexisting with the usual phases formed by simply hydrated pope, at different phs. for investigation of the charge/polarity, we turned to the popc/2,6bath system (smaller charge/polarity). despite decrease in temperature of phase transition and dimensions of the lattice, the structures were the same as in hydrated lipid, illustrating a less significant effect than on the similar lipid pope. another additive, 2,5ath, mixed with pope showed the effect of multiple thioalky chains. no morphology change was seen, compared to pure lipid. interestingly, despite both additives differ by one thioalkyl chain only, their influence on the pope matrix is so different. cross-linking of phospholipid membranes by calcium-sensitive synaptotagmins synaptotagmins are vesicular proteins implicated in many membrane trafficking events. they are highly conserved in evolution and the mammalian family contains 16 isoforms. we now show that the tandem c2 domains of several calcium-sensitive synaptotagmin isoforms tested, including drosophila synaptotagmin, rapidly cross-link phospholipid membranes. in contrast to the tandem structure, individual c2 domains failed to trigger membrane cross-linking in several novel assays. large-scale liposomal aggregation driven by tandem c2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic lightscattering and both confocal and negative stain electron microscopy. high-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem c2 domains results in a constant distance of approximately 9 nm between the apposed membranes. our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem c2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. membrane proteins can be challenging samples to work with and as such often require the use of multiple techniques. here we present an insight into the structure of the antimicrobial peptide melittin in lipid membranes using various techniques. we explore the conformational changes of membrane-bound melittin and its interaction with model membrane lipid systems with a series of spectroscopic methods that can be used in parallel. the techniques used include linear dichroism and ft-ir for orientation information and circular dichroism for conformation information. we use dynamic light scattering for molecular sizing, fluorescence emission for information on peptide environment, thin-layer chromatography for lipid identification, analytical ultracentrifugation to identify oligomerisation state and calorimetry to investigate the thermodynamics. we observe how the physical properties of both the peptide and the membranes affect the insertion kinetics of the peptide in the membrane. phospholipid membranes dynamics: molecular dynamics vs neutron scattering v. conti nibali 1 , m. tarek 2 , u. wanderlingh 1 , g. d'angelo 1 1 department of physics, faculty of science, university of messina, italy, 2 unité mixte de recherche cnrs/uhp 7565, université henri poincaré, nancy, france collective dynamics and single-particle dynamics of hydrated multilamellar phospholipid bilayers (1,2-dimyristoylsn-glycero-3-phosphatidylcholine, dmpc) have been studied by means of all-atom molecular dynamics simulations. here we report results of a gel phase bilayer at 283 k and of a liquid crystal phase bilayer at 303 k. coherent and incoherent dynamic structure factors and meansquare displacements have been calculated from the trajectories for both the inplane and out-of-plane lipid dynamics. moreover, the results have been compared to recent quasi-elastic and inelastic neutron scattering data. optical tweezers allow trapping of particles of different types in a wide range of sizes [1] . among these, unilamellar vesicles are of interest as they are known to be effective vectors in drug delivery and they are also studied as models for cell membranes. this work focuses on the interactions between optically confined unilamellar vesicles and their cross-linking by proteins [2] . synaptotagmins are vesicular proteins implicated in many membrane trafficking events having an accelerating effect on the membrane fusion. calcium-sensitive synaptotagmins are thought to confer calcium sensitivity to the fusion of secretory vesicles with target membranes [3] . the novel optical assay reported here allowed us to visualize the cross-linking of 600 nm liposomes mediated by synaptotagmin and calcium. the use of the optical tweezers approach to investigate the function of other fusogenic proteins related to exocytosis (i.e., snare proteins) is discussed as well. antimicrobial peptides (amps) have received increasing interest as the search for new potential antibiotics has become imperative due to increasing bacterial multiresistance. acylating the natural occurring polymyxin b (pmb) significantly enhances antibacterial activity towards two representative gram-negative bacteria e. coli and k. pneumonia compared to the nonacylated pmb. the aim of the presented work was to study various biophysical parameters, such as partitioning coefficients, zeta potentials and effective charges of a selected amp, mastoparan-x (mpx) and a propanoylated (pampx) and octanoylated (oampx) analogue, respectively. fluorescence spectroscopy, isothermal titration calorimetry and ζ-potential measurements were the main techniques used for the measurements. we employed 2 different luv model systems; a partially charged (popg/popc 1:3) and a neutral system (popc). for the neutral luvs there was an increase in partitioning with increasing length of the acyl chain, whereas partioning into the partially charged luvs was governed by a balance of hydrophobic and electrostatic contributions. the selectivity for the partially charged luvs over the neutral luvs was in the order pampx, mpx and oampx. the modeled effective charge for the peptide followed the same trend as the partitioning coefficient for the three peptides for the respective luv systems. does the lysosomal membrane need triglycerides? a spectroscopic study of a simple model membrane l. duelund 1 , k. pakkanen 2 , m. vuento 2 , j. h. ipsen 1 1 memphys -center for biomembrane physics, university of southern denmark, odense, denmark, 2 nanoscience center, university of jyväskylä, jyväskylä, finland lysosomes are intracellular organelles in which proteins and other macromolecules are degraded. morphological and functional changes in different compartments of the endocytic pathway are connected to several diseases. a crucial step in understanding biogenesis of lysosomes and their role in disease conditions, is to characterise the properties of the lysosomal membrane. by the use of tlc we have found that lysosomes contain non-neglible amounts of triglycerides (tg). to investigate how the presence of tgs could influence the lysosomal membrane, we have investigated the properties of a mixture of popc and triolein, as a simple model for the lysosomal membrane. we found the system to form two types of popc-rich membranes. these were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. by using epr and fluorescence spectroscopy the physical properties, including order, fluidity and water penetration, of these phases were found to differ markedly despite of their almost identical composition. the results suggest that presence of tgs on lysosomal membranes could have a crucial effect in the barrier functions and thus, the integrity of the organelle. model membranes with the capacity to align in magnetic fields such as bicelles and magnetically sensitive vesicles are of high interest, since their macroscopically alignment allows for the determination of structure, dynamics and topology of molecules within the membrane [1] . we performed 31 p solid state nmr on a magnetically sensitive lipid possessing a large positive magnetic anisotropy introduced in form of a biphenyl unit during lipid synthesis in one of its acyl chains (1-tetradecanoyl-2-(4-(4biphenyl) butanoyl)-snglycero-3-phosphocholine (tbbpc)). the phosphorus lineshapes of tbbpc mlvs studied at various magnetic fields (7.1t, 9.4t, 11.7t, 16.4t), revealed a drastic change in shape upon exposure to fields > 9.4 tesla: resonances resulting from phospholipid molecules oriented perpendicular to the magnetic field decrease whereas resonances resulting from parallel oriented molecules increase. this is a sign for magnetically induced vesicle deformation from vesicle to oblate ellipsoid shape. factors influencing this drastic deformation, such as magnetic anisotropy, membrane elasticity, lipid chain length and field dependency are discussed based on existing theories [2] . hepatitis g virus (gbv-c/hgv) is an enveloped viruses belonging to the flaviviridae family. clinical findings have suggested that in people co-infected with gbv-c/hgv and human immunodeficiency virus (hiv), delayed progression of aids has been observed (1). the mechanism by which this virus may inhibit the progression of aids remain to be elucidated. enveloped viruses acquire their lipid membranes by budding through host cellular membranes (2) , in this process; fusion peptides play an important role. study of the interaction of hgv-c peptides with lipid membranes could lead us useful information about the mechanism that takes place. in this work we present a study on the effects of e1 peptides on the fluidity of and polarizability of model membranes (dmpc and dppc liposomes) by dsc and fluorescence polarization. both techniques showed that the presence of the studied sequences in phospholipid mixtures already affected the thermotropic properties of the gel to liquid-crystalline phase transition. systems were thermodynamically characsmall angle neutron scattering (sans) studies have been performed to study the structural changes induced in membranes of vesicles prepared from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations of the anti-fungal drug, amphotericin b (amb).the vesicles, sonicated to a mean size∼100nm, were prepared using dimyristoylphosphatidylcholine(dmpc) or dmpc-cholesterol or dmpc-ergosterol mixtures -with both of the mixed systems involving 30mol% sterol. analysis of the sans data show that when the concentration of amb added is just above the drug's cmc (∼1µm) there is an increase in the membrane thickness of both the dmpc-chol and dmpc-erg vesicles (both cases + 4å), but the thickness of the pure dmpc vesicle membranes remains the same as in the absence of amb. when amb is added at a concentration in excess of its cmc (∼10µm), the mixed-sterol vesicles show the same changes in membrane thickness as observed with the lower amb concentration, and the pure dmpc vesicles again remain unaffected. on the basis of these studies, therefore, there appears to be no difference in the structural changes induced by the insertion of amb into the model fungal cell membranes (mimicked by dmpc-erg vesicles and those resulting from its insertion into the model mammalian cell membranes (mimicked by dmpc-chol vesicles). the third transmembrane helix of bacteriorhodopsin, also known as phlip, is a unique model system for studying the interactions of a natural transmembrane domain with lipid membranes: depending on ph, the water-soluble peptide either adsorbs superficially or inserts as a transmembrane helix on addition of lipid vesicles [1] . published values for the free energies of these processes were based on a stoichiometric model invoking two distinct sets of binding sites [2] . however, discrepancies between data obtained from different experimental techniques and inconsistencies between experimental and expected temperature dependencies suggest that these values should be taken with caution. we therefore reassessed membrane interactions of phlip using titration calorimetry and fluorescence spectroscopy. if electrostatic effects at the membrane surface are taken into account, the data can be described quantitatively by a partition equilibrium, but not by a stoichiometric binding model. the thermodynamics of membrane partitioning differ substantially from those determined previously [2] and draw a different picture of peptide-lipid interactions. beyond deepening our insights into the first step of the two-stage model of membrane protein folding, this also sheds light on the ability of phlip to drag cargo molecules across lipid membranes. adsorption of monolysine and polylysines at the surface of lipid membranes of varied composition was studied by two methods. both methods -the electrokinetic one, measured the surface potential of liposomes, and the intramembranouse field compensation sensitive to boundary potential of planar bilayer lipid membranes -detected the positive changes of the potentials for membranes with negatively charged component (cardiolipin, cl). neither monolysine, nor polylysines adsorbed at neutral membranes (phosphatidylcholine, pc). pentalysine show the difference between these potentials for the membranes composed from cl/pc mixtures. this difference is attributed to dipole part of boundary potential and indicates the changes in lipid packing. polylysines show high affinity to the membrane and saturation with plateau. these saturation levels correspond to surface charge densities 0.005 and 0.016 c/m 2 for oligomers with 5 and 12 units, and 0.032 c/m 2 for polymers with 130 and 1435 units. these values do not depend on the ionic strength of background electrolyte but proportional to the content of negatively charged components in the lipid bilayer. the polylysine layer at the mica surface was studied by atomic-force microscope (afm) technique. it was shown that pentalysine molecules cover the surface by the layer of 0.8 nm thickness and polylysines of high molecular weight by the layer up to 4 nm. effects of lysolipids on the mechanical stability of lipid membranes j. r. henriksen 1 , l. feldborg 1 , j. h. ipsen 2 , t. l. andresen 1 1 technical university of denmark, dtu nanotech, frederiksborgvej 399, b124, 4000 roskilde, denmark., 2 university of southern denmark, department of physics and chemistry, memphys center, campusvej 55, 5230 odense m, denmark lysolipids (lpcs) and fatty acids (fas) are the natural products formed by the hydrolysis of phospholipids. lpcs and fas form micelles in solution and thus act as detergents in the presence of lipid membranes. in the present study we investigate the detergent strength of a homologous series of lysolipids (lpcs) on popc lipid membranes by use of isothermal titration calorimetry (itc) and vesicle fluctuation analysis (vfa). the membrane partition coefficient (k) and critical micelle concentration (cmc) are determined and found to obey an inverse proportionality relation (cmc x k ∼ constant). the partition coefficient and critical micelle concentration are used for the analysis of lpc's effect on the membrane bending rigidity. the dependence of the bending rigidity on the lpc membrane coverage has been analyzed in terms a phenomenological model based on continuum elastic theory which yield information about the curvature inducing properties of the lpc molecule. the results reveal: (i) an increase in the partition coefficient with lpc acyl-chain length and (ii) the degree in acyl chain mismatch between lpc and popc determines the magnitude of the membrane mechanical perturbation per lpc molecule on the membrane. patch clamp electrophysiology remains the gold-standard for ion channel research because of the information richness of the data produced. automated planar patch clamp devices, with their higher throughput and high data information content, have made the technique accessible to a wider audience. nanion technologies offers two planar patch clamp workstations combining higher throughput with high data quality. the port-a-patch records from a single cell at a time and the patchliner from up to 8 cells simultaneously with high success rates (typically 60-80%). both, the port-a-patch and the patchliner are bench top patch clamp rigs which uses a planar borosilicate glass chip for obtaining a giga-seal for electrophysiological recordings. suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. both workstations have been successfully used for whole cell, perforated patch, and cell attached recordings as well as for guv-bilayer recordings. due to the versatility of nanion`s products it is possible to study a wide variety of ion channels including nav, kv, cav, ligandgated, herg or reconstituted proteins such as ksca, cx26, ompc. special features unique to nanion products, including but not limited to internal solution exchange and temperature control, expand the experimental possibilities. waves on lipid monolayers j. griesbauer, s. boessinger, a. wixforth, s. f. matthias univiversity of augsburg, experimental physics i, biological physics group, augsburg, germany " for the sake of illustration we shall try to provide a physical basis for the equations, but must emphasize that the interpretation given is unlikely to provide a correct picture of the membrane." [ hodgkin&huxley, 1952 ] to explain the occurrence of reversible heat production during nerve pulse propagation it has been suggested by multiple authors [wilke,kaufmann,heimburg] that travelling sound waves and not ion channels may offer a better explanation than the hodgkin&huxley theory. therefore, if sound waves are an essential feature of the nerve membrane they should also appear on lipid monolayers where ion conductivity is evidently absent. here we demonstrate that sound waves can be excited on a lipid monolayer by using a set of planar electrodes incorporated into the monolayer and driven by an alternating voltage. not only do our results indicate propagating waves on lipid monolayers in accordance with their thermodynamic predictions, but importantly, no significant attenuation is detected proposing an adiabatic phenomenon. in order to provide evidence that our physical explanation provides a correct picture of the membrane, direct detection of the waves was done, whereas a clear transduction of signals was shown. finally, the impact of toxins, neuropharmaca and anaesthetics needs to be integrated in our physical picture of the nerve membranes, what can easily be done now and could deliver a new approach for understanding their physical mechanism. our earlier studies showed that organometallic compounds (otc) in the presence of uvc radiation enhanced the degree of phosphatidylcholine (pc) liposome oxidation, whereas quercetin effectively protected the membrane. the present investigation is concerned with the effect of uvb and otc (chlorides of diphenyl-and dibutyltin, triphenyl-and tributyltin), both in combined action and separately, on oxidation of erythrocyte proteins, pc liposome and albumin. the degree of oxidation of proteins and liposomes induced by otc and radiation, also in the presence of selected antioxidants (trolox, quercetin) was determined on the basis of changes in the number of sulfhydryl groups, carbonyl groups and malone dialdehyde, respectively. the studies indicate that uvb induces pc liposome and erythrocyte oxidation, whereas in the case of albumin it causes both an increase in the number of c=o groups and free sh groups (most probably, braking sulphonic bridges). otc compounds interact with membrane biomolecules both as weak pro-and antioxidants, also in combined action (uvb plus otc). the prooxidative effects are markedly diminished by application of antioxidants. the action of quercetin results from its ability to incorporate into membranes and formation of complexes with otc (liposome>erythrocyte>albumine). this work was supported by grant n n3364 34. a phospholipase c/ sphingomyelinase from pseudomonas aeruginosa has been assayed on giant unilamellar vesicles (guv) consisting of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol, at equimolar ratios. the enzyme activity modifies the chemical composition, thus the physical properties of the bilayer, and conversely the latter influence the enzyme activity. biochemical assays of enzyme activity, together with confocal fluorescence microscopy examination of guv provide novel information about the system. the original lipid composition in the absence of enzyme gives rise to lateral phase separation of liquid-ordered and liquid-disordered domains in the guv. the two enzyme end-products, diacylglycerol and ceramide, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase. morphological examination of individual guv shows that the enzyme binds preferentially the more fluid (or more disordered) domains, and that, in most cases, it causes the fluidification (disordering) of the other domains.. cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. to this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures (33-75 mol% cholesterol) have been measured and compared with the original mixture before lipid hydration. there is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the bilayers is much lower than expected. the maximum solubility of chol in bilayers containing saturated pc or pc with less than four unsaturations is 50-60mol%, while in polyunsaturated pc, e. g. dic18:4, and in sphingomyelin the maximum chol contents is 23mol%. a quantitative analysis of the vesicles is thus required before any experimental study is undertaken. membrane fusion assay based on poresuspending lipid bilayers i. höfer, c. steinem institute of organic and biomolecular chemistry, university of göttingen, germany membrane fusion has attracted significant interest due to its biological relevance. thus in recent years a great variety of fusion assays has been developed. since the process of membrane fusion is not yet fully understood, our aim is to develop a new fusion assay based on pore-spanning membranes, which have been proven mechanically stable, highly insulating and rather tension free. the application of these so-called micro-blms should allow simultaneous monitoring of lipid mixing, content release as well as electrical readouts. furthermore both membrane sides can be addressed individually to apply transmembrane potential or fusion modulating compounds. first results show that the micro-blms provide the opportunity to investigate lipid bilayer fusion by means of fluorescence microscopy. the formation of porespanning membranes is achieved by the painting technique of dphpc doped with oregon green dhpe dissolved in ndecane. the addition of large unilamellar vesicles doped with texas red dhpe allows direct observation of single fusion events. lipid mixing during fusion leads to a decrease of the donor fluorescence (oregon green) and an increase in acceptor fluorescence intensity (texas red) in the plane of the planar bilayer due to fret. in future work simultaneous monitoring of lipid-mixing and content release combined with electrical measurements are planned to gain further insight into different intermediate steps of membrane fusion. the kinetics of membrane-peptide folding and orientation m. r. hicks department of chemistry, university of warwick, uk. understanding interactions of peptides with lipid membranes is essential if we are to be able to design new and better antibiotic peptides. there are different steps in these interactions and following the kinetics of these processes requires that we combine the information from different biophysical techniques. here we present data on the kinetics of peptidemembrane interactions using circular dichroism to report on conformation and linear dichroism to report on orientation of the peptides in/on membranes. these are combined with other techniques such as dynamic light scattering and fluorescence spectroscopy to elucidate the mechanisms of action of the peptides. one of the most important aspects of membrane-active peptide design is that of specificity. we investigate specificity for different types of membranes by using libraries of lipids with different properties of charge, chain saturation and curvature stress. in this way one can test for effects of e.g. negatively charged head groups found in bacterial membranes or cholesterol found in animal and human membranes. using these approaches it is proposed that we will be able to modify peptide sequences, test what part of the kinetic processes are affected and subsequently use this information to design new and better antibiotics. k. kubica, h. misiak wroclaw university of technology, wroclaw, poland reversible membrane electroporation, that can be observed as a effect of electric field influence on biological membranes, finds application in cell delivery of biologically active compounds. the membrane susceptibility for creation of stable pores, depends on electric field parameters applied to the membrane as well as on the membrane environment (ionic strength, ph, temperature) and membrane molecular composition. we have already shown that the changes of van der waals lipid chain interaction effect the process of pore formation. there are known factors which increase or decrease the membrane respond to electric field. because pores appear in lipid membrane matrix it make sense to study this phenomenon using model planar lipid membranes. as lipids are very receptive on oxidizing factors we decided to study the relation between lipid oxidation and membrane reaction on electric field. with the use of four electrode galvanostat we are examining the influence of factors initiating lipid oxidation (uv, fe 2+ ) and the efficiency of some antioxidant compounds of lipid protection on electric properties of lipid membrane. the product of lipid oxidation is determined by characteristic reaction with tiobarbituric acid. we want to determine the role of polyunsaturated fatty acids on membrane properties also. results of our work will be useful in optimization of medicine distribution, aided by electric field, into cells. budding of giant phospholipid vesicles induced by β2-glycoprotein i j. kovacic, b. bozic, s. svetina institute of biophysics, faculty of medicine, university of ljubljana, 1000 ljubljana, slovenia β 2 -glycoprotein i (β 2 gpi) is classified among amphitropic proteins: in its inactive form it is dissolved in plasma and in its active form it is bound to membrane. in comparison to other amphitropic proteins it exhibits a distinct membrane interaction behavior. a patch of positively charged amino acid residues contacts anionic phospholipids via electrostatic interactions, and a hydrophobic loop anchores the protein into the outer leaflet of the membrane via hydrophobic interactions. the binding constant depends on physical properties of the membrane such as membrane lipid composition, surface potential, lipid packing density and curvature. the binding of an amphitropic protein alters the membrane's spontaneous curvature as well as the difference between the equilibrium areas of membrane leaflets. theoretical model has been developed to predict vesicles shape transformations realized by the formation of buds. it was shown that the effects are stronger for flaccid vesicles which were therefore chosen for our experimental investigations. vesicles were composed of 20 % pops and 80 % popc, and flaccidity was achieved by an osmotic adjustment. the solution containing β 2 gpi was subsequently injected to a chamber with flaccid vesicles by a syringe pump. observation took place under a phase-contrast microscope. experiments showed a concentration dependent occurrence of buds. their number and size were related to the degree of vesicle flaccidness. the interplay between detergent cohesion and peptide adsorption on the structure and dynamics of a glycophorin a tm-detergent complex j. khao, j.-p. duneau, j. n. sturgis lism, cnrs -aix marseille university, marseille, france in spite of the major interest of membrane proteins at functional, genomic and therapeutic scales, their biochemical and structural study remain challenging as they require, solubilization in detergent micelles. the complexity of this task arises from the structural dependence of membrane proteins on their anisotropic environment and in particular by a delicate balance between different physico-chemical properties. in order to study these properties in a small protein detergent complex, we have used molecular dynamics simulations on the transmembrane part of glycophorin a (gpatm) solubilized in micelles of the detergent di-hexanoyl-phosphatidylcholine(d6pc). we show that the molecular aggregates organizes to give distinct populations of detergent molecules. those molecules which loosely interact with the peptide are preferentially involved in highly cohesive inter-detergent interactions that impose a global bilayer structure to the micelle. interaction profiles of other detergents with gpatm depend upon the nature of residues along the surface of the peptide. this topology dependence leads to different modes and strength of interactions that ultimately constrain the orientation of the micelles around the peptide. this simple model illustrates how differential detergent selectivity for faces and strong constraints coming from purely environmental features could influence transmembrane helix packing, membrane protein structure and assembly. in this paper we present a study of a new family of bolaamphiphiles. these amphiphiles are unsymmetrical bolalipids having one sugar polar head and the second glycine betaine polar head. they are potentially useful for pharmaceutical or cosmetics applications as vectors for drugs. therefore it is important to investigate their self-assembled properties. the chemical variations that we introduced in this new family concern the length of the main chain that connects two polar heads as well as the length of the side chain placed on the position 1 of the sugar moiety. another variation concerns the introduction of a diacetylenic unit into the main chain in order to rigidify it. we have performed the saxs (small angle x-ray scattering) measurements on the dehydrated compounds as a function of temperature and observed the lamellar liquid crystalline structures. we also measured the saxs spectra of aqueous solutions of these compounds that have shown lamellar l α structure in all cases. these measurements are compared with polarised optical microscopy measurements that confirmed our interpretation. formation of membrane tubular structures induced by phase separation in giant vesicles y. li, r. lipowsky, r. dimova max planck institute of colloids and interfaces, science park golm, 14424 potsdam, germany tubular membrane structures (also known as tethers) exist widely in eukaryotes. abundant work has been done on tube extrusion from cells and model membranes under the application of external forces. we present a novel system allowing tube formation in the absence of external forces. aqueous solutions of two chemically dissimilar polymers, polyethylene glycol (peg) and dextran are encapsulated in giant vesicles, a cell-size model system. the exposure of vesicles to hypertonic solutions induces phase separation of the internal aqueous polymer solution. the excess membrane area created by this vesicle deflation, engages in the formation of tubular structures. membrane tube formation and phase separation are coupled processes. hydrodynamic flows and changes in the membrane spontaneous curvature during phase separation might be the driving forces for tube formation. the tubes are rather stable: without external perturbation, they can exist for several days. they prefer to be located in the peg-rich phase at low polymer concentration. at high concentration, they are absorbed at the interface of the liquid phases to lower the surface energy of the system by decreasing the contact area between the liquid phases. the membrane tubes can be retracted back to the vesicle surface by increasing the membrane tension via vesicle aspiration. membrane tubes, which can form and be retracted easily, might be relevant to lipid storage in cells. insight into the antimicrobial mechanism of a de novo auto-assembling peptide b. legrand 1 , m. laurencin 2 , e. duval 3 , c. zatylny 3 , j. henry 3 , m. baudy-floc´h 2 , a. bondon 1 1 rmn-ilp, umr cnrs 6026 -univ. de rennes1, france, 2 icmv, umr cnrs 6226 -univ. de rennes1, france, 3 pemm, umr ifremer 100 -univ. de caen, france a short (14 residues) de novo antimicrobial peptide (k4) composed of a cationic polar head and a hydrophobic tail was studied. it exhibits a broad spectrum of antimicrobial activity on bacteria. no haemolytic activity or cytotoxicity on eukaryotic cells are observed at mic. when bacteria are lysed by the k4, spherical objects are observed on the sem micrograph. k4 was structurally studied using various membrane mimetic media such as different micelles and small unilamellar vesicles (suv) of different composition. cd revealed that k4 adopt various structures (random, β-turn, α-helix) in the presence or the absence of detergents or phospholipids. nmr structures confirmed the α-helical structure of k4 hydrophobic tail in presence of sds. k4 self-assembles at high concentration as observed by sem and dls. we suggest that k4 may act as a surfactant building mixed microsomes composed of peptide and lipids. this destabilization mechanism of the bacterial membrane support the "detergent like model" previously described in the literature. oxidative stress and the membrane dipole potential; modulation with tocopherol s. le-nen-davey 1 , b. m. davis 2 , j. l. richens 2 , k. a. vere 2 , m. w. tilley 2 , p. g. petrov 1 , c. p. winlove 1 , p. o'shea 2 1 biomedical physics group, university of exeter, uk, 2 cell biophysics group, university of nottingham, uk tocopherol, a component of vitamin e well know for its antioxidant properties, has recently been thought also to influence the structure of cellular membranes. tocopherol treatment reduces hyperglycaemia induced oxidative stress and the associated endothelial dysfunction which is a precursor to the vascular complications of diabetes. tocopherol and insulin interactions are also modified by hyperglycaemia. it is unclear whether these clinically important effects arise from the anti-oxidant and/or structural properties of tocopherol. we have therefore measured the dipole and surface potentials of phosphatidylcholine vesicles containing different amounts of cholesterol, ketocholesterol and tocopherol. we have also investigated the effects of hyperglycaemia, ketocholesterol and tocopherol, alone and in combination, on microdomain formation and interactions of insulin within the membranes of cultured endothelial cells. both sets of experiments indicate that tocopherol causes significant modifications of the membrane dipole and surface potentials. the physiological significance of these changes will be discussed. t. d. lazzara 1 , a. janshoff 2 , c. steinem 1 1 georg-august university göttingen, institute for organic and biomolecular chemistry, tammannstr. 2, 37077, germany, 2 georg-august university göttingen, institute for physical chemistry, tammannstr. 6, 37077, germany cell penetrating peptides (cpp) have been shown to penetrate cellular membranes. they have been of interest for their ability to translocate not only themselves through cellular membranes, but also carry along with them, cargo as large as iron nanoparticles. the exact entry mechanism remains unclear, but has been shown to vary with peptide sequence. their translocation properties have been demonstrated through different experiments involving vesicles, cells and living animals. we plan on using nano-black lipid membranes (nano-blm), which span the pores of nanoporous materials as a model system to study the interaction between cpp and lipid membranes. the nanopores can be used as cellular containers whose interior surface can be functionalized with receptors for biotin-streptavidin recognition. we hope that this system will provide greater control over experimental variables, such as the type of cpp and lipid used, as well as provide kinetic data that can be used to evaluate cpp activity and the kinetics of cargo transport. ethanol induces phospholipid acyl chain interdigitation. while much is known, important issues remain unclear, such as the role of lipid domains. the main purpose of this study was to follow, in real time, the changes induced by ethanol on supported lipid bilayers with nano to microdomains by in situ atomic force microscopy. to this goal, a pure lipid in the fluid phase (dopc), a pure lipid in the gel phase (dppc), binary phospholipid mixtures with gel/fluid phase coexistence, and cholesterol-containing, raft -forming mixtures (dopc/dppc/cholesterol and dopc/sphingomyelin/cholesterol) were investigated. from the height differences observed upon ethanol addition to pure lipids (dppc and dopc), and to dopc/dppc mixtures, it is shown that in the binary system the interdigitation of the fluid phase occurs prior to the gel phase. however, for the lipid rafts mixtures the simultaneous interdigitation of both raft and non raft portions of the membrane is observed both in mica and silicon substrates. for all compositions studied, domain formation or rearrangement accompanied by lipid bilayer expansion occurs as a consequence of interdigitation. these results show the ability of ethanol to influence the bilayer properties in different ways according to membrane composition. ethanol may exert its biological effects by reducing bilayer thickness, and also by changing membrane proteins conformation and lateral distribution, as a consequence of the altered properties of the lipid bilayer. interactions between non-steroidal anti-inflammatory drugs and a pc/cholesterol bilayer m. markiewicz 1 , t. librowski 2 , p. serda 3 , m. pasenkiewicz-gierula 1 1 faculty of biochemistry, biophysics and biotechnology, jagiellonian university " 2 department of pharmacodynamics, medical college, jagiellonian university " 3 regional laboratory, jagiellonian university, krakow, poland. the non-steroidal anti-inflammatory drugs (nsaids) are among the most frequently prescribed and used drugs [1] . there are several side effects connected with frequent use of nsaid, mainly gastrointestinal ulcers and bleedings. a plausible molecular mechanism of these effects are direct interactions of nsaids with gastric phospholipids [2, 3] . the influence of three well-known non-steroidal antiinflammatory drugs with a diverse gastric toxicity (aspirin, ketoprofen and piroxicam) and three newly-synthesized xanthone derivatives (belonging to the nsaids) on the structure and dynamics of lipid bilayers was studied using small angle x-ray scattering and molecular dynamics simulations. the results showed some correlation between nsaid toxicity and its binding to the lipid polar groups. this binding increases membrane fluidity by reducing its density due to an increased membrane surface area. a reduced lipid packing in the membrane most likely increases gastric mucosa permeability, which can result in a decreased resistance of the gastric mucosa to luminal acid. we studied the partition of the anionic amphiphile 1pyrenesulfonate (psa) into mlv and luv (produced by extrusion), composed by zwitterionic popc and zwitterionic/anionic mixtures with pops (until 10 mol %). psa is an anionic amphiphile that mimics several xenobiotics (e.g. pharmaceuticals, pesticides) and endogenous substrates that interact with biological membranes. we found increasing b. lorenz, s. schuy, a. janshoff georg august university, göttingen, germany cell-cell and cell-virus interactions are ubiquitous in living organisms. the analysis of the forces acting between two cells or a cell and a virus gives insight into details of these interaction processes such as their stochastics, cooperativity, reversibility, and energy landscape. using colloidal probe microscopy in conjunction with solid-supported lipid bilayer techniques, we mimic the contact between two membranous compounds displaying sponge glycolipids or viral peptides on their surfaces. after spreading functionalized lipid bilayers on both -a colloidal probe and a silicon wafer surface -the molecular interactions are quantified by means of force-distance curves. by probing the dynamic interaction strength between the viral peptides n36 and c34 we aim for a deeper understanding of the role of these peptides in the complex process of the formation of the prehairpin intermediate as the key step in retroviral fusion. as far as the cellular interaction of marine sponge cells is concerned, we intend to investigate the strength, specificity, and the ca 2+ dependency of the self-recognition between sponge glycans. the systems advantages are the flexibility of the membrane composition and the control over the distribution of receptor molecules in the membranes. since adhesion in biological systems relies heavily on cluster formation within the biomembrane, we plan to mimic the clustering by "printing" interaction domains and comparing the results to homogeneous samples. development of video-rate imaging microscope using laurdan and its applications to lipid raft t. ohba, k.-i. muto, t. kiuchi, k. ohki department of physics, graduate school of science, aobaku, sendai, japan 980-8578, ohki@bio.phys.tohoku.ac.jp various biological functions are located on cell membranes, and the biomembranes maintain heterogeneity as microdomain even in its dynamic structure. existence of such domain was reported as phase-separated 'rafts' in a cell. and the bio-functions of membrane protein are affected by physical property of its surrounding lipid bilayers. laurdan is a useful fluorescence dye to monitor membrane fluidity, and we have developed an instrument to image spatial and temporal change in membrane fluidity by use of laurdan. in order to examine role of 'micro-domain' in biomembrane, we applied this imaging instrument to a giant liposome, cho cells and pc12d cells. fluorescence microscope was equipped with a home-build dual-view optical unit. microscopic image of membranes stained with laurdan is separated into an image at 440 nm and an image at 490 nm by the combination of monochromatic filters and dichroic mirrors. the each image is focused on an image plane of a ccd camera side by side. and generalized polarization (g.p.) image is calculated from those images by personal computer according to the definition of g.p. the g.p. imaging at video rate was applied to a giant liposome of composed of dmpc and dmpe in order to observe phase separation. it was also examined that specific interaction of sphingomyelin and cholesterol in living cho cells and neuritis protrusion from raft region of pc12d cells stimulated by neuron growth factor. current fluctuations in biological lipid membranes from human cell lines s. nuschele, a. wixforth, m. f. schneider university of augsburg, experimental physics 1, univer-sitätsstrasse 1, d -86159 augsburg, germany lipid membranes can undergo phase transitions at physiological temperatures. not only do single lipid component membranes show melting behaviour but also biological lipid membranes from eukaryotes as well as from prokaryotes. performing calorimetric and monolayer studies we are able to detect phase transitions in extracted membranes from different human cell lines (e.g. keratinocytes and melanoma cells). close to and in the melting transition regime we measured distinct channel like current fluctuations. the opening times of these current fluctuations can be predicted based on the lateral compressibility measured from the monolayer isotherm and agree well with the experimental data [*]. the applied method of extraction excludes the presence of functional proteins in the membrane rendering the lipid bilayer as the source of the observed current fluctuations. the molecules are composed of single hydrophobic tail and two hydrophilic aldonamide-type groupings (gluconyl c12-dga or lactobionyl c12-dla) linked by the propylene chain at the nitrogen atom. the micellization processes of c12-dga and c12-dla were studied by means of itc. the critical micelle concentrations, the enthalpies (∆h m ) and the entropies (∆s m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (∆g m 0 (hy)) were calculated. qspr analysis was also used to predict cmc of studied compounds. the interactions of c12-dga and c12-dla with model membranes (dppc and dppc/chol. bilayers) were studied by means of dsc. using quantum computations some basic molecular properties were calculated. the conformational space was explored using molecular mechanics. obtained results were compared with those for analogical compounds with single head groups, e.g. with also synthesised by us n-alkanoyl-nmethyllactitolamines (c n mela) and common sugar-based surfactants c 12 gluc and mega-10. enfuvirtide (t-20) was the first hiv-1 fusion inhibitor peptide approved for clinical use. t-1249 is an inhibitor still under evaluation. previous studies, based on tryptophan intrinsic fluorescence, showed that the peptides interact differentially with membrane model systems (luv) with different lipid compositions. studies with human blood cells were necessary to further establish the role of membranes on these peptides mode of action. an experimental strategy was applied based on the membrane dipole potential, as measured by the fluorescent probe di-8-anepps. human erythrocytes and peripheral blood mononuclear cells (pbmc) were successfully labeled. for both systems, a fusion inhibitor concentration-dependent decrease on di-8-anepps fluorescence excitation ratio (a measure of the spectral shift and dipole potential) was observed. the quantitative analysis of these variations indicated that t-1249 has an approximately ten-fold higher affinity towards erythrocyte and pbmc, when compared with enfuvirtide. this is in agreement with the previously known adsorption of t-1249 on cholesterol-rich membrane domains and with its higher partition constants. hiv associates with erythrocytes in vivo, which can constitute a route to deliver peptide to the viral membranes (also rich in cholesterol). lymphocyte membranes can concentrate and accelerate the drug interactions with its molecular target, gp41 in its exposed conformation. oxidation of low density lipoprotein (ldl) is known to be a key step in atherogenesis, leading to inflammation, proliferation and apoptosis of cells of the arterial wall. these effects are largely exerted by oxidatively fragmented phospholipids, which are highly exchangeable between cells, tissues and lipoproteins. in particular, pgpc has been identified in minimally modified ldl and has been reported to elicit a wide range of pathophysiological responses in vascular cells, e.g. the activation of apoptotic signaling pathways. we investigated here the behavior of the fluorescent oxidized pgpe-alexa647 compared to dhpe-bodipy in artificial supported lipid bilayers with different cholesterol contents. the two labeled lipids differ in the type of membrane insertion: while dhpe-bodipy is anchored to the membrane via two fatty acids, pgpe is incorporated with only one fatty acid. the second chain is an acyl fragment in sn-2 position, which represents the oxidation product of an unsaturated acyl chain. with increasing cholesterol content we observed a decrease in the diffusion coefficient for both lipids. interestingly, the diffusion of the oxidized lipid was reduced in a higher degree compared to that of the non-oxidized lipid. the calculated ratios of the diffusion constants of pgpe-alexa647 and dhpe-bodipy suggest a different type of interaction with cholesterol. membrane proteins account for a third of all proteins encoded for by the human genome, and play a vital role in a number of cellular processes. few membrane protein structures have been determined to date in comparison to soluble proteins. this discrepancy is due to experimental difficulties in preparing membrane protein samples for structural analysis. traditional techniques for studying membrane proteins by x-ray crystallography or solution nmr use detergent solubilised proteins which can differ from their native confirmations. solid-state nmr allows the study of transmembrane proteins in lipid bilayers, representing a more native like environment in which to obtain biologically relevant structural information. we have been working on the development of reliable methods for reconstitution of transmembrane peptide into liposomes using glycophorin a as a model tm protein. using reconstitution methods based on the removal of detergent by bio-beads, we have used electron microscopy to screen the ideal conditions for insertion of gpa into liposomes in preparation for mas nmr experiments. em has allowed us to identify conditions favourable for insertion of peptide into lipid vesicles and those that result in aggregation. in order to confirm the secondary structure and insertion of gpa into lipid vesicles, techniques such as cd, ocd, ftir and dls have been used to provide quantitative information in addition to the visual results from em. nonesterified fatty acids regulate a broad spectrum of metabolic activities and are involved in many physiological, pathological and/or pharmacological processes in living cells. they spontaneously transfer between donors and acceptors such as fatty acid binding proteins and lipid membranes. we focus on protein-lipid dispersions of human serum albumin (hsa) and sterically stabilized liposomes (ssl) composed of dppc and appropriate amount of peg:2000-dppe in which stearic acids (sa) are inserted either in the protein or in the ssl. exploiting the fact that hsa has a single tryptophan residue and that the intrinsic trp-fluorescence emission signal is quenced by the presence of sa, the kinetics of sa transport between hsa and peg:2000-grafted dppc membranes is studied by means of fluorescence. it is found that the transfer of sa between hsa and ssl is a first-order process and the kinetics of transfer depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers), and on the grafting density of the peg-lipids at the protein/lipid interface. indeed, in the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer and it is faster for the passage from dppc bilayers to hsa. the presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer-chains, especially for lipid membranes in the fluid phase. a. orth 1 , w. römer 2 , l. johannes 2 , c. steinem 1 1 institute for organic and biomolecular chemistry, university of goettingen, germany, 2 laboratoire trafic et signalisation, institut curie, paris, france shiga toxin (stx) from shigella dysenteriae is an ab 5 -class bacterial toxin. infections with stx lead to the haemolyticuraemic syndrome, which is known to be a major cause for renal failure at an early age. the interaction of the homopentameric b-subunit (stxb), which is responsible for binding and intracellular transport of the holotoxin, with its cellular receptor, the glycosphingolipid gb 3 , is the first step for endocytosis of the toxin. one b-subunit can bind up to 15 gb 3 -molecules to form stxb-gb 3 -clusters causing negative curvature of a membrane. the binding of stxb to giant unilamellar vesicles, composed of dopc, cholesterol and gb 3 induces tubular membrane invaginations, which were also found in experiments with energy-depleted hela-cells. in recent studies, protein and lipid reorganization processes after attaching stxb to solid supported membranes, composed of dopc/sphingomyelin/cholesterol/gb 3 have been investigated. the compaction of gb 3 -molecules led to an additional stxb-gb 3 -enriched phase, which was also observed in lipid monolayers at the air/water interface. in this study, the influence of stxb on model membranes will be investigated combining the advantages of free-standing lipid membranes with those of ssms. the impact of stxb on pore-suspending membranes will be followed by confocal laser scanning mi croscopy and atomic force microscopy. a. robaszkiewicz 1 , c. spickett 2 , p. sicinska 1 , g. bartosz 1 , m. soszynski 1 1 department of molecular biophysics, university of lodz, lodz, poland, 2 strathclyde institute of pharmacy and biomedical sciences, glasgow, u.k. hypochlorite generated in vivo under pathological conditions is a known oxidant, able to initiate lipid peroxidation process, which affects the stability of biological membranes. the aim of this study was the analysis of the products formed during the reaction of hypochlorite with phosphatidylcholines containing unsuturated fatty acid residues (1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, 1stearoyl-2-arachidonylphosphatidylcholine) and their effects on the human erythrocytes. using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in the decrease of the rotational correlation time and rotational motion freedom of liposomes estimated by epr using spin probes (16-and 5doxylstearic acid). unilamellar chlorohydrin liposomes had lower diffusion coefficient for calcein than liposomes made of parent lipids. flow cytometry demonstrated fast incorporation of uni-and multilamellar chlorohydrin liposomes labeled with nbd-pe into erythrocytes. this effect was accompanied by the formation of the erythrocyte subpopulations of higher volume, decrease of the rate of fluorescein diacetate hydrolysis, estimated by flow cytometry, and increase of affinity and maximal velocity of the membrane enzyme acetylcholinesterase. extensive bilayer perforation coupled with the phase transition region of an anionic phospholipid k. a. riske 1 , l. q. amaral 2 , m. t. lamy 2 1 depto. biofisica, universidade federal de sao paulo, sao paulo, brazil, 2 instituto de fisica, universidade de sao paulo, sao paulo, brazil at low ionic strength dimyristoylphosphatidylglycerol (dmpg) exhibits a broad phase transition region characterized by several superimposed calorimetric peaks. peculiar properties, such as sample transparency, are observed only in the transition region. we use differential scanning calorimetry, turbidity and optical microscopy to study the narrowing of the transition region with the increase of ionic strength. upon addition of salt, the temperature extension of the transition region is reduced and the number of calorimetric peaks decreases until a single cooperative event is observed in the presence of 500 mm nacl. the transition region is always coupled with a decrease in turbidity, but a transparent region is detected within the melting process only in the presence of up to 20 mm nacl. optical microscopy of giant vesicles shows that bilayers first rupture when the transition region is reached and subsequently lose optical contrast. fluorescence microscopy reveals a blurry image in the transparent region, suggesting a different lipid self-assembly. overall sample turbidity can be related to the bilayer optical contrast. our observations are discussed in terms of the bilayer being perforated along the transition region. in the transparent region the perforation is extensive and the bilayer completely loses the optical contrast. financial support: fapesp. label-free imaging of biological membranes using surface imaging techniques j. l. richens, k.-a. vere, p. o'shea cell biophysics group, university of nottingham, nottingham, uk surface plasmon resonance (spr) is a detection technique traditionally used for specific protein detection, which is now exploited routinely as a generic label-free sensor. spr responds to changes on a metal surface conditioned to sense the binding of analytes. thus, the composition of the external medium and metal surface will be fundamental to the signal output. here we use a model phospholipid membrane system to investigate the effect of altered buffer and surface compositions on spr signals. comparisons are undertaken between continuous gold surfaces and gold nanoparticles. we demonstrate that surface electrostatics and the salt composition and molarity of a buffer all have significant impacts on spr output. the association of respiratory syncytial virus matrix protein with membrane microdomains the association of the matrix protein from respiratory syncytical virus with membranes has been characterised by tensiometry, brewster angle microscopy, and atomic force microscopy following deposition of langmuir monolayers onto modified silica substrates. association of the protein with monolayers containing phosphocholines and cholesterol leads to the formation of materials with new properties that differ from those of either of the pure components. the behaviour of the protein in monolayers rich in cholesterol and sphingomyelin exhibits a significant concentration dependence. at low concentrations, the protein exhibits a simple partioning behaviour. at higher concentrations, lipids are extruded from the monolayer below a critical surface area. these findings are discussed in relation to the recently published structure of the protein (pnas, 2009, 106, 4441-4446) , the documented formation of viral filaments during key stages of the infection cycle and the isolation of the protein from detergent-resistant membrane fractions. structure and dynamics of closed melting membranes v. m. rondelli 1 , s. santorsola 1 , p. brocca 1 , e. del favero 1 , l. cantù 1 , m. zimbone 2 1 dep. of chemistry, biochemistry and biotechnologies for medicine, university of milan, segrate, italy., 2 dep. of physics, university of catania, catania, italy. we studied the dynamical and structural properties of large unilamellar vesicles (≈ 120 nm luvs) of phospholipids (dmpc, dc 15 pc and dmpc : dc 15 pc = 1 : 1 molar mixture) in the temperature range around the chain-melting transition, ≈ 3 • c wide, with 0.1 • c resolution and 0.01 • c accuracy. small-angle (saxs) and wide-angle x-ray scattering (waxs) measurements show that across the transition the vesicle behaves as an 'evolving membrane', passing through several different states, each of them being characterized by different proportions of coexisting fluid-and gelchains molecules. noteworthy, no kinetics has been detected. on the same samples, a unique and very sensitive laser light scattering technique allows to determine the characteristic times of thermally induced shape fluctuations, connected to the elastic properties of the membranes. results indicate a clear softening of the membranes in correspondence to the chain-melting transition, as indicated by a manifold increase of the corresponding fluctuation characteristic time. meanwhile the overall size of the vesicle is not sensibly changed. this softening is likely to be due to the presence of structural defects, eventually driving to local morphological modifications. thermodynamics characterization of isolated lung surfactant assembled as lamellar bodies e. x. rodriguez 1 , r. alvarez 1 , r. barrio 2 , c. irles 3 , a. ortega 1 1 biochemistry depto., school of medicine, 2 inst. of physics unam, 3 nat. inst. of perinatology, mexico city, mexico lungs are a large extension of ∼80m 2 of one layer cells, which is structured as compacted sacs called alveoli, where lung function takes place: the gas exchange. alveolar endotelium is formed by two kinds of cells: neumocyte type i (nti), which covers ∼97% of the alveolar surface where gas exchange occurs and, ntii where lung surfactant (ls) is produced. ls are a mixture that lay over a water film in the luminal surface of the alveoli and it is thought to decrease the surface tension to avoid alveolar collapse during expiration. ls are made of phospholipids and proteins, which are determinant for the structure of ls under particular conditions of pressure and temperature. ls inside the cell is packed in organelles called lamellar bodies (lb), and is released to the water/air interphase in other conformation. in the present study we isolate lb from pig's lung and study lb thermodynamic characteristics by microcalorimetry in the presence and in the absence of structural proteins. phase transition profile of lb with and without proteins is basically the same; while ls in the alveolar lumen have a higher transition temperature (tm). mayor changes in tm are observed between lb and ls from alveolar lumen. although ls lipid composition in and outside the cell is assumed to be the same, the ground for the differences in tm under these two conditions is unknown. m. rodrigues 1 , g. rádis-baptista 3 , b. g. de la torre 2 , m. castanho 1 , d. andreu 2 , n. c. santos 1 1 instituto de medicina molecular, portugal, 2 pompeu fabra university, spain, 3 federal university of cearà, brasil nucleolar-targeting peptides (nrtps) have been recently designed by structural dissection of crotamine, a toxin from the venom of a south america rattlesnake (radis-baptista et al. 2008. j med chem 51:7041) . at µm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work is to decipher the molecular mechanism for the translocation of nrtps into cells. quantification of partition into membranes was carried out, based on intrinsic tyrosine fluorescence. the role of the bilayer phase, anionic lipids, reducing agents and peptide concentration on the extent and kinetics of partition were studied. both nrtp1 and nrtp2 exhibited high partition to popc (neutral) lipid vesicles (k p ≈3×10 3 ), which was enhanced by the anionic lipid popg for nrtp1, but not nrtp2. the peptides showed a decrease in partition for popc:cholesterol (liquid ordered state) or dppc (gel) membranes. depending on the lipid composition, the peptides either increased or decreased their quantum yield upon membrane insertion. quenching experiments with acrylamide showed no peptide aggregation in solution. once the translocation mechanism is fully understood we will test nrtps as carriers of relevant cellular cargos, evaluating their potential clinical application in drug delivery or gene therapy among other applications. the ingestion of trans fatty acids (tfa) formed during the partial hydrogenation of vegetable oils has been linked to a detrimental impact on health by an, as yet, unknown mechanism. we synthesized deuterated analogs of 1-elaidoyl-2-stearoylphosphatidylcholine (t18:1-18:0pc) that contains a single "unnatural" trans double bond and 1-oleoyl-2-stearoylphosphatidylcholine (c18:1-18:0pc) that contains a single "natural" cis double bond. solid state 2 h nmr, complemented by molecular dynamics (md) simulations, was then employed to compare molecular organization in model membranes prepared from these isomeric molecules. analysis of spectra recorded as a function of temperature reveals a higher chain melting temperature for the trans isomer, indicating tighter molecular packing in the gel state. in the liquid crystalline, however, the difference between the trans and cis isomers is subtle. order as probed by the perdeuterated [ 2 h 31 ]18:0 sn-2 chain, and corroborated by computer simulation, coincides within <5%. only in the conformation of the double bond is an appreciable difference implied. thus, our results contradict the conventional view that tfa resemble saturated fatty acids, which is >20% more ordered. (supported by acs, prf 43281-ac7.) photooxidation and lateral membrane diffusion of dipole molecules v. s. sokolov 1 , e. a. sokolenko 1 , a. a. lents 2 , p. pohl 2 1 a.n frumkin institute of physical chemistry and electrochemistry, russian academy of science, moscow, russia, 2 institut fuer biophysik, johannes kepler universitaet linz, austria the photodynamic oxidation of phloridzin, of the styryl dye di-8-anepps and of unsaturated lipids was monitored "online" by measuring the collapse of the dipole potential which has been introduced to the membrane by these molecules. their photodamage occurred at different rates when the target molecules and the singlet oxygen generating photosensitizer (phthalocyanin) were adsorbed to the same or to opposite sides of the planar lipid bilayer. the difference in the oxidation rates were attribute to singlet oxygen transport through lipid bilayer and therefore we were able to estimate the permeability of lipid bilayers to singlet oxygen. however, the apparent permeabilities derived from experiments with different targets were differ from each other. therefore, we tested the hypothesis that the lateral membrane diffusion of target molecules and oxidation product may have biased our analysis. in line with this anticipation we found that the apparent permeability is dependent on the size of the planar bilayer. the development of a new mathematical model, which takes the mobility of all reacting species in the aqueous and lipid environments into account, allowed estimation of the real membrane permeability to singlet oxygen. it appeared to be very close to that of oxygen in the ground state. a number of complex three-dimensional lyotropic liquid crystal phases are already known, such as the bicontinuous cubic phases, but so far only a single example has been found -a cubic phase of spacegroup fd3m -of a structure based upon a complex close packing of inverse micelles (1) . we now report the discovery (2) of a novel lyotropic liquid crystal phase, of space group, p6 3 /mmc, whose structure is based upon a hexagonal close packing of identical quasi-spherical inverse micelles. the model membrane system consists of a hydrated mixture of dioleoylphosphatidylcholine, dioleoylglycerol, and cholesterol. this novel phase has a number of unique features which may render it useful for a range of applications. firstly, it is the only known self-assembled lyotropic phase whose structure consists of a periodic close packing of identical inverse micelles. secondly, it is stable in excess aqueous solution, which is very important for potential biological or biomedical applications. references (1) v. luzzati, v., r. vargas, a. gulik, p. mariani, j.m. seddon, and e. rivas, biochemistry 31, 279-285 (1992) . dystrophin is a rod-shaped muscular subsarcolemal protein. its deficiency is one of the root causes of duchenne muscular dystrophy. dystrophin rod domain contains 24 homologous repeats, where the sub-domain constituted by the repeats 11 to 15 (r11-15) was reported to bind actin and membrane lipids. we analyzed the interaction of r11-15 with lipid monolayers. to better understand the assembly mechanism of this protein with lipids, we studied its adsorption behavior at the air-liquid and lipid/liquid interface using ellipsometry, surface pressure, and atomic force microscopy (afm). using two different mixtures of phospholipids, ellipsometry and pressure surface data show that r11-15 interacts with the lipid monolayers, but is inserted into the monolayer formed by dopc-dops while it lies below the monolayer of dopc-dope. this indicates that r11-15 interacts more strongly with dopc-dops than with dopc-dope. afm images show that the pressure of lipid monolayer influences the organization of r11-15. when the lipid surface pressure is 30mn/m, r11-15 forms a striking network, indicating a protein-protein interaction in addition to the protein-lipid interaction. this unique behavior of one part of the central domain of dystrophin may explain its key role in muscle cell. studies on polyphenol extracts from helichrysum l., fagopyrum mill., crataegus l. and hypericum l. were performed in order to find if they may be applied as a natural free radical scavengers protecting biological membranes against peroxidation. ghosts erythrocytes were used in the experiments. they were suspended in tris-edta solution, ph 7,4, then.irradiated with a bactericidal lamp without (control) or with a proper amount of the extracts studied. the product of lipid peroxidation was malonic dialdehyde (ma). the colour reaction of ma with thiobarbituric acid (tba) enables to determine the concentration of ma spectrophotometrically. it was found that peroxidation increased with the irradiation time. however, it significantly decreased when concentrations of poliphenols increased. the best antioxidtive property was found for hypericum l. the antioxidative efficiency sequence of the plant extracts studied was the following: hypericum l. > crataegus l. > fagopyrum mill. > helichrysum l. the results obtained indicate that polyphenol extracts exhibit excelent antioxidative properties that make them good free radical scavengers for efficient protection of biological membranes. this work was sponsored by ministry of science and education, scientific project no. n n305 337034. interaction of cationic porphyrins with neutral and negatively charged liposomes i. voszka 1 , g. corradi 2 , p. maillard 3 , h.-j. steinhoff 4 , g. csík 1 1 institute of biophysics and radiation biology, semmelweis university budapest, hungary, 2 research institute for solid state physics and optics, hungarian academy of sciences, budapest, hungary, 3 institute curie, section de biologie, orsay, france, 4 fachbereich physik, universitat osnabrück, germany porphyrin derivatives are used in photodynamic therapy of tumors. the knowledge of the photosensitizer location within the cell is important. the effect of porphyrin derivatives containing cationic side groups was examined on neutral and negatively charged liposomes by esr. the esr signal was first examined as a function of temperature and porphyrin concentration in the dark. a significant change related to the appearance of quasi free spin labels was obtained for spin probes at the 12 th carbon atom and was more expressed for the asymmetrical derivative. illuminating the samples the esr amplitude decreased for all positions of the spin probe but to different extent. the effect was more expressed in case of the symmetrical derivative, especially for label positions 5 and 12. for the asymmetrical derivative the effect changed from moderate to weak from the 5 th to the 16 th position. this indicates that the asymmetrical derivative is incorporated nearer to the lipid head groups while the symmetrical one may be located deeper in the membrane. under oxygen-free conditions both derivatives showed weaker but still pronounced effects.. single channel recording of α-hemolysin in nanopore-spanning tethered bilayer lipid membranes n. t. thet 1 , i. pfeiffer 2 , w. knoll 1 , i. köper 1 1 max planck institute for polymer research (mpi-p), ackermannweg 10, 55128 mainz, 2 austrian research centers gmbh -arc, tech gate vienna, 1220 vienna, austria artificial lipid bilayer membranes mimic biological cell membranes in many aspects and can be used to study functional processes such as ion channeling, signal transducing, transport of nutrients etc. as most of the functions of a cell are accomplished by membrane proteins, research has been ongoing in studying and characterization of membrane proteins embedded in model lipid bilayer membranes. black lipid membranes (blms) were studied for decades but their potential for practical applications is hindered, mainly due to their lack of stability and limited lifespan. recently, tethered bilayer lipid membranes (tblms) proved to have long life span of up to several months without significant changes in membrane resistance and capacitance. in order to combine advantages of blms and tblms, we have designed a membrane system which is freely spanned across a single nanopore in a silicon nitride membrane. this system not only mimics cell membranes, but also it allows control over the chemical composition of buffer with unlimited ionic reservoirs on both sides of the membrane. this freestanding tblm maintains structural stability and lifetime of up to 120 hours without significant decrease in its structural integrity and electrical sealing. for the validation of the tblm formation, we have inserted well known α-hl pores. we are able to control the amount of α-hl pores insertion and measure single channel ion transport across the tblm by α-hl pores using a capacitor feedback amplifier. the language of shape: biological reactions are dramatically affected by the shape of lipid membrane d. stamou university of copenhagen, copenhagen, denmark a plethora of biological process are taking place on the surface of lipid membranes. as a rule membranes in vivo are curved in a variety of complex geometries. here i will present a quantitave study on the influence of membrane curvature on protein-membrane and membrane-membrane interactions. to gain systematic access to a continuum of membrane curvatures we immobilized liposomes on a surface at dilute densities. using confocal fluorescence microscopy we imaged single liposomes of different size, and therefore different curvature, and monitored their interaction with a binding partner (proteins or other liposomes). i will discuss unpublished data on two important classes of biomolecular interactions that exhibited dramatic curvature dependence: a) snare-mediated docking and fusion b) anchoring of peripheral proteins. the following references provide partial information on the single-liposome assay: a. zettergren, c. gudmundsson, t. nylander, e. sparr physical chemistry1, lund university, 22100 lund, sweden there is accumulating evidence of substantial amounts of phospholipids in the cell nuclei 1 , although the function of these lipids is still not fully understood. it has been shown that the chromatin complex composed of dna, rna and proteins also includes phospholipids, and that rna colocalize with these 2 . although the rna-phospholipid interactions may have important implications to biological function, in gene therapy and in medicine, very little work has been dedicated to the characterization of rna interaction with phospholipids. the objective of this work is to investigate the adsorption behavior of short single stranded 10 bases long rna (ssrna 10 ) molecules (similar to mirna) to lipid monolayers at the air-water interface as well as to study how the presence of rna affect the domain formation in the monolayers using fluorescence microscopy. monolayer studies have shown adsorption of ssrna 10 to monolayers consisting of zwitterionic dppc as well as to monolayers consisting of cationic dodab. the adsorption behavior of these very short nucleic acids differ significantly from the adsorption process for longer nucleic acids as for example a 2000 base pairs long ds dna (dsdna 2000 ) which has been used as a reference system 3 . the presence of ssrna 10 significantly changes the compression isotherm of both dppc and dodab monolayers. plant defensins are cysteine-rich antimicrobial peptides found in various plant species. they share a common threedimensional structure, stabilized by eight disulphide-linked cysteines consisting of three antiparallel β-strands and one α-helix. most plant defensins show antifungal activity with no effect on mammalian and plant cells. expression of these peptides in plant tissue is induced by pathogen infection. the mechanism of defensin action is based on membrane permeabilization. this occurs through an interaction with high affinity binding sites on fungal membranes, resulting in alteration of membrane potential. the genes encoding for a peach ppdfn1 and a grape vvamp defensins were expressed in e. coli and purified to homogeneity. they were tested for antimicrobial activity against some fungi and showed to have an inhibitory effect on the spore germination. biophysical analysis showed that defensins were able to interact with artificial membranes. binding of defensins to membranes was dependent on lipid composition, increasing with the sphingolipids content. interaction between peptides and sphingolipids could lead to insertion of the defensins into the membrane resulting in its destabilization. extracts of the plant perilla frutescens have many uses in the asian kitchen, e.g. as a popular garnish used in sushi. the plant is also employed in eastern traditional medicine to treat a variety of ailments including colds, food allergy and depression. two of the main constituents of the plant are limonene and perilla aldehyde. by bio-oxidation, these molecules can be converted to perillic alcohol and perillic acid. these cyclic terpenes possess antibacterial and anticarcinogenic properties. the modes of action for these compounds are at present not understood, but their remarkably diverse pharmacological properties suggest that they might target the phospholipid matrix of the cellular lipid membrane. indeed, the cyclic terpenes can bind to, and alter the physiochemical properties of the lipid bilayer of the membrane, the effects of which can cascade down to several essential cellular processes. here, we use molecular dynamics (md) simulations to investigate the effect of limonene and its derivatives on the properties of lipid bilayers including the changes in acyl chain order parameters, bilayer thickness and the area per lipid. md can afford molecular-scale dynamic information, often not easily accessible from experimental measurements. this information can be used to interpret existing experimental data obtained by e.g. isothermal titration calorimetry, electron paramagnetic spectroscopy and differential scanning calorimetry. catalysis in the membrane: interfacial mechanism of phospholipase a2 h. p. wacklin 1 , r. k. thomas 2 1 institut laue langevin, grenoble, france, 2 physical and theoretical chemistry laboratory, oxford university, u.k. phospholipase a2 cleaves the sn-2 acyl chains of membrane phospholipids and performs a range of physiological functions. one of the least well understood aspects of the its mechanism is how its activity is regulated by the interaction with the substrate membrane. we have used neutron reflection to monitor changes in membrane structure during lipid hydrolysis [1, 2] . the penetration depth of pla2 depends on lipid packing and increases during the lag phase of porcine pancreatic pla2. by using a selectively deuterated lipid substrate, d31-popc, we determined the relative membrane-water partitioning of the lipid products in-situ. the lyso-lipid product partitions into the solution phase, while fatty acid accumulates in the membrane and increases the affinity of pla2 [2] . pla2 is inhibited at ph 5, which is consistent with protonation of the catalytic histidine. however, irrespective of ph, pla2 is fully activated by me-betacyclodextrin, which facilitates the release of the lyso-lipid from the enzyme-substrate complex. me-beta-cyclodextrin does not interact directly with the membrane surface or the substrate lipids, indicating that product release occurs outside the immediate membrane-water interface. diffraction limits resolution in optical microscopy, but many interesting biological problems occur on shorter (molecular) length scales. recently, methods to circumvent the diffraction limit have been presented. fluorescence photoactivation localization microscopy (fpalm) uses activation of many small subsets of photoactivatable or photoswitchable fluorescent probes (pafps) to generate images with effective resolution in the tens of nanometers. pafp molecules are photoactivated, imaged, localized, and photobleached in small numbers. the process is repeated for many subsets to build up data on thousands to many hundreds of thousands of molecules. the positions of all localized molecules are used to construct an image of the sample with resolution limited not by diffraction, but by the localization precision and molecular density. results will be shown from a variety of biological systems, including live and fixed cells expressing a variety of pafp-tagged proteins. bi-plane fpalm can image in three dimensions with demonstrated resolution of 30 nm x 30 nm x 70 nm. polarization fpalm can image both molecular positions and anisotropies simultaneously with lateral resolution of ∼20-30 nm. using these powerful capabilities, many potentially interesting biological problems can be addressed. binding of the hiv-1 ncp7 on oligonucleotides at the single-molecule level j. godet, p. didier, a. jouonang, y. arntz, y. mély laboratory of biophotonics and pharmacology, umr cnrs 7213, university of strasbourg, france the nucleocapsid protein (ncp7) of the hiv-1 is a small basic protein which plays key functions in the viral life cycle. the activity of ncp7 mainly rely on its potent rna-and dna-chaperone activities that direct the rearrangement of numerous nucleic acid molecules into their most stable conformation. two main features of nc's chaperone activity are its abilities to aggregate and destabilize nucleic acids. in addition, the rapid kinetics of ncp7 interaction with nucleic acids was recently proposed as another major component of nc's chaperone function based on the apparent correlation between an indirect measurement of the nucleic acid dissociation kinetics of ncp7 and its overall chaperone activity. but so far, no direct measurement of the on/off rates of ncp7 binding to oligonucleotides was performed. in the present work, we realized single molecule fluorescence resonance energy transfer (smfret) measurements to probe the transient interactions of a tmr-labelled ncp7 with a short cy5-labelled dna oligonucleotide confined into nanovesicles. after confirming the efficiency of the ncp7/odn complex encapsulation into the nanovesicles by fcs, the vesicles were tethered to the surface for immobilization. integrity of the entrapping vesicles attached on the surface was confirmed by afm. finally, the association and dissociation constants retrieved from these smfret measurements were discussed in the context of the dna-chaperoning activity of the protein. high-resolution spatiotemporal organization of the integrin lfa-1 m. f. garcia-parajo 1 , t. s. van zanten 1 , g.-j. bakker 1 , r. diez-ahedo 1 , a. cambi 2 , c. g. figdor 2 1 bionanophotonics group; ibec, barcelona, spain, 2 tumour immunology dept; ncmls, nijmegen, the netherlands lfa-1 is a leukocyte-specific integrin involved in different steps of the immune response. on monocytes, lfa-1 plays a key role in the regulation of monocyte-endothelial interaction during rolling, arrest and extravasation into the underlying tissue. i n-vivo experiments showed that blood borne lymphocytes can 'switch' within seconds from rolling to arrest. furthermore, tem observations of pro-active, ligandindependent nanoclusters confirmed that affinity and clustering are complementary processes required in adhesion. yet, the mechanisms leading to fast-switching remain obscure. in our group, we used a combination of single molecule fluorescence techniques to study the spatiotemporal organization of lfa-1 on monocytes. we performed optical nanoimaging of lfa-1 nanoclusters in relation to membrane rafts with a resolution of 70nm and accuracy of ∼3nm. in quiescent cells, lfa-1 nanoclusters do not associate with membrane rafts and diffuse freely on the membrane. binding of the integrin to its ligand icam-1 induces the formation of microclusters that further associate with rafts and exhibit reduced mobility, consistent with cytoskeleton interactions. our work highlights the markedly different spatiotemporal organization of lfa-1 that might explain its concerted action to form larger and stable platforms on the cell surface required for rapid and effective cell adhesion. c. ciobanasu, u. kubitscheck rheinische friedrich-wilhelms-universität bonn, germany cell penetrating peptides like the hiv1 tat peptide have the property to rapidly translocate the cell membranes and the capability to deliver a wide range of cargoes. the mechanism of the membrane translocation is still under investigation and object of considerable controversy. we applied and single-molecule and confocal laser scanning microscopy (lsm) to study peptide-membrane interactions. electron-multiplying ccd cameras yield images of single fluorescent molecules with a time resolution in the range of a few milliseconds only, which allows the tracking of fluorescently labelled peptides and lipids at bio-interfaces in realtime with a localization precision of a few nanometers. we formed giant unilamellar vesicles (guvs) from different lipid mixtures and examined their interaction with fluorescently labeled tat peptides. we found that the passive peptide internalization process depends on lipid composition, charge of the lipid bilayer, and the ionic properties of the medium. a translocation of cationic tat peptides was observed in membranes containing at least 20 mol% of lipids with a phosphatidyl ethanolamine or a high mol fraction of the phosphatidyl serine head group. in salt-free solution tat efficiently bound to guvs, however, in a physiological nacl solution tat binding was completely abrogated, but the peptides efficiently equilibrated across the guv membrane. new approaches to measure interactions in the live cell plasma membrane g. j. schütz biophysics institute, johannes kepler university linz, austria in my lecture, i will show examples how to obtain insights into the organization of the cellular nanocosm by single molecule experiments. our primary goal is an understanding of the role of such structures for immune recognition. brightness and single molecule colocalization analysis allows us to study stable or transient molecular associations in vivo. in particular, i will present results on the interaction between antigen-loaded mhc and the t cell receptor directly in the interface region of a t cell with a surrogate antigenpresenting cell. in addition, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions. the method allows identifying and characterizing interactions between an arbitrary fluorescence labeled protein ("prey") and a membrane protein ("bait") directly in living cells. cells transfected with a fluorescent fusion protein of the prey are plated on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of the bait; the fluorescence copatterning is used as readout for the interaction. we applied this tool for the study of the interaction between cd4 -the major coreceptor for t cell activation -and lck, an important tyrosine kinase in early t cell signaling. in addition to the well-known zinc-clasp structure, we found strong contributions of lck membrane anchorage for the binding of the two proteins. developing a fluorescent redox sensor for monitoring metal-ion mediated catalysis in biosystems a. rybina 1 , a. kiel 1 , b. thaler 2 , a. sprödefeld 2 , r. krämer 2 , d. p. herten 1 1 bioquant and, 2 department of inorganic chemistry, heidelberg university, germany a fluorescent redox sensor is an electron photo-switching device that can be used for the characterization of the redox state of a given environment. it combines a fluorescent fragment with a redox-active unit that senses the media by a redox reaction and controls the light emitting properties of the fluorophore. such reversible sensor can help to examine the electrochemical state and changes in biological systems during biochemical processes in real time. recently a new fluorescent molecular sensor with a redox-active hydroquinoneunit covalently linked to fluorophore rhodamine b was developed. (kierat r.m. et al., bioorg. med. chem. lett., 2009 -accepted) . the reduced hydroquinone-form of the sensor is fluorescent while its oxidation to benzoquinone-derivative leads to a significant decrease of the fluorescence. although the above method shows great promise for applications in biological systems, the exact mechanism of this process is not fully understood yet. we use fluorescence spectroscopy to investigate oxidation reactions on the ensemble and single-molecule level and study kinetic rates. the proposed strategy is to use cu (ii) complex as oxidation mediator immobilized on surface via dna linker to examine the oxidation mechanism. fluorescently labeled atp as a probe of the outer mitochondrial membrane barrier: role of vdac fluorescence correlation spectroscopy (fcs) was applied for studying the distribution of fluorescently labeled atp (bodipy-atp) in isolated mitochondria. the setup and peak intensity analysis (pia) was described in our recent paper (perevoshchikova et al. 2008 biochim.biophys.acta 1778 :2182 . the binding of bodipy-atp to mitochondria was maximal in the non-energized state, whereas the addition of succinate (respiratoty substrate) or atractyloside (adenine nucleotide translocase inhibitor) led to a decrease in the binding. nadh reduced the fcs signal from bodipy-atp added to non-energized mitochondria more than nad+ did under the same conditions suggesting the control of nucleotide transport through voltage-dependent anion channel (vdac) residing in the outer membrane. konig's polyanion also decreased the bodipy-atp binding to mitochondria with the effect being reduced by alamethicin or digitonin. control experiments showed that bodipy-atp did not bind to liposomes showing minor role of unspecific binding. it was suggested that bodipy-atp in combination with fcs can be used to monitor the functional state of mitochondrial vdac which is considered to be a principal regulator of mitochondrial function. fluorescence correlation spectroscopy studies of lysozyme partition to phospholipid vesicles a. m. melo 2 , a. coutinho 2 , m. prieto 1 1 cqfm and in, ist, 1049-001 lisboa, portugal, 2 dqb, fcul, 1749-016, lisboa, portugal binding to membrane lipids has been increasingly recognized as an important step in the aggregation and cytotoxicity of several amyloidogenic proteins [1] . in addition, it has been recently reported that membranes containing negatively-charged phospholipids can also trigger rapid amyloid-like fiber formation by a variety of several nonamyloidogenic proteins, such as cytochrome c and lysozyme [2] . our study aims to elucidate the factors that govern the formation of these lipid-protein complexes. given the importance of electrostatic interactions between the proteins and the acidic phospholipids in the putative membrane-induced protein misfolding step, it is essential to first characterize quantitatively the protein partition behavior towards liposomes prepared with variable anionic lipid content. in this study, lysozyme was chosen as a model protein and fluorescence correlation spectroscopy (fcs) was used to monitor its binding to liposomes after its conjugation to alexa fluor 488. most organic dyes labelling techniques produce a mixture of populations of molecules labelled with a different number of fluorophores. the influence of this poli-dispersity of labelled molecules on the protein partition behaviour will be explored, namely the ability of the fcs technique to detect the production of non-competent membrane-binding species. t. wohland 1 , p. liu 1 , x. shi 1 , y. h. foo 1 , t. sudhaharan 2 , s.-w. chong 3 , v. korzh 3 , s. ahmed 2 1 chemistry dept., singapore nat. univ., 2 medical biology inst., singapore, 3 molecular & cell biology inst., singapore biomolecular interactions have been measured mostly under in vitro conditions because of higher accuracy and ease of measurement. however, it has become clear that the cellular environment has an important influence on these interactions. it was therefore necessary to develop new tools to allow the measurement of interactions in cells and organisms. recently, we have developed a modality of fluorescence cross-correlation spectroscopy (fccs) called single wavelength fccs (sw-fccs), which uses one-photon excitation to excite two fluorophores with overlapping absorption but separable emission spectra. sw-fccs has been used to determine e.g. dimer fractions of proteins in live cells. here we aim to extend the use of sw-fccs to cells and organisms. in the first part we determine the dissociation constants of a small rho-gtpase (cdc42) with an effector domain (crib) and two effectors (n-wasp or irsp53). in the second part we measure the binding between cdc42 and a scaffolding protein (iqgap1) in dependence of their expression levels in cho cells and in live zebrafish embryos. by using gfp/mrfp fusion proteins we can excite both fluorophores at 514 nm and detect them separately in two different wavelength channels. we quantitatively determine the dissociation constants and compare their differences in vitro, in cells, and in embryos. these experiments demonstrate that sw-fccs is a powerful biophysical tool for the quantitation of biomolecular interactions in cells and organisms. addressing plasma membrane structure at the nanoscopic length-scale s. wieser, m. axmann, g. j. schütz biophysics institute, johannes kepler university linz, altenbergerstr.69, a-4040 linz, austria there is increasing interest in a detailed understanding of the structure and dynamics of the cellular plasma membrane, primarily based on recognizing its essential role for controlling cellular signaling processes. we employed single molecule fluorescence microscopy to study diffusion of cd59, a gpi-anchored protein, in the plasma membrane of living t24 cells at sub-wavelength resolution, both on the cell body and on tunneling nanotubules connecting cells. the lateral motion of this single fluorescence labeled molecule was imaged on a millisecond time scale. within the experimental errors, no indications for confined diffusion for cd59 on the cell body in t24 cells have been found. furthermore by separating longitudinal and transversal mobility, we found isotropic diffusion behavior on the surface of tunneling nanotubules. in both studies we analyzed the mean square displacement as a function of the time-lag and the distribution of displacement steps. however, a closed analytical theory for these analysis is only available for the simplest models. to address a suspected diffusion process we reasoned that a full analytical description may not be required; it may well be sufficient to compare the experimental data with monte carlo simulations of the process. we demonstrated the working principle for this simulation based analysis for free diffusion, hop diffusion and transient binding of the tracer molecule to slowly moving receptors. n. chakroun 1 , f. fraternali 1 , m. malfois 2 , h. rezaei 3 , c. a. dreiss 1 1 king's college london, u.k., 2 diamond light source, didcot, oxfordshire, u.k., 3 inra, jouy-en-josas, france prion(prp) diseases are fatal neurodegenerative diseases affecting mammals including human and sheep.they are characterized by the accumulation of extracellular βrich fibrillar deposits of a structurally modified form (prp sc ) of the cellular prp c .despite the increasing interest for prp diseases,the mechanism of prp c /prp sc conversion is still unknown.studies on prp diseases suggest that neurotoxicity arises from small pre-fibrillar oligomers.we have used a range of biophysical techniques combined with molecular dynamics simulations (md) to resolve the oligomerization pathways of sheep prp (sprp).we have shown that under well established conditions, sprp oligomerizes into three oligomers, which form in parallel.in addition, we have now identified the minimal region of sprp leading to the same oligomerization profile of the entire sprp,namely h2h3.low resolution shapes of sprp, h2h3 and resulting oligomers have been determined by small-angle x-ray scattering.time-resolved studies have been used to follow the oligomerization of sprp and h2h3 monomers into the oligomers.the conversion of sprp sc at the molecular scale was studied by md.simulations of the h2h3 region recreating experimental conditions revealed a complete unfolding of h2 helix followed by h3 helix.these crucial steps are followed by the formation of β-sheet structures leading to a stable βrich double hairpin structure. single-molecule force spectroscopy investigation of the conformational equilibria of alphasynuclein m. brucale 1 , a. rampioni 1 , m. sandal 1 , i. tessari 2 , l. tosatto 2 , l. bubacco 2 , b. samorì 1 1 istituto di biochimica g.moruzzi, università di bologna (italy), 2 dipartimento di biologia, università di padova (italy) alpha-synuclein (asyn) is an abundant intrinsically disordered protein (idp) primarily located at presynaptic terminals. mutations in the gene encoding asyn have been linked to early-onset parkinson's disease (pd). by means of single molecule force spectroscopy (smfs) experiment, we show how the conformational equilibrium of monomeric wild type (wt) asyn shifts toward more compact structures in several unrelated conditions linked to pd pathogenicity [1] . the conformational heterogeneity of pathological alpha-syn mutants a30p, a53t and e46k has also been characterized, revealing marked differences in the conformational behaviors of the mutants with respect to wt asyn [2] . all the mutants show a distinctively higher propensity, with respect to wt, to acquire a monomeric compact conformation that is compatible with the acquiring of beta structure. the same smfs experimental methodology is then used to characterize the conformational behavior of wt and mutant asyn in a variety of conditions, in an attempt to gain insight about the multiple and contrasting parameters controlling the equilibrium. in vitro protein folding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble proteins. this is not the case for integral membrane proteins, which constitute about one third of all eukaryotic proteins and more than half of all validated and potential drug targets. fully reversible denaturant-induced unfolding remains limited to a few β-barrel porins, whereas the much larger and more relevant class of α-helical membrane proteins has thus far evaded this approach. we report here the first example of an α-helical membrane protein that can be unfolded completely and reversibly by a chemical denaturant: mistic, a 110-residue protein from bacillus subtilis [1] , dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. using spectroscopic and microcalorimetric techniques, we exploited this unique property to provide (i) a quantitative comparison of membrane protein stability in different membrane-mimetic systems; (ii) an experimental test of controversial predictions [2] regarding the folding core of mistic; and (iii) a convenient setup to study the spontaneous, translocon-independent membrane insertion of this unusual membrane protein. the mechanical functioning of biological tissues is important from many viewpoints such as diseases, clothing and even food. the protein collagen makes up the greater part of these tissues, and is remarkable for its many uses in the body, however there are at least two other major components. one of the most interesting properties of these tissues is their non-linear behavior under stress. this behavior is essential to prevent a catastrophic failure such as an aneurysm. at least three major theories have been proposed within the past few years to explain this behavior, but have been impossible to verify. in order to determine a correct description of the mechanical structure of the tissue we have been using cutting edge technological solutions to address the single molecules within the extra cellular matrix. this technique combines optical methods with single molecule force spectroscopy, allowing stiffness measurements over the nanoscale as well as determining the individual protein tensions within the extra cellular matrix. the results show that this method can be used to determine the network properties even in the complicated aortic wall enabling better understanding of disease states, which in this case include marfan's syndrome and ascending aortic aneurysms. beta amyloid peptide abetapy3-42 shows a faster aggregation kinetics than abeta1-42 c. d'arrigo 1 , m. tabaton 2 , a. perico 1 1 institute for macromolecular studies, national research council, 16149 genova, italy, 2 department of neurosciences, university of genova, 16132 genova, italy we test directly the differences in the aggregation kinetics of three important beta amyloid peptides, the full-length abeta1-42 and the two n-terminal truncated and pyroglutamil modified abetapy3-42 and abetapy11-42, found in different relative concentrations in the brains in normal aging and in alzheimer disease. 1 we find by following the cd signal and the tht fluorescence of the solution in phosphate buffer, a substantial faster aggregation kinetics for abetapy3-42. this behavior is due to the particular sequence of this peptide which is also responsible of the specific oligomeric aggregation states, found by tem, during the fibrillization process which are very different from those of abeta1-42, more prone to fibril formation. in addition abetapy3-42 is found here to have an inhibitory effect on abeta1-42 fibrillogenesis, coherently with its known greater infective power. this is an indication of the important role of this peptide in the aggregation process of beta-peptides in alzheimer disease. the puzzle of the anomalously long denaturation kinetics of green fluorescent protein (gfp) mutants still is largely unveiled. in this study we have followed the effect of mutation h148g on the stability of gfpmut2 (mut2g) in the presence of guanidinium hydrochloride (gdnhcl). different techniques of fluorescence spectroscopy have been employed in order to obtain information concerning the unfolding event: time resolved fluorescence, fluorescence correlation spectroscopy (fcs), and fluorescence anisotropy. the substitution of the histidine with glycine affects protein stability versus ph: in particular mut2g kinetics is not ph dependent and at basic ph values the protein is less stable. the fluorescence properties (quantum yield, lifetime) and the rotational correlation time are unchanged during the unfolding dynamics, while the number of fluorescent proteins decreases exponentially. an extrinsic probe, bound to cysteine 48, has been employed in order to gain more insights on the unfolding process, monitoring the stability of a different region of the protein. in particular, it has been found that a softer region is present around cysteine 48 in both gfp variants, showing that the unfolding process does not follow a simple two step mechanism. recently, negatively-charged membranes were reported to catalyze amyloid fiber formation by amyloidogenic peptides/proteins and also to induce formation of "amyloidlike" fibrils by nonamyloidogenic proteins. here, we used an approach which combines steady-state and time-resolved fluorescence measurements to obtain structural information about these supramolecular assemblies and to gain insights about the factors that control their formation. by exploring a wide range of lipid concentrations, the interaction of alexa488-lysozyme with phosphatidylserine-containing membranes was found to be a complex multi-step process, critically dependent upon the protein-to-lipid molar ratio (p/l) used. upon increasing the total lipid concentration in solution, there was a balance between an increased overall protein binding to the lipid vesicles and a progressive protein dilution on the membrane surface. as the surface potential of the vesicles decreased upon increasing the protein interfacial coverage of the liposomes, the protein binding mode was found to switch from a peripheral binding of lysozyme to the anionic headgroups (at low to intermediate p/l) to a partial insertion of the basic protein into the hydrophobic core of the membrane (at a high p/l). it is hypothesized that disruption of the protein tertiary structure might be a stepwise process beginning with loosening of the structure caused by its deeper insertion in the membrane bilayer. unexpected scaling laws in the mechanical unfolding of single protein molecules m. clusel, e. i. corwin, h. lannon, j. brujic center for soft matter research, physics department, new york university, new york, ny, usa it is a question of fundamental importance to understand the response of proteins to a stretching force, particularly in the case of mechanically active proteins, such as those in muscle fibers. we aim to understand how the structure and topology of a protein affect its resilience to external forces and presumably its function. owing to recent advances in single molecule force-clamp spectroscopy using the atomic force microscope (afm), we are now able to probe the structure and dynamics of single proteins under a constant stretching force by measuring their end-to-end length over time. the probability distribution of unfolding times at a given force allows us to estimate the strength of the protein in terms of a characteristic energy barrier, while the shape of the distribution provides a window into the microscopic mechanism by which the protein breaks apart. here we show a novel scaling of the unfolding kinetics with the stretching force, which deviates significantly from the currently accepted bell model. instead, we propose a physical picture for forced unfolding that is analogous to the mechanics of fracture. v. garcía-gonzález, j. mas-oliva instituto de fisiología celular. universidad nacional autónoma de méxico. 04510 méxico, d.f. méxico. studies focused on the thermodynamic and kinetic analysis have demonstrated that transfer of neutral lipids such as cholesterol esters through an aqueous phase is a highly costly biophysical event. therefore, nature has developed a series of lipid transfer proteins such as the cholesterylester transfer protein (cetp) designed to efficiently lower the energy barrier for transfer of cholesterol-esters between lipoproteins through an aqueous environment. employing circular dichroism we evaluated the secondary structure stability of a small peptide derived from the carboxy-end of cetp (helix y ) in a wide range of ph's. the percentage of α-helix is diminished only at extreme temperatures and acidic ph's in a reversible way. we report that while a mixture of phosphatidylcholine/cholesteryl-ester forms large aggregated particles independently of ph, inclusion of helix y to the mixtures close to neutral ph's allows the formation of small micellar-like structures confirmed by dynamic light scattering and electron microscopy. these results suggest that helix y when close to physiological ph values presents the ability to organize a micellar structure around itself. this type of organization allows the process to dramatically lower the energetic barrier for lipid transfer through aqueous media, phenomenon directly related with the facilitation of lipid transfer between lipoproteins. mimicking metastasis by a novel microfluidic approach there is increasing evidence that cancer metastasis shares commonalities with thrombosis. the von-willebrand-factor (vwf), a mechanical active blood clotting protein appears to be a particular potent candidate to bridge the gap between clotting and cancer extravasation. modeling the crucial physiological conditions of the blood circulatory system, for an in situ study of blood clotting-metastasis connections is not only, absolutely necessary, but also a challenging task. here, we present acoustically driven flow as a novel microfluidics method for mimicking the blood flow. this method enjoys very beneficial advantages of possibility of handling very little volumes of fluids, together with freedom to model most of the geometries present in our microcirculatory system. one technologically challenging, yet physiologically important factor, is the hydrodynamic condition in a bifurcated vessel, where complex shear profiles arise. we present a model to mimic these conditions and discuss the impact of hydrodynamics on vwf mediated cancer cell adhesion in bifurcated vessels of our microcirculatory system. protein structural changes occurring in flows stresses inherent to viscous fluid flow have previously been associated with protein unfolding, although structural changes have not been well documented as a function of relevant hydrodynamic parameters. we have used raman spectroscopy to monitor the structure of various protein solutions in situ for multiple flow scenarios within a concentric cylinder fluidic device (1) . the flows, which ranged from circular couette to wavy taylor-couette flow, were characterised experimentally using particle image velocimetry. several proteins were observed to change conformation when exposed to these flows, although the nature of these changes was protein specific. shearing hen egg white lysozyme in water altered the protein backbone structure, while similar shear rates in a 95% glycerol, 5% water solution affected the solvent exposure of the side chain residues near the exterior of the α-domain. the solventdependent response may be due to the flow topology, viscous stress, or the surface hydration properties. comparison of spectra acquired at different time points, including before and after flow, confirmed that the observed changes are reversible and independent of fluid stress exposure time. the scripps research institute, la jolla, ca, usa. intrinsically disordered proteins are increasingly found to play major roles in cell biology and disease. we are utilizing single-molecule fluorescence methods to probe these complex and highly dynamic molecules, allowing more direct measurements of structural distributions and dynamics, while avoiding loss of information due to ensemble averaging. in one example, we investigated the structural dynamics of sup35-nm, whose regulatable amyloid formation is believed to have functional significance in yeast. using a combination of single-molecule fret as a molecular ruler, coincidence to interrogate intermolecular interactions, and correlation analysis to probe conformational dynamics, we showed that the monomeric protein populates an ensemble of compact and rapidly interconverting conformations. a particularly interesting feature of intrinsically disordered proteins is that they are relatively unstructured in isolation, but can gain stable structure by interaction with binding partners. in this context, we used single-molecule fluorescence to characterize the complex folding pathway for the parkinson's-related idp alpha-synuclein induced by binding to a lipid-mimic. this combined single-molecule fluorescence methodology provides a powerful approach for detailed studies of the coupling of folding and dynamics with interactions and biology of this important class of proteins. m. ito 1 , j. johansson 2 , r. stromberg 1 , l. nilsson 1 1 department of biosciences and nutrirtion, karolinska institutet, huddinge, sweden, 2 department of anatomy, physiology and biochemistry, swedish university of agricultural sciences, the biomedical centre, uppsala, sweden amyloid β-peptide (aβ) is a 39-42 amino acid polypeptide and known to aggregate and form insoluble amyloid fibril, which is regarded as a primary cause of alzheimer's disease (ad). the discovery of practical and effective treatments and drugs for ad has been waited eagerly. in a recent experimental study, it was suggested that stabilization of the helical conformation of the aβ middle region, which strongly favors collapsed coil formation in the extracellular environment, would reduce the aβ fibril formation. based on the experimental evidence, inhibition of the unfolding of the aβ α-helix can be a forceful strategy to repress the aβ fibril formation resulting in prevention of ad. however, the detailed mechanism of the unfolding of the aβ α-helix has remained unclear, because the x-ray or the nmr structure of the aβ α-helix in aqueous solution has not been reported due to its instability. the aim of this study is to find effective ways to inhibit the unfolding of the aβ middle region, which is a prerequisite for the aβ fibril formation. in this study, we attempted to elucidate the molecular mechanism of the aβ unfolding by molecular modeling and molecular dynamics (md) simulations. the md simulations were performed for α-helical structures of a wild-type aβ(13-26) model and a mutant aβ(13-26) model. linker average hydrophilicity as a tool to discriminate between extended and non-extended calcium binding proteins a. isvoran 1 , e. quiniou 2 , c. craescu 2 , l. mouawad 2 1 west university of timisoara, department of chemistry, pestalozzi 16, 300115 timisoara, romania, 2 inserm u759, centre universitaire paris-sud, bâtiment 112, 91405 orsay, france the ef-hand calcium binding proteins (cabps) may exist either in an extended or a compact conformation, sometimes correlated with their functions. for the cabps with know structure and function, calcium sensors are usually extended and calcium buffers compact, hence the interest to predict the form of the protein starting from its sequence. in this study we used two different procedures, the sosuidumbbell algorithm and a novel procedure that is based on the linker average hydrophilicity (lah). the two procedures were tested on 17 known-structure cabps and then applied to 59 unknown-structure centrins. the so-suidumbbell algorithm yielded the right conformations for 15 of the 17 known-structure proteins and predicted that all centrins should are compact. the lah procedure discriminated well between the extended and non-extended forms of all the known-structure cabps and it reflected well the phylogenetic classification of centrins being a simple and powerful means to discriminate between extended and nonextended forms of cabps. only few residues that constitute the linker are responsible for the form of the cabp, showing that this form is mainly governed by short-range interactions. (http://u759.curie.u-psud.fr/modelisation/lah) lipid and protein organization of hepatitis b antigen characterized by fluorescence spectroscopy v. greiner 1 , c. egelé 1 , s. oncul 1 , f. ronzon 2 , c. manin 2 , a. klymchenko 1 , y. mély 1 1 laboratoire de biophotonique et pharmacologie, umr 7213 cnrs, faculté de pharmacie, université de strasbourg, france, 2 sanofi pasteur, 1541 av. marcel mérieux, 69290 marcy l'étoile, france. hepatitis b surface antigen (hbsag) particles are 20 nm lipoprotein particles, mainly composed of the major s surface viral protein containing 13 trp residues and yeast-derived lipids. since the structure of these particles is still missing, we further characterized them by fluorescence techniques. fcs indicated that the particles diffuse mainly as monomers and contain about 80 proteins per particle. fluorescence quenching and time-resolved fluorescence experiments showed that the fluorescence signal is largely dominated by the trp residues at the protein surface. moreover, time-resolved anisotropy measurements indicate that the protein motion is restricted and that the surface trp residues exhibit both local and segmental motions. the lipid part of the particles has been studied by environment sensitive 3-hydroxyflavone probes and viscosity-sensitive dphbased probes, and compared to lipid bilayers and low density lipoproteins (ldls), taken as models. the results suggest that the lipid part of hbsag is closer to ldls than to model lipid bilayers. we present an extensive calorimetric study of bovine alphalactalbumin for various ca++ content. equilibrium dsc raw data are analyzed and the melting temperature tm, the specific heat jump deltacp, the heat of unfolding deltahm are directly extracted. the binding of calcium on the native (n) state greatly stabilizes the protein, essentially by the enthalpic difference between the unfolded (u) and n states. we show that subsequent addition of calcium in the mm range stabilizes further the n state. the equilibrium calorimetric measurements are completed with out of equilibrium stopped flow refolding kinetics by cd spectroscopy performed at different temperature and ca++ concentrations. we discuss the possible stabilization mechanisms compatible with our measurements. protein unfolding/refolding in cellular compartments: application of luciferase constructs our studies show that a reporter enzyme, firefly luciferase, can be used for evaluation of the stress-induced proteotoxicity within different cellular compartments such as the nucleus, cytoplasm or mitochondria. in transfected mammalian cells, firefly luciferase is localized in microsomes. we engineered plasmid constructs encoding luciferase with inserted specific sequences that ensure its cytoplasmic or intranuclear, or intramitochondrial localization. in addition, we fused luciferase to the green fluorescent protein (gfp) that enables to visualize patterns of the compartment-targeted product. using such gfp-labeled constructs we had a possibility to monitor protein unfolding, aggregation and refolding in the cytoplasm, nucleus and mitochondria of transfectants exposed to either stressful conditions. gfp-luciferase expressed in mammalian cells behaves as a relatively labile protein which can undergo reversible unfolding and aggregation in response to heat shock, atp depletion or action of toxic agents. in the case of cell recovery, refolding of denatured luciferase is carried out at the chaperone machine. we explored unfolding/refolding of gfp-luciferase in the cytoplasm, nucleus and mitochondria of ischemia-stressed rat cardiac cells and in several cancer cell lines treated with hyperthermia or some chemotherapeutic drugs. the results obtained have revealed intriguing correlations between the proteotoxic impact within either compartment and the viability of treated cells. amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid bilayers s. jha 1 , d. sellin 1 , r. seidel 2 , r. winter 1 1 biophysical chemistry, department of chemistry, tu dortmund university, otto-hahn str. 6, d-44227, dortmund, 2 max-planck-institute for molecular physiology, otto-hahn str. 11, d-44227, dortmund, germany the islet amyloid polypeptide (iapp), which is considered as the primary culprit for β-cell loss in type 2 diabetes mellitus patients, is synthesized in the β-cells of the pancreas from its precursor, the pro-islet amyloid polypeptide (proiapp). proiapp is co-processed in the secretory granules and co-secreted to the extracellular matrix together with insulin as iapp. here, we compare the amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. the two peptides show an enhanced amyloidogenic propensity in the presence of negatively charged membranes and similar secondary structural properties. however, proiapp shows a much less amyloidogenic propensity, probably due to the increased net charge on proiapp, compared to iapp. unlike iapp, proiapp forms small oligomeric structures at the lipid interface, having heights of ∼3.5 nm. this morphological distinction can be attributed to the presence of the pro-region, flanking the amyloidogenic iapp. the addition of proiapp to iapp marginally delays iapp fibrillation, probably by interfering with the interaction between amyloidogenic iapp cores of distinct iapp molecules. thus, it appears reasonable to speculate that the pro-region of the proiapp could serve to delay the fibrillogenesis of iapp at negatively charged lipid bilayers. the role of transmembrane domain interactions in the kinetics and folding of cpt 1 z. a. jenei 1 , k. borthwick 2 , v. a. zammit 2 , a. m. dixon 1 1 chemistry dept., univ. of warwick, coventry, uk, 2 clinicalȃsciencesȃres.ȃinst., warwick univ., coventry, uk carnitine palmitoyltransferase i (cpt 1) enzymes are polytopic integral membrane proteins in the outer membrane of mitochondria (omm). cpt 1 controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for βoxidation. the two catalytically active isoforms, cpt 1a and cpt 1b, are different in their inhibitor binding kinetics and structure (interaction between n-and c-segments, interactions of transmembrane domains (tmd)). it has been suggested that inter-and intramolecular tmds interactions are important for cpt 1a, but not for cpt 1b function. cpt 1a has also been implicated in formation of oligomeric complexes through its tm segments. the study of tm helix-helix interactions in cpt 1 isoforms could lead to a better understanding of their function and inhibitor binding kinetics, and will contribute towards the design of pharmacological strategies aimed at modulating the activities of cpt 1 enzymes in conditions such as diabetes. to investigate the ability of the tmd in cpt 1 to self-associate and the order of any oligomers formed, several biochemical and biophysical techniques have been used. we found the self-association of rcpt 1a tmds (tm1, tm2) to be different as measured using the in vivo tox-cat assay. chemical cross-linking and analytical ultracentrifugation studies demonstrated formation of both trimers and hexamers by the rcpt 1a tm2 peptide. these results provide further evidence that tm2 plays role in formation of a channel in the omm. self-assembly of transmembrane domains in cpt1: role of gxxxg(a) motif in possible channel formation z. a. jenei 1 , k. borthwick 2 , v. a. zammit 2 , a. m. dixon 1 1 department of chemistry and, 2 warwick medical school, university of warwick, coventry, uk carnitine palmitoyltransferase 1a (cpt1a), a membrane protein that controls the rate of oxidation of long-chain fatty acids, is of key importance in diabetes and has recently been reported to exist as an oligomer in vivo. we have investigated full-length cpt1a and find that the protein exists as a hexamer in liver mitochondria. using mutants of cpt1a expressed in yeast mitochondria, we have localised key protein interactions in the hexamer to the transmembrane (tm) domains of the protein. detailed study of the tm domains in isolation, in both e.coli membranes and detergent micelles, demonstrated that while tm1 shows little self-assembly, tm2 displayed significant self-association. biophysical analyses of a tm2-derived synthetic peptide revealed oligomerization behaviour identical to native cpt1a in mitochondria, providing a strong link between tm2 helixhelix interactions and cpt1a hexamer formation. this is significant in light of a recent suggestion that cpt1a oligomerization may lead to formation of a channel in the mitochondrial outer membrane through which acylcarnitine gains access to the inter-membrane space. our data supports this new theory, and we go on to demonstrate experimentally the structural determinants of hexamer (channel) formation, specifically gxxxg(a) motifs in the tm domain which pack favourably in the hexamer and stabilize the oligomer. investigation of flexible loop role in structure and thermodynamic stability of firefly luciferase p. maghami, b. ranjbar, s. hosseinkhani department of biochemistry and biophysics, faculty of basic sciences, tarbiat modares university, tehran, iran protein folding, like any chemical process, consists of two fundamental components, thermodynamics and kinetics, which determine the stability of the folded state and the pathway of folding, respectively. these processes are currently too difficult to be solved de novo by purely computational methods. experimental evidence is required to simplify the problem via protein engineering .in this research, the wild type firefly luciferase (photinus pyralis) and some of its mutants were over expressed and purified. then their unfolding thermodynamics were examined, using circular dichroism and conventional fluorescence measurements. the unfolding equilibrium constant were measured over a complete rang of denaturant conditions. the measurements were shown structural and physico-chemical changes between wild-type and mutant proteins. exploring intrinsic disorder of unstructured membrane proteins by surface polymer physics intrinsically unstructured proteins (iups) are considered as a separate class within the protein world because they lack a well-defined folded structure. because iup's function is indeed directly linked to structural disorder, they are assumed to be natively unfolded. several physicochemical techniques are available to discriminate the degree of disorder. however a clear structural classification is still lacking. in this context, polymer physics emerges as a powerful tool for getting inside on the conformational abilities directly related to structural disordered of iups. in the present contribution, surface pressure and ellipsometry experiments in conjunction with polymer physics have been used to infer structural data in terms of molecular conformation and flexibility of a membrane protein essential for bacterial division, zipa. this protein has been pointed to posses a high molecular flexibility and to adopt a random coil conformation. folding dynamics of peptides studied by timeresolved infrared spectroscopy c. krejtschi 1 , o. ridderbusch 1 , r. huang 2 , l. wu 2 , t. a. keiderling 2 , k. hauser 1 1 institute of biophysics, university of frankfurt, germany, 2 department of chemistry, university of illinois at chicago, usa peptides are ideal model systems to study protein folding mechanisms. ir techniques provide both the necessary time resolution as well as the structural sensitivity. we initiate rapid heating by laser-excited ns temperature jumps (∼10 • c) and study fast ns-to-µs relaxation dynamics [1] . the dynamics of the α-helix to random coil transition of polyglutamic acid was analyzed under reversible folding/refolding ph-conditions. the observed relaxation kinetics allowed separation of the folding and unfolding process with additional use of ftir measurements in thermal equilibrium. sitespecific dynamics were monitored by use of isotopic labeling for a β-hairpin peptide whose conformation is stabilized by a hydrophobic core. various single and cross-strand isotopically labeled variants were analyzed. the isotope-edited kinetics show variations in local structural stability of the hairpin backbone. our data support a multistate dynamic behavior, and the site-specific kinetics are consistent with a hydrophobic collapse hypothesis for hairpin folding [2] . small heat shock protein hsp22 was predicted to belong to the family of intrinsically disordered proteins. one of the features of these proteins is that they do not demonstrate cooperative thermal transitions on heating. we applied different methods (dsc, ftir and intrinsic tryptophan fluorescence) to investigate the thermal unfolding of hsp22. dsc results have shown that thermal denaturation of hsp22 begins from 25 o c and occurs, with very low cooperativity, within a broad temperature region (up to 80 o c and above). the thermal unfolding of hsp22 is fully reversible. the ftir data show that heating of hsp22 from 20 to 70 o c results in complete disappearance of α-helices (from 8-12% to 0) and the decrease in β-sheets content from 42 to 30%. studies on the temperature dependences of tryptophan fluorescence have shown a significant red shift of the spectrum. these changes occurred within temperature region from 30 to 80 o c with midpoint at ∼56 o c. probably, this transition can be explained by destruction of β-sheets around trp96, the only trp residue of the α-crystallin domain of hsp22 (other 3 trp residues of hsp22 are localized in the n-terminal domain). the data obtained confirm the suggestion that hsp22 is a protein, whose significant part is intrinsically disordered. we propose that, on heating, the α-crystallin domain containing β-sheets melts at higher temperature than the n-terminal domain containing the most of α-helices. t. rosenkranz 1 , a. katranidis 1 , d. atta 1 , j. enderlein 2 , i. gregor 2 , m. grzelakowski 3 , p. rigler 3 , w. meier 3 , j. fitter 1 1 isb-2: molecular biophysics, research centre jülich, germany, 2 institute of physics, biophysics/complex systems, georg august university, göttingen, germany, 3 institut für physikalische chemie; universität basel, basel, swizerland the protein folding mechanism is the missing link in the biological flow of information from the dna to its specific function. since most of proteins within a cell consist of more than one domain studies on this protein class are of major importance. it is a common feature of multidomain proteins to aggregate under refolding conditions, which hinders a refolding. molecular encapsulation of single molecules prevents aggregation. by immobilizing the nanocapsule the observation period in a wide field microscope will be extended, so that slow or rare folding events can be detected. a major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies [1] . polymer vesicles maintain their structural integrity even under harsh unfolding conditions. furthermore the nanocontainer proved to be permeable to guanidinium hydrochloride. using encapsulated phoshoglycerate kinase, labeled with atto-655, a dye which experiences fluorescence quenching by photo-induced electron transfer (pet) with tryptophans, we demonstrate the remarkable properties of polymeric nanocontainers. applying pet as a folding probe we detected multiple unfolding/refolding transitions of single proteins. proteins frequently become irreversibly modified by carbonylation, a process of introducing the carbonyl group (carbon monoxide) in a reaction with reactive oxygen species (ros) such as superoxide, peroxide or ozone. the main targets for carbonylation in proteins are amino-acid side chains of lysine, arginine and proline. products of carbonylation are aminoadipic semialdehyde from lysine (asa) and glutamic semialdehyde (gsa) from arginine and proline. importantly, carbonylated proteins are marked for proteolysis by the proteasome, but can escape degradation and form aggregates that can be cytotoxic. carbonylation increases with the age of cells and it is associated with ageing and age related disorders such as alzheimer's disease, parkinson's disease and cancer. we have used the molecular dynamics method to study the stability of carbonylated proteins villin headpiece and ubiquitin. simulations were run after mutations of arginine, proline and lysine into gsa and asa had been performed. in addition, we have used thermodynamic integration on lysine, arginine, proline, asa and gsa residues in order to estimate their solvation free energy (related to relative hydrophobicity and hydrophilicity). our results suggest that carbonylation markedly decreases the overall stability of proteins, and that one potential reason for that may be a disruption of the balance between hydrophilic and hydrophobic regions in the protein. a. martino, d. crane, i. m. feavers, b. bolgiano division of bacteriology, national institute for biological standards and control, potters bar, uk the sensitivity to protein's secondary structure and progress in computational calculations have made circular dichroism (cd) an attractive technique to explore the optical properties of three promising vaccine candidates to neisseria meningitidis. clinical batches of a c-term deleted form of nada (genome-derived neisseria antigen -gna1994) and the fusion proteins gna2132-1030 (fp-1) and gna2091-1870 (fp-2) were therefore studied. increases in temperature and denaturant concentration on secondary structures and folding/unfolding profile were monitored by cd in the far and near uv regions and complemented by trp fluorescence spectroscopy data. furthermore, epitope conformational changes on binding activities to immune-sera were investigated. the calculated secondary structure content was in broad agreement with the available predicted or solved protein structures. upon temperature incubation, a structural transition from a highly α-helical nada to a more unordered conformation, with a mid point at ∼40 • c, was observed. fp-1 and fp-2 maintained their conformation up to 50 • c or 6m guhcl in the case of fp-1. unfolding was not always reversible. reductions in binding to monoclonal ab titrated along with increasing unfolding. folding/unfolding studies have proven useful in better understanding the solution behaviour and extent of folding of proteins. cold denaturation of yfh1 offers the clue to understand the effect of alcohols on protein stability s. r. martin 1 , v. esposito 1 , p. de los rios 2 , a. pastore 1 , p. a. temussi 3 1 national institute for medical research, the ridgeway, nw7 1aa london, u.k., 2 laboratoire de biophysique statistique, sb/itp, ecole polytechnique fédérale de lausanne (epfl), ch-1015, lausanne, switzerland, 3 dipartimento di chimica, università di napoli federico ii, via cinthia, i-80126 napoli, italy although alcohols are well known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. here we present a study of the effects of alcohols on protein stability using yfh1. exploiting the unusual property of this protein of undergoing cold denaturation around 0 • c without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol,ethanol methanol. in all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. we suggest that alcohols, at low concentration and physiological ph, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation. biomolecular sciences and biotechnology tor vergata moro 5, 00185 rome, italy cholesterol plays an important role in regulating the structural properties of phospholipid and non-phospholipid membranes. in this study we have applied in situ energy dispersive x-ray diffraction (edxd) to investigate the effect of cholesterol on the structure of different phospholipid and non-phospholipid oriented membranes. in detail, phosphatidylcholine (pc) bilayers and niosomal membranes, made of a non-ionic surfactant centre for bioactive chemistry, department of chemistry department of chemistry this process is initiated at nuclear envelope remnants (ners) in the presence of atp and gtp. the mvs can be divided in two main populations: mv1 and mv2. mv2 has a classical lipid composition while mv1 is enriched in phosphoinositides (pips: pi, pip, pip 2 and pip 3 ). ners have an unusual lipid composition, enriched both in cholesterol and pips. physicochemical properties of the pips were investigated as a function of ph and temperature (t) using nmr, saxs and dls to map out their phase state. pips-water dispersions are observed in lamellar, hexagonal or isotropic phases depending on t and ph. in parallel, model membranes mimicking mv1 and ners lipid composition were studied by 2 h and 31 p nmr. mv1 modelling shows a complex behaviour of pips on pc membranes: they order or disorder membranes, whereas the order of pc/pi/pip/pip 2 membrane is lower than that of pc or pc/pi membranes c. manzo, t. s. van zanten, m. f. garcia-parajo bionanophotonics group, ibec-institut de bioenginyeria de catalunya, barcelona, spain membrane proteins play a fundamental role in intra-and inter-cellular functions. in particular, the proteins lateral mobility in the fluid membrane environment is crucial for the regulation of several mechanisms, as receptor-mediated signal transduction and establishment of immunological synapses. these mechanisms are controlled through protein crowding and reduced lateral diffusion, which induce macromolecular associations and limit the application of conventional single molecule fluorescence techniques. to measure proteins mobility on living cells membrane, we developed a fluorescent correlation spectroscopy (fcs) setup in which the sample illumination is obtained through near-field scanning optical microscopy (nsom) probes. the use of nsom probes is particularly suited for the observation of dynamics on the cell membrane and overcomes the drawbacks of other techniques. in fact, through a shear-force-based position control, the probe is kept at a fixed distance from the membrane and its sub-wavelength aperture (∼100 nm) reduces the illumination area, allowing the observation of highly crowded regions of the membrane. on the basis of preliminary results, the nsom-fcs is expected to provide an additional insight on the proteins trafficking at the membrane level. the technique also presents several potential developments, as the further reduction of the illumination area and two-colors correlation. single molecule fluorescence microscopy of the store-operated calcium channel subunit orai1 j. madl, j. weghuber, d. bergmair, m. fahrner, m. muik, c. romanin, g. j. schütz johannes kepler university, institute for biophysics, linz, austria store-operated calcium entry (soce) is essential for many cellular signalling processes. the essential pore forming subunit of soce channels in the plasma membrane is orai1. here we present single molecule fluorescence microscopy of orai1 which was performed in order to directly visualize the stoichiometry of mobile orai1 pores. the protein was fluorescently labeled with monomeric gfp. a novel single molecule fluorescence approach, toccsl (thinning out clusters while conserving the stoichiometry of labeling), was used for the determination of the stoichiometry. this technique allows reducing the density of fluorescently labeled molecules without affecting the stoichiometry of labeling. density reduction is achieved by completely photobleaching a defined area within the plasma membrane; nonbleached gfp-orai1 aggregates enter the bleached region subsequently by diffusion. our data indicate that there are different populations of orai1 present in the cell: most of orai1 is located in the plasma membrane. a second population of orai1-mgfp was found to be localized in intracellular vesicles. a significant fraction of the plasma membrane orai1 exhibits a diffusive movement. we found by analyzing the bleaching characteristics of single orai1-mgfp aggregates that in resting cells mobile orai1 is predominantly dimeric. a. kobitski 1 , a. nierth 2 , m. helm 2 , a. jäschke 2 , g. u. nienhaus 3 1 university of ulm, germany, 2 university of heidelberg, germany, 3 university of karlsruhe, germany rna molecules have attracted enormous attention in recent years, and various novel roles were revealed for rna in biological processes. ribozymes are a class of rna molecules capable of catalyzing chemical reactions. we have studied a diels-alderase (dase) ribozyme, a small artificial 49-mer ribozyme, which is capable of catalyzing carbon-carbon bond formation between an anthracene diene and a maleimide dienophile in multiple turnovers. single-molecule fluorescence resonance energy transfer was employed to investigate the intramolecular dynamics of this rna molecule as a function of mg 2+ ion concentration. folding into a functional state occurs via an intermediate state, and continuous fluctuations between these two states were observed on the 100ms time-scale at the midpoint concentration of mg 2+ ions. an effect of substrates binding on the folding and catalytic reaction of the dase ribozyme is in the focus of our recent research with the ultimate goal to obtain a detailed structural view of the single-molecule conformational changes that accompany the catalytic reaction. a. katranidis 1 , r. schlesinger 1 , k. nierhaus 2 , i. gregor 3 , m. gerrits 4 , g. bueldt 1 , j. numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins. here we present an approach employing a two color single molecule sensitive fluorescence wide-field microscope in order to visualize surface tethered fluorescently labeled ribosomes and de novo synthesized gfp molecules in real time [1] . fluorescence of co-translational folded proteins was observed from mature fluorescent gfp molecules which carry 31 additional amino acids at the c terminus remaining linked to the ribosome. thus it was possible to co-localize fluorescence from labeled ribosomes and from gfp molecules. we demonstrate that the green fluorescence protein mutant gfp emerald is produced with a characteristic time of five minutes. the fastest gfp molecules appeared already within one minute. processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. in fluorescence spectroscopy, photobleaching is a process which leads to irreversible loss of fluorescent properties of a dye molecule, usually due to photochemical reactions. it is especially important for fcs experiments on slow-diffusion systems since for high excitation intensities it can have a strong impact on fluorescence intensity correlation function. usually it is observed as apparent shortening of the mean diffusion time of the dye molecules. the behavior of tmr-labeled fd-virus rods in water solution under various excitation conditions was investigated. the experiments were conducted for low (1:1) and high (625:1) tmr:virus ratios and for increasing laser intensities. the correlation function was measured in the experiments. the results were fitted using origin software to estimate the influence of photobleaching, and compared with computer simulations. a strong effect of photobleaching was visible for rods labeled with a single dye molecule, while rods labeled with 625 tmr molecules showed little to no bleaching. a prolongation of characteristic diffusion times for highly labeled virus rods in comparison to low-labeled ones was also observed. k. toth 1 , a. gansen 1 , a. valeri 2 , v. böhm 1 , c. a. seidel 2 , j. langowski 1 1 abt. biophysik der makromoleküle, deutsches krebsforschungszentrum, heidelberg, germany, 2 lehrstuhl für molekulare physikalische chemie, heinrich heine universität, düsseldorf, germanythe nucleosome has a central role in the compaction of genomic dna and the control of dna accessibility for transcription and replication. we studied the effect of dna sequence and selective histone acetylation on the structure, stability and disassembly of the mononucleosomes. quantitative single molecule fret measurements between dyes attached to different parts of the nucleosome permitted us to detect the equilibrium between several subpopulations of reconstituted nucleosomes in solution. we obtained that the heterogeneity and stability of the samples are correlated with each other and influenced both by the dna sequence and the histone acetylation. the path of the linker dna is more sensitive to all studied effects than the dna on the core. intermediates of the disassembly pathway were identified and characterized. j. strömqvist 1 , s. johansson 1 , y. ohsugi 3 , k. andersson 2 , l. xu 1 , m. kinjo 3 , p. höglund 2 , j. widengren 1 1 experimental biomolecular physics, kth, stockholm, sweden, 2 department of microbiology and cell biology, karolinska institutet, stockholm, sweden, 3 laboratory of molecular cell dynamics, hokkaido university, sapporo, japan dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly49a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined.here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. dynamic multiple-target tracing probes spatiotemporal cartography of cell membranes in order to decipher the non random and non homogeneity of the plasma membrane organization, we had performed fluorescence correlation spectroscopy measurements on live cells. this allowed us to establish the presence of nanoscale confining structures 1 and to demonstrate their implication in signaling process 2 . complementing these studies, we present here a new analytical method, namely multiple-target tracing (mtt) 3 which takes advantage of the high resolution provided by singlemolecule sensitivity to generate dynamic maps at high densities of tracked particles. introducing deflation by subtracting detected peaks allows detecting peaks of lower intensity. we achieved an exhaustive detection of particles with performances reaching theoretical limits, and a reconnection of trajectories integrating the statistical information from past trajectories. we demonstrate the potential of this new method of analysis by applying it to the epidermal growth factor receptor labeled with quantum dots, in the plasma membrane of live cells. this has allowed us to build up a global representation of molecular dynamics in cell membranes. dual polarisation interferometry (dpi) is a surface analytical technique capable of dynamically measuring biophysical parameters of conformational change in biomolecular interactions. the technique measures three key parameters, namely layer thickness, layer density (ri) and mass, thereby enabling the resolution of conformational changes involved during binding. a number of different applications are presented. protein-protein interactions: understanding the biophysical nature of protein interactions can deliver insights into the mechanisms by which proteins interact, thereby elucidating protein function. dpi enables correlation between binding affinity and conformational change, greatly enhancing the study of structure-function relationships. lipid layers: the birefringence mode of dpi can be used to study the formation of lipid bilayers and biomolecular self-assembly. it is possible to use a combination of bilayer refringence and mass to study phase transitions associated with protein or peptide binding to the lipid bilayer. the individual stages of adsorption, absorption and micellisation can be distinguished. carbohydrate interactions: dpi uses a planar glass surface and a wide range of coupling chemistries. a carbohydratespecific surface can be used to study a wide range of biomolecular interactions, such as lectins, acidic proteins, extracellular matrix signaling and interactions with complex membranes. the experimental characterization of the elementary conformational steps constituting the protein folding pathway remains a big challenge. quenching of the triplet state of tryptophan by close contact with cysteine has been shown to provide a new tool for measuring the rate of intramolecular contact formation -one of the most elementary events in the folding process -in peptides and proteins using only natural probes. here we show an extensive study on a stabilized mutant of the second beta-hairpin of gb1 domain. steady state fluorescence and kinetics of contact formation between a natural tryptophan and a cysteine added to the c-terminal are measured for different temperatures and solvent conditions. we separately address the contributions of different structural elements to the overall stability of the hairpin. we extract kinetics parameter for contact formations in the unfolded state, formation of the hydrophobic core and tails pairing in the folded state. by means of a fragment peptide terminated with a tryptophan and a cysteine, we also estimate the structural propensity of the turn region. the data coherently combine with a simple model previously developed to describe the dynamics of unstructured chain [biophys. j. 96, 1515 (2009)], here modified with the addition of attractive interactions between specific residues. catalytic power of partially denatured enzymes: implementation of molten-globule-like states m. shushanyan 2 , d. e. khoshtariya 1 , m. makharadze 1 , t. tretyakova 2 , r. van eldik 3 1 institute of molecular biology and biophysics, gotua 12, 0160 tbilisi, georgia, 2 department of physics, i. javakhishvili tbilisi state university, 0128 tbilisi, georgia, 3 department of chemistry and pharmacy, university of erlangen-nürnberg, germany impact of nonspecific moderate denaturants, urea and dmso, on the kinetic (functional) and thermodynamic (stability) patterns of a hydrolytic enzyme, α-chymotrypsin (α-ct) has been investigated. furthermore, an impact of urea and guhcl in combination with of temperature on the kinetic pattern of carboxypeptidase a has been examined. for the case of α-ct, in particular, we have observed about tenfold increase of the apparent mickaelis constant when going from 0 to 6 m urea (25 o c), whereas the value of catalytic constant remained almost unchanged, indicating that the protein is not unfolded even under those severe conditions. the matching microcalorimetric experiments revealed that both the temperature-induced melting temperature and transition enthalpy decrease gradually with the increase of the additive concentration. with 6 m urea the peak-shaped calorimetric feature disappears totally. however, catalytic power of α-ct was preserved owing to its catalytic constant. for other studied enzyme/substrate/denaturant arrays diverse kinetic peculiarities due to mgls have been observed. rubredoxins are a class of iron-containing proteins whose biological role on electron transfer processes and metal binding is still unclear. the unfolding dynamics of the rubredoxin mutant rda51c from the mesophile desulfovibrio vulgaris (dv) was studied on the temperature range from 25 • c to 87 • c and along time at 87 • c. resonance energy transfer (ret) was used to determine the donor (d; trp37) to acceptor (a; 1,5-iaedans) distance. from 25 • c to 87 • c the d-a distance increased 4å. however the random coil expected d-a distance was only achieved after heating the protein solution during 2.5 hours at 87 • c. from uv-vis absorption data it's clear that this protein is capable of maintaining its iron-sulfur center at 90 • c. by melting the protein at the same temperature all iron-centers disintegrate and the protein unfold after 2.5 hour. the trp37 fluorescence also shifts 13 nm to the red reflecting the partial exposition of the indole ring to the solvent. from fluorescence and anisotropy decay curves a breathing type movement of the protein structure was observed between 30 • c to 60 • c without lost of significant secondary structure. this structure flexibility should play an important role on the thermal stability of the dvrd the antimicrobial peptide novicidin (nc) -modified from the sheep self defense peptide smap-29, for reduced mammalian cytotoxicity -is a cationic peptide (net charge +∼7.5) that adopts random coil structure in solution, but an α-helix in the presence of lipid vesicles. the conformation of nc in presence of the lipids dlpc, dlpg, dmpc, dmpg, dopc, dopg, dope, and dops in different combinations reveal the lipid selectivity, affinity, and phase dependent changes with varying l/p ratios and temperatures, observed from cd spectroscopy. it is understood that the conformational change is dependent on chain length and head group of the lipid. apart from the in vivo results on the nc activity, studies using qcm-d, dual polarisation interferometry, and calcein release assay reveal the kinetics and concentration dependent activity of nc in lipid bilayers and vesicles. preliminary studies on orientation of nc in various lipid environments using ssnmr, lsnmr, and molecular dynamics simulations apparently suggest that nc may form toroidal pores/detergent effects depending on the chain length of the lipids. further experiments on nc in presence of lipids using dsc, itc, ssnmr, oriented cd, ld, and confocal microscopy to determine the structure, thermal stability, orientation in lipid bilayers, and thereof the action of nc will help in proposing a comprehensive model for its mechanism of action in model membranes. dielectric method for measuring glass transition and denaturation temperatures of hydrated proteins g. e. thomas 1 , s. bone 1 , g. drago 2 1 institute for bioelectronic and molecular microsystems, bangor university, gwynedd, uk., 2 applied enzyme technology, pontypool, uk.the flexibility of protein structures is important in allowing the variety of motions, covering a wide range of magnitudes and frequencies, essential to biological activity. high frequency dielectric measurements can be used to study the flexibility of proteins by probing the relaxation of dipolar constituents of their structures. hydrated proteins exhibit a broad dielectric loss extending over the frequency range from 1mhz to 10ghz which can be decomposed into a number of constituent dispersions. one of these dispersions, with a relaxation time of ∼18ns, has been attributed to the relaxation of protein backbone peptide groups in the protein interior. in the work reported here, this dielectric dispersion was investigated as a function of temperature for the enzyme glucose oxidase. two critical temperatures were identified as the glass transition and denaturation temperatures, both of which were found to decrease with increasing protein water content. the results were consistent with a scheme in which the hydrated glassy protein undergoes a change in structural mobility at the glass transition temperature and experiences an irreversible change in conformation at a higher denaturation temperature. both glass transition and denaturation temperatures are key indicators of protein stability and are important in the production and storage of protein based pharmaceuticals. a. szymańska, k. kacprzyk, g.ślósarek department of molecular biophysics, a. mickiewicz university, umultowska 85, 61-614 poznań, poland aggregation dynamics of proteins plays an important role in molecular biology and medicine as it permits explanation of several disease states. an interesting problem is to find out in which conditions the interactions of the protein molecules lead to formation of ordered structures and in which to disordered ones. in this study, dynamic light scattering, circular dichroism and also congo red dye experiments were performed to analyze various structural states of lysozyme induced under different concentration of ethanol solvent. at low ethanol concentration the attractive interaction between the protein macromolecules dominate. after addition of more ethanol solvent, the translational diffusion coefficient was much smaller than that for lysozyme solution at zero ethanol concentration. it can be explained by the structural transformation of the polypeptide chain leading to a partially folded conformation needed for oligomerization and fibrillation process. on the basis of the cd experiments we concluded that the ethanol solvent induces changes in secondary structure of lysozyme solution. on addition of 75% v/v ethanol solution the intramolecular hydrogen bonds were destabilized. above this ethanol concentration, β -sheet were the dominant secondary structure of lysozyme in solution. the phase diagram illustrating the formation of: monomers, oligomers at various structural states, protofilament formation state, protofilament and amyloid fibrils was constructed. key: cord-008777-i2reanan authors: nan title: ecb12: 12th european congess on biotechnology date: 2005-07-19 journal: j biotechnol doi: 10.1016/j.jbiotec.2005.06.005 sha: doc_id: 8777 cord_uid: i2reanan nan in the last 20 years biotechnology has made tremendous progress in its different application fields: red biotechnology, the use of biological methods for medical purposes, is firmly established in the development of new drugs. the use of plant or green biotechnology is under controversial discussion in politics and public. nevertheless, genetically modified herbicide and insect resistant crops are cultivated to a large extent. industrial biotechnology, now often named white biotechnology, seems widely underestimated in the public perception. it includes all industrial processes for the production of chemical products and enzymes, which fully or partly rely on the biological toolbox of nature. white biotechnology processes are carried out in a contained environment, typically in a bioreactor in a dedicated industrial plant. well-known examples are the fermentative productions of antibiotics, amino acids, vitamins and enzymes, products related to medical, food and feed applications. many products like the amino acids glutamic acid, lysine, threonine and tryptophane are exclusively produced using microbes in large scale industrial processes. in other cases, like the water soluble vitamin b2, biotechnological processes successfully replaced chemical productions, due to lower costs and improved ecoefficiency. in contrast to this, most industrial chemicals and polymers are produced by chemical synthesis based on oil and gas. however, there are some examples for bioproducts among industrial chemicals. the solvents acetone and butanol, for instance, were manufactured by fermentation for several decades in the last century. since the 1950s these fermentations have been replaced by more efficient and cheaper chemical synthesis. recently, new pilot and production processes for biopolymers like pha or biomonomers like 1,3-propanediol or lactic acid were announced by different companies. currently, ethanol is by far the largest white biotech product by volume. in brazil, where bioethanol is used as liquid transportation fuel, the annual production is in the range of 15 mio m 3 . bioethanol is of growing importance also in the united states. business consultants predict a tremendous growth of biotechnological products within the chemical industry. high prices for crude oil, dropping prices for renewable resources, and scientific progresses nourish the expectation that industrial biotechnology will replace many bulk chemicals. is this realistic? will we switch from a petrochemical industry to a biobased chemistry within the next years? based on economic considerations it can be stated that this is a long term goal. to achieve this it remains a scientific challenge to make renewable raw materials available for competitive bioproduction of bulk chemicals at low costs. conversion of lignocellulosic material to fermentation sugar may be a solution. also green biotechnology can contribute to the supply of cheap fermentation raw materials. innovative ideas for downstream processing or further chemical conversion of fermentation products are required to enter the chemical value chains. furthermore, the identification of new higher value bioproducts is a chance for short term successes in white biotechnology. enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering can contribute to develop new metabolic pathways, may be even for unnatural compounds. by continuously increasing the efficiency and throughput of dna sequencing we, together with colleagues, have sequenced the human genome and the genomes of all the major model organisms. the challenge now centers on understanding these vast instruction sets. our ability to read these instructions must be enhanced through collection of key additional data sets. one productive path for delineating the functional sequences and inferring their function is comparative sequence. the mouse genome sequence, for example, led to estimates that only 5% of the human genome is functional. sequencing of an extensive set of additional mammalian genomes promises to define these functional sequences with a resolution of less than 10 base pairs. on a different course, we have sequenced the chimpanzee genome to learn what has changed in the evolution of humans. beyond providing for the first time a catalog of the differences between the two genomes, the comparison of the chimpanzee and human genomes reveals the patterns of neutral mutation and regions that deviate from that. the talk will summarize these and related findings. the sequence of additional primate genomes will help delineate what has changed specifically in humans and add power to the analysis. ultimately, capturing human sequence variation and correlating with phenotypic variation will be required to understand function. but learning what these functional elements do requires new sets of experimental data. for this, we have turned to the nematode c. elegans. in this simple system most of the ∼20k genes have been defined and experimentally confirmed. beyond the hundreds of genes with already known mutants, two centers are systematically producing gene knockouts or rnai can be used to inhibit any gene temporarily. sequences of three caenorhabditis species are already available, and two more are underway. expression data has been collected for all the genes under many conditions and time points through development. to enhance the resolution of expression data and to simplify phenotypic analysis of embryonic mutants, we are developing a system that will automatically trace the cell lineage and assign gene expression to precise cells with high temporal resolution. the latest results with the system will be described. in the longer term, this and similar datasets should provide an understanding of how the genome specifies the form and behavior of the worm. uhlen department of biotechnlogy, albanova university center, royal institute of technology (kth), stockholm, sweden here, we present a new protein atlas database (www.proteinatlas.org) showing the expression and localization of human protein in normal and cancer tissues. the atlas is based on the use of antibodies (agaton et al., 2003) to generate high-resolution immunohistochemistry images representing 48 normal tissues and 20 different cancer types (uhlen and ponten, 2005) . each antibody is used to generate more than 500 individual images and each image has been annotated by a pathologist (kampf et al., in press) . the database has been created by the swedish human proteome resource (hpr) and the program has been set-up to allow the exploration of the human proteome with antibody-based proteomics (nilsson et al., in press) . the basic concept is to generate, in a systematic and high-throughput manner (uhlen and ponten, 2005) , specific antibodies to all human proteins, and subsequently used these for functional analysis of the corresponding proteins in a wide range of assay platforms, including (i) a protein atlas for tissue profiles (kampf et al., in press) , (ii) specific probes to evaluate the functional role of individual proteins, and (iii) affinity reagents for purification of the specific proteins and their associated complexes for structural and biochemical analyses. ments, most effective source of variation was perturbation in growth medium, followed by perturbation in growth rate. effect of gene deletion on data variation was found to be less apparent when compared to other perturbations. a significant similarity in variation of metabolome and mrna data was observed, which may be used as the key point for integration of these two sets of data in functional analysis of genes. projection to latent structures (partial least squares, pls) is used for integration of transcriptome and metabolome data. comparison of pca and pls shows that linear model constructed via pls to predict the metabolome data does not make use of all the variation in transcriptome data. thus, pls allows the discrimination between the portion of gene expression change that affects the metabolome profile and the portion that is not directly effective on metabolome. both pca and pls can be used to detect the open reading frames (orfs) which are the main sources of variation in transcriptome data and/or effective on metabolome profile. extracellular metabolomics to accelerate the discovery of key genes involved in fibre degradation silas g. villas-bôas, geoffrey lane, graeme attwood, adrian cookson agresearch limited, grasslands research centre, tennent drive, private bag 11008, palmerston north, new zealand. e-mail: silas.villas-boas@agresearch.co.nz (s.g. villas-bôas) the genome of the hemicellulose-degrading microbe clostridium proteoclasticum is been sequenced and an array of candidate genes with diverse activity relevant to fibre degradation have been identified by automated gene annotation methods. c. proteoclasticum falls within the butyrivibrio-pseudobutyrivibrio assemblage of rumen bacteria which are though to play an important role in the degradation of plant hemicellulose-lignin complexes which limit fibre degradation in the rumen. for new zealand it makes strategic sense to invest in microbial genomics efforts applied to agriculture where the country holds a strong competitive advantage and where ruminants constitute the vast majority of farmed animals. in conjunction with dna sequencing, proteomics and transcriptomics (micro-array analysis) we are using metabolomics as an additional functional genomics tool for gene discovery. we have established a footprinting approach for microbial metabolome analysis focused mainly on metabolic intermediates of polysaccharide degradation to provide quantitative information on end products of fibre-degrading enzymes. a gc-ms method has been developed that is able to resolve complex biological mixtures containing mono-, di, and oligosaccharides, in addition to a series of organic acids. we are currently phenotyping a series of c. proteoclasticum mutants to validate our analytical methodology and we are going to fully characterize the fibrolytic ability of c. proteoclasticum to be compared with other fibre-degrading microbes. we believe that our metabolomics data will complement current proteomic analysis of fibre-degrading enzymes and micro-array analysis of gene expression from a series of mutants by providing direct evidence of the metabolic function of key genes involved in fibre-degradation processes. many gram-negative bacteria utilize cell-to-cell communication systems that rely on diffusible n-acyl homoserine lactone (ahl) signal molecules to monitor the size of the population in a process known as quorum sensing (qs). in human pathogens this form of gene regulation ensures that the cells remain invisible to the immune system of the host until the pathogen has reached a critical population density sufficient to overwhelm host defenses and to establish the infection. the qs regulon of pseudomonas aeruginosa and burkholderia cepacia, two important pathogens for patients suffering from cystic fibrosis, has been studied by proteome analyses. comparative twodimensional gelelectrophoresis of pre-fractionated protein mixtures (extra-, surface-, and intracellular proteins) coupled to mass spectrometry analysis or n-terminal sequencing has been employed to recognize and identify qs-controlled proteins. our findings strongly support the importance of ahl-mediated cell-cell-communication as a global regulatory system and suggest that qs control also operates via post-translational mechanisms. as qs has been proven to be a central regulator for the expression of pathogenic traits and biofilm formation in opportunistic human pathogens it represents a highly attractive target for the development of novel anti-infective compounds. functional genomics technologies (transcriptomics and proteomics) have been exploited to validate the target specificity of natural and synthetic qs inhibitors, thus having a great potential as alternative therapeutics for the treatment of bacterial infections. modeling cell cycle complex formation from high-throughput data sets lars juhl jensen european molecular biology laboratory, meyerhofstrasse 1, 69117 heidelberg, germany. e-mail: jensen@embl. de to analyze the dynamics of protein complexes during the mitotic cell cycle, we integrated data on protein interactions and gene expression. the resulting time-dependent interaction network for the first time places both periodically and constitutively expressed proteins in a temporal cell cycle context, thereby revealing novel components and modules. we discover that most complexes consist of both periodically and constitutively expressed subunits, suggesting that the former control complex activity by a mechanism of just-in-time assembly. consistent with this, we show that additional regulation through targeted degradation and phosphorylation by cdk1 (cdc28p) specifically affects the periodically expressed proteins. alessandra luchini, andrea callegaro, silvio bicciato department of chemical engineering processes, university of padova, padova, italy. e-mail: alessandra.luchini@unipd.it (a. luchini) since transcriptional control is the result of complex networks, analyzing dynamical states of gene expression is of paramount importance to detect the multivariate nature of biological mechanisms. although hundreds of studies fully demonstrated the relevancy of microarrays in describing different physiological conditions, to reconstruct complex interaction pathways it is necessary to analyze the temporal evolution of transcriptional states. however, a robust experimental design for identifying differentially expressed genes over a temporal window would require large amounts of microarrays. unfortunately, replicates for each time point and experimental condition are not always available, because of cost limitations and/or biological samples scarcity. in addition, common data analysis tools, like anova, require replicates and disregard correlation structure among times. we present a method for the identification of differentially expressed genes in un-replicated time-course experiments. the procedure does not assume any model or distribution function, takes into account the correlation of data, and does not require sample replicates at the various time points, other than the presence of an initial time point for all analyzed conditions. the identification of differentially expressed genes as the result of a system perturbation is formally stated as a hypothesis testing problem in which a defined statistic is used to rank transcripts in order of evidence against the null hypothesis. specifically, (i) data are structured so that measurements are correlated in time, within the same biological condition; (ii) the null hypothesis is formulated so that changes in expression levels at different time points are equivalent; (iii) time point t0 represents the system before the perturbation. therefore, modulated genes are detected testing the statistical significance of expression differences between physiological states at each time point, once corrected by the variability at t0, and given an empirical null distribution constructed using permutations. statistical significance is assessed by the q-value. the method has been tested on time-course microarray experiments aimed at studying the temporal changes of gene expression in: (i) skeletal muscle cells treated with a histone deacetylase inhibitor (iezzi et al., 2004) and (ii) immature mouse dendritic cells (dc) exposed to larval and egg stages of s. mansoni (trottein et al., 2004) . differentially expressed genes, identified using the proposed algorithm, have been compared with results obtained from anova model and sam paired test. the biological significance and soundness of selected transcripts was also verified using global functional profiling by means of ontotools. results demonstrate that this novel procedure allows the identification of biologically relevant genes using half of the replicates required by standard model-based approaches. carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y2 have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. the calcium-dependent antibiotic (cda) is a lipopeptide synthesised non-ribosomally and produced by streptomyces coelicolor a3(2). cda contains several non-proteinogenic amino acid residues. hydroxyphenylglycine (4-hpg) is one of the unusual amino acids in the structure of the cda and vancomycin groups of antibiotics. for the members of the vancomycin group of antibiotics, the 4-hpg residue plays crucial roles in the structure and function of the final glycopeptide antibiotic. to reveal the putative biosynthetic pathway of this amino acid in cda, a standard "double crossover replacement strategy" was used to delete 4-hydroxymandelic acid synthase (4-hmas, encoded by hpd) from s. coelicolor mt1110 and 2377, using the delivery plasmid pzmh3. there was no cda production in the disrupted strains. plates containing a gradient of hydroxymandelic acid were used to restore cda production in both s. coelicolor mt1110 hpd and 2377 hpd. exogenous supply of 4-hydroxyl phenylglyoxylate and 4-hydroxyphenylglycine reestablished cda production by the hpd mutant. feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues (mutasynthesis). a cxcl2 tandem repeat promoter polymorphism is associated with susceptibility to severe sepsis in the spanish population n. maca-meyer 1 , c. flores 1 , l. pérez-méndez 1 , r. sangüesa 2 , e. espinosa 2 , j. villar 1 : 1 research institute, hospital universitario n.s. de candelaria, s/c tenerife 38010, spain; 2 department of anesthesiology, hospital universitario n. s. de candelaria, s/c tenerife 38010, spain. e-mail: nmacame@ull.es (n. maca-meyer) sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria, and remains an important cause of mortality in the intensive care unit. cxcl2 chemokine (or mip-2) exhibits a pivotal role in the immune response, and several functional studies in animal models of sepsis have catalogued cxcl2 as a candidate gene for the development of sepsis. we have performed a case-control association study of cxcl2 gene variants and susceptibility to severe sepsis in 179 hospitalised patients and 364 healthy individuals. after the examination of linkage disequilibrium in the region, we analysed whether two promoter polymorphisms (snp rs3806792 and a newly described polymorphic short tandem repeat d4s3454) were associated with the syndrome. we found a significant association of common variants at d4s3454 with the development of severe sepsis (heterozygote carriers or 2.82; 95% ci 1.10-7.24, and homozygote carriers or 3.65; 95% ci 1.41-9.43; mantel-haenszel χ 2 test for linear trend p = 0.0006). the risks remained significant even after a genomic control adjustment, based on 20 additional genotyped polymorphisms not linked to the candidate gene. these preliminary results suggest that cxcl2 gene variants may contribute to the development of severe sepsis. kasper møller 1 , ana paula oliveira 1 , jens nielsen 2 , mark johnston 1 : 1 center for microbial biotechnology, biocentrum, technical university of denmark, denmark; 2 department of genetics, school of medicine, washington university, st. louis, usa glucose is the preferred carbon and energy source for most cells. in saccharomyces cerevisiae, a complex regulatory network ensures that s. cerevisiae ferments glucose to ethanol even in the presence of oxygen. to obtain a better understanding of this crabtree effect and the logic of the glucose signalling network in s. cerevisiae, we are analyzing glucose sensing and signalling in the related species saccharomyces kluyveri, which exhibits much less of a crabtree effect (it prefers not to ferment glucose when oxygen is available). we show that there are only two major glucose transporters in s. kluyveri, and that these are regulated in response to the availability of glucose via a glucose sensor and a signalling pathway similar to the glucose induction (rgt2/snf3-rgt1) pathway in s. cerevisiae. we have used dna-microarrays for s. kluyveri to find targets of the s. kluyveri glucose induction pathway, as well as to evaluate the global response to a change in environment from growth on ethanol to growth on glucose. this study identifies a number of differences in the regulation of glucose uptake and global responses to glucose between s. kluyveri and s. cerevisiae, which may contribute to their different glucose metabolism. detection and analysis of microrna using lna probes nana jacobsen, christian lomholt, peter mouritzen, peter stein nielsen, mikkel noerholm exiqon a/s, bygstubben 9, dk-2950 vedbaek, denmark. e-mail: mouritzen@exiqon.com (p. mouritzen) micrornas are a class of short endogenous rnas that act as post-transcriptional modulators of gene expression. growing evidence suggest that micrornas exhibit a wide variety of regulatory functions and exert significant effects on cell growth, development, and differentiation. recent studies have shown that human microrna genes are frequently located in cancer associated genomic regions and perturbed microrna expression patterns have been observed in many malignant tumors. we have exploited the significantly improved hybridization properties of lna oligonucleotides against rna targets to design lna-modified dna probes for detection of different micrornas in animal and plants by northern blot analysis, microarray hybridization and in situ hybridization. we will describe the results obtained from detection and analysis of different micrornas in c. elegans, zebrafish, mouse, and plants. in addition, we will describe a novel lna-based method for expression profiling of mature micrornas by quantitative rt-pcr. expression profile of the sty and paa genes in pseudomonas sp. y2 by means of dna microarrays david bartolomé-martín 1 , david juck 2 , m a teresa del peso-santos 1 , charles w. greer 2 , julián perera 1 : 1 departamento de bioquímica y biología molecular i, facultad de ciencias biológicas, universidad complutense de madrid, 28040 madrid, spain; 2 environmental microbiology group, biotechnology research institute, national research council canada, montréal, que., canada h4p 2r2. e-mail: perera@bio.ucm.es (j. perera) dna microarrays are a new and powerful tool to study gene expression in very diverse systems. environmental biotechnology and biodegradation are some of the fields of research where this technology may be very promising. pseudomonas sp. y2 is a bacterium able to grow in minimal medium plus either styrene (sty) or phenylacetic acid (paa) as the sole carbon and energy sources. this bacterium is the only organism where the genes that code for both the upper (sty genes) and the lower (paa genes) catabolic pathways for the styrene degradation have been described till now. it is unique in having two active copies of the genes encoding the lower pathway (paa1 and paa2 gene clusters). we have designed a dna microarray with the sty and paa genes in order to analyse their expression in the wild type pseudomonas sp. y2, in p. sp. y2 t2 (a paa2 deletion mutant) and in p. sp. y2 c1 (a crc gene mutant). this analysis has been performed on bacterial cultures grown in media with different carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y2 have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. dynamics in induced repression of phosphomannose isomerase pmi40 gene of saccharomyces cerevisiae anssi törmä 1,2 , juha-pekka pitkänen 1,2 , laura huopaniemi 2 , risto renkonen 2 : 1 medicel ltd., haartmaninkatu 8, 00290 helsinki, finland; 2 rational drug design program, department of bacteriology and immunology, haartman institute and biomedicum, university of helsinki, p.o. box 63, 00014 helsinki, finland. e-mail: juhapekka.pitkanen@medicel.com (j.-p. pitkänen) gdp-mannose is the precursor of cell wall biosynthesis in s. cerevisiae. to understand the system level role of gdp-mannose, we studied a conditional knock-out strain of the key enzyme in its synthesis; pmi40. the experimental procedure allowed us to study the order of mechanisms the cells launch in order to adjust to a sudden malfunction in the metabolic machinery. we collected 100 samples from continuous cultivations over 80 h and measured genome-wide gene expression levels, 10 enzyme activities, and concentrations of 30 intracellular metabolites. for sampling we have built a sample robot, which automatically takes and preserves the samples. in order to carry out this magnitude of experimentations and generated data, we have constructed a proprietary software platform to handle all the phases from project management in wet-lab to workflow and pathway management in in silico. after normalization and clustering, significantly changed genes and metabolites were searched for enrichment in biological processes and molecular complexes. further, gene expression levels, metabolite concentrations, and enzyme activities were searched against each other for causality over time. overall, we focused on thorough analysis of our own data and known database data in order to reward our efforts with knowledge. at the transcriptome level, repression of pmi40 led to two major types of activation profiles, one peaking at the time when pmi40p activity and gdp-mannose were depleted and the other later during recovery from the perturbation. the primary response was most enriched with genes known to play roles in mating and filamentous growth and associated with the transcription factors ste12p, tec1p, dig1p, and mcm1p, whereas the secondary response consisted of genes involved in carbon metabolism and associated with the general stress response regulators msn2p and msn4p. skn7p, a high-level transcription factor was associated with both the primary and the secondary response, consistent with its suggested role of coordinating environmental responses and developmental processes. transcriptome of pig ovarian cells: discriminant genes involved in follicular development bonnet a., le cao k.a., low-so g., san cristobal m., tosser-klopp g., hatey f. laboratoire de génétique cellulaire, centre inra de toulouse, castanet-tolosan 31326, france in order to identify genes and gene networks involved in pig ovarian follicular development, we built subtractive suppressive hybridization libraries (ssh) from granulosa cells of healthy follicles (small, medium or large). the rna isolated from these cells was used to hybridize cdna nylon micro-arrays. data analysis using a gaussian linear mixed model showed that 83% of the variability is due to the genes. two hundred fifty one regulated genes (from the 956 expressed) were identified and clustered into three groups according to the follicle size. moreover, we found previously identified genes such as aromatase, igfbp2 which supported the validity of our experimental model. ramdom forest analysis put forward the most discriminant 11 genes between the three follicle classes. this study put forward gene sets such as those involved in cell modeling, regulation of transcription, apoptosis during follicle growth. the next step will be to describe more precisely the spatio-temporal expression patterns at the mrna levels of the genes identified by these experiments. microalgae constitute a significant source of valuable natural products, e.g. sulfated polysaccharides, polyunsaturated fatty acids, and phycobiliproteins that find applications in wide range of industries, including food, pharmaceutical, agricultural and cosmetics. however, genomic and molecular genetic studies of microalgae lag far behind those of higher plants. in order to accelerate red microalgal genomic studies by taking advantage of current genomics and post-genomic technologies, we have generated expressed sequence tag (est) databases of two red microalgae porphyridium sp. and dixoniella grisea grown under various physiological conditions. to date we have sequenced 7210 and 6231 ests of porphyridium sp. and d. grisea, respectively. the sequence assembly resulted into, ca. 2000 non-redundant unigenes for each microalga, only 40% of which were identified by similarity to sequences in the public databases. porphyridium sp. and d. grisea unigenes were compared with the whole-genome predicted proteomes of three microalgae and those of representative eukaryotic and prokaryotic organisms. both microalgae have highest similarity to the red microalga cyanidioschyzon merolae. the order of sequence similarity to other organisms examined was arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, thalassiosira pseudonana (diatom), saccharomyces cerevisiae, caenorhabditis elegans, archaea and cyanobacteria. although red microalgae are considered as phylogenetic bridge between prokaryotes and eukaryotes, our data show that the red microalgae have strong similarity to eukaryotes and only distant similarity to prokaryotes. gene expression profiles were studied by analyzing cdna and subtraction libraries constructed from algae grown under various physiological conditions. we observed that top three most abundant ests in the stationary phase of porphyridium sp. were adp ribosylation factor like-1, flavohemoglobin and adp ribosylation factor-1. in addition, we have identified several genes which were specific to nitrate-and sulfate starvation. the sarco(endo)plasmic reticulum ca 2+ -atpase (serca, "the calcium pump"), is responsible for pumping the ca 2+ released into the cytoplasm during muscle contraction back into the sarcoplasmic reticulum store while proton are pumped the opposite way as counter-cations. these transport processes go against the concentration gradients and are therefore energy consuming. the energy is derived from atp hydrolysis via formation and break-down of a phospho-enzyme intermediate. over the last year a number of new crystal structures have been published which have added to our understanding of how this task is accomplished, which provides an impressive insight to the mechanism of a molecular pump. rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. this work was supported principally by embo and the mrc. structure and target-specificity of thioredoxin h kenji maeda 1 , anette henriksen 2 , per hägglund 1 , christine finnie 1 , birte svensson 1 : 1 biochemistry and nutrition group, biocentrum-dtu, technical university of denmark, dk-2800 kgs. lyngby, denmark; 2 biostructure group, carlsberg laboratory, dk-2500 valby, denmark. e-mail: kenji@biocentrum.dtu.dk (k. maeda) thioredoxins are ubiquitous small proteins with protein disulphide reductase activity. thioredoxins can alter the structures and the activities of various target proteins by reducing their disulphide bonds. seeds of several plants are abundant in cytosolic thioredoxins referred as h-type. in barley, two thioredoxin h isoforms, hvtrxh1 and hvtrxh2 that share 51% sequence identity but differ in temporal and spatial distributions were previously identified and characterised. in the present study, the relationship between structures and targetspecificities of h-type thioredoxins are analysed. the 3d-structures of hvtrxh1 and hvtrxh2 are determined by x-ray crystallography as the first crystal structures of thioredoxin h. comparison of the structures shows that the majority of solvent exposed residues near the active sites are conserved between the two isoforms. this is in agreement with previously observed similarity in target-specificity of the two isoforms. thioredoxins from organisms distantly related to barley, such as e. coli, have highly similar folds but different surface charge distributions compared to barley thioredoxins. a comparison of the target-specificities of hvtrxh1, hvtrxh2, e. coli thioredoxin and several thioredoxin mutants will be attempted to reveal the structural features that influence specificity of barley thioredoxin h isoforms. enbrel is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human 75 kda (p75) tumor necrosis factor receptor (tnfr) linked to the fc portion of human igg1. the cho-expressed molecule contains both n-and o-linked oligosaccharides with a total carbohydrate content of 20% by mass. the o-linked oligosaccharides were released by hydrazinolysis and their structure determined by exoglycosidase sequencing and maldi-tof mass spectrometry. to locate precisely the o-linked sites, the glycosylation heterogeneity of tnfr:fc was simplified by treatment with n-acetyl neuraminidase and n-glycanase. the remaining molecule, which only carries core o-linked glycan structures, was cleaved by trypsin and analyzed by lc-ms. precise localization of o-glycosylation sites was determined based on the concept of a specific modification of the o-glycosylated serine into 2aminopropenoic acid and o-glycosylated threonine into 2-amino-2butenoic acid. the deficit in mass resulting from this transformation was the marker used to localize the modified residues on the peptides by tandem mass spectrometry sequencing (ms-ms). ms-ms spectrum of enbrel glycopeptides were interpreted based on the presence of 2-aminopropenoic acid and 2-amino-2-butenoic acid, resulting in a complete map of o-linked glycans precisely located at 10 different sites. anu mursula, beatrix fahnert, sari krapu, eija-riitta hämäläinen, ritva isomäki, peter neubauer bioprocess engineering laboratory and biocenter oulu, university of oulu, oulu, finland. e-mail: anu.mursula@oulu.fi (a. mursula) wnt proteins form a highly conserved family of secreted glycoproteins important in cell-cell signaling events during embryogenesis and adult tissue maintenance. impairments within this complex signaling pathway can lead for example to developmental defects in embryos, degenerative diseases and cancer. respectively, wnt proteins can be used as tools in basic research concerning wnt function, developmental biology, screening for interacting compounds, and for medical applications (e.g. therapeutics, stem cells). hence, recombinant wnts provide a valuable basis for these purposes. however, production of recombinant wnt proteins is challenging, because they contain multiple disulfide bonds making the folding very difficult. a process for production of murine wnt-1 in e. coli has been developed in our laboratory. the knowledge obtained from this research has also been applied to the expression of other wnts, namely wnt-4 and wnt-6, and can be used to approach other cysteine-rich proteins as well. since the expression level of wnt proteins is rather low so far, tools for monitoring and optimizing the production process have been established. by means of this sandwich hybridization method the level of target (wnt) mrna can be measured. the technique has already been applied to analyzing wnt-1 mrna levels. probes also for wnt-4 and wnt-6 have been generated. thus, transcription of wnt genes in all kind of cells (e.g. tissue, recombinant hosts) in general as well as kinetics of transcription can be studied using these tools. in growth factor signaling, stimulation of cell-surface receptors first triggers activation of the receptor itself and then of a large number of intracellular effector molecules. the stimulus is integrated with a host of other cellular processes, leading to cytoskeletal changes, activating transcriptional programs in the nucleus and ultimately resulting in cell proliferation, differentiation or motility. classical signaling pathways and networks depict potential protein-protein interactions only in a static form. in the cell, these interactions are dynamic and occur in an ordered fashion. here, we apply a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance in order to study the global dynamics of signaling events. briefly, three cell populations are metabolically labeled with either normal arginine or a 13 c substituted form, or a 13 c 15 n variant (stable isotope labeling by amino acids in cell culture, silac). each population was then stimulated with egf for a different time period and tyrosine phosphorylated proteins were affinity purified with anti-phosphotyrosine antibodies. the proteins from the precipitated complexes were quantitatively analyzed and identified using lc-ms/ms. arginine containing peptides occurred in three forms, directly indicating protein activation at the corre-sponding time point. combination of two experiments sharing one common time point of activation then generated five-point dynamic profiles. from the 202 proteins quantified, we identified 81 signaling proteins, including virtually all known egfr substrates and 31 novel effectors, and the time course of their activation upon egf stimulation. discriminating proteins involved in the signaling network from unspecific binders was straightforward as these presented an activation profile. we have now further extended this study by directly measuring in vivo phosphorylation sites in response to growth factor stimulation and monitoring the time evolution of the phosphorylation events. finally, we determined and quantitatively compared the global egf and pdgf tyrosine phosphoproteomes in human mesenchymal stem cells and revealed a control point in their differentiation into bone-forming cells. such global activation profiles provide a novel perspective in cell signaling and will be crucial to model the highly dynamic signaling networks in a systems biology approach. klaus schneider 1 , dave g smith 2 , steven skaper 2 , alastair d. reith 1 : 1 discovery research, glaxosmithkline, coldharbour road, harlow, essex, uk; 2 neurology & gastrointestinal cedd, glaxo-smithkline, coldharbour road, harlow, essex, uk over the last 15 years, progress in signal transduction research has revealed an astonishing degree of complexity in cell signalling which is manifested in positive and negative regulations and feedback loops within signalling pathways and by cross-talks between pathways, all of which are highly cell-type dependent. it has become evident that protein phosphorylation by protein kinases plays a major role in this complex regulation of cell signalling (hunter, 2000) . due to the importance of signal transduction in disease processes, many protein kinases may constitute key targets for disease intervention. yet, the lack of a full understanding of the regulation, the activation and, importantly, of downstream substrates of particular protein kinases requires often more detailed studies before initiation of resource-intensive efforts to find disease-modifying molecules. technologies for the study of protein kinase signalling include 32 p labelling, mutational and knock-out studies and more recently rna interference. these tools are complemented by approaches that are based on proteomic technologies developed over the course of the last 10 years. in this presentation, the scope of proteomics technologies to contribute to an understanding of kinase signalling will be discussed. an overview of available technologies will be given and results will be presented from a proteomic study of glycogen-synthase kinase 3 (gsk3) signalling (coghlan et al., 2000) . novel findings will be presented on a study of gsk3 inhibition in a primary neuronal cell line by differential 2d gel electrophoresis, which resulted in the identification of more than 40 proteins that were significantly regulated. proteome analysis is typically done by nanospray lc/ms in order to achieve higher sensitivity and thus a greater number of protein identifications. however, nano-scale lc systems can be more challenging to use and maintain. to obtain the best chromatographic performance, connections must be made carefully to minimize band broadening. improved chromatographic performance can enhance the mass spectrometric results by tandem ms as a greater number of peptides can be detected. a microfluidic chip-based system has been developed (yin et al., 2004) that minimizes the number of connections and the delay volumes. this work evaluates the performance of this device against the traditional nanospray approach. a yeast extract sample was separated by sds-page and bands were excised from the gel for further analysis. after in-gel digestion, the sample was analyzed by both traditional nanospray and the microfluidicbased chip device. after protein database searching, the identified proteins and the protein sequence coverage's were compared for the two approaches. the microfluidic device was demonstrated to be equivalent or better compared to the traditional approach. yin, h., killeen, k., brennen, r., et al., 2004. anal. chem. 77, 527-533 . plant cytochromes p450 (p450s) play key roles in the biosynthesis of most bioactive compounds with agronomic and therapeutic applications. a collection of about 120 plant p450s was expressed in yeast. the cdnas were isolated from the model plant with a sequenced genome arabidopsis thaliana, some others from wheat, helianthus tuberosus or vicia sativa. they were expressed under the control of a galactose-inducible promoter in an engineered strain of saccharomyces cerevisiae in which the gene of the native p450 reductase was replaced with the gene of a p450 reductase from a. thaliana under the control of the same galactose-inducible promoter, in order to provide an optimal context for plant p450 expression and activity (pompon et al., 1996) . an original procedure was designed for the high-throughput functional screening of this enzyme collection. it is based on the detection of oxygen consumed during the catalytic reaction by a fluorochrome embedded in the bottom of the microwell plates. this method was validated using several recombinant p450s of known activity. it also allows for a very efficient screening for enzyme inhibitors. the advantages and limits of the method will be discussed. this work was carried out with the support of génoplante programme (no 2001004) . reference pompon, d., louerat, b., bronine, a., urban, p., 1996. methods enzymol 272, 51-64. 3 folding of a bacterial membrane protein studied by protein engineering daniel e.otzen, pankaj sehgal, peter a. christensen department of life sciences, aalborg university, sohngaardsholmsvej 49, dk -9000 aalborg. e-mail: dao@bio.aau.dk (d.e. otzen) we have carried out a kinetic analysis of the folding of the 4-helix transmembrane protein dsbb in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate and dodecyl maltoside. this analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. the analysis also takes into account the composition of the mixed micelles, which is different from the bulk detergent composition. refolding and unfolding are consistent with a three-state folding scheme involving the sdsdenatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of sds. the temperature-dependence of the folding reaction displays an unusual decrease in heat capacity accompanying unfolding, which probably reflects the amphiphilic environment of the membrane protein. destabilization of dsbb by different short-chain alcohols correlates very well with the alcohols' respective hydrophobicities. data from a series of ala-scanning mutants tentatively identify a nucleus for folding, which is relatively diffuse and involves all four helices. we are currently complementing this work with studies of the association of peptides corresponding to individual transmembrane segments of dsbb. the reca protein of e. coli plays a crucial role in homologous recombination and dna repair. the recombination process takes place in a filamentous complex, in which the protein monomers are arranged in a helical manner around a single-stranded dna (ss-dna). in the presence of atp the filament can accommodate a second, double-stranded dna (ds-dna) and the strand exchange reaction can occur. the three-dimensional structure of reca itself and its complex with adp have been determined by x-ray crystallography. the active nucleoprotein filament, however, has only been studied at low resolution. both electron microscopy (em) and small-angle neutron scattering (sans) indicate significant differences between the structures of the active nucleoprotein filament and the compressed, inactive filament of only reca. we have presented a structural model of the reca protein in its active filament with ss-dna, using data obtained by linear dichroism (ld) polarized-light spectroscopy, based on a technique, we call "site-specific linear dichroism", which allows the orientation of a set of amino acids to be determined from ld data by systematic modification of the protein. here, we show that ld data of the nucleoprotein filament with ds-dna is over all similar to the data of the complex with ss-dna, indicating that the orientation as well as internal structure of reca in the active filament is not significantly altered when the bound dna is changed from single-stranded to double-stranded. this result supports the idea that the strand exchange reaction occurs without large conformational change of the reca protein. the choline-binding modules: a powerful biotechnological tool jesús m. sanz instituto de biología molecular y celular, universidad miguel hernández, elche, spain choline-binding modules (chbms) are present in some virulence factors of streptococcus pneumoniae (pneumococcus). the most extensively studied chbm is c-lyta, the carboxy-terminal domain of the pneumococcal cell-wall amidase lyta. the three-dimensional structure of choline-ligated c-lyta is built up from six loop-hairpin structures ("choline binding repeats", chbrs) forming a left-handed ␤-solenoid with four choline binding sites. although the structure of the ligand-free form is not yet known, our folding studies suggest that it is more loosely packed, with a partially unfolded amino-terminal region and a stable carboxy-terminal moiety that is extremely resistant to chemical denaturation (maestro and sanz, 2005) . the affinity of c-lyta for choline and other structural analogues allows its use as an efficient affinity tag for overexpression, immobilization and single-step purification of proteins of biomedical interest (c-lytag fusion protein purification system). this system presents many advantages when compared to current commercial methods, namely simplicity, compatibility with buffers and robustness. the availability of multiple supports that specifically bind chbms (such as multiwell plates) has recently allowed the development of a new procedure for the immobilization of c-lyta-containing hybrid proteins that may be used in proteomics, diagnostics and peptide display. in this communication, we present our last results about the stability, folding and engineering of c-lyta, together with a compendium of the current biotechnological potential of this protein, and highlight the productive link between basic molecular studies and their application. many of the modern approaches for studying disease compare steady state functions, such as repair, growth, and regulated gene expression within the various biological compartments organised by specialized function, be it mitochondria or blood vessels. the assignment of protein identities, which are linked to key biological mechanisms, which are associated with disease processes and disease progressions are an important area of this work (marko-varga and fehniger, 2004) . today, the technology available for studying proteome expression and resolving exact protein and peptide identities in complex mixtures of biological samples allows global protein expression within cells, fluids, and tissue to be approached with confidence. this confidence is due in part to reproducible repetitive sampling and analysis technologies including robotics data acquisition and high level mass spectrometry including both laser-desorbtion and electro spray ionisation. the precision in defining differences between normal and diseased steady states is aided by the creation of compiled reference and master data sets and by new methods for multiplexing the analysis of samples in groups. the establishment of key representative reference proteome systems representing the dynamic changes in protein expression during disease will be vital to the interpretation of changes observed in specific samplings of disease states and specific cells obtained from these samples. the creation of reference databases of proteins linked to disease pathways will play an important role in furthering our understanding of the "proteome of disease". examples will be given where protein expression patterns have been generated from compartments within tissue sections. marko-varga, g., fehniger, t.e., 2004. j. proteome res. 3, 167178. 2 adaptation of the saccharomyces cerevisiae proteome to nutrient limitations studied by metabolic stable isotope labeling and mass spectrometry albert j.r. heck netherlands proteomics centre and utrecht university, the netherlands. e-mail: heck@npc.genomics.nl. url: www.netherlandsproteomicscentre.nl one of the major aims of proteomics is to provide quantitative data on differential protein expression levels. recently, mass spectrometry-based methods have been introduced that can provide quantitative data on differential protein expression, mostly using stable isotope labeling (goshe and smith, 2003) . we opted for metabolic labeling as this provides efficient means to quantify differential protein expression, and has the advantage that all proteins are labeled universally (romijn et al., 2003) . in their natural habitat microorganisms encounter non-optimal growth conditions and often growth is limited by one nutrient. microorganisms need to respond rapidly to changes in the environment in order to survive. in the present study, we investigate the proteome response of chemostat cultivated wildtype saccharomyces cerevisiae to two different nutrient limitations, namely carbon and nitrogen limitation. yeast was metabolically labeled in well-controlled chemostat cultures. 14 n and 15 n labeled proteins were separated using 1d gel electrophoresis followed by rp-lc-esi-ms on a lc-q. relative quantification was performed by using relex software (maccoss et al., 2003) . we quantified 759 proteins, using on average 8 peptide peak pairs per protein. this analysis revealed that 419 proteins showed a significant increase/decrease in expression level. the functional annotation of these proteins revealed that the yeast cells change expression levels of enzymes involved in metabolism of the growth-limiting compound. the protein expression ratios were compared with corresponding transcript levels. moreover, we compared the accuracy of quantifica-profiles mainly reflected differences in cellular origins in addition to different functional roles. mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute phase proteins, antioxidant proteins and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization and in lipid metabolism. several proteins not previously implicated in nerve regeneration were identified, e.g. translationally-controlled tumor protein, annexin a9/31, vitamin d-binding protein, ␣-crystallin b, ␣-synuclein, dimethylargininases and reticulocalbin. real-time pcr analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. in conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. yeasts plasma membrane macromolecular components involved in stress resistance paola branduardi, paola paganoni, danilo porro dipartimento di biotecnologie e bioscienze, università degli studi di milano-bicocca, piazza della scienza, 2-20126 milano, italy. e-mail: paola.branduardi@unimib.it (p. branduardi) the plasma membrane is a universal structure of living cells constituting an essential barrier dividing and defining the intracellular from the extracellular environment. it is consequently easy to deduce the crucial role played by said structure for any cell of any living organism, and especially for unicellular organisms, since all the information deriving from the external environment as well as many of the consequent cellular responses have to pass through this barrier. unicellular organisms, thanks to easy manipulation and cultivation techniques, can represent a very useful model for studying the plasma membrane function and response. in addition microorganisms, and among them yeasts, can be considered advantageous cell factories for recombinant productions. in this contest, the implementation of any process of production has to take into account, among others, the response and the tolerance of the host to the external environment. from these considerations derives the interest of our group to analyse the main macromolecular components of yeasts plasma membranes (proteins, lipoproteins and lipids), isolated from cells grown under different stress conditions, with particular attention to acidic environments. here, we present our recent data about separation and identification (by sequencing analyses) of lipoproteins isolated from the conventional yeast saccharomyces cerevisiae as well as from the non-conventional and acid tolerant yeast zygosaccharomyces bailii cell cultures grown in different conditions. in parallel, the protein fraction is under evaluation through a differential 2d proteomic approach and consequent analyses. effect of fungal polysaccharides on the expression of pancreatic proteins in streptozotocin-induced diabetic rats sang woo kim 1 , hye jin hwang 1 , kwang bon koo 2 , jang won choi 2 , jong won yun 1* : 1 department of biotechnology; 2 department of bioindustry, daegu university, kyungsan, in an attempt to search novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of pancreatic proteins in streptozotocin-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited excellent hypoglycemic effect, lowering the average plasma glucose level of the diabetic rats to 52.3%. pancreatic proteome were analyzed by 2-de system, which separated more than 2000 individual spots. the 2-de analysis demonstrated that thirty-four proteins from a total of about 500 matched spots were differentially expressed, of which 26 spots were identified as the proteins whose expression has previously been associated with diabetes. twenty-two overexpressed and twelve underexpressed proteins were significant (p < 0.05) between the healthy and diabetic rats, and the altered proteins were restored (p < 0.05) upon eps treatment. it was first found that carbonyl reductase (18.6-fold, p < 0.001) and mawdbp (31.4-fold, p < 0.01) were surprisingly upregulated upon diabetes induction, and then those two protein concentrations were completely restored by eps treatment. moreover, we obtained eight unidentified proteins that have not been reported to be related with diabetes mellitus. these results evidenced the effect of fungal eps on searching potential markers for diagnosis and therapeutic manipulation of diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. the model established in our experiment is expected to mimic human diabetic status, which will help us to interpret the roles of biomarkers in diabetic state. the use of polyol-responsive monoclonal antibodies in immunoaffinity chromatography and as a probe for unfolding of wild-type and altered (t103i) amidase from pseudomonas aeruginosa s. martins 1 , j. andrade 1 , a. karmali 1 , a.i. custódio 2 , m.l. since immunoaffinity chromatography is a powerful protein purification technique of interest in proteomics, monoclonal antibodies (mabs) against mutant (t103i) amidase from p. aeruginosa were raised by hybridoma technology. in order to identify mabs that bind t103i amidase tightly but release under gentle conditions, hybridoma clones secreting polyol-responsive mabs (pr-mabs) were previously screened. nearly 10% of elisa assay-positive hybridoma produced clones secreting pr-mabs with potential application as ligands for immunoaffinity chromatography. to select the optimal conditions for amidase elution, an elisa-elution assay was carried out, with two of these clones (f6g7; e2a6). the dissociation of ag-ab complex required 10% of propylene glycol and either 0.25 m (nh 4 ) 2 so 4 or 0.25 m nacl. the binding of purified mab of igm class (e2a6) to wild-type and mutant amidases was investigate by direct elisa, which revealed that it recognised specifically a common epitope on both amidases. conformational changes on antigen molecule were studied. mab e2a6 showed a higher affinity for heat denatured forms than for native forms as revealed by affinity constants suggesting that the mab recognizes a cryptic epitope. the effect of mab e2a6 on amidase activity was also investigated. the binding of mab to wild-type and mutant amidases exhibited an inhibition and activation of 60% as a function of time, respectively. this pr-mab is useful as a probe to detect conformational changes in native and denatured amidases as well as a ligand in immunoaffinity chromatography, which is of great interest in protein purification and proteomics. fragility and solubility of non-classical inclusion bodieš s. peternel 1 , a. ristič 1,2 , v. gaberc-porekar 1 , v. menart 1,2 : 1 national institute of chemistry, ljubljana, si-1000; 2 lek pharmaceuticals d.d., ljubljana, si-1000. e-mail: spela.peternel@ki.si (š. peternel) human granulocyte colony stimulating factor (g-csf) is a pharmaceutically important cytokine. when overexpressed in escherichia coli, it is usually accumulated in the form of inclusion bodies (ibs) . when produced at 42 • c classical insoluble ibs are formed while at 25 • c non-classical ibs containing a high amount of correctly folded g-csf are formed. as higher fragility and solubility of non-classical ibs were noticed, we decided to check whether bacterial cell disruption method has any influence on their mechanical stability and solubility. enzymatic lysis, sonication and homogenization, methods often used for disruption of bacterial cells during the isolation of ibs were compared. lysozyme treatment of bacterial cells appears to be mild enough disruption method not influencing the integrity of ibs. homogenization of bacterial cells at high pressure (100.000-120.000 kpa) shows no impact on classical ibs while some loss of target protein from the non-classical ibs is observed. sonication seems to be most harmful as even at rather low sonication altitudes, noticeable disassembling and solubilization of non-classical ibs occurs while no effect on classical ibs is perceived. our studies show that non-classical ibs are much more fragile and soluble than classical ones. therefore, one should extremely carefully choose the method for cell disruption to avoid undesirable loss of the target protein. the danish tick ixodes ricinus parasitize three different hosts both mammals and birds during the 3-year life cycle. the aim of this study was to identify the last blood host being the host, which the nymph had parasitized before molting to the adult instar. the reason for the study was to reveal the origin of the host contributing the most to the life cycle of the tick and thereby the maintenance of tick-borne diseases in denmark. the most common tick-borne diseases are lyme borreliosis and tick-borne encephalitis (tbe) causing illness in both animals and humans. we analyzed adult ticks, which were collected from known hosts. the analysis was performed at different heat stable proteins, which could be detected during the off host period by elisa. we found that heat stable proteins could be used as identification markers for host recognition. mushroom polysaccharides alter the expression of diabetesassociated proteins in the liver of streptozotocin-induced diabetic rats hye-jin hwang 1 , sang-woo kim 1 , kwang-bon koo 2 , jang-won choi 2 , jong-won yun 1* : 1 department of biotechnology, daegu university, kyungsan, kyungbuk 712-714, korea; 2 department of bioindustry, daegu university, kyungsan, kyungbuk 712-714, korea. e-mail: jwyun@daegu.ac.kr (j.-w. yun) in the present study, we investigated the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of liver proteins in streptozotocin (stz)-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in eps-fed rats to 52.3%. in the next step, we analyzed the differential expression patterns of rat liver proteins from each group, to discover potent candidates for diabetesassociated proteins. a total of 69 proteins of the 2-de gel were expressed differentially between diabetic and healthy rats. among them, 34 proteins were upregulated and 35 proteins were downregulated upon diabetes induction. many of these changes were in accordance with observations in previously published studies. surprisingly, the altered levels of most proteins in diabetic group were fully or partially restored to those of non-diabetic control group by eps treatment. moreover, we obtained 13 unidentified proteins that have not been reported to be related with diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. potential is still limited by the differences observed in the structure of plant and mammalian n-glycans. indeed, theses differences and particularly the presence of ␤1,2-xylose and ␣1,3-fucose glycoepitopes are responsible for the immunogenicity of plant n-glycans. in order to reduce the structural differences between plant and mammalian n-glycans, current strategies are to knock out plant-specific glycosyltransferases or to humanize plant n-glycans by expression of mammalian glycosyltransferases in plants. in the present study, we have expressed a human ␤1,4-galactosyltransferase in alfalfa. in order to further increase the efficiency of the human ␤1,4-galactosyltransferase in the plant golgi apparatus, we have exchanged the endogenous targeting signal of this human glycosyltransferase for the ones from plant glycosyltransferases recently characterized in our laboratory. we will illustrate this approach of targeted expression with the results obtained by fusion of the catalytic domain of human ␤1,4-galactosyltransferase with the n-terminal sequence of a plant glycosyltransferase that targets the fusion to the very early compartments of the golgi apparatus. the efficiency of natural versus targeted expression of human ␤1,4galactosyltransferase in alfalfa will be compared in term of n-glycan humanization. altogether, our results clearly illustrate that we are now on the way to get perfect copy of mammalian glycoproteins in alfalfa plants. construction of recb-recd gene fusion and analysis of fusion enzyme activities oytun portakal 1 , gerald r. smith 2 , pakize dogan 1 : 1 department of biochemistry, hacettepe university medical school, 06100, ankara, turkey; 2 divisions of basic sciences, fred hutchinson cancer research center, seattle, wa 98109-1024, usa. e-mail: oytun@hacettepe.edu.tr (o. portakal) protein folding is a fundamental process to gain protein function. in an oligomeric protein, the interaction between polypeptides affects the folding process and assembly to the holoenzyme. recbcd is a heterotrimeric and multifunctional enzyme that plays an essential role for major pathway of homologous recombination in e. coli. it is composed of recb, recc and recd gene products. recd is the fast motor unit of the recbcd enzyme. recd also plays a role for high affinity dsdna binding, nuclease activity and chidependent regulation. this study was designed to test the hypothesis that recd polypeptide regulates the essential reca loading activity. the approaching of the study was to fuse recd gene to subsequent recb gene and to observe the changes in enzyme activity and structure. for these purpose two genetic fusion mutations, two-nucleotide deletion and three-codon substitution were created at the overlap sites (ta) of recb and recd genes. fusion mutations were constructed by phage-mediated recombination system, which is called recombineering. this technology requires red function but not host reca protein function. here, we showed the recbd fusion polypeptides in crude extracts. genetic characterization tests were revealed that both fusion enzymes are recombination proficient and have wild-type phenotype. biochemical assays demonstrated that recbdc fusion heterotrimers have dsdna exonuclease, unwinding and chi cutting activities. they were also resistant to dna damaging agents. western blot analysis also detected a wild type length recd polypeptide together with recbd fusion polypeptides and a decreased heterotrimer compared to wild type. our findings suggest that recb-recd genetic fusions may affect recd assembling to the heterotrimer, but not affect it's native folding. sandwich immunoassay-a simple strategy for enhancement of the sensitivity and the specificity in prostate specific antigen detection based on surface plasmon resonance cuong cao, sang jun sim department of chemical engineering sungkyunkwan university, 300 chunchun-dong, jangan-gu suwon, prostate cancer is a deadly disease in men. prostate specific antigen (psa) has been proved to be the most reliable and specific biomarker in preoperative diagnosis, monitoring and followup of patients with prostate cancer. in this study, a biochip based on surface plasmon resonance was fabricated to detect psa at concentrations ranging from 1 to 1000 ng/ml. to reduce nonspecific binding, the chemical surface of sensor was constructed by using various ethyleneglycol mixtures of different molar ratios of hs(ch 2 ) 11 (och 2 ch 2 ) 6 cooh and hs(ch 2 ) 11 (och 2 ch 2 ) 3 oh. we also biotinylated the sams surface to enhance the orientation of protein immobilization. by using this surface, spr-based psa detection gave a positive ru value at the fist response in the whole range of psa concentrations. however, this ru value could get better and more reliable by simply applying a secondary interactant, the psa polyclonal antibody, in sandwich immunoassay. the results shown this approach could satisfy our purpose without modify the secondary interactant, which has usually been done by the other report. expression of epitopic domains of human coagulation factor viii in escherichia coli amir amiri yekta 1,2 , naser amirizadeh 3 , alireza zomorodipour 1* , fariba ataei 1 : 1 department of mol genet. national institute for genet eng & biotechnol tehran-iran p.o. box: 14155-634, tehran, iran; 2 islamic azad university of jahrom, jahrom, iran; 3 department of hematol, faculty of med, tarbiat modarres university, tehran, iran. e-mails: amir amiriyekta@yahoo.com (a.a. yekta), * zomorodi@nrcgeb.ac.ir (a. zomorodipour) human factor viii (hfviii) plays major role in the intrinsic pathway of blood coagulation and is used to treat individuals with hemophilia a for bleeding episodes. many researches have been focused on the molecular aspects of this protein. in this regard, epitopes of hfviii as well as their corresponding antibodies have many important applications. bacterially produced fviii-epitopes are capable to neutralize the alloantibodies that inhibit hfviii activity. the purpose of present study was to over-express two epitope-containing fragments of fviii in e. coli under t7 promoter (novagen). two dna fragments from light-and heavy-chains of hfviii (942bp-c1c2 and 1644bp-a1a2, respectively) were subcloned in the expression vector. the use of his 6 -tagged tail was also considered for detection and purification purposes. in each of the examined clones, a protein of expected size was detectable. in the c1c2-expressing clone the specificity of the over-expressed protein was confirmed by its reaction with the rabbit serum directed against native hfviii as well as anti-his-tag antibody. in the heavy chain-related-expressing clone, the expression level was low, but it was detectable by immunoblotting experiments. manipulations of the growth as well as induction may be required. the over-expression of the other epitopes reported in the heavy chain may be achievable by the expression of (a) sub-fragment(s) of this region. the over-expressed his-tagged c1c2-related protein was appeared to be trapped in the cell as non-soluble inclusion bodies. therefore, after homogenizing of the induced recombinant cells, the nonsoluble fraction was dissolved in a solution of denaturant (guanidine hydrochloride) and subjected for the purification, using a ni-nta resin (qiagen) followed by protein measurement. accordingly, an expression level of 5 mg/l (of culture) of the purified c1c2-related peptide was obtained. the recombinant hfviii c1c2-derived peptide has provided useful mean for further experimental and medical applications. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red 646 and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed 2d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with 0.6% w/v epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than 75 consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph 8.1) or chloride (ph 8.5) as leading ion and -amino-caproic acid (ph 8.9) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic 2danalysis. development of strategies for heterologous expression of glucose dehydrogenase from the halophilic archaeon halobacterium sp. nrc-1 juan carlos cruz-jiménez 1 , lorenzo saliceti-piazza 1 , rafael montalvo 2 : 1 chemical engineering, university of puerto rico-mayagüez campus, mayagüez 00680, puerto rico; 2 biology, university of puerto rico-mayagüez campus, mayagüez 00680, puerto rico. e-mail: juancruzj@hotmail.com (j.c. cruz-jiménez) halophilic archaea are excellent model organisms and valuable for biotechnology applications; they are easy to culture in the lab, genetically tractable, and exhibit a variety of interesting and useful characteristics. most halophilic archaea require 1.5 m nacl to sustain growth and structural integrity. among the 2.630 genes in halobacterium, we are studying the gene encoding a glucose dehydrogenase, gene id is 446, located between the 345205 and 346272 bases (halobacterium sp. nrc-1 genome project). this extremozyme is bioengineerable, and its use as a model for studying biocatalysis in aqueous/organic and nonaqueous media has not been explored to date. the utilization of enzymes in organic solvents has several potential advantages over aqueous systems. a major benefit is the increased solubility of many substrates, resulting in higher concentrations of reactant and products, hence reducing and purification costs and simplifying recovery protocols. cells were grown aerobically during seven days at 37 • c in a complex medium, harvested by centrifugation and their genomic dna extracted. for cloning of the gene, primers were designed based on the sequence recently published by the halobacterium sp. nrc-1 genome project. the forward (5 -ccgcatgcgcc cacagtccc-3 ) and reverse (5 -ccggcctctagaacggcctgg-3 ) primers were designed to incorporate restriction sites for sph i and xba i, respectively (in bold). we are pcr amplifying the genomic dna and developing methods for the heterologous expression using the mesophilic escherichia coli, as well as purifying the enzyme. the purification procedure will be carried out using high resolution methods based on the protein's halophilicity. bioinformatics methods will be used to facilitate conforming of protein function and for comparison with a native enzyme. quantitative measurements by mass spectrometry of hundreds of proteins simultaneously using the new proteinchip systemseries 4000 p. iversen, e. fernvik ciphergen biosystems inc., symbion research park, fruebjergvej 3, dk-2100 copenhagen, denmark. e-mail: piversen@ciphergen.com (p. iversen) most mass spectrometry methods used in proteomics allow for the identification of multiple proteins in a limited number of complex samples, but lack the ability to assess the quantity of the proteins and their modifications. however, mounting evidence shows specific cleavage of well-known proteins as being strong candidates for specific biomarkers, and in order to discover these biomarkers one has to be able to monitor the quantity and mass of hundreds of proteins from hundreds of complex samples reproducibly. the new series 4000 instrument in connection with proteinchip arrays ® from ciphergen biosystems enables this. the new 4000 series instrument is optimized for sensitivity, reproducibility and quantitation. new ion optics allows the use of higher acceleration voltages thus increasing the sensitivity, but without lowering the resolution. a new method of blanking the detector in connection with a non-linear gain of the detector also increases the sensitivity to the effect that igg can be detected down to 0.2 fmol. furthermore, the unique design of the instrument permits the detection of proteins with great variation in both mass and concentration and thus making it ideal for proteomics studies. a unique feature of the 4000 series instrument is the possibility to normalize the output by controlling the laser and detector so that results can be read with equal precision on different instruments, which is not often possible in mass spectrometry where individual instruments yield different results. this feature is vital in the validation of research results beyond individual laboratories. the coupling of liquid chromatography with mass spectrometry is now firmly established as a routine method for the identification of proteins that have been subjected to enzymatic digestion. in an on-line lc-ms experiment, the column eluent is coupled to the electrospray source via an emitter and any tryptic peptides present in the mixture are mass analyses as they elute from the hplc column. should there be any co-eluting species in the eluent, these will be separated in the mass analyser by their mass-to-charge ratio. it has become increasingly clear that relative quantification of protein expression changes is important in modern biology and medicine. several current approaches have been developed that utilise stable isotope labelling of samples in combination with separation and subsequent analysis by mass spectrometry. however, we have recently described an lc-ms strategy where quantification is achieved via normalisation of the ms datasets and comparison of the peptide intensities (of the observed tryptic peptides) across samples is performed. in this case, it is desirable to perform replicate injections and hence reduce statistical errors. this approach places a requirement upon good chromatography, especially in terms of retention time reproducibility. in addition exact mass measurement of the eluting ions is required as well as the ability to generate reproducible and reliable peak intensity, or area, calculations for the eluting tryptic peptides. the ability to measure the mass to charge ratios of ions accurately, across injections and across samples, increases confidence that the same ions have been matched from each sample injection. in this presentation our current strategy for the relative quantification of proteins will be discussed using, as examples, complex protein mixtures from salmonella enterica, eschericia coli and human serum. proteomic analysis for the production of rhctla4ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen 4-immunoglobulin (rhctla4ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using 2-d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy2, cy3 and cy5 dyes and run within a single dige gel. using decyder tm software, 2218 spots were detected with two-fold thresholds with 95% confidence and it was found that 60 proteins underwent significant change during the production of rhctla4ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla4ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. study of substrate specificity of rnr-exoribonucelases using hybrid proteins ana barbas, mónica amblar, cecília m. arraiano instituto de tecnologia química e biológica, ean, 2784-505 oeiras, portugal. e-mail: ab@itqb.unl.pt (a. barbas) the ribonucleases are essential enzymes responsible for the regulation of gene expression and have shown to be important for biotechnology purposes. for instance, commercial mutants deficient in ribonucleases have been quite relevant for the over-production of recombinant proteins. escherichia coli rnase ii is a processive 3 -5 exoribonuclease prototype of the rnr family that has homologues widespread in the majority of the sequenced genomes. by sequence alignment it has been proposed for the rnr type proteins the existence of three different domains: an n-terminal cold shock nucleotide binding domain (csd), a rnb catalytic domain, and a c-terminal s1 nucleotide binding domain. we have constructed several rnase ii deletion mutants to enable the characterization of each domain. these studies have allowed us to determine that both csd and s1 are involved in the binding of the enzyme to the rna substrate, being the s1 domain the most important. in rna-binding proteins it has been shown that the s1 domain's conformation is highly conserved. however, it is not known whether the substrate specificity is s1-dependent. in order to characterize the s1 domain and verify if it is directly related to substrate specificity, we have constructed rnase ii hybrid proteins in which the s1 domain was substituted by the s1 of two other exoribonucleases, rnase r (rnii-rnr) and pnpase (rnii-pnp). preliminary results have demonstrated that both quimeric proteins are capable of binding and degrading various rna substrates. in addition, studies are currently being carried out to verify the possibility that s1 domain of pnp in the hybrid protein might be involved in multimerization and/or interaction with other proteins. the murine monoclonal antibody igg1, anti-digoxin was produced in a rolling bottle fermentor. purification was performed on a protein g column. cd spectra were recorded on a jasco-810 spectropolarimeter. protein concentrations of 20-50 g/ml and path length of 1 cm were used for measurements in a far uv region. all measurements were performed in a cell holder thermostand with an accuracy of ±0.2 at 25 • c. at this temperature the predominance of ␤-strands is indicated. large conformational changes occur at 78 • c. at this temperature the spectra tense to irregular ␤-strands and unordered structures. these evidences confirming temperaturedependent conformational changes of protein and also high thermal stability of mentioned monoclonal antibody. generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling janni kristensen, kim kusk mortensen, hans peter sørensen 1 laboratory of biodesign, department of molecular biology, aarhus university, gustav wieds vej 10 c, dk-8000 aarhus c, denmark. e-mail: hans.peter.sorensen@teknologisk.dk (h.p. sørensen) we recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of escherichia coli. recombinant proteins were covalently coupled to the e. coli ribosome by fusing them to ribosomal protein 23 (rpl23) followed by expression in an rpl23 deficient strain of e. coli. this allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpl23 and the target protein. to assess the efficiency of separation of target protein from ribosomes, by site specific proteolysis, we required monoclonal antibodies directed against rpl23 and gfp. we therefore purified rpl23-gfp-his, rpl23-his and gfp from e. coli recombinants using affinity, ion-exchange and hydrophobic interaction chromatography. these proteins could be purified with yields of 150, 150 and 1500 g per gram cellular wet weight, respectively. however, rpl23-gfp-his could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. the purified rpl23-gfp-his fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by elisa using rpl23-his and gfp to select for monoclonal antibodies specific for each protein. this resulted in 20 antibodies directed against rpl23 and 3 antibodies directed against gfp. antibodies were screened for isotypes and their efficiency in western immunoblots. the most efficient antibody against rpl23 and gfp were purified by protein g sepharose affinity chromatography. the purified antibodies were used to evaluate the separation of ribosomes from gfp, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1a by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. proteomic analysis for the production of rhctla4ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen 4-immunoglobulin (rhctla4ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using 2-d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy2, cy3 and cy5 dyes and run within a single dige gel. using decydertm software, 2218 spots were detected with two-fold thresholds with 95% confidence and it was found that 60 proteins underwent significant change during the production of rhctla4ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla4ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. similarity searches and multiple alignment of s 1 and s 2 protein of sars-cov for modeling 3d structure and its evolution (origin) mohammad soltany rezaee rad, iman tavassoly, negar mottaghi, banafsheh rezaee. e-mail: mohammad.soltany@gmail.com (m.s.r. rad) aims: the exact origin of the cause of severe acute syndrome (sars) is still an open question. nowadays 8 recombinant origins for this virus have been found. s 1 and s 2 subunit of spike protein of this virus are the most important proteins responsible for severe acute respiratory syndrome. in fact they are glycoproteins of this virus exist on its surface. they are responsible for mediating fusion of viral and cellular membrane. the classification and modeling 3d structure of this virus can help us to suggest new ideas about its charististics and function, which may lead to new therapeutic and preventing modalities. methods: we used nucleotide sequence of s 1 and s 2 subunit of s (spike) protein for multiple alignments. we have done multiple alignments with different bioinformatics software (clusterx, entrez) for comparing the sequence with the other viruses and, we used weblab view software for modeling and identifying 3d structure of these proteins. findings: the similarity searches on nucleotide sequence of this protein with the 30 single strands rna (ssrna) shows the virus belong to a known classification named coronaviridae. these 3d structures show the responsibility of s protein in this syndrome. another findings based on these alignments is an important similarity between these subunits and genome of hiv-1 showing they have familiar mechanism in pathogenesis. discussion: multiple alignments are powerful tool in classification of new recombinational virus and emerging infection. 3d structure model of this virus is an important guide to understand the mechanism of this virus. the shape of glycoprotein that modeled with bioinformatics software can help us in understanding mechanism of binding this virus to human cells. this fact can be used in designing drug and vaccine to cure and prevent the sars. blocking these origins and sites leads to inhibiting the virus attachment. also the similarity between this virus and hiv-1 shows us that both of them have similar proteins that cause pathogenesis of these viruses. the simulated moving bed (smb) technology is a continuous countercurrent chromatographic separation technique that has been applied successfully in the last years to a number of significant problems. an smb consists of a series of fixed bed chromatographic columns connected in a loop, and outperforms column chromatography in terms of productivity and solvent consumption. the use of smb instead of batch processes for bioseparations, i.e. separations involving large and rather complex molecules with multiple 3d configurations depending on parameters such as ph, temperature, etc., is becoming of greater and greater interest. examples of these are therapeutic proteins, antibodies and plasmid dna among others. for all chromatographic processes in this field, one of the most crucial issues is the cleaning of the chromatographic media with a special solvent system, an operation usually referred to as cleaning in place (cip). in single column chromatography this is easily done off-line, but this is not compatible with standard smb operation. in order to overcome this limitation, the standard smb configuration has been modified by adding a dedicated section plus an additional section for the re-equilibration of the freshly cleaned column with the working solvent before it is re-inserted into the smb loop. in such a way, cip according to gmp criteria can be incorporated into the smb unit and operation, which is then called cip-smb. this new smb configuration is also related to the three fraction separation unit called 3f-smb that has been recently introduced and applied to the separation of nucleosides. in this work we apply cip-smb using a size exclusion stationary phase to the separation of plasmid dna from the filtered cell lysate solution. plasmid purification has become a key issue in the last years as a result of the advances in gene therapy, whereas traditional laboratory methods are not always suitable for therapeutic purposes. we report about separation performances, which are then discussed in the light of smb design criteria and compared to column chromatography performance. computer guided optimization of adsorptive bioseparation processes bernt nilsson department of chemical engineering, lund university, 221 00 lund, sweden. e-mail: bernt.nilsson@chemeng.lth.se separation processes like chromatography can be highly nonlinear and the behavior can sometimes be hard to predict. optimization of preparative chromatography is often done experimentally, which is both time consuming and expensive. a model-based approach to optimization is therefore an attractive and challenging way to overcome some drawbacks in the traditional working procedure in biotechnical industry. efficient model-based optimization for industrial needs requires three parts; models, methods and tools. model-based methodology requires a set of chromatography column model structures, which can capture the phenomenon of interest. for instance they have to capture column load variations, elution profile changes, operation condition disturbances, column configurations and stationary phase properties. to derive a reliable model for optimization it has to be calibrated and validated to experimental data, which requires an efficient calibration procedure. different calibration procedures are discussed and compared. after validation the model can be used in the design of a separation step. to do a robust design a set of requirements have to be fulfilled. the column size and operation conditions are used to optimize the performance of the step, which requires a constraint nonlinear optimization method. the choice of objective function for optimization and corresponding constraints are not obvious and the resulting operation conditions are often not robust. therefore there have to be additional methods available for analysis of the performance, like sensitivity and robustness analysis. optimization of the purification of antibodies is discussed and exemplified. the work with mathematical models and numerical methods has to be supported by a set of computer tools of different kinds in order to solve industrial problems effective. there is a need for different kinds of tools; customized tool to solve specific problems by a non skilled user and general toolbox for the expert. an example of a toolbox is presented. tina tarmann, alois jungbauer department of biotechnology, university of natural resources and applied life sciences, vienna, austria plasmids and viruses are the contemporary vehicles for genetherapy and genetic vaccination. extremely promising results have been reported from in-vitro, in-vivo and clinical studies. currently a lot of these compounds are manufactured with a technology which has been directly transferred from laboratory to pilot scale without further engineering. membrane based separations, chromatographic separations and precipitation have been employed for separation of plasmids and viruses. chromatographic separation have been designed with aim of protein separation. thus such processes suffer from either mass transfer limitations or low capacity. monoliths without intraskeleton mesopores and chromatography particles with giga pores are excellently suited for adsorption and separation of plasmids and viruses. low mass transfer resistance and high capacity compared to conventional beaded materials can be observed. adsorption kinetics were derived from infinite and finite bath methods and isotherms were constructed. these data also suggest that a conformational change of the plasmids takes place upon adsorption. discussion of the mass transfer properties and an example of scale up of a chromatographic separation process using these novel materials will be shown and discussed in respect to already existing processes. the recent developments in molecular therapies such as non-viral gene therapy and dna vaccination have fostered the development of efficient plasmid dna (pdna) purification processes. the separation of supercoiled (sc) and open circular (oc) isoforms is one of the key steps in the large scale purification of pdna vectors intended for a therapeutic use. although escherichia coli produces mainly the more compact sc pdna isoform, oc, linear and denatured pdna isoforms are usually present and are likely to be less efficient in transferring gene expression. for this reason, regulatory agencies specify that more than 90% of pdna in a therapeutic product is in the sc isoform. in this work histidine-base recognition is explored as a mean to separate pdna isoforms. the agarose gel used here combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. chromatographic profiles were obtained by injection of native plasmid (sc + oc) samples in the histidine-agarose support showing an efficient and baseline separation of both isoforms. the high resolution obtained with this support indicates that the method is potentially applicable to the separation of pdna at preparative and analytical scale. affinity ligand development with a novel encoded bead screening technology ib johannsen, versamatrix a/s, gamle carlsberg vej 10, dk-2500 valby, denmark. e-mail: www.versamatrix.com the presentation describes a new invention for fast development of affinity ligands, where up to 20,000 ligands can be screened onbead and identified in a few hours. combinatorial synthesis by the split and mix procedure is a powerful technique for generating vast numbers of diverse chemical compounds on polymer beads with relatively little effort. traditionally, the technique is hampered by the laborious spectroscopic and chemical analysis, needed to determine the exact structures of the ligand on selected beads. in this way, 6-12 month analysis time could easily be spent just to analyze a tiny fraction of the library. in the versaffin tm technology each bead is encoded, individually tracked, and identified during the synthesis and screening. this decreases the whole ligand development time from months to weeks and increases the amount of information significantly. the bead code further enables evaluation of the ligandprotein binding under varying binding and elution conditions. the instrument for reading the encoded beads and for quantifying the amount of bound protein is presented. the encoded beads we use are based on functional cross-linked polyethylenglycol (peg), which is compatible with water as well as most organic solvents. thus, the combinatorial synthesis can be carried out in organic solvents and the resulting compounds can be evaluated, still bound to the parent beads, under aqueous conditions. a further advantage of using peg based beads for on-bead screening is the fact that peg is biologically inert and therefore does not interfere in a bioassay. in the biopharmaceutical industry, pressure is mounting to shorten development times and thereby time to market, e.g. in the field of monoclonal antibodies, generic processes have been established which allow for more rapid development from gene to production of pre-clinical and proof of concept/phase i material. for non-mab products from various expression systems, productspecific approaches still prevail. however, for most product types similar issues like clearance of process-and product-related impurities, overall purity and yield, or manufacturing issues have to be dealt with in downstream process development. integrated and timely approaches based on process science and developed orthogonal analytical tools are often hampered by tight time frames and limited resources available. on the other hand, thorough understanding and analytical characterization of product characteristics but also of (process-related) impurities are pre-requisites for fully exploiting separation power and for achieving final purities way above 95% in a robust and cost-effective manner. from primary separation to polishing steps, we have made several attempts to improve the efficiencies of process steps themselves but also of ways to develop them. the strategies applied comprise implementation of innovative processing tools, rational streamlining and optimization of a sequence of unit operations, tech transfer and scale up considerations. also in this context, the applicability of scale down and ultra scale down models for process development and optimization purposes, their potential for speeding up and their limitations will be discussed. chromatographic monoliths are rather new chromatographic stationary phases. they consist of a single piece of a highly porous material. the pores are interconnected forming a network of channels. since the transport mechanism is predominantly based on convection, mass transfer between mobile and stationary phase is significantly enhanced resulting in short separation times. because of that they seem to be an ideal support for separation and purification of extremely large molecules like proteins, dna or even viruses. in this talk, various features of the monoliths like high porosity, fast mass transfer, surface accessibility and dynamic binding capacity will be described. effect of each feature on the separation and purification efficiency will be discussed in terms of molecular size and properties. while chromatographic monoliths are already widely accepted in microchip fluid devices, capillary columns as well as analytical columns, very few reports about preparative monolithic columns can be found. reasons for lack of preparative chromatographic columns will be elucidated and preparation strategy for construction of several liter volume methacrylate based monoliths will be presented. finally, several examples of biomolecule purification like large proteins, plasmid and genomic dna and viruses on cim convective interaction media ® monolithic columns will be given. further, their application as bioreactors and supports for solid state synthesis will be demonstrated. hubbuch institute of biotechnology, forschungszentrum juelich, 52425 juelich, germany. e-mail: m.schroeder@fz-juelich. de (m. schroeder) the intraparticle diffusion coefficient is an important parameter for modeling of chromatographic separation processes. a new method based on dynamic measurements of intraparticle concentration profiles of proteins in process chromatographic media with a confocal laser scanning microscope is presented. the diffusion coefficient is determined by fitting experimental data to a spherical diffusion model. excellent agreement of experimental data with simulation results is obtained. the diffusion coefficient is measured for seven proteins in sepharose 6 ff, spanning molecular weights from 14.3 to 160 kda. in addition, multicomponent diffusion processes for combination of differently sized proteins are analyzed and the influence of adsorbed proteins on the diffusion coefficient is measured in sp or q sepharose ff. taken together the presented method allows measuring the diffusion coefficient of proteins in process chromatographic media in a packed column. use of automated docking for predicting chromatographic behavior of proteins in hydrophobic interaction chromatography andrea mahn, m. elena. lienqueo contreras university of chile, santiago, chile in the present work, we have extended and automated the methodology proposed by mahn et al., 2005 for predicting protein behavior in hydrophobic interaction chromatography (hic). this methodology is based on the good correlation level between the average surface hydrophobicity of the interfacial zone (local hydrophobicity lh) and protein retention time in hic, for only three different ribonucleases. for determining the lh it is necessary to select the most probable protein-ligand conformation. in this work, we have determined the most probable conformation, of more than 12 proteins, using (i) first, the module insight ii affinity by accelrys for providing automated docking (grid method) of ligands (phenyl) to the proteins (100 conformations); (ii) then, the different probable docked protein-ligand conformations were automatically scored using the module insight ii ludi by accelrys; (iii) after that, each conformation was clustered and each cluster was scored by using the average score of each cluster (iv) finally, the most probable conformation was selected using a function based on the number of cluster components, and the average score value. then, when the most probable conformation was selected, the local hydrophobicity (lh) was calculated using the graphical representation and analysis of structural properties (grasp) program. the results have shown an acceptable correlation level (r > 0.90) between lh and the experimental dimensionless retention time (drt). in view of these results, we consider that this methodology could be used to adequately represent the chromatographic behavior in hic for all kinds of proteins (with a heterogeneous and homogeneous surface hydrophobicity distribution) and without a large number of tedious experiments, but only using computational simulation and adequate score criterion. potato tuber proteins are nutritious and show potential as functional ingredient in food systems. however, the present bulk processing technology can only recover byproduct protein for animal feed use. an expanded bed adsorption (eba) process for isolating functional food-grade protein from crude potato starch effluent was previously developed. moderate capture efficiency (20-25%) of the total crude protein was most likely caused by diffusion limitations and aggregated protein, inaccessible for adsorption. we employed the same adsorption ligand attached to agarose-tungsten carbide beads to create stable beds of 2.0-2.7× expansion using flow rates at 400-750 cm h −1 . a pilot scale eba process was run in a commercial processing plant over a three month campaign of starch production from potatoes of mixed variety. fresh crude effluent (150-300 l/cycle) was applied to a column (20 cm × 2 m) containing 20 l of resin. protein capture by eba was reliable in operation, producing a refined protein material, which after dewatering and gentle drying, showed improved functionality over heat-coagulated protein produced at the same plant. overall productivity increased. however, finding a robust operating window of predictable productivity is challenging since the potato fruit water is complex and deteriorates easily. from breakthrough curves, it is observed that the major bulk protein, patatin, displays non-langmuir adsorption behavior. this may indicate a range of interactions for different species of the same protein. chlorogenic acid (ca), the main polyphenolic substance in potato tuber, causes enzymatic browning and undesirable flavor changes, but polyphenols can also react with protein. assessing the effects of interacting cell components therefore applies to the bioprocessing of plant material. at present the acceptance of biochip technology for on site use, e.g. diagnosis or environmental control is hindered by rather expensive and complex instrumental systems. there is a need to provide reliable and cost-effective systems that can be operated with minimal training. the construction of electronic biochip microarrays using semiconductor technology enables the construction of compact systems with high integration at acceptable production costs. the key feature of the fully electrical biochip technology are micro arrays made in advanced si-technology and carrying several array positions with interdigitated nanometer gold electrodes on its surface. the chips are fabricated by standard silicon fabrication methods allow-ing high volume production and to minimise the cost per chip. the advantage of fully electronic microarrays is the intrinsic high spatial resolution and direct signal coupling of the biosensing element and the transducer. the function of fully electronic biochips is also based on the electrochemical transduction and quantification of the formation of affinity complexes on the chip surface. a portable device for field applications and point of care diagnosis have been designed and manufactured. the amperometric device enables the recognition of biomolecular interactions by measuring the redox recycling products of elisa enzyme labels. the highly sensitive signal transduction is achieved with a 16-channel interdigitated ultramicroelectrode array. one major advantage of fully electronic microarrays is the direct signal coupling of the biosensing element and the resulting robustness and opportunity for miniaturisation. those electrical biochip arrays have been adapted for the detection of all types of affinity complexes, such as for dna, rna, proteins and haptens. self assembling of capture oligonucleotides via thiol-gold coupling have been used to construct the array chip nucleic acid interface. thus e.g. pathogenic micro organisms have been identified and quantified via their genomic dna or ribosomal rna respectively. another application based on immobilized antibodies is shown to sense extreme low concentration of bioagent toxins. for processing the assay formats and the electrical read out of the detection of affinity complexes a modular fully automated measurement system has been developed. it is manufactured in industrial lines and available at market. dynamics and self-assembly of organic molecules on surfaces revealed by high-resolution, fast-scanning stm flemming besenbacher interdisciplinary nanoscience center (inano), university of aarhus, dk-8000 aarhus c, denmark. e-mail: fbe@inano.dk in the interdisciplinary area of nanoscience and nanotechnology, the adsorption and self-assembly of organic molecules on singlecrystal surfaces have attracted much attention lately due to the potential applications in fields ranging from molecular electronics to biocompatible interfaces. the supramolecular structures formed upon deposition of molecular species on solid surfaces depend on the molecular architecture and the distribution of functional groups on one hand, which determines the thermodynamically stable molecular arrangement, and on the other hand, on kinetic factors like thermal diffusion, spontaneous rotations and conformational dynamics. i will show how the unique aspect of our aarhus stm and the time-resolved, high-resolution stm imaging can be used to obtain important new insight into the dynamics, and can provide very important new information on the atomic-scale realm and on the dynamics of molecular nanostructures. the time-resolved stm data are visualized in the form of stm movies (see www.inano.dk/spm) which can subsequently be analyzed in order to extract quantitative information on the activation energy, the prefactors and the adsorbate-promoted diffusion. i will specifically discuss: (i) the self-assembly of guanine quartets on au(1 1 1) and the influence of cooperative hydrogen bonds, and (ii) the molecular recognition in binary mixtures of dna bases. g molecules are found to self-assemble into a hydrogen-bonded network of g-quartets, whose structure corresponds perfectly with the quartet structure of telomeric dna determined by x-ray crystallography. the strong preference of g molecules to form quartets can be explained by a cooperative effect that strengthens the hydrogen bonds within the g-quartet network over the hydrogen bonds in isolated dimers. by means of a combination of stm experiments and dft calculations we compare the 2d molecular networks formed on deposition of the binary mixtures g-c (purine-pyrimidine pair of complementary bases) and a-c (purine-pyrimidine pair of non-complementary bases). we find that the non-complementary bases segregate into islands of pure a and a network of pure c, whereas the complementary bases g and c form a network that cannot be separated by annealing up to the desorption temperature for c. high-resolution stm images allow us to identify the structures for the enhanced thermal stability as structures that contain g-c bonds, possibly with the same structure as the watson-crick pairs in dna molecules. kühnle, r., et al., 2002 . nature 415, 891. otero, r., et al., 2004 . angewandte chemie int. ed. 43, 2092 . otero, r., et al., 2004 . nat. mater. 3, 779. otero, r., et al., 2005 . angewandte chemie int. ed. 44, 2. rosei, f., et al., 2002 . science 296, 328. schunack, m., et al., 2002 . biomedical and pharmaceutical companies are using an increasing number of carbohydrate polymers in the formulation of drugs. one such polymer with highly attractive features is chitosan that can be produced from crustacean shells. chitosan is non-toxic, biocompatible and biodegradable. chitosan can be formulated as nanoparticles or membranes and have enhanced several bioprocesses. among the well-documented features are enhanced drug uptake by tight junction relaxation and enhanced in vivo uptake and protection of nucleic acid formulations. chitosan research has increased throughout this decade. research programs are addressing the potential of chitosan applications but preparations with variable molecular size and charge are not easily available. specifically companies working with the development of new drugs and enhancement of drug functionality are in need of formulation technology that provides well characterized biocompatible material. in the chitosan innovation consortium the danish companies, coloplast, novozymes biopolymers, pipeline biotech, and zgene, together with the research center inano (aarhus university) and bioneer a/s have developed a series of chitosan preparations suitable for research of biopharmaceutical applications (www.chitosan.dk). the ability to obtain functional formulations is currently being tested in both in vitro and in vivo experiments. the consortium participants have established a platform from which chitosan processing, characterization and formulation technology can be extracted. by providing specified chitosan preparations the polymer feature can be adjusted to fit specialized biopharmaceutical applications. high throughput bioprocessing govind rao center for advanced sensor technology, umbc, baltimore, md 21250, usa the post genome era holds a great deal of promise. an enormous number of new proteins await study. these will require sophisticated culture techniques, as cells will have to be grown under large numbers of environmental conditions to elucidate expression triggers. unfortunately, unlike molecular biology, bioreactor technology is little changed since its inception. the primary reason has been a lack of sensor technology that can be readily employed to monitor the cellular environment. we will take a look at the current status of the technology and report on promising optical sensor technology that permits low-cost high throughput cell culture and fermentation. noninvasive sensors that monitor ph, po 2 and pco 2 and high sensitivity solutions for glucose and glutamine measurements will be presented. in addition, the mixing characteristics that determine bioreactor performance will be examined at the small scale and their relevance to the large scale will be demonstrated. on-line monitoring and fed-batch operation in shake flask and micro titre plate cultures jochen büchs 1 , frank kensy 1 , markus jeude 1 , tibor anderlei 2 , barbara dittrich 3 , doris klee 3 : 1 biochemical engineering, rwth aachen university, aachen, germany; 2 ac biotec, jülich, germany; 3 textile chemistry and macromolecular chemistry, rwth aachen university, aachen, germany although methods of molecular biology has led to rational design of micro-organisms to suit our requirements, screening of large numbers of strains and media is still one of the most important tasks in biotechnology. batch operation of shaken bioreactors and absence of on-line monitoring is still the general state of the art for that purpose. it is also a very common practice in screening projects that only the final product titre is measured at the end of the culture for the evaluation of the "best performers". in the recent years several approaches were introduced to follow microbial cultures also in shaken bioreactors like shake flasks or micro titre plates (mtp's). it became obvious that the cultures can behave quite unexpected and most relevant and essential information is lost, if only the final product titre at the end of the cultures is utilised for evaluation. as a result, the screening may be directed to an unknown and non-intended direction or may even fail. this is demonstrated with some examples in this contribution. new methods and techniques are introduced to measure the oxygen and carbon dioxide transfer rate and the respiratory quotient in shake flasks and the optical density, nadh fluorescence, ph and do 2 in mtp's. if the desired product can be fused to a fluorescence protein, like gfp or yfp, also the product formation can be monitored on-line in mtp's. it is of utmost importance that the operating conditions of the applied shaking bioreactors are suitable and shaking motion is not stopped during the measurement. otherwise, e.g. power input, mixing and oxygen supply is interrupted and the micro-organisms will ongoingly rearrange their metabolism to cope with these disturbances of their environmental conditions. another problem of screening is the commonly applied batch operation mode. a lot of microbial systems display an overflow metabolism, substrate or osmotic inhibition or are characterised by a catabolite repressed product formation. in all these cases, batch operation is not the preferred operation mode and, therefore, these cultures are run in fed-batch in larger scales. in particular, it is nearly impossible to screen systems, which are catabolite repressed by the carbon source, in batch mode in defined mineral media. after initial growth has led to a nearly complete consumption of the carbon source, the product formation is derepressed. but then no more carbon source is available to continue with production. it is quite questionable whether in batch operation mode suitable strains can be selected for later fed-batch operation or not. this consideration has resulted in the development of a new technique which allows to run the screening in fed-batch operation mode. this technique is applicable in shake flasks as well as in mtp's. dramatic increases in product titre between 4-and 400-folds were observed under these conditions compared to conventional batch screenings. mikkel nordkvist, john villadsen center for microbial biotechnology, technical university of denmark, dk-2800 lyngby. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) efficient mixing and mass transfer are highly important in the chemical industry and in the fermentation industry. poor mixing can result in low yield and variable product quality in a number of cultivation processes, and mass transfer can easily become the limiting step in aerobic cultivations, especially at high cell density. we have tested a new tank reactor system, where liquid is withdrawn from the bottom of a tank, rapidly circulated, and injected back into the bulk liquid through the nozzles of rotary jet heads. liquid feed as well as gas is added in the recirculation loop and thereby distributed via the rotary jet heads. solid feed in powder form can also be added in the loop with advantage, and heat is efficiently removed in a plate-type heat exchanger, which is part of the loop. the system has a very simple design with no internal baffles or heat exchange area, and between batches the rotary jet heads are used for cleaning in place. a number of applications ranging from dispersion of liquid and powder to mass transfer will be presented. mass transfer applications include baker's yeast cultivation and oxidation of lactose to lactobionic acid by a carbohydrate oxidase. fast and accurate analytical information that can be used to rapidly evaluate the interactions between biological systems and bioprocess operations is essential for optimization of biological production processes. we have researched and developed a multiplexed microbioreactor system for the parallel operation of multiple microbial fermentations. the microbioreactors have working volumes from 5 to 150 l, and are instrumented for real-time monitoring of dissolved oxygen, ph and optical density. the growth profiles obtained with escherichia coli compare favorably to results obtained from conventional 500 ml batch bioreactors. we also demonstrate the use of our microbioreactors coupled to dna microarray analy-sis, as a tool for accelerated discovery and elucidation of metabolic pathways and gene expression profiles. the multiplexed system represents a significant step towards high-throughput data acquisition and has the potential to replace current instrumented bioreactors, which are bulky, expensive to run, and require many mechanical manipulations. design of a laboratory scale bioreactor to study solid-state tobacco fermentation m. di giacomo, l. nappi, d. silvestro, m. paolino, d. parente r&d biology department, british american tobacco italia, naples 80126, italy italian toscano cigar production is based on the fermentation of dark fire-cured tobacco. the process starts with the rise of leaf moisture to levels of water activities assuring development of the wild phylloplane microflora in the absence of free water. the intense growth of microorganisms modifies leaf characteristics (ph rise from acidic to alkaline condition) contributing to define the toscano typical smoke profile. tobacco fermentation takes place in great bulks of 500 kg which cause considerable amount of heat evolution as a function of the metabolic activities of the microorganisms. this heat accumulates and temperature can rise to as high as 70 • c. a laboratory cylindrical packed-bed bioreactor was designed to work under isothermal conditions. the reactor was ideal to ferment small quantities of tobacco (200 g) and was made up of a column aerated from the bottom with humidified air and placed in a thermoregulated room. experiments were conducted with constant temperature and air flow. moreover, bioenrichment experiments were conducted in the presence of different microbial starter cultures. fermentation courses were monitored by measuring microbial counts and chemical/physical modification of the substrate. with this laboratory scale system we obtained different kinds of information on the role and dynamics of the microorganisms involved in the fermentation process and on the influence of different environmental conditions. for the future, the design of an adiabatic device for tobacco fermentation is planned. on-line liquid chromatography as a process analytical technology for monitoring and control of biotech processes rick e. cooley 1,2 : 1 process analytics center of excellence, dionex corporation, sunnyvale, ca, usa; 2 eli lilly and company, indianapolis, in, usa (retired) biotech processes, used to produce an active pharmaceutical ingredient (api), generally differ from small molecule api manufacturing processes in that the starting materials tend to be more variable, more complex, and the product of interest in lower concentration due to the fact that they originate from a biological rather than a chemical process. this complexity and low starting concentration has generated a high level of interest in developing technologies that can be utilized to increase yield and reduce variability in the initial bioreactor phase, as well as, downstream isolation and purification operations. the use of process analytics, or on-line analytical measurements, is a technology approach that can contribute to increased process understanding and control. this presentation will provide examples of how various analytical measurement technologies have been utilized to monitor typical bioprocess unit operations leading to increased automation and control. examples of the use on-line liquid chromatography to monitor bioreactors, process scale chromatography columns, and enzymatic reactions will be presented in more detail. application of advanced monitoring strategies for recombinant protein production karl bayer department of biotechnology, university of natural resources and applied life sciences, vienna a-1190, austria high yield in combination with the required quality are the key objectives of large-scale production of recombinant proteins using different host/vector systems. however, in the past the efficient production of recombinant proteins was frequently limited due to inadequate exploitation of the cell factory and deficiencies in process design. to achieve optimal exploitation of microbial cell factories the key requirement is to enhance the monitoring capabilities to improve the insight into the host metabolism dynamics and to cope with limited understanding of the interaction of recombinant protein synthesis with host cell metabolism. since each protein exerts an individual influence on the host/vector system, the selection of appropriate analytical methods is even more important. in order to overcome these problems high throughput technology platforms, such as dna microarrays for transcriptome and differential 2-d electrophoresis (dige) for proteome analysis provide extensive data to screen for significant analytes and provide an appropriate basis for in silico modelling and reverse engineering of regulatory networks. taking advantage from such an iterative process experimental design will be improved and further aid to increase the performance of modelling. in addition, transcriptome data are used to screen fast stress responsive promoters to set up gfp based reporter gene fusions for in-situ monitoring of the metabolic load due to recombinant gene expression. moreover chemometric methods are frequently applied to model complex data from easily obtainable on-line data sets to overcome the limited monitoring capabilities due to the high complexity and nonlinearity of biological reactions and reaction networks. this strategy has been successfully applied to model bdm (bacterial dry matter), pcn (plasmid copy number) and the amount of recombinant protein using data sets acquired from off-gas analysis (o 2 consumption, co 2 evolution), alkaline consumption rate, in-situ capacitance and multi-wavelength fluorescence measurements. at-line monitoring of bioprocess-relevant marker genes using an electric dna-chip britta jürgen 1 , daniel pioch 1 , le thi hoi 1 , jörg albers 2 , rainer hintsche 2 , stefan evers 3 , karl-heinz-maurer 2 , michael hecker 4 , thomas schweder 1 : 1 institut für pharmazie, ernst-moritz-arndt-universität, 17487 greifswald, germany; 2 ebiochipsystems, 25524 itzehoe, germany; 3 vtb-enzymtechnologie, henkel kgaa, 40191 düsseldorf, germany; 4 institut für mikrobiologie, ernst-moritz-arndt-universität, 17487 greifswald, germany. e-mail: britta.juergen@uni-greifswald.de (b. jürgen) the gram-positive bacteria bacillus licheniformis and bacillus subtilis represent important industrial hosts for the production of enzymes (e.g., proteases and amylases) or antibiotics (e.g., bacitracin). both organisms are attractive for this purpose because of their apathogenity and their classification as gras organisms (generally regarded as save). moreover, their easy cultivation and their high natural capacity to secrete proteins into the growth medium qualify them for the industrial overproduction of homologous or heterologous proteins (simonen and palva, 1993) . for the control of the physiological state and the productivity of the production cells efficient analysis tools are of great interest. a prerequisite for the evaluation of the physiological state of cells during industrial fermentation processes is the analysis of so-called process-relevant marker genes, the expression of which indicates unfavorable growth and production conditions (schweder and hecker, 2004) . by means of proteome and transcriptome analyses we have identified critical process-relevant genes of b. licheniformis and b. subtilis cells under different nutrient limitation conditions and during industrialclose bioprocesses. dna-chips with probes for such process-relevant marker genes could be valuable diagnostic tools for the monitoring of the cellular physiology during microbial bioprocesses. in order to provide reliable tools for the monitoring of the cell physiology during microbial bioprocesses, we have developed a fast mrna analytical approach, which allows an at-line monitoring of the transcriptional activity of selected marker genes during bioprocesses. this approach is based on an easy, fast and reliable rna isolation procedure and the measurement of specific mrnas by means of an electric dna-chip barken et al., 2004 a robust bioprocess is crucial to ensure the consistent process performance and provide the high quality of product for drug manufacturing in biopharmaceutical industries. existing methodologies for bioprocess design do not involve establishing mechanisms to achieve the desirable robust bioprocesses and have low capacity in handling uncertainty in the product manufacturing. also, the solutions are often obtained step wise and do not account for interactions between the steps. despite its importance, the robustness of a bioprocess has not been properly defined and studies carried out in statistic sense are often retrospective. in addition, the computational cost is expensive due to using a line search algorithm for finding an optimal operating solution. finally, the existing methodologies are difficult to apply to the whole bioprocess in biopharmaceutical industries. this paper attempts to define rigorously a measure for process robustness and presents a new methodology for evaluating the robustness of bioprocess operations and their performance. the methodology is based on the concept of 'windows of operation' which shows the whole range of possible operating regions. the methodology also establishes a lower bound for the largest variation of a design variable to ensure the performance. these bounds are achieved by min-max optimization techniques. a direct search algorithm has been developed and its computational cost is much lower than the line search algorithm. results include visualization of robust operating regions and a set of indices which compare the performance of different operating strategies. the capabilities and efficiency of this methodology are illustrated by applying it to the centrifuge selection for the clarification of high solids density cell broths. the research work will impact considerably upon robust bioprocess operation. when grown on methanol, pichia pastoris is able to synthesize proteins to high titres as well as secreting and glycosylation, thereby making this organism a very interesting host for the production of recombinant drugs at large scale. the methanol residual concentration has been reported to strongly influence the specific productivity, the optimum concentration being around 3 g/l. a suitable monitoring and control technique is therefore necessary to study and improve the productivity of p. pastoris fermentations. the current research aims at showing how a mid-infrared spectrometer (atr-ftir) can be calibrated in-situ in order to monitor and control p. pastoris fermentations. this method is simple and fast, and eliminates the need of both standards preparation and off-line calibration. it is based on the observation that during fed-batch processes, only substrate and biomass concentrations vary significantly. the method therefore consists in adding a known amount of methanol at the beginning of the process, just after inoculation, and subsequently calibrating the instrument. financial support for the swiss national science foundation is gratefully acknowledged. implantable biomaterials are subjected to inflammatory responses mediated by adherent phagocytes such as monocytes and macrophages. these cellular responses and behavior have been shown to be dependent on the type of protein that adsorbs to the surface. surface modification is necessary to control and prevent protein adsorption, and thus modulate the inflammatory responses. hydrophilic surfaces that adsorb minimal amounts of protein are considered useful for minimizing the inflammatory reactions to biomaterials. in this study we have used two routes to modify polyethylene terephthalate (pet) films: (1) a wet-chemical method for attachment of linear polyethylene glycol chains (mpeg); and (2) gas-phase plasma polymerisation of diethylene glycol vinyl ether (degve) to generate peg-like surfaces. the surface chemistry was assessed by x-ray photoelctron spectroscopy (xps), fourier transform infrared spectroscopy (ftir) and time-flight secondary ion mass spectrometry (tof-sims). the two pegylated surfaces were compared for their ability to minimise both fibrinogen adsorption and the adhesion and activation of macrophage-like human leukocytes. adsorbed fibrinogen has been shown to be one of the key proteins in stimulating inflammatory responses to biomaterials. adsorption was investigated quantitatively using 125 i-radiolabeled human fibrinogen. in addition, the conformation of the adsorbed protein was tested using an antifibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. the results showed that pegylated surfaces adsorbed up to 90% less fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mpeg layers than on the peg-like plasma polymer surfaces. adhesion of in-vitro differentiated macrophage-like u937 cells was reduced on both the peg-like plasma polymer surfaces and the linear mpeg layers compared to the unmodified pet surface, but cells adhering to the peg-like plasma polymer surfaces secreted less tumor necrosis factor-␣ (tnf-␣) than cells adhering to the linear mpeg layers. thus, the linear mpeg surface is relatively efficient at reducing adhesion of macrophage-like cells, but those cells that do attach are in a more activated and proinflammatory state. analysis of ceramides from biological samples m. budvytiene 1 , j. liesiene 1 , b. niemeyer 2 , n. ceramides in human skin play an important role in the regulation of cell growth, differentiation and apoptosis. moreover, they are involved in the numerous signaling pathways. the growing interest in the investigations of ceramides physiological functions requires efficient separation methods of ceramides from biological resources and sensitive analytical methods. in this work some sensitive and selective methods, involving thin layer chromatography (tlc), high performance liquid chromatography (hplc), mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) were employed for the separation and characterization of ceramides from human foreskin. epidermal lipids were extracted from human foreskin for 24 h at room temperature using three solvent mixtures (chloroform/ethanol/water 1:2:0.5, v/v/v); (chloroform/ethanol 1:1, v/v), and (chloroform/ethanol 2:1, v/v). ceramides retention char-acteristics in tlc and hplc were compared with the retention of commercial standards. the best separation was obtained using normal phase column packed with hilic silica 3 m. the elution was performed using mixture of chloroform and ethanol 50/50 (v/v) as an eluent. two commercial standards n-stearoyl-sphingosine cer(ns), r f = 0.51, and n-palmitoyl-sphingosine cer(np), r f = 0.50, were selectively separated with hplc system in above mentioned conditions. the retention time of cer(np) and cer(ns) was 4.31 and 5.19 min, respectively. the same lipids were detected by hplc in the human foreskin extracts. the structure of the lipids from collected fractions was confirmed by means mass spectrometry and nmr. the physiological functions of the separated ceramides are investigated. production recombinant human thymosin-␣ 1 overexpressed as intein fusion protein in e. coli roman s. esipov, vasily n. stepanenko, anatoly i. miroshnikov, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya 16/10, moscow, 117997 russia. e-mail: esipov@ibch.ru (roman s. esipov) medicines based on polypeptides consisting of 30 and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin-␣ 1 production system intein mediated purification with affinity chitin (impac system)-binding tag has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin-␣ 1 . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl 2 and we have found that, in case of intein-thymosin-␣ 1 , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of 0.5-1 mm zinc chloride in buffers on all stages of thymosin ␣ 1 isolation. the structure of recombinant thymosin-␣ 1 of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. we present a microbioreactor with thermoelectric cooling to inactivate cellular metabolism by cell culture freezing. small-scale cultivation methods have gained increased importance and their development has been supported by advances in bioprocess monitoring methods. yet efficient sampling methods for off-line analysis remain important where in-situ real-time measurements are difficult, for example for intracellular metabolite concentration or enzyme activity measurements, and for all methods which are invasive by nature, such as protein purification. freeze-stop measurements of metabolite levels are ideal, because they are inert, i.e. do not require the addition of a chemical. in large systems, the chilling time is often the limiting factor, and alternative methods for cell metabolism inactivation are required, such as the spraying of the cell suspension in 60% methanol at a temperature of −40 • c, or the use of boiling buffered ethanol. due to the small thermal mass of microsystems, shorter chilling times can be expected. sample cooling to 4 • c in a microbioreactor has been presented previously. in this contribution, we investigate sample freezing to completely inactivate the cell metabolism from microbioreactor working volumes of approximately 100 l. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic 1 , alvin w. nienow 1 , ian w. taylor 2 , ryan hicks 2 , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w3110). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. when choosing an expression host for production of a specific recombinant protein, one can essentially select from a multiplicity of different systems. while e. coli bacterium is usually the starting point for any cloning and expression effort, there is no universal expression host that would work optimally for all proteins. practical issues to consider include e.g. need for post-translational modifications and protease activity of the host. we have produced recombinant hiv-1 nef in different host cell systems: e. coli, p. pastoris yeast and stable drosophila s2 insect cells. using strain/cell line development, production and purification data from practical experiments, we were able to conduct a techno-economical comparison of the different host cell systems. the annual production goal was set at 100 mg of high-purity nef. this was supposed to be produced campaign-wise in 1-2 batches using laboratory-scale bioreactors and other equipment. in this study, it was shown that although the production costs of the different systems were in the same range, the production in e. coli was most inexpensive, and the s2 cell system was the most expensive. regardless of the selected host system, the labour costs incurred the most expenses. when comparing different stages of the work (strain/cell line development, bioreactor production and down-stream processing), the strain development, the most man-hours demanding stage, involved approximately half of the costs of every production system. although e. coli was the most inexpensive host system for producing nef, it has some definite disadvantages: e.g. the production of endotoxins and the disability to perform post-translational modifications. if these disadvantages are of importance, the production must be done using more expensive system. modelling and control of industrial fermentation j.k. rasmussen, s.b. jørgensen capec, department of chemical engineering, technical university of denmark, dk-2800 lyngby, denmark. e-mail: jkr@kt.dtu.dk (j.k. rasmussen) fed-batch processes play a very important role in chemical and biochemical industry. fermentations in biochemical industry are most often carried out as fed-batch processes. present control schemes do not utilize the full potential of the production facilities and may fail to achieve uniform product quality and optimal productivity. the introduction of model based control strategies is considered difficult because suitable models are not readily available. first principle engineering models can be used but the usually limited knowledge of the regulatory network in the micro-organism makes model development very time consuming. another strategy is to use a purely data-driven approach where only limited prior knowledge of the process is required. a framework for generation of such blackbox models is used in this project. this framework is called "grid of linear models" (golm), it uses a large number of linear models which each describes the behaviour of the process within a certain time interval. the combination of these models results in a model which covers the entire time span of the fermentation and approximates the nonlinear time varying behaviour. a procedure for deriving golm models from operational data has been developed and because they consist of a large set of linear models it makes them suitable for model predictive control implementation with iterative learning capabilities from batch to batch. iterative learning model predictive control (ilmpc) based on a golm model is being implemented on a fermentor at novozymes a/s. the results will be evaluated in terms of the controller's capability to ensure uniform product quality and reject both intra and inter batch process disturbances. the model based approach renders optimization of the process recipe possible by using the ilmpc capability. this opportunity provides a great potential for increase of productivity and reduction of cost. j.m. viader-salvadó, j.a. fuentes-garibay, l.j. galán-wong, m. guerrero-olazarán institute of biotechnology, biological science school, autonomous university of nuevo león, san nicolás de los garza, n.l., méxico, the production of recombinant trypsin and trypsinogen has been reported as difficult due to a probably toxicity on host or its instability. in an effort to attain high-level production of shrimp trypsin for aquaculture applications, we have evaluated shrimp trypsinogen production by recombinant pichia pastoris strains in 5-l bioreactors. a p. pastoris gs115 mut+ containing in its genome the litopenaeus vannamei trypsinogen cdna fused in frame to saccharomyces cerevisiae ␣-factor secretion signal, previously constructed in our laboratory, was used. four three-step fermentations (glycerol batch, glycerol and methanol fed-batch) were carried out. the glycerol batch step (2 l of basal salts medium, 50 g/l glycerol, 8.8 ml biotin 0.02%, 8.8 ml ptm1, 250 l 289 antifoam, ph 5 adjusted to with 28% nh 4 oh) was carried out until glycerol was completely exhausted (21 h). the glycerol fed-batch step was carried out feeding with 50% glycerol (12 ml/l biotin 0.02% and ptm1) at 0.8 ml/min by 9 h and 45 min of posterior starvation. the methanol fed-batch step was carried out feeding with 100% methanol (12 ml/l biotin 0.02% and ptm1) by 133 h using a methanol concentration on/off feedback control to maintain constant the methanol concentration in the culture medium to 1 g/l. in all the fermentations the air flow rate and the agitation were set at 5 l/min and 800-1000 rpm, respectively. with the four fermentation assays, the influence of the ph and temperature in the production phase to the recombinant shrimp trypsinogen production were evaluated. in the four fermentations, at the end of the second step a biomass of 250 g/l wet weight were obtained (od600 230). the methanol demand in the four fermentations surprisingly was not increasing rather initially it was 0.37 ml/min, after 32 h decreased 2.5 times for 29 h, increased to 0.49 ml/min for 23 h and afterwards it was decreased manually to a constant value of 0.3 ml/min for that the dissolved oxygen will not decrease to values less than 20% (last 48 h). the total protein amount in the culture medium supernatant increased during the production step until values of 1.6 g/l (assay at ph 6), 6.5 times more than the worst assay (ph 3) recombinant shrimp trypsinogen production was confirmed by sds-page (about 500 mg/l) and trypsin enzymatic activity was detected using bapna as substrate after trypsinogen activation with shrimp hepatopancreas extract. large conformational change on giant dna induced by ascorbic acid: a nobel scheme on its antioxidative activity yuko yoshikawa, emi sakai, yoshiko oda department of food and nutrition, nagoya bunri college, nagoya 451-0077, japan ascorbic acid is often regarded as an antioxidant in vivo, where it protects against dna damage by scavenging reactive oxygen species. in the present, we will show another potent scenario on the protective effect of ascorbic acid through a significant structural change of giant dna. recently, we examined the effect of ascorbic acid on the higher order structure of dna through single molecular observation with fluorescence microscopy, and found that ascorbic acid generates a pearling structure in giant dna molecules, where elongated and compact parts coexist along a molecular chain. the results of observations with atomic force microscopy indicate that the compact parts assume a loosely packed conformation. as the extension, here we study the protective effect against double-strand breaks by reactive oxygen at different concentrations of ascorbic acid, in relation to the change of the higher order structure of giant dna. we have performed a real time observation on the doublestrand breaks on individual dna molecules by use of fluorescence microscopy. we have found that the double-strand break is markedly protected when ascorbic acid exists over millimolar concentrations. it is found that such a protective effect of ascorbic acid corresponds well to the above mentioned change on the higher order structure of dna. it has been reported that human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations. therefore, it is expected that the ascorbic acid concentration that induces the large conformational change on dna may be of physiological significance. , y., et al., 2004 . febs lett. 566, 39-42. yoshikawa, y., et al., 2003 . plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. there are many process both industrial and food based which employ protein hydrolysis and hydrolytic products have a wide variety of applications from industrial fermentation media to food additives. traditionally, proteins are hydrolysed by chemical means. acid hydrolysates of protein are used to produce food ingredients and flavour compounds. however, hydrolysis by chemical reagents produce potentially hazardous byproducts and these non-selective hydrolysis products cannot easily be defined. the use of enzymes allows for selective hydrolysis of protein and thus produces a potentially safer and more defined material. the present investigation describes the effects of substrate, enzyme and hydrolysate concentration on the hydrolysis of corn gluten. the corn gluten was hydrolysed by a commercial protease preparation neutrase. the protein hydrolysis reactions were carried out in 0.2 l of aqueous solutions at the temperature of 50 • c and ph 7 and were monitored by using ph-stat method. the degree of hydrolysis (%) and soluble protein concentration depending on time were investigated by using 1, 2, 4, 6, 8 and 10% (w/v) substrate concentrations; and 0.25, 0.4, 0.5, 0.6 and 0.75, 1% (v/v) enzyme concentrations; and 25, 50, 75 and 100% (v/v) this paper describes medium optimization for urease production by aspergillus niger ptcc 5011 by one-factor-at-a-time and orthogonal array design methods. the one-factor-at-a-time method was used to study the effects of carbon and nitrogen sources on urease production. among various carbon and nitrogen sources used, sucrose and yeast extract were the most suitable for urease production, respectively. subsequently, the concentration of sucrose, yeast extract and mineral sources were optimized using the orthogonal array method in two stages. the effects of nutritional components for urease production by a. niger ptcc 5011 in the first and second stages were in order of urea > niso4 > sucrose >kh 2 po 4 > k 2 hpo 4 > cacl 2 > yeast extract > mgso 4 and yeast extract > sucrose > k 2 hpo 4 > kh 2 po 4 > urea > cacl 2 > niso 4, respectively. the optimal concentrations of nutritional components for improved urease production were determined as 20g/l sucrose, 0.85 g/l urea, 3.4 g/l yeast extract, 0.03g/l niso 4 ·6h 2 o, 0.5 g/l mgso 4 ·7h 2 o, 0.04 g/l cacl 2 , 0.35 g/l kh 2 po 4 , and 0.35 g/l k 2 hpo 4 . these results showed that urea, niso 4, yeast extract and sucrose had significant effect on urease production by a. niger ptcc 5011. tween 80 and mgso 4 had negligible effect on urease production by this strain. the subsequent confirmation experiments determined the validity of the models. maximum urease activity in optimized media by onefactor-at-a-time and orthogonal array methods were about 1.14 and 2.74 times greater than with the basal medium, respectively. carbon sources create fingerprint fermentation characteristics pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 ib lab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e. glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e. serine alkaline protease (sap; ec 3.4.21.62),) and ␤-lactamase (ec 3.5.2.6), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. 1.5-2-fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide ç alık bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey ␣-amylase (e.c. 3.2.1.1) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣-1,4 glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b-14396) which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in 0.5 dm 3 airfiltered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs1). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled 1.0 and 3.5 dm 3 systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc28s ultracentrifuge, ␣-amylase activity was measured by the dns method (bernfeld, 1955) . amino acid concentrations were determined with a hplc (waters), protein and organic acid concentrations were measured with a hpce (waters, quanta 4000e) (ç alık et al., 1998) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method (rainer, 1990) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources i.e., glucose, fructose, maltose, lactose and soluble starch; n sources i.e., (nh 4 ) 2 hpo 4 , (nh 4 ) 2 so 4, and nh 4 cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and byproduct concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar ç alık iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc18::bal carrying recombinant e. coli on a defined medium with 8.0 kg/m 3 glucose were investigated in order to finetune the bioreactor performance, in v = 3 dm 3 batch bioreactors at five different conditions with the parameters at, i.e. q 0 /v r = 0.5 vvm and n = 250, 375, 500 and 750 min −1 and; q 0 /v r = 0.7 vvm and n = 750 min −1 . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph 0 7.25 are optimum for maximum bal activity, i.e. 860 u/cm 3 at 12 h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains 102 metabolites and 133 reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e.coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. the detection and diagnosis of pathogenic bacteria causing many diseases to the human body is an area of important research to public welfare. food is the most important energy source to humans, but it can give rise to disease caused by pathogenic bacteria not performing adequate detection tests. oligonucleotide-based microarrays are becoming increasingly useful for the analysis of expression profiles and polymorphisms among interested genes. here, we checked the possibility of development of oligonucleotide-based microarrays for detection and diagnosis of foodborne pathogenic bacteria. the oligonucleotide chip technology was applied to one control strain and seven foodborne pathogenic bacteria strains. it was designed repeated spots of eight hyperspecific and two highly conserved (control) capture probes from 16s rdna sequences. in order to validate experimental quality and to certificate specificities among specific spots at a glance by 2d and 3d views, quantitative visualization tool was developed. using the proposed oligonucleotide chip, we could classify and diagnose species and even subtypes of some pathogens. induction of in vitro neuro-muscular junctions using neuroblastoma and fibroblast cell lines for facilitating receptor-binding studies with botulinum toxin arindam chaudhury, bal ram singh department of chemistry and biochemistry, university of massachusetts dartmouth, 285 old westport road, north dartmouth, ma 02747-2300, usa. e-mail: g achaudhury@umassd.edu (a. chaudhury) botulinum toxin (bont), the most potent biological toxin known, is responsible for botulism, a fatal paralytic disease of the neuromuscular transmission. it blocks the release of acetylcholine at the neurotransmitter junction of the synapse. the objective of the current study was to induce in vitro neuro-muscular junctions through co-culturing of nerve and precursor-muscle cell lines. presently no known primary cultures or cell lines are available for nerve-muscle co-culture, thus validating the current work. j2-3t3 fibroblast cell line was first adapted to grow in media conducive for growth of sh-sy5y neuroblastoma cell line. the two cell lines were then splitted and co-cultured and observed for junction formations. light and fluorescent microscopic studies revealed en plaque (twitch-type) and en grappe (bulbous nerve endings) nerve-muscle junctions. growth rate of j2-3t3 cells decreased substantially when the media was initially changed. structurally they were more spindle-shaped than the normal reticular shapes of j2-3t3 cells, when grown in a media tailor-made for them. the formation of nerve-muscle junctions were confirmed using markers specific for each cell type. future work is focusing on receptor identification for the botulinum toxin in the established in vitro neuro-muscular junctions and also the transcellular translocation of the toxin. fourier-transform infrared (ftir) spectrometers have recently enjoyed widespread popularity in bioprocess monitoring applications due to their non-invasiveness and in-situ sterilizability. their online applicability creates an interesting opportunity for process control and optimization. however, the precision and accuracy of the predicted analyte concentration values directly depend on the quality of the measured signal and the robustness of the calibration model. instability and time drift in the measured spectra are currently one of the main obstacles in ftir monitoring. the intensity of the detected signal is influenced both by random noise and structural drifts and offsets. as a result, it is often necessary to scale the measured spectrum with respect to a constant reference spectrum, a technique similar to the internal standard approach used in analytical assays, such as hplc. applying this technique has lead to a noticeable decrease in the standard error of prediction in the monitoring of an anaerobic s43 fermentation of the saccharomyces cerevisiae yeast. in order to test the robustness of the calibration model and to increase its resistance to signal instability, random spikes of known amounts of analytes were introduced into the measured medium. this approach can be used to fine-tune the calibration model on-line and is currently one of the aspects investigated in this laboratory. the effect of the stringent response induction on l-valine biosynthesis by corynebacterium glutamicum ilze denina 1,2 , longina paegle 2 , liga zala 1,2 , maija ruklisha 2 : 1 faculty of biology, university of latvia, kronvalda blvd. 4, riga lv-1586, latvia; 2 institute of microbiology and biotechnology, university of latvia, kronvalda blvd. 4, riga lv-1586, latvia. e-mail: ilzede@hotmail.com (i. denina) the present study was focused on methods of the stringent response induction and on investigation of its effect on valine overproduction by isoleucine auxotrophs of corynebacterium glutamicum. the intracellular level of guanosine 5 -diphosphate 3diphosphate (ppgpp) increased and bacterial growth rate (µ) decreased during the short-term experiments when the exponentially growing cells were exposed to isoleucine limited conditions. the induction of the cellular stringent response resulted in a drastic increase in the activity of acetoxydroxy acid synthase (ahas), also by a significant increase in valine production. in contrast, an increase in ahas activity and valine synthesis by c. glutamicum was not achieved when bacterial growth was down-regulated in a ppgpp-independent manner. these results demonstrated that induction of the ppgpp-mediated stringent response might be significant in order to increase valine overproduction by c. glutamicum. infections with human cytomegalovirus (hcmv) continue to be an important health problem in certain patient populations, such as newborns, graft recipients of solid organs, or bone marrow and aids patients. in these groups, hcmv is a major cause of morbidity and mortality. the complex biology of hcmv necessarily begins with an initial interaction between the envelope of the infectious virus and the host cell. glycoprotein b (gb) is the major antigen, on the envelope of hcmv, for the induction of neutralizing antibodies. the region between aa 552 and 635 of hcmv gb (termed antigenic domain 1, ad-1) has been identified as the immunodominant target for the humoral immune response following natural infection. screening methods for detection of neutralizing antibodies have not been used because they are costly and labor intensive and thus far are not feasible for use on a large scale. for the development of reliable and inexpensive serodiagnostic tests the ad-1 of hcmv glycoprotein gp58, which are known to bind neutralizing antibodies, was expressed in prokaryotic systems. in this work, one prokaryotically expressed fusion protein which codifies ad-1 with ␤-galactosidase was used. the influence of different process conditions, on the production of the fusion protein containing the ad-1 as well as sugars addition to the fermentation medium was investigated. in order to analyze the expression of fusion protein, the ␤-galactosidase activity was followed throughout the fermentation. lysis process was also optimized and some final confirmation tests about protein antigenicity were performed. polyenzymic systems for the preparation of drugs based on modified nucleosides d. chuvikovsky, r. esipov, t. muravyova, a. miroshnikov shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya 16/10, moscow 117997, russia. e-mail: esipov@ibch.ru (r. esipov) considerable progress in the preparation of nucleoside analogues was achieved by combination of chemical and biochemical transformations. enzyme-catalyzed chemical transformation is now widely recognized as practical alternative to traditional organic synthesis in pharmaceutical and chemical industries. pentofuranosyltransfer reaction catalyzed by nucleoside phosphorylases was successfully employed for the synthesis of a variety of base-and sugar-modified nucleosides. enzymes involved in the metabolism of ribose phosphate and deoxyribose phosphate, such as ribokinase and phosphopentomutase, were used for the preparation of sugar-modified nucleosides. nucleosides phosphorylases (thymidine phosphorylase (tp), uridine phosphorylase (up) and purine nucleoside phosphorylase (pnp)), ribokinase and phosphopentomutase from e. coli have been cloned and overexpressed in e. coli. fast and efficient methods for the purification of nucleosides phosphorylases have been developed. the amount of purified protein was about 140 mg/l of cell culture, corresponding to 6300, 16,800 and 4200 units, respectively, of the up, tp and pnp. synthesis of medicinal drugs ribavirin (1-(␤-d-ribofuranosyl)-1,2,4-triazole-3-carboxamide), cladribine (2-chloroadenine-9-␤-d-2 -deoxyribofuranoside) and fludarabine (2-fluoroadenine-9-␤-darabinofuranoside) with the use of recombinant enzymes were studied. several important factors affecting the modified nucleosides production (ph, temperature, enzyme concentration, donor/acceptor ratio) were investigated and optimized. under optimum conditions ribavirin, cladribine and fludarabine produced in the reaction mixture in yields of 84, 85 and 80%, referred to 1,2,4-triazole-3-carboxamide, 2-chloroadenosine and 2-fluoroadenosine, respectively. aggregation and adsorption of fibroin molecules in aqueous solution won hur school of biotechnology and bioengineering, kangwon national university, chunchon 200-701, korea. e-mail: wonhur@kangwon.ac.kr fibroin, the structural protein from bombyx mori, is composed of heavy chain (generally called 'fibroin') and light chain polypeptides of about 370 and 25 kda, respectively. this study investigated the aggregation of fibroin and the adsorption between fibroin and surfaces. the variations of particle size and zeta potential were investigated by electrophoretic light scattering spectrophotometer (els). the adsorption of fibroin on surface was investigated in a continuous flow system by biacore applied surface plasmon resonance(spr) technique. the particle size and zeta potential range of aqueous fibroin were 140 nm, ±20 mv, respectively. iso-electric point(ph iep )of fibroin was ph 4.29. the amount of fibroin adsorbed on a gold surface was less than 0.1 g/ml even in the presence of high concentration of fibroin. the modification of gold surface was accomplished by applying chemicals known to form self-assembled monolayer, those are carrying nh 3 + , coo − , benzene ring and peptide that similar structure with fibroin. the adsorbed amount of fibroin on the self-assembly monolayers (sams) increased in the following order: nh 3 + > benzene ring > coo − > peptide surface. the deposition of fibroin in aqueous solution on non-waven fabric was affected by nacl and high temperature. ph influences metabolite profiling of ␤-lactamse producing b. licheniformis nazari̇leri 1 , pınar ç alık 1 , ali şengül 2 : 1 ib lab, department of chemical engineering, metu, 06531 ankara, turkey; 2 gülhane sch med, dept immunol, 06018 ankara, turkey. e-mail: e115715@metu.edu.tr (n.i̇leri) ph conditions in the bioreactor affect product and by-product formations by influencing metabolic pathways and changing metabolic fluxes, based on its influence on, i.e. dna transcription, protein synthesis, transport mechanism, atp generation and cellular energetics. whereupon, some fermentation processes favours uncontrolled-ph conditions while others favours controlled-ph conditions. on the bases of the interactions between the cell and the bioreactor through a process, carried out at either uncontrolled-or controlled-ph conditions, intracellular ph can be widely different and variable during the fermentation process. consequently, if one aims towards a quantitative understanding of the cell metabolism, one has to take into account the time variations of the intracellular ph and its effects on the in-vivo kinetics of the metabolic steps involved. moreover, since the presence of dormant or dead cells in the cultivation medium have negative effect on the synthesis of the production of desired product; the physiological state of the culture has great importance. in this context, the effects of ph on the regulation of intracellular ph, transport mechanism, and metabolic activity of b. licheniformis during production of ␤-lactamase (ec 3.5.2.6), an industrial enzyme catalyzing the hydrolysis of beta-lactam ring in beta-lactam antibiotics, was investigated. in addition, the physiological state of the organism and its effect on the production were observed. the optimal controlled-ph operation was found to be ph 6.75 with 54 u/cm 3 enzyme activity. the intracellular and extracellular na + , k + ion concentrations increased significantly throughout the process with increasing ph. on the other hand, the intracellular nh 4 + ion concentration was relatively constant. isolation and characterization of angiotensin-i converting enzyme inhibitory peptides by use of anti-peptide antibody fida hasan 1 , megumi kitagawa 2 , yoichi kumada 1 , naoya hashimoto 1 , masami shiiba 2 , shigeo katoh 1 , masaaki terashima 2 : 1 graduate school of science and technology, kobe university, nada, kobe 657-8501, japan; 2 department of human science, kobe college, okadayama, nishinomiya 662-8505, japan inhibitory peptides against angiotensin-i converting enzyme can be promising bio-functional peptides as natural alternatives for the non-peptide ace inhibitory drugs. these peptides are inactive within sequences of parent proteins and can be released during enzymatic digestion or food processing. immunointeraction is very effective for the purification of proteins and peptides with high purity. in this study, ace inhibitory peptides from hydrolysate of bonito meat were isolated by an anti-peptide antibody column and hplc, and kinetics of production of these ace inhibitory peptides was studies. an anti-peptide antibody against an ace inhibitory peptide, which was found by kohama et al. from tuna was obtained by immunization of the antigen peptide pc-iace (kkpthikwgd). water extract of bonito meat was digested at 37 • c in a modified gastric juice, 1.76 mg/ml nacl containing 312 g/ml pepsin (ph 2). peptides in hydrolysates were purified by use of an affinity column coupled with the antipeptide antibody and separated by hplc equipped with a reverse phase column (cosmosil 5c18-ms-ii, 4.6 cm × 150 cm). amino acid sequences and ic 50 values of the potent ace inhibitory peptides were determined. sds-page and western blotting experiments clarified that bonito protein contained peptides having similar sequence to the antigen peptide. a fraction of retention time 18-28 min in hplc purification samples showed high inhibitory activity, and several peptides in this fraction were separated. after 48 h digestion, two major inhibitory peptides, herdpthikwgd and pthikwgd, were found to be relatively stable in the gastric juice. kluyveromyces marxianus physiology on several levels of carbon, nitrogen sources and oxygenation during inulinase production silva-santisteban yépez, o. bernardo, francisco maugeri department of food eng./unicamp, 13081-970, campinas, sp-brazil. email: maugeri@fea.unicamp.br (f. maugeri) inulinase produced by yeasts is an interesting alternative compared with the one produced by filamentous molds, as culture conditions can be better controlled. during the assays, it was observed that inulinase production levels varied with nutritional conditions in batch culture. kluyveromyces marxianus atcc 16045 culture is described by two main phases, the first one being the growth phase, where substrate consumption and basal inulinase production were performed, and the second one being the phase where some metabolites are uptaken and high inulinase production is observed. the metabolic fluxes analyses were used to describe the cell physiology in the first phase, in a variety of conditions of sucrose and ammonium sulfate concentration and aeration condition. the metabolic network included the main metabolic pathways such as glycolisis, pentose phosphate pathway, krebs cycle, oxidative phosphorylation and biomass biosynthesis. the physiology in this phase was correlated with high inulinase production in the second phase. it was also noticed that inulinase production diminished when sucrose was in high concentration, leading, additionally, to ethanol production. in these terms, it was unveiled a kind of crabtree effect performed by this strain. forward extraction of l-aspartic acid from fermentation broths by reverse micelles and backward extraction by gas hydrate methodö. aydogan 1 , e. bayraktar 1 ,ü. mehmetoglu 1 , m. parlaktuna 2 , t. mehmetoglu 2 : 1 department of chemical engineering, faculty of engineering, ankara university, tandogan, ankara 06100, turkey; 2 department of petroleum and natural gas engineering, middle east technical university, ankara 06531, turkey. e-mail: mehmet@eng.ankara.edu.tr (ü. mehmetoglu) recently gas hydrate method has been applied as a technique for backward extraction of amino acids from reverse micelle systems. in this study, backward extraction of l-aspartic acid was investigated by gas hydrate method. at the first stage, production of l-aspartic acid was carried out using 50 ml of 0.5 m ammonium fumarate (ph 9.5) as substrate at 37 circc in an orbital shaker at 150 rpm for 4 h. e. coli (atcc 11303) was used as biocatalyst. at the end of reaction excess fumaric acid was extracted in reverse micelle phase. then forward extraction of l-aspartic acid was carried out with injection method in reverse micelle phase. for back extraction, co 2 is used to form gas hydrates crystalline structure. back extraction experiments were carried out between 35-37 bar g pressure and at 2 circc. at the end of the back extraction l-aspartic acid was obtained in crystalline form. the results indicate that recovery of l-aspartic acid from reverse micelles by forming gas hydrate can be achieved with a yield of 55.3%. consequently, gas hydrate method can be used as a new technique for backward extraction of amino acids from reverse micelles. aerobic and anaerobic cultivations of aspergillus niger on different nitrogen sources susan meijer, gianni panagiotou, lisbeth olsson and jens nielsen center of microbial biotechnology, biocentrum-dtu, technical university of denmark, dk-2800 lyngby, denmark in this study, we aim at creating a succinic acid producing strain of a. niger. a. niger is known to be a strictly aerobic organism, meaning it is not able to use the reductive part of the tca cycle to produce succinate. during aerobic growth a. niger uses oxygen as electron acceptor in the respiratory chain, thereby reoxidizing the produced nadh to nad + . however, under anaerobic conditions other compounds than oxygen, such as no 3 − are required as electron acceptor (denitrification). this process consists of no 3 − reduction to nh 4 + coupled to substrate-level phosphorylation that supports growth under anaerobic conditions. in the present study, our aim was to investigate the effect of different nitrogen sources on the physiology of a. niger during growth under aerobic and anaerobic conditions. aerobic growth experiments on three different nitrogen sources; ammonium, nitrate and nitrite, showed that ammonium and nitrate could be consumed by the filamentous fungus. nitrite on the other hand could not facilitate growth, indicating the absence of a nitrite uptake system. however, under anaerobic conditions notable growth was only observed on nitrate. these data support the hypothesis of the existence of an alternative electron acceptor that might facilitate anaerobic growth of a. niger. among the therapeutic proteins derived from mammalian cells, recombinant antibodies received a great deal of attention as a prominent product through biotech pipelines toward the marketplace. they now occupy about 25% of the estimated medicines in clinical development and many more antibodies which lead the value of the market going forward are reported. there are various environmental factors affecting rcho cell cultures such as medium components, temperature, ph, and byproducts (ammonia, lactate, and, etc.) . because most of mammalian cells are very sensitive to their environmental change, appropriate control of environmental parameters is a very important consideration to enhance cell growth and production of target proteins. balanced addition of limiting medium components plays an essential role on improvement of cell density and product concentration. temperature and ph are easily adjustable process parameters, being reported to influence cell growth and recombinant protein production. ammonia and lactate are well-known byproducts which have an inhibitory effect on cell growth when their concentrations exceed a specific level. in this work, effects of various environmental factors including temperature, ph, amino acids, vitamins, hormones, and metabolic byproducts on cell growth and recombinant antibody production were investigated in the cultivation of recombinant chinese hamster ovary cells. the most suitable condition of each environmental condition was proposed for enhancement of the cell growth and the productivity of recombinant antibody. the present study was carried out in order to assess the protective effects of calycosin-7-o-␤-d-glucopyranoside isolated from astragali radix (ar) on hyaluronidase (haase) and the recombinant human interleukin-1␤ (il-1␤) induced matrix degradation in human articular cartilage and chondrocytes. we isolated the active component from the n-butanol soluble fraction of ar as haase inhibitor and structurally identified as calycosin-7-o-␤-d-glucopyranoside by lc-ms, ir, 1 h nmr, and 13 c nmr analyses. the protective effect of arbu on the matrix gene expression of immortalized chondrocyte cell line c-28/i2 treated with haase was investigated using a reverse transcription polymerase chain reaction (rt-pcr). its effect on haase and il-1␤-induced matrix degradation in human articular cartilage was determined by using a staining method and calculating the amount of degraded glycosaminoglycan (gag) from the cultured media. pretreatment with calycosin-7-o-␤-d-glucopyranoside effectively protected against matrix degradation of the human chondrocytes and articular cartilage. therefore, it would appear that calycosin-7-o-␤-d-glucopyranoside from ar is a potential natural ant-inflammatory or anti-osteoarthritis agent and can be effectively used to protect against proteoglycan (pg) degradation. catechol-o-methyltransferase (comt) is an enzyme that catalyses a variety of endogenous and exogenous catechol substrates by transferring a methyl group from s-adenosylmethionine (sam) to either the meta-or the para-hydroxyl group of the catechol ring. the enzyme has a physiological role in the metabolism of the catechol estrogens, inactivation of the catecholamine neurotransmitters such as dopamine and epinephrine and detoxification of a variety of xenobiotic catechols. comt activity has been identified in various tissues; however with the developments in molecular biology and gene technology, the production and purification of large amounts of recombinant comt is a good option for biochemical, pharmacological and structural studies. in this work, cultures of recombinant e. coli harbouring a model plasmid were grown in a 500 ml shake-flask containing 125 ml of complex medium. the influence of medium composition and induction time on comt production, recovery and clarification by sonication, ammonium sulphate precipitation and purification by hydrophobic interaction chromatography onto a butyl-sepharose column will be presented and discussed. bioactive bacterial exopolysaccharides corinne sinquin 1 , karim senni 2 , jacqueline ratiskol 1 , farida guéniche 2 , jean guézennec 1 , gaston godeau 2 , sylvia colliec-jouault 1 : 1 ifremer, 44311 nantes cedex 3, france; 2 ea2496 université rené descartes, 92120 montrouge, france. e-mail: corinne.sinquin@ifremer.fr (c. sinquin) interest in mass culture of microorganisms from the marine environment has increased considerably, representing an innovative approach to the biotechnological use of under-exploited resources. marine bacteria associated with deep-sea hydrothermal conditions have demonstrated their ability to produce in an aerobic carbohydrate-based medium, unusual extracellular polymers (guezennec, 2002; colliec-jouault et al., 2004) . these exopolysaccharides (eps) present original structural features that can be modified to design innovative bioactive compounds and improve their specificity. with the aim of promoting biological activities, chemical modifications (depolymerization and substitution reactions) of one eps produced by vibrio diabolicus have been undertaken (raguenes et al., 1997) . the structure of the native eps has been described (rougeaux et al., 1999) the potential of the eps derivatives as therapeutical agents will be presented. physiological responses of e. coli to glucose and oxygen shifts in fed-batch fermentations jaakko soini 1 , christina saarimaa 1 , arne matzen 2 , peter neubauer 1 : 1 bioprocess engineering laboratory, department of process and environmental engineering, university of oulu, oulu fi-90014, finland; 2 sanofi-aventis, germany. e-mail: jaakko.soini@oulu.fi (j. soini) in high-cell density fermentations e. coli cells are often subjects of transient changes in microenvironment around them. these changes can be, for example, medium component gradients or differences in oxygen availability. we have studied the physiological response of e. coli w3110 cells to simultaneous oxygen limitation and overfeeding of glucose. the aim is to obtain more information of physiological changes for better understanding of the bottlenecks in such processes. the response of the cells for glucose and oxygen shifts was studied by analyzing key metabolites and proteins and mrna transcript levels. the transcript levels were measured using a sandwich hybridization technique (rautio et al., 2003) proteomic analysis was carried out by 2d-electrophoresis and the metabolite analysis by hplc. the main focus of this study is to monitor the expression patterns of marker genes involved in mixed acid fermentation, glycolytic pathway and tricarbonic acid cycle. rautio et al., 2003 . sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates. microb. cell fact. influence of ner on genetic instability of the (ctg/cag) tracts in bacterial chromosome sylwia szwarocka 1 , paweł parniewski 2 : 1 department of microbiology and immunology, university of łódź, 90-237 łódź, banacha 12/16, poland; 2 centre for medical biology, polish academy of sciences, 93-232 łódź, lodowa 106, poland many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy and friedreich's ataxia, are associated with expansions of triplet repeat sequences (trs) (cgg/ccg, ctg/cag and gaa/ttc) in or near specific genes. mechanisms that mediate the expansions and deletions of trs include dna replication, repair and recombination. many investigations suggest that the structural properties of the trs play a consequential role in their genetic instabilities. nucleotide excision repair (ner) is the major cellular system in both prokaryotes and eukaryotes and recognises damages due to distortion of the dna helix. involvement of ner in the hairpin loop repair that can form within ctg tracts has been reported. the participation of this repair systems in the trs instability was investigated in e. coli only on multicopy plasmids. the results showed that deficiency of some ner functions dramatically affects the stability of long (ctg/cag) inserts. in this work we present a chromosomal model to study the instability of the trs in e. coli. we introduced the (ctg/cag)n tracts into the chromosome of e. coli and used strains with some deficiency of the ner and investigated genetic stability of these tracts after multiple recultivations. in general, our results show that the (ctg/cag)n repeats are much more stable in the chromosome than in plasmids. these data may suggest that instability of trs in plasmids is associated with interaction between repetitive tracts on different plasmid molecules inside the cell. however, mutations of ner genes may increase (uvra and uvrb mutants) or decrease (uvrc and uvrd mutants) stability of the trs in the e. coli chromosome. this study was partially funded by the kbn grant 2 p05a 01927. performance analyses of a multi-stage integrated fermentation process for lactic acid production hsun-tung lin, feng-sheng wang department of chemical engineering, national chung cheng university, chia-yi 621-02, taiwan. e-mail: chmfsw@ccu.edu.tw (f.s. wang) in this work, we considered a multi-stage integrated continuous fermentation process for producing lactic acid. each stage consists of a mixing tank, a fermenter, a cell recycle unit and an extractor. the generalized kinetic model is first applied to formulate the integrated process. we have compared the overall productivity and conversion of the integrated process with those of two simplified processes. from the design equations, we obtain that three processes have the identical overall conversion. however, the proposed process has the greatest overall productivity. the specific kinetic model for lactic production (youssef et al., 2000) was applied to the integrated process in order to find the maximum overall productivity. two optimization problems are respectively considered to determine the optimal stages, operating conditions and design variables. the first problem supposes that the integrated process has the equal working volume ratio for each fermenter. such a process requires four stages to yield the maximum overall productivity and the nearly complete overall conversion. however, if the working volume ratio for each stage is considered as the decision variables in the second optimization problem, three stages is enough to achieve the identical overall productivity. youssef, c.b., guillou, v., olmos-dichara, a., 2000. contr. eng. pract. 8, 1297-1307. modelling of the binding of ligands to macromolecules jørgen m. mollerup department of chemical engineering, building 229, dtu, 2800 lyngby, denmark a variety of factors that govern the properties of proteins are utilized in the development of chromatographic processes for the recovery of biological products including the binding and release of protons, the non-covalent association with non-polar groups (often hydrophobic interactions), the association of small ions (ion exchange) and the highly specific antigen-antibody interaction (affinity interactions). the fulcrum point in the understanding and modelling a chromatographic separation is the adsorption isotherm that determines the peak shape at preparative load. to enable an efficient chromatographic process development strategy it is necessary to conduct theoretical and experimental investigations of the adsorptive behaviour of proteins. thermodynamically consistent models for ion exchange chromatography and hydrophobic interaction chromatography have been developed and can be utilised in the simulation of a chromatographic separation. besides, measurements on hic media can be utilised to determine the cohn salting-out coefficient. the lig-and binding process can frequently be coupled to associated structure changes in the protein, the ligand or both. this gives rise to nonlinear adsorptive behaviour known as cooperativity which cannot be modelled using conventional models which displays convex behaviour. examples of cooperative behaviour are the reversible binding of oxygen and carbon monoxide to haemoglobins and the binding of nad + to yeast glyceraldehydes 3-phosphate dehydrogenase. in the paper we discuss the modelling of reversible binding of mobile as well as immobilised ligands to macromolecules and compare modelling to experiment. comparative analysis of the temperature policy for processes with a deactivating native enzyme i. grubecki, m. wojcik department of chemical and biochemical engineering, university of technology and agriculture, 85-326 bydgoszcz, ul. seminaryjna 3, poland a comparative analysis of the temperature policy for an enzymatic reaction with michaelis-menten kinetics in a batch reactor has been carried out. both isothermal and optimal temperature policies for processes with deactivating native enzyme have been considered. in the model, the thermal deactivation was described by a first-order reaction, and the arrhenius-type dependence between rate parameters and temperature was assumed. as an indicator for a direct comparison between the isothermal and optimal temperature policies the quotient of conversions under identical initial and final condition was used. a method was presented to calculate this indicator, which is based on the analytical and numerical solutions. this method can be of great importance for the industrial practice. application of changeable temperature policy could result in significant increase in conversion when ratio of activation energy for deactivation and activation energy for reaction is high. studies on the impact of mixing during brewing using near and mid-infrared spectroscopy georgina mcleod 1 , alvin w. nienow 1 , graham poulter 2 , reg wilson 3 , henri tapp 3 , christopher j. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w3110). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. template refolding utilizing biospecific interactions shigeo katoh 1 , yoichi kumada 1 , nanae maeshima 1 , daisuke nohara 2 : 1 graduate school of science and technologykobe university, kobe 657-8501, japan; 2 department of biomolecular sciencegifu university, gifu 501-1193, japan. e-mail: katoh@kobe-u.ac.jp (s. katoh) recombinant proteins over-expressed in e. coli are often accumulated as insoluble particles called inclusion bodies. proteins in inclusion bodies must be solubilized by a denaturing agent, such as urea and guanidine hydrochloride, and refolded to recover their native structures having biological activities. in bioprocesses it is important to obtain high refolding efficiencies and high throughputs at high protein concentrations. in refolding operation, a denatured protein solution is usually added batch-wise into a large volume of a refolding buffer in order to start refolding by reducing the concentration of a denaturant and to prevent aggregate formation of renaturing molecules. thus, a large volume of a stirred tank is required, and the concentration of protein after renaturation becomes low. biointeractions between a pair of biomolecules, such as enzyme-inhibitor, antigen-antibody and hormone-receptor, are highly specific and have been used for detection and separation of biomolecules. these interactions may be used as templates for refolding of target molecules, which can be captured with the templates and are prevented from aggregate formation and, in the case of proteases, from autoproteolysis. the specific interaction might promote refolding of the target molecules. these might improve the refolding efficiency. the biointeractions between antigen-antibody and enzyme-inhibitor were used for efficient refolding in packed columns, in which template ligands (antibody, inhibitor) were coupled on gel support. denatured solutions of target molecules (carbonic anhydrase and s. griseus trypsin) were mixed with refolding buffer and supplied to the affinity column coupled with the template ligands for refolding. with refolding in the column, higher refolding efficiencies were obtained than those by the batch dilution method with relatively low concentrations of denaturants. by increasing the adsorption capacity of the column, throughput of refolding can be increased without decrease in the refolding efficiency. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic 1 , alvin w. nienow 1 , ian w. taylor 2 , ryan hicks 2 , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. carbon sources create fingerprint fermentation characteristics pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 1 bre lab, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 ib lab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e., glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e., serine alkaline protease (sap; ec 3.4.21.62),) and ␤-lactamase (ec 3.5.2.6), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. 1.5-2-fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. kinetic resolution of racemic benzoin with different lyophilized microorganisms ç . babaarslan 1 ,ü. mehmetoglu 1 , a.s. demir 2 : 1 ankara university, faculty of engineering, department of chemical engineering, 06100, ankara, turkey; 2 middle east technical university, department of chemistry, 06531 ankara, turkey. e-mail: barslan@eng.ankara.edu.tr (ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the 2-hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at 30 • c and 150 rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs 112-07 as ee s = 16% and ee p = 30% (conversion = 35%) using thf as solvent and vinyl acetate as acyl donor. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box 6121, campinas, cep 13083-862, são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during 3, 5 and 7 days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the mediun was composed by, cwb:cr were mixed with distilled water and transferred into 250 ml capacity erlenmeyers flasks and autoclaved at 120 • c for 20 min. the medium was then inoculated with spores (5.0 × 10 7 ) and the flaks were incubated at 32 • c. tannase was assayed according to the methodology of mondal et al. (2001) . acording to the statist analyses, the optimum conditions to produce tannase was the range of temperature (29-34 • c); tannic acid (8.5-14%); residues % (coffe: wheat bran) (50:50) and 5 days fermentation time. the enzyme production increased 8.6 times more enzyme production than that was obtained before this optimization. how to cope with fda's pat-initiative with respect to fermentation process monitoring and control marco jenzsch 1 , andreas luebbert 1 , rimvydas simutis 2 : 1 institute of bioengineering, martin-luther-university halle-wittenberg, halle (saale), germany; 2 process control department, kaunas university of technology, kaunas, lithuania with its pat initiative, fda forces drug manufacturers to increase their activities in innovative manufacturing techniques, and, more than previously, to focus on quality assurance. the agency particularly places emphasis on making use of modern process supervision and control techniques such as up-to-date process analytics, multivariate data acquisition and analysis tools in order to improve process monitoring and control. in this contribution we show by means of practical examples how this guidance can be applied to cultivations of genetically modified microorganisms. a comparison of different multivariate state estimation techniques will be presented and compared with more knowledge-based techniques such as the extended kalman filter. the comparison was made for the model system gfp expressed from e. coli bacteria (bl21/de3/gfp) for which more than 40 full data sets are available. all these techniques have already been used during real protein formation at productionscale fermenters, with the same success. hence, the requirements expressed in the pat initiative can immediately be put into practice. feedback control of the recombinant protein production processes based on such estimations is show for several cultivation systems. simple parameter adaptive controllers are compared with model supported controllers, for instance, generic model controllers and model predictive controllers. the results clearly show that we have at hand a rather extended arsenal of feedback control procedures that can be used successfully to tightly control the processes even along set-point profiles of physiological variables such as the specific growth rate (µ). again, fda's suggestion with respect to "control in the engineering sense" can be applied immediately to reduce batch-to-batch variances and thus to increase process quality. extending life by alternative respiration? alexander kern, franz hartner, anton glieder institute of biotechnology, graz university of technology, a-8010 graz, austria. e-mail: a.kern@tugraz.at (a. kern) alternative oxidase transfers electrons directly from the ubiquinol pool in mitochondria to oxygen, allowing cell respiration in presence of complexs iii and iv inhibitors like antimycin a or cyanide. electron transfer by alternative oxidase is not coupled with proton transfer across the mitochondrial membrane, thereby uncoupling the supply of small metabolic intermediates by the central metabolic pathway from energy production in the cell. alternative oxidase is present in mitochondria of plants, many fungi and a few, mostly crabtree-negative yeasts, but not in p. angusta (hansenula polymorpha) and s. cerevisiae. alternative oxidase has multiple functions in different organisms. it is involved in stress answers, in programmed cell death, maintenance of the cellular redox balance, and also citric acid accumulation in a. niger. we isolated the alternative oxidase gene from the methylotrophic yeast p. pastoris in order to study its effects on the cellular energy content, respiratory activity, its protective role against oxidative stress. our results indicate the importance of an exact regulation of the alternative oxidase due to its impact on many cellular functions. new types of energy efficient fermenters with better mass transport, mixing and cooling properties than the current crop of rushton turbine derived tank bioreactors are likely to be required in the future. such fermenters will be needed in order to meet the increasing pressure on costs for low price commodity type products such as single cell protein or food and technical grade enzymes, and to meet the demands of the new wave of white biotech, in which bio-produced chemicals must be made at prices competitive with those of the traditional chemical industry. with this in mind, a prototype pilot scale (500 l) u-loop fermenter has recently been commissioned at biocentrum-dtu. in this fermenter, liquid circulation is driven by a propeller pump through a vertical u-shaped pipe, which is connected at the top with a de-gassing tank. we present here a study of liquid mixing and dispersion in the prototype u-loop fermenter. sub-sequently we show that the results can be described with the tanks in series model. mixing was characterised using pulses of nacl tracer, which were detected with a conductivity probe in various parts of the fermenter. bodenstein numbers (bo) were determined for flow rates corresponding to a linear fluid velocity of 1.05 m/s in the 'legs' of the reactor and showed that the majority of the mixing occurred in the top degassing part (bo = 29) rather than in the u-loop section (bo = 422). it was also observed that the time for mixing to 90% homogeneity after tracer pulse addition was a function of the number of cycles through the reactor (3-3.5) within the range of flow velocities (u) studied (u = 0.41 m/s to u = 1.74 m/s). the mixing time to 90% homogeneity was between 44.6 s (at u = 1.74 m/s) and 181 s (at u = 0.41 m/s). today many biotechnological processes are operated at suboptimal conditions and according to best practice. however, the current industrial development is towards analyzing more parameters and in particular there is a large interest in analysis of biological/biochemical variables. the quality of products and also the possibility to optimize production in submerged cultivations would be greatly enhanced if more on-line/real-time information were at hand. the present investigation was undertaken with the aim of evaluating the potential in using multi-wavelength fluorescence for monitoring and control of filamentous fungi fed-batch cultivations. a recombinant a. oryzae expressing a heterologous lipase was applied as model system. spectra of multi-wavelength fluorescence were collected every five minutes with the bioview ® system (delta, denmark) and both explorative and predictive models, correlating the fluorescence data with the important biological parameters cell mass and lipase activity, were built. the models will be presented, furthermore, advantages and disadvantages of multiwavelength fluorescence for monitoring of cultivation processes will be discussed. moving from r&d to pharmaceutical development is a costly process. it is therefore of paramount importance to design a manufacturing process that combines robust and well-documented technological platforms. therapeutic recombinant proteins designed for human administration should be as close to the authentic product as possible. here, the use of a scalable process and an economically sound affinity tag can be a relevant choice. the tagzyme tm system has been designed to allow for the precise removal of amino terminal affinity tags. the system is based on the use of recombinant aminopeptidases including dipeptidyl peptidase i (dapase tm ). dapase tm is currently produced under cgmp providing a suitable strategy for its use in pharmaceutical production. the tagzyme tm system is superior to other methods since: (1) it is based on exopeptidases, precluding, e.g., unspecific protein cleavage reported when using so-called site-specific endoproteases. (2) it has been tested for production of more than 200 recombinant proteins. (3) it is easily scalable from lab scale to kg of processed protein. (4) it allows the use of his-tags for commercial production without patent infringment, due to our ipr position. (5) the commercial use of tagzyme tm does not require any licensing, only purchase of the enzyme(s). (6) the use of aminopeptidases for pharmaceutical production has been extensively documented for approved drugs. (7) a number of therapeutics is currently being developed using tagzyme tm . (8) unizyme can assist in the optimization of the dsp to enable further cost reduction in the process. these aspects will be discussed and illustrated in the presented poster. website sphingolipids are biologically active molecules involved in the regulation of a large quantity of biological responses. they function in cell proliferation, survival and death (apoptosis) as messengers. dysregulation of apoptosis has significance in numerous pathological conditions including cancer. several anticancer agents act by increasing tumor cell ceramide (a kind of sphingolipid) content. so, a novel approach to cancer therapy would be the pharmacological manipulation of sphingolipid metabolism. in this study, sphingolipid metabolism in baker's yeast s. cerevisiae is used as a model system as many of its sphingolipid related genes and proteins have been characterized. gepasi-biochemical kinetics simulator was used for metabolic control analysis (mca) of the above-specified system. the concentration control coefficients (ccc), flux control coefficients (fcc) and elasticity coefficients were calculated, and their significance in identification of anticancer drug targets is determined. elementary flux modes were also identified and metabolic pathway analysis (mpa) was performed. quantitatively, control effective flux (cef) values were used for potential drug target identification. the results from mca and mpa indicate almost the same potential drug targets: serine palmitoyl transferase, ceramide synthase and ceramidase. drugs against these targets are in preclinical and clinical development. for the identification of new potential drug targets, the cccs, fccs, cefs and elasticity coefficients were examined with an objective function of maximizing the cell ceramide concentrations. it was found that manipulation of inositol-1-phosphate synthase and phosphoinositide kinase activities have considerable effects on ceramide concentrations. if a drug targeting the two enzymes at the same time is designed, it might give a better outcome in terms of cancer therapy. in recent years, there is a growing interest in utilization of airlift reactors (alrs) to biotechnological processes. nevertheless, their industrial application still remains limited because of a lack of reliable studies on transfer phenomena and mixing enabling a suggestion of suitable scale-up procedure. the way to more widely utilization of alrs to biological processes lies in experimental research (on a model medium as well as on a real fermentation medium) followed by mathematical modelling and scaling-up of the processes. this paper deals with a modelling of a glucose-gluconic acid fermentation by a. niger in an internal loop airlift reactor. knowledge of the stoichiometric relationship in the key reaction provides a good opportunity for estimation of substrate and product concentration. the model is based on material balance equations and has been adjusted to experimental data obtained from three internal loop airlift reactors (10.5, 40 and 200 l) . in the model, the alr is divided into ideal stirred tanks in series. in each zone (tank) of the alr the material balance is calculated in two phases (the gas and the liquid phase). this work was supported by the slovak scientific grand agency, grant number vega 1/0066/03 alkaline phosphatase (ap, e.c. 3.1.3.1) is a thermolabile enzyme which is indigenous to all dairy products. it has an inactivation temperature slightly above the value that is required to destroy the most resistant pathogenic microorganism likely to be found in milk. due to that feature, this enzyme is used as an indicator of proper pasteurization. the effect of temperature treatment on the activity of ap was investigated in raw cow's and goat's milk. the stability of alkaline phosphatase in raw milk was compared with the stability of this enzyme in a 0.1 m potassium phosphate buffer with ph 6.6. the ph value of the buffer was approximately the same as that of raw milk. the inactivation curves were measured in the temperature range from 54 to 69 • c. ap in cow's milk was completely inactivated at 69 • c during 60 s but approximately 30% of activity remained at 54 • c after 100 min of treatment. the time required for a complete inactivation of the enzyme in the raw cow's milk was reduced from 90 to 1 min as the temperature increased by 11 • c. heat treatment of goat's milk caused the decrease of activity of the enzyme in the same temperature range as in the case of cow's milk. the increase of temperature from 58 to 68 • c reduced the inactivation time from 35 min to 40 s. the study of thermal stability of the alkaline phosphatase in the buffer solution showed that the time required for inactivation of enzyme was significantly shorter than in milk. milk thus had a protective effect on the activity of alkaline phosphatase. the experi-mental curves were fitted simultaneously using kinetic models where the initial heating period was considered. this work was supported by a grant of 6th framework program of eu, project foodpro, no. sme-2003-1-508374. during the process of separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale, the chromatographic unit operations have an important role. three different protein-binding modes are employed: ion-exchange, hydrophobic and affinity binding. two adsorbent properties are of uppermost importance: a high selectivity and adsorption capacity. in the case of ion-exchange/hydrophobic chromatography, the binding of charged proteins can be affected by ph and ionic strength. in this work, the adsorption capacity of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros 50a, prosep-va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was measured as a function of ph. as a model mab and contaminant proteins, human immunoglobulin (igg), human serum albumin (hsa) and horse skeletal muscle myoglobin (myo) were used. the resin properties were investigated within the range of ph 4-8. the experiments were conducted in a batch-mode, individually for each model protein. the results showed that ion-exchange and hydrophobic resins provided the best selectivity for igg at ph 6. the selectivity of affinity adsorbents was essentially unaffected by ph, however, the highest capacity for igg was at ph 7. another investigated aspect was the dynamics of protein binding. the solution of individual protein in contact with tested adsorbent was circulated through an uv spectrophotometer, what enabled the measurement of time-dependent decrease of protein concentration. the results indicated that affinity adsorbents with a rigid matrix needed approximately four times shorter time to reach the adsorption equilibrium with igg in comparison with a gel. the gels, however, provided higher adsorption capacity. at ion-exchange resins, the time necessary to adsorb 99% of total amount of igg was about 0.5-2 h. the affinity adsorbents were highly selective and therefore they adsorb very small amount of tested contaminant proteins (hsa, myo). the adsorption capacity was saturated by 50% in less than 25 min in all cases of dynamic adsorption measurements. this work was supported by a grant of 6th framework program of eu, project aims, no. nmp3-ct-2004-500160. microtechnology has for several years been applied within chemical reaction engineering. the advantages of microtechnology are that it makes it possible to develop light weight and compact systems, and the systems enable large surface-to-volume ratio, which results in low mass-transfer distances. in addition, parameters like pressure, temperature, residence time, and flow rate are more easily controlled. the use of microtechnology is also beginning to find its ways into the field of biotechnology. what we are aiming at is the development of a microreactor that can be applied as a production tool in industry as an alternative to conventional enzymatic reactors. our strategy is to use a small plate of a suitable material with microchannels fabricated into its surface, and the approach is to covalently couple enzymes into the microchannels. substrate can then be pumped through the channels and the enzymatic conversion will take place within the channels. as model enzyme in the development of the microreactor we are applying celb, a thermostable ␤-glycosidase from pyrococcus furiosus. kinetic resolution of racemic benzoin with different lyophilized microorganisms ç . babaarslan 1 ,ü. mehmetoglu 1 , a.s. demir 2 : 1 ankara university, faculty of engineering, department of chemical engineering, 06100, ankara, turkey; 2 middle east technical university, department of chemistry, 06531, ankara, turkey. email: barslan@eng.ankara.edu.tr (ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the 2-hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at 30 • c and 150 rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs 112-07 as ee s = 16% and ee p = 30% (conversion = 35%) using thf as solvent and vinyl acetate as acyl donor. chromatography is one of the most important unit operations at separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale. since these proteins belong to the group of immunoglobulins, their molecular weight (about 150,000 g/mol) or hydrodynamic radius (10.7 nm), respectively, is relatively large. the adsorbents used in ion-exchange/affinity chromatography of these biomolecules should thus provide a high pore accessibility coupled with a high value of specific surface area in order to ensure a sufficient ligand density and a high binding capacity. in this study, the pore accessibility of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros 50a, prosep -va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was investigated via size exclusion of standard-sized molecules (glucose, sucrose and dextrans with molar weight range 1200-40 × 10 6 g/mol) at nonbinding conditions. the experiments were conducted in a batch and column-mode. the batch experiments provided absolute partition coefficients, which were calculated from a mass balance and represent the fraction of pore water accessible to a solute. it was found that several adsorbents contained a small fraction of very small pores (less than 1 nm), whereas some adsorbents contained a significant fraction of pores larger than 100 nm. the column measurements provided relative partition coefficients, which were calculated from the retention volumes of solutes and represented the relative accessibility of pores, scaled between the accessibility of the smallest and largest solute used. when the absolute partition coefficients were recalculated into the relative form, it was found that the coefficients obtained by both methods correlated very well. the relative partition coefficients of solutes with the hydrodynamic radius of 10 nm (corresponding to mabs) was about 0.2-0.4 at ion-exchange and hydrophobic adsorbents and 0.6-0.8 at affinity adsorbents. the relation between hydrodynamic radius of the solutes and their partition coefficients was successfully described with the giddings random plane model. this work was supported by a grant of 6th framework program of eu, project aims, no. nmp3-ct-2004-500160. the transfer of laboratory results to a larger scale is often a critical step in process development to industrial application. the objective of this study was to scale-up the bioreactor for the fructosyltransferase production based on the results obtained in a 3 dm 3 stirred bioreactor. the investigations made in this bioreactor provided a clear picture of the effect of medium composition on the obtained fructosyltransferase (ftase) activity but the influence of mixing intensity was unequivocal. the increase of agitation rate had a positive effect up to a certain level where both fructosyltransferase and biomass production increased. the final optimal yield factor of ftase per dry cell mass obtained in the laboratory bioreactor was 10,300 u g −1 . we studied the effect of oxygen transfer on the process of ftase production at a larger scale, in 12 and 100 dm 3 mechanically stirred bioreactors and in air-lift bioreactors, 20 and 60 dm 3 whilst the medium composition was kept constant. the yield factors of ftase were comparable in both mechanically stirred bioreactors and they were about 7500 u g −1 . this decrease compared to that in the laboratory bioreactor could be explained by a slower cell growth. this fact was also confirmed by that glucose was not depleted till the end of fermentation and free fructose concentration was also lower. the yield factor of ftase was 7400 u g −1 in the 60 dm 3 air-lift reactor and 6100 u g −1 in the 20 dm 3 air-lift bioreactor. the lower yield of ftase in 20 dm 3 bioreactor was caused by a better biomass growth. this work was supported by slovak scientific grand agency, grant numbers vega 1/0066/03 and 1/0065/03 and by science and technology assistance agency, grant number apvt-20-025704. the poster gives an overview of the objectives and achieved results of an interdisciplinary project on direct product isolation from crude feedstocks using magnetic micro adsorbents in combination with suitable magnet technology. the project was funded by the deutsche bundesstiftung umwelt and was running between august 2002 and november 2004. in the course of the project several milestones could be met, which can be looked at as critical key points on a route towards an industrial realization of the process. among these milestones are: (i) the production of magnetic micro adsorbents with high capacity and selectivity in batches up to 50-100 g; (ii) the proof that the micro adsorbents can be reused many times; (iii) generation of a variety of recombinant tagged, active enzymes; and (iv) the design, assembly and operation of a fully automated pilot plant capable of generating approx. 5 g/h (≈95% purity) protein. the process was also simulated by help of the software tool superpro designer and simple mass balance and sorption equilibrium approaches were used to derive rules for estimating optimum process parameters and productivities. finally an environmental performance evaluation was conducted externally by the german dechema. in this study the effect of fed-batch cysteine addition to a culture of a high-gsh-accumulating yeast strain on the metabolism of glutathione was investigated. it is known that cysteine is the rate limiting amino acid in the biosynthesis of gsh. the influence of the consumption rate of cysteine on glutathione metabolism and growth of s. cerevisiae mt-32 was determined. the results show that for rates of consumption below a critical value the microorganism growth is similar to a culture without feed of cysteine, but glutathione production is increased two-fold. on the other hand, if cysteine consumption rate is above the critical value the changes of cell metabolism implies ethanol accumulation in the extracellular media which diminishes biomass synthesis. the maximum specific glutathione production in this case is maintained at two-fold; however, gamma-glutamylcysteine accumulation is increased. cysteine present in culture media directs cell metabolism to a greater synthesis of ammonia and amino acids. hydrophobic interaction chromatography (hic) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. in contrast to reversed phase chromatography this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. when proteins are injected on hic columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. the higher the salt concentration the lower is the amount eluted protein. the rest can be desorbed with a buffer of low salt concentration or water. it has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. this has been also corroborated by physicochemical measurements. the retention data of five different model proteins and 10 different stationary phases were evaluated. partial unfolding of proteins upon adsorption on surfaces of hic-media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. furthermore we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain. stationary phases for bioseparation of glycoproteins j. aniulyte 1 , j. liesiene 1 , b. niemeyer 2 : 1 department of chemical technology, kaunas university of technology (ktu), radvilenu pl. 19, 50254 kaunas, lithuania; 2 institute of thermodynamic, helmut-schmidt-university/university of the federal armed forces hamburg, holstenhofweg 85, hamburg, germany d-22043 nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated.three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of 18 atoms) and carbonyldiimidazole activation.cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg per ml support and a high recovery (up to 93%). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affinity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately 1 × 10 −6 m, and 0.4 × 10 −5 m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. cell disruption and chromatography are key unit operations in the downstream processing of an intracellular product. the cost involved in the extraction and purification of intracellular products can be reduced by selective release of proteins and reduction in the number of steps involved in the purification. the extent of disruption can be varied to provide a selective release, limiting the release of the contaminant proteins. the particle size distribution of the cell debris in the resulting suspension depends on the extent of disruption. expanded bed adsorption chromatography allows for the direct capture of the proteins from an unclarified suspension. this technique allows for the integration of solid-liquid separation, concentration and preliminary purification in one unit operation. a perfectly stable expanded bed can be obtained by choosing the appropriate flow conditions and a suitable adsorbent. the difference in the density between the adsorbent and the cell debris in the suspension, permits the cell debris to flow through the column without blocking, whilst the protein molecules in the suspension are adsorbed onto the adsorbent. after sample application, the bed is washed with buffer and the proteins eluted from the column in the packed bed mode. the presence of the cell debris in the feedstock influences the expansion of the bed and the adsorption of protein molecules. the physical properties of the suspension obtained after cell disruption depends on the extent of disruption. the particle size distribution of the cell debris, the viscosity and the release of soluble proteins and other intracellular components are influenced by the extent of disruption. the influence of the extent of disruption of e. coli on the expansion of the bed and the adsorption of ß-galactosidase is presented in the current study. e.coli cells were disrupted at different operating pressure using a high pressure homogenizer. the resulting crude homogenate is subjected to expanded bed adsorption chromatography using streamline deae as adsorbent. the disrupted suspension was characterised in terms of viscosity, density, particle size distribution of the cell debris and the extent of protein and ß-galactosidase released. the interaction between cell debris and adsorbent was quantified as the cell transmission index (ratio of the amount of cells present in the sample before and after passing through the bed). the expansion of the bed at a constant settled bed height and flow rate was measured. the influence of the cell debris on the extent of adsorption of ß-galactosidase has been quantified in terms of dynamic binding capacity (dbc) at 10% of the inlet concentration. the dbc of ß-galactosidase that was released by disruption at 7500 psi (5%, w/v, w/w, 1 pass) was found to be 100 u/ml of adsorbent while dbc of samples disrupted at 2500 psi (5% w/v, w/w, 1 pass) was 67 u/ml of adsorbent. the extent of disruption of e. coli over a wide range and its effect on the expansion and adsorption will be presented. study of dna binding during expanded bed adsorption and factors affecting adsorbent aggregation ayyoob arpanaei 1 , niels mathiasen 1 , timothy hobley 1 , owen rt thomas 1,2 : 1 center for microbial biotechnology, building 223, biocentrum-dtu, technical university of denmark, 2800, lyngby, denmark; 2 department of chemical engineering, university of birmingham, edgbaston, b15 2tt, uk. e-mail: aa@biocentrum.dtu.dk (a. arpanaei) the adsorption of sonicated calf thymus dna (as a model dna molecule) to biosepra q hyper z adsorbents was evaluated in batch and expanded bed modes. stability of the expanded bed during feedstock loading was also studied. two batches of prototype q hyper z (batch 1 and 2) were examined, which had ionic capacities measured to be 122 and 147 mmol cl − /ml support respectively. in all adsorption experiments a 50 mm tris-hcl ph 8 buffer was used. maximum static binding capacities of adsorbent batches 1 and 2 were determined to be 20.9 and 23.8 mg dna/ml particle, respectively. dynamic binding capacity at 10% breakthrough (dbc 10% ) was measured in a 1-cm diameter eba column containing 6.7±0.5 cm settled bed with a feed of 20 g/ml dna. dbc 10% of the adsorbents were 7.4 and 12.7 mg dna/ml support for batches 1 and 2, respectively in buffer containing no salt. however, the maximum dbc 10% for batch 1 (10.1 mg dna/ml support) and 2 (18.9 mg dna/ml support) were obtained in buffers containing 0.25 and 0.35 m nacl, respectively. further increases in salt concentration led to a decrease in dbc 10% for both adsorbent batches. the bed compression during loading that was observed in experiments at high conductivities (achieved by adding salt) was less than that seen with low conductivity (2 ms/cm) solutions. aggregation of adsorbent particles and channeling of flow were not observed in the presence of salt concentrations more than 0.1 m. the effect of different concentrations of dna during loading in the presence of 0.15 m nacl was studied. it was found that increasing dna concentrations in the feed from 20 to 40 g/ml, 60 to 80 g/ml resulted in a decrease of dbc 10% by 16, 24 and 30%, respectively. the bed compressed slower during loading of feedstock with low dna concentrations compared to that for higher concentrations. the expanded bed showed a partly reversible compression behavior during feedstock loading. this is attributed to the electrostatic interaction between dna adsorbed on the particles surface and rearrangements of dna strands as the number of free ligands on the adsorbent surfaces decrease during loading. large-scale production of plasmid dna for gene therapy and dna vaccination applications has become necessary as a result of the increasing number of approved protocols using non-viral vectors for gene delivery. a major challenge of large-scale plasmid production is to establish a robust cgmp manufacturing capable of producing hundreds of milligrams or grams of a pharmaceutical grade product. alcohol and salt precipitation are operations largely used in the early steps of plasmid downstream processes. however, there are few systematic studies on the influence of these precipitation agents in the final plasmid recovery and purity. in this work, alcohol and salt precipitation steps used in a plasmid purification process developed by our group have been optimized aiming at large-scale production. the optimization of alcohol precipitation indicated that almost 100% of the pdna precipitated when 0.6 vol. of isopropanol were used. the studies also indicated that the precipitation profile was strongly influenced by pdna initial concentration. finally, the final plasmid recovery and purity after a sequential alcohol and salt precipitation were strongly dependent on the concentrations of these precipitation agents. thus, a commitment between high recovery and purity level should be made during the development of the downstream processes. comparison of novel and conventional processes for protein refolding and initial purification h. ferré 1,2 , u. jørgensen 1 , l. scale down of downstream processing unit operations is convenient for assessing process alternatives, particularly if feedstock is scarce. in this study it was imperative to use the smallest possible scale for comparison of a new system for continuous protein refolding and direct expanded bed adsorption (eba) capture with a traditional process composed of discrete operations of batch renaturation, centrifugation, microfiltration and packed bed chromatography (pbc). minimisation of the scale was restricted by the eba step: the smallest practical scale being a 1cm diameter column with 5-6 cm of settled bed, expanded two fold. in order to permit a fair comparison a similar column diameter and adsorbent volume was used in the packed bed process. in both alternatives, chelating media charged with cu 2+ was used and a feedstock of denatured hat-tagged human beta-2 microglobulin (hat-h␤2m). following batch refolding and clarification, the performance of the packed column was severely hampered due to fouling of the top adapter. reducing the protein loaded to the packed bed to 50% of dbc working lead to a recovery of 68.5% at a purity of 87% and 4.6-fold concentration. the eba-based process performed unimpeded and productivity was calculated to be 8% higher than for that employing a packed bed. however, due to the severe scale restrictions placed on the eba process, which limited optimisation, significant productivity improvements of eba over packed bed are expected at larger scale. high gradient magnetic filtration has the potential for rapid processing of large volumes of crude bioprocess liquors when magnetic adsorbents are employed. the binding of a protein to a superparamagnetic solid support provides a unique selective 'handle'. typically the focus is placed on using the magnetic handle for direct capture of a protein from a fermentation broth. however, magnetic adsorbents may provide solutions to a range of downstream processing problems and in this presentation we illustrate this with a number of case studies. using whey as a model system, we show that the extent of the tryptic hydrolysis (ca. 0.2 mg/ml added enzyme) of proteins could be controlled by adding benzamidine-linked magnetic adsorbents after a given period (4-15 min), followed by removal of the loaded adsorbents using a magnetic filter. hydrolysis was stopped effectively and approx. 50% of the added trypsin could be recovered. a coupled process was devised for the refolding and purification of inclusion body proteins. solubilised (in 8 m urea) inclusion bodies of recombinant histidine affinity tagged human beta 2 microglobulin (hat-h-beta2m), were refolded by dilution in a pipe reactor (14 s), then captured directly on cu(ii) charged magnetic immobilised metal affinity adsorbents in a second pipe reactor (10 s residence time). loaded adsorbents were retained in a magnetic filter, then washed and the protein eluted. a generic framework for the prediction of scale-up when using compressible chromatographic packings r. tran 1 , j. joseph 1 , a. sinclair 2 , y. zhou 1 , n. packed bed chromatography is the pre-eminent technique in the downstream purification of many biological products. the aspect ratio of a packed bed has a significant effect on the column pressure drop by virtue of wall support which is reduced at low aspect ratios. this can result in unexpectedly high pressures during manufacturing caused by the compression of the matrix via drag forces due to fluid flow through the bed. the need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation so that maximum throughput can be achieved. several studies have generated correlations which allow for the prediction of column pressure drops but have either been mathematically complex, which makes their practical use unfeasible, or they have used highly specific empirical constants and hence require a large amount of experiments to be performed before they can be used. in this study, we have established relationships to link the critical velocity of operation, to bed height (l), column diameter (d c ), feed viscosity (µ) and also to the matrix rigidity through the level of agarose cross-linking (a%). the correlation is straight forward to use and involves very few system-specific constants thus significantly reducing the need for any preceding laboratory-scale experimentation. this paper describes the series of experiments that were performed to establish the correlation, using a range of cross-linked agarose matrices (2-10%), at various aspect ratios, fluid flow rates and varying viscosities (0.9-1.85 mpas). a mathematical model was developed where parameter estimation for multi-variables was achieved by least squares optimisation. the model can be used to predict the extent of compression in industrial chromatography applications and will be useful in the development of chromatographic operations and for column sizing. institute of process engineering, swiss federal institute of technology, zurich. e-mail: makart@ipe.mavt.ethz.ch (s. makart) simulated moving bed (smb) technology receives increasing attention in biotechnology and in the biopharmaceutical industry as it enables an increase in productivity per unit mass of stationary phase, reduced solvent consumption, and fast and reliable scale-up. combining continuous chromatographic separation unit and reactor should enable the production of biopharmaceuticals and fine chemicals with high purity and yield at the same time. due to the increasing demand of enantiopure intermediates in the pharmaceutical industry, biocatalytic processes gain more and more importance because of their excellent enantioselectivity. yet the application of biocatalytic carbon-carbon bond formation on process scale is often hampered by an unfavourable equilibrium position and difficult downstream processing due to substrate/product mixtures. coupling a continuous separation unit to such a process would improve the feasibility by driving the reaction to completion and thus increasing the overall yield. we will discuss the design of such an integrated biocatalytic/smb process, taking the formation of l-allo-threonine from glycine and acetaldehyde, catalysed by the glycine-dependent aldolase glya from e. coli, as a model reaction. the enzyme exhibits absolute stereoselctivity at the c-alpha atom, whereas selectivity is less strict at c-beta. in situ product removal, by the integration of an smb unit, would aid to maintain a high diastereomeric excess as it shortens the residence time of the products in the reactor, in addition to shifting the reaction to the product side. the in-line coupling of the chromatographic unit to the enzyme reactor requires the use of the same solvents for reaction and separation, so the choice is limited to aqueous solutions close to physiological ph, limiting in turn the possible stationary phase materials. in a screening of different cation exchangers, amberlite cg-120 ii gave promising results: threonine is more retained than glycine, acetaldehyde is poorly and the cofactor plp is not retained. adsorption isotherms were determined by the retention time method and a smb under process conditions was simulated. by improving the packing of the column, i.e. achieving a more even particle size distribution, we tried to further increase the efficiency of the separation step. the application of enzymes for the synthesis of optically active substances is nowadays of growing importance in the pharmaceutical industry. this requires a proper cultivation of the microorganism as well as a posterior isolation process yielding a constant catalyst quality at high purity. goal of this project is the development of an integrated process for the production and isolation of a lipase from trichosporon beigilie and its posterior application for the enantioselective synthesis of pharmaceutical products. the cultivation of the microorganism is optimised in a laboratory and pilot-scale fermenter in a fed-batch mode. parameters like media composition, temperature, ph and aeration rate are set up. taking advantage of the localisation of the enzyme (covalently attached to the cell membrane) the first step of product isolation consists of a continuous cell disruption. optimal results are achieved with the continuous bead mill disruption process (68% enzyme release with a specific activity of 0.8 u/mg of protein). the non disrupted cells are recycled as inoculums for a new cultivation, increasing the yield of the overall process (by 5-times in the pilot-scale fermenter). in order to isolate the product two different process sequences are considered. the first one consists of an extraction (peg and phosphate buffer) coupled to an ion-exchange chromatography (q-sepharose ff). the second one applies a precipitation step with ammonia sulphate followed by a hydrophobic interaction chromatography (sepharose-hic) provid-ing a lipase yield of 72% (8-times higher than the one provided by combining extraction-chromatography). an ultrafiltration process is used in order to concentrate the lipase and its final properties (molecular weight, isoelectric point, activity, stability and kinetic data) are studied using p-nitrophenylacetate as model substrate. the relevance of the obtained product for its application in the pharmaceutical industry is proven by transforming (r,s)-naproxen-methylester into (s)-naproxen acid with an enantiomeric excess of >99% (after 24 h). biotensides (sugar fatty acid esters, sfaes) find nowadays a wide range of applications in pharmaceutical, personal care and food industry because of their biocompatibility, biodegradability and special surfactant properties. goal of this project is the development and optimisation of an integrated process for the enzymatic synthesis of sfaes from renewable sources to be used in cosmetic formulations. the following figure shows the scheme of the overall process. commercial and also new screened lipases are applied in the reaction between sugar and fatty acid. the mixture grade of the initial reaction system is increased by ultrasounds taking into account the influence on the catalyst characteristics and also the necessity of an organic solvent as adjuvant. the reaction takes place in an enzymatic membrane reactor (emr) equipped with an ultrafiltration membrane which retains the catalyst. the separation of the by-product (water) from the rest of the components can be achieved by means of a pervaporation unit which coupling to the emr allows the semi-batch process. in order to separate the esters from the fatty acid a stepwise elution chromatography method is developed using silica as adsorbent and ethyl acetate and methanol as eluents. with this system 91% of the dimer is isolated with purity (hplc) of 93%. the application of a dialysis membrane technique allows the separation of 80% of the fatty acid by building ester micelles changing the polarity of the organic solvent used as eluent. solubility and crystallisation properties of recombinant bacillus halmapalus ␣-amylase cornelius faber centre for microbial biotechnology, biocentrum dtu, building 223, 2800 lyngby, denmark a comprehensive knowledge of solubility properties is a prerequisite for the efficient design and operation of bulk enzyme recovery processes, however, complete phase diagrams are only available for very few proteins, in particular lysozyme of high purity. here, we present the results of detailed solubility studies in aqueous solutions of an industrially relevant ␣-amylase of technical grade. experiments were conducted in small scale batch mode (working volume of 1 ml). the influence of selected cations and anions from the hofmeister series on the stability of the ␣-amylase was examined. the hofmeister series for anions was followed in the correct order at all salt concentrations studied, i.e. from 0 to 1 m, whereas the series was reversed for monovalent cations at concentrations up to 0.5 m, with the exception of lithium. to further investigate why the position for lithium was different to the hofmeister series established for lysozyme, the zeta potential of protein solutions at low concentrations of selected salts was determined. the results of these measurements indicate a pronounced effect of lithium on the zeta potential, as compared to other salts. in particular, the ph of zero zeta potential (i.e. the pi) was shifted approximately 0.5 ph units towards alkaline conditions in the presence of lithium, whereas the pi stayed almost constant for sodium and potassium. since the solubility exhibits a minimum at ph-values at or near the protein's pi, shifts in ph caused by salt addition are important to identify and quantify to avoid uncontrolled phase separation. the measurement of the zeta potential of proteins in solution holds significant promise as an attractive tool for understanding and controlling processes that are operated close to the solubility limit and which are often plagued by uncontrolled precipitation or crystallisation and thus rely on carefully chosen operating conditions. polyphenolic interactions with potato proteins during industrial expanded bed adsorption processing sissel løkra 1 , knut olav straetkvern 1 , bjørg egelandsdal 2 , gerd vegarud 2 : 1 department of natural science & technology, hedmark university college, n-2317 hamar, norway; 2 norwegian university of life sciences, 1432 as, norway. e-mail: sissel.lokra@lnb.hihm.no (s. løkra) in plant extracts it has been shown that polyphenols have a tendency to react with proteins, either by covalent or non-covalent interactions. these reactions can induce changes in the surface properties of the proteins, and, e.g. cause proteins to be insoluble and precipitate at ph-values below their isoelectric point. potato proteins have a high nutritional quality and show interesting functional properties in food systems. moreover, chlorogenic acid (ca) and caffeic acid constitute about 90% of the total polyphenol content of potato tuber. we have experienced expanded bed adsorption (eba) chromatography to be a method well suited for recovering industrial proteins from potato starch effluent. the process separates proteins from polyphenolic pigments, fiber and minerals. during the adsorption step, patatin, the major potato tuber protein shows complex binding kinetics demonstrated by breakthrough curves. in addition to diffusion limitations in the eba resin, changes in protein structure and surface properties probably are likely to affect this adsorption behavior. reactions between ca and patatin might result in a range of interactions for different species of the same protein. this project therefore aims to assess the interactions between ca, patatin and other major tuber protein fractions and how these changes affect the protein capture in eba. changes in size and charge are screened in 2-d electrophoresis and analyzed further. samples of different protein fractions are taken from breakthrough curves and dynamic binding capacity experiments in model systems with real feedstock. sandwich hybridisation assay for analysis of brewery contaminants s. huhtamella 1 , m. leinonen 1 , t. nieminen 1 , a. breitenstein 2 , p. neubauer 1 : 1 bioprocess engineering laboratory, university of oulu, finland; 2 scanbec gmbh, halle, germany. email: peter.neubauer@oulu.fi (p. neubauer) here we describe the development of a sensitive, cultivationindependent analytical method for the analysis of brewery contaminants which can be performed within three hours in crude sample extracts. the method is based on 16s rrna detection by a paramagnetic bead based sandwich hybridization assay (sha) with two oligonucleotide probes designed to either detect the species or a group of contaminants. the signals were read out either by a fluorimeter (rautio et al., 2003; leskelä et al., 2005) or potentiometrically with an electric biochip instrument (ebiochip systems) . this assay is advantageous over rt-pcr becasue it only detects viable cells and the method can be directly applied to crude cell extracts without prior purification. we describe the principle of designing and evaluating a series of groupspecific lactobacillus probes and the optimisation towards effective cell lysis and high assay sensitivity. the applicability of the sha was evaluated with real brewery samples and the results were compared to routine tests. in all steps of the evaluation the reliability and usability of the method was prioritised. the optimised method combined with a 24 h pre-enrichment period gave reliable results, had a detection limits of about 10 4 -10 5 cells per assay and was easily applicable in a brewery environment. biodesulfurization is one of the possibilities studied by the researchers to attain the maximum sulfur levels imposed for a near future by governments (european directive, 2003) . rhodococcus erythropolis igts8 is a natural and strictly aerobic microorganism able to remove the sulfur atom from dibenzothiophene (dbt) in a selective way (4s route (oldfield et al., 1997)), obtaining 2hydroxibifenyl (hbp) and sulfate. growth is carried out using the experimental procedure performed in previous works dealing with the inoculum built up, media composition and operational conditions (del olmo et al., 2005a (del olmo et al., , 2005b . this work is focused to determine the oxygen uptake rate during the production of the biocatalyst. four experiments were carried out at a biostat b fermentor (braun biotech.) using as only variable the constant stirrer speed used: 150, 250, 400 and 550 rpm. oxygen uptake rate have been determined by means of two methods: dynamic technique at different times during growth for few seconds to ovoid influences and from oxygen profile when the term dealing with oxygen transfer rate is known (predicted by the model proposed in a previous work (garcía-ochoa and gómez, 2004)). our values obtained from the techniques used present the same tendency in all the runs carried out: our values from dynamic technique is always lower than our values obtained from the oxygen profile. these values are modeled and the difference observed is explained due to the cellular economy principle: during the seconds employed in the dynamic technique determinations microorganism do not produce 4s route enzymes. it was studied different methods to recover a p. salmonis antigenic protein from recombinant e. coli cells. this protein has shown be highly effective in vivo vaccine. it has the ability to stimulate salmon immune system protecting them against of aggressive disease salmonid rickettsial syndrome resolving by this way a great problem of salmonid aquaculture. biomass obtained from iptg induced e. coli bl21 (de3) codon plus culture was used for soluble and insoluble antigenic protein recovery. it was evaluated recuperation by glass bead mill, freezing and thawing, osmotic shock and lisozyme/edta treatments, all of them applied in single or combined way. biomass was measured by dry weight of cells, soluble protein concentration was quantified according to bradford method, and antigenic protein was identified by sds-page and western-blot analysis. cells treated with lisozyme/osmotic shock and then glass bead mill the soluble protein was a 62.9% of the dry weight cell mass whereas using lisozyme/edta and glass bead mill as a single treatment only a 24.4 and 22.6% were obtained respectively. the freezing and thawing disruption treatment released less than 5% of soluble protein, as well as the osmotic shock procedure too. the sds-page and west-ernblot analysis revealed that the antigenic protein must be purified from the insoluble cell fraction when physical or mechanical disruption methods were employed and from the soluble cell fraction when chemical or enzymatic treatments were used. we propose investigate in further studies the inclusion bodies formation to design an efficient purification procedure for the target protein. the iso-peroxidase pox2 from garlic bulb allium sativum that represented the major peroxidase activity was purified to homogeneity. the enzyme is monomeric and has a molecular mass of 36 kda, and a pi around 9. the optimum temperature ranged between 25 and 40 • c, while optimum ph was around 5. pox2 appeared remarkably thermostable since it retained 50% of its activity at 50 • c for at least 6 h. in addition, the enzyme was stable at a ph range from 3, 5 to 11. kinetic constants were calculated, the apparent k m values were 500 and 150 m for gaïacol and h 2 o 2 , respectively. the high thermostability of pox2 may represent clear advantages in a number of processes including immobilizing peroxidase and use it as a biosensor to detect oxidant component as h 2 o 2 and other peroxides. immobilization of pox2 was achieved by binding covalently the enzyme to a sepharose matrix (bead and membrane va epoxy). the immobilized peroxidase showed great stability at heat and storage than the soluble enzyme. the native enzyme retained 55% of its activity at 60 • c for 10 mn while the immobilized pox2 retained full activity for 35 mn at the same temperature. in other side, the free enzyme retained full activity for at least one month and a half during storage at 25 • c, and lost 50 m of its activity after 2 months. the immobilized form of pox2 retained complete activity for 2 months at the same temperature. the immobilized enzyme was used to detect h 2 o 2 in some food components such as milk and fruit juices. in a second study, same experiments were performed in order to detect the smaller quantity of added h 2 o 2 to the farming milk. purpose: a new research field has been created to begin to address protein function at level of regulation of enzyme activity. this new area has been given the name chemical proteomics, or activity-based proteomics (abps), and makes use of small molecules that can covalently attach to catalytic residues in an enzyme active site. the selectivity of the chemically reactive group allows specific proteins or protein subset to be tagged, purified and analyzed. methods: this molecule (abps) has three subsets: tag, linker, and warhead. warhead is a nucleophile and attach to active site. linker is a polypeptide that makes a simple connection between warhead and tag. tag is fluorcent or radioactive material that facilitates the detection of drugs. findings: several diseases such as cancer, rheumatoid arthritis and osteoporosis are associated with elevated levels of protease activity. serine hydrolyses abps have been used to profile enzyme activity in a diverse range of cancer cell lines. in studies comparing metastatic and non-metastatic human breast cancer models, it was shown that the former exhibited a higher activity of a ␥-glutathione-s-transferase, an enzyme that has not previously been associated with breast cancer. discussion: additionally, abps can be used to develop robust screens for small molecule inhibitors of a specific enzyme target within a large family of related enzymes. this method of inhibitor screening allows compounds to be assayed for both potency and selectivity against a set of related in complex biological samples. this technique is able to identify novel enzymatic proteins and drugs and has the potential to accelerate the discovery of new drug target. a cyclodextrin glycosyltransferase (cgtase) from a new isolated strain from bacillus clausii e16, was purified through q-sepharose, gel filtration chromatography and deae-sephadex a-50. the mw of the pure enzyme was 75 kda with sds-page. the enzyme displayed optimum ph value and ph stability at ph 6.0 and in range of 6.0-11.0, respectively. the optimum temperature and thermostability were at 55 • c and up to 60 • c by 1 h, respectively. the k m and v max were 2.85 mg/ml and 80.0 mol/min mg and 0.83 mg/ml 13.45 mol/min mg using maltodextrin and soluble starch, respectively. the isoeletric point was 4.8 and the n-terminal region of the pure enzyme was sequencing by maldi-tof-ms. the ratio of ␣-, ␤and ␥-cd was 0.29:1.00:0.79 and 0:1:0 with maltodextrin and soluble starch at 2.5%. application of magnetic separation technology for recovery of immobilised lipases nadja schultz 1 , anke neumann 1 , george metreveli 3 , matthias franzreb 2 , christoph syldatk 1 1 chair of technical biology, university of karlsruhe, engler bunte ring 1, d-76131 karlsruhe; 2 forschungszentrum karlsruhe, institute of technical chemistry, water-and geotechnology; 3 inst. für wasserchemie, engler bunte ring 1,76131 ka. e-mail: nadja.schultz@ciw.uni-karlsruhe.de (n. schultz). url: www.fzk.de/itc-wgt (m. franzreb) first results on the development of the magnetic separation technology for the recovery of immobilised lipase from a 2-phase-system, which should be suitable for a large scale use in future, known as high gradient magnetic separation (hgms) are presented. the application of immobilised lipases makes the reuse of the enzyme in a process possible and is therefore interesting for industrial applications. in this study immobilised lipase is used in a 2-phase-system. here the new approach to recycle and reuse the lipase, immobilised on magnetic particles, from a 2-phase-system with the help of the new high gradient magnetic separator (hgms) is examined in 30 ml scale. as model enzyme for the immobilisation on magnetic microparticles (polyvinyl alcohol (pva), 1-2 m) the commercially available (novonordisc) lipase a (cala) from candida antarctica was used. necessary for screening of immobilisation methods and characterisation of the immobilised lipase (candida antarctica) was the development of a robust, simple and rapid chromophoric activity assay. therefore the pnpp-lipase assay was optimised for direct application on immobilised lipases (in preparation schultz et al., 2005) . further more a ph-stat-assay for measuring the activity of free and immobilised cala in a 2-phase-system of tributyrate and buffer was optimized for this system. another important basis for the realisation of the recovery of immobilised lipases was to optimise the immobilization technique of the lipase. furthermore approaches for the explication of generally empirical based immobilization techniques on insoluble support were made. hereby we successfully applied the zeta potential measurement on the immobilization behaviour of the lipase cala. for to determine the operating temperature for the biomagnetic separation procedure we studied stability analysis of free and immobilised lipase cala at different temperatures (37, 25, 4, −20 • c) and at different ph values (ph 6, 7 and 8). the optimal temperature and ph value for the free and immobilised lipase was determined. presently and constructively on the so far developed methods and techniques we intensively work on the demonstration and feasibility of the recovery of immobilized lipase from a 2-phase-system. challenging approaches and first results on the recovery of immobilized lipase from a 2-phase-system in 30 ml scale were shown already. nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated. three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of 18 atoms) and carbonyldiimidazole activation. cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg per ml support and a high recovery (up to 93%). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affin-ity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately 1 × 10 −6 , and 0.4 × 10 −5 m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. quantitative methods in high throughput screening of aqueous two phase systems matthias bensch, björn selbach, jürgen hubbuch institute of biotechnologie 2, forschungszentrum jülich, germany purification of biopharmaceuticals is one of the most expensive and at the same time least understood steps in bioprocesses. during the process development for protein production, short time to market and the demand for cheap processes dominate today's process development. one way of reducing process costs is to implement integrative processes. aqueous two phase systems (atps) combine the advantages of removing cell debris and simultaneously purifying and concentrating the target protein, however, to the cost of highly complex systems which are difficult to predict and optimize. using high throughput screening techniques in the development of atps processes thus seems to be an ideal candidate for achieving both a reduced development time and an economical process without the need for preliminarily well characterized systems. in this study, we use the robotic system tecan freedom evo tm as an automation platform for the evaluation of aqueous two phase systems. central to this workstation are the integrated hardware as liquid handler, gripper, reader and centrifuge. we have created high throughput methods for rapid parameter estimations. as a first step, pipetting and mixing had to be calibrated for the use of highly viscous polymer solutions which are common in atps. the focus of the current work lies on the integration of the automatic preparation and analysis of two phase systems in microtiter plate scale. the robotic platform can now automatically create aqueous two phase systems and measure characteristic values such as binodal curves, protein concentrations and protein distributions between the two phases. the major bottleneck of hts processes, namely the rapid analysis of impure systems, is tackled by using automated elisa tools. depending on the intended use, the high number of measured partition coefficients and yields can be used for modelling or rapid process design. today, most optimisations of chromatography separations are based on experimental work and rule of thumb. the pat initiative has opened up for a model-based approach for downstream processing of pharmaceutical substances. this work uses a nonlinear chromatography model to optimize an ion exchange separation step. the general rate model with langmuir mpm kinetics described the behaviour of the components in the column. the optimal operating points using both productivity and yield as objective functions were found. the optimizations were run with both igg and bsa as target proteins respectively to compare their optimal operating points. the requirement on the optimal operating point was a purity of at least 99%. this requirement was added to the optimization problem as a nonlinear inequality constraint. flow rate, loading volume, start salt concentration in elution, elution gradient and cut points were used as decision variables in the optimization. the more retained component, bsa, was much easier to separate from igg with a gradient elution than igg from bsa while still retaining a high productivity and yield. the higher load volume at the optimal operating point, with bsa as target protein, causes a displacement of igg and thereby improving the separation. a high productivity at the yield optimum was still possible with bsa as target protein. both a lower productivity and yield was obtained with igg as target protein. optimisation and robustness analysis of a hydrophobic interaction chromatography step niklas jakobsson, marcus degerman, bernt nilsson department of chemical engineering, lund university, p.o. box 124, se-221 00 lund, sweden process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. the present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. factorial experimental design is common practice in industry today for robustness analysis. the method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. in addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. the methodology consists of threes consecutive steps. firstly, screening experiments are performed using a factorial design. secondly a kinetic-dispersive model is calibrated using gradient elution and column load experiments. finally the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. the process studied in this work is the separation of polyclonal igg from bsa using hydrophobic interaction chromatography. department of biochemistry and microbiology, ict prague, technicka 3, prague cz-166 28, czech republic the display of novel metal binding sites on the surface of the biosorbent represents potent tool to increase its binding capacity and improve selectivity. in this study, the 15.8 kda transcriptional regulator merr of mercury-inducible mer operon of tn21 exhibiting high affinity and selectivity towards hg 2+ , was displayed on the surface of s. cerevisiae. to achieve this, merr was genetically fused with gene encoding c-terminal domain of ␣-agglutinin which resulted in covalent attachment of the of the fusion protein on the cell wall glucan via glycosylphosphatidylinositol anchor. to evaluate the performance of such modified whole-cell biosorbent with specific regard to hg 2+ , we constructed a new biosensor e. coli strain, which utilizes kanamycine resistance gene as a reporter under the control of mer promoter. it allowed determination of hg 2+ in a range of 5-100 nm by simply monitoring the growth in the hg 2+ /kanamycine-containing media. the effect of genetic engineering of s. cerevisiae surface by merr became significantly pronounced in biosorption experiments with solutions containing 10 m hg 2+ when modified cells accumulated 2.5-fold more hg 2+ than the control strain expressing mere anchoring domain. sensitivity analysis of amino acids in simulated moving bed chromatography ju weon lee, chong ho lee, yoon mo koo center for advanced bioseparation technology, inha university, inchon 402-751, korea. e-mail: ymkoo@inha.ac.kr (y.m. koo) the difficulty of simulated moving bed (smb) design is that the optimization of the operation conditions relies on the determination of accurate adsorption isotherms. most smb chromatograph is carried out under nonlinear conditions, and the nonlinear behavior should be considered properly in the equilibrium isotherms. the other difficulty is the smb operation which has the characteristics of continuous process, all flow rates and switching time of valves should be maintained during the operation of smb. if the disturbances of operating conditions and isotherm parameters are occurred, it affects the zone flow rates and the migration velocity of the solutes, and these effects change the internal profiles of the solutes. therefore, it is the reason of decreasing the purity and the yield of products the objective of this work is to consider the sensitivity of isotherm parameters and operating parameters in smb chromatography process. two amino acids, phenylalanine and tryptophan, separation by smb process is selected as control system. application of ph and po 2 probes during bacillus caldolyticus fermentation: an additional approach in improving a feeding strategy johannes bader 1 , boris neumann 2 , karima schwab 1 , milan popovic 1 , rakesh bajpai 3 : 1 studiengang biotechnologie, fachbereich v, tfh-berlin, seestr., 13347 berlin, germany 2 proteome factory ag, dorotheenstr. 94, 10117 berlin, germany; 3 department of chemical engineering, university of missouri-columbia, w2061 ebe, columbia, mo, usa. e-mail: popovic@tfh-berlin.de (m. popovic) bacillus caldolyticus,a thermophilic microorganism, is a good producer of thermostable liquefying ␣-amylase. during optimisation of initial and feeding media for fed-batch fermentation a two component feeding containing starch and casitone was found advantageous. to approach the optimal feeding rate the method published by akesson et al., 2001 was extended to two component feeding. the key idea, discussed in this presentation, was using the po 2 and ph probing signals to determine if the feeding of one or the other component should be increased or decreased. each of the probes offers information of different areas of feeding condition. to prevent excessive feeding of starch the ph probe is preferable. in case of excessive casitone feeding the po 2 probe responds in very authentic way enabling together with the ph signal reliable and reproducible evaluation of feeding strategy. however a congruent response of po 2 and ph probes means the approaching of the optimum feeding rate for both components. akesson, m., hagander, p., axelsson, j.p., 2001. probing control of fed-batch cultivations: analysis and tuning. contr. eng. pract. 9, 709-723. antibody immobilization by using the plasma polymerized acrylic acid r. jafari, m.tatoulian, f. arefi-khonsari laboratoire de génie des procédés plasmas et traitement de surface, enscp, upmc, 11 rue pierre et marie curie, 75005 paris, france the objective of this work is therefore to produce a surface containing a high density of cooh functions on the polymer beads (ps) for the covalent immobilization of antibodies. we have investigated the plasma polymerization of acrylic acid in a fluidized bed reactor the polystyrene (ps) beads. for such application, there is a strong need to obtain stable plasma polymerized acrylic acid (ppaa) coating, resistant to washing with water. different physico-chemical analyses have been used (water contact angle measurements (wca), xps and sem analysis) to characterize the ppaa coating deposited on ps beads under different experimental conditions. the xps results showed that the pretreatment of surface of the beads before deposition of acrylic acid plays an important role on the stability of ppaa layer. the instability of the coating is partially due the fact that under certain conditions the coatings are soluble in water and secondly due to the bad adhesion of the polymer beads which are hydrophobic to the growing ppaa coatings. xps as well as tof-sims gives evidence of the immobilization of the antibody. xps results as well as static sims allows to detect nitrogen on the surface of the treated beads which proves the presence of the immobilized antibodies. under optimum condition the ppaa coatings provides the possibility to show a nitrogen uptake which varies between 6.5 and 9% of the apparent stoicheiometry of the surface. holst department of medical physiology, the panum institute, university of copenhagen, dk-2200 copenhagen, denmark glp-1 (glucagon-like peptide-1), a peptide of 30 amino acids secreted by endocrine cells in the gut in response to meal ingestion, was discovered during a systematic search for gut factors capable of enhancing insulin secretion. it turned out to be the most efficacious insulin releaser known, and unlike other factors, was shown to retain its insulinotropic activity also in patients with type 2 diabetes. subsequent research has documented that the peptide not only releases insulin from the beta cells, but also enhance all steps of insulin biosynthesis, up-regulates beta cell gene transcription, and has trophic effects on the beta cells. the latter includes both proliferation of existing cells, neogenesis from ductal precursor cells, and inhibition of apoptosis. the peptide also inhibits glucagon secretion, reduces gastric emptying and reduces appetite and food intake. because of these actions, glp-1 administered to patients with type 2 diabetes dramatically lowers blood glucose as well as glycated hemoglobin levels, and reduces body weight. however, natural glp-1 is extremely rapidly metabolized in the body, and the problem has been how to convert the unstable peptide into a clinically useful agent. the two main problems are its susceptibility to enzymatic degradation by ubiquitous dipeptidylpeptide peptidase iv (dpp-iv) and its rapid renal elimination. a related peptide, isolated from the saliva of a lizard, exendin-4, was found to be a full agonist of the glp-1 receptor, to be resistant to dpp-iv and to be cleared more slowly by the kidneys. this peptide was highly effective in clinical studies and has now (30/4) been approved for diabetes treatment by the fda. other approaches include acylation of glp-1 whereby it attaches to albumin in the body and acquires resistance to dpp-iv as well as a slow renal elimination. also this analogue (liraglutide) has favourable clinical effects. fusion proteins of glp-1 and larger, slowly eliminated proteins in the body are currently being evaluated. small molecule, orally available inhibitors of dpp-iv have been demonstrated to protect endogenous glp-1 from degradation and to be efficacious in both experimental and clinical diabetes, and numerous inhibitors are currently in clinical development. the incretin hormones are released from gut endocrine cells upon meal ingestion. they enhance glucose-induced insulin secretion and nay be responsible for up to 70% of postprandial insulin secretion. the incretin hormones are glucagon-like peptide-1 (glp-1) and glucosedependent insulinotropic polypeptide (gip). in patients with type 2 diabetes (2dm) the incretin effect is severely reduced or absent. in 2dm patients the secretion of gip is normal, but its effect on insulin secretion is almost completely lost. glp-1 secretion, on the other hand, may be impaired, but its insulinotropic actions are preserved and it may restore insulin secretion to near normal levels. substitution therapy with glp-1 might therefore be possible. glp-1 is a product of the glucagon gene and its actions include: (1) potentiation of glucose-induced insulin secretion; (2) stimulation of the expression of ␤-cell genes essential for insulin secretion, including the insulin gene; (3) stimulation of ␤-cell proliferation and neogenesis (by enhancing endocrine differentiation of duct cells) and inhibition of ␤-cell apoptosis; (4) inhibition of glucagon secretion; (5) inhibition of gastrointestinal secretion and motility, notably gastric emptying; and (6) inhibition of appetite and food intake. these actions make glp-1 particularly attractive as a therapeutic agent for 2dm. thus, continuous subcutaneous administration of glp-1 for 6 weeks resulted in a 5 mmol/l reduction in mean plasma glucose and a reduction in hgba1c of 1.3%; a weight loss of 2 kg; improved insulin sensitivity; improved ␤-cell function; and the treatment was associated with no significant side effects. unfortunately, glp-1 is rapidly destroyed in the body by the ubiquitous enzyme, dipeptidylpeptidase iv (dpp-iv). clinical strategies therefore include: (1) the development of metabolically stable analogues of glp-1 viz. activators of the glp-1 receptor; and (2) inhibition of dpp-iv. orally active dpp-iv inhibitors have proven successful in experimental diabetes and several companies are now trying to develop clinically suitable inhibitors. so far the clinical experience is limited, but recent clinical studies have provided proof of concept. metabolically stable analogues/activators include the structurally related lizard peptide, exendin-4 or analogues thereof, as well as glp-1 derived molecules that bind to albumin and thereby assume the pharmacokinetics of albumin. these molecules are effective in animal experimental models of type 2 diabetes, and have been employed in clinical studies of up to 52 weeks' duration. on the basis of these studies it can be concluded that a therapy of type 2 diabetes mellitus based on stimulation of glp-1 receptors is likely to be effective and to become a clinical reality within the not too distant future(1-4). recombinant activated coagulation factor vii (rfviia) was developed to treat bleedings in hemophilia patients, who have developed inhibitors against fviii or fix, and has been demonstrated to have an efficacy rate of 80-90% in major surgery as well as in serious bleedings in such patients. to use rfviia as a hemostatic agent in severe hemophilia is a new concept of treatment, not being a substitution therapy, but using a pharmacological dose of exogeneous rfviia to compensate for the lack of fviii or fix. the administration of extra rfviia has been found not only to bind to tissue factor (tf), but also to the negatively charged phospholipids surface of thrombin activated platelets. hemostasis occurs on surfaces being initiated on the tf-expressing cells as a result of exposure of tf, not normally exposed to the circulating blood, following an injury to the vessel wall. tf is a true receptor protein with an intramembraneous part and an intracellular tail. its ligand is fvii/fviia. as soon as tf is being exposed to the blood, it forms complexes with fviia already present in the circulation. these complexes activates fx and provide the initial limited amount of thrombin molecules activating the co-factors, fviii and fv, as well as fxi and platelets. following the thrombin activation of platelets, negatively charged phospholipids are being exposed on the outer surface of the platelets. on this surface most coagulation proteins bind tightly, facilitating the conversion of fx into fxa and the full thrombin burst, necessary for the formation of a tight fibrin hemostatic plug resistant against premature lysis. in hemophilia patients the initiation of hemostasis is essentially normal, but, since they lack fviii or fix, they do not form the fviiia-fixa complex necessary for full thrombin generation on the activated platelet surface. as a consequence the fibrin plug formed in hemophilia is loose, fagile and easily dissolved resulting in continuous bleeding. pharmacological doses of rfviia have been demonstrated to mediate direct binding of rfviia to the negatively charged thrombin activated platelet surface, thereby generating thrombin formation in the absence of fviii/fix. through this mechanism hemostasis is generated in hemophilia patients independent of fviii/fix. furthermore, by generating more thrombin at an increased rate the formation of stable, tight fibrin hemostatic plugs are facilitated. such fibrin plugs are more resistant against premature lysis and help not only to initiate but also to maintain hemostasis. based on its capacity of enhancing thrombin generation locally on the activated platelet surface, rfviia has been used to ensure hemostasis also in other situations than hemophilia, such as platelet defects including thrombocytopenia. recently, rfviia was shown to be hemostatically effective in patients with profuse bleedings as a result of vast trauma and tissue damage. in these patients with a complex hemostasis pattern including a host of changes leading to an impaired hemostatic function, extra rfviia seems to help generate a burst of thrombin resulting in the formation of a stable hemostatic plug more resistant against the ongoing lysis. in patients with intracerebral haemorrhage, one single dose of rfviia recently was found to limit the expansion of the haemorrhage and thereby leading to improved functional outcome. institute for medical microbiology and immunology, panum 18.3.12, blegdamsvej 3, dk-2200 copenhagen n, denmark. email: s.buus@immi.ku.dk complete genomes from several species including many pathogenic microorganisms are rapidly becoming available along with the corresponding "proteomes". even at the peptide level, the diversity of proteome is enormous and easily represents a unique imprint of the originating organism. it is perhaps not surprising that the immune system considers peptides as key targets. recent immunological advances have shown that mhc molecules act as peptide selectors for immune recognition. we have proposed to generate accurate predictions of peptide binding to mhc and used these to identify immunogenic epitopes directly from genomic data. we have developed an iterative data-driven immunobioinformatics approach where data is used to generate predictors, and predictors are used to select new and complementary data for the next iteration. we have demonstrated the superior performance of this approach compared to a random data selection approach. we have also developed an efficient approach to select the most informative mhc molecules to investigate. the resulting, immunobioinformatics resource represents an immediate and powerful application and interpretation of genomic data, and will enable a rational approach to immunotherapy in the future. allergen specific immunotherapy is a causal treatment for igemediated allergic diseases such as hay-fever, and it has relied traditionally on preparations derived from aqueous extracts of various natural allergenic source materials. the cloning and production of an increasing number of allergens through the use of dna technology has not only facilitated the characterisation and analysis of the allergenic proteins, but also provided the opportunity to use these recombinant proteins instead of natural allergen extracts for the diagnosis and therapy of allergic disease. detailed physicochemical, biochemical and immunological characterisation are essential for the comparison of natural and recombinant proteins, and also provide a basis for developing derivatives. chemically modified allergens with attenuated ige-reactivity are currently used for immunotherapy in order to enable high doses to be achieved with a minimized risk of inducing allergic side reactions. dna technology provides the opportunity to develop and produce hypoallergenic allergen variants using strategies including gene mutation. the design of such variants must ensure that t cell reactivity and immunogenic activity are retained in order to preserve therapeutic potential. the recombinant allergens and their derivatives have several advantages over natural allergen extracts. they are relatively easy to produce in consistent pharmaceutical quality; the problems of natural extract standardisation can be avoided completely; the relative concentrations of the individual allergens can be controlled to obtain optimal dosages; nonallergenic proteins are excluded; the possible risks of contamination are avoided. the first clinical trials with grass pollen allergens and birch pollen hypoallergenic variants have yielded very encouraging results. the use of recombinant polyclonal antibodies (pabs) may improve the treatment of disease caused by complex targets such as infectious agents, when compared to monoclonal antibody therapy. symphogen has developed a method for reproducible production of target-specific fully human pab compositions, so-called symphobodies. the antibody genes are first isolated from donors with an immune response against the target and antibodies are screened for specificity. subsequently, the pabs are expressed in mammalian cells using the sympress technology, which is based on site-specific integration. this procedure ensures that each of the expression constructs encoding the antibody genes are stably integrated at the same site in each of the host cells, thereby eliminating genomic position effects and differential growth and production rates. further, the sympress technology comprises the generation of a polyclonal working cell bank (pwcb) which is used as inoculation material for the manufacturing. these cells display sufficient genetic stability to enable a controlled gmp production of recombinant polyclonal antibodies. symphogen's first product, sym001, is a recombinant human polyclonal rhesus d-specific symphobody preparation consisting of 25 different anti-rhesus d antibodies. this product is intended to be used for treatment of idiopathic thrombocytopenia purpura and prevention of hemolytic disease in newborns. recombinant anti-rhesus d symphobodies were produced and shown to be biologically active against rhesus d. the expression technology provided a compositional reproducibility between batches which is sufficient for manufacturing of such a polyclonal product for clinical use. scaledup production for clinical trials is currently ongoing. stem cells play an important role in renewing tissues such as skin and cornea. they are responsible for the continuous generation of the differentiated epithelium. we have characterized stem cells of the skin and cornea in situ and their fate in vitro in human skin reconstructed by tissue engineering using keratin (k)19. in the outer root sheath of the hair follicle, stem cells (label-retaining cells) present in the basal layer of the bulge area express k19 and present a loosely arranged keratin filament network and low levels of k14 protein in their cytoplasm. in addition, another stem cell population (also labelretaining) is present in the first suprabasal layers. these cells exhibit a very dense keratin network and express k17. three-dimensional tissue constructs (dermis and epidermis) obtained by the self-assembly approach of tissue engineering allow the preservation of k19 positive stem cells in the basal layer of the epithelium. in the eye, the stem cells are located in the limbal part but not in central cornea and they express k19. the epithelium of reconstructed cornea is thinner compared to reconstructed skin and more transparent. the characterization of stem cells in reconstructed tissue is essential to evaluate the long-term survival of these tissues in vitro but also after grafting. these human reconstructed tissues are developed for fundamental (physiological, toxicological studies) and clinical applications such as transplantation for the permanent replacement of damaged organs. lg is holder of the canadian research chair (cihr) on stem cells and tissue engineering. alessandra gliozzi physical department, university of genoa, 16146 genoa, italy hollow nanometer-sized capsules can be prepared by means of different techniques. first "nanocapsules" were liposomes, however they are too unstable for many medical or pharmaceutical applications. in contrast, recently developed polyelectrolyte capsules prepared by means of the layer-by-layer technique are much more stable and seem to be a very promising way for coating living cells or tissues in order to prevent or reduce their immune rejection after implantation. several observations on single living cells encapsulated by the alternative adsorption of oppositely charged polyelectrolytes will be presented. the most relevant result is that cell preserve their metabolic activity, are still capable of dividing and performing specific functions. moreover, a technique to immobilize in defined arrays coated cells expressing green fluorescent protein by using a microcontact printing of polyelectrolytes will be presented. finally, tests performed to study the induction of fibrosis and vascularization by nanocapsules implanted in rat kidney and liver will be presented. over the past decade we have developed methods to generate spontaneously and synchronously beating tissue equivalents from neonatal rat heart cells in the culture dish. these tissue equivalents display the key morphological and functional features of intact myocardium and have been termed engineered heart tissue (eht). to generate ehts, heart cells are mixed with freshly neutralized, liquid collagen i, matrigel and growth supplements and grown in a circular casting mold around a central cylinder, which subjects the cells to a continuous mechanical load. this process is enforced by cyclic mechanical stretch. we use eht mainly for two purposes, as a test bed for the effects of pharmacological or genetic manipulations and for cardiac repair. as a cell culture model, ehts compare with standard 2d monolayer cultures of neonatal rat cardiac myocytes and freshly isolated adult cardiac myocytes. advantages of ehts are their functional similarities with intact heart muscles, the ability to easily measure force of contraction under mechanical load, the pos-sibility to transfect cardiac myocytes inside ehts with adenovirus at high efficiency and the reproducibility in large series. a disadvantage is that contractile function as measured at the end of the culture period also integrates influences on tissue development, cell-cellconnections, extracellular matrix production and on non-myocytes. at present we are working on downscaling the eht method to a 96well format for screening purposes. to use ehts for cardiac repair we created multi-looped ehts from five circular ehts large enough to cover the infarct scar 14 days after coronary artery ligation in rats. ehts survived and formed a layer of muscle tissue on top of the infarct scar. ehts restored undelayed anterograde impulse propagation over the scar, prevented further ventricular dilatation, normalized enddiastolic pressure and relaxation, and partly restored contraction of the scar. thus, the study provides evidence that implanting ehts onto infarcted hearts can improve cardiac contractile function after myocardial infarction. the goal of tissue engineering is the development of skin, bones and even organs to restore, maintain and improve tissue function within the body. the current paper focuses on the investigation of invitro growth of osteoblast cells in different types of scaffolds. three of the scaffolds were made of pcl (polycaprolactone) 10% glycerol and 10% hca(hydroxylapetite). two of the scaffolds were made by compression molding, and one was made by fused deposition modeling utilizing the stratasys. the fourth cerabio was a commercially available product totally ceramic. the pores in compression molding were obtained by putting in 75% volume of sugar either 150 and 595 m which was later removed by leaching. the fused deposition scaffold was made by placing the filaments in a predetermined arrangement. the stratasys system was a computer designed model. the scaffolds were seeded with hfob 1.19 human fetal osteoblast cell line with vigorous shaking overnight and incubating at 37 • c and 5% co 2 . observation of the seeded scaffolds was made after 3 days and 9 days of incubation. the seeded cells were stained with bcip/nbp at 37 • c overnight. the cell proliferation in the 595 and 150 m scaffolds appeared approximately the same with a possible advantage of 595 m. the cerabio sample demonstrated the greatest proliferation among the four scaffolds studied and the stratasys sample exhibited a different type of cell adhesion with the cells were clustered in the interstices of the structure. denise freimark, ruth freitag, valérie jérôme chair of process biotechnology, university of bayreuth, d-95448 bayreuth, germany. e-mail: denise.freimark@uni-bayreuth. de (d. freimark) tissue engineering is emerging as an alternative to bone grafts for the regeneration of defects that do not heal spontaneously. ultimately, the development of an optimum carrier and the identification of ideal inductive factors and cells may enable tissue engineering to provide an improvement over bone grafts in the future. bone formation and repair require a complex cascade involving growth factors, cytokines and angiogenesis. at present the complexity of the molecular mechanisms that control gene expression in bone forming cells in embryo as well as in adult is not fully understood. several factors like bone morphogenetic proteins (bmps), transforming growth factor beta (tgf-␤), vascular endothelial growth factor (vegf) and insuline-like growth factor (igf) have been identified and their ability to stimulate bone formation in vitro and in vivo has been investigated. while much is known about these factors per se, less is known about genetic regulation of artificially stimulated osteogenesis. interestingly, some in vitro investigations showed that only optimal growth factors concentrations lead to effective bone formation whereas higher concentrations had deleterious effects which suggest some variation in the activated regulation pathways. therefore, one of our goals is to analyze kinetics, dose-dependence and synergistic effects of growth factors and cytokines on regulation pathways of bone formation. moreover, the optimal vascularization of the scaffold is a major hurdle in the development of engineered bone. it is well known that: (i) vascular invasion precedes bone growth and (ii) osteogenesis takes place in the vicinity of newly formed vessels. thus, inadequate bone vascularization is associated with decreased bone formation. further analysis of the intercommunication between endothelial cells and osteoprogenitors in co-culture systems could provide key information that could be thereafter used to solve this problem. we propose to add some new knowledge to this complicated puzzle. a first step in our investigation is the production of some of the growth factors mentioned above in recombinant form. these factors are expressed in a novel vector (ptriex tm ; novagen) which allows recombinant protein production in prokaryotic or in eukaryotic systems with a single plasmid. afterwards, we analyze potential synergistic effects of these factors as well as kinetic and dose-depend parameters on the genetic regulation of downstream pathways in osteoblasts culture. in parallel, we develop an in vitro system allowing us to investigate the intercommunication between endothelial cells and osteoprogenitors. there has been significant interest in the therapeutic and scientific potential of human embryonic stem (es) cells since they were first isolated in 1998. if human es cells could be differentiated into suitable cell types, stem cells might be used in cell replacement therapies for degenerative diseases such as type i diabetes and parkinson's disease, or to repopulate the heart following myocardial damage. however, there is a significant shortage of high quality human es cell lines and few research groups have experience in the propagation and manipulation of these cells. we are addressing this important issue using the combined expertise of the stem cell biology laboratory and the assisted conception unit at king's college, london. with local ethical approval and under licence from the uk human fertilisation and embryology authority, we have been establishing high quality human es cell lines from a novel source of human embryos. to date, we have derived three human es cell lines and are now focused on the generation of therapeutically important cell populations, including cells that may have clinical application in degenerative and traumatic injury to the brain and spinal cord, heart, retina and other target organs. wallenberg neuroscience center, department of physiological sciences, lund university, bmc a11, s-221 84 lund, sweden cell replacement therapy for parkinson's disease is based on the idea that implanted dopamine neurons may be able to substitute for the lost nigrostriatal neurons. in rodent and primate models of parkinson's disease it has been shown that transplanted dopamine neuroblasts can re-establish a functional innervation and restore dopaminergic neurotransmission in the area of the striatum reached by the outgrowing axons; that the grafted neurons are spontaneously active and release dopamine in an impulse-dependent manner, at both synaptic and non-synaptic sites; and that they can reverse or ameliorate some of the parkinson-like motor impairments induced by damage to the nigrostriatal system. clinical trials in patients with advanced parkinson's disease have shown that dopamine neuroblasts obtained from fetal human mesencephalic tissue can survive and function also in the brains of pd patients, restore striatal dopamine release, and ameliorate impairments in motor behavior. the principal limitation of this approach is the problems associated with the use of tissue derived from aborted human fetuses, and the large numbers of donors needed to obtain good therapeutic effects. until now, transplantation of dopamine neurons has focused primarily on differentiated neuroblasts and young postmitotic neurons, at the stage of neuronal development that is optimal for survival and growth of the grafted cells. however, progenitors taken at earlier stages of development might prove more effective. efforts are now made to expand multipotent neural stem-or progenitor cells in vitro, and control their phenotypic differentiation into a dopaminergic neuronal fate. initial results suggest that in vitro expanded cells can survive and function after transplantation to the striatum in the rat pd model, but the overall yield of surviving dopamine neurons has been very low. with further development, expanded progenitors or dopamine neuron precursors, possibly in combination with cell engineering techniques, may offer new sources of cells for replacement therapy in pd. stem cell therapy has been very much in vogue for several years now. like gene therapy before it, it has raised unrealistic hopes of cures being available imminently. unlike gene therapy, it can cite proof of principle in the well established practice of bone marrow transplantation which is actually a good example of stem cell therapy. however, most of the uses that are now touted as targets for stem cell therapy, envisage the conversion of the stem cells into lineage restricted progenitor cells or more commonly, the final differentiated cell type. such conversions are extremely difficult to initiate and control. the procedures involve the manipulation, differentiation, and expansion of cell cultures in the laboratory, with unknown long term effects on the genetics and physiology of the cells. these issues are compounded when one considers as source material, human embryonic stem cells, where not only the final cell product requires significant scrutiny, but also there are safety issues surrounding the persistence of undifferentiated cells. on top of all these challenges are commercial (for stem cell companies), clinical, and regulatory pressures which will impact heavily on the pace of progress. nonetheless, despite all these hurdles, various academic groups and companies are making significant progress and examples of such developments in diabetes and cardiovascular repair will be given stem cells have the unique ability to perpetuate themselves while continually replenishing tissues throughout the life of an organism. the era of cellular and tissue regeneration for the treatment of disease and the effects of aging has indeed begun. it has been known that mechanical factors play an important role in the regulation of cell physiology. it is therefore reasonable to believe that mechanical factors also play a significant role in the metabolic activity and differentiation of mscs. in this study, we investigated the viscoelasticity of individual bone marrow-derived adult human mesenchymal stem cells (hmscs), and the role of specific cytoskeletal component -f-actin microfilaments on the mechanical properties of individual hmscs. the mechanical properties of hmscs were determined using the micropipette aspiration technique coupled with a viscoelastic solid model of the cell. for the hmscs under control conditions the instantaneous young"s modulus e 0 was found to be 886 ± 289 (pa), the equilibrium young"s modulus e ∞ 372 ± 125 (pa), and the apparent viscosity 2714 ± 1626 (pas). after exposed to 2 m of chemical agent-cytochalasin d that disrupt the f-actin microfilaments, the young's moduli of hmscs decreased by up to 72% and the apparent viscosity increased by 167%. these findings suggest that microfilaments are crucial in providing the viscoelastic properties of the hmscs, and changes in the structure and properties of them may influence the mechanical properties of hmscs significantly. pharmacologic transcription control of desired transgenes is essential for gene-function analysis, drug discovery, biopharmaceutical manufacturing, design of complex artificial regulatory networks, precise and timely reprogramming of key cell characteristics for gene therapy and engineering of preferred cell phenotypes for tissue engineering. capitalizing on our recent advances in the design of small molecule-responsive transcription control modalities we have used conditional molecular interventions for (i) improvement of specific productivity in biopharmaceutical manufacturing, (ii) transdifferentiation of therapeutically relevant cell phenotypes and (iii) design of artificial microtissues. we will also report on a completely new dimension of transgene control as well as engineering of hysteretic and epigenetic gene networks in mammalian cells. chronic diseases are a growing burden for the individual and society alike. one key factor driving this increase is an ageing population. currently, there are 600 million persons aged 60 years or over and this number is predicted to triple by the middle of the 21st century. effective prevention of irreversible damage to major organ systems requires early diagnosis and treatment yielding significant quality of life to the individual and sparing valuable health care resources. even when safe and effective medicines are available, a remaining problem to successful therapy are issues of patient compliance. historically, vaccines have been one of the major advances towards the longevity we enjoy today, with compliance rates close to 100%. hence, vaccines for early treatment of chronic diseases are ideally positioned for long-term therapy, and will take away the burden of self-medication associated with orally active drugs. here we will discuss a new generation of therapeutic vaccines based on virus-like particles (vlps). by displaying target molecules in a highly repetitive manner on vlps, it is possible to break b cell unresponsiveness in experimental animals as well as in humans. using such vaccines in animals, chronic diseases such as hypertension, alzheimer's disease, obesity and rheumatoid arthritis could be treated. furthermore, vaccination against nicotine resulted in high nictotine-specific antibody titers in humans, greatly facilitating smoking cessation in immunized individuals. interdependence of the impact of methanol and oxygen supply on protein production with recombinant pichia pastoris n.k. khatri, f. hoffmann martin-luther-university halle-wittenberg, institute for biotechnology, halle d-06120, germany. e-mail: f.hoffmann@biochemtech.uni-halle.de (f. hoofmann) the methylotrophic yeast pichia pastoris is a potent expression system for secretion of recombinant proteins. methanol as inductor of the foreign gene expression is also a substrate with high oxygen demand, which can lead to sudden oxygen depletion upon induction. thus, supply rates of methanol and oxygen are major process parameter during protein production with recombinant pichia pastoris. limiting dosage of methanol allowed maintenance of oxygen sufficient conditions during production of a single chain antibody fragment, but the product was degraded from the c-terminal end. in contrast, full-length product accumulated with controlled methanol concentrations despite oxygen limitation. the volumetric methanol uptake rate are limited by the oxygen transfer capacity of the reactor. higher methanol concentrations decreased the biomass yield and thereby increased the specific methanol uptake rate. this enabled prolonged production and yielded fivefold higher product concentrations. at the same time, the accumulation of small molecular weight contaminants was reduced. dosage of pure oxygen accelerated methanol uptake, grow and production. switching to dostat mode upon oxygen depletion led to an arrest of product accumulation, in contrast to persistent methanol feeding. the productivity was tenfold higher than without oxygen. combined with high methanol concentrations, however, fast methanol uptake led to toxication of the cells and early stop of production. flow cytometry revealed that perturbation of oxygen metabolism was followed by partial lysis of the culture. recombinant production of therapeutic proteins poses severe challenges due to their complexity (cystines, subunits, size). formation of the correct disulphide bridges is a prerequisite for activity but difficult to achieve in a prokaryotic host. there proteins mainly fold post-translationally as opposed to eukaryotic co-translational folding. thus the recombinant products are often not soluble and/or active. that is why different production parameters (e.g. host, induction conditions, temperature, compartment, proteinaceous fusion partners, co-expression of chaperones, foldases) are applied in order to gain functional recombinant proteins. the impact of all these strategies cannot be predicted and every problem of the production (expression, solubility, activity) might need to be solved for every target protein separately. nevertheless, much effort has been put into this for almost 3 decades, because they are important targets for the pharmaceutical industry. human growth factors influencing cellular proliferation and/or differentiation are one example. this case study gives an overview of strategies tested within the development of two processes leading to an optimised yield of active protein. murine wnt-1 (wnt family) possesses 23 conserved cysteines (most likely all involved in disulphide bridge formation and one in posttranslational modification) and could only be successfully produced in e. coli (fahnert, 2004) recently despite various attempts for many years. the other target protein is human collagen prolyl-4-hydroxylase being a heterotetramer consisting of two ␣-subunits and ␤-subunits each. the ␣-subunit strongly aggregates if produced separately whereas the ␤-subunit is pdi and is suggested to have a chaperone function. therefore a sequential induction strategy was proposed for this protein . the moss physcomitrella patens has been recently recognized as an ideal producer of recombinant proteins with respect to glycosylation. due to the elaborated post-translational capabilities of moss cells, the glycosylation patterns have been manipulated to obtain similar proteins to those found in animal cells. the protein expression using moss in suspension offers important advantages in comparison to other systems e.g. cho cells. the recombinant proteins can be targeted into the mineral medium, simplifying the down stream processing. moreover, there are neither known moss viruses nor plant viruses that are pathogenic for humans. the moss are cultivated axenically in a filamentous stage, the so called protonema. a pilot 30 l tubular photoreactor is used to characterize the response of p. patens to variations on the culture conditions. the phototrophic culture in bioreactors is systematically investigated, where light quantity and quality, stress, concentration of phytohormones, and moss morphology influence the differentiation, growth, and protein expression. a tight control of the moss morphology in suspension, quantified by image analysis, has shown to be advantageous in order to delay the cell differentiation and maintain the carbon dioxide uptake in long bioreactor runs. the introduced perfused culture system by means of cross flow filtration allowed for a continuous product separation and concentration, and feed back of the productive cells. the characterization of this highly controlled culture system is presented and the potential of p. patens as an alternative tool for molecular farming is discussed. (rnai) is an evolutionarily conserved, endogenous mechanism for sequence-specific gene silencing that uses small double-stranded rnas (called short interfering rnas or sirnas) to direct cleavage or prevent translation of homologous mrnas. harnessing rnai for therapy presents an opportunity for potentially treating a wide variety of diseases. the main obstacle is delivering sirnas into the cytosol of target cells in vivo. although we were able to protect mice from autoimmune hepatitis by hydrodynamic tail vein injection of sirnas targeting fas, this delivery method is unlikely to be adaptable for human use. alternate strategies to deliver sirnas in vivo as small molecule drugs using currently available, clinically acceptable injection methods that have shown promise in mouse models will be discussed. these include local delivery to mucosal surfaces and delivery into specific cells via cell surface receptors using an antibody fragment fused to protamine. these sirna complexes silence gene expression in vivo only in cells bearing the targeted receptor. experiments showing efficient, effective and cell-specific delivery in a mouse tumor model will be discussed. in addition, encouraging preliminary data using rnai for a microbicide to prevent sexually transmitted infection will be presented. rna interference (rnai) holds significant progress as a therapeutic approach to siolence disease-causing genes, particularly those that encode "non-druggable" targets. the key hurdle for rnai therapeutics is in vivo delivery. a critical requirement for achieving systemic rnai in vivo is the introduction of "drug-like" properties, such as stability, cellular delivery and tissue biodistribution, into synthetic sirnas. our progress in achieving in vivo silencing of endogenous genes with chemically modified sirnas will be discussed. hiv-1 replication in human t cells can be inhibited by stable expression of a short hairpin rna targeting the viral nef gene (shrna-nef). however, hiv-1 escape variants emerge after prolonged culturing, and all but one escape mutant acquire a mutation in the shrna-nef target sequence. we observed single and multiple nucleotide substitutions, but also partial or complete deletion of the target sequence. these results demonstrate the sequencespecificity of this antiviral approach. we observed an inverse correlation between the level of resistance and the stability of the shrna/target-rna duplex for most of the escape mutants. however, two escape variants did not follow this pattern, including an escape mutant with a single point mutation at position −7 upstream of the target sequence. these mutants provide a much higher level of resistance than expected based on duplex stability, which is obviously not affected in the −7 mutant. we demonstrate that these mutants adopt an alternative rna secondary structure that occludes the target sequence. this results in reduced shrna-nef binding and provides a novel mechanism for rnai-resistance. to avoid viral escape, one should ideally target hiv-1 with multiple effective shrnas against conserved genome sequences. we performed a large scale screening to identify such targets, and we have identified at least nine genome segments that can be targeted effectively with shrnas. these potent antivirals are currently being assembled in a lentiviral vector for gene therapy applications in hiv-infected individuals. furthermore, we will describe approaches to forecast viral escape routes and to effectively block such evolutionary paths with additional rnai measures. in this study we analyzed the effect of antibodies against electronegative ldl on the development of atherosclerotic lesions in low-density receptor-deficient (ldlr −/− ) mice. two groups of (ldlr −/− ) mice (eight females) fed 0.5% cholesterol-enriched chow were used. the first group received a monoclonal antibody against electronegative ldl (100 g) and the second one received pbs (controls). additionally, other two groups (eight males) of (ldlr −/− ) mice were treated with a polyclonal antibody against electronegative ldl (100 g) or pbs (controls). antibodies were administered by intravenous route one week before starting the hypercholesterolemic diet and then every week over an experimental time of 21 days. afterwards, quantification of atherosclerotic plaque area of heart and aortic arch was done by analysis of the slices stained with oil red/hematoxolin/light green with the image propus software. the passive immunization with either monoclonal or polyclonal antibodies against electronegative ldl significantly reduced the atherosclerotic plaque areas in atherosclerosis-prone ldlr −/− mice. in conclusion, antibodies against electronegative ldl administered by intravenous route may play a protective role in atherosclerosis. supported by fundação de amparoà pesquisa do estado de são paulo (fapesp, scholarships to d.m.g., l.s. and a.b. and grants to m.h.k. and d.s.p.a.). the enzyme asparaginase from the procaryote escherichia coli or erwinia crysanthemi is used for the treatment of lymphoblastic leukaemia. the drug causes immunological reactions in despite of the treatment efficiency. asparaginase may also be obtained from saccharomyces cerevisiae and this enzyme could provide an alternative to its bacterial counterparts. in this study, the periplasmic nitrogen regulated asparaginase ii from s. cerevisiae, that is coded by the asp3 gene, was cloned and expressed in the methylotrophic yeast pichia pastoris under the control of the aox1 gene promoter. the recombinant p. pastoris strain was cultured in shake flasks and in a 2 l instrumented bioreactor. in both cases it was observed specific enzyme yields seven fold higher in comparison to that using a nitrogen derepressed strain of s. cerevisiae, reaching 800 u/g dry cell mass. high cell density cultures carried out in the 2 l bioreactor, in which it was attained 107 g dry cell mass/l, resulted in a dramatic improvement in asparaginase fermentation. as such, it was measured enzyme yields of 85,600 u/l and productivities of 1083 u/l h. department of biochemistry and food chemistry, biotechnology, university of turku, tykistokatu 6, biocity 6th floor, 20540 turku, finland. e-mails: lorenzo.galluzzi@utu.fi; deadoc@libero.it; deadoc@aliceposta.it (l. galluzzi) the bacterial luciferase operon from the bacterium photorhabdus luminescens has been used, since its first description, for exceptionally different applications. these ranged from the environmental monitoring to the cell tagging, from the analysis of cellular metabolism to the high-throughput screening of novel compounds. the wild-type luxcdabe operon has been engineered in countless ways (for instance by changing the order of the constituent genes, by optimizing the codon usage and by coupling it to many promoters) and has been expressed in prokaryotic and eukaryotic organisms in order to meet precise research and commercial needs. upon the operon expression light is emitted as the side product of a chemical reaction catalyzed by the luciferase enzyme, an ␣␤ heterodimer encoded in luxa and luxb genes. the reaction involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (fmnh 2 ) with the liberation of excess free energy in the form of a blue-green light at 490 nm. the luxcde genes code for the polypeptides (transferase, synthetase, and reductase) forming the fatty acid reductase complex that catalyzes the conversion of fatty acids into the long-chain aldehyde required for the luminescent reaction. noteworthy is that for the production of the substrates for the luciferase both atp and nadph are required, while neither is involved in the actual light emitting reaction (wilson and hastings, 1998) . recently, the coupling of the luciferase operon to regulated promoters lead to the construction of genetically modified bacteria able to sense the presence of chemicals and to respond, in a dosespecific manner, with light emission. this approach has been applied to the detection of antibiotics in samples from the food industry as well as to the detection of heavy metal ions in environmental samples (kurittu et al., 2000; bechor et al., 2002) . in addition, it opened the possibility of screening wide libraries of new compounds looking for molecules with pre-determined features, able to induce the bioluminescent response by de-repressing the lux operon transcription when incubated with the appropriate bacterial sensor. the high throughput and low costs are the main advantages of this system, which shows as well a certain degree of specificity (galluzzi and karp, 2003; galluzzi et al., 2004) . nevertheless, since the in vivo bioluminescence relies upon a complex network of biochemical reactions, under certain circumstances the light emission is not a direct consequence of the lux operon transcriptional induction but it more likely originates at a posttranslational stage. as a matter of fact, one can suppose that a change in the light emission will be observed whenever the concentration of one or more substrates for the lux␣␤ reaction occurs. consequently, all the molecules sharing the ability to impair the delicate chemical equilibrium regarding the compounds involved in bioluminescence will be sensed by the bacteria as inducing compounds. this, in turn, will result in a loss of specificity of the assay. we investigated this aspect of the whole-cell assays based upon the bacterial luciferase operon for drug discovery by means of a reporter plasmid in which the luxabcde genes, rearranged and optimized for the after 60 min (black downward arrow) of incubation at 37 • c under vigorous shaking the following concentrations of trimethoprim were added to the growing cells: 25 g/ml (triangles), 50 g/ml (circles) and 250 g/ml (rumbles). water was administered to control cultures (squares). light emission was quantified every 30 min by means of the wallac victor 2 multilabel counter (perkin-elmer, turku, finland) and normalized to the absorbance, measured at 600 nm with the same device. multi-96 white-walled transparent-bottomed plates were used for the assays (nalge nunc, usa). between the measurements the plates were kept at +37 • c under vigorous shaking. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). expression in gram + organisms, are under the control of the qacr regulatory region from staphylococcus aureus . non-pathogenic s. aureus rn4220 cells bearing the pqaclux plasmid (cultivated in lb broth supplemented with 0,5% d-glucose and 10 g/ml chloramphenicol) emit light upon the specific transcriptional induction with quaternary ammonium compounds, widely used as surface disinfectants and in many over-the-counter drugs . however, the incubation of the same cells with an inhibitor of the dihydrofolate reductase enzyme, trimethoprim (sigma-aldrich chemie, steinheim, germany), enhanced in a dosedependant manner the light emission observed upon the induction with the optimal concentration (1 g/ml) of benzalkonium chloride (bc). the extent of this increase in luminescence varied from 5-10% to more than 200%, according to the trimethoprim concentration and to the measurement time. interestingly, when the same plasmid is carried by escherichia coli xl1 cells, the lux operon is expressed constitutively (since the qacr regulatory region is not functional in xl1 cells) and at much higher levels than in induced rn4220 cells. also in this experimental system the incubation with trimethoprim results in a dramatic increase of the luminescent signal from the cultures. the explanation for these observations can be found in the molecular mode of action of trimethoprim. the inhibition of dihydrofolate reductase, indeed, directly leads to the accumulation of its substrates, among which is nadph, deeply entangled in the biochemical network of reactions centred on the light emission from the lux operon. nadph provides the reducing power to restore the reduced flavin mononucleotide pool and it is as well involved in fig. 2 . xl1/pqaclux growing cells were incubated with the following concentration of trimethoprim: 25 g/ml (triangles), 50 g/ml (circles) and 250 g/ml (rumbles). water was administered to control cultures (squares). light emission and absorbance measurements were performed as previously described for rn4220 cells. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). the diverse level of growth observed for rn4220 and xl1 cells can be accounted by the different antimicrobial activity exerted by trimethoprim towards gram− and gram+ cells and by the lower activity of the promoter which in pqaclux plasmid drives the transcription of the selection marker (chloramphenicol acetyl transferase). the production of the long-chain aldehyde, both substrates of the lux␣␤ heterodimer (wilson and hastings, 1998) . in conclusion, here we demonstrate that the use of light emission from the bacterial luciferase as a transcriptional reporter has to be very carefully controlled, since some molecules (here trimethoprim) could mimic to some extent a specific promoter activation by impairing the delicate intracellular biochemical equilibrium. on the reverse side of the coin, fig. 3 . simplified scheme depicting the bacterial folate metabolic pathway. only part of the reactions and compounds are reported. trimethoprim inhibits the nadph-dependant reduction of dihydrofolic acid into tetrahydrofolic acid catalyzed by dihydrofolate reductase. this results in the accumulation of both substrates, which become available for other reactions, and in the depletion of tetrahydrofolate, the major c 1 carrier in the synthesis of purines, thymidine, glycine, methionine, and pantothenate in bacteria. for antimicrobial purposes trimethoprim is often associated with sulfonamides, with which it displays a synergistic activity (since they act on the same pathway but at an earlier reaction) (scholar and pratt, 2000) . however, it has to be noted that the lux reporter system could be used in many different in vivo experimental setups, where the change in the intracellular concentration of nadph, atp or other metabolites would be sensibly detected. we have carried out the construction of five pichia pastoris strains harboring in their respective genome three different growth hormones cdna's (22 and 20 kda human growth hormones and bovine growth hormone), a shrimp (litopenaeus vannamei) trypsinogen cdna and a bacterial (bacillus subtilis) phytase gene. in all the cases, the same kind of expression vector and the same transformation technique were used. each dna was fused in frame to saccharomyces cerevisiae alpha-factor secretion signal to lead the secretion of the foreign protein into the culture medium. the induction of each recombinant strain, all with his + and mut + phenotype, was carried out in shake flaks using methanol buffered minimal medium (bmm) and growth rates on methanol determined. production level of secreted proteins was evaluated by protein analysis by sds-page. furthermore, the expression with three of the constructed strains was performed in a 5-l bioreactor and the level of secreted protein determined in each case. both human growth hormones (hghs) were secreted into the culture medium with a high degree of purity obtaining up to 64% of hghs of total proteins in crude fermentation medium and 3-18 mg/l of hghs. neither bovine growth hormone, shrimp trypsinogen or bacterial phytase were detected in methanol induced cultures of the respective strains, in spite of the same culture conditions were used for all p. pastoris strains. the growth rates on methanol were different between some strains. in fermentor cultures the hghs production were increased and shrimp trypsinogen was produced and secreted into the culture medium after improving the culture conditions. bovine growth hormone was detected only when the culture conditions were modified. the protein production level for each strain was affected by the proprieties of each recombinant protein produced. competitive advantages of the diagnostic method for invasive amoebiasis using preserved antigenic extracts without using enzymatic inhibitors m.s. flores 1,2* , e. tamez we have patented a method to diagnose invasive amoebiasis using a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors (ic:mc). the available tests for serologic diagnosis of invasive amoebiasis like elisa and indirect haemaglutination (iha) do not have consistent results in endemic zones. here we show the advantages of the assay and the validation of this diagnostic test for invasive amoebiasis. we demonstrated the reduction of proteolytic activity of ic:mc compared with the proteolytic activity of crude extract and crude extract with enzymatic inhibitors using assays. we displayed the ic:mc sds-page pattern and the western blot (wb) pattern useful for diagnosis. to search the clinical utility of this test we examined the wb obtained with sera from patients with different liver diseases; 90 patients had invasive amoebiasis and 45 patients had other liver diseases. the results were compared with those of iha test. also we have tested the accuracy of wb using sera from people with multiple intestinal parasites, like giardia lamblia, hymenolepis nana, blastocystis hominis, entamoeba coli, etc. the sensibility of the wb using the preserved amoebic antigens was 99%, specificity was 100%, positive predictive value was 100%, negative predictive value was 98% and accuracy was 99%. the wb did not exhibit cross reactions with sera from persons with intestinal parasites. our test was better than the (iha) test commonly used in endemic zones. these results show the improvement of using the preserved amoebic antigens in diagnostic tests. also they prove the diagnostic accuracy of our new wb test. antibacterial and antifungal activity of heracleum sphondylium subsp. artvinense yasemin kaçar 1 , sema tan 2 , aysun ergene 2 , perihan güler 2 , semra mirici 3 , ergin hamzaoglu 2 , ahmet duran 4 , sinem yildirim 2 : 1 mersin university, faculty of science and literature, department of biology, 33800 mersin, turkey; 2 kırıkkale university, faculty of science and literature, department of biology, 71450 yahsihan-kırıkkale, turkey; 3 akdeniz university, faculty of education, department of biology education, 07400 antalya, turkey; 4 selcuk university, faculty of education, department of biology education, 42300 konya, turkey turkey is covered yearly with a huge number of plant species. about 9222 species are condenced on the region that between asia and europe. many plant species have been used in folkloric medicine to treat various ailments. heracleum l (apiaceae) is include over than 70 species on the world. this variety is represent with 17 species in turkey that seven species are endemic. heracleum sphondylium subsp. artvinense is endemic species for turkey. ethanolic and aquous extract of heracleum sphondylium subsp. artvinense were investigated for their antimicrobial activities against six bacterial species (e. feacalis, e. coli, s. aureus, p. aeruginosa, l. monocytogenes, shigella) and two yeast (c. albicans, c. krusei). both ethanolic and aqueous extract of heracleum sphondylium subsp. artvinense showed antimicrobial activity against the gram negative bacterium (shigella) and gave the best activity against c.albicans. to develop artificial vectors allowing nucleic acid to transfect into mammalian cells are crucial for extending gene therapy. synthetic vectors based on lipid molecules are particularly attractive because of their potential safety. however, the low encapsulation efficiency of nucleic acid is one of the problem to be solved. recent advances in dna-lipid complex have improved this drawback, and now some lipid molecules are used as transfection agents. in particular, cationic lipids interact with negatively-charged cell surfaces and nucleic acids. the former interaction results in delivery of the nucleic acids directly across the cell membrane. on the other hand, the latter interaction improves the efficiency of nucleic acids entrapment. in this study, we developed a preparation method of "nanovesicle" containing nucleic acids by using reverse micellar solubilization. since dna interacts spontaneously with cationic amphiphiles, complete extraction of the dna molecules into an organic phase using dimethyl distearyl ammonium bromide (2c18ab), an oil-soluble cationic surfactant, is achieved. the re-encapsulation of the reverse micellar droplets solubilizing dna by water-soluble surfactants facilitates the formation of nanovesicles. a high salt concentration at the re-encapsulation step promotes the production of nanovesicles containing dna molecules. eventually, more than 80% of dna was encapsulated in this nanovesicles under the condition of 6m nacl and 5% (w/v) cetyltridecyl ammonium bromide (ctab). in addition, the nanovesicles prepared at 45 • c was smaller than that prepared at room temperature. the resultant 2c18ab/ctab nanovesicle was ca. 16 nm. asymmetric and chemically-modifiable vesicle surface are effective to design gene delivery system. moreover, such a small dna (or rna) carrier has a potential for novel gene therapy application from skin. the key players in clinically important inflammatory diseases, endothelial cells and leukocytes, communicate through membranebound cell adhesion molecules (cam's). obvious strategies for therapeutic intervention include specific means to affect the expression of cam's on the cells involved. rna-interference (rnai) is a well known means to achieve specific gene-inhibition. for this study, two cam's were chosen, namely vascular cell adhesion molecule-1 (vcam-1) as a target for inhibition, and intercellular adhesion molecule-1 (icam-1) as a non-target reference. we designed short interfering rna (sirna) oligos for vcam-1 in order to selectively inhibit the expression of this cam in cultured human vascular endothelial cells (huvec). real-time rt-pcr showed an 80% down-regulation of vcam-1 mrna while the expression of icam-1 remained unaffected. neither cam was affected by non-specific sirna. in order to further substantiate the potential use of sirna for therapeutic purposes, we have set out to investigate two things: (1) does vcam-1-specific sirna affect the amount of vcam-1 on the surface of huvec? (2) is adhesion between leukocytes and endothelial cells affected by vcam-1-specific sirna? the manufacturing of plasmid dna (pdna) is crucial to obtain a consistent product for gene therapy applications. although flowsheets for pdna production are established on the basis of experience, simulation tools provide a valuable help for evaluating alternatives. a process designed to produce 23 kg pdna/year is analysed with the superpro designer tool. the target pdna is amplified in escherichia coli. after harvest, alkaline lysis is used to disrupt cells and release pdna and impurities. precipitations with isopropanol and ammonium sulphate are performed to concentrate/pre-purify pdna prior to hydrophobic interaction chromatography. experimental data is used as input for simulation. inventory analysis identified water, yeast extract, tryptone, isopropanol and ammonium sulphate as the major raw materials. major raw material costs (50%) are related to fermentation components. economic analysis indicates a unit production cost of $ 375/g pdna. for a selling price of $ 1667/g, the payback time was 1.2 years and the roi was 88.6%. an environmental analysis highlighted the replacement of the isopropanol precipitation for a microfiltration step as a benefit which would: (i) reduce the cost of raw materials (13.8%), (ii) reduce the environmental impact associated with isopropanol (70%) and (iii) reduce costs associated with the treatment/disposal of liquid waste (32.3%). preparation of chitosan microspheres for controlled release of somatotropin s. simsek 1 , j. introduction: proteins and peptides have received extensive interest for their therapeutic applications in clinical applications. in order to achieve high administration efficacy of proteins, polymeric particulate carriers have been developed as an effective way to control the drug release profile and to protect the protein molecules from degradation. somatotropin also known growth hormone is a protein hormone of about 190 amino acids. growth hormone is of considerable interest as a drug used in both humans and animals. chitosan a natural linear biopolyaminosaccharide is obtained by alkaline deacetylation of chitin. properties such as biodegradability, low toxicity and good biocompatibility make it suitable for use in biomedical and pharmaceutical formulations. the aim of this study was to prepare chitosan microspheres containing somatotropin and to investigate these microsphere formulations in-vitro release properties. methods: somatotropin-chitosan microspheres were prepared as follows: chitosan was dissolved in acidic solution (2%) containing polysorbate 80. sodium sulphate solution (20% w/v) containing somatotropin was added into the chitosan solution and mixed 500 rpm for a hour. resulting suspension was centrifuged 15,000 rpm 15 min at 4 • c. the formed microspheres were freeze-dried and sieved. in-vitro release studies were performed in ph 7.4 phosphate buffer and time interval samples were removed and analysed by bradford protein assay method. results: microspheres were obtained by using chitosan. protein encapsulation efficiency was between 95 and 99%. average particle size of microspheres was between 48 and 57 m. during to in-vitro release studies burst effect was observed with chitosan microspheres. for decreasing the burst effect gluteraldehit, betacyclodextrin and poly ethylene oxide were added to formulations. conclusion: according to our results modified chitosan microspheres are promising vehicles for controlled release somatotropine delivery. cancer immunotherapy using hyperthermia with magnetic nanoparticles and dendritic cells k. tanaka, a. ito, t. kobayashi, t. kawamura, s. shimada, k. matsumoto, t. saida, h. honda department of biotechnology, school of engineering, nagoya university, nagoya, aichi 464-8603, japan. e-mail: h041306d@mbox.nagoyau.ac.jp (k. tanaka) our hyperthermia system utilizes magnetic nanoparticle covered with lipid layer including cationic lipid (magnetite cationic liposomes, mcls) as a heating mediator and necrotic cell death is induced by means of locally generating heat. in this process, heat shock proteins (hsps) are strongly induced and released. dendritic cells (dcs) are potent antigen-presenting cells (apcs) that play a pivotal role in regulating immune responses in cancer, which is in carrying antigens to apcs and in the maturation of dcs by acting as a danger signal. in the present study, we investigated the therapeutic effects of dc therapy combined with mcl-induced hyperthermia on b16 melanoma. in an in vitro study, when immature dcs were pulsed with b16 cells heated at 43 • c for 30 min, mhc class i/ii, costimulatory molecules cd80/cd86, and chemokine receptor ccr7 in the dcs were up-regulated, thus resulting in dc maturation. c57bl/6 mice bearing a b16 melanoma nodule were subjected to combination therapy using hyperthermia and dc immunotherapy. mice were divided into four groups: group i (control), group ii (hyperthermia), group iii (dc therapy), group iv (hyperthermia + dc therapy). complete regression of tumors was observed in 60% of mice in group iv, while no tumor regression was seen among mice in the other groups. increased ctl and nk cell activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of b16 melanoma cells. this study has important implications for the application of mcl-induced hyperthermia plus dc therapy in patients with advanced malignancies as a novel cancer therapy. fermentation of a marine bacterium for the production of cytotoxic compounds vicky webb 1 , els maas 1 , eiichi akaho 2 , hiroto kambara 2 , debbie hulston 1 , anna kilimnik 1 : 1 marine biotechnology, national institute for water and atmospheric research ltd, kilbirnie, wellington new zealand; 2 faculty of pharmaceutical sciences, kobe gakuin university, kobe 651-2180, japan marine bacteria are a potential source of novel compounds for the pharmaceutical industry. new zealand marine bacteria were isolated from a variety of sources and initially bacterial supernatants were screened using a cell based mtt cytotoxic assay. two bacteria were chosen for further fermentations in different media to optimise cytotoxic compound production. the media used were selected for their diverse ingredients. they were either carbon rich, nitrogen rich, starch rich or a basic seawater medium. the fermentations were extracted using ethyl acetate and methanol prior to assaying. the results showed that cytotoxic compound production was enhanced ten fold in the starch rich medium compared to the other media. the levels of cytoxicity appeared to be depended on the cell line used, with the epithelial lung carcinoma cell line a549 being more sensitive to the cytotoxic compounds than the human fibroblast cell line mrc-5. isolation and identification of marine bacteria from deep-sea sediments els maas 1 , cara brosnahan 1 , vicky webb 1 , helen neil 2 , phil sutton 2 : 1 marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington new zealand; 2 oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand polystyrene micro plate activated by utilizing a common co-60 gamma source with a crotonic acid. the well surface have been studied in term of optical quality, protein (h-igg) binding capacity and stability. a significantly enhanced total capacity and strength of binding to grafted surfaces was demonstrated as compared to passive adsorption of the proteins to untreated surfaces.the majority routine laboratory tests for the measurement of rheumatoid factor (rf) are semi quantitative and in order to achieve accurate, sensitive and specific rf assay, we are developed enzyme linked immuno sorbent assay (elisa) for human igm-rf kit. stable and reproducible binding of antigen (human-igg) to well of micro titer plate is a prerequisite for rf-elisa kit. the principle of the assay was that the antibodies in serum of patients with rheumatoid arthritis (ra), commonly rheumatoid factor (rf) are directed to the fc part of human igg. in most cases rf belong to the igm class, the well of micro titer plate are coated with antigen (h-igm), and antibodies (h-igg) binding to immobilized antigen is detected by adding enzyme conjugate (anti human-igm-hrp-enzyme) to the wells, substrate was used for color reaction.the performance characteristics of the assay was, the coefficient of variation (c.v) of intra and inter assay was 4.4% and 2.2% respectively, the linearity is ranging from 86 to 112%, and the recovery for three different sera is ranging from 89 to 106%. the data presented in this paper indicated that the activation of polystyrene micro titer plate by the gamma rays could be use for preparation of igm-rf kit and may be others immunoassay techniques. overcoming the nuclease barrier to gene expression during trafficking of plasmid dna vectors a.r. azzoni 1 , a. tavares 2 , g.a. monteiro 1 , d.m.f. prazeres 1 : 1 centro de engenharia biológica e química (cebq), instituto superior técnico, 1049-001 lisboa, portugal; 2 instituto gulbenkian de ciência, rua da quinta grande 6, 2780-156 oeiras, portugal. e-mail: azzoni@ist.utl.pt (a.r. azzoni) inefficient nuclear delivery of plasmid dna (pdna) vectors is thought to be a bottleneck to gene transfer in gene therapy and dna vaccination utilizing non-viral delivery systems. one of the main barriers found by pdna vectors during trafficking to the nucleus is degradation by a population of endo/exo-nucleases. this barrier may be partially circumvented by shielding the pdna from the nucleaserich cell environment with adjuvants or by using nuclease inhibitors. a different approach that has been studied at the cebq is the generation of pdna vectors that are more resistant to nuclease action a priori. in this work, the nuclease barriers to gene expression are being studied aiming at the generation of pdna with improved resistance to nucleases and thus higher transfection efficiency. by engineering the plasmid labile sequences, new plasmid vectors with an improved resistance to physical-chemical and biological degradation are being generated. this was indicated by an extended half-life of the supercoiled isoforms during storage and when the plasmids were exposed to nucleases present at eukaryotic cell lysates and mice plasma. the intracellular trafficking of the new plasmid vectors through the cytosol of mammalian cells was then assessed by fluorescence in situ hybridisation (fish) and the expression of the reporter protein (egfp) was detected by fluorescence microscopy. identification and evaluation of antibacterial phytochemicals of fishbone fern (nephrolepis cordifolia) rikhia chakraborty 1,2 , promod kumar verma 2 : 1 department of cancer biology, lerner research institute, 9500 euclid avenue cleveland, oh 44195, usa, 2 department of biotechnology, guru nanak dev university, amritsar, punjab 143005, india. e-mail: riar5400@rediffmail.com (r. chakraborty) in this study, different aqueous and organic extracts from the fern, nephrolepis cordifolia, were used for screening tests for antibacterial effects. protein and lipid extracts were first tested for antibacterial activity. subsequently, crude extracts of leaves, roots, and stems were prepared in methanol, ethanol, chloroform, hexane, petroleum ether, diethyl ether, and water using optimized standard protocols. each fraction was tested for anti-microbial effect through agar well-diffusion assay, and paper disc method. the antibacterial spectrum against which the fern is active was thus determined. dosagedetermination for optimum activity was also determined for each of the extracts. bacillus and staphylococcus were used as the indicator test-organisms. the results were very encouraging; being effective even at the 54th day, thus showing that the antibacterial properties were not due to any changes in external factors and physiological effects. ethanol, methanol and chloroform extracts from the subaerial portions had strong anti-microbial properties. agar-well diffusion assay and paper-disc diffusion assay done for different solvent fractions were giving comparable results. 2.5 mg was adequate for maximum effect against b. circulans, s. aureus, s. epididermis, and streptococcus sp. 7.5 mg was adequate as effective dosage for kleb-siella pneumoniae. 10 mg was required for mycobacterium bovis, e. coli. pseudomonas was not showing any susceptibility. given the broad spectrum of activity, especially towards the gram-negative bacteria, nephrolepis cordifolia is definitely a promising plant having pharmacological importance. for a full interpretation of the present results further investigations are necessary to elucidate the different physical and chemical parameters of the active principles and also to determine the mechanism of action. the present work highlights n. cordifolia as a plant having a broad spectrum of antimicrobial activity, a phenomenon very rarely observed in the plant kingdom. bacteria (escherichia, salmonella, proteus, staphylococci and bacillus) were isolated from hospital soil using selective enrichment and growth on selective and differential medium, viz. macconkey's agar, clyed medium and baird parkers medium. confirmation and species identification was carried out by biochemical and serological tests. from these isolates, three different pathogens were used to study multiple antibiotic resistances. e. coli bj 83 showed resistance to ampicillin, streptomycin and cefurixime. s. typhi and s. aureus showed resistance to ampicillin and cefuroxime. assay using octadisc using e. coli and s. typhi showed a broader resistance pattern to antibiotics including amoxicillin, clavulanic acid, cephalexin, chloramphenicol, ciprofloxacin, and cotrimoxazole. the r plasmid profile was studied to understand the mechanism of drug resistance. to combat the problem of drug resistance, a strategy of combined antibiotic response of cultures were studied. such a synergistic combination would possibly have the effect of overcoming multiple antibiotic resistances. for e.g. kanamycin resistance strains were inhibited in presence of kanamycin and cefotaxime. further, the bactericidal activities of antimicrobials in honey and garlic were also tested. the mic of honey was observed to be 4%, while that of garlic was between 0.1 and 1%. honey and garlic were also found to inhibit the growth of organisms in the presence of antibiotics. kanamycinresistant e. coli was unable to grow in presence of kanamycin and 0.08% garlic. these traditional agents, long used in ayurvedic system of medicine in india, could be further explored for potent antimicrobial properties. hepatitis b virus (hbv) infection is s global health problem. assays for hbv antigens and antibodies are widely available and standardized. extremely sensitive qualitative pcr kits are also available for detection of hbv in serum. hbv pcr kit may be useful in assessment of occult hepatitis b in hbcab positive alone subjects and carriers. a positive pcr results show presence of virus articles in serum without considering serologic results. but there are differences in efficiency of hbv dna amplification kits. in this study we compare two commercially available hbv pcr kits for evaluation viremia of hbsag positive carriers and hbcab alone positive subjects. material and methods: of the 368 randomly selected subjects serologically examined for hbv, 49 and 43 were positive for hbsag and hbcab alone respectively. both later groups were tested for hbv dna by two commercial kits, hbv pcr detection kit (cinnagen, iran) and hbv pcr test (pazhohesh azma, iran). dna extraction kits recommended by each manufacturer were used for hbv dna extraction. amplicons in both kits were a highly overlapped fragment in 5 conserved sequence of viral genome. results: of the 49 hbsag positive carriers, hbv dna was detected in 37.4 and 65.3% using kit1 and kit2 respectively. only 28.6% were positive in both kits. in hbcab positive subjects (n = 43), 23.3% were positive by kit 2 and all samples were negative when tested by kit 1. there was a significant difference between two kits. sensitivity of kit 1 and kit 2 were 48.6 and 91.4%, respectively. overall, kit 2 increased the detection rate of hbv dna by 88.2%. discussion: our study show there is a significant variation between these two commercial kits especially in hbcab positive subjects that the copy of virus is very low. from these results, it can be concluded that the unstandardized kits have not compatible results, and pcr test interpretation should be done with great care. . the antibacterial activity of the extracts was evaluated based on the inhibition zone using plate diffusion method. most of the extracts were active against both gram positive and gram-negative bacteria, but pseudomonas aerugenosa, bacillus subtilis and sarcina marcescens, were more susceptible to almost all the extracts. finally, further research will be done to elucidate the nature of the active compound and investigate for peptides by using protein gel immobilization bioassay. short interfering rna delivery and gene silencing using polymeric nanocarrier systems k.a. howard 1 , x. liu 1 , d. oupicky 2 , f. besenbacher 1 , j. kjems 1 : 1 inano, university of aarhus, denmark, 2 department of pharmaceutical sciences, wayne state university, detroit, usa the effectiveness of a drug is determined by the ability to migrate through the body and reach target sites in therapeutically relevant levels. nanocarriers for delivery of bioactive agents are being developed at inano to maximise drug payload at target sites. the inclusion of "biological triggers" into the nanocarrier design is used for modulation of cellular nucleic acid trafficking and increased target interaction. chitosan and peptide-based polymers were used to formulate nanocarriers in the size range 30-250 nm containing small interfering rnas (sirnas) for gene silencing applications. page analysis showed the structural integrity of the sirna was maintained during particle formation. in systems composed of bioresponsive polymers, nanocarrier disassembly and sirna release under cellular conditions were shown, using atomic force microscopy. the time course for sirna uptake into nih cells was visualised using confocal microscopy. in addition, sirna localisation within cells could be modulated by the composition of the polymer used. the ability of the nanocarrier system to mediate gene expression was investigated in a cell line stably expressing enhanced green fluorescent protein (egfp). furthermore, the various delivery systems were tested in a mouse model stably expressing the egfp protein using both nasal and intravenous delivery routes. the use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. however, the presence of xenoreactive antibodies in humans directed against swine gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute immunological reaction. the graft of genetically modified organ of a swine depleted of enzyme ␣1,3-galactosyltransferase that is responsible for gal antigen origin, would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. to prevent hyperacute rejection it is also possible to modify swine genome by human genes controlling enzymatic cascade of complement or modifying the set of donor's cell surface proteins. for this purpose genetic constructs containing inactivated ␣1,3-galactosyltransferase gene, human cd59, cd55 and cd46 genes controlling complement activation and human genes encoding ␣1,2-fucosyltransferase and ␣-galactosidase enzymes modifying cell surface proteins were prepared. these genetic constructs were transfected into the pig foetal fibroblast using lipofection method. after selection, molecular and cytogenetic characteristic of cells with transgene integrated into the host genome were performed. introduction: protease 2a(2a-pro) of coxsackievirus b3 (cvb3) plays major role in viral replication. in case of infection, viral proteins are being synthesized from viral mrna using host biosynthesis machinery. 2a-pro of virus, after being synthesized, exhibit two critical functions, cleavage of viral proteins and breaking eif4g (eukaryotic initiation factor 4g-formerly called p220) which leads to host cell translational system shot-off. the enzyme plays essential role in viral replication and cellular damage. to understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure 2apro enzyme. in this study an expression system with efficient and high yields was obtained. methods: cdna of 2apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pet22b(+) and since 2a-pro is a toxic product, naturally before induction its expression will act on the host and damage the cells. for this different hosts were checked and finally, blr(de3) plyss which carries an extra-plasmid for lysozyme expression that minimizes unwanted target protein production (leakage) was selected. on the other hand for biological activity assay, polyclonal antibodies against antigenic sites of p220 was prepared by synthesizing small peptides, corresponding to antigenic site of p220 coupling to klh and injecting subcutanously to rabbit. then, the enzyme and its substrate (hela cells lysate that contain p220) were incubated together for different times intervals. results: the recombinant product was confirmed by sds polyacrylamide gel electrophoresis and immunoblot analysis. also p220 cleavage by 2apro was assessed by sds-page and western blot analysis. cleavage of p220 by r2a-pro was prominent after 24 h. so recombinat 2a-pro with good activity was prepared. application of bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system on production of glycoprotein in larvae of silkworm ayano kageshima, tatsuya kato, misun kwon, enoch y. park department of applied biological chemistry, shizuoka university, ohya 836, suruga-ku, shizuoka 422-8529, japan bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system was applied on production of glycoprotein in larvae of silkworm. the bacmid system of autographa californica nuclear polyhedrosis virus (acnpv) has already been established and widely used. since the acnpv does not have a potential to infect silkworm we developed the first practical bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system directly applicable for the protein expression of silkworm. by using this system, the green fluorescence protein and glycoprotein, human ␤1,3-n-acetylglucosaminyltransferase 2 were successfully expressed in silkworm larvae not only by infection of its recombinant virus but also by direct injection of its bacmid dna. three different kinds of signal sequences were tested for the secretion of glycoprotein into hemolymph of silkworm. signal peptides of prophenoloxidase-activating enzyme and bombyxin: insulin-like brain secretory peptide showed the highest secretion ratio, 99% of total expressed ␤1,3-n-acetylglucosaminyltransferase 2 was secreted into hemolymph of silkworm. using bacmid system 50 mu/ml of ␤1,3-n-acetylglucosaminyltransferase 2 was expressed in hemolymph of silkworm, which was 2-three times higher than that of insect cell. silkworm is one of the most attractive hosts for large-scale productions of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. this method provides the rapid protein production in silkworm, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. we have established an efficient system for foreign gene expression in lily (lilium longiflorum) pollen in a transient mode. pollen was transformed using agrobacterium via vacuum filtration for 20 min. the pollen germinated for 24 h was analyzed to confirm its foreign gene expression in molecular analysis. mouse fed the transgenic pollen culture for 8 weeks showed immune reaction specific for pollen-derived recombinant protein. and the igg level was highly elevated by one time boosting injection afterwards. the lily pollen system may be suggested as a novel type of biofactory for producing edible vaccine with rapidity. anti-apoptosis engineering with the 30kc6 gene obtained from silkworm shin sik choi, won jong rhee, tai hyun park school of chemical and biological eng., seoul national university, seoul 151-744, korea the chinese hamster ovary (cho) cell line producing recombinant human erythropoietin (epo) was manipulated to express the 30kc6 gene, which was originally obtained from a silkworm. the expression of 30kc6 inhibited serum deprivation-induced apoptosis and increased the cell density and epo expression level per unit cell by five-and two-folds, respectively. an increase in these two factors resulted in a 10-fold increase in the volumetric productivity of epo. compared with the controls, the oligosaccharide structures of the epo synthesized by the cells expressing 30kc6 showed greater homogeneity. the terminal sialylation of the glycans of epo were promoted by the expression of 30kc6. the positive effects of 30kc6 expression on the cell viability and productivity were attributable to the stable maintenance of the mitochondrial activity. these results demonstrate that the cho cell line genetically engineered with the 30kc6 gene has a great potential for use in the production of therapeutic proteins. evaluation of fucoidan-chitosan hydrogels on superficial dermal burn healing in rabbit: an in vivo study a.d. sezer 1 , f. hatipoglu 2 , z. ogurtan 3 , a.l. baş 4 , j. introduction: healing of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the pharmaceutical biotechnology. fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. although a great number of studies on the different pharmacological properties of fucoidan are present, there is very limited information on the fucoidan-based system used in dermal burns. the aim of this study was to prepare chitosan hydrogel containing fucoidan and to investigate this hydrogel formulation for treatment of dermal burns on rabbits. methods: fucoidan-chitosan hydrogels were prepared as follows: the polymers were dissolved in acidic solution and sonicated for removing the air-bubbles then the gels were stored at +4 • c for in vivo studies. seven adult male new zealand white rabbits (mean weight, 3.8 ± 0.6 kg) were used for the evaluation of the gels on superficial dermal burns. the back of the rabbits were depilated and sedated. the wounds were made by circular stamp aluminium caps (3.8 cm 2 ) at 80 • c. four wounds were formed for each rabbit; (a) was treated with fucoidan-chitosan gel, (b) was treated with fucoidan solution, (c) was treated with chitosan gel (without fucoidan) and (d) as a negative control group. biopsy samples were taken at 7, 14 and 21st days at the beginning of the study and each wound site was macroscopically observed and evaluated histopathologically. results: oedema was not observed in all groups after 3 days treatment except controls. after 7 days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan gel and fucoidan solution. the best regenerate dermal papillary formation and the fastest closure of wounds were observed in group a after 14 days treatment. the wound epithel elongation and thickness values were measured; a (5566 and 179 m), b (3586 and, 146 m), c (3666 and, 162 m), d (3533 and, 134 m) at the end of the study. conclusion: re-epithelization and contraction of the wound area which was treated with fucoidan-chitosan hydrogel were faster than the other groups. the fucoidan-chitosan hydrogel formulations can be suitable for the treatment of dermal burns. aim: to analyze the neutralizing -related activity of antibodies against e1 region of hcv, specific polyclonal antibody was raised by immunized rabbits with synthetic peptide that had been derived from e1 region of hcv with the amino acid sequence e1 antibody [ghrmawdmm). materials and methods: hyper-immune hcv e1 antibodies were incubated over night at 4 • c with serum samples from patients positive for hcv rna, with different viral load, ranged 7-11 million copes/ml, then incubated 90 min to hepg2 cells. rt-pcr and flow cytometry were used to study the inhibition binding and entry effect of e1 antibody. direct immunostaining of e1 antibody conjugated with fitic and flow cytometry analysis showed reduced the mean fluorescence intensity in the samples pre-incubated with e1 antibody compared with samples without e1. of 18 positive serum samples, 13 (72%) samples showed completely inhibition of infectivity as detected by rt-pcr. conclusion: in house produced e1 antibody, blocks binding and entry of hcv virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. isolation of these antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his7tnf␤ and n19tnf␤ were expressed in e. coli. high solubility of n19tnf␤ was expected, however, both analogs his7tnf␤ and n19tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n19tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his7tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with 0.2% nls, while his7tnf␤ and n19tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. enzymatic modification of sphingomyelin long zhang, lars hellgren, xuebing xu biocentrum-dtu. e-mail: lz@biocentrum.dtu.dk (l. zhang) due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient, high yield production methods are of great interest. in the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin (sm) have been studied. sm is a ubiquitous membrane-lipid and rich in dairy products or by-products. in present study, we have optimized the production of ceramide from sm using phospholipase c from clostridium perfringens. water and enzyme amount had the biggest influence on sm hydrolysis in the system. botulinum neurotoxins constitute a family of bacterial toxins for botulism syndrome in human. the toxins bind with high affinity to nerve cells where they cause a complete inhibition and release of neurotransmitters and thereby produce flaccid paralysis. in this work we have reported isolation of the binding domain of type e neurotoxin by pcr and expressed in a proper expression vector. the output of this investigation can be used as a tool to study the mechanism of binding of holotoxins and also can be useful to study the antibody production against botulism syndrome. the synaptobrevin (vamp2) a protein which play a key role in the fusion and exocytosis of the vesicle of mammalian nerves terminals this protein is substrate different serotypes of botulinum neurotoxins. light chain of clostridium botulinum type b, d, f and g cleave end of neurons. due to intraction of clostridium botulinum neurotoxin with vamp2, this protein can be used as one of the toolsin the detection of poisings cause by this bacteria in the clinical laboratory using the enzyme with proof reading activity the above gene amplified by pcr technique for the production of the recombinant protein, the prokaryotic expression vectors (pet system) was used. a recombinant protein developed on poly acrylamide gel analyzed by western blotting and eliza. most children with down syndrome (ds) are born to younger mothers (<35 years). recent reports linking down syndrome (ds) to maternal polymorphisms at the methylenetetrahydrofolate reductase (mthfr) gene locus have generated great interest among investigators in the field. in this study, forty mothers with their affected outcomes and 100 control mothers were included. all mothers were subjected to complete medical and nutritional history with special emphasis on folate intake through food or oral supplementation. estimation of blood homocysteine level was done. also we examined the two polymorphisms in genes encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (mthfr), namely, 677c > t and 1298a > c. folic acid intake from food and from vitamin supplements was significantly low (below the recommended daily allowance) in the group of mothers with ds children compared to control mothers (p < 0.01) using student t-test. blood homocysteine was normal in both control and ds mothers. the prevalence of the two polymorphisms, namely, 677c > t and 1298a > c in mothers of ds children (case mothers) (n = 40) was compared with controls (n = 100). frequencies of mthfr genotypes (cc, ct, and tt) at position 677 demonstrated no difference between the case and control groups. genotype frequencies of mthfr at position 1298a (aa, ac, and cc) were different among the case and control mothers. we here report the first study on a possible relation between ds with mthfr 1298a > c genotypes in egypt. our results showed that mthfr 1298a > c polymorphism is a remarkable genetic entity among egyptian females with d.s. children. sufficient folate intake and supplementation is an important preventive strategy in overcoming the risk of nondisjunction. homologous recombination (hr) is the mechanism that permits the creation of genetically engineered strains through gene targeting. in order to further develop gene targeting techniques, notably for higher eukaryotes such as filamentous fungi, it is of crucial importance to fully understand the molecular mechanisms behind mitotic hr. in saccharomyces cerevisiae, a dna double strand break (dsb) is an essential intermediate in meiotic hr. however, as hr occurs at a low rate in mitotic cells, it has been difficult to determine the nature of the event(s) that triggers it. rad52 is a key protein involved in hr and is evolutionarily conserved from yeast to human. to shed light on the molecular events in hr, we have generated a large collection of defined rad52 mutant strains in the yeast s.cerevisiae. a screen of these mutants led to the identification of strains that fail to repair dna dsbs, yet are proficient for homologous recombination. this result strongly suggests that dsbs may not be the major cause of spontaneous mitotic hr and gives new perspectives in respect to novel potential gene targeting substrates. we have analyzed these separation of function mutants in a variety of new assays to obtain a more detailed understanding of their controversial phenotype. our latest results will be presented. the outcome of interferone plus ribavirine treatment of hepatitis c virus (hcv) genotype 4 is unfortunately poor. development of alternative therapy for this genotype is of a paramount importance. inhibition of hcv gene expression in vitro by the use of antisense phosphorothioate oligodeoxynucleotides (s-odn) against internal ribosomal entry site (ires) elements were associated with favorable results. to assess s-odn activity, previous studies utilized viral subgenomic or full cdna fragments linked to reporter genes and transfected into adhered cells or in a cell free system. in the present study we utilized hepg2 cells infected with native hcv rna of genotype 4. the culture system presented herein was shown to support hcv replication on the following bases (1) consistent detection of both plus and minus rna strands for 4 weeks in cells and in fresh culture supernatent, (2) ability of supernatent to infect naive hepg2 cells (3) consistent expression of core and e1 envelope proteins in infected cells throughout the 4 week culture. s-odns against aug translation start site (s-odn-1, nt 326-348) of the viral polyprotein precursor and stem loop iiid within the ires region (s-odn2, nt 264-282) were added to infected cells. intracellular viral replication was monitored by nested rt-pcr of plus and minus strands. the results of these experiments demonstrated that intracellular replication of hcv genotype 4 was completely arrested after 48 h in culture using either s-odn molecule (with more efficacy of s-odn1 than s-odn2) at concentrations as low as 1 m. the inhibitory effect of s-odn appeared to be specific to hcv replication since equal levels of human glyceralehyde 3-phosphate dehydrogenase (gapdh) gene expression were noted pre and post supplementation of s-odns. in conclusion, the present study provides evidence antisense phosphorothioate oligonucleotides have potent inhibitory effect on genomic replication of hcv genotype 4, the most common type in egypt. a sensitive and specific pcr-elisa was developed to detect shigella dysentery in food. the assay was based on the incorporation of degoxigenin-labeled dutp and a biotin-labeled primer specific for shiga toxin genes during pcr amplification. the labeled pcr product were bound to streptoavidin-coated wells of a microtiter plat and detected by an elisa. the elisa detecting system was able to increase the sensitivity of the pcr assay by up to 100-fold, compared with a conventional gel electrophoresis. the detection limit of the pcr-elisa was 0.1-10 cfu dependent upon shigella dysentery serotypes and genotypes of shigatoxin. the entire procedure took about 4 h. isolation, cloning, expression and purification of snap-25 as a substrate of botulinum neurotoxin type a and e m.l. mossavi 1 , f. ebrahimi 1 , j. amani 1* , h. basiry 1 , z. ahmadi 2 : 1 department of biology, faculty of science, imam hussein university, tehran, iran; 2 department of biology, faculty of medicine, bageatallah university, tehran, iran. e-mail: kpjamani@ihu.ac.ir (j. amani) clostridial neurotoxin inhibit neurotransmitter relase by selective and specific intracellular proteolysis of synaptosomal associated protein of 25 kda (snap-25); synaptobrevin/vamp-2 and syntaxin. snap-25 is one of the components that form docking complex in synaptic ends. this protein is substrate for botulinum neurotoxins type a and e. each of these toxin serotypes specifically cleave snap-25 in particular position and there by block docking and synaptic vesicle membrane fusion and finally prevent neurotransmitter exocytosis and transition of neurotic signals. in order to use the protein as a substrate for detection of different type of clostridium neurotoxin in vitro test, the protein was produced by recombinant technique. the dna encoding snap-25 was isolated from rat brain by pcr using the two primer the amplified fragment colonel into expression vector pet32a.the expression protein was purified by affinity chromatography. confirm by different method the his tag fusion protein was digested with entrokinase. the present work deals with the preparation of pure alpha-1antitrypsin (aat) protein from healthy subjects, which can be used in preparing its corresponding monospecific antibody in albino rabbits. this antibody was found very useful in the immuno-diagnosis of pulmonary emphysema. this study has been also concerned with the biochemical changes associated with aat deficiency in pulmonary diseases. to fulfill this work, a group of healthy blood donors was selected for separation of pure aat antigen from blood. the pure aat was used for the preparation of anti-aat, the purity and potency of antibody was checked by titration methods. the biochemical changes were studied in thirty subjects clinically divided into three groups including, control, heavy cigarette smokers with pulmonary emphysema, and non-smoking subjects with pulmonary emphysema. the activity of elastase and hydroxyproline (hp) level as a marker of elastin and collagen breakdown were assayed in bronchoalveolar lavage (bal) fluid. the activity of aat and to inhibit proteases as represented by tryptic inhibitory capacity (tic) was evaluated. serum ceruloplasmin, transferrin and iga level as well as thiobarbituric acid reactive substances (tbars) were estimated in all groups. our data revealed that aat and its tic showed very highly significant decreased levels in all patients with emphysema as compared to control, while the elastase activity and hp level in bal fluid were significantly increased in these patients. serum tbars was significantly increased in such patients associated with increasing level of both ceruloplasmin and transferrin. while, serum iga was significantly increased. furthermore, the biochemical changes were markedly changed in smokers with emphysema than non-smoking subjects. in conclusion, the preparation of anti-aat on the local level is very important where less expensive and less time consuming and can be useful in immuno-diagnosis and prognosis of pulmonary diseases. a new method combined with boosting and projective adaptive resonance theory for analysis of gene expression data from cancer patients hiro takahashi, yasuyuki murase, hiroyuki honda department of biotechnology, school of engineering, nagoya university, nagoya 464-8603, japan. e-mail: h041305d@mbox.nagoyau.ac.jp (h. takahashi) an optimal and individualized treatment protocol based on accurate diagnosis is urgently required for the adequate treatment of patients. for this purpose, it is important to develop that a sophisticated algorithm that can manage large amount of data, such as gene expression data from dna microarray, for optimal and individualized diagnosis. in our previous study, we developed the projective adaptive resonance theory (part) as a gene filtering method and boosted fuzzy classifier with sweep operator (bfcs) as a modeling method. in the present study, we applied the combination of part and bfcs (part-bfcs method) to microarray data of brain tumor (central nervous system tumor) obtained from the website. the method enabled the selection of 14 important genes related to the prognosis of the tumor, i.e., sensitivity for combined therapy with surgery, radiotherapy, and chemotherapy mainly with vincristine, cisplatin, and cytoxan. the constructed model showed about 20% higher accuracy than that of the conventional method. genomic signal analysis of hiv variability based on rt gene p.d. cristea, rodica tuduce d. otelea biomedical engineering center, university "politehnica" of bucharest, romania. e-mail: pcristea@dsp.pub.ro (p.d. cristea) dna sequences genotyped from 60 clade f hiv-1 isolates from romanian patients in the laboratory of the national institute of infectious diseases "matei bals", bucharest, romania, have been studied. the symbolic sequences have been converted into digital genomic signals by using a complex quadrantal representation of the nucleotides described earlier. the cumulated phase and unwrapped phase of a complex genomic signal reflect the statistical distribution of bases and base-pairs, respectively. independent component analysis of the genomic signals has been used to characterize the variability of the f subtype hiv strains isolated in romania. the sequenced segment is of (about) 1302 base pairs, approximately aligning with the standard sequence of hiv-1 (accession nc001802 in genbank) over the interval 1799-2430 bp. this segment, which is currently used for the standard identification and assessment of hiv-1 strains, comprises the protease (pr) gene and two thirds of the reverse transcriptase (rt) gene. only results referring to the analysis of the rt gene region are presented here and used for extracting features of virion isolates and for establishing phylogenetic trees of the studied strains. the analyzed rt encoding segment has the length 1005 bp and is located in the second interval (298-1302 bp) of the analyzed dna segment, respectively along the 2096-3100 bp region of nc001802. taking into account the mutations identified in these sequences, the samples were classified in three groups from the point of view of their resistance to current antiretroviral compounds: sensitive, resistant and multiresistant. the paper presents results for the isolates in which mutations leading to multiple drug resistance have been identified. over expression of secb protein in e. coli enhances the periplasmic expression of human growth hormone m. ghafari 1,2 , a. zomorodipour 2 : 1 islamic azad university of jahrom, tehran, iran; 2 national institute for genet eng and biotechnol., tehran, iran. email: maryamghafari2001@yahoo.com (m. ghafari) among several proteins involved in the secretion pathway of proteins in e. coli, secb plays a key-important role in solubilization of preproteins before processing. in order to increase processing of a human growth hormone precursor (pelb::hgh) which appeared to have problem in processing efficiency, as a possible solution a regulated co-expression of a secb gene was considered. in this regard, we designed an arabinose-regulated secb expressing plasmid compatible with an iptg/lactose-regulated pelb::hgh expressing plasmid. for the construction of the secb expressing plasmid the origin of replication and antibiotic resistant gene (amp) of a pbad vector was replaced by a p15a-ori and a kanamycin resistant gene, respectively. the expression and processing of pelb::hgh preprotein in the two-plasmid containing bacteria in a secb over-expression state was compared to that of the pelb::hgh expression in normal bacteria. although a decline in total expression level of hgh during the overexpression of secb was observable, probably due to presence of two different expressing plasmids, but both the processing efficiency of pelb::hgh and the transport of mature protein into the periplasmic space was enhanced during prolonged arabinose induction. current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts. natural allergen extracts contain a mixture of allergenic and non-allergenic components that are difficult to standardize. recombinant allergens can improve diagnosis and de-sensitization against single component. major cat allergen is a heterodimer composed of disulfide linked 7.8 and 10.1 kda polypeptide chains. both chains of feldi protein were obtained in e.coli system. purification of recombinant proteins was performed in denaturing conditions using immobilized metal affinity chromatography specific for proteins with histidine tag. from 1000 ml culture approximately 13 mg protein for feldi chain 1 and 43 mg of feldi chain 2 were obtained. after purification histidine tag was removed by hydrolysis with thrombin. immunological activity of feldi against serum of patients allergic to cat was narrowed to subgroup of patients allergic to feldi protein by surface plasmon resonance. immunological activity of each chain and renatured heterodimer was also tested using immunoprecipitation techniques against serum of population group. subdoligranulum variabile -a novel member of the human gut micro flora with a high prevalence kim holmstrøm, trine møller bioneer a/s, hørsholm, dk-2970, denmark in 2003 we isolated and cultured for the first time a bacterium from a human fecal sample representing a hitherto unknown member of the clostridium leptum rrna supra generic cluster. the c. leptum rrna supra generic cluster represents one of the 3 major phylogenetic lineages within the human gut microbiota, and is characterized by having only a small proportion of its members actually identified by cultivation compared to the estimated numbers of bacteria contained in this group from culture-independent gut flora analyses. s. variabile is an obligate anaerobe gram negative bacterium with a characteristic pleiomorphic coccoid-droplet-like cellular morphology. its closest previously cultivated relative based on a 16s rdna phylogenetic analysis is faecalibacterium prausnitzii, a rod-shaped and therefore easily distinguishable gram negative bacterium present in high numbers in the human fecal micro flora. based on 16s rdna sequence we designed a s. variabile specific oligonucleotide probe for use in fish analysis to estimate the prevalence of this "new" bacterium in fecal samples collected from healthy human beings. interestingly, we observed a high proportion of s. variabile present in all tested samples, and in some instances we observed a higher prevalence than the more well-known group of bifidobacteria equally estimated by fish analysis. documentation of these results will be presented. several species of sea cucumbers, long an incumbent of traditional medicines were selected as the source of animal based antibiotic compounds. swabs of the inner surface and coelomic fluid (inner fluid) samples from sea cucumber (holothuria atra jaeger) were taken. thirty strains of bacteria were isolated. these strains were grown in different antibiotic production media. nine human pathogenic bacterial species were used as test agents and they are, k. pneumoniae, mrsa, m. luteus, s. thyphimurium, s. epidermitis, s. saprophyticus, b. subtillis, p. aeruginosa and s. pyogenes; only four bacterial strains showed mild antibiotic activity against s. pyogenes and s. thyphimurium. similar testing on two other species, h. scabra and s. variegatus will be carried out. different media, especially antibiotic production enrichment media will also be used. characterization will be done upon obtaining an antibiotic compound, which shows moderate to high activity against at least one of the nine human pathogens used. for plant based medicines, three rhizomes, "cekor," "jerangau" and "bonglai" were analyzed. solvent extraction using ethanol, methanol and acetone was carried out, at a concentration of 20-50 mg per ml solvent. the filtrates were used for the antibiotic testing stage. all the three plant species showed moderate antibiotic activity against m. luteus, s. epidermitis, s. pyogenes and s. saprophyticus. interestingly, the antibiotic activity increased when combinations of the herbal extracts were used. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx 5cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx 5cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited 5% to 8% of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax7 and the musclespecific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. metabolic engineering design of an extracellular hgh synthesis system birgül şentürk 1 , pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre lab, department of chemical engng, ankara university, 06100 ankara, turkey; 2 iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.özdamar) metabolic engineering design of an extracellular human growth hormone (hgh) synthesis system is based on cloning the dna encoding human therapeutic protein together with the signal dna sequence of an extracellular enzyme gene into a host-vector system. in this context, extracellular protease (subc) signal dna sequence, i.e. pre-signal dna sequence, was fused into the frame in front of hgh mature dna sequence, by the use of four primers designed using pcr-based gene splicing by overlap extension method. b. licheniformis chromosomal dna and plasmid carrying hgh cdna were used as templates in pcrs, respectively, for the amplification of the subc signal dna sequence and hgh mature peptide sequence. for the fusion of two target genes, i.e. mature peptide sequence of hgh and, signal dna sequences were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their 5 ends that are complementary to 3 portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at 3 end. extension of this overlap by dna polymerase has yielded the recombinant hybrid-gene; and hybrid-gene serve as template for the continuation of reactions for the increase of the concentration in the microreactors. the hybrid gene fragment was first cloned into puc19, and then sub-cloned to pmk4 e.coli-bacillus shuttle plasmid. thus, a new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis 168 (spo − ). the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the proposed metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, hgh, amino acids and organic acids concentrations as the constraints. on the basis of the intracellular bioreaction rates and the interactions with the bioreactor operation parameters, an in-depth insight will be provided for further metabolic engineering design for the extracellular hgh production in r-b.subtilis. medicines based on polypeptides consisting of 30 and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin ␣ 1 production impac system (intein mediated purification with affinity chitin-binding tag) has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin ␣ 1 . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl 2 and we have found that, in case of intein-thymosin ␣ 1 , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of 0.5-1 mm zinc chloride in buffers on all stages of thymosin ␣ 1 isolation. the structure of recombinant thymosin ␣ 1 of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-d) or 1 mg/l naphtalenacetic acid (naa). the developed calli and regenerated plants were maintained on 2,4-d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + 2,4 d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his7tnf␤ and n19tnf␤ were expressed in e. coli. high solubility of n19tnf␤ was expected, however, both analogs his7tnf␤ and n19tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n19tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his7tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with 0.2% nls, while his7tnf␤ and n19tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. high level expression of recombinant growth hormones in e.coli faces common problems such as protein aggregation and inclusion body formation. discussions are raised whether it is more beneficial to obtain soluble protein but to loose expression rates. here we describe an experiment based on the hypothesis that slower expression should result in at least partially soluble recombinant protein. experiments were performed on bovine, chicken and mink growth hormones. expression rate was controlled externally by adjusting cultivation temperature, media, inducer amount, and both induction and cultivation times. another approach to the problem was performed through genetic manipulation. we changed strong t7 promoter to e. coli promoter consensus sequence thus reducing expression rate. recombinant growth hormone was still found to form aggregates, even when expressed at extremely low levelsseveral (2-8) percent of total intracellular protein. we developed optimization scheme of insoluble protein production and showed that expression rate minimization is not influencing recombinant growth hormone solubility in vivo thus suggesting an idea of sequence specific aggregation. to optimize the recombinant protein production in small-scale shake-flask system and high cell density fermentation, new tools have been developed at our laboratory which helps to get knowledge about the physiological state of the cell culture. these tools include (i) a quantitative monitoring system for cellular mrnas based on a sandwich hybridization technique, and (ii) a wireless online monitoring tool (senbit), applicable for standard sensors such as ph, po 2 and temperature for the continuous data collection from shake flasks. the senbit system is a new tool supplying valuable information for the optimisation of the expression of recombinant genes in shake flasks and allowing conclusions towards the reproducibility of shake flask cultures. the presentation will focus on use of the sensitive sandwich hybridization technology for the quantitative analysis of process relevant marker genes in different kind of microbial cell cultures with a focus on the production of recombinant proteins. samples from shake flask cultures and high cell density fed-batch fermentations of the yeast pichia patoris, have been analysed. additionally the mrna analysis was combined with the application of the senbit wireless system to study the production of a recombinant protein in shake-flask cultures of p. pastoris. aside from p. pastoris, mrna sandwich hybridization also was used to monitor product expression in fed-batch fermentation of e. coli for the production of a protein with two subunits by sequential induction. quantitative rna analysis as a tool for optimization of tetrameric collagen prolyl 4-hydroxylase production in e. coli a. neubauer 1 , m. bollok 2 , j. myllyharju 1 , p. collagen prolyl 4-hydroxylase (p4h) involved in the biosynthesis of collagens is an ␣ 2 ␤ 2 tetramer. recombinant expression of p4h in e. coli was described recently . the construct for cytoplasmic expression contains the genes of both subunits in one plasmid under control of different promoters. the ␣ subunit forms inactive aggregates, when expressed separately. in mammalian cells the ␤ subunit is available in large excess and keeps the ␣ subunit in a soluble active form. to mimic this in the bacterial system, we induced both genes sequentially. after induction of the ␤ subunit with iptg, expression of the ␣ subunit was initiated with anhydrotetracycline. here we use the analysis of the product mrnas with a bead based sandwich hybridisation assay (sha) (rautio et al., 2003) for optimization of the fermentation procedure. a high p4h activity was obtained if a high mrna level of the ␣ subunit could be maintained over a longer time. the obtained results illustrate the importance of the second induction for a high level expression of the p4h tetramer. the cells need to be in a "healthy state" with low metabolic load to react efficiently to the second induction. the data illustrate the optimization of a fermentation process by monitoring mrna levels which is of general interest for optimization of products which are difficult to detect. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx 5cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx 5cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited 5-8% of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax7 and the muscle-specific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. collagen and its derived product gelatin are attractive mammalian proteins to be used as model for the production of complex heterologous proteins in plants. the availability of a recombinant product will provide a safer, more homogeneous product than the current animal-derived material. the aim of the project is to investigate the feasibility of a production system for the accumulation of recombinant collagen for conversion to gelatin using barley. the 5 -end of the cocksfoot mottle virus (cfmv; genus sobemovirus) genomic rna sequence, called cfmv -element, has been shown to enhance recombinant protein synthesis in barley (wo 01/55298, mäkinen et al., 1995) . the -element will be used to study whether accumulation levels of complex mammalian proteins can be further increased, using collagen as a model that will serve as basis for exploring the expression of other complex proteins. this system can be study the production of barley-derived recombinant collagen for conversion to gelatin. classical swine fever virus (csfv) is an animal pestivirus which can be used as a surrogate model to elucidate the role of envelope glycoproteins of closely related human hepatitis c virus (hcv). the necessity to use the surrogate models for hcv is due to the fact that this virus cannot be grown in vitro cultures. csfv genome codes for three major antigenic glycoproteins which are located in the same cluster of genes; they are designated as e2 and e0 (e rns ) and e1. glycoproteins form heterodimeric and homodimeric complexes on the external part of viral particles. it is generally accepted that envelope glycoproteins play a major role in the initial stages of viral infection both for csfv and hcv. formation of complexes is needed to effectively infect host cells. we have investigated the formation of glycoprotein dimers by immunoperoxidase monolayer assay and by immunoblotting (western blotting). immunoblotting is a very useful technique in these studies because the complexes are formed via cysteine-cysteine disulphide bonds and they are retained during sds-page under non-reducing conditions. by modifying the glycoprotein genes and by arresting n-glycosylation of e2 and e0 we have investigated which factors influence the formation of complexes. it has been found that some glycosylation inhibitors which act at the early stages of glycan chain processing influence, not only glycosylation, but also the stability of e2 protein, effectively inhibiting the formation of glycoprotein complexes and the yield of the virus. these inhibitors are potential agents for arresting the multiplication and spread of csfv, and its relative -human hcv. recombinant proteins have been produced in a variety of heterologous protein expression systems. eukaryotic unicellular algae have distinct advantages, e.g. it can synthesize complex protein that requires post-translational modification. furthermore, microalgae can be grown in confined environment and thus prevents leakage of genes to the environment. our group has developed a platform technology for the production of recombinant proteins in red microalga porphyridium sp. we have constructed algal transformation plasmid vectors containing a camv 35s promoter and polya signal site. a streptoalloteichus hindustanus bleomycin-resistant gene was used as the selective marker. we have expressed ovalbumin and hepatitis-b surface antigen (hbsag) as model proteins. transformation was carried out by agitating algal cells and vector dna with glass bead. transgenic lines were selected by growing algal cells on agar plate containing 6 g/ml zeocin. positive transgenic lines were selected by screening the colonies by pcr and confirmed by dna sequencing. expression of ovalbumin and hbsag protein was examined by western blot analysis. ovalbumin was found to be expressed inside the algal cells while small hbsag was secreted into the medium due to presence of signal peptide. these findings indicate that red microalgae are capable of producing heterologous proteins. life-material exhibition as ethical interpretation chang shih-lung biotechnology industry study center, taiwan institute of economic research, taipei 106, taiwan. email: schang@tier.org.tw in this thesis we aim to explore the medical-related life-material exhibitions within ntu hospital-the humanity building, and taipei mackay hospital-the historical showroom as abecedarian clues to understand the burgeoning phenomenon-medical museums in taiwan. following these clues, we treat the whole context of medical museums, which transform values through situational construction, as the background to interpret the ethical implications of group val-ues that are transformed in the medical profession. in this thesis, we see medical museums, as the social prescription transforming medical profession, format the ritual context of situation ethics with the cultural construction of life-material. multi-disciplinary interactions and visiting itinerary can be transferred to the exploring horizon of research approach through description and interpretation. among them, we observe that humanistic elements have become essential equipment (or mat'eriel) of medical profession. though humanistic equipment (or mat'eriel) has its bottleneck in the museum situation, it can also unblock new possibilities for museum exhibitions, ethical practice or life ethics. as part of a wide research program aimed at developing new antitumoral agents, we present herein a series of stereoisomeric derivatives of fused tetrahydrofuranes (fthf) substituted with diverse protecting groups either at the primary or secondary hydroxyl groups. unprotected derivatives were also synthesised to investigate the influence of substituents on the in vitro activity of fthf. data on the synthesis, chemosensitivity and apoptosis induction by this series of fthf will be correlated to substitution pattern, stereochemistry and protecting groups, as an aid to the rational design of novel antitumour drugs. establishment of in vitro test systems for pulmonary edema resorption by peptide drug candidates dominik geiger, aswin mangerich, rudolf lucas, klaus p. schäfer, inge mühldorfer 1 department of biotechnology, altana pharma ag, konstanz, germany; 2 imc university of applied sciences, krems, austria tnf-␣ was found to up-regulate the rate of lung liquid clearance (llc) in several animal models by the activity of its lectin-like tip domain. this activity can be mimicked by a circular peptide designated tip. tip was shown to induce llc and consequently pulmonary edema resorption in different animal models. in order to study the mechanism of action of tip, we established two in vitro test systems for edema resorption with the human lung epithelial cell line calu-3: (1) the ability of calu-3 cells for spontaneous dome formation within confluent monolayers was utilized for quantitative examination of tip's activity on active transepithelial fluid transport ("dome assay"). (2) the effects of tip on bioelectrical properties of polarized cell monolayers were studied by using the transepithelial electrical resistance (teer) technology ("teer assay"). dome assay experiments confirmed that dome formation is a sodium dependent process and that tip is able to increase this process. teer assay experiments proofed that tip acts in a polarized and dose dependent manner. in conclusion, there is strong evidence that the dome and the teer assays are suitable systems for in vitro activity testing of tip and other anti-edema peptide drug candidates and are useful for studies on their mechanism of action. increasing safety concerns in gene therapy result in more stringent regulatory requirements. those cover the complete process chain of cell banking, fermentation, and purification. a wide range of applications for pdna requires gram to kilogram amounts for clinical trials and market supply. economic, productive and robust processes are a prerequisite for low cost of goods (cogs). therefore manufacturer of biopharmaceuticals need to address these considerations by developing new production processes meeting the new standards. boehringer ingelheim austria developed a novel pdna production process suitable for large-scale cgmp production. the process is based on e. coli fermentation. the process contains no components from animal origin. the optimized fermentation process yields up to 1 g pdna/l fermentation volume. for isolation of pdna from e. coli alkaline lysis in glass bottles or stirred tanks is commonly used. cell wall structure is destroyed by a combination of alkaline ph and detergents. in our process alkaline lysis is operated in a closed, continuous system directly connected to clarification without using enzymes. we developed a scalable process for pdna purification based on 3 different chromatographic principles. the capture step is carried out by hydrophobic interaction chromatography followed by anion exchange chromatography as intermediate step. final polishing is carried out by conventional size exclusion chromatography in a group separation mode providing also buffer exchange and desalting for the final formulation. the process results in a pdna drug substance of highest quality containing a low level of impurities (genomic dna, rna, proteins endotoxines) suitable for therapeutic applications. depending on the conditions during fermentation and the used host homogeneities of greater than 95% or even 98% are possible, while a high over all yield can be achieved (∼50%). during the development monolithic chromatography supports (cim ® ) were compared with conventional resins and evaluated as potential alternatives. the complete process is monitored by a set of analysis covering cell banking to final purification. new sensitive methods based on hpce, hpiex and fluorometric measurement were developed. whereas many natural amino acids are currently produced by very cost efficient biological processes, the manufacture of methionine is still performed by traditional chemical synthesis. this was mainly due to the poor performances of the currently available producing strains that inhibited the development and commercialization of a biological process to l-methionine. metabolic explorer has recently reinvestigated and developed an efficient biological process that employs an engineered microorganism and utilizes a renewable starting material (corn sugar) as its feedstock and converts glucose into l-methionine. we will describe (a) the general scheme for the engineering of the host organisms and (b) the general approach to maximize carbon and reducing equivalent throughput to l-methionine. to highlight this effort, we will present the global approach developed to improve the process combining metabolic flux analysis, traditional protein biochemistry, molecular biology and fermentation optimisation. saccharomyces cerevisiae is an established 'work horse' of the fermentation industry and modern biotechnology has led to a spectacular expansion of the range of products that can be produced by this yeast. however, for the large-scale sustainable production of chemicals, it is equally important that the range of carbohydrate feedstocks be expanded. especially relevant in this respect is the ability to consume the pentose sugars d-xylose and l-arabinose, which make up a substantial part of plant carbohydrates. wild-type s. cerevisiae strains cannot metabolise d-xylose, but are capable of slowly metabolising its keto-isomer, d-xylulose. therefore, efficient conversion of d-xylose into d-xylulose has long been a key issue in yeast metabolic engineering. non-saccharomyces yeasts that is capable of growing on d-xylose use two enzymes, xylose reductase and xylitol dehydrogenase for this purpose. while both enzymes have been successfully expressed in s. cerevisiae, this is not always compatible with efficient product formation. for example, in the case of ethanol production, the different cofactor specificities of these two oxidoreductases cause massive byproduct formation. theoretically, introduction of a xylose isomerase, which catalyses the interconversion of xylose and xylulose, might circumvent these problems. however, it is notoriously difficult to express bacterial and archaeal xylose isomerases in s. cerevisiae and, until recently, activities of heterologous xylose isomerases expressed in s. cerevisiae were vanishingly low, at least under physiological conditions. a breakthrough was reached when, in 2003, a xylose isomerase gene from the anaerobic fungus piromyces was expressed in s. cerevisiae. while this led to high activities of xylose isomerase, these were not enough to enable fast growth or product (ethanol) formation. in this presentation, we will discuss how a combination of metabolic and evolutionary engineering led to fast and efficient xylose utilization by engineered saccharomyces cerevisiae strains under aerobic as well as anaerobic conditions. furthermore, we will illustrate how evolutionary approaches can be applied to facilitate the utilization of mixed substrates. hyaluronic acid (ha) is a natural and linear polymer composed of ␤-1,3-n-acetyl glucosamine and ␤-1,4-glucuronic acid repeating disaccharide units with a molecular weight (mw) up to 6 mda. it is a major constituent of the extracellular matrices and the synovial fluid. in the last decades, various fields of application including cosmetics, ophthalmology, rheumatology, tissue engineering and drug delivery have been explored, owing to the many important biological functions of ha and its unique physico-chemical properties. however, for some specific applications, the relatively high mw of ha is a limiting factor and the availability of low mw fractions would be highly desired. for food applications, low mw ha has been shown to penetrate the gastrointestinal barrier, thereby increasing the ha bioavailability. moreover, low mw ha fractions are able to re-establish the ha content in the skin and can thus be used in cosmetics as anti-aging and anti-wrinkle agents. finally, low mw ha has shown to prevent oxygen free radical damage in granu-lation tissue during wound healing. a range of methods has been described for the depolymerization of ha to low mw fractions. these techniques involve heat treatment, ultrasonication, uv/gamma irradiation, chemical and enzymatic degradation. we present results on a degradation process of ha originating from bacillus subtilis fermentation into well-defined low mw fractions. the process developed in lab scale is safe, well-controlled and produce low mw ha fractions with narrow polydispersity. moreover, the process is readily up-scalable. the low mw ha fractions are being evaluated in various cosmetic applications. metabolic pathway manipulation for improving the properties and productivity of microorganisms is becoming an established concept. metabolic engineering can be defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant dna technology. a detailed analysis of the physiological means of the different pathways is needed to be able to introduce modifications aimed to the production of not only important metabolites, but also to understand the fundamentals of cell biology. aimed to produce single compounds, metabolic engineering necessarily includes the modification of the cellular pathway(s) as well as the redirection of the energy toward the production itself. the existing metabolic engineering applications are the culmination of more than two decades of global experience developing processes for the production of fine chemicals, vitamins, nutraceuticals and animal nutritional aids such as amino acids. based on the relative low complexity, the first biotechnological applications have been developed from microorganisms. our laboratory has been engaged in this field since different years. yeasts like saccharomyces cerevisiae, kluyveromyces lactis and zygosaccharomyces bailii have been developed for the production of fine chemicals like lactic and ascorbic acids from d-glucose. in this contribution, we will present the last data obtained. since 40 years amino acid production is in the focus of industrial microbiology. l-glutamate and l-lysine are produced with corynebacterium glutamicum, while escherichia coli is used for l-threonine production. the worldwide market of threonine drastically increases: in 1996 the amount of threonine produced worldwide was 15,000 t and raised to 30,000 t in 2000 and 45,000 t in 2004. concerning predictions of experts the demand of threonine will rise with a two-digit rate of economic growth within the next few years. meanwhile the prices declined. these conditions enforce very efficient production processes. beside the strain development and an optimised downstream procedure the fermentation process is an important target for productivity improvement. strain development is dependent on detailed knowledge of the production strains. with innovative methods we are able to get a close look inside the cells under different culture conditions. these methods have been called 'omics' in recent literature. knowledge about genome, transcriptome, proteome, phosphoproteome, metabolome and fluxome leads to new ideas for strain improvements. data generated by these methods must be based on clearly defined culture conditions. therefore, highly parallel fermentations have to be performed to generate biological parallel samples. cutting-edge technical equipment is the basic requirement for experiments like this. other requirements are optimised sampling for different analysis, technical parallels of analytical steps and a detailed statistical analysis of data. these procedures guarantee distinction between real data and data noise. integration of all these data to a holistic model of the cell is the challenge for the future. by combination of a new strain, process and downstream improvements the plant productivity was increased drastically. we ended up in an optimized, fast and high yield process to scope challenges worldwide market comes up with. rieping, m., hermann, t. (2002); fermentation process for the preparation of l-threonine; wo/0218543. ) are potentially quite useful biocatalysts, as they allow for the regioselective and stereoselective hydroxylation of activated as well as non-activated carbon atoms. in addition, the large number of members of the p450 superfamily exhibits a wide diversity of specificities from which a useful biocatalyst may be selected. from a technical point of view, however, they have significant drawbacks. thus, they usually cannot be produced in large quantities nor recovered or stored without a severe loss in activity. their catalytic activity is mostly quite low, and their operational stability leaves much to be desired. most p450 enzymes require a complex protein/phospholipid machinery for activity, and the final electron donor in the reaction cascade, usually nadph, does require extensive recycling to arrive at a commercially satisfactory process. recently, the use of bacterial cytochromes as hydroxylation biocatalysts has received considerable attention. some of them are natural fusion proteins, which contain the heme and the reductase domains on a single polypeptide chain. they are catalytically much more active compared to, e.g. cytochromes occurring in human tissue. we thus have set out to further improve the technical applicability of these enzymes, and have centered our activities around several bacterial cytochromes. it proved very useful to apply rational mutagenesis and directed evolution to these enzymes, leading to a surprising compatibility of mutant enzymes with a wide variety of substrates. mechanisms for the limited stability of the enzymes were explored, leading to hybrid enzymes with enhanced stability, and the cofactor problem was alleviated using auxiliary enzymes or mediator-based technologies. as a result, a bioreactor based on microbial cytochromes was built and operated for several days. baeyer-villiger monooxygenases represent useful biocatalytic tools as they can catalyze reactions, which are difficult to achieve using chemical means. however, so far only a limited number of these monooxygenases were available in recombinant form kamerbeek et al. (2003) . using a recently described protein sequence motif fraaije et al. (2002) and the available genome sequence information, we were able to identify and overexpress a number of novel bacterial bvmos. one of the overexpressed bvmos was found to be relatively stable as it originates from thermobifida fusca, which grows at ∼60 • c. the enzyme was shown to be active on a broad range of substrates, preferring aromatic ketones fraaije et al. (2005) . the best substrate discovered so far is phenylacetone, hence its name: phenylacetone monooxygenase. we have solved the crystal structure of phenylacetone monooxygenase, which represents the first structure of a bvmo malito et al. (2004) . the crystal structure provides insight into the complex mechanism of catalysis mediated by fadcontaining bvmos. by site-directed mutagenesis we have probed the role of several active-site residues. a crucial role is played by an arginine residue. as phenylacetone monooxygenase shares significant sequence identity (>40%) with all known nadph-dependent bvmos, many of the observed structural features seem to be conserved within this class of atypical monooxygenases. by homology modeling using the phenylacetone monooxygenase structure, catalytic properties of other baeyer-villiger monooxygenases can be explained or predicted. screening for fungal baeyer-villiger monooxygenases l. butinar 1 , j. friedrich 1 , v. alphand 2 : 1 laboratory of biotechnology, national institute of chemistry, ljubljana si-1001, slovenia; 2 groupe biocatalyse et chimie fine cnrs fre2712, université de la méditerranée, marseille, france the asymmetric form of the baeyer-villiger (bv) oxidation (transformation of ketones into lactones) is an important challenge for organic chemistry since the obtained lactones are valuable building blocks for synthesis of countless biologically active products. to date, enzymatic or microbial bv oxidations appears as more successful than their chemical counter-parts. (ten brink et al.) whereas most active bv monooxygenases are produced by bacteria (among which the well-studied enzyme of acinetobacter calcoaceticus), only a few fungal strains expressing bvmo were described (alphand et al., carnell and willetts) . in order to increase the number of available biocatalysts which perform such an asymmetric biotransformations, a screening of fungi belonging to major groups of zygo-, ascoand basidiomycetes was conducted using bicycloheptenone as testsubstrate. surprisingly, a large number of the tested fungi were able to transform the substrate into one to four different lactone isomers. the yields, the enantio-and regio-selectivity of the reaction depended on the fungal strain. alphand, v., furstoss, r., 2000. j. mol. catal. b 9, 209-17. carnell, a., willetts, a.,1992 . biotechnol. lett. 14, 17-21. ten brink, g.j., et al., 2004 . pyranose oxidase (p2ox) is a periplasmic enzyme that widely occurs in basidiomycetes. it catalyses the c-2 oxidation of several aldopyranoses to the respective 2-keto derivatives, transferring electrons to molecular oxygen to yield h 2 o 2 . p2ox is of interest for carbohydrate conversions, as its reaction products (2-keto sugars) can be attractive intermediates in the production of food ingredients. we cloned the gene encoding p2ox, and subsequently amplified a cdna clone by rt-pcr. the cdna was inserted into a bacterial expression vector and successfully expressed in e. coli. properties of the heterologous protein were compared to those of the native enzyme showing that they are essentially identical. both the native as well as the recombinant enzyme were used in biotransformations of sugars. recently, the 3d-structure of this tetrameric enzyme was elucidated. based on structural information, several enzyme variants containing point mutations were constructed and further characterised. two of these variants (e542k and e542r) displayed improvements in stability and certain kinetic properties thus making them attractive for biocatalytic applications. lactones are important compounds for the fragrance and flavour industry. right now the production of lactones is dependant on the import of crude materials from tropical countries. in this project, we want to tackle the manufacture of lactones via a biocatalytic route using p450 monooxygenases. cytochrome p450 monooxygenases catalyse the oxyfunctionalization of non-activated c-atoms. cyp102a1 from bacillus megaterium, cyp102a2 and cyp102a3 from bacillus subtilis are soluble fusion proteins comprising p450 monooxygenase and fad/fmn reductase domains in one polypeptide chain. all three enzymes are highly homologous fatty acid hydroxylases. especially, cyp102a1 also known as p450 bm-3 is well characterized and shows high activity compared to other p450 monooxygenases. the aim of the work is to change selectivity but conserve the high activity that is typical for those enzymes. using methods of structure modelling, rational protein design and directed evolution new mutants of these enzymes with changed regioselectivity are obtained. products of conversion with monooxygenases are intermediates in the production of lactones. the interface of biology and materials science has led to new materials with unique structural and functional properties, and new process technologies with the ability to produce, from "bottoms up", a wide range of biomimetic structures. these materials and their designs have broad application as catalysts, sensors, and devices for use in synthesis, cell and tissue engineering, bioanalysis and screening, and nanoelectronics. we have focused on the generation of sugar-based nanostructures, complete with tailored selectivities and biocatalytic activities at the molecular and nanoscales. these include biocatalytically-generated carbohydrate derivatives that selfassemble with high precision to give novel architectures with functional and responsive properties. izumoring: a strategy for total production of rare sugars ken izumori rare sugar research center, kagawa university, kagawa 761-0795, japan. e-mail: izumori@ag.kagawa-u.ac.jp we found a new enzyme, d-tagatose 3-epimerase (dte), that epimerize all ketohexoses at c-3 position. this epimerase catalyze not only between d-tagatose and d-sorbose, but also d-fructose = dpsicose, l-sorbose = l-tagatose, and l-psicose = l-fructose. this new enzyme offered us a useful key tool to connect all ketohexoses using hexitols as intermediates. the figure shows that all eight ketohexoses can be connected with dte and polyol dehydrogenases (pdh) in a ring. using this ring, we can easily find the pathway to transfer d-fructose to d-tagatose via d-psicose using dte and pdh. various aldose isomerases transform ketohexoses to the corresponding aldohexoses. so, we can connect all 16 aldohexoses with 8 ketohexoses using the enzyme. finally, all hexoses, 8 ketohexoses, 10 hexitols and 16 aldohexoses are connected using enzyme reactions in a ring structure (not shown). this kind of strategy is effective also on transformation of tetroses and pentoses. now, we can produce all monosaccharides; tetroses, pentoses and hexoses by enzyme reac-tions. the bioproduction strategy of all rare sugars (monosaccharides that are rare in nature) is illustrated using ring form structures named as izumoring. we have already succeeded to produce d-psicose in large scale and are now in the progress of mass production of various rare sugars from natural and cheap sugars using izumoring. bioprocess development for chiral intermediates christian wandrey institute of biotechnology, forschungszentrum jülich gmbh, jülich d-52425, germany chiral alcohols, diols, amino alcohols and chiral acids (e.g. hydroxy acids and amino acids) play an important role in pharma and agro synthesis. in the past such chiral intermediates were obtained by racemic resolution via chiral reductions using prochiral precursors. here the problem of cofactor regeneration arises. this problem could be solved by enzyme-coupled or substrate-coupled cofactor regeneration using formate or isopropanol as reducing agent. alternatively, whole cell bioreductions were developed where glucose is used as the reducing agent. in recent years "designer microorganisms" were developed in which oxidoreductases (e.g. alcohol dehydrogenases) were over-expressed in escherichia coli. in such cases, cofactor regeneration was achieved intracellularly with isopropanol as the reducing agent or by coexpression of a formate dehydrogenase, so that once again format could be used for reduction. another route to obtain chiral intermediates is a fermentative approach using classical pathways (like the aromatic amino acid pathway). here, the pathway is interrupted after the intracellular production of chorismate. new chiral intermediates can be obtained by over expression of additional genes, which catalyzed the production of chorismate derivatives leading to cyclohexadiene-transdiols and the corresponding amino cyclitols. the last example can be regarded as an example of industrial biotechnology where glucose is used as starting material (white biotechnology). here bioprocess development is carried out in an integrated approach, in which molecular biochemical engi-neering cares for the optimal intracellular metabolic network and the classical biochemical engineering cares for the optimal environment of the cell in a fermenter. examples will be given which reach (in cooperation with industrial partners) up to kilogram scale. biotransformations are usually involved in just one or very few separate reactions in organic syntheses. the development of a cell-free "system of biotransformations" (sbt), in which a set of enzymes acts in a coordinated fashion in a one-pot synthesis, lead to increased catalytic complexity, selectivity and yield, as well as facilitated operation at reduced costs. the example chosen to prove the usefulness of the sbt-approach is the production of dihydroxyacetone phosphate (dhap). dhap, a c3-compound from glycolysis, is an important precursor for asymmetric c-c-bond formation. so far, the production of dhap is difficult and expensive. for the construction of the dhap-producing sbt, e. coli's glycolysis is isolated from the metabolism to an as large as possible extent by the construction of a multi-ko-mutant. a culture is grown in an appropriate medium, homogenized in the production buffer, and used as the catalytic system. high production yields can be achieved since the production pathway is almost completely isolated from the metabolic network. the employed dhap-producing sbt provides not only a path from glucose to the product, but also an integrated atp-regeneration and nad-recycling system. in first experiments with a tpi-ko-mutant, a dhap-production yield on glucose of 32% could be achieved, without optimizing the system. the system remained active for more than 24 h. up to now, atp cannot be applied in catalytic concentrations, but has to be present in equimolar amounts to glucose. the production yield could be increased by 10% through the addition of phosphate ions as substrate to the reaction, enabling the system to utilize atp more efficiently. these experiments indicate that the sbt-approach is viable and a large potential remains to improve the dhap-producing sbt. for some years novozymes have manufactured a pectate lyase for scouring of textile as an ecological alternative to the traditional harsh chemical treatment, and recently, we at novozymes discovered additional applications for pectate lyases. however, to be commercially attractive more robust pectate lyses had to be developed. in this paper, we will demonstrate how we for two different pectate lyases have improved their stability significantly. as the two enzymes are quite similar in sequence and structure, it was a new discovery for us to find that different concepts of protein engineering had to be used in our attempt to stabilize each individual pectate lyase. the stability of one enzyme was improved by substitutions in the internal of the structure whereas the stability of the other pectate lyase was primarily improved by changing surface residues. starting with knowledge from structural analysis, we have applied rational based protein engineering resulting in few selected variants. also random based protein engineering combined with screening of hundreds of thousands variants was used. in conclusion: the project team showed that by synergistic use of the two approaches, we were able to move faster towards a solution and eventually we succeeded finding new stabilized pectate lyase variants, applicable for new business areas. the importance energy independence as a national goal equals or exceeds that of the moon landing in 1990. the development of a new industry to produce fuel ethanol from woody biomass would increase national security, improve employment and the environment, and provide substantial relief from the debt of imported petroleum. costs associated with the rapid development of this new industry (∼$1.4 billion per year) could be paid by re-assigning 1 cent per gallon from existing federal gasoline taxes, a small price to pay for future energy independence. the corn-to-ethanol industry continues to make a remarkable contribution to our liquid fuel needs through expansion. today, one row of every six rows of corn is converted into fuel ethanol in the u.s. however, this expansion will be limited to 3-4% of total automotive fuel by the economics of corn costs and production. corn can do more! corn stover is the single most abundant agricultural residue in the us and can be used as a feedstock to produce 60-80 gallons of ethanol per dry ton. further expansion with other biomass feedstocks such as agricultural and municipal residues (lignocellulose, woody biomass) could produce over 100 billion gallons of fuel ethanol annually according at a recent joint report by the usda and doe (april, 2005) . current technology has been demonstrated at pilot scale for the production of fermentable sugars from hemicellulose by dilute acid hydrolysis and for the hydrolysis cellulose using fungal cellulose enzymes. biocatalysts such as recombinant escherichia coli have been developed and demonstrated for the efficient conversion of all sugar constituents of biomass to ethanol. a national goal for the full-scale deployment of current technology to produce biomass-based fuel ethanol will allow the us to reduce imported petroleum by 50%. together with increased efficiencies of hybrid vehicles, energy independence could be achieved within 10-20 years. similar gains could be realized by many nations around the world to provide new manufacturing and employment, redistributing wealth and ensuring a cleaner, healthier environment. bioethanol production using thermophilic bacteria marie just mikkelsen, birgitte k. ahring emab, biocentrum-dtu, 2800 lyngby, denmark the industry of bioethanol production is facing the challenge of redirecting the process from fermentation of relatively easily convertible but expensive starchy materials, to complex but inexpensive lignocellulosic biomass. on lignocellulosic hydrolysates, gram-positive thermophilic bacteria have unique advantages over the conventional ethanol production strains. the primary advantages are their natural broad substrate specificities, and in some strains, a high tolerance to lignocellulosic hydrolysates. moreover, ethanol fermentation at high temperatures also has the advantages of high productivities and substrate conversions, low risk of contamination and facilitated product recovery. some thermophilic bacteria naturally produce primarily ethanol from most sugar monomers present in lignocellulosics, but modifications are still necessary to increase ethanol yields. the release of useable sugars from lignocellulose biomass for industrial fuel-ethanol fermentation is often facilitated by a weak acid hydrolysis step. as a consequence, inhibitors such as furfural and 5-hydroxymethylfurfural (hmf) are formed as degradation products of xylose and glucose, respectively. moreover, the fermentative end-product of ethanol is also inhibitory. these, and other inhibitors present an environment, which elicits the expression of stress-related genes in saccharomyces cerevisiae. recently, 65 s. cerevisiae genes have been identified as important in furfural stress tolerance. when furfural is present, yeast with these genes disrupted grows poorly compared to wild-type yeast. a sub-class of these genes suggests that yeast grown under furfural-induced stress may rely upon similar pathways as cells grown under various other stresses, including oxidative, heat, and sorbate. to investigate this link further, we analyzed stress-induced phenotypes such as ros activity, dna damage, and membrane damage in wild-type and mutant yeast exposed to furfural or hmf stress. moreover, we investigated whether overexpression of this sub-class of genes would provide protection from furfural-induced stress and oxidative damage. micro-organism to be used in fermentation of lignocellulose hydrolyzates should preferably have three characters: (a) high ethanol tolerance, (b) resistance to inhibitors found in the hydrolyzate, and (c) a broad substrate utilization range, since the hydrolyzate contains several sugars. in addition to the possibility of controlling the level of potential inhibitors, fed-batch fermentations also permit the parallel uptake of several different monomeric sugars. two strains of saccharomyces cerevisiae, cbs 8066 (a commonly used laboratory strain) and tmb 3000 (a strain isolated from a spent sulfite liquor fermentation plant), were characterized in batch and fed-batch fermentation of a dilute-acid hydrolyzate from spruce. the strains had different abilities to ferment spruce hydrolyzate. the study suggests that the furan reduction capacity of a yeast strain is a key factor for its performance in fermentation of lignocellulosic hydrolyzate. polyketides constitute a structurally highly diverse group of natural products that possess broad ranges of pharmacological properties and represent a major source for novel cancer therapeutics. however, these compounds may be sub-optimal in regard of activity, selectivity, availability and unwanted side effects. in addition, the sustainable production of these valuable metabolites can be a challenge. studying the molecular basis of the biosynthetic pathways may set the basis for improving the production and for rationally engineering derivatives with altered bioactivity profiles, e.g. through targeted knockouts, mutasynthesis ziehl et al. (2005) , and swapping of pathway genes. our results in elucidating and manipulating the biosynthesis of selected antitumoral polyketide metabolites from bacteria (aureothin, chartreusin) and fungi (cytochalasines, rhizoxin) are presented. analyses at the genetic and biochemical levels provided new insights into several unusual biosynthetic features, e.g. non-linear polyketide assembly for the nitroaryl-substituted polyketide aureothin he and hertweck (2003, 2005) , an oxidative rearrangement cascade in the chartreusin pathway xu et al. (2005) , and a fungal iterative pks-nrps hybrid synthase schuemann and hertweck (2005) involved in cytochalasin biosynthesis. the most surprising result was obtained from elaborating the biogenesis of the antimitotic agent rhizoxin from rhizopus sp., which allowed for a significant improvement in large-scale production partida-martinez and hertweck (2005). he, j., hertweck, c., chem. biol. 2003 , 10, 1225 -1232 chem. bio. chem. 2005, 6, glycopeptides such as vancomycin and teicoplanin are the drugs of last resort for the treatment of severe infections caused by antibiotic resistant gram-positive bacteria. glycopeptides inhibit the peptidoglycan biosynthesis by binding as dimers to the d-ala-d-ala termini of the cell wall precursors. amycolatopsis balhimycina synthesizes the vancomycin-type glycopeptide balhimycin, whose structure and biological properties greatly resemble vancomycin and which only differs by its glycosylation pattern. using a "reverse genetics" approach we have identified the 66-kb gene cluster encoding the biosynthesis of balhimycin. by a combination of genetics, biochemistry and analytical organic chemistry, we were able to elucidate the biosynthetic pathway and to assign functions to almost all genes of the cluster. the biosynthesis starts with the pathway-specific provision of the non-proteinogenic amino acids ␤-hydroxytyrosine (␤-ht), hydroxyphenylglycine (hpg) and dihydroxyphenylglycine (dpg) which form together with (n-methyl)-leucine and asparagine the heptapeptide backbone of balhimycin. dpg is synthesized via a polyketide synthase mechanism (pksiii) similar to that known from plant chalcon/stilben synthases (pfeifer et al., 2001) . for the ␤-ht synthesis three genes are essential which form an operon (puk et al., 2004) : bpsd, an nrps binds a tyrosine molecule, which is then hydroxylated by the p450 monooxygenase oxyd. the perhydrolase bhp is required for the release of ␤-ht. subsequently bhaa, a nadh/fad-dependent halogenase catalyzes the chlorination of ␤-ht to form chloro-␤-hydroxytyrosine (puk et al., 2002) , which is needed to stabilize the dimerization. the amino acids are linked by non-ribosomal peptide synthetases (recktenwald et al., 2002) , and the aromatic side chains are interconnected by p450 monooxygenases; a series of reactions which lead to the first antibiotically active intermediate. inactivation of the oxygenase genes revealed the order of the cyclization steps (bischoff et al., 2001) : the oxygenases act in a stepwise fashion in the sequence oxyb, oxya and oxyc. the resulting cross-linked heptapeptide is then finally modified by methylation and glycosylation. the biosynthesis is regulated by the strr-type regulator bbr, which was shown to bind in front of different operons of the balhimycin gene cluster. this ensures the coordinated expression of the biosynthetic genes. the non-producing mutants, defective in the supply of the non-proteinogenic amino acids, were used as recipients in cloning experiments as well as in approaches of precursor-directed biosynthesis by feeding chemically synthesized alternative precursors. thus, novel balhimycin derivatives were generated (weist et al., 2002 (weist et al., , 2004 . bischoff et al., 2001 . angew. chem. int. ed. 40, 4688-4691. pfeifer et al., 2001 . j. biol. chem. 276, 38370-38377. puk et al., 2004 . j. bacteriol. 186, 6093-6100. puk et al., 2002 . chem. biol. 9, 225-235. recktenwald et al., 2002 . microbiology 148, 1105 -1118 . weist et al., 2002 . angew. chem. int. ed. 41, 3383-3385. weist et al., 2004 spectroscopy guided discovery of novel bioactive microbial natural products thomas ostenfeld larsen, michael edberg hansen cmb biocentrum-dtu, technical university of denmark, 2800 lyngby, denmark the task of finding novel bioactive natural products is usually bioassay driven. often a certain type of compound (e.g. polyketide, alkaloid) turns out to be active in an assay. when having generated a promising hit in a bioassay the normal procedure in the drug discovery process usually is to produce a large number of structurally analogous compounds either by traditional chemical synthesis or by combinatorial chemistry in order to study structure activity relation-ships and to find even more active lead compounds. alternatively to chemical synthesis of analogues nature can be explored for structurally similar compounds by uv-spectroscopy guided screening. this work will present a new method for the systematic and automated computer assisted search of full uv spectra in large number of datafiles for both dereplication of known and discovery of new natural products based on the use of the new mathematical algorithm x-hitting. exploring the substrate spectrum of the antibiotic producing bacteria saccharopolyspora erythraea p. krabben, p. oliveira, f. baganz, j. ward department of biochemical engineering, university college london, london wc1e 7je, uk knowledge of substrate utilisation capabilities play an important role in the development of genome scale metabolic models (borodina et al., 2005) and refinement of first generation annotations. furthermore, knowledge of the product formation during catabolism of different substrates provides valuable information about the distribution of metabolic fluxes and thereby forms a basis for rational strain improvement. we present here, data on the substrate utilisation capabilities and the corresponding product formation of s. erythraea. this analysis will help in improving the production of erythromycin and provide clues to the activation of the cryptic secondary metabolic pathways present in the s. erythraea genome. reference borodina, i., et al., (2005) . genome-scale analysis of streptomyces coelicolor a3 (2) modeling of growth and product formation on complex media containing multiple substitutable substrates is a challenge. complex media offers the organism multiple choices of carbon and nitrogen substrates including free amino acids, peptides, soluble and insoluble proteins in addition to the defined sources such as glucose and ammonium sulfate. we present a structured model that accounts for growth and product formation kinetics of rifamycin b fermentation in a multi-substrate complex medium. the model considers the organism to be an optimal strategist with a mechanism to regulate the uptake of the substrate combinations. further, we assume that the uptake of a substrate depends on the level of a key enzyme, which may be inducible. the model also considers control parameters as fraction of flux through a given metabolic branch. the control parameters are obtained using a simple multi-variable constrained optimization. the model parameters were rigorously estimated via a specifically designed experimental plan. the model correctly predicts the experimentally observed growth and product formation kinetics and the regulated simultaneous uptake of the substitutable substrates under different fermentation conditions. the model and the model parameters provide useful insights into the growth and product formation strategy of this industrially important process. this presentation will describe the experimental results, the model development and the relevant model parameters for a. mediterranei s699. the recent surge in oil price and the increasing concern on our environment have generated much interest in the production of chemicals from renewable resources. succinic acid, also called as amber acid, is a four-carbon dicarboxylic acid, which can be used as a precursor of numerous products including biodegradable polymers, green solvents, pharmaceuticals, and bulk and fine chemicals. a new capnophilic bacterium named mannheimia succiniciproducens mbel55e was isolated from the rumen of korean cow. this bacterium can produce large amounts of succinic acid along with some other metabolites such as lactic, formic and acetic acids. we have completely sequenced the genome of m. succiniciproducens and charaterized its genome content in the context of metabolic pathways. we then constructed the genome scale in silico metabolic network for metabolic flux analyses, and carried out metabolic flux analysis under varying environmental conditions. based on the in silico analyses results, we selected several target genes to be manipulated for enhanced succinic acid production. detailed results of metabolic engineering based on genome-scale information will be reported. we have been developing tools for inverse metabolic engineering in order to identify gene targets that improve the phenotype of industrial strains and cells for medical applications. to this end, we create genomic fragment libraries from a source organism and use it to transform the host organism. cells are properly selected in environments that favor the phenotype of interest and genes enriched in these cells are sequenced and used in follow up transformations of cells with specific genetic backgrounds. this overall strategy is complemented with additional tools for modulating gene over-expression, gene deletion, and high throughput clone isolation. we will demonstrate applications of this strategy to the identification of gene targets for improved xylose assimilation in recombinant saccharomyces cerevisiae and improved lycopene production in escherichia coli. based on assumed reaction network structures, nadph availability has been proposed to be a key constraint in ␤-lactam production by penicillium chrysogenum. in this study, nadph metabolism was investigated in glucose-limited chemostat cultures of an industrial p. chrysogenum strain. enzyme assays confirmed the nadpspecificity of the dehydrogenases of the pentose-phosphate pathway and the presence of nadp-dependent isocitrate dehydrogenase. pyruvate decarboxylase/ nadp-linked acetaldehyde dehydrogenase and nadp-linked glyceraldehyde-3-phosphate dehydrogenase were not detected. although the nadph requirement of penicillin-gproducing chemostat cultures was calculated to be 1.5-1.7-fold higher than that of non-producing cultures, activities of the major nadph-providing enzymes were the same. isolated mitochondria showed high rates of antimycin a-sensitive respiration of nadph, thus indicating the presence of a mitochondrial nadph dehydrogenase that oxidizes cytosolic nadph. the presence of this enzyme in p. chrysogenum has important implications for stoichiometric modelling of central carbon metabolism and ␤-lactam production and may provide an interesting target for metabolic engineering. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf-21 cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf21 infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp486 coding for am-cyan coral protein, which emits natural green fluorescence. complete elucidation of the genetic control of a metabolic flux requires the availability of fine-grained expression levels of the gene(s) of interest. we developed a collection of promoters of varying strength for tuning gene expression in the yeast s. cerevisiae. engineered promoters were obtained through random mutagenesis of the constitutive tef1 promoter. eleven mutated promoters were selected by fluorescence-activated cell sorting (facs) spanning gradually increasing activities between about 8 and 120% compared to the native tef1 promoter. data were also confirmed at the level of mrna via rt-pcr. by introducing selectable markers in front of the different tef1 promoter mutations, we provided plasmid collections, which can be directly used to amplify promoter replacement cassettes for genomic integration of the fine-grained promoter collection in front of any yeast gene. l-arabinose, widely distributed in plant kingdom, is a component of plant cell wall. l-arabinose does not abundantly exist in free state in plants, but usually in corn hull, sugar beet pulp, gum arabic, mesquite gum, as the polysaccharide such as arabinoxylan and arabinogalactan. to produce arabinose from agricultural wastes, we screened arabinogalactan degradable strain from compost. thereafter, putative arabinase gene from this strain was cloned (b1029 ts2-8). as a result of spectrometric assay using -nitrophenyl ␣l arabinofuranoside, recombinant showed 3-4-fold higher activity than wild type e. coli strain. after enzymatic reaction with corn fiber, b1029 ts2-8 produced 2.15 g/l of l-arabinose, which was detected on hplc. however, the enzyme activity was very low. so, we are transferring the gene into expression vector system. further characterization study and enzyme engineering to enhance the activity toward corn fiber will be presented in poster. there are only a limited number of hypersaline areas all over the world, which include several locations in turkey such as van lake located in eastern region of turkey. isolation and identification of halophilic and hyperhalophilic microorganisms from such locations is essential for the determination of biodiversity in turkey. high-level production of extremozymes from these microorganisms has also many economical advantages due to their stability at extreme reaction conditions. proteolytic enzymes are the most important group of enzymes produced commercially. of these, proteases produced by alkalophilic microorganisms are investigated not only in scientific areas such as protein chemistry and protein engineering but also find wide application in food, pharmaceutical, leather and detergent industries. in this study, 24 microorganisms isolated from van lake were screened for the presence of extracellular alkaline protease activity. the optimum screening temperature and ph were determined as 37 • c and ph 9.5, respectively. one of the isolates that could grow at 0-20% salinity reached highest levels of extracellular alkaline protease activity. this best producer, which was identified as the halotolerant bacillus pumilus, was found to produce alkaline protease both in the presence and absence of nacl. to improve enzyme production yields, culture conditions such as medium composition, growth ph and temperature were optimized. the effect of different carbon sources, organic and inorganic nitrogen sources on the production of alkaline protease was studied. whereas a mixture of inorganic and organic nitrogen sources induced high protease production, use of only an organic nitrogen source supported poor enzyme production. halotolerant bacillus pumilus produced maximum alkaline protease activity when maltose, yeast extract and sodium nitrate were used as carbon source, organic and inorganic nitrogen sources, respectively. this project was supported by tubitak through project tbag 2321-103t069. in the market of biochemical products a very important role is played by heterologous proteins production, and despite recent advances in mammalian cells exploitation, yeasts can still present advantages as host systems. among them, the spoilage yeasts belonging to the zygosaccharomyces genus have become, due to some peculiar properties, significantly attractive. in particular, z. bailii is characterized by acid resistance, osmotolerance to high sugar and ethanol concentration combined with high biomass yield. despite still little is known about its genetics and cellular biology, our group is working on its development and exploitation for recombinant productions with an integrated approach coupling physiological study with the creation of molecular tools for heterologous proteins production. we previously described and did a patent application regarding the first techniques necessary to transform this yeast and to express and secrete different proteins derived from different sources. here we present and discuss the last advances in optimization of heterol-ogous protein expression in particular, on one side we present a reproducible strategy for target gene deletion, leading to the first z. bailii auxotrophic mutant, and on the other we show the improvement of gene dosage and plasmid stability by building a set of multicopy expression vectors based on the sequences of the z. bailii 2 -like endogenous plasmid psb2 and an integrative plasmid. all the known ␥-butyrolactone autoregulator receptors are highly conserved in the dna binding motif present in their n-terminal portions and have been proposed to play roles as transcriptional regulators in antibiotic production and/or morphological differentiation. previously, kim et al. reported that the cloned scar in streptomyces clavuligerus has several characteristics of the autoregulator receptors in the genus streptomyces. in this study, to clarify the in vivo function of scar, a scar-disrupted strain was constructed by means of homologous recombination after introducing a scar-disruption construct via transconjugation from e. coli. no difference in morphology was found between the wild-type strain and the scar disruptant. however, the scar disruptant showed a 1.8-fold higher production of clavulanic acid than the wild-type strain. the phenotype was restored to the original wild-type phenotype by complementation with intact scar. therefore, the autoregulator receptor, scar, acts as a negative regulator of biosynthesis of clavulanic acid but plays no role in cytodifferentiation of s. clavuligerus. lactate dehydrogenase catalyses the production of lactate from pyruvate. it is the first target for many researches on lactic acid producer microorganisms like rhizopus oryzae. in the present study based on the known sequences of r. oryzae ldha and ldhb genes skory (2000), they were cloned and expressed in a citric acid producer fungus aspergillus niger. the aspergillus niger strains expressing ldha or ldhb gene resulted in increased production of lactate in aspergillus niger. among 50 transformants tested 4 ldha and 5 ldhb expressing strains were found to have higher lactate dehydrogenase activity compared to wild type in the conditions tested. the highest specific activity obtained with ldha transformants was only 2.5 times of the wild type while this was 10 times for one of the transformants expressing ldhb. in addition to increased lactate production citric acid production was also increased. however, gluconic acid production ceased in the ldha or ldhb expressing a. niger strains. the production of lactic acid in a. niger transformants and lactate dehydrogenase a and lactate dehydrogenase b enzymes are being investigated in the chosen strains. selection of n source suitable for production of rhodococcus sp. biomass for the purposes of microbial transformation of 5␣h-epoxypregnanolone (5␣h) and 5 -epoxy-pregnenolone ( 5 ) into their 9␣-hydroxy-derivatives was carried out. three dehydrated and three non-dehydrated n sources were tested. the transformation reaction was carried out in phosphate buffered medium containing 1 g l −1 of the steroid substrate and 0.1 g l −1 cells. the steroids were determined by hplc. the transformation resulted in formation of three derivatives appearing in the reaction medium in the sequence: 4 -3-keto-; 9␣-hydroxy-and 9␣,20␤-hydroxy-epoxy-pregnenolone. a strong influence of the n source on the hydroxylating activity of the biomass was observed. triptose (difco) gave a cell depot actively hydroxylating 5␣h without any significant accumulation of the 4 -3-keto-derivative. the most effective accumulation of hydroxylated derivatives of 5 was observed with biomass grown on freshly prepared meat extract while the commercial products triptose (difco), meat extract (difco) and lactalbumin (flika) gave valuable information about the dynamics of the transformation process. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene 1 , nazif kolankaya 2 : 1 kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; 2 hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth (1% soytone, 4% d-glucose and 0.5% cellulose pulp). maximal extracellular ligninase production was detected after7 day (7 nkat). the optimum biobleaching conditions are 30 • c and ph: 4.8, with 10 days. in this condition p. versicolor decreased the kapa number from 38.55 to 19.42 and increased brigthness from 28 to 32.7 in 10 day treatment. xylanase production, purification and characterization from a soil isolate, bacillus m-13 ayşegül ersayin 1 , aytaç kocabaş 1 , b. zümrüt ogel 2 , ufuk bakir 3 : 1 biotechnology department, middle east technical university, ankara, turkey; 2 food engineering department, middle east technical university, ankara, turkey; 3 chemical engineering department, middle east technical university, ankara, turkey, 06531. e-mail: ubakir@metu.edu.tr (u. bakir) xylan is a major component of the plant cell wall, representing up to 35% of the dry weight. xylan molecule is a complex polymer consisting of a ␤-d-1,4-linked xylanopyranoside backbone substituted with acetyl, arabinosyl and glucuronosyl side chains. hydrolysis of the xylan backbone is mainly catalysed by endo-␤-1,4-xylanases (ec 3.2.1.8). many bacterial and fungal species are able to utilize xylan as a carbon source. interest in the enzymology of xylan hyrdolysis has increased because use of xylanases in bioconversion of agricultural wastes to valuable products like single cell protein, xylo-oligosaccharides and fuel, in bio-bleaching processes, food and animal feed industries. in this study, xylanolytic nature of a soil isolate bacillus spp., bacillus m-13, has been shown. bacillus m-13 produced multiple xylanases when grown on a liquid medium containing agricultural wastes as the sole carbon source. various agricultural wastes including corn-cobs and cotton waste, with and without pretreatments were used to maximize enzyme production. the major xylanase having molecular weight of 20 kda upon sds-page and a pi of 9.1 upon ief was partially purified by liquid chromatographic techniques 150-fold with 40% recovery, including gel filtration, ion exchange and hydrophobic interaction chromatography. enzymes are important constituents in the laundry detergents due to their contribution to shortening washing times, reduction of energy and water consumption by lowering washing temperatures, provision of environmentally friendlier wash-water effluents and fabric care. however, they can loose a significant part of their activity in the chemically-hostile detergent matrix over a time period of several weeks. therefore, improving the storage stability of enzyme granulates is the main challenge in the development of a new product. the complexity of the detergent matrix implies the presence of a complicated mechanism involved in the inactivation of the enzymes. a combination of factors, such as oxidation by h 2 o 2 released by the bleaching agents, humidity, high temperature, autolysis of enzymes, high local ph in a granule, oxygen, and other detergent components, plays a role in the activity loss. an experimental investigation on the inactivation of the solid-state enzyme during storage has been initiated. the release rate of h 2 o 2 from the bleaching agent, sodium percarbonate, was determined using a simple and accurate method for measuring the gas phase h 2 o 2 concentration. the deactivation kinetics of pure enzyme was determined as a function of gas phase h 2 o 2 concentration and humidity. the preliminary results indicate that humidity plays a significant role in the inactivation mechanism of the detergent enzyme due to a possible increase in the mobility of the enzyme molecule and the surface area exposed to destructive agents. the effect of main granulate ingredients on the stability of the enzyme was investigated and the extent of the protection of each component was estimated. the study is important for the revealing of the phenomena occurring in the detergent matrix during storage. understanding the inactivation mechanism provides a valuable tool for the development of more effective protective coatings and stabilizers. the use of biosurfactants in cosmetic industry has attracted great attention of biotechnological researchers because they consist of two inexpensive, renewable and easily accessible starting agricultural materials: sugar and oil/fat. carbohydrate based products are non-toxic, biocompatible and biodegradable. in addition, the enzymatic processes present many advantages in comparison with the chemical methods, which employ high temperatures in the presense of alkalin catalysts, high-energy consuption and low selectivity of products. sugar esters present vast application, such as for antibiotics, biomaterials, surfactants, cosmetics and so on. we investigated the synthesis of sugar vinyl esters, using protease-catalysed transesterefication method applying protease from bacillus subtilis. sucrose 0.125 m and vinyl ester 0.5 m has been mixed in dimethylformamide at 160 rpm of agitation. at first, we have studied the effects of protease from bacillus subtilis concentrations (5, 10, 20 and 40 mg/ml) as catalyst. afterwards, the influence of the temperature (30 and 50 • c). after that, the influence of the molar ratios (1:1; 1:2; 1:4 m/m) between vinyl laurate (ch3(ch2)10cooch ch2) and sucrose. subsequently, we investigated the effects of water amount, using 0, 5, 10 and 20% of water in dmf. the conversion ratios of sucroseto-sucrose esters were determined decreasing sucrose measurement with hplc. the results showed that the best conditions to produce high activity on the enzymatic reaction was by using 40mg/ml of protease from bacillus subtilis at 30 • c, molar ratio of 1:4 (vinyl laurate:sucrose) and adding 10% of water in dmf. finally, we succeeded in the characterization of vinyl sugar ester, which was produced after 25 hours of reaction by 13 c nmr. the results confirmed the c1substituted sugar mono-ester (1 -o-vinyl lauroyl sucrose). effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide ç alık bre lab, department of chemical engng, ankara university, 06100 ankara, turkey ␣-amylase (e.c. 3.2.1.1) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣-1,4 glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b-14396), which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in 0.5 dm 3 air-filtered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs1). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled 1.0 and 3.5 dm 3 systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc28s ultracentrifuge, ␣-amylase activity was measured by the dns method bernfeld (1955) . amino acid concentrations were determined with a hplc (waters), pro-tein and organic acid concentrations were measured with a hpce (waters, quanta 4000e) ç alik et al. (1998) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method rainer (1990) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources, i.e. glucose, fructose, maltose, lactose and soluble starch; n sources, i.e. (nh 4 ) 2 hpo 4 , (nh 4 ) 2 so 4, and nh 4 cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and by-product concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. bernfeld, p., 1955 . methods in enzymol. 1:149-159. ç alık, p., ç alık, g.,özdamar, t.h., 1998 . enzyme microb. technol. 23, 451-461. rainer, b.w., 1990 . geldanamycin is a benzoquinone ansamycin produced as a secondary metabolite by the actinomycete, streptomyces hygroscopicus var. geldanus, in submerged culture. it is a broad-spectrum antibiotic and exhibits an anti-tumour activity through its interaction with the heat shock protein 90 family of chaperone proteins. the optimal recovery of geldanamycin from fermentation broths is the focus of the presented work. the application of adsorbent resins was assessed and the viability of developing a solid phase extraction process for geldanamycin was determined. it was found that recovery of geldanamycin from fermentation broth was possible using adsorbent resins and the use of resins facilitated the recovery of a product stream of high purity. the composition of the fermentation broth had an impact on the performance of the resins and it was found that assessing performance on the basis of experimentally derived data was more apt than studying the kinetics of adsorption alone. adsorptive processes are, by their nature, difficult to optimise and this was found to be the case when optimising the recovery of geldanamycin from partially clarified fermentation broth. considerable effort was required to optimise geldanamycin adsorption, via examining the effect of environmental conditions and process system configuration, and geldanamycin desorption, via examining the effect of environmental conditions and investigating selective elution patterns. it is well known that halophilic eubacteria synthesize compatible solutes in order to face the high ionic strength environment in which they proliferate. these biomolecules are gaining more and more importance as biotechnological tools in a wide array of applications, and the recently developed novel bioprocesses enabled large-scale production of these compounds and therefore commercial distribution. however, there is still interest in the optimization of the production process of sole hydroxyectoine that was demonstrated to have a superior stabilization capacity. in this research project, we optimized growth conditions of marinococcus m52 to obtain high yield of hydroxyectoine. their production proved faster in the batch experiments at a higher oxygen supply, even if the stationary phase was comparable in all cases. the mf experiments showed a final biomass which was 5-fold that obtained in the corresponding batch process. in addition the monitoring of compatible solutes production showed that in the last 24h experiment hydroxyectoine accounted for 80-90% of the total content, accumulating up to 13-15% of the cell dry weight. studies for improving downstream process for ectoine and hydroxyectoine recovery showed that short permeabilization cycles in water are effective in a temperature range between 45 • c and 55 • c using a ratio 1:4/biomass:water. moreover, we evaluated the ability of ectoine to stabilize lactic acid bacteria during freeze-drying and to protect human cells from heat stress. in particular, the compatible solutes were added to the medium of confluent keratinocytes before subjecting the cells to heat stress, or lps insult. rt-pcr and western blot analysis demonstrated the hsp70b' gene over-expression in heat stressed human keratinocytes treated with ectoine. finally, we demonstrated that even at low concentration (50 mm) these compatible solutes are able to diminish cell death in lactic acid bacteria due to lyophilization procedure. among all existing alternative energy sources, biomass-derived bioethanol is especially advantageous since it is clean, sustainable and potentially inexpensive. the actual production of bioethanol is divided into a pre-treatment step, an enzymatic hydrolysis step and a fermentation step. while fermentation has been practiced by humans for centuries, our knowledge of enzymatic hydrolysis is still limited. nevertheless, it is well accepted that hydrolysis is a synergism among three classes of enzymes, ␤-glucosidase, endoglucanase and cellobiohydrolase. furthermore, a complete and efficient hydrolysis is only achieved when the enzymes are in the correct proportion. the common enzyme proportions have so far been based on the natural enzyme abundance as produced by the microorganism or mainly been determined by a trial-and-error approach. in this study, however, we used metabolic control analysis (mca) as a modelling tool to gain fundamental knowledge about enzymatic hydrolysis and to design an optimal enzyme mixture. using gepasi, a free software, the degree of control of each reaction step or each enzyme towards the overall hydrolysis can be calculated. our hypothesis is that the amount of each enzyme used for hydrolysis should be proportional to the degree of control of the enzyme. with mca, a significant amount of time, labour and reagents can be saved on developing hydrolysis enzyme mixture. furthermore, this study should demonstrate the usefulness of mca on understanding enzyme-catalyzed reactions outside the cell. process optimization for fed-batch fermentation of bacillus thuringiensis subsp. israelensis arindam chaudhury, gopinathan c department of biotechnology, university of calicut, calicut, kerala 673635 india. e-mail: g achaudhury@umassd.edu (a. chaudhury) bacillus thuringiensis (bt) is a desirable biopesticide because of its low cost and lack of toxicity. the use of bt in developing countries is limited due to process complications and economic non-feasibility of the fermentation process. in the present study, we have shown how regional production, using inexpensive alternatives for carbon and protein sources, can effectively reduce the cost of mass production of bt. while using alternative media supplements, the biomass production, nor the larvicidal activity was hampered. in addition, the positive effects of sparged aeration and the indispensable role of yeast extract were also proved. this work provides the first experimental proof of delineating the sporulation process and delta-endotoxin production. the role of various buffering agents and additives in increasing biomass production and early sporulation were also investigated. for the production of coenzyme q 10 (coq 10 ), an electron carrier in the respiration chain with antioxidant activity. with decrease of dissolved oxygen level from 20 to 5%, the intracellular coq 10 content increased about 4-fold, yielding 2 mg per g-dry cell weight at 5% dissolved oxygen level. azide significantly increased the intracellular coq 10 content, with the highest value of 5.3 mg per g dry cell weight in the presence of 0.45 mm of sodium azide. however, dnp (up to 200 m) and h 2 o 2 (up to 10 m) did not affect the intracellular coq 10 content, indicating proton gradient release and oxidative stress do not affect the synthesis of coq 10 . these results show that restricted electron flux by limited oxygen supply and the addition of azide increases the intracellular coq 10 content. fourier transform infrared spectroscopy (ft-ir), combined with in situ heat sterilizable attenuated total reflection (atr) probes, constitutes a promising and versatile technique for on-line monitoring of bioprocesses. the ft-ir enables rapid determinations of the medium composition without the requirement of sample withdrawal and preparation. in this work the concentration levels of the substrates glycerol and methanol were monitored on-line in pichia pastoris cultivation. partial least squares (pls) models were used for obtaining the concentration readings. the glycerol concentration measurement proved to be very reliable and reproducible in the glycerol batch phase. however, the on-line information regarding the glycerol concentration was not utilized for any process control purposes. on the other hand, the availability of on-line information about the methanol concentration proved to be crucial for the successful implementation of the cultivations. the temperature strategy in the methanol fed-batch phase utilized temperatures as low as 10 • c. in order to keep the metabolic activity at a reasonable level the culture was therefore pushed towards the maximal substrate consumption rate, rather than being a conventional substrate limited fed-batch. as a consequence methanol accumulation occurred on occasions. without on-line information about the concentration this accumulation, if sustained, would have resulted in a poisoning of the culture, either from methanol itself, or perhaps more importantly from formaldehyde. therefore, it can be concluded that the ft-ir/atr instrument was very useful in this application. jørgensen department of chemical engineering, denmark technical university, building 229, dk-2800 lyngby, denamrk. e-mail: fpd@kt.dtu.dk (f.p. davidescu) modeling biochemical reaction network in microorganisms still represents a challenge due to the very large number of enzyme catalyzed biochemical reactions, to the very complex system and to the many feed-forward and feedback regulation mechanisms. the presented approach to model such a system is based on the stochastic grey-box modeling framework proposed by kristensen et al. (2003) . this methodology consists of parameters estimation based on a prediction error method followed by different statistical tests for parameter significance and for model (in-) validity. the methodology furthermore allows estimation of unknown functional relations, e.g. kinetic rates. a set of experimental data zangirolami (1998) obtained during continuous cultures of a high enzyme producing aspergillus oryzae strain. the oxygen concentration was decreased stepwise and the substrate concentration was modified from one experiment to other. a model proposed by agger et al. (1998) is investigated on these data. the primary interest is to develop a physiologically feasible model, also at the low oxygen concentrations often found in industrial practice. microbially produced secondary metabolites such as antibiotics have tremendous economic importance. streptomyces spp. have long been identified as sources of antibiotics and chemotherapeutic compounds, synthesising over 4000 bioactive compounds. geldanamycin is a novel chemotherapeutic agent produced by streptomyces hygroscopicus var. geldanus in submerged fermentation. initial studies have focused on optimisation of media design through understanding and controlling metabolic routes of biosynthesis within the cell. geldanamycin is a by-product of the shikimate or aromatic amino acid biosynthesis pathway. stimulation of this pathway and concomitant production of geldanamycin is achievable through amino acid control. increasing concentrations of primary carbon source greatly influence biomass generation and product formation, as does the inclusion of cations such as magnesium and calcium to the fermentation media. optimisation of production media through balancing minerals, nitrogen, and carbon sources has significantly improved antibiotic yields in shake flask cultures and the development process will be extended into pilot scale through the use of bioreactors. microbiology and biotechnology research group, school of life sciences, napier university, edinburgh, eh10 5dt, scotland. email: m.el-mansi@napier.ac.uk (m. el-mansi) synopsis: during growth of corynebacterium glutamicum on glucose or other glycolytic intermediates, pep carboxylase fulfils an anaplerotic function as it replenishes intermediary metabolism with biosynthetic precursors that are essential for growth and glutamate production. under these conditions, pep carboxylase plays a central role and this in turn is characterised by a high flux control coefficient thus rendering this enzyme an ideal target for metabolic interventions. further analysis in silico revealed that any increases in the concentration of the enzyme was accompanied by increases in flux through the enzyme itself as well as glutamate formation, presumably as a consequence of sustaining a high intracellular level of ␣-ketoglutarate; the immediate precursor for glutamate biosynthesis. a combined approach to enhance periplasmic expression of human growth hormone in escherichia coli, using a modified signal peptide from alpha amylase gene of bacillus licheniformis s.k. falsafi 1,2 , a. zomorodipour 2 : 1 islamic azad university of jahrom, iran; 2 department of mol genet. national inst for genet eng & biotechnol., tehran, iran. e-mail: soheil falsafi@yahoo.com (s.k. falsafi) the alpha amylase gene signal peptide, originated from a strain of bacillus licheniformis, was shown to be able to transport its native protein, when expressed in e. coli the competence of the fusion protein being processed and translocated through the inner membrane is highly dependent on the amino acid sequences in the signal peptide. therefore, in order to increase the expression efficiency of bla signal peptide, we reconstructed the bla signal peptide coding fragment with the following modifications. two rare codons of arg 6 (cgg) and arg 10 (cga) and codons for leu 15 (tta) and pro 23 (cct), in the signal peptide were substituted with their corresponding e. coli major codons. two other changes, including phe 20 (ttc) → leu 20 (ctg) and ala 28 (gcg) → met 28 (atg), were also introduced to increase the processing efficiency. the hgh-expressing plasmid equipped with the modified bla (blaf2) was subjected for further expression analysis in a t7-based expression system. the results obtained from the protein patterns of the induced bacteria indicates in high expression level of hgh preprotein (hgh::blaf2) followed by efficient transfer of the mature hgh to e. coli periplasm. (ip6) has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip3) and inositol tetrakisphosphate (ip4) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip6 is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia1 was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia1 converted ip6 into ip5 (myoinositol 1,2,3,5,6 pentakisphosphates) and another isomer, which is yet to be elucidated. characterization of the novel ␤-peptidyl aminopeptidase (bapa) from sphingomonas sp. 3-2w4 that cleaves synthetic ␤peptides birgit geueke, hans-peter e. kohler environmental microbiology, eawag, 8600 duebendorf, switzerland. e-mail: birgit.geueke@eawag.ch (b. geueke) non-natural peptides, which are capable of evoking a specific biological response, are currently receiving much attention. oligomers of ␤-amino acids (␤-peptides) are representing a group of pharmaceutically interesting peptides because of their very high stability towards enzymatic degradation and their ability to mimic the structure of naturally occurring biologically active peptides. the pharmaceutical potential on the one hand and the high stability on the other hand aroused interest for studies on the environmental fate and the degradation behaviour of this class of compounds. a novel bacterial strain (sphingomonas sp. 3-2w4) that was capable of degrading short ␤-peptides was isolated from an enrichment culture. the ␤peptide degrading enzyme was purified and its gene sequence was determined (bapa). the gene encodes a ␤-peptidyl aminopeptidase (bapa) of 402 amino acids that is synthesized as preprotein with a signal sequence of 29 amino acids. it belongs to the n-terminal nucleophile (ntn) hydrolase superfamily and is the first peptidase that is capable of cleaving amide bonds in ␤-peptides composed of synthetic ␤-amino acids. the biochemical properties of recombinant bapa were investigated regarding its substrate specificity and possible application in the synthesis of ␤-peptides. to produce efficient strains of agaricus bitorquis (quel.) saccardo, which are resistant to high temperatures p. guler, a. ergene, s. tan kirikkale university, faculty of science and literature, department of biology, yahsihan-kirikkale in this study, the culture mushroom agaricus bitorquis (quel.) sacc. the growth of the mycelium and the fructifications under high temperature is examined. the spores taken from the mushrooms that were collected from nature were grouped as a, b, c, d, e. the spores were inoculated into malt extract agar and incubated at 30 • c and primer mycelium was produced. the mycelium discus taken from primer mycelium in 8 mm diameter were inoculated into the center of malt extract agar and incubated at 30, 32, 34, 36 and 38 • c separately. during the incubation period the growth of the mycelium were measured. during the growing period the radial growth speed of the mycelium were taken as criteria. the best mycelium growth for all groups was seen at 30 • c. at 36 • c the e group mycelium and at 38 • c other group's mycelium did not grow. these temperatures were determined as thermal lethal point for the groups. from all the mycelium produced from all temperatures spawn was prepared and with the results taken from these, spawn calendar is prepared. in this research, the spawn was inoculated to compost with mixing system and separately put in culture rooms, temperatures as 30 and 32 • c. at this level the culture mushroom production techniques were used. the harvested mushrooms were inspected morphologically. at this morphological inspections the cap width, cap tissue thickness, stalk thickness and stalk long ness was taken as criteria. in the study the best growth was seen at d group mushrooms and this group mushrooms tyrosinase's activities were measured and graphics were made. introduction: viral contamination of biological products; cause many problems in viral diagnostic laboratories, blood transfusion organizations, and biological producers. bovine viral diarrhea virus (bvdv), from the pestivirus genus, is the most common viral contamination in (fetal) bovine serums (fbs). also, bvdv used as a module, for study hepatitis c virus inactivation due to its similarity in structure and genome. pulsed uv lights (puvls) have this potential to inactivate known and unknown or reemerging viruses as well as prions. two puvl with the wavelengths of 355 and 266 nm, were produced by q-switched nd 3+ :yag laser in its third and forth harmonic, respectively. the energy of each pulse for 355 nm was 12.7 mj/cm 2 and for 266 nm was 35.2 mj/cm 2 . bvdv were produced and titrated in mdbk cell line. mdbk and fbs were already checked for non-cytopathic or cytopathic pestiviruses, using related ag-elisa kit. bvdv suspended in solution with the dilution of 1:2 before exposure. the quartz tube with the minimum uv-absorption in compare with air, used as a container for exposed solutions. calculation of the virus titer, 10 4.3 tcid 50 /ml, was done based on the reed and muench method. bvdv suspended in pbs was exposed into the 3.52-352 j/cm 2 of puvls with the wavelength of 355 nm and also, was exposed into the 1.27-92.25 j/cm 2 of puvls with the wavelength of 266 nm. furthermore, bvdv suspended in fbs was exposed into the 88, 176, 352 and 704 j/cm 2 of puvls with the wavelength of 355 nm and also, was exposed into the 6. 35, 25.4, 63.5, 127 and 190 .5 j/cm 2 of puvls with the wavelength of 266 nm. results: the minimum dose for inactivation of bvdv suspended in pbs with the 355 and 266 nm wavelengths of puvls, were 352 and 92.25 j/cm 2 , respectively. also, the minimum dose for inactivation of bvdv suspended in fbs with the 355 and 266 nm wavelengths of puvls, were 704 and 127 j/cm 2 , respectively. to evaluate the fbs quality to support cell culture, treated fbs with the dose of 190.5 j/cm 2 of 266 nm puvls was used to grow vero cell line in 12 successive passages. the viability of cells in two study groups was identical. the statistical evaluation of two treated groups showed no significant difference, in 12 passages. conclusion: because inexpensive equipment can be used to produce puvls capable of handling different volumes of biologics with operational ease, this viral inactivation technique is cost effective for relevant industries. the procedure has the potential to be combined synergically with other inactivation method. puvls offer a new, nonadditive and chemically safe alternative for the treatment of fbs to inactivate adventitious viruses and to preserve the biological activity necessary for the propagation of cell culture. characterization and gene cloning of the g-resorcylic acid decarboxylase for application to selective production of g-resorcylic acid y. iwasaki 1 , y. ishii 2 , k. kino 1 , k. kirimura 1 : 1 dept. appl. chem., sch. sci. eng., waseda univ., tokyo, japan; 2 bme, asmew, waseda univ., tokyo, japan. e-mail: iwasaki@moegi.waseda.jp (y. iwasaki) for selective production of ␥-resorcylic acid (␥-ra, 2,6-dihydroxy-benzoic acid) from resorcinol (re, 1,3dihydroxybenzene) under mild conditions, we screened various microorganisms and found the reversible ␥-ra decarboxylase (rdc) as a novel enzyme applicable to carboxylation of re to form ␥-ra, in a bacterial strain rhizobium radiobacter wu-0108 1) . rdc catalyzed the decarboxylation of ␥-ra, and regio-selective carboxylation of re to form ␥-ra, without formation of ␣-ra and ␤-ra. the molecular weight of rdc was estimated to be 130 kda by gel-filtration, and that of the subunit was determined to be 34 kda by sds-page, suggesting that rdc is a homotetrameric structure. the gene encoding rdc was sequenced, and a site-directed mutagenesis study revealed that the two histidine residues at positions of 164 and 218 in rdc are essential for the catalytic activity of rdc. through the reactions using e. coli cells highly expressing rdc, 6.7 mm ␥-ra was produced from 15 mm re at 30 • c for 16 h, with a yield of 45%. ishii, y. et al., 2004. biochem. biophys. res. commun., 324, 611-620. laccase biosynthesis in stirred fermenters teresa jamroz, stanislaw ledakowicz, barbara sencio department of bioprocess engineering, technical university, lodz pl 90-924, poland industrial applicability of enzymes is closely related to development of efficient methods of their production. currently, significant interest in lignolytic enzymes, including laccase, has been observed. laccase is an enzyme applied in various industrial branches and environmental processes. broad laccase applicability induces researchers to develop urgently efficient methods for its commercial production. laccase (ec.1.10.3.2. p-diphenol oxidase) is produced by cap mushrooms from the class basidomycetes. this is the so-called white rot fungi which in natural conditions appears on both living and dead wood. as shown in the practice of biotechnological processes, high-efficient strains have low resistance to destructive factors in bioreactors. hence, to preserve a proper morphology and physiological state of an organism, strictly determined culture conditions must be obeyed. this is very important in the case of basidomycetes for which submerged culture in the liquid phase is not a natural habitat. results of studies on laccase production from cerrena unicolor family are discussed. cultivation of active biomass was carried out in stirred tank and rotating disc bioreactors of different volume (b. braun of working volume 12 dm 3 ; fas-01 of working volume 3.5 dm 3 ). experiments in both fermenters were made at impeller revolutions 200 and 300 min −1 , on a modified lindberg substrate. significant differences in the rate and yield of laccase production were reported. an almost three times higher values of laccase activity were obtained in the b. braun fermenter, at rotational speed 300 min −1 . to retain suspended cells in bioreactor a filtration process can be used. the biomass is concentrated by withdrawing cell-free culture broth. if the desired product is dissolved in the broth (extracellular production), the procedure enables the continuous harvest in the cellfree permeate. an application test of filtration system for suspended biomass of aspergillus niger in submerged single stage continuous culture was presented in this report. the system is easy to construct and there is a possibility of its sterile exchange during cultivation. the culture medium contained the following substances (g/dm 3 ): white sugar, 150.0; nh 4 no 3 , 1.5; mgso 4 ·7h 2 o, 0.2; kh 2 po 4 , 0.2; feso 4 ·7h 2 o, 0.05. fermentations were carried out in the lab bioreactor biomer 10. the bioreactor was a standard cstr (continuous stirred tank bioreactor) with working volume of 5 dm 3 . high citric acid concentration in culture medium (p = 108.9 g/dm 3 ), high yield of citric acid (y p/s = 72.6%) and high efficiency coefficient (k ef = 71.1) were observed in single stage continuous culture with biomass retention. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar ç alık, iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc18::bal carrying recombinant escherichia coli on a defined medium with 8.0 kg/m 3 glucose were investigated in order to fine-tune the bioreactor performance, in v = 3 dm 3 batch bioreactors at five different conditions with the parameters at, i.e. q o /v r = 0.5 vvm and n = 250, 375, 500, 750 min −1 and; q o /v r = 0.7 vvm and n = 750 min −1 . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph o = 7.25 are optimum for maximum bal activity, i.e. 860 u/cm 3 at 12 h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains 102 metabolites and 133 reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e. coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. saprophytic mycobacterium strains belong to the best known microorganisms which have been applied to the pharmaceutical industry for the production of steroid drugs. the mycobacterial cell wall is the permeation barrier to chemical compounds, including lipophiles. using isoniazid (inh), the inhibitor of the mycolic acids biosynthesis, we were able to demonstrate increased ad production and susceptibility to antimicrobial agents. the process of sterol transformation and products accumulation was monitored using gas chromatography. isoniazid was shown to intensify ␤-sitosterol side-chain degradation by mycobacterium sp., and accumulation of 4-androstene-3,17-dione (ad) and 1,4-androstadien-3,17-dione (add), which are the starting materials in the biotechnology of medically important steroids. to confirm these results, the sensitivity of the bacteria to antimycobacterial drugs was performed. the minimum inhibitory concentration mic 50 of rifampicin and erythromycin decreased markedly in the presence of inh. this work was supported by grant nr 3p04c 06923 of the committee for scientific research. for the purpose of high-throughput screening and to reduce experiments with animals in pharma biotechnology biosensor systems gain importance. the principle of a biosensor is the combination of cultured cells and a sensorchip device, which allows the monitoring of cellular activity. in contrast to traditional analytics with a biosensor you can measure on-line cellular activity change caused by an effector as well as the restored activity after privation of the effector (re-native activity). cmos technology can be used for the realisation of various biological sensorchips such as adhesion sensorchips, metabolical sensorchips and electrophysiological sensorchips. the standard cmos technology allows a high reproducibility of the chips, the integration of electronic components on the chip, which reduces the amount of external devices and the combination of different sensors on one chip. in cooperation with the semiconductor company micronas and the biotech company bionas we have realised different types of cmos sensorchips to measure adhesion of a cellular monolayer with interdigitated electrodes (ides), metabolical activity via acidification with ion-sensitive field effect transistors (isfet) and sponteneous neuronal network activity with passive palladium electrodes. microbial agents have been applied to the different stages of pulp and paper processing. the work presented describes a study on the effect of applying ligninolytic enzymes, such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and subsequently subjecting these to different ageing processes. industrial pulps were obtained from different portuguese pulp and paper companies. the pulps used were (1) unbleached pine pulp from portucel tejo; (2) unbleached eucalyptus pulp from portucel setúbal; (3) bleached eucalyptus pulp from portucel setúbal; and (4) pulp made from recycled paper from renova s.a. several types of handsheets were produced with 2 different grammage namely, 60 and 180 g/m 2 . the prepared handsheets were subject to an aging sequence in three different chambers: ultraviolet radiation (wavelength of 280 nm), temperature (19 • c) and moisture (70%); and thick saline fog at a concentration of 1% and temperature of 35 • c. in order to evaluate the effect of moisture cycles and temperature, two aging sequences were used for each type of handsheet. in the first, the moisture varied (60, 80 and 100%), while the temperature was held constant (25 • c); in the second the temperature varied (60, 70 and 80 • c) and the moisture was held constant (50%). following the aging phase, the handsheets were subject to several chemical (viscosity and index kappa) and physico-mechanical (colour, tensile breaking strength, stretch and the bursting strength) tests in order to characterize the effect of the aging conditions. results will be presented describing the effect of application of the laccase-mediator system on the optical and mechanical properties of the prepared handsheets. fundação para a ciência e a tecnologia, project pocti/ agr/47309/02. aspergillus niger is a filamentous fungi widely used in industry. its growth as freely dispersed hyphae leads to an increase in the medium viscosity and to problem of mass transfer, especially oxygen transfer. oxygen acts both as final electron acceptor in the mitochondrial chain and as nutrient for the biosynthesis of unsaturated fatty acids and sterols. thereby, a lack of oxygen affects the nadh/nad ratio, the atp production, the growth and has a strong influence on the physiology of the microrganism. in the present study, the metabolic changes of a. niger in response to a lack of oxygen was investigated using oxygen limited chemostats combined with nitrogen pulse. under these conditions, the main consequence of a sudden decrease of oxygen availability is an increase in the mannitol production. this work showed that the mannitol biosynthesis, involving the enzyme mannitol-1-p dehydrogenase, helps the reoxidation of nadh when the final electron transport acceptor, oxygen, is limiting. investigation of the lipase activity of the bacteria isolated from olive mill wastewater sevgi ertugrul 1 , nur koçberber 1 , gönül dönmez 1 , serpil takaç 2 1 department of biology faculty of science ankara university 06100 beşevler ankara turkey; 2 department of chemical engineering faculty of engineering ankara university 06100 tandogan, ankara, turkey the bacteria that could grow on media containing olive mill wastewater (omw) were isolated and their lipase production capacities were investigated. the strain possesing the highest lipase activity among 17 strains grown on tributryin agar medium was identified as bacillus sp. the effect of ph on the lipase activity of the strain was investigated in tributryin medium and ph 6 was found to be the optimal. the liquid medium composition was improved by adding different carbon sources and fatty acids into tributryin medium -omitted tributryin -to increase the enzyme activity. the cultivations were performed at 30 • c and ph 6. lipase activity of the bacillus sp. was measured spectrophotometrically through the hydrolysis of p-nitrophenol palmitate. among the media containing different compositions of tricapryn, trimyristin, tributyrin, triacetin, tween 80, glycerol-trioleate, glycerol-trioctanoate, glycerol-tridodecanoate, omw, glucose, and whey; the medium consisted of 20% whey + 1% glycerol-trioleate was found to give the highest lipase activity. cultivation of bacillus sp. in the optimum medium at ph = 6 and 30 • c for 64 h was resulted in the extracellular and intracelluar lipase activities of 15 and 168u/ml, respectively. this study was supported by ankara university biotechnology institute (project no: 2004-151 and 2005-164) . under different abiotic stresses, cell growth and metabolic activity are highly influenced in all types of living organisms medium osmolality is usually one of those factors affecting different types of biological systems in different ways. however, even in the same organ of higher eukaryotes the degree of osmoregulation mechanism is highly variable in different types of cells. therefore, studying the effect of osmotic stress on mammalian cell is very important subject for particular cell line. the effect of hyperosmotic pressure on the kinetics of cell growth of and metabolic activity of mesenchymal stem cells (mscs) and two industrially important cell lines, hybridoma cells and human embryonic kidney cell (hek) were investigated in batch cultures at different osmotic pressures in the range from 325 to 500 mosm kg −1 . in case of mscs cells, the maximal specific growth rate [] of 0.029 [h −1 ] associated with the highest specific glucose consumption rate [-q gluc ] of 0.1129 × 10 −6 [mol cells −1 h −1 ] was obtained in medium of 375 mosm kg −1 . in case of hybridoma cells, osmotic pressure showed not only influence on the kinetics of cell growth and metabolism but also on the monoclonal antibody production. the maximal mab production was obtained in case of cells cultivated under osmotic pressure of 375 mosm kg −1 . further increase in osmotic pressure resulted in significant reduction in growth rate as well as mab production. on the other hand, hek cells were more sensitive to osmotic pressure in industrially used serum free medium and the addition of serum decreased the inhibitory effect of high osmotic pressure on the cells. gustavo g. fonseca 1,2 , andreas k. gombert 2 , elmar heinzle 1 , christoph wittmann 1 : 1 biochemical engineering, saarland university, saarbrücken, germany; 2 chemical engineering, são paulo university, brazil kluyveromyces marxianus cbs 6556 is a potentially interesting yeast strain characterized by a high capacity of conversion of substrate into biomass. however, this yeast has been only marginally studied so far. therefore, we performed a metabolic characterization in batch and chemostat cultures at dilution rates of 0.10, 0.25 and 0.5 h −1 . the specific rate of o 2 consumption (qo 2 ) increased with dilution rate from 2.87 to 11.09 mmol (g dw) −1 h −1 . the respiratory coefficient remained almost stable around 1.0 for all metabolic states investigated. even at the dilution rate of 0.5 h −1 , which is close to the maximum growth rate of the strain of 0.56 h −1 , no significant overflow metabolism was observed. the concentration of extracellular metabolites increased with the dilution rate, but remained below 6% of the carbon consumed as glucose. all carbon balances closed near 100% underlining the consistency of the data. in contrast to s. cerevisiae the respiratory capacity of k. marxianus cbs 6556 is not strongly influenced by the dilution rate in aerobic chemostat or batch cultures, indicating its high potential for biomass-directed applications. a thermostable l-arabinose isomerase for enzymatic production of d-tagatose o. hansen, f. jørgensen, p. stougaard department of enzyme technology, bioneer a/s, kogle allé 2, dk-2970 hørsholm, denmark. e-mail: och@bioneer.dk (o. hansen) d-tagatose, an isomer of d-fructose, is a low-calorie bulk sweetener with a sweetness equivalent to sucrose. d-tagatose has obtained gras approval for use as a food ingredient, and is currently produced by chemical isomerization of d-galactose, which may readily be obtained by hydrolysis of lactose. structurally, d-galactose is closely related to l-arabinose, and it has previously been shown that some variants of l-arabinose isomerase (araa) may catalyze the conversion of d-galactose to d-tagatose, in addition to the metabolic conversion of l-arabinose to l-ribulose. we have screened a number of bacterial araa enzymes for their ability to catalyze the isomerization of d-galactose to d-tagatose. the best enzyme was found in the thermophilic bacterium thermoanaerobacter mathranii (dsm11426). the araa gene of t. mathranii was cloned, sequenced and expressed in e. coli. amino acid sequence comparisons of the t. mathranii sequence and other known araa sequences showed a relatively low sequence identity of about 30%, indicating a distant phylogenetic relationship to the other members of the l-arabinose isomerase group. the t. mathranii enzyme was thermostable with optimal activity at 65 • c and it required manganese ions. unlike other araa variants, the t. mathranii enzyme showed k m values in the same order of magnitude for l-arabinose and d-galactose, suggesting that this enzyme is a versatile isomerase capable of isomerizing structurally related aldoses. the enzyme was immobilized by chemical cross-linking of a crude e. coli cell homogenate, and the immobilized enzyme efficiently converted d-galactose into d-tagatose. currently, we are developing an enzymatic method for industrial production of d-tagatose using the immobilized enzyme. the agricultural production can be negatively affected by different pest insects (pi) and the use of chemical insecticides (chi) has been the traditional method for controlling pi during decades. nevertheless, there are various ecological implications due to the extensive application of chi. a viable alternative for the use of chi in certain agro-systems, is the use of entomopathogenic nematodes (epn) of the genera steinernema and heterorhabditis that are natural pathogens for different pi; besides, the presence of a symbiotic bacterium is necessary for an effective entomopathogenic activity can take place. the nematode/bacterium complex does not represent a risk for the environment. different authors propose that the best alternative for the massive production of epn is the submerged culture within bioreactors; nevertheless, more research is required to have really robust processes. particularly, information regarding the actual hydrodynamics during epn production and its relation with the epn productivities are scarce, among other aspects. the present study deals with the hydrodynamic characterisation during the production of the epn steinernema carpocapsae and its symbiont bacterium xenorhabdus nematophilus, in submerged monoxenic culture in an internal-loop air-lift bioreactor (v l = 4.5 l) using two culture media: one of them containing whey and the other one, agave juice, aguamiel, (agave spp.). process viscosity of the culture broth was determined along the time, exhibiting a maximum value of 20 mpa s. moreover, it was determined that the hydrodynamic conditions were always located within the laminar region (re < 500). at the experimental conditions tested, it can be inferred that the epn productivity are more sensitive to changes in the culture medium composition than on the prevailing hydrodynamic conditions during the fermentations. bacillus thuringiensis is a gram-positive bacterium used as a biological pest control agent. moreover, it is able to produce, several biologically active molecules such as bacteriocins and hydrolytic enzymes among which chitinases that play double roles, fungicide and improving the insecticidal effect of b. thuringiensis deltaendotoxins. a newly isolated b. thuringiensis subsp. kurstaki strain bupm4, was shown to produce a novel bacteriocin named bacthuricin f4. the highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. upon purification of bacthuricin f4, the specific activity was increased 100-fold. this bacteriocin was heat-stable up to 70 • c and resisted up to ph 3.0. its molecular mass, determined by mass spectrometry was 3160.05 da. direct n-terminal sequencing of bacthuricin f4 revealed the following sequence: dwtxwsxl. the latter was unique in the databases. bacthuricin f4 was active against bacillus species while it had little or no effect on gram-negative bacteria. the bacteriocin produced by the b. thuringiensis strain bupm4 respond to both criteria of thermostability and stability to low phs. thus, it could be used as a source of bacteriocin active against related species of bacillus harmful for agricultural products and as food preservative. the other example of antimicrobial compound produced by b. thuringiensis is a chitinase. we describe the selection of b. thuringiensis high chitinase-producing strain bupm255, and the characterization and the heterologous expression of a novel chitinase encoding gene. the cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of b. thuringiensis. the identification of chitin hydrolysis products resulting from the activity, exhibited by chi255 through heterologous expression in e. coli revealed that this enzyme is a chitobiosidase. the addition of the sequence of chi255 to the few sequenced b. thuringiensis chi genes might contribute to a better investigation of the chitinase "structure-function" relation. cloning and characterization of s-adenosyl-l-methionine synthetase from pichia ciferrii dscc 7-25 kwon-hye ko 1 , gee-sun yoon 1 , gi-sub choi 1 , joo-won suh 2 , yeon-woo ryu s-adenosyl-l-methionine(sam) has an important role for dna methylation and cell signaling. sam was synthesized from methionine and atp by sam synthetase and play an pivotal function in the primary and secondary metabolism of cells. recent studies have revealed in the effect of sam in case of morphological differentiation in both eukaryotes and prokaryotes. the p. ciferrii produces large quantities of sphingoid base. tetraacetylphytosphingosine(taps), which is a precursor of sphingolipid, could be used for the production of pharmaceuticals and cosmetics. we isolated sam gene from p. ciferrii and cloned it into expression vector for e. coli and p. pastoris, respectively. an 1.2 kb sam-s gene fragment was isolated by low-strigency pcr using degenerated primer. by the analysed primary sequence deduced from dna sequence, this gene included conserved domains similar with other well-known sam synthetase. first of all, sam synthetase gene cloned pgem-t vector and subcloned into histidine tagging system to purify the expressed protein using metal chelating resin. typical characteristic analysis of this enzyme is underway. metabolic networks offer a large variety of different synthesis pathways starting from cheap substrates and leading to interesting high-value compounds, i.e. metabolites. in case an interesting pathway can be disconnected from the remaining metabolic network, the perforated cell or the crude extract could be used for a one-pot multi-step synthesis of the desired compound. pathway isolation, achieved by deletion of genes encoding gene products enabling side reactions, interferes with the viability of the organism, which is a requisite for the production of the system of biotransformation (sbt). in this work, a rational systems biology-derived approach is presented for the design of a sbt. it is illustrated for a sbt allowing for production of dihydroxyacetone phosphate. the design procedure comprises three steps: (i) a production pathway is identified in the metabolic network of e. coli. the e. coli pathway is complemented by two additional enzymes in order to obtain a fully energy and redox balanced production pathway. (ii) an optimal combination of gene knockouts is designed and a suitable growth medium composition is identified, both by a model-based approach: flux balance analysis of a genome-scale metabolic network is used to predict enzyme expression within the wild-type organism on different media, while a mixed-integer optimisation is employed to identify viable mutants as this approach strongly depends on the quality of the fba prediction, available regulatory information on the usage of metabolic pathways and thermodynamic constraints were taken into account. (iii) thermodynamic analysis of the obtained, partially branched sbt reaction cascade revealed the extent of loss in yield by the remaining side reactions. in summary, this systems biology-driven approach potentially enables the substitution of a elaborative multi-step synthesis process by a one-pot enzyme reaction cascade. institute of industrial biotechnology, inha university, incheon 402-751, korea. e-mail: leecg@inha.ac.kr (c.-g. lee) algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. currently, its application is being extended to the areas of wastewater treatment and agriculture. however, lack of suitable photobioreactors (pbrs) makes the cost of algally-derived compounds higher than those derived by chemical synthesis and thus has prevented widespread use of algal cultures. the culture of algae prior to the late 1940s was apparently restricted to laboratory scale operations. experiments on outdoor algal mass production began in the late 1940s with nearly concurrent development of experimental culture facilities in germany and the united states. for the next two decades, outdoor mass culture of algae was undoubtedly the hottest topic in the algal biotechnology area. recent developments of high-density pbrs enable the production of valuable biologically active compounds by algal mass cultures. however, light is almost always the limiting factor in high-density photobioreactors. key factors for successful photobioreactors will be discussed and various photobioreactors will be analyzed and compared for their advantages and disadvantages. the new techniques, such as pigment redcution and application of flashing light and lumostatic operation, will be discussed for possible solutions to overcome the light limitation in high-density microalgal cultures. a quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host. southern analysis is laborious and time consuming. several colony hybridisation methods have been developed for the analysis of a large number of transformants. unfortunately these methods suffer from different disadvantages such as non-specific binding, a limited usability to screen for specific integration and the fact that these procedures always take a few days (van zeijl et al., 1998) . recently, methods for pcr-based analysis of fungal transformants have been described. most of these methods require high quality dna. many methods for dna extraction from fungi have been described in the past few years. these methods often are tedious, time consuming, costly or limited to a small number of samples each time. most of the available protocols include the growth of mycelium in a liquid culture, followed by freeze-drying or maceration in liquid nitrogen and grinding of the frozen material to break the cell walls (cassago et al., 2002) . lately a few methods have been described to isolate dna from fungi suitable exclusively for pcr and appropriate for the simultaneous treatment of a large number of samples. some methods also describe the direct use of mycelium (cooke et al., 1997) in the pcr-reaction mixture. we compared the applicability of a few rapid dna extraction methods for myrothecium gramineum and tested the resulting dna samples on there suitability for pcr-applications. myrothecium gramineum is a filamentous ascomycete used in ongoing research as a new cloning and expression host. five methods were tested. in four of these methods dna was extracted from mycelium (goodwin et al., 1993 and aljanabi et al., 1997) or spores (ferreira et al., 1997 and xu et al., 1995) prior to pcr. a fifth assay used mycelium straight in the pcr-reaction mixture. only this last method seemed useful for myrothecium to isolate dna suitable for pcr. fragments up to 2000bp were amplified. cheese whey is a liquid effluent from cheese-making processes. there is an increased interest in the economic utilization of whey produced by the dairy industries, because the whey is a pollutant, due mainly to its lactose content. the goal of this work was to find the most suitable values of some fermentation parameters for lactic acid production from whey by a lactic acid bacterium, lactobacillus helveticus (atcc 15009). the effects of lactose content, temperature, ph and the supplementation with yeast extract were investigated using surface response methodology. a composite central design was used with three center points, making a total of 27 operational conditions. the region of maximum production is outlined by the following intervals: temperature around 40 • c; lactose concentration between 70 and 85 g/l; concentration of yeast extract between 20 and 25 g/l; ph between 7 and 7.5. fermentation studies on a continuous fermentative process coupled to a vacuum flash evaporator were carried out in lab scale equipment. the phases of this work consisted in an assembly and instrumentation of the prototype and elaboration of a supervisory system coded in labview 6.1, which allows the data acquisition and control through personal computers. the experiments in continuous fermentation used saccharomyces cerevisiae and sugar cane molasses as substrate. the analytical follow up was done through analysis of total reducing sugars, ethanol, glycerol, dry mass and viable cells. the system worked for months uninterruptedly, producing an alcoholic solution at the condenser with 50 • gl. the fermentation operated with concentrations of ethanol at 5 • gl, which is a weakly inhibitory value for the yeast of the process, even when fed with concentrated cane molasses, containing up to 330 g/l of sugar. the result meets the initial goal, which was to operate the system with low level of ethanol and to guarantee high productivity, even in high concentrations of sugar in the feeding. the results showed that system productivity was superior to that of the conventional continuous process. lactic acid (la) is a versatile chemical, used as an acidulant, flavor and preservative in the food, pharmaceutical, leather and textile industries, and for production of biodegradable poly lactic acid (pla). l(+)lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. in this research, lactic acid production was improved on 20 l fermentor. in our experience, among six strains of lactobacillus were examined for the production of l(+) lactic acid, lactobacillus casei ssp. casei atcc 39392 was selected as a highest l(+) lactic acid producer. optimized medium used for lactic acid production contained (per l) 80 g glucose, 50 g whey powder and 20 g corn steep powder. for a homofermentative process, ph 6.0 was found to be optimal. in order to avoid product inhibition, the produced lactic acid was neutralized using calcium hydroxide. maximum production and productivity of lactic acid in batch system, were 81 g and 1.35 g/lh, but in fed batch system, after 3 feeds of glucose, production and productivity increased up to 360 g and 4 g/lh. saleh a. mohamed molecular biology dept., national research centre, cairo, egypt an extracellular polygalacturonase (pgii) from trichoderma harzianum was purified to homogeneity by two chromatography steps using deae-sepharose and sephacryl s-200. the molecular weight of t. harzianum pgii was 31,000 da by gel filtration and sds-page. pgii had isoelectric point of 4.5 and optimum ph of 5.0. pgii was very stable at the ph 5.0. the extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (de). pgii had very low activity toward nonpectic polysaccharides. the apparent km value and kcat value for hydrolyzing polygalacturonic acid (pga) were 3.4 mg/ml and 592 s −1 , respectively. pgii was found to have temperature optimum at 40 • c and was approximately stable up to 30 • c for 60 min of incubation. all the examined metal cations showed inhibitory effects on the enzyme activity. 1, 10 phenanthroline, tween 20, tween 80, triton x-100 and sds had no effect on the enzyme activity. the rate of enzyme catalyzed reduction of viscosity of solutions of pga or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. the storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to one year. these properties of t. harzianum pgii with appreciable activity would be potentially novel source of enzyme for food processing. tarek m. mohamed, biochemistry division, chemistry department, tanta university, tanta, egypt preparation of peroxidase from horseradish, which could be used for commercial applications such as diagnostic kits, was occurred through a simple reproducible method consisting of extraction, ammonium sulphate precipitation, filtration through non-binding protein filter and lyophilization. the purification method was developed allow the preparation of 33 mg of enzyme from 1 kg of horseradish roots. the one mg of enzyme contains 900 units of peroxidase. this value is similar to that produced by sigma (50-1000 unit mg −1 powder). the final preparation is salt free reddish brown powder with free ammonia content less than 0.01 g −1 units. the rz value (a400/a280) of the enzyme, which is a good criterion of purity and heme content, was 2.6. the lyophilized enzyme was stable at −20 • c for at least one year. the liquid form of the enzyme in presence of 0.1% sodium azide was stable up to 25 days at 4 • c, while it lost most of activity at room temperature in the same period. the properties of horseradish peroxidase including km, optimal ph and temperature, activation energy, thermal stability and effect of different compounds were studied. the applicability of this enzyme in determination of serum glucose was performed. the analysis of glucose in human sera gave results using the kit containing the prepared peroxidase similar to those obtained with a commercial glucose kit. lactobionic acid production using lactose oxidase: from laboratory to 600 l scale mikkel nordkvist 1 , per munk nielsen 2 , peter budtz 3 , john villadsen 1 : 1 center for microbial biotechnology, technical university of denmark, dk-2800 lyngby, denmark; 2 novozymes a/s, dk-2880 bagsvaerd, denmark; 3 chr. hansen a/s, dk-2970 hørsholm, denmark. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) currently, lactobionic acid is mainly a high-price specialty product used, e.g. in solutions for organ stabilization. however, lactobionic acid can also be used as a biodegradable cobuilder in detergents, and it has several applications in food technology. with lower production costs it has the potential to become a bulk chemical. the kinetics for the oxidation of lactose to lactobionic acid by a new carbohydrate oxidase was studied in a 1 l bio-reactor with control of ph, temperature, and dissolved oxygen. the byproduct hydrogen peroxide has a negative influence on the lactose oxidase enzyme, and hence additional experiments were made with addition of catalase to remove hydrogen peroxide, thereby also providing extra oxygen. on the basis of the experiments in 1 l scale, experiments were performed in a 600 l reactor equipped with a new system for dispersion of air to supply the necessary oxygen for the oxidation. the aeration system in the large scale reactor was able to supply oxygen sufficiently fast to give the same production rate, at low values of the air flow rate and the energy input, as was obtained in the high-performance laboratory reactor. the non-characterized gene previously proposed as d-tagatose 3-epimerase from agrobacterium tumefaciens was cloned and expressed in escherichia coli. the expressed enzyme was purified by affinity chromatography on histrap hp, desalting chromatography on hiprep 16/60, and gel filtration chromatography on sephacryl s-300 hr with a final specific activity of 8.89 u/mg. using maldi-tof-ms, the native protein was estimated to have a molecular mass of 32,600 da and a monomeric structure. the purified enzyme exhibited maximal activity at 50 • c and ph 7.5 without the addition of metal ions and at 60 • c and ph 7.0 with 1.0 mm mn 2+ . among various metal ions, mn 2+ was the most effective divalent cation for d-fructose epimerization activity. the addition of mn 2+ significantly increased the thermal stability and the epimerization activity with other ketoses such as d-psicose, d-tagatose, d-ribulose, d-sorbose, and d-xylulose. the activity, substrate affinity, maximum velocity, and catalytic efficiency (k cat /k m ) of the enzyme for d-psicose were higher than those for d-tagatose, which suggests that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. the equilibrium ratio between d-psicose and d-fructose was 37:63 at 60 • c with 1.0 mm mn 2+ . when the enzyme was used at 14 u/ml, dpsicose was produced at 211 g/l from 700 g/l d-fructose containing 1 mm mn 2+ after 120 min, corresponding to a conversion yield of 30.2%. the role of ammonium ions in glucosamine formation during the citric acid fermentation process by aspergillus niger m. papagianni 1 , f. wayman 2 , m. mattey 2 : 1 department of hygiene and technology of food of animal origin, school of veterinary medicine, university of thessaloniki, thessaloniki 54006, greece; 2 department of bioscience, university of strathclyde, glasgow, g1 1xw, uk. e-mail: mp2000@vet.auth.gr (m. papagianni) stoichiometric modeling of the citric acid fermentation process by aspergillus niger, in 12-l stirred tank reactor, indicates that nh 4 + ions combine with a c-containing metabolite inside the cell to form a nitrogen compound which is then excreted by the mycelium. the close correlation between calculated and experimental profiles indicates that this metabolic process is rapid and takes place before the c-structure of the glucose has been greatly altered by glycolysis or the pentose phosphate pathway. hplc analysis identified glucosamine as the product of this relationship. a clear effect of medium concentration of nh 4 + on glucosamine formation was observed when fermentations carried out with optimal and sub-optimal ammonium concentrations. (nh 4 ) 2 so 4 addition when medium nitrogen was depleted, enhanced the formation of new cells from the tips of fragmented hyphae and led to glucosamine accumulation in amounts depending to pulse concentration. the fungus reacts in excess ammonium by converting it to glucosamine, to be utilized later when a regeneration process takes place with fragmentation of vacuolated hyphae and subsequent regrowth, depending always on the culturing conditions. however, depending on carbon and ammonium concentration in medium, glucosamine can be secreted in concentrations as high as 50 g/l. about 1000 microorganisms originated from traditional korean food origin were screened for efficient palatinose production. an isolate designated fmb1 was exceptionally efficient in sucrose-palatinose conversion activity. conversion of sucrose into palatinose by fmb1 was much faster than a reference strain of erwinia rhapontici. fmb1 is a gram negative, facultatively anaerobic, motile, noncapsulate, and straight rod-shaped bacterium producing acid from glucose. based on api and 16s rdna analyses, fmb1 was determined to be enterobacter sp. the maximum conversion of 10% sucrose to palatinose and trehalulose by enterobacter sp. fmb1 was achieved within 6 h. the preliminary dna sequencing result of the gene corresponding to sucrose isomerase of enterobacter sp. fmb1 revealed that it showed 87% similarity to that of klebsiella sp. (??). within the scope of an r&d project developed in collaboration with leather tanning portuguese industrial partners a screening of new proteases to be used in the industrial process was performed. a bacillus subtilis strain isolated from alkaline spent purge liquor was shown to be a promising protease producer. microorganism growth was studied for optimisation of temperature, agitation, ph and medium composition either for biomass or proteases production. optimal growth temperature is different for maximum biomass growth (40 • c) and optimal proteolytic activity (43 • c) yielding biomass specific growth rate of 1.6 and 1.4 h −1 , respectively. the achieved proteolytic activities were 5.7 and 7.2 u/ml of protease, respectively. optimised medium composition (7 g/l beef extract, 4 g/l yeast extract, 5 g/l peptone and 0.4 g/l cacl 2 ) yielded a specific growth rate of 1.5 h −1 and 13.9 ku/l of protease, in shake flask experiments. bioreactor experiments (from 1 to 16 l) with the selected medium were performed at 43 • c in order to test aeration rate (1 and 2 vvm), stirring (300-700 rpm) and ph (uncontrolled, controlled at 7 and 8). best protease activity was 64 u/ml in 2 l bioreactor without ph control at 500 rpm and 2 vvm. the proteolytic extract was characterized and compared to commercial bates. results indicate that these proteases can be employed in the purge phase of the leather tanning process in industry. the gram positive bacterium bacillus megaterium is known for its capacity to produce exoenzymes including amylases, proteinases, and penicillin amidase at industrial scale. here, we describe the development of various vectors for the production and export of recombinant heterologous proteins employing b. megaterium signal peptides. the target gene can be cloned directly adjacent to the signal peptide coding sequence (bart et al., 2005) . this arrangement allows for a correct n-terminal sequence of the mature protein after processing by the signal peptidase sipm. using this newly developed protein production and export system lactobacillus reuteri 121 levansucrase (van hijum et al., 2001) was secreted in significant amounts (∼4 mg/l) into the growth medium. fusion of the recombinant levansucrase to affinity tags allowed one-step purification of the recombinant protein from the growth medium. however, fused peptide tags resulted in a decreased secretion of the fusion protein. 1.4 mg his 6 -tagged levan-sucrase were purified per litre of culture. the system was further enhanced via coexpression of a gene for the signal peptidase sipm (malten et al., 2005a) and deletion of the gene for the extracellular protease nprm. developed new tools allow for various strategies of integrated high level production, export and purification of heterologous proteins in b. megaterium. methods for high-throughput screening of secreted enzymes are under development. the determined sequence of the b. megaterium genome, studies using high-cell density cultivations (malten et al., 2005b) and proteome data from batch fermentations implicate new targets for directed genetic optimization of b. megaterium production and secretion strains. novel strong and inducible promoters are currently under investigation. toru matsui 1 , naoya shinzato 1 , hisashi saeki 2 , hitoshi matsuda 2 : 1 center of molecular biosciences, university of the ryukyus, okinawa 903-0213, japan; 2 japan energy co., japan. e-mail: tmatsui@comb.u-ryukyu.ac.jp (t. matsui) optically active epoxides are considered as the potential intermediate for chiral drugs synthesis. although s-styrene oxide (so) have been extensively investigated using styrene monooxygenase from pseudomonas sp., microbial production of r-so with high enantiomeric excess was hardly examined. in this study, r-so producing bacteria from styrene was screened using various alkene assimilating bacteria. r-so with the highest ee (ca. 100%ee) was obtained using ethene utilizing bacteria, identified as mycobacteirum sp., while produced relatively lower at around 70%ee when using propene utilizing bacteria. the alkene monooxygenase gene homologue sequence amplified from the genomic dna revealed a significant similarity to that of etnabc. these bacteria also showed stereoselective degradation of racemic so, suggesting that the produced so might be further stereoselectively degraded to increase the ee. the ethene utilizing bacteira produced not only r-so but also s-epichrolhydrin at high ee.when using arylchloride as the substrate. this research was supported by nagase science and technology foundation. the secretion efficiency of the escherichia coli sec pathway is dependent on the growth phase but not on protein size f.j.m. mergulhão, g.a. monteiro centro de engenharia biológica e química, instituto superior técnico, av. rovisco pais, 1049-001 lisbon, portugal. e-mail: filipem@alfa.ist.utl.pt (f. mergulhão) the secretion efficiency of the escherichia coli sec pathway was evaluated through the expression of green fluorescent protein and human proinsulin fusion proteins. translocation to the periplasm is dependent on the growth phase of the bacterial culture and the highest secretion efficiency is attained in mid-exponential phase. secretion performance is independent of protein size (17-42 kda) and even when the amino acid composition of the secreted proteins is very similar, the amino acid distribution within the protein can affect translocation. in silico prediction analysis suggests that proteins that are prone to form ␣-helix structures are more efficiently translocated. culture medium composition plays an important role on secretion performance with the highest secretion results being obtained in minimal medium. streptokinase is a common fibrinolytic drug. that is used in thrombolytic therapy for long time. to compare with another thermbolytic drugs like tpa, it has lot of advantages. in this present research dna was extracted from s. equisimilis h46a for the first time in iran. streptokinase gene was amplified by using two forward primers and one reverse primer. a common restriction enzyme, bamh-i, was used for cloning. both ends of the pcr products (full length: 1323 bp and mature section: 1245 bp) and the restriction site on mcs of pqe-30 vector were digested. in this study, pqe-30 expression vector was used with high level expression ability for production of recombinant fusion streptokinase with simplifying the purification by employing affinity-metal chromatography method. in addition, the cloning results were controlled by double digestion and sequencing. takesono oxidation of short-chain iso-alkanes was studied with propanegrown resting mycelia of scedsporium sp. a-4. isobutane was oxidized to tert-butanol, but not to isobutanol. isobutanol was used for growth, but both isobutene and tert-butanol were not used for growth. isopentane was oxidized to 3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-2-butanol but not to 2-methyl-1-butanol. 2-methylpentane was oxidized to 4-methyl-1-pentanol, 2-methyl-2pentanol, and 4-methyl-2-pentanol but not to 2-methyl-1-pentanol or 2-methyl-3-pentanol. 3-methylpentane was not oxidized. oxidation of branched alcohols was also studied. application of nadph-dependent 2.5-diketo-gluconic acid reductase for production of l-ascorbic acid claudia pacher 1,2 : 1 division of food biotechnology, department of food sciences and technology, boku, university of natural resources and applied life sciences, muthgasse 18, a-1190 vienna, austria; 2 research centre applied biocatalysis, petersgasse 14, a-8010 graz, austria. e-mail: claudia.pacher@boku.ac.at ascorbic acid is an organic acid with various applications in the food and pharmaceutical industries. at present, the majority of commercially manufactured vitamin c is synthesized via the reichstein process, which is highly energy-consuming, involves considerable quantities of organic solvents and gives an overall yield of about 50%. therefore, during the past decades a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic or fermentative means, which show some advantages regarding costs and environmental friendliness. one of the fermentation routes runs via 2.5-diketo-d-gluconic acid (2.5-kdg), produced by pectobacter (erwinia) cypripedii. this compound has been reduced by a nadph-dependent 2.5-diketogluconic acid reductase (dkr) from corynebacterium glutamicum to the key intermediate 2-keto-l-gulonic acid (2-klg) before chemical rearrangement leads to the final product. for economical reasons we wanted to express dkr heterologously. based on our long term experience with coenzyme regeneration we wanted also to perform the reaction in homogeneous solution. the spent coenzyme of nadph-dependent dkr has been regenerated by a second isolated nadp-dependent enzyme like glucose dehydrogenase. we describe here the recombinant production, purification and characterization of dkr and the results of enzymatic 2-klg formation by using the recombinant enzyme in a homogeneous system with conjugated coenzyme regeneration. grp78 residing in endoplasmic reticulum (er) functions as a molecular chaperon by associating transiently with incipient proteins as they traverse the er and aiding in their folding and transport. furthermore, the protein can also be induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, blockage of vesicular trafficking by brefeldin a and er-calcium-atpase pump inhibition by thapsigargin. thus, substances that directly down and up-regulate grp78 transcription are expected to be useful for treatment of cancer and alzheimer's disease, respectively. in the course of our screening program to obtain substances, which regulate grp78 expression, we first constructed an assay system monitored by the expression of a reporter gene. hela cells, which are transformed with luciferase gene under the control of grp78 promoter designated as hela 78c6 cells, respond sensitively to luciferase grp78 induction by er stress such as treatment of tunicamycin. by using this screening system, we isolated pyrisulfoxin as an up-regulator of grp78, and valinomycin, citreoviridin and alternariol as down-regulators. detailed studies on other biological activities were now under way. pdh is an enzyme that was described only several years ago in a number of ecologically related litter-decomposing fungi (agaricales, gasteromycetales). it catalyzes the c-3 and/or c-2 oxidation of several aldopyranoses to the respective keto sugar derivates. pdh shows a very broad substrate range, oxidizing almost all major sugar components of wood polysaccharides, and is implicated to play a role in lignocellulose degradation. agaricus bisporus, the white button mushroom, is an economically significant agricultural crop. the cultivation, which is done by solid-substrate fermentation on straw-and hay-based composted substrate, is sometimes seen as one of only few economically feasible methods for bioconversion of lignocellulosic agricultural waste material. deeper insight in the physiological role of pdh may provide help for mushroom growers to increase yield, improve quality or make new sources of raw materials utilizable. we amplified a fragment of the pdh gene with degenerated primers derived from internal peptide sequences. the screening of a genomic library led to the isolation of the pdh gene. subsequently we amplified a cdna clone by rt-pcr and investigated the transcriptional regulation by different carbon sources on a defined minimal medium. optimization of monoclonal antibody production processes with simulation and scheduling tools demetri petrides, charles siletti intelligen inc., scotch plains, nj 07076, usa. e-mail: dpetrides@intelligen.com (d. petrides) this presentation will review the state of the art in batch process simulation and scheduling tools and their applications in the design and debottlenecking of integrated biopharmaceutical processes. a systematic methodology will be presented for identifying and eliminating size, time, and throughput bottlenecks that limit production in single and multi-product facilities. the methodology will be illustrated with an industrial case study dealing with the optimization of a multi-product facility that produces therapeutic monoclonal antibodies (mabs). mab processes are characterized by a long bio-reaction time (e.g., 1.5-2 weeks for fed-batch operation and 1-2 months for perfusion operation). the cycle time for processing a lot in the recovery and purification train typically takes 3-4 days. consequently, one way of increasing throughput is by installing extra bioreactors that operate in staggered mode and utilize the same recovery train. the result is that multiple batches may be at different stages of completion at any given time. since cleaning equipment (e.g., cip skids) and buffer preparation and holding tanks are shared by multiple steps across many batches, this type of operation leads to time/scheduling bottlenecks that constrain the cycle time and the throughput of a process. the problem becomes more challenging in the context of multi-product facilities and when constraints imposed by the limited availability of resources are considered. our methodology and its computer implementation will illustrate how to systematically identify and eliminate such bottlenecks. the industrial case study will provide a real world example of the methodology. application of two stage continuous cultures of aspergillus niger for citric acid biosynthesis jerzy j. pietkiewicz, malgorzata janczar, wladyslaw lesniak food biotechnology department, university of economics, wroclaw 53-345, poland. e-mail: jerzy.pietkiewicz@ae.wroc.pl (j.j. pietkiewicz) the aim of the work was application test of submerged two stage single stream continuous cultures of aspergillus niger for citric acid production from sucrose. studies were carried out in lab fermenters with working volume of 5 dm 3 . the bioreactors were standard cstrs. in two stage continuous cultures (tscc) high mycelium growth and high citric acid production were observed in the first bioreactor. there was almost four times lower growth of biomass rate and about three times lower citric acid production rate in the second bioreactor. studies on influence of dilution rate in race from 0.0098 to 0.0230 dm 3 /(dm 3 h) on course and efficiency of tscc showed, that the highest citric acid yield (y p/s = 86.3%), high volumetric rate of its production (r pc = 0.958 g/(dm 3 h)) and the highest biosynthesis efficiency coefficient (k ef = 82.7) were obtained with dilution rate d = 0.0148 dm 3 /(dm 3 h). there was also high citric acid concentration (p = 129.5 g/dm 3 ) and low residual sugar concentration (s k = 6.9 g/dm 3 ) in the medium flowing out the second bioreactor in those cultures. the beta-galactosidases in commercial use are of different origins and yeast and fungal lactases present the greatest interest. the yeast lactases present neutral optima ph and are suitable for the hydrolysis of lactose in milk. in this work, the aim was to study the influence of aeration in the production of beta-galactosidase in batch fermentations with kluyveromyces marxianus atcc 46537. the medium composition for culture was as follows (in g/l): lactose pa 50, yeast extract 5, (nh 4 ) 2 so 4 4 and kh 2 po 4 2. the fermentations was carried out at 30 • c, ph 5.5, at 200 rpm starting with an initial cellular concentration of 1 × 10 7 cels/ml, with different aeration rates. the cells were disruped with chloroform 2% (v/v) as solvent. the enzymatic activity was determined as initial rate of lactose hydrolysis at defined conditions. the studies have revealed the importance of aeration on kluyveromyces marxianus in the growth and beta-galactosidase synthesis. the enzymatic activity of fermented medium with 0.5 vvm was 50% higher than one without aeration. furthermore, the cellular growth was faster in the aerobic fermentation than in the anaerobic one. the aeration has taken an important place in the enzymatic synthesis and in the cellular growth, however the results have shown that the aeration rate increase of 0.5-1.5 vvm has not implied a increase in the cellular growth neither in the enzymatic reached activity. the lactose presents in the milk has a solubility of only 20% at 30 • c, and a high percentage of the world population presents intolerance to this sugar, due to the low or absence of the activity of the lactase enzyme in the organism. to minimize such problems, the most viable alternative for nourishing dairy products is the enzymatic hydrolysis of milk, although it is an expensive process due to the high cost of the beta-galactosidase enzyme. an alternative that has been greatly studied is the immobilization of this enzyme, originated from many different sources. there are several immobilization procedures for this enzyme, however, a procedure considered ideal was not obtained yet. the objective of this work was to study the immobilization process of beta-galactosidase from aspegillus oryzae in sodium alginate with commercial gelatin. in the immobilization process was studied the glutaraldehyde influence for 1, 3 and 5%, in the presence of commercial gelatin at the concentration of 2% at the immobilization medium. the activities of the immobilized enzymes were obtained in a stirred micro-reactor, at the temperature 30 • c, ph 4.5 with a 50 gl −1 lactose solution in acetate buffer. the experimental results showed that the immobilized biocatalyst that presented the larger initial activity was the one obtained at the immobiliza-tion medium that contained 5% of glutaraldehyde. after 20 daily determinations of enzymatic activities, a fall of 30, 40 and 24% was verified in the enzymatic activities for the immobilized biocatalysts using glutaraldehyde at 1, 3 and 5%, respectively, however, in all cases, the enzymatic activity reached the half of their initial activity after 10 determinations. hydrolysis of sucrose by immobilized beta-fructofuranosidase in silica eloízio júlio ribeiro, ubirajara coutinho filho faculdade de engenharia química, universidade federal de uberlândia, uberlândia 38400-902, brazil. e-mail: ejribeiro@ufu.br (e.j. invertase, known as beta-fructofuranosidase (ec 3.2.1.26), plays a catalytic role in the conversion of sucrose into glucose and fructose. it is largely used in the food industry to prevent the crystallization of sucrose in sugar mixtures and can be used in enzyme reactors for hydrolysis of sucrose. the objective of this work was to study the kinetic of sucrose hydrolysis by immobilized betafructofuranosidase in a continuous recirculation reactor, evaluated the enzyme stability and determine the effective half-life of immobilized enzyme. invertase was covalently immobilized on sillanized controlled pore silica. nonlinear fitting were used to determine the kinetic parameters for substrate and product inhibition observed in the enzymatic hydrolysis of sucrose. the kinetics studies of immobilized invertase were carried out in a continuous recirculating reactor. the half-time of enzyme inactivation (t 1/2 ) was calculated from the initial rates of the remaining enzyme activity. the model of inhibition by substrate and product adequately represented the enzymatic hydrolysis. the fructose effect was competitive inhibition (k f = 3.1022.10 −4 mol/ml) and the glucose effect was noncompetitive inhibition (k g = 2.2521.10 −4 mol/ml). the effective diffusivity of sucrose into the support was shown to be the same as for sucrose in dilute solution (0.75 × 10 −5 cm 2 /s at 40 • c). the half-time of enzyme inactivation (t 1/2 ) was 1656 h. controlled pore silica showed to be an excellent immobilizing support. the immobilized invertase was very stable at temperatures lower than 50 • c. the intrinsic parameters (k i , k f , k g and v m ) were shown to be similar to the apparent values. the low permeability of mycobacterial cell wall envelopes is a result of the unique composition and organization of the cell wall lipids. the permeability of mycobacterial cell wall can be changed by means of partial disintegration of its compounds. the aim of present work was to characterize the changes in the cell wall skeleton (cws) and non covalently bound free lipids under the influence of isoniazid, the inhibitor of mycolic acids biosynthesis. fatty acid (fames) and mycolic acid methyl esters (mames) obtained from all tested preparations were analyzed by gc/ms analysis. the analysis of free lipids and cws revealed distinct changes in the composition of the frac-tions obtained from the cells exposed to action of the isoniazid. the changes in the quantity of fatty acids in the inh-treated cells indicates that inh interferes with the synthesis of lipidic compounds of the mycobacterial cell wall also. the decreased amount of covalently bound mycolic acids in the cws is responsible for the enhanced penetration of hydrophobic compounds through the cell wall. this work was supported by grant nr 3p04c 06923 of the committee for scientific research. barbara sencio, teresa jamroz, stanislaw ledakowicz department of bioprocess engineering, technical university, lodz, poland the enzyme laccase (ec.1.10.3.2. p-diphenol oxidase) is a subject of research in many centres dealing with improvement of bioprocesses with the use of different white rot fungi species. most strains that produce this enzyme in vitro require inductors initiating its biosynthesis. when cerrena unicolor was applied, it was found that the strain produced laccase very efficiently without additional toxic compounds. to specify optimum conditions for laccase production in a submerged culture, research was undertaken to obtain the most efficient inoculum c. unicolor. the goal of this research was to determine the effect of form and incubation time of inoculum on enzymatic activity of the laccase producing strain. the experimental inoculum was the mycelium prepared on a solid and liquid substrate. basing on results obtained, it was found that the laccase yield was the highest in the cultures where the mycelium was grown on a solid substrate. maximum activity of the c. unicolor strain was achieved on the 16th day of culture, and the amount of laccase produced was higher by, ca. 30% as compared to the mycelium obtained from the liquid substrate. results of these experiments were used to continue studies on the impact of inoculum age. experiments were carried out using an inoculum incubated for 1-3 weeks at the temperature 30 • c in a certomat bs1 shaker at 110 rpm. the best results in the c. unicolor strain culture were achieved using a 7-day-old inoculum. effect of alcohol treatment on hydrolytic activity of candida rugosa lipase serpil takaç, a. ezgiünlü department of chemical engineering, institute of biotechnology, ankara university, 06100 tandogan, ankara, turkey candida rugosa lipase (crl) was treated with 20, 40, and 60% concentrations of methanol (m), ethanol (e), 2-propanol (2p) and 1-butanol (1b) to investigate the changes in its hydrolytic activity toward p-nitrophenylacetate. the treatment included the following steps at +4 • c: (i) stirring crl with phosphate buffer for 24 h; (ii) treating the solutions with alcohols; (iii) stirring treated-crl for 24 h; (iv) centrifugation at 10,000 rpm for 30 min; (iv) dialysis the supernatant against bidistilled water for 39 h. the activity of crls was followed for 120 h at 37 • c in the presence and absence of isooctane. the enzyme activity was measured spectrophotometrically and the protein concentration was measured by lowry's method. it was found that the recovered protein did not change considerably with the type of alcohol; however, decreased with alcohol concentration. in the presence of isooctane, specific activities of the untreated and treated-crls increased compared with those obtained in the absence of isooctane. 1b-crls and e-crls showed higher activities than m-crls and 2p-crls whereas untreated-crl exhibited higher activity than m-crls, e-crls and 2p-crls. the highest and the lowest activities were obtained with 20% 1b-crl and 60% 2p-crl, respectively. the changes occur in the structure of crl after treatments were investigated by electrophoretic analysis. this study was supported by ankara university biotechnology institute (project no: 89) . different genera, species and strains of microorganisms were found to posses different cryoresistance. optimal ways for cryopreservation of microorganisms-producers of antibiotics, microorganisms, used in food industry, agriculture and veterinary have been developed. it was demonstrated, that non-lethal damages could occur in cryopreserved microorganisms after their returning to physiological culture conditions, which were manifested in streptomyces' hypha fragmentation, that of cyanobacteria's, streptococci's chains as a result there was an increase in a number of colony-forming units, a reversible inhibition of microorganisms' proliferative activity in bacillus thuringiensis and lactic streptococci, stimulation of the enzyme processes and antibiotic production. non-lethal damages are repaired during microorganism culturing in the first passage. the cause of non-lethal damages is a reversible inhibition of biosyntheses of protein and nucleic acids respiratory activity. the repairing of non-lethal damages is accompanied by the production of stressproteins, different from heat shock proteins. effect of ph in the 2-propanol treatment of candida rugosa lipase on its enantioselectivity in the hydrolysis of racemic naproxen methyl ester serpil takaç, a. ezgiünlü department of chemical engineering, ankara university, 06100 tandogan, ankara, turkey candida rugosa lipase (crl) was treated with 2-propanol (2p) at the ph values of 1.5, 4, 6, 7.5, 9 and 12 to investigate the changes in its enantioselectivity in the hydrolysis of racemic naproxen methyl ester. the treatment included the following steps at +4 • c: (i) stirring crl with different buffer solutions to maintain the desired ph values for 24 h; (ii) treating the solutions with 40% 2p; (iii) stirring treated-crls for 24 h; (iv) centrifugation at 10,000 rpm for 30 min; (iv) dialysis the supernatant against bidistilled water for 39 h. hydrolyses of racemic naproxen methyl ester to form s-naproxen were performed in shaking flasks at 200 rpm and 37 • c for 192 h in isooctane-phosphate buffer solution biphasic system using treated-crls with the activity of 7.75 u. the concentrations of the enantiomers of naproxen methyl ester and naproxen were determined with hplc. it was found that the treatment ph has an important role on the enantioselectivity and conversion. the highest enantiomeric excess for the substrate, for the product, enantiomeric ratio, and con-version were obtained with crl treated at ph 1.5 as 39, 98, 181 and 29%, respectively. these values were followed with 2p treated crl at ph 12 as 35, 98, 121 and 27%. however, lower enantiomeric excesses, conversions and enantiomeric ratios were obtained at the treatment ph values between 1.5 and 12. the effects of fatty acids, nitrogen (as nh 4 no 3 ), phosphorus (as kh 2 po 4 ), ph value, manganese (mn 2+ ), iron (fe 2+ ) and methanol concentration on growth and production of oxalic acid from post refining fatty acids by a mutant of aspergillus niger xp in submerged fermentation experiments was studied. of the a. niger strains screened, a. niger xp was identified as the best oxalate producer on lipids. the influence of the ph on oxalic acid formation shows that the maximum production rate and higher concentration of product are observed at the ph ranging from 4 to 5. with a medium containing 50 g fatty acids/l, the production reached a maximum of 68 g oxalic acid/l after 7 days. the addition of 1.5% (w/v) methanol to seed culture increased the product yield and concentration of oxalic acid but decreased the amount of an undesired by-product (citric acid). under this condition, the maximum oxalate productivity (14-18 g/l days) was maintained for 2-4 days of fermentation. other results of the experiments show that supplementation of the production medium with manganese and iron enhances oxalate production. fatty acids proved to be a very good substrate for oxalic acid production by a. niger xp giving excellent yields and productivity at low ph. the results are very promising as they may lead to cheap alternative processes for oxalic acid production from renewable lipid resources. department of food engineering, middle east technical university, ankara 06531, turkey. e-mail: banuy@metu.edu.tr (b. yalcindag) laccase (e.c. 1.10.3.2, p-benzenediol:oxygen oxidoreductase), which is an enzyme belonging to the multi-copper oxidase family, catalyzes the oxidation of a broad variety of polyphenols with a preference for p-isomers, which are converted to p-quinones. fungi generally contain several laccases which have been found to be involved in delignification, melanin synthesis and pathogenesis. laccase has also important potential application areas especially in food and chemical industries. after aspergillus fumigatus genome data were released, research on functional analysis of laccases has been initiated in our laboratory. laccase genes of aspergillus nidulans, ya and tila, and laccase and multi-copper oxidase genes of aspergillus fumigatus, abr2 and abr1, were used to analyze a. fumigatus genome for laccases. this sequence analysis resulted in 4 probable laccase genes, one of which was the previously cloned abr2 gene. in this study, one of these genes (aflac1) was further characterized. after sequence alignment and characterization studies, aflac1 was predicted to have 2128 bp having six introns, which makes the protein 606 amino acid long, and the predicted protein sequence showed 63% homology with the dihydrogeodin oxidase of aspergillus terreus and 38% homology to the laccase 2 of botryotinia fuckeliana. aflac1 gene is found within an uncharacterized gene cluster containing genes with homology to glutathione-s-transferase, polyketide synthase, o-methyl transferase, and others. the information obtained from sequence analysis was employed in designing pcr-primers to amplify the aflac1 gene, followed by cloning onto pan52-1 and pan52-4 vectors for heterelogous expression in aspergillus sojae. in addition, by the use of rt-pcr, aflac1 cdna will be cloned and expressed in escherichia coli. furthermore, gene silencing studies will be performed to enlighten the function of aflac1 and associated gene cluster. stability of growth rate of photosynthetic cells is an important factor in designing of effective photobioreactors especially in long term operations. in our experiments, in order to keep operational parameters almost constant, a semi-continuous culture method was developed. in this method, a part of culture broth containing grown cells was repeatedly replaced by fresh medium at a predetermined time interval. the replacement of broth with fresh media could keep the cell concentration, volume of broth and distribution of light intensity constant at initial values throughout the cultivation. it was shown that in one side illumination with a halogen lamp, if the ratio of the light intensity at the front side of a flat plate photobioreactor to that at the rear side was kept lower than 4, the growth rates was sustained in constant levels. however, at higher ratios the growth was followed by rapid decrease after 5-6 h. supplemental illumination with a fluorescent lamp from the rear side of the flat plate photobioreactor could sustain almost stable growth rate. beside of the illumination conditions, increased ferrous ion concentrations in medium could keep the stability of growth rate even in unstable illumination conditions, while consumed ferrous ion was slight. glutathione (gsh) plays a pivotal role in protecting cells from by-products generated by oxidative metabolism. these characteristics make this active tripeptide an important drug for the treatment of liver diseases and is of interest in the food additive industry, therapeutics and sport nutrition. in the first part of the research, a screening was carried out among 48 yeast strains, to find out those able to accumulate higher gsh intracellular levels. two saccharomyces cerevisiae strains proved to be the best gsh producers (1.3%dw), in every samples the presence of s-adenosyl-methionine in traces (0.2% dw) was also evidenced. s-adenosyl-methionine (sam) plays a role in the immune system, maintains cell membranes, participates in detoxification reactions and in the manufacture of brain chemicals and cartilage. the second part of the research was aimed at increasing, in a post fermentative procedure, gsh levels present inside the cells at the end of the growth phase. moreover, time course of sam intracellular levels, to be related with accumulated gsh, was also monitored. cells were then suspended in an appropriate solution containing mineral salts, glucose and the aminoacids, precursors of the two studied molecules. according to this procedure, gsh intracellular levels reached 4.6% dw after 48 h incubation. moreover, gsh levels can be related to sam production (up to 1.3% dw). the presence, in several samples, of intermediate metabolites, such as cystathionine and omocysteine, proved the establishement of an intracellular equilibrium between gsh and sam; this behaviour represents a promising starting point for the set-up of a microbial process for the simultaneous production of the two studied molecules. three acetate mutants of y. lipolytica yeasts, which varied in colony morphology (rough and smooth), were employed for continuous citric acid production from glucose and fructose syrup in a membrane reactor with cell recycle. the strains were compared for their product yields, specific acid production rates and ratios of citric acid to isocitric acid. experiments shoved that glucose syrup was a better substrate for citric acid production by y. lipolytica. citric acid concentration in the effluent ranged from 80 to 120 g/l, depending on the yeast strain used. all y. lipolytica strains produced very low amounts of isocitric acid. its concentration did not exceed 3 g/l. based on the results of these experiments, smooth strain awg-7 was found to be the most suitable for citrate production both from glucose and fructose syrup during long time continuous processes (500 h). in the steady state, the highest citrate productivity (1.3 g/lh) was obtained with this strain, when the feed medium contained 200 g/l of glucose and dilution rate (d) was d = 0.013 1/h. supplementation of the feed medium with bacto-pepton improved the productivity, citric acid yield and stability of the continuous process in the cell recycle fermentation system. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. glycine oxidase is the product of the yjbr gene of bacillus subtilis that was predicted by sequence homology to be a flavoprotein similar to sarcosine oxidase. glycine oxidase catalyzes the oxidative deamination of various primary and secondary amino acids (e.g. sarcosine, n-ethylglycine, and glycine) and d-amino acids to form the corresponding ␣-keto acids and hydrogen peroxide. previous investigations reported on the cloning and production of the glycine oxidase gene in escherichia coli was up to 1 u/g cell. the present works has improved the expression of the recombinant his-tagged glycine oxidase by 15-fold by using pet28a and rosetta cells under the optimal iptg, temperature and time of induction. the protein obtained represented 30% of total soluble proteins in crude extract. the enzyme was purified to near homogeneity using imac with a 95% recovery and with and specific activity of 1.21 u/mg protein. the enzyme was active towards glycine, sarcosine and different d-amino acids, having in general, a basic ph optimum. the kinetic parameters were also studied, showing a km range from 0.3 to 300 mm. the enzyme was immobilized, and used to obtain pyruvic acid (␣-keto acid) from d-alanine with a good yield. the enzymatic synthesis of lipophilic derivatives of various natural antioxidants including flavonoid glycosides, as well as derivatives of cinnamic acid, was performed using various immobilized lipases in ionic liquids such as 1-butyl-3-methylimidazolium tetrafluoroborate (bmim-bf 4 ) and 1-butyl-3-methylimidazolium hexafluorophosphate (bmim-pf 6 ). the influence of various reaction parameters on the catalytic behavior and the selectivity of lipases was pointed out. a response surface methodology was applied for the optimization of the yield and the productivity of the biocatalytic process. the antioxidant activity of the biocatalytically prepared lipophilic derivatives of natural antioxidants, as expressed on cu 2+ -induced oxidation of low-density lipoprotein (ldl) and total serum, was investigated. process strategy for reduction of proteolysis in pichia pastoris fermentations jan weegar 1 , john dahlbacka 1 , noora sirén 2 , niklas von weymarn 2 , kaj fagervik 1 : 1 faculty of chemical engineering,åbo akademi university, finland; 2 laboratory of bioprocess engineering, helsinki university of technology, finland. e-mail: jan.weegar@abo.fi (j. weegar) the yeast pichia pastoris is a popular host organism for production of recombinant proteins. it is, however, common that the products are degraded by proteases towards the end of the fermentation, resulting in productivity and purity decreases. proteolysis of recombinant proteins in p. pastoris fermentations is affected by the temperature and ph of the growth medium. in this study, it was shown that decreasing the temperature from 30 to 10 • c during the induction phase effectively prohibited proteolysis. on the other hand, the temperature decrease resulted in a reduced maximal methanol consumption rate, which subsequently resulted in a culture highly sensitive to residual methanol. the temperature was slowly decreased according to a predetermined trajectory. as the temperature reached values below 12 • c, the methanol concentration had to be closely monitored and the substrate feed rate adjusted in order to prohibit methanol poisoning as well as to maintain the culture as a substrate limited fed-batch. measurement of protease concentrations revealed that proteases were present at 10 • c, but at this temperature the proteolysis rate was evidently effectively reduced. the recombinant protein produced could almost totally be recovered (i.e. high purity) with this process strategy compared to a constant high temperature culture (30 • c) where severe breakdown of the product was observed. with the growing of an ecological conscience in the public opinion, more and more industrial processes are analyzed for a possible ecologically beneficial alternative. for the production of ascorbic acid, which is nowadays done by the reichstein process, a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic means, which show some advantages regarding costs and environmentalfriendliness. besides of two-stage fermentation, our approach is to design a tailor-made organism that produces 2-keto-l-gulonic acid, which is the direct precursor of ascorbic acid from glucose or gluconic acid and which can easily be converted to the final product by conventional methods. erwinia (pectobacter) cypripedii, which is a natural producer of 2,5-diketo-d-gluconic acid, was selected as a suitable host for a 2,5-diketo-d-gluconate reductase from corynebacterium glutamicum. to increase the yield of the desired compound we investigate two 2-keto-reductases in the host organism that diminish the yield of 2-keto-l-gulonate by reducing the compound to l-idonic acid, or by metabolisation of intermediates. these two enzymes were investigated with vari-ous biochemical and molecular biological methods, which will be presented. overproduction of bioinsecticides by heat and salt stress and control of dissolved oxygen in cheap media of bacillus thuringiensis nabil zouari, dhouha ghribi, samir jaoua laboratoire des biopesticides, centre of biotechnology of sfax, tunisia, bp:k, 3038 sfax, tunisia bioinsecticides based on preparations of spores and insecticidal crystal-proteins (icps) produced by the bacterium bacillus thuringiensis (bt) proved to be a high tool for fighting some agricultural pests and vectors of diseases. however, the use of bt preparations as commercial insecticides would be prohibitively expensive because it is not easy to reach cheap overproduction of icps during large-scale fermentation. here, we report possibilities to improve delta-endotoxins production as a consequence of responses of bt strains to low levels of heat and salt stress. each stressor results differently in the improvement of delta-endotoxins production, but both were shown to be most efficient at the beginning or the midexponential phase of the cultures which become resistant at the stationary or the sporulation steps. heat stress caused increase of 84% of synthesis yields of the sporulating cells, in contrast, salt caused increase of 25% of spores counts, corresponding to 28% toxins production improvement. combined effects of both stressors lead to toxins production improvement of 66%, yield improvement of 40%. we focused on the overcome of carbon repression catabolite, closely related to oxidative metabolism, by an adequate control of dissolved oxygen in the cheap media we formulated for bt insecticides production. we showed that an equilibrium between the high density of vegetative cells and their ability to synthesize toxins during their sporulation was necessary to take into account. 40% increase of icps production was reached into 3 l fermenter combination of mutagenesis, heat and salt stress and oxidative metabolism control allowed more than 100% improvement of delta-endotoxins. these results are of great importance in practical point of view, since high bioinsecticides concentrations could be produced without decrease of the yields of their production. mechanism and function of the intramembrane-cleaving protease rhomboid marius lemberg 1 , javier menendez 2 , christopher koth 2 , matthew freeman 1 : 1 mrc laboratory of molecular biology, cambridge, uk; 2 ontario center for structural proteomics, toronto, canada rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. oligosaccharides and 3-keto-glycosides. availability of the enzyme is, however, hampered by the very slow growth and low production rates of the fungus. cloning of the encoding gene and production of the protein by heterologous expression are therefore a prerequisite not only for any biotechnological application, but further scientific investigations as well. on the basis of peptide sequences degenerated primers were designed, and the resulting pcr fragment was used as a probe to isolate the gene from a genomic library. two very similar genes encoding previously uncharacterized proteins were also found, and flanking regions were amplified using rage-pcr. furthermore cdna clones of all genes were isolated by rt-pcr. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. the behavior of phytate degrading enzymes isolated from malaysian zea mays root in rice bran media anis shobirin meor hussin 1 , abd-elaziem farouk 1 , hamzah mohd salleh 1 , ralf greiner 2 : 1 biomolecular engineering research group, department of biotechnology engineering, kulliyyah of engineering, international islamic university malaysia, jalan gombak, 53100 kuala lumpur, malaysia; 2 centre for molecular biology federal research centre for nutrition and foods, haid-und-neu-straße 9, d-76131 karlsruhe, germany phytate degrading enzymes catalyze the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen. they are widespread in nature, occurring in plants and microorganisms, as well as in some animal tissues. phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for phytate degradation and their application for animal feed and human health. from over isolate 140 screened isolates of phytate degrading enzymes, three isolates from the root malaysian maize plantation have shown phytate degrading activity. the production of phytate degrading enzyme was studied using different concentrations [%, w/v] of rice bran media during different stages of cultivation. the dephosphorylation of phosphate from rice bran phytate has shown regulatory effect on the secretion of bacterial phytases. in this conference, we will present data for the characterization of the enzymes. in this paper we present the properties of a phytase purified from a bacterium isolated from malaysian wastewater, which might find application as an animal feed supplement. the phytase described herein is a periplasmic enzyme. the phytase was purified about 180fold to apparent homogeneity using ion-exchange chromatography and gel-filtration with a recovery of 10% referred to the phytatedegrading activity in the crude extract. the enzyme exhibits an activity of about 1106 u mg −1 . gel filtration of the native enzyme on a calibrated sephacryl s-200 column gave a molecular mass of the phytase of 42,000 ± 1500 da with elution position being measured by determination of enzyme activity. lower molecular mass species or higher molecular mass aggregates were not observed. the phytase appeared homogeneous by polyacrylamide gel electrophoresis under non-denaturing conditions at ph 8.3 and 4.8 and gave a single protein band upon sds gel electrophoresis after coomassie staining of the gels. these results indicate that the phytase could be regarded as homogeneous. the estimated molecular mass after sds-page indicated that the phytase having a molecular mass of 41,500 ± 2500 da. consequently, this enzyme is a monomeric protein. the purified enzyme had a single ph optimum at ph 4.5 and was virtually inactive above ph 7.0. at ph 3.0, 40% and at ph 2.5, 20% of the activity at optimal ph was observed. the effect on enzyme stability was studied in the ph range 1.0-9.0 at 4 • c. within 14 days the phytase did not lose any activity in the ph range from 3.0 to 8.0, but at ph values below 2.0 a rapid decline in activity was observed. at ph 1.5, 72% and at ph 9.0, 65% of the initial activity was lost during 24 h. in the range of temperatures studied, 10-80 • c, the optimum temperature for the enzyme was found to be 65 • c. the apparent activation energy was estimated at ph 4.5 from the slope of log v max versus 1/t. the data showed excellent linearity from 15 to 65 • c. the arrhenius activation energy for the hydrolysis of phytate was calculated to be 37.5 kj/mol. in order to check thermal stability, the purified enzyme was incubated at different temperatures, cooled to 4 • c and assayed using the standard phytase assay. the enzyme lost no activity in 10 min at temperatures up to 65 • c. when exposed for 10 min at 70 • c, it retained 50% and at 80 • c 12% of the initial activity. in order to determine the substrate selectivity of the purified phytase, several phosphorylated compounds in addition to phytate, were used for k m and v max estimation by detecting the release of the phosphate ion during hydrolysis using formation of a soluble phospho-molybdate complex in an acidic water-acetone mixture. only phytate was identified as a substrate. the kinetic parameters for the hydrolysis of phytate were determined to be k m = 0.15 mmol l −1 and k cat = 1164 s −1 at ph 4.5 and 37 • c. like other bacterial phytatedegrading enzymes, the purified enzyme showed substrate inhibition. the activity of the purified enzyme was inhibited at substrate concentrations >7 mm. the study of the effect of metal ions on enzyme activity showed that none of them had an activating effect when used at a concentration between 10 −4 and 10 −3 m. mg 2+ , ca 2+ , mn 2+ , co 2+ , ag + , hg 2+ , and cu 2+ had little or no effect on enzyme activity, while zn 2+ , fe 2+ , and fe 3+ showed strong inhibitory effects. the reduced phytate-degrading activity in the presence of fe 2+ and fe 3+ is attributed to a lower phytate concentration in the enzyme assay because of the appearance of a fe-phytate precipitate. when compounds which tend to chelate metal ions, such as o-phenanthroline, edta, oxalate, citrate or tartrate, were tested for their effect on enzyme activity, it was noted that none of them was inhibitory at a concentration from 10 −4 to 10 −3 m. fluoride, a known inhibitor of different phytate-degrading enzymes from bacteria and the hydrolysis product phosphate as well as its structural analogs molybdate, wolframate and vanadate were found to be strong inhibitors of the purified enzyme. flouride inhibited the hydrolysis of phytate with a k i value of 112 mol l −1 . several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (amps) in recombinant bacterial expression systems. in the present work, we investigated the use of the baculoviral polyhedrin (polh) protein as a novel fusion partner for production of a model amp (halocidin 18 subunit; hal18) in escherichia coli. the recombinant hal18 amp could then be hydroxylamine cleaved from the fusion protein and easily recovered by simple dialysis and centrifugation. this was facilitated by the fact that polh was soluble in the alkaline cleavage reaction but became insoluble during dialysis at a neutral ph. importantly, recombinant and synthetic hal18 peptides showed nearly identical antimicrobial activities against e. coli and staphylococcus aureus, which were used as representative gram-negative and -positive bacteria, respectively. these results demonstrated that baculoviral polh can provide an efficient and facile platform for production or functional study of target amps. extensive industrial and food additive applications of succinate have attracted much effort towards finding an environment-friendly alternative to the petrochemical production processes. it is very attractive to engineer s. cerevisiae for succinate production because of its generally regarded as safe (gras) status, ease of genetic manipulation and fermentation. we approached this metabolic engineering problem with a two-step methodology combining modern computational as well as molecular biology tools. in the first step we identified potential metabolic engineering targets leading to high succinate yield and productivity, with the aid of genome scale metabolic model and a bi-level optimization framework using flux balance analysis and quadratic programming. in the next step, various deletion mutants are being constructed and characterized for physiology and succinate production. results from these experiments then will be used to improve the predictions in computational models. so far, we have constructed a saccharomyces cerevisiae mutant deleted in sdh3, which encodes a major subunit of sdh-complex converting succinate to fumarate in mitochondria. the physiology of sdh3 mutant has been characterized in aerobic and anaerobic batch cultivations and in glucose limited chemostat at dilution rate as low as 0.027 h −1 . in aerobic batch fermentations, the mutant showed reduced maximum specific growth rate as compared to the wild-type, and it was incapable of growing on ethanol as sole carbon source, as predicted from the model. interestingly, the mutant showed much higher specific growth rate in anaerobic conditions, close to the wildtype strain. moreover, the chemostat cultivations indicate that the critical dilution rate of the mutant is below 0.027 h −1 . this opens further opportunities to investigate interesting behavior of the mutant and the underlying regulatory processes to improve our understanding of yeast mitochondrial metabolism. department of chemical engineering, yıldız technical university, davutpaşa campus, 34210 esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) lactose is the dominant carbohydrate in milks which are, in turn, the only significant natural sources of lactose. a large number of people do not digest lactose properly due to a lack or inactivity of the intestinal beta-galactosidase and they suffer from intestinal dysfunctions -gas, abdominal pain and diarrhea -if their diet contains lactose. moreover, lactose is a sugar with a high bod, low sweetness, low solubility, when compared to the products of its hydrolysis (glucose and galactose) and being a hygroscopic sugar has a strong tendency to adsorb flavours and odours. the hydrolysis of this sugar is very attractive towards the improvement of processes for the production of ice cream and other refrigerated dairy products and it could be very interesting for the development of additives for animal and human alimentation. the enzymatic hydrolysis of lactose is carried out by beta-galactosidases, enzymes that are widely distributed in nature, appearing in micro-organisms, plants and animal tissues. the present investigation describes the effects of the sonication process parameters on enzymatic hydrolysis of milk lactose and enzyme stability. bandelin sonopuls sonicator was used for the lactose hydrolysis experiments. ␤-galactosidase enzyme used is produced from kluyveromyces marxianus. the reactions were carried out in 250 ml of milk. the process variables for the sonicator are duty cycle, acoustic power and enzyme concentration. the amount of residual lactose concentration (g/l) and residual enzyme activity (%) against time were investigated versus process variables. beside of this; the mathematical models depending on the operating conditions were also derived by using the experimental data of lactose concentration and enzyme activity. kinözbek department of chemical engineering, yıldız technical university, davutpaşa campus, 34210 esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) over the last decade, the use of plant protein hydrolysates alternative to animal protein hydrolysates in human nutrition has broadly expanded. protein hydrolysates are often used in different nutritional formulations, such as supplementation of drinks to enhance their nutritional and functional properties, or special medical diets. there are many processes which employ protein hydrolysis and hydrolytic products. among these processes, the use of enzymes allows selective hydrolysis of protein and produces a potentially safer and more defined material. in the present study, the effect of the temperature, ph and viscosity on the hydrolysis of corn gluten was investigated using a stirred batch reactor system. the corn gluten was hydrolysed by using neutrase enzyme, a bacterial protease produced by a selected strain of bacillus amyloliquefacien. the reactions were carried out in 0.2 l of aqueous solutions containing 1% (w/v) corn gluten and 0.4% (v/v) enzyme. the degree of hydrolysis (%) and soluble protein concentration depending on the time were investigated at the temperatures 40, 45, 50, 55, and 60 • c; and at the ph values 6.5, 7, 7.5, and 8. to investigate the effect of viscosity, the various amounts of glycerol was added to the reaction solutions to produce viscosities in the range of 1.415-13.43 cp. the degree of hydrolysis (dh) was computed by using ph-stat method. for the soluble protein determination in the hydrolysates samples, the folin-lowry method (1951) was used. polyhydroxyalkanoates (pha), one of the most promising bioplastics for the partial replacement of synthetic polymers like polypropylene, are polyesters produced by bacteria as intracellular storage reserves of carbon and energy. the industrial production of pha is achieved by pure cultures in its natural state or using genetically engineered organisms. the main obstacle to the replacement of synthetic plastics by biopolymers is their great cost difference. research on the field of biopolymers synthesis using mixed cultures and waste organic carbon sources as substrates prove to decrease substantially the production costs of pha. the optimization of pha production under aerobic feeding conditions (adf) was achieved recently in our group, obtaining the highest value of pha content stored by mixed cultures (79.2% of cell dry weight). in this work only a homopolymer of polyhydroxybutyrate (phb) was obtained and since it is a highly crystalline and brittle material its application field is limited. the mechanical and thermal properties of pha can be varied to a great extend by adjusting the monomer composition. the incorporation of different monomeric units, other than hb, in the polymer chain, originates copolymers with improved mechanical properties. optimization of pha production from propionate by a mixed culture was studied varying the carbon and ammonia concentrations. propionate only, acetate alone or a mixture of acetate and propionate were tested. copolymers of hydroxybutyrate and hydroxyvalerate, p(hb/hv), with different compositions were obtained. consequently polymer properties could be manipulated by feeding the selected volatile fatty acid composition. the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of 6 weeks. mean transformation frequency ranged from 27% (for 8196 up to 31% (for 15834). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba 9402 tl-dna and the 35s gus gene showed an average of more than 35%. these obtained root cultures were additionly elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g.uralensis were obtained by infection of a. rhizogenes 8196 have produced gl at an yield of 4.5% dry weight on the period of culture as a 30 days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels (3.42 g/l) of the total flavonoids production have been identificated on the strains which transformed by lba 9402. this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. production, purification and characterization of scytalidium thermophilum phenol oxidases didem sutay 1 , ufuk bakir 1 , zumrut b. ogel 2 : 1 chemical engineering department, middle east technical university, inonu bulvari, 06531 ankara, turkey; 2 food engineering department, middle east technical university, inonu bulvari, 06531 ankara, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) phenol oxidases (pos) are a group of enzymes which are responsible for oxidation of various phenolic compounds in the presence of molecular oxygen. there are different types of pos present in nature and three major groups of these enzymes are laccases (e.c. 1.10.3.2, p-benzenediol: oxygen oxidoreductase), catechol oxidases (e.c. 1.10.3.1, o-diphenol oxidoreductase) and tyrosinases (e.c. 1.14.18.1, monophenol monooxygenase). another group of enzymes, peroxidases (e.c. 1.11.1.7), can also be considered as a member of po family. pos have very wide substrate range and final oxidation products of these substrates are quinones, which are highly reactive molecules and polymerize into brown, red or black waterinsoluble compounds. pos are very common in nature, they can be found in almost all plants, animals and microorganisms. pigmentation and protection from the environment are main functions of these enzymes. pos have different applications in food, pharmaceutical, textile industries and waste-water treatment systems. the objective of this study was po production by the thermophilic fungus, scytalidium thermophilum, purification and characterization of the enzyme. for this purpose, enzyme production was performed either in a shaker-incubator or a temperature, ph and dissolved oxygen controlled 2 l bioreactor (probiotem) to optimize enzyme production medium composition and bioreactor parameters. as the carbon and nitrogen sources, 4% glucose and 0.4% yeast extract were determined as the optimal concentrations, respectively. copper, gallic acid and tannic acid were determined to increase enzyme production. purification was performed by using membrane and chromatographic techniques. hydrophobic, ion exchange and gel filtration columns were used by using a fplc system;äkta prime (amersham biosciences). especially the phenyl sepharose tm high performance column appeared to be very efficient for po purification from scytalidium thermophilum. purified po have been characterized by electrophoretic techniques and kinetic studies. isolation of lipolytic microorganisms from subtropical soils. cloning, purification and characterization of a novel esterase from strain pseudomonas sp. cr-611 núria prim, cristian ruiz, cristina bofill, f.i. javier pastor, pilar diaz department microbiology, university of barcelona. av. diagonal 645, microorganisms or their enzymes are used in a wide range of biotechnological activities such as polymer hydrolysis, synthesis of added-value compounds, sample decontamination, etc. thus, there is an increasing interest for isolating new enzymes and new enzymeproducing organisms for their use in industrial conversions (cherry and fidantsef, 2003) . among these enzymes, lipases, esterases, cellulases, xylanases and pectinases play an important role in many biotechnological processes like those related to pulp and paper processing. three samples of subtropical forest soil from puerto iguazú (argentina) were used for the isolation of autoctonous microorganisms growing in an organic matter-rich environment. a total of 724 pure cultures of bacteria and fungi were obtained and their hydrolytic activities on polysaccharide and lipidic substrates were assayed using olive oil, tributyrin, cholesterol esters, xylan, cellulose and pectin as substrates. among the isolates analysed, 449 were active on one or more of the substrates evaluated, and 43 of them degraded all substrates. nearly half of the strains displayed lipolytic activity, whereas the number of strains active on xylan, cellulose, pectin and cholesterol esters, was much lower. the 76 strains bearing the highest hydrolytic activities were selected and stored for further characterization (ruiz et al., 2005) . among them, strain cr-611, one of the most active isolates on tributyrin, was selected for identification and characterization of its lipolytic system. lipolytic strain cr-611 was identified by morphological, physiological and phylogenetic tests, as a pseudomonas sp., closely related to p. fluorescens. sds-page and zymogram analysis (diaz et al., 1999) of cell extracts and supernatants from the strain revealed a complex lipolytic system consisting of at least two lipolytic enzymes. sequence alignment and clustering of previously described pseudomonas lipases and esterases allowed the design of different sets of primers for the isolation of the lipase/esterase coding genes. a gene coding for an esterase with homology to family vi bacterial lipases (arpigny and jaeger, 1999) was isolated and cloned in escherichia coli. the cloned enzyme was further purified and characterized, showing preference for short fatty acid esters and displaying a typical michaelis-menten kinetics, with no interfacial activation. the substrate profile, together with the kinetic behaviour and sequence similarity of the cloned enzyme to family vi bacterial esterases, allowed to identify this enzyme as an esterase and was named esta6. maximum activity was achieved on muf-butyrate at 55 • c and ph 8.5, suggesting that it could be of interest for biotechnological purposes. microbial xylitol production from agricultural wastes has recently attracted much attention from industries because it has potentials to realize the cheaper production of xylitol with low environmental impact (tada et al., 2004) . in order to realize the effective xylitol production by a xylose utilizing yeast, the oxygen supply is a key for maximizing xylitol yield over consumed xylose (y xl ) because the intracellular xylitol metabolism is strongly influenced by the amount of available oxygen. in the present work, we tried to apply a metabolic reaction model in order to determine the optimal oxygen transfer rate (otr) in a fermentor for maximizing xylitol yield. corn cob hydrolysates containing 25 g-xylose/l was employed as medium for xylitol production by computer-controlled batch cultures using candida magnoliae (ferm p-16522, aist). a metabolic reaction model considering main xylitol metabolisms including glycolysis, pentose-phosphate pathway, tca cycle and cell synthesis was developed. the model allows to estimate various intracellular metabolic flux distributions including a xylitol production rate. the oxygen uptake rate to maximize the ratio of xylitol production rate over xylose consumption rate corresponds to the otr condition to maximize a xylitol yield over xylose consumed. based on the metabolic reaction model, the otr was optimized by a linear programming, the optimal otr and the maximum xylitol yield were estimated as 0.5 mmol o 2 /l h and 0.81 g-xylitol/g-xylose, respectively. the experimental verification using the optimal otr demonstrated that the xylitol yield was greatly improved to 0.75 g-xylitol/g-xylose from 0.6 g-xylitol/g-xylose in our previous study. expression of a bacterial sugar phosphate transporter in s. cerevisiae to release l-glycerol 3-phosphate accumulated by metabolic engineering almut popp, huyen thi thanh nguyen, ulf stahl, elke nevoigt department of microbiology and genetics, university of technology, 13353 berlin, germany. e-mail: a.popp@lb.tu-berlin. de (a. popp) in contrast to glycerol, its phosphorylated precursor l-glycerol-3phosphate (l-g3p) is retained by the plasma membrane. therefore, engineered yeast strains accumulate l-g3p in the cytosol resulting in low overall yield of the desired product and laborious downstream processing. a suitable sugar phosphate transporter in the yeast plasma membrane would overcome these limitations. the glycerol-3-phosphate transporter (glpt) of e.coli is an antiporter and naturally mediates the uptake of l-g3p in exchange with inorganic phosphate. we assume that this transporter would also mediate the excretion of accumulated l-g3p into a phosphate-rich medium if it was present in the plasma membrane of engineered yeast. despite many inconsistencies in codon usage, we were able to express the bacterial glpt gene in yeast. expression was monitored by a c-myc tag added to the c-or n-terminal hydrophilic tail, respectively. the quantity of the construct with n-terminal tag clearly exceeds the quantity of the construct with c-terminal tag. both gene products are located in the endoplasmic reticulum, as shown by immunofluorescence microscopy. obviously, yeast's transmembrane protein sorting machinery does not recognise it as a substrate for the secretory pathway. metabolic flux analysis of c-and p-limited shikimic acid producing e. coli gaspard lequeux 1 , louise johansson 2 , jo maertens 1 , peter vanrolleghem 1 , gunnar lidén 2 : 1 biomath, ghent university, coupure links 653, 9000 gent, belgium; 2 department of chemical engineering, lund university, p.o. box 124, 22100 lund, sweden. e-mail: gaspard.lequeux@biomath.ugent.be (g. lequeux) metabolic flux analysis (mfa) was applied to decipher why plimited e. coli fermentations are more optimal for shikimic acid production in comparison with glucose-limited fermentations. as mfa allows obtaining insight in the intracellular flux distribution over different pathways by only measuring net production and consumption rates of metabolites, under the condition that parallell pathways are removed. to this end a detailed metabolic network model was created and checked for consistency, dead-ends, and parallell pathways. several fermentations were performed at different dilution rates (ranging from 0.05 to 0.3 h −1 ) and different limitations (phosphate and glucose). the e. coli strain used was w3110 with genetic modifications in the aromatic amino acid pathway to enhance shikimic acid production. for each dilution rate, a metabolic model was solved. this way, the evolution of each flux can be followed with respect to the dilution rate. the p-limited cultures showed a better yield which can be explained by the diminished excretion of dehydro-shikimic acid (as is known from literature) and a reduction of the hydrolysis of atp. takasumi hattori, kuniki kino, kohtaro kirimura department of applied chemistry, school of science and engineering, waseda university, tokyo, japan. e-mail: takasumi@suou.waseda.jp (t. hattori) alternative oxidase is a terminal oxidase in respiration chain, which is a branched chain of cytochrome pathway, and inhibited by salicylhydroxamic acid (sham), but not by cyanide. the citric acid-producing fungus aspergillus niger wu-2223l has a cyanide-insensitive and sham-sensitive respiration catalyzed by the alternative oxidase (kirimure et al., 1999) and did not produce citric acid when cultivated with sham (kirimure et al., 2000) . in this study, the transcript levels of alternative oxidase gene (aox1) (kirimure et al., 1999) and activities of alternative oxidase under the conditions of citric acid production were examined during 9 days-cultivation. the amount of aox1 mrna was determined by northern blot analysis, and the specific activity of alternative oxidase as that of duroquinol oxidase. the transcript level and the activity were highest at 2 days at log-phase, decreased during 2 and 4 days, and thereafter maintained at low levels. however, the transcript and alternative oxidase activity was constitutively detected during whole the cultivation periods under the conditions of citric acid production. the sequence analysis of aox1 chromosomal dna revealed the presence of potential binding site of cyclic amp responsive element (cre), stress responsive element (stre) and heat shock factor (hsf) in its upstream region. these results indicated that the expression of alternative oxidase was regulated in the transcription level and alternative oxidase contributes as the main respiration chain during citric acid production. kirimura, k., et al., 1999 . curr. genet. 34, 472-477. kirimura, k., et al., 2000 . biosci. biotechnol. biochem. 64, 2034 -2039 . trichoderma strains are considered to be among the most useful fungi in industrial enzyme production, agriculture and bioremediation. metabolic versatility displayed by these fungi makes it a very amenable source of new gene products. functional analysis of candidate genes goes by two complementary ways: gene overexpression and loss-of-fuction mutants generation. a few expression systems are available mainly to direct constitutive gene expression in catabolite repression conditions. following a genomic approach, we have recently cloned some gene promoters with high expression in glucose in trichoderma harzianum cect2413. a main goal in a gene functional analysis is the generation of knock-out mutants. up to date, there is no reference about successful gene disruptions in t. harzianum mainly due to a very low homolog recombination frequency. rna-mediated gene silencing has been shown as an efficient tool to diminish or totally abolish specific gene expression. especially those strategies based on the use of hairpin constructs allow rapid and easy generation of strains with reduced levels of mrna from genes of interest. we have used the t. harzianum cect2413 tss1 promoter to direct the expression of a hairpin dna construct that induced the appearance of small-interfering rnas (sirnas) and the silencing of the uida reporter gene in a previous uida overexpressing strain. reduced levels of mrna and gus activity correlated with the presence of sirnas. this is the first report on rna-mediated gene silencing in trichoderma and constitutes a useful and promising tool for functional genomic studies in fungal systems. analysis of the metabolic response of escherichia coli to quantitative modulations of the glucose-6-phosphate dehydrogenase based on 13 c-labelling experiments cécile nicolas, fabien létisse, stéphane massou, philippe soucaille, jean-charles portais. e-mail: fabien.letisse@insa-toulouse.fr (f. létisse) microorganisms have an efficient capacity for adapting their metabolism in response to genetic or environmental changes, and understanding metabolic robustness has become an emergent issue. part of the robustness originates from the network organization of metabolic systems, where the interplay between all available biochemical reactions provides alternative mechanisms for compensating the perturbations. recently, 13 c-metabolic flux analysis ( 13 c-mfa) has been applied to escherichia coli knock-out mutants lacking key enzymes to determine the phenotypic effects of structural changes in the metabolic network, providing further evidences for compensatory phenomena. the aim of our on-going work is to understand how the central metabolism in e. coli responds to quantitative alterations at a specific key-point of the metabolic network. the glucose-6-phosphate dehydrogenase (g6pdh), a key enzyme in the central metabolism for which the effects of deleting the gene (zwf) has been already described (zhao et al., 2004) , was chosen as the target. to this aim we have generated a set of expression mutants, i.e. mutants having each a fixed level of expression of the zwf gene. four different levels of expression, leading respectively to g6pdh activity 2; 2.9; 5.7 and 14 times higher than in the wt strain, have been obtained. for each mutant, transcriptomics analysis will be carried out and compared to both the zwf-and wt strains to detect changes in the network structure, and the distribution of fluxes will be measured using 13 c-mfa. the flux maps obtained for the various strains will be compared to evaluate the quantitative response of the central metabolic network to imposed and increased g6pdh activity. metabolic control analysis will be applied to provide insights onto the sensitivity of the measurable metabolic fluxes to g6pdh activity. combination of transcriptomics and fluxomics approaches will provide information on the nature and extent of the compensatory mechanisms. because the activity of a single enzyme is tuned at different levels in knock-out and expression mutants, this investigation provides a situation that mimics gene-level regulation of metabolism. , j., et al., 2004, metab. eng., 6, 164 . with the depletion of the world's petroleum supply, there has been an increasing worldwide interest in ethanol as an alternative, nonpetroleum source of energy. this fact caused increased interest in the new ethanol technology fermentation process research. as reported before, bacteria zymomonas mobilis possesses more advantages than saccharomyces cerevisiae, microorganism used for ethanol production in industrial scale. for that reason, we have focused on the fermentation studies of this facultative bacterium in free and immobilized form. the immobilization of the cells into polyvinylalcohol (pva) hydrogel lens-shaped capsules lentikats, improved the batch fermentation process efficiency nine times. starch, the substrate considered as one of the best of renewable energy source is considered as fuel ethanol feedstock. due to z. mobilis disability of maltose, maltotriose and dextrin utilization, the starch has to be converted into glucose monomers. this pre-fermentation step can overcharged whole ethanol production process. this ineffective part of the process was resolved with immobilization of glucoamylase into lentikats. the system with immobilized enzyme and cell was stabile in continuous mode for 60 days without any significant change in the system efficiency. the combination of cell and enzyme immobilization can significantly improve the efficiency and the cost of ethanol production in industrial scale. fermentation of an inhibitory dilute acid-hydrolysate from spruce using a fed-batch procedure combined with cell-reuse andreas rudolf, gunnar lidén department of chemical engineering, box 124, lund university, se-221 00 lund, sweden. e-mail: andreas.rudolf@chemeng.lth.se (a. rudolf) a well-controlled addition of hydrolysate to the fermentation has proved very efficient in reducing yeast inhibition due to compounds formed during lignocellulose hydrolysis. furthermore, using high cell mass concentrations has been another way of avoiding the negative impact of the inhibitory compounds. if possible, the yeast should therefore be re-used in the process. in the present work a dilute-acid hydrolysate from spruce was fermented using a fed-batch procedure with reutilization of yeast. the fermentation procedure worked satisfactorily, with more than 98% of fermentable sugars consumed in each of the four consecutive fed-batch fermentations performed. the ethanol yields on fermentable sugars reached 0.45 g/g. there was continued cell growth in the repeated fed-batch experiments, with an average cell yield on fermentable sugars of 0.06 g/g. in contrast, only about 20% of the fermentable sugars were consumed within 24 h, when the fermentation of the hydrolysate was run in a batch process. the work shows the potential to re-use the yeast in a suitably designed process. metabolic engineering is defined by bailey in his seminal 1991 paper as "the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant dna technology". the manipulation of these functions ultimately results in the manipulation of metabolism, which is the purpose of many biotechnological processes. metabolic engineering has sought its methods and tools in mathematics and the physical sciences, and later in information technology (it) leading to the proliferation of bioinformatics. in this work we propose a novel approach to metabolic engineering that regards it as a business process reengineering (bpr) endeavour. hammer and champy in their celebrated 1993 book define bpr as "the fundamental rethinking and radical redesign of business processes to achieve dramatic improvements in critical contemporary measures of performance, such as cost, quality, service and speed". our thesis is that metabolic engineering with its goal of reengineering the metabolism of the microorganism, is equivalent to business process reengineering (bpr) in business and management. indeed this is essentially what metabolic engineering does to the cell through the use of recombinant dna technology, which can be viewed as a radical redesign of the metabolic process. after all, it causes changes that cannot be attained otherwise, and whose purpose is to achieve dramatic improvements in cellular activities such as the increase in production of some metabolites by orders of magnitude. the cost incurred by the process, which is metabolism in this case, can be, for example, energy requirements or change to a cheaper substrate. in this study we elucidate this parallelism between the two with emphasis on modelling of metabolism and how the concepts of business process modelling can be applied to it. the purpose is not to produce a model, but rather to introduce the modelling methodology and demonstrate its utilisation and benefits and outline its limitations and challenges. we believe that the novelty of our work lies in applying a new paradigm in approaching metabolic engineering that has not been considered previously. thermophilic ethanol production from wheat straw hydrolysate in continuous culture tania i. georgieva, birgitte k. ahring biocentrum-dtu, technical university of denmark, building 227, dk-2800 lyngby, denmark. e-mail: tig@biocentrum.dtu.dk (t. georgieva) ethanol production from lignocellulosic biomass has attracted widespread attention as an unlimited low cost renewable source of energy to transportation fuels due to increasing petroleum use and air pollution towards greenhouse gases. wheat straw available as agricultural residue has been considered as a potential lignocellulosic substrate for industrial bioethanol production. a major technical obstacle to commercialize bioethanol production form lignocellulose (such as wheat straw) is associated with a lack of microorganism able to rapidly and efficiently ferment both hexose and pentose sugars into ethanol and to tolerate the inhibitors present in undetoxified hydrolysates. currently used industrial mesophilic microorganisms (saccharomyces cerevisiae and zymomonas mobilis) are excellent ethanol producer from glucose, however, they are not able to ferment other sugars such as xylose, which is the second most abundant sugar in lignocellulose. thermophilic anaerobic bacteria have been considered for ethanol production from lignocellulosic biomass as an alternative to mesophilic ethanol producing strains, predominantly because of their abilities naturally to ferment the whole diversity of sugars found in lignocellulosic biomass. an increase attention to thermophilies for ethanol production have also arise from broad spectrum of advantages regarding industrial scale ethanol fermentation such as high growth and metabolic rates, low oxygen solubility, reduced risk of reactor contamination, and cost savings via mixing, cooling and facilitated product recovery. in addition, simultaneous co-fermentation of glucose and xylose in a single operation unit could substantially reduced the ethanol production cost. research has been attempted to study the potential of using a thermophilic anaerobic bacterium for continuous ethanol fermentation of lignocellulosic biomass, with particular emphasis on effectiveness of our strain to ferment undetoxified wet oxidized wheat straw hydrolysate with respect to sugar (glucose and xylose) conversion and ethanol yield. the experiment was carried out in a lab-scale reactor operated at 70 • c with wheat straw hydrolysate as a substrate in concentrations from 20 to 80 wt.% equivalent to total sugar mixture of 11-38 g/l. wheat straw hydrolysate (woh) [200 g/l wheat straw, 92.4% dry matter (dm)] was prepared using wet oxidation pretreatment process followed by enzymatic saccharification with commercial enzymes mixture of celluclast1.5, and novozym188 (novozymes, denmark). both xylose and glucose sugars were simultaneously converted to ethanol. the sugar utilization was higher than 90%, and high ethanol yields were achieved. reactor shows good long-term performance (124 days) in terms of operation stability and reactor contamination. maltotriose is the second most abundant fermentable sugar in wort and due to incomplete fermentation, residual maltotriose in beer may cause problems in the brewing industry. to study genes that might improve utilization of maltotriose we used a library with dna from brewer's strains and a laboratory strain and identified a new transporter encoded by mtt1. mtt1 gave lager strain a15 the ability to grow on yp/2% maltotriose in the presence of 3 mg/l of the respiratory inhibitor antimycin a. this transporter gene shares 74% similarity with mph2 and mph3, 62% similarity with agt1 and 91% similarity with mal61 and mal31. moreover, mtt1 shares even higher similarity (98%) with the s. pastorianus mty1 gene (m. salema-oom, unpublished, ncbi accession number aj491328). purified radiolabeled maltotriose and radiolabeled maltose were used to study sugar uptake of lager strains a15 and ws34/70, and of a15 containing mtt1 or mtt1alt, a more efficient, altered version of this gene lacking the 66 basepairs from the 3 end and containing 57 base-pairs of vector sequences. these transport studies show that mtt1 and, especially, mtt1alt encode maltose transporters with relatively high activity towards maltotriose compared to maltose. this study is part of a multi-disciplinary project, funded by the european union (contract no. qlk1-ct-2001-01066) focusing on the development of high-gravity resistant brewer's yeast strains. metabolic engineering of l-phenylalanine pathway in bacillus subtilis yasemin demirci 1 , pınar ç alık 2 , güzide ç alık 1 , tunçer h.özdamar 1 : 1 bre laboratory, department of chemical engineering, ankara university, 06100 ankara, turkey; 2 iblab, department of chemical engineering, metu, 06531 ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.özdamar) metabolic engineering design of a recombinant l-phenylalanine (phe) production system is based on coordinated overexpression of the flux-controlling genes in the aromatic-amino acid pathway. based on the insights gained by the work carried out in our laboratories (özçelik et al., 2004) , aroh for the reaction r96 at the branch-point chorismate that connects the preceding reactions of the aromatic group amino acid pathway to the proceeding reactions towards phe, was predicted as the first-, and aroa for dahp synthase (r89) predicted as second-metabolic engineering sites. aroa gene was cloned next to aroh, by the use of four primers designed using pcr-based gene splicing by overlap extension method. the genes were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their 5 ends that are complementary to the 3 portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at 3 end. extension of this overlap by dna polymerase has yielded the recombinant two-gene product; and the two-gene product serve as template for the continuation of reactions for the increase of the concentration in the micro-reactors. the two-gene fragment was first cloned into puc19, and then sub-cloned to pmk4 e. coli -bacillus shuttle plasmid. the new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis. the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, phe and other amino acids, and organic acids concentrations as the constraints. on the bases of calculated intracellular fluxes of recombinant b. subtilis carrying pmk4::aroa::aroh, an in-depth analyses of the metabolic engineering design will be presented. ozçelik,i̇., ç alık, p., ç alık, g.,özdamar,t.h., 2004. metabolic engineering of aromatic group amino acid pathway in bacillus subtilis for l-phenylalanine production. chem. eng. sci. 59 (22-23), 5019-5026. the 100% respiratory-deficient nuclear petite amylolytic saccharomyces cerevisiae npb-g strain capable of excreting a hybrid protein possessing both ␣-amylase and glucoamylase enzyme activities was generated and its employment for direct fermentation of starch into ethanol was investigated under both shake flask and controlled bioreactor cultivation conditions. when compared with a standard host strain, higher ethanol concentrations and yields were achieved with the nuclear petite strain under both cultivation conditions. further improvement in ethanol production was achieved by the use of an initial glucose supplement. comparison of the ethanol fermentation performances of the respiratory-deficient npb-g and the parental respiratory-sufficient wtpb-g strain showed an increase of, ca. 48% in both ethanol production yields and ethanol productivities with the respiratory-deficient strain. response surface methodology (rsm) was used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both strains with concomitant improvement in their amylolytic potentials. high ethanol yields on substrate values of the bioreactor cultures, that were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient strain developed in this study was very effective in the direct fermentation of starch into ethanol. establishing a biotechnology educational framework to support a knowledge-based economy in puerto rico rosa buxeda, lorenzo saliceti-piazza industrial biotechnology program, university of puerto rico, mayagüez campus, mayaguez 00680, puerto rico. e-mail: rbuxeda@uprm.edu (r. buxeda) industrial biotechnology has been identified as a major thrust area of economic development within the past five years for the island of puerto rico. the portfolio of biotechnology manufacturing investments in the island has passed the two billion dollar mark. world known companies like amgen, abbott and eli-lilly lead these investments, which have catalyzed a strong technology transfer to the island. a strong collaboration between industry and academia was needed to provide a well-trained workforce for these company startups. as a result, the university of puerto rico, mayagüez campus (upr-m) developed four initiatives which are part of the educational pipeline in biotechnology. these are: (i) an industrial biotechnology program, a 5-year bs degree which contains a novel curriculum with courses from science and engineering with undergraduate research and industrial internships as part of the degree requirements; (ii) an industrial biotechnology learning center, which provides customized biotechnology and bioprocessing training modules, including lectures and hands-on experiences to train and develop the workforce needed in the biotechnology manufacturing plants; (iii) a biotechnology summer camp, which addresses the high school student population and its main purpose is to educate and advise on the different career paths that can be followed in the field of biotechnology; and (iv) a biotechnology center for research and training in bioprocessing, that will address the development of corporatesponsored research projects to strengthen links between industry and academia in order to build up a knowledge-based economy. our paper will describe each initiative in detail as well as its outcomes and impact on puerto rico's knowledge-based economy goals. isolation of acid phosphatase from sweet potato and immobilization using different adsorbent d. omay, y. güvenilir, n. deveci istanbul technical university, department of chemical engineering, maslak 34769, istanbul, phosphatase enzymes occur in a wide range of plant and animal tissues. they catalyze the hydrolysis of phosphate bonds in organic phosphates, between the phosphate group and rest of the molecule. immobilization of enzymes and biological compounds is currently gaining importance due to its wide variety of applications in the food and pharmaceutical industries and also its biomedical applications. it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan. the aim of this experimental study was determined as partial purification and isolation of acid phosphatase enzyme and its immobilization. the purification was realized by applying centrifugation, ammonium sulfate precipitation and dialysis respectively. the specific activity of the supernatant was 0.1 u/mg and after 80% saturation this value increased 0.64 u/mg. furthermore, acid phosphatase was investigated using different adsorbent (chitin, chitosan, synthetic zeolite and raw zeolite) and evaluated the storage stability and re-usability of the immobilized acid phosphatase. it was estimated that, acid phosphatase activity was shielded the ratio of 94, 96, 99, and 92% by using raw zeolite, synthetic zeolite, chitin and chitosan respectively under 12 h operation condition. øyvind m. jakobsen 1,2 , michael c. flickinger 3 , svein valla 1 , trond e. ellingsen 2 , trygve brautaset 1 : 1 department of biotechnology, norwegian university of science and technology, norway; 2 sintef applied chemistry, sintef, norway; 3 biotechnol. institute, department of biochemistry, molecular biology and biophysics, university of minnesota, usa aerobic methylotrophs can utilize one-carbon (c 1 ) compounds such as methane and methanol as a sole c-source for growth and energy. the majority of research on these organisms has focused on their biochemical novelity and commercial viability. for the industrial production of bulk products such as the amino acids lysine and glutamate raw material costs and abundance are important, and c 1 sources are thus attractive compared to sugars. bacillus methanolicus can secrete up to 55 g/l of glutamate upon methanol growth at 50 • c (thermotolerant and methylotroph) and mutants producing 35-40 g/l of l-lysine have been selected. we study the genetics for conversion of methanol into biosynthesis of glutamate and lysine, and in the present report we unravel the regulation of genes impor-tant for the consumption and tolerance level for c 1 compounds by b. methanolicus. b. methanolicus has a methanol dehydrogenase gene (mdh) for oxidation of methanol into formaldehyde and a ribulose monophosphate (rump) pathway for assimilation of formaldehyde. we recently discovered that mdh and five rump genes are carried by natural plasmid pbm19 in this bacterium and this represented the first documentation of plasmid-dependent methylotrophy in any microorganism. we here use real-time pcr to analyse the regulation of plasmid-and chromosomally located rump genes, in cells upon methylotrophic and non-methylotrophic growth. high methanol concentrations in the growth medium is cell toxic and the mechanisms for this sensitivity of b. methanolicus is poorly understood. our results indicate that plasmid pbm19 plays a fundamental role for this trait as well and the impact of our results on the biotechnological applications of this bacterium is discusssed. department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, 3.9 (t1), 7.8 (t2) and 11.7 (t3) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period (15 days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t3 showed more less firm after 15 days of storage being 20.17 g/100 g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t2 and t3 of 433 and 479 mpa s respectively, compared with control of 299 mpa s at 15 days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t2 gained the highest scores (85 points) followed by the control (81.5 points) after 15 days of storage, while yogurt of t3 showed a low scoring being 75. from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of 7.8 units of plant proteinases/ml milk. the penicillium chrysogenum oat1 gene encoding a class iii omega-aminotransferase has been cloned and characterized. this enzyme that converts lysine into 2-aminoadipic semialdehyde is important in providing 2-aminoadipic acid, a precursor of penicillin and other ␤-lactam antibiotics. the enzyme is related to ornithine-5-aminotransferases and to lysine-6-aminotransferases encoded by the lat gene located in the bacterial cephamycin gene clusters. expression of oat1 is induced by lysine, ornithine and arginine and repressed by ammonium ions. area-binding consensus sequences and an 8-bp direct repeat associated with arginine induction in emericella (aspergillus nidulans) have been found in the oat1 promoter region. deletion of the oat1 gene resulted in the loss of omegaaminotransferase activity. the deletion mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed a reduced growth on lysine. complementation of the deleted mutant with the oat1 gene restored growth on ornithine, arginine and lysine to the levels of the parental strain and omega-aminotransferase activity. the strong expression of oat1 gene after induction by the basic amino acids may provide additional 2-aminoadipic acid for the formation of the 2-aminoadipyl-cysteinyl-valine tripeptide for ␤-lactam biosynthesis. morphological characterisation of two high producing strains of penicillium chrysogenum carrying a disruption in the nadph-dependent glutamate dehydrogenase k. rueksomtawin, j. thykaer, h. noorman, j. nielsen center for microbial biotechnology, biocentrum-dtu, technical university of denmark, dk-2800 lyngby, denmark. e-mail: kr@biocentrum.dtu.dk (k. rueksomtawin) metabolic engineering has proven to be useful in optimisation of ␤-lactam production, e.g. constructing superior strains with multiple copies of the ␤-lactam gene cluster. it is however, of equal importance to gain insight into other aspects of the metabolism to establish a general overview in order to apply metabolic engineering for further improvement of the production strains. in that context, the redox metabolism is essential, as it functions as a tightly controlled connection between the different parts of the metabolism. in order to investigate this role of the redox metabolism in more detail, the gdha-gene, encoding the nadph-dependent glutamate dehydrogenase, was disrupted in two industrial strains of penicillium chrysogenum. during physiological characterisation of the two strains it became apparent that considerable changes in the morphology had occurred due to the genetic alteration. since the morphology is an important parameter in process optimisation, an examination of the morphology of the two strains was undertaken. in this work, the morphological differences between the gdha-disrupted strains and the reference strains were comprehensively investigated both during growth on solid media and submerged growth in a flow-through growth chamber. with the advance development of computerized image analysis techniques, the key morphological properties of the individual hyphal elements were quantified. in comparison to the reference strains, the disruption of the gdha gene resulted in a morphological change from short hyperbranched hyphal elements to long elongated hyphal elements with less branches. in the parallel studies with aspergillus nidulans and its corresponding gdha-deleted strain, no difference in the morphology was observed. polyketides (pk) represent one of the largest groups of natural products and are found in fungi, bacteria and plants. since many useful polyketides either originate from sources that are difficult or even impossible to cultivate or are produced in inadequate amounts, we are interested in expressing polyketide synthases (pkss) in heterologous hosts. saccharomyces cerevisiae, aspergillus niger and aspergillus nidulans were chosen as initial hosts, because the techniques necessary to cultivate and manipulate these strains genetically are well established. 6-methylsalicylic acid synthase (6-msas) was chosen as a model pks. replicative plasmids carrying the genes encoding 6-msas from penicillium patulum and phosphopantetheinyl transferase (pptase), respectively, were transformed into s. cerevisiae. in addition, an integrative vector was designed and the gene encoding 6-msas was integrated in the yeast chromosome. batch cultivations on galactose minimal media were performed. the results are presented and in particular the effect of expression mode and type of pptase (bacterial versus fungal) is discussed. furthermore, the progress of the work on expressing 6-msas in a. niger and a. nidulans using an integrative vector system is presented and discussed. the valorisation of functionalized chemicals from biomass resources compared to the conventional fossil production route ben brehmer, wageningen ur agrotechnology & food sciences, workgroup: valorisation of plant production chains, p.o. box 17, 6700 aa wageningen, the netherlands. e-mail: ben.brehmer@wur.nl at some undisclosed point in the foreseeable future, cheap fossil fuels resources will become depleted and the industry will be forced to pursue more difficult reserves with increasingly high extraction costs. most alternatives available and under research do not consider price as the main motivation for replacement, but focus solely on sustainability and environmental benefits. sustainability is an interesting word as there are enough fossil resources scattered around the world to be sustainable in quantity but not sustainable in price. seeing that fossil fuels are derived from prehistoric biomass it is not at all presumptuous to assume that every application and product can be replaced by the biomass of today. in fact, many highly specialised pharmaceutical chemicals already have a biomass origin. yet, not all of the uses of fossil fuels need to be replaced by a comparable carbon based source, such as biomass. energy and transportation in particular do not necessary need to rely on carbon cleavage, whereas practically all of the petrochemicals contain a carbon backbone. the main stipulation in substituting fossil-based chemicals with bio-based chemicals is availability and cost. it is proposed that already today, by utilising existing, recently developed and developing technology, it is economically advantageous for many chemicals to derive from biomass, in particular the functionalized chemicals. the only way to validate this conjecture is to develop a complete comparative life cycle analysis. as opposed to a traditional lca, the "multicriterion" developed here will revolve around energy flows and process efficiency in terms of exergy. the aim is to assess the optimum route with the best production options along the whole production chain while determining any possible limiting factors. using this tool, a systematic production matrix relating several logical source crops and a few key chemicals of varying derivative levels can be created and compared to the conventional fossil routes. combined with economic considerations and some unambiguous environmental factors, the investigation will provide all the information relevant to the industry. the goal is to create an objective and reliable simulation system ratifying the economic and environmental feasibility of exploiting biobased chemicals today and indicate the steps necessary for further improvement. biosynthesis of multi-enzymatic preparation from aspergillus niger ibt-90 useful in textile fabric treatment rita pyc 1 , jadwiga sojka-ledakowicz 2 , tadeusz antczak 1 , joanna lichawska 2 : 1 institute of technical biochemistry, the technical university of lodz, lodz 90-924, poland; 2 textile research institute, lodz 92-103, poland among many methods of producing enzymatic preparations, i.e. by liquid surface fermentation -lsf, submerged fermentation -smf or solid state fermentation -ssf, this last is most advantageous. cultivation in solid state means fermentation on a matrix formed by industrial and agricultural wastes. most often filamentous fungi -due to the lack of available water in the foundation -are the efficient, competitive microorganisms applied in solid state bio-conversion. the aim of research works carried out at the institute of technical biochemistry of the technical university of lodz and at textile research institute, lodz was defining optimal conditions for biosynthesis and testing the possibility of applying multi-enzymatic preparation from aspergillus niger ibt-90 in the treatment of woven fabrics made of natural cellulose fibres. as the result of biosynthesis optimization process, malt sprouts, wheat barn and beet pulp were selected as the best media to obtain enzymes of high pectinolytic activity maintaining at the same time high activity of cellulolytic enzymes and xylanase. the highest obtained activities reached: 2000 0 pm for total pectinolytic activity, 7 j/ml for endoglucanase and 527 j/ml for endoxylanase and they were 2.4-3.0 times higher than those achieved before optimizing process. performed research works demonstrated that the optimum activity of applied enzymatic system is obtained in the range of ph 4.6-4.8. woven fabrics made of flax and cotton fibre blends, subjected to bio-pre-treatment, were evaluated with reference to their sorption properties. comparative evaluation of liquid sorption by woven fabrics allowed to notice efficient enhancement of fibres' sorption capabilities after pre-treatment using enzymes system from aspergillus niger ibt-90. this offers the possibility of substitution of alkali scouring of linen-cotton woven fabrics before their bleaching by bio-treatment. the phylum actinobacter includes many bacteria of industrial importance both for accumulation of primary and secondary metabolites. both primary and secondary metabolites are dependent on precursors and cofactors that are provided by the central carbon metabolism of microorganisms. there are alternative pathways for catabolism of carbon either via the embden meyerhof parnas (emp) and pentose phosphate (pp) pathway or through the entner doudoroff (ed) pathway. the emp pathway is energetically more favorable and has therefore been presumed to be the dominating route for carbon metabolism in bacteria producing secondary metabolites. however, primary metabolism is poorly studied for most actinobacter species as focus traditionally has been on secondary metabolism. with the aim to gain more knowledge about the diversity of central carbon metabolism within the phylum actinobacter, 17 different strains were collected from various sources and strain collections. the strains were grown in minimal medium with supplement of standard vitamin solution and [1-13 c] glucose as carbon source. the 13 c-labeling patterns of proteinogenic amino acids were determined by gc-ms analysis. through this method, the fluxes in the central carbon metabolism during balanced growth were estimated and pathways for carbon metabolism were determined. in particular the labeling patterns of alanine and valine were of interest as they are derived from pyruvate and therefore can be used to distinguish between whether glucose is metabolized through the ed pathway or the emp pathway. nobacter, amycolatopsis balhimycina produces the glycopeptide balhimycin. the balhimycin aglycone is identical to the aglycon of vancomycin, which is a commercial glycopeptide. as a. balhimycina appears to be accessible to genetic modifications and the biosynthetic cluster responsible for balhimycin production is published, this genetically well-characterised academic strain can serve as a platform strain for production of vancomycin analogues derived through combinatorial biosynthesis. the understanding of the physiology of this microorganism is essential for the efficient accumulation of potentially commercial secondary metabolites. the bacterium is capable of growth in a fully defined minimal medium, with the production of balhimycin. the strain was grown at either nitrogen or phosphate limitation and balhimycin accumulation was followed at these conditions. flux analysis of balhimycin production by amycolatopsis balhimycina based on 13 c labelling experiments was performed. the zygomycetes blakeslea trispora and phycomyces blakesleeanus accumulate beta-carotene, ubiquinone (coenzyme q), various different sterols, and other terpenoids, all of them produced via the mevalonate pathway. these fungi are used or could be used for the industrial production of these terpenoids, edible oil, chitosan, and various organic acids. by measuring the specific radioactivity of terpenoids made from radioactive mevalonate, leucine or acetate in the presence of excess glucose in wild types and mutant strains we have concluded that these fungi have separate subcellular compartments for the production of carotene, sterols and triacylglycerols. the terpenoid moiety of ubiquinone is synthesized in the same compartment as ergosterol. these compartments contain separate pools of all their common metabolites, beginning from acetyl-coa. mevalonate carbon atoms do not find their way back to general metabolism, i.e., these fungi lack the "shunt" pathway. the compartments are regulated independently. the very large variations in carotene content caused by many environmental and genetic changes are not accompanied by variations in the ubiquinone content. the ubiquinone content increases when the cultures grow on leucine or acetate as carbon sources and is not affected by illumination. phycomyces, but not blakeslea, increases the production of ubiquinone in presence of oligomycin. lincomycin, produced by streptomyces lincolnensis, is important, clinically used antibiotic. its gene cluster consists of 27 putative open reading frames with biosynthetic or regulatory functions (lmb genes) and three resistance genes (lmra, lmrb, lmrc). the organization of transcription units was determined. the analysis of the lincomycin biosynthetic gene transcripts in various cultivation stages revealed the genes with putative regulatory functions which are transcribed in early stages of cultivation. previous analysis of biosynthetic pathways of lincomycin and functionally different anthramycin antibiotics (anthramycin, sibiromycin, tomaymycin, mazetharmycin and porothramycin) indicates that the genetic information on the lincomycin and anthramycin biosynthesis should share common elements (genes), both biosynthetic and regulatory. hybridization experiments demonstrated presence of several analogues of lmb genes involved in the biosynthesis of anthramycin produced by streptomyces refuineus and porothramycin produced by streptomyces albus. effect of various calcium salts on the erythromycin production by saccharopolyspora erythraea m. rostamza 1 , a. noohi 1 , j. hamedi 2* : 1 department of biology, faculty of science, science and research branch, islamic azad university, tehran, iran; 2 microbial biotechnology lab., department of biology, faculty of science, university of tehran, tehran, iran. e-mail: jhamedi@khayam.ut.ac.ir (j. hamedi) calcium carbonate has the positive effect on the erythromycin, however, because of its low water solubility, caused to clogging the spargers of fermenters and fouling the microfilters. in this research, various soluble calcium salts was added to the fermentation medium and their effect the growth of saccharopolyspora erythraea and erythromycin production was studied in the complex medium consisted soy bean meal, dextrin and starch as major ingredients. the fermentation conditions were 220 rpm, 8 days at 30 • c. the results obtained showed that there is no significant difference between erythromycin concentrations in the medium containing calcium lactate and calcium carbonate. however, erythromycin concentrations in the other calcium salts containing media were less than to calcium carbonate containing medium. optimum concentration of calcium lactate for erythromycin production was 10 g/l. lincosamides and its derivatives are clinically important antibiotics. comparison of gene cluster coding for lincomycin biosynthesis and newly identified cluster of genes for celesticetin biosynthesis revealed new information on functions of several genes common for both biosynthetic pathways. the celesticetin gene cluster was identified by screening the cosmid library of streptomyces caelestis with heterologous probes based on lincomycin biosynthetic genes involved in a part of biosynthesis shared by both antibiotics. sequence analysis of the cluster revealed 24 putative orfs, out of which 18 are lincomycin biosynthetic genes analogues, four are specific for celesticetin biosynthesis and one codes for resistance. the gene cluster is bounded with transposase genes on both sides. in order to clarify function of three putative regulatory genes of lincomycin biosynthesis, the insertional inactivation with the pcr targeting system in streptomyces lincolnensis was done and resulted in differently reduced production of lincomycin. larsen cmb biocentrum-dtu, technical university of denmark, 2800 kgs. lyngby, denmark we present a new algorithm called "x-hitting" for automatic identification of novel bioactive compounds based on full spectroscopic characters of highly complex mixtures of natural product. one of the most dramatic advances in recent drug discovery has been the increase in screening capacity throughput and data handling. therefore, analysis has become the bottleneck in the drug discovery process. the algorithm presented here is investigated and demonstrated on identifying potentially new bioactive compounds. in addition method is shown to have a high performance for automatic identification of known structures. these tasks are referred to as new-hitting and cross-hitting, respectively. finally, the receiver operating characteristics (roc) is introduced to the research field as an important tool for evaluating the performance of the "compound predictor". through examples it is shown, that known cross-hits are identified with high proficiency, and that the new-hitting works on finding new targets represented by analogues and structurally new compounds. a gene encoding a ␥-butyrolactone autoregulator receptor that have a common activity as dna-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation in streptomyces was cloned from a natamycin producer, streptomyces natalensis, and its function was evaluated by in vivo. pcr using the primers designed from two highly conserved regions of streptomyces autoregulator receptors gave a 102-bp band, the sequence of which revealed high similarity to the expected region of a receptor gene. by genomic southern hybridization with 102-bp insert as a probe, a 687-bp intact receptor gene (sngr) was obtained from s. natalensis. in vivo to clarify the function of sngr, a sngrdisrupted strain was constructed, and a phenotype was compared with the wild-type strain. the sngr disruptant started natamycin production 6 h earlier and showed a 4.6-fold-higher production of natamycin than the wild-type strain. in addition, sporulation was earlier and 10-fold abundance. the phenotype indicates that the autoregulator receptor protein of s. natalensis acts as a primary negative regulator of the biosynthesis of natamycin and is related to regulation of sporulation. exploring the biocatalytic potential of the novel thermostable baeyer-villiger monooxygenase: phenylacetone monooxygenase daniel e. torres pazmiño 1 , gonzalo de gonzalo 2 , gianluca ottolina 2 , giacomo carrea 2 , dick b. janssen 2 , marco w. fraaije 1 , 1 biochemical laboratory, groningen biomolecular sciences and biotechnology institute, university of groningen, nijenbaeyer-villiger monooxygenases (bvmos) represent useful biocatalytic tools as they can catalyze reactions which are difficult to achieve using chemical means. however, only a limited number of these atypical monooxygenases are available in recombinant form. using a recently described protein sequence motif, a putative bvmo was identified in the genome of the thermophilic actinomycete thermobifida fusca. the nadph-dependent and fad-containing monooxygenase is active with a wide range of aromatic and aliphatic ketones and sulfides. genetic and kinetic data suggest that phenylacetone is the physiological substrate of the enzyme. previously, it was reported that this bvmo exhibits only a moderate enantioselectivity with (r,s)-␣-methylphenylacetone. this poster we will show an overview of the biocatalytic potential of the enzyme as explored so far. interestingly the enzyme has been found to perform highly enantioselective oxidations with a range of ketones and sulfides. this again indicates that this novel thermostable oxidative biocatalyst can be a useful tool for the synthesis of chiral building blocks. the enzyme s-adenosylhomocysteine hydrolase (adohcyase) catalyzes hydrolysis of s-adenosylhomocysteine (adohcy), an inhibitor of transmethylation reactions, into adenosine (ado) and homocysteine (hcy). the catalysed reaction is reversible, and the equilibrium is strongly displaced in direction of the synthesis of adohcy when reaction occurs in vitro. nevertheless, its biotechnological application resides in the synthesis of antivirals. for it, we selected between different producing microorganisms of the enzyme, the gram positive bacterium corynebacterium glutamicum. after designing the specific oligonucleotides, the gene was expressed in escherichia coli using the expression system pet28a+ with iptg. the electrophoretic analysis under denaturing conditions, shows a clear induction and over-expression of a protein with a mw of 49 kda. on the other hand, the immobilization of this recombinant enzyme in a solid support allows to use it as a catalyst for the synthesis of adohcy. the enzyme was purified by imac thanks to the presence of n-terminal 6 × his tag end, and immobilized in eupergit c for the optimization of the production of adohcy, a product of high value. sequencing, cloning, expression, purification and characterization of a novel cytochrome p450 monooxygenase from rhodococcus rubber luo liu, rolf d. schmid, vlada b. urlacher institute of technical biochemistry, stuttgart university, allmandring 31, 70569 stuttgart, germany. e-mail: itbvur@itb.unistuttgart.de (l. liu) the cytochrome p450 monooxygenases are heme-containing proteins, which catalyze a wide range of oxidative reactions (werck-reichhart and feyereisen, 2000) . a monooxygenation activity was observed for the strain rhodococcus ruber dsm 44319. a p450-like gene fragment was amplified by pcr using degenerated primers. for identification of regions that flank this p450-like dna fragment, the method "directional genome walking using pcr" was applied (mishra et al., 2002) . the full size gene encoding a cytochrome p450 enzyme was amplified by pcr from genomic dna and cloned into the vector pet28a(+) for heterologous expression in escherichia coli bl21(de3) cells. the enzyme was purified using metal affinity chromatography. the primary protein structure suggests, that this enzyme is a natural self-sufficient fusion protein consisting of a ferredoxin, a reductase and a p450 monooxygenase. the reductase activity was determined using an exogenous electron acceptor cytochrome c. the reductase domain of this p450 monooxygenase showed a strong preference for nadph over nadh. the substrate spetrum was investigated. in the presence of nadph the p450 enzyme shows hydroxylation activity towards 7-ethoxycoumarin, naphthalene, indene, acenaphthene, toluene and fluorene. mishra, r.n., singla-pareek, s.l., nair, s., sopory, s.k., reddy, m.k., 2002 . directional genome walking using pcr. biotechniques 33, 830-834. werck-reichhart, d., feyereisen, r., 2000. cytochromes p450: a success story. genome biol. 1 (6), 3003.1-3003.9. selective production of monoglyceride consisted of conjugated linoleic acid by penicillium lipase yomi watanabe 1,2 , yoshie yamauchi-sato 3 , toshihiro nagao 1 , satoshi negishi 3 , tadamasa terai 4 , takashi kobayashi 1 , rolf d. schmid 2 , yuji shimada 1 : 1 osaka municipal technical research institute, osaka, japan; 2 stuttgart university, stuttgart, germany; 3 the nisshin oillio group, ltd, yokosuka, japan; 4 osaka institute of technology, osaka, japan conjugated linoleic acid (cla) is a group of c18 fatty acid (fa) containing two conjugated double bonds. it is expected to prevent cancer, adipositas, atherosclerosis etc, and is commertially available in the primary form of free fa, containing almost equal amounts of 9cis,11trans-and 10trans,12cis-cla. it is therefore desired to be converted to a palatable form. for this purpose, we have previously proposed two enzymatic ways to convert cla to monoglyceride, an emulsifier, with penicillium camembertii lipase; (1) sequencial esterification-glycerolysis, (2) esterification at low tem-perature. these methods, however, are time-and energy-consuming. esterification of cla with glycerol under ambient pressure by the lipase produces equal amounts of mono-and diglycerides. in contrast, it was newly found that the reaction under reduced pressure supressed the formation of diglycerides and achieved to produce 90% monoglyceride at 95% esterification. improving the thermal stability of cellobiohydrolases i (cel7a) from t. reesei by site directed evolution frits goedegebuur 1 , lydia dankmeyer 1 , peter gualfetti 2 , brad kelemen 2 , edmundo larenas 2 , paulien neefe 1 , pauline teunissen 1 , colin mitchinson 2 : 1 genencor international bv, archimedesweg 30, 2333cn leiden, the netherlands; 2 genencor international inc., 925 page mill road, palo alto, ca 94304, usa genencor international has been working to produce improved enzyme products for economic conversion of ligno-cellulosic biomass to fermentable sugars. most of this work was performed under a subcontract with the u.s. department of energy for cellulose cost reduction for biomass conversion. cellulolytic biomass conversion is performed in nature by a complex mixture of enzymes. cellobiohydrolases play a key role and all effective cellulase mixtures contain a large excess of cellobiohydrolases over endoglucanases, suggesting that it is the exoglucanase activity that is limiting. the fundamental dependence of reaction rate on temperature predicts that large increases in performance, and decreased enzyme cost, would be achieved if the enzymatic conversion could be operated at elevated temperatures. industrial strains of trichoderma reesei produce cellulases at very high levels and low cost. however, t. reesei cbh1 (hypocrea jecorina cel7a) does not have sufficient stability to survive and perform at high temperatures. this poster shows the thermal stability improvement in t. reesei cbh1 by site directed evolution. sites with increased thermal stability properties were combined and evolved in high temperature stable cbhi variants. the evaluation of lipases as biocatalysts for organic chemistry can be carried out, at laboratory scale, by using soluble enzymes for biotransformations in aqueous media. however, the industrial exploitation of such an enormous potential should require a suitable protocol for immobilization of lipases. the binding of lipases on suitable pre-existing supports should greatly improve the performance of industrial reactors allowing us a continuous use or re-use of such interesting biocatalysts. in addition, lipases, like most enzymes, are not perfect chemical catalysts. lipases may be unstable and they may not have the optimal activity nor the optimal enantio or regioselectivities. in this way, immobilization of lipases, together with its relevance for the performance of each different industrial reactor, could be also used as a tool to improve and optimize some of these parameters. that is, immobilization of lipases, far from an already solved problem, constitutes an exciting field of research in the promising area of industrial bio-organic chemistry. in this work we would like to present useful immobilization methods of several lipases. the lipase immobilised were used for enzymatic hydrolysis of peptidomimetics of structure a. type of immobilzation used can changed the enantioselectivity of biocatalyst prepared. the absolute configuration of products b and c obtained in enzymatic reactions were assigned by chemical correlation. two-component flavin-dependent monooxygenases form an interesting class of flavoenzymes. they consist of two separate proteins; a monooxygenase component, which catalyses an oxygenation reaction in the presence of reduced flavin, and a flavin reducing component, which reduces flavin (fad or fmn) using nad(p)h as an electron donor. a well-known example of this class of monooxygenases is styrene monooxygenase (otto et al., 2004) . due to the ability to form enantiopure epoxides, which are relevant building blocks for the pharmaceutical industry, styrene monooxygenases form a valuable class of enzymes for biocatalysis. while screening a metagenomic library for oxidative enzymes, an indigo-producing clone was found. sequencing the particular clone revealed an inserted fragment of environmental dna encoding a two-component monooxygenase ( many investigations over the recent years have been directed to the production of natural aroma compounds. through biotransformation and bioconversion, aroma compounds considered as "natural" can be produced starting from monoterpenes, generating high value products as rose oxide. rose oxide is found in small amounts in some essential oils such as bulgarian rose oil and geranium oil. (−)-rose oxide is an impacting flavor compound and has a small threshold: 0.5 ppb. application of agro-industrial residues in bioprocess on one hand provides alternative substrates, and on the other hand helps solving pollution problems, that might be caused by the disposal of this residue in nature. the liquid cassava waste, is originated by the pressing of cassava roots. it is considered as a "harmful" pollutant waste due to its high organic charge and presence of cyanide. on the other hand, can be considered rich in nutrients that can be used in other applications. it was found that sporulated surface cultures of penicillium sp. were able to convert citronellol into cis-and trans-rose oxides. other bioproducts were 3,7-dimethyl-5-octen-1,7diol and 3,7-dimethyl-6,7-epoxy-1-octanol. no chemical oxidation or auto-oxidation products were detected in liquid control broths. the experiments were conducted at 30 • c and 160 rpm. when the medium was cassava, the production of rose oxide, 3,7-dimethyl-5octen-1,7-diol and 3,7-dimethyl-6,7-epoxy-1-octanol were insignificant reaching trace amounts. but when the mycelium developed in cassava medium and than transferred to mineral medium (citronellol as c-source) the concentrations of rose oxide increased dramatically, reaching 70 mg/l for the cis-isomer and 30 mg/l for trans-isomer. a mechanistic mathematical model of enzymatic degradation of avicel and phosphoric acid swollen cellulose (pasc) has been proposed. the model is based on the degree of polymerization (dp) of starting substrate, and follows its decline with time, to the final end product -glucose. three enzyme classes, namely, endoglucanase (eg), cellobiohydrolase (cbh) and ␤-glucosidase (bg) are all individually incorporated in the model. the model is, additionally, taking into account cooperative action of the involved enzymes, as well as effects of enzyme inhibition by end-products, cellobiose and glucose. to be able to describe the complex process of enzymatic hydrolysis with a set of differential equations certain assumptions needed to be introduced. those assumptions represent the simplification of an up-to-date knowledge of both substrate and enzyme structure, but also enzyme mode of action. for example, one of the often asked questions is: "what is happening with shorter cellooligosacharides (dp 7 and up) laying on the surface of cellulose chain? are they being adsorbed to the core cellulose chain or partly solubilized to a hydrolysis broth?" to give answers to these questions and confirm mathematical modeling real enzyme hydrolysis data are needed. in this work, four well characterized, highly purified mono-component enzymes from humicola insolens (two eg and two cbh) and one bg from aspergillus niger were used to hydrolyze avicel and pasc. by careful choice of catalyst, some enzyme specific characteristics like presence or absence of cellulose binding domain will also be incorporated into the model. hydrolysis experiments were initially performed by distinct mono-component enzymes, to confirm the basic characteristics of each of the enzyme classes. soluble hydrolysis products (dp 1-6) were analyzed by hplc and detection of non-soluble, higher-dp polysaccharides was performed by technique of polysaccharide analysis using carbohydrate gel electrophoresis. optimisation of halogenase enzyme activity k. muffler 1 , m. retzlaff 2 , k.-h. van pée 3 , r. ulber 1 : 1 technische universität kaiserslautern, germany; 2 technische universität münchen, germany; 3 technische universität dresden, germany. e-mail: muffler@rhrk.uni-kl.de (k. muffler) halogenases provide the opportunity of a regioselective and stereospecific halogenation of organic subtrates in contrast to the class of haloperoxidases. these enzymes allow a gentle synthesis of halogenated organic molecules and are capable to halogenate in specific positions (hammer et al., 1997; keller et al., 2000) , whereas traditional organic synthesis often failures or mainly leads to byproducts (hasegawa et al., 1999) , e.g. halogenation of tryptophan in other positions than position 3. our current research is focussed on tryptophan-5-halogenases, because the 5-br/cl-tryptophan could be applied as a pharmacologically attractive precursor of serotonin. in our work we describe the optimization of an enzyme assay respectively the enzyme activity. for this purpose we use a genetic algorithm. the responsible gene of the fadh 2 dependent enzyme was cloned from streptomyces sp. origin into a pseudomonas fluorescens strain. however, the optimization procedure was done with the purified his-tagged tryptophan-5-halogenase, which was easily obtained from the crude extract of the lysed cells by application of immobilized metal affinity chromatography. the application of algorithms allows an optimization of the multidimensional search problem leading to a global optimum in the search space in contrast to the traditional used one-factor-at-a-time method. latter often failures, because this method does not reflect possible influences the parameters can have on each other. effect of organic solvent type on the enantioselectivity of candida rugosa lipase in the hydrolysis of racemic naproxen methyl ester in biphasic reaction system serpil takaç, deniz mutlu department of chemical engineering, institute of biotechnology, ankara university, 06100 tandogan, ankara, turkey hydrolysis of racemic naproxen methyl ester to produce s-naproxen was carried out in the biphasic system using isooctane, cyclohexane, hexane and toluene with candida rugosa lipase after stirring in the phosphate buffer (ph 7.5, 0.02 m) for 2 h at +4 • c and centrifuging. the hydrolyses were carried out in shaking flasks for 120 h at 200 rpm and 37 • c with the initial substrate concentration of 0.034 m. the concentrations of the enantiomers of racemic naproxen methyl ester in organic solvents and those of naproxen in buffer solution were determined with hplc. it was found that the enantiomeric excess for substrate (ee s ), enantiomeric ratio (e) and conversion (x) decreased in the following order: isooctane > cyclohexane > hexane > toluene. the enantiomeric excess for product (ee p ) was found to be the same for isooctane, cyclohexane and hexane where the lowest ee p was obtained in toluene. the highest ee s , ee p , e and x values achieved in isooctane at the residence time of 120 h were 91, 95, 130, and 49%, respectively. this study was supported by ankara university biotechnology institute (project no: 89). fusarium fujikuroi (gibberella fujikuroi mating group c) produces multiple secondary metabolites such as gibberellins and bikaverin. gibberellins are terpenoid hormones that induce growth and regulate various stages of development in plants. they have numerous applications in agriculture industry. bikaverins are polyketides that have toxicity against different organisms because they inhibit respiration. regulation of polyketide and gibberellin synthesis by nitrogen has been intensively studied in fusarium but little is known about their regulation by carbon source. our main interest is to understand the regulation of biosynthesis of these compounds in f. fujikuroi imi 58289. to investigate this regulation the organism was grown in high nitrogen medium under submerged conditions and then transferred to nitrogen-free media having various concentrations of different carbon sources. the gibberellin production was not affected significantly. on the other hand bikaverin was synthesized enormously when sucrose was used as the only carbon source. high production in sucrose required a minimal amount of the sugar, but did not change appreciably above this threshold along a wide range of concentration. the bikaverin synthesis was repressed when glucose coexisted with sucrose in the medium. the effect of the c source on the expression of key genes, cps/ks (copalyl diphosphate synthase/kaurene synthase) for gibberellin and pks4 (polyketide synthase) for bikaverin biosynthesis is currently under investigation. ment of dormancy and loss of viability in seeds with the passage of time, it lacks any systematic propagation from seeds and is typically propagated through rhizomes. this restricts large scale cultivation of this plant. in vitro propagation of plants is an effective means for rapid multiplication of species, in which conventional methods have limitations. in the pesent study we have analysed the role of various growth promoters and the effects of dark and light incubation on germination of n. alba seeds. the results indicate that in vitro propagation of n. alba from seeds can be applied as an efficient method of multiplication. the study was funded by the state planning commisson (dpt) turkey and the university of ankara vide projects no. 120640. this research was performed for developing of biological treatment process of odor gas such as mek, h 2 s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over 93% was obtained by biofilm formation. at 400 ppm of inlet odor gas concentration and 10 s of retention time, the removal efficiency was 76 and 93% in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over 97% at the operational conditions above 15 s of retention time. post soviet countries are going through the transition stage and are extremely sensitive to new technology, economics or social changes and globalization processes. that is why decision-making and system of regulation of the use of gmo's are very sensitive to number of factors. three levels of factors, the most influential to decision-making: global, regional and local; are identified at the research paper. global level depends on external policy of leaders of gmo regulations. usa and eu have the biggest influence on transition countries, though their positions completely differ. as usa was leader in inventing gmos, it is lobbing newly created biotechnological industry. in eu and other european countries lobbing of biotechnological industry was not as strong as in usa. thus, their national law is stricter. world trade organization and international agreements are also part of global level. regional level. geographical position of country is also very important because every regulation system depends a lot on regulation that is implemented in neighboring countries. countries do not exist in vacuum; they are linked territorially, politically, economically and socially with neighboring states. regulation systems of the transition countries in the eastern europe can be divided into three types: those who have no system of regulation of gmo (belarus, romania, hungary and ukraine); who approved some variety that are treated as safe to the market (poland, moldavia and georgia) and who approved all of the gmos (bulgaria, croatia and russia [before 2004]). but even if a country declares not to use gmo it is rather difficult to control import of such products, because of the lack of the testing laboratories, corruption of the state employees, agreements on intellectual property, and institutional country problems. in spite of global and regional tendency of gmos related regulation the most important part is the local level, namely the national regulation system. depending on national level we are choosing priorities at higher levels. as an example of post soviet countries ukraine is taken, as ukraine is one of the largest countries and one of the biggest exporters of agricultural products in europe. research includes the analysis of attitude to gm product, their potential risks and benefits of three categories that influence decision-making the most: farmers, gm experts and non-governmental organizations. recombinant microorganism development for extracellular benzaldehyde lyase production hande kaya 1 , pınar ç alık 1 , tunçer h. ozdamar 2 : 1 iblab, department of chemical engineering, metu, 06531 ankara, turkey; 2 bre laboratory, department of chemical engng, ankara university, 06100 ankara, turkey. e-mail: e119497@metu.edu.tr (h. kaya) in this study, the extracellular production of the benzaldehyde lyase (bal, ec 4.1.2.38) that catalyses the synthesis the enantiopure 2-hydroxy ketones for drug syntheses, by bacillus sp., was aimed. for this purpose, the signal dna sequence of an extracellular bacillus enzyme, i.e., serine alkaline protease, was fused in front of the bal gene (accession number ax349268) from pseudomonas fluorescens biovar i, using pcr-based gene splicing by overlap extension (soe) method. b. licheniformis (dsm 1969) chromosomal dna was used as sap gene (accession number x03341) template for the synthesis of sap signal sequence. bal gene was amplified by using the plasmid carrying bal gene, puc18::bal. thereafter, the signal peptide of sap with its own promoter was fused in front of the bal gene by soe method. the hybrid gene first cloned into puc19 plasmid, thereafter sub-cloned into pbr373, pmk4 and phv1431 shuttle vectors. the escherichia coli-bacillus plasmids carrying the hybrid gene pre(subc)-bal was transferred into bacillus subtilis npr− apr−, and bacillus licheniformis. the influence of the host bacillus species on bal production on a defined medium with glucose was investigated in bioreactor systems. for each of the recombinant (r-) bacillus species, effects of initial glucose concentration on cell growth and bal production were investigated; and, physiological differences and similarities between the wild-type and r-bacillus species are discussed. thereafter, the benzaldehyde lyase production capacities of recombinant e. coli and b. subtilis are compared in terms of cell concentration and bal volumetric and specific activities. for the comparison bal gene was cloned into prseta vector which is under the control of strong t7 promoter and expressed in e. coli bl21 (de3) plyss strain. the variations in by-product distributions with each recombinant organism and yields are also discussed. phosphoketolases (ec 4.1.2.9) are thiamine diphosphate (thdp)-dependent enzymes, that play a crucial role in the pentose phosphate pathway (ppp) of heterofermentative and facultative homofermentative lactic acid bacteria, and of the d-fructose 6phosphate shunt of bifidobacteria. reports affirm that cellulomonas flavigena can use the ppp when is cultured under anaerobic conditions. a genomic library of c. flavigena constructed in -zap express vector was screened. four positive clones were isolated, in vivo excised and the resulting pbk-cmv phagemids, each containing a 4.0 kb insert, were characterized by restriction analysis and dna sequencing. the open reading frame (orf) of the dxylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase gene, xpkl, was located from nucleotide 54 to 2466. the xpkl orf encoded a 804 amino acids-residue polypeptide (xpkl) with a calculated molecular mass of 89,000 da, a value coincident with that estimated by comparative sds-page (about 90,000 da). a putative ribosome binding site (gggagc) is present 11-5 nucleotide upstream of the translational start of the xpkl polypeptide. the c. flavigena xpkl polypeptide sequence was 66% identical to dxylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase from bifidobacterium sp., bifidobacterium gallinarum, gloeobacter violaceus and bifidobacterium adolescentis. this analysis also revealed highly conserved regions. lactococcus lactis is a main diacetyl-producing bacteria by citrate metabolism in dairy products. the transport of citrate in these bacteria is dependent on citrate permease that is encoded by citp gene. previous studies of the citqrp operon in escherichia coli mutants showed that citp message is considerably stabilized in rnase iii mutant. so, in the context of the citrate metabolism research, the characterization of the lactococcal rnase iii enzyme is very important for the dairy industry. rnase iii is an endoribonuclease which has an important role in rrna processing and control of gene expression. with the aim of studying lactococcal rnase iii we have cloned the rnc gene from l. lactis ssp lactis il1403 in the broad host range pls1rgfp vector. this plasmid includes the gfp gene, encoding the green fluorescent protein (gfp), cloned under the control of the pm promoter, that is inducible by maltose. maltose induction of the lactococcal rnc expression showed a 5-fold increase of rnc transcription from this plasmid. activity assays for lactococcal rnase iii were standardized using crude extracts and a substrate specific for b. subtillis rnase iii. the results showed that this substrate was specifically cleaved by lactococcal rnase iii and its activity induced by maltose. lac-rnc was cloned in pet15 vector and the corresponding six-histidine-tagged rnase iii protein was overproduced in e. coli bl21 (de3) strain by iptg induction. the protein was purified by affinity chromatography using hplc system and was shown to be active by in vitro activity assays using the lac-rnase iii specific substrate mentioned above. we have also cloned lactococcal rnc gene and studied its expression in an e. coli rnc deletion mutant ( rnc). complementation assays performed in e. coli demonstrate that the lactococcal rnase iii (lac-rnase iii) is able to process rrnas and to regulate the levels of polynucleotide phosphorylase (pnpase). these results demonstrate that the lactococcal enzyme is able to substitute the ec-rnase iii not only in the rrna processing, but also in the processing of mrnas. the amount of lactococcal rnc transcript in an e. coli rnc strain was 3.3-fold higher than in the wild type strain, suggesting that the e. coli rnase iii triggers the degradation of the heterologous rnc mrnas. the results obtained have shown that lac-rnase iii is an interesting enzyme for biotechnological purposes. objectives: the pharmaceutical and food industry has an increasing demand for selectively glycolized active agents. in our application isomaltose can be synthesized by immobilized dextransucrase, which transfers a glycosyl residue from sucrose (substrate) to glucose (acceptor). as the reaction proceeds, isomaltose can act as an acceptor and is converted into undesired follow-up products called isomalto-oligosaccharides, imos. we investigate on two approaches to avoid imo formation, selective adsorption of isomaltose (ergezinger et al., 2005) and the co-entrapment of dextranase adsorbate, which breaks imos down to isomaltose. results and conclusions: the first part of our research concerns the adsorption of dextranase on bentonite, which complies with langmuir model. at complete saturation (0.8 g g −1 ) our immobilisate exhibits an activity of 16,000 u g −1 . a kinetic analysis does not reveal significant differences between the adsorbed and free form of enzyme (k m,bentonit : 14.1 ± 0.7 m versus k m,free : 13.0 ± 0.7 m). thus, bentonite displays a high binding capacity paired with favorable kinetic properties. beyond that we investigate the activity of dextransucrase in co-immobilisates, which is reduced during coimmobilization due to interactions with the adsorbate. among various co-immobilisates, the one containing dextranase bound to preblotted bentonite imparts the highest activity (40% as compared to control: immob. dextransucrase). the molar yield coefficient of coimmobilisates y isomaltose/sucrose surpasses coefficient of control by 13%. further on we will characterize mass transfer of dextranase substrate into alginate matrix as well as bentonite-dextransucrase interactions. and kefir. a second approach is to use yeast as a production organism to produce natural folates for fortification. here we investigate and discuss the folate content in skq2n, a diploid strain of saccharomyces cerevisiae, when cultured in different media and at different stages of growth. the aim is to gain a basal knowledge of the folate production profile, forms of folate produced and degree of leakage to the surrounding medium, in relation to the culturing medium and physiological state of the cells. danisco innovation, danisco a/s, langebrogade 1, po box 17, dk 1001 copenhagen k, denmark we at danisco a/s (copenhagen, denmark) have revealed a new starch degrading pathway by the discovering several new enzymes and metabolites in fungi and algae. these new enzymes include glucan lyases, dehydratases and tautomerases, which proved to be useful in biocatalysis. these new metabolites proved to be useful as both antioxidants and antimicrobials for food and non-food applications. this pathway is named as anhydrofructose pathway of starch and glycogen degradation. this technology is referred to as the anhydrofructose technology. diet is evolving from nourishing populations via providing essential nutrients to improving health of individuals through nutrition. modern nutritional research focuses on health promotion and disease prevention, on protection against toxicity and stress, and on performance improvement. as a consequence of these ambitious objectives, the disciplines "nutrigenetics" and "nutrigenomics" have evolved. nutrigenetics asks the question how individual genetic disposition, manifesting as single-nucleotide polymorphisms (snps), copy-number polymorphisms (cnps) and epigenetic phenomena, affects susceptibility to diet. nutrigenomics addresses the inverse relationship, i.e. how diet influences gene transcription, protein expression and metabolism. the mid-term objective of nutrigenomics is integrating genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (global protein analysis) and metabolomics (metabolite profiling) to define a "healthy" phenotype. the long-term deliverable of nutrigenomics is personalised nutrition for maintenance of individual health and prevention of disease. the major challenges for -omics in nutrition and health still lie ahead of us, some of which apply to -omic disciplines in general while others are specific for -omic discovery in the food context: (i) the integration of gene-and protein expression profiles with metabolic fingerprints is still in its infancy as we need to understand how to (a) select relevant sub-sets of information to be merged, and (b) resolve the issue of the different time-scales, at which transcripts, proteins and metabolites appear and act; (ii) the definition of health and comfort is less of a clear-cut case than the one of disease; (iii) -omics in nutrition must be particularly sensitive: it has to reveal rather many subtle than a few abundant signals to detect early deviations from normality; (iv) in the food context, health cannot be uncoupled from pleasure, that is, food preference and nutritional status are interconnected. transcriptomics serves to put proteomic and metabolomic markers into a larger biological perspective and is suitable for a first "round of discovery" in regulatory networks. metabolomics, the comprehensive analysis of metabolites, is an excellent diagnostic tool for consumer classification. the great asset of this platform is the quantitative, non-invasive analysis of easily accessible human body fluids like urine, blood and saliva. this feature also holds true to some extent for proteomics, with the constraint that proteomics is more complex in terms of absolute number, chemical properties and dynamic range of compounds present. proteomics in the context of nutrition and health has the potential to (a) deliver biomarkers for health and comfort, (b) reveal early indicators for disease disposition, (c) assist in differentiating dietary responders from non-responders, and, last but not least, (d) discover bioactive, beneficial food components. independent of the context of application, proteomics represents the only platform that delivers not only markers for disposition or condition but also targets of intervention: the only way to intervene in a biological condition and to modulate its outcome is interfering with the proteins involved. it is evident that not only comprehensive analyses with one discovery platform (lateral integration of information) are required but also vertical integration between different -omic levels are indispensable for a deeper understanding of disposition, health, environment and diet (desiere, 2004) . a major "vertical integration issue", to date unresolved, is given by different timescales of transcript production, protein expression and metabolite generation (nicholson et al., 2004) . the transcript machinery usually responds fast to an external stimulus (seconds to minutes), the proteins may be expressed within minutes to hours (and have a halflife from minutes to even months) and metabolites vary significantly during the day and depend on latest dietary input. this means that data, which seem to correlate qualitatively (e.g. reflecting the same pathway), may not necessarily be related time-wise. rather, they may represent different responses at different time points and, possibly, to different stimuli. comprehensive -omic analyses is an essential building block of "systems biology", which can be defined as follows (clish et al., 2004) : systems biology is the comprehensive analysis of the dynamic functioning of a biological system (cell, organ, organism or even ecosystem) at gene, protein and metabolite (or higher organizational) level, achieved by comparison of two defined biological states of this system, typically before and after perturbation. while a comprehensive list of components (genes, proteins, metabolites) of a given biological system is a pre-requisite for this kind of research, the main reasoning for the "system view" is that only information on the interactions between the components gives clues to function of the entire network. a systems biology approach has recently demonstrated the power of proteomics to dissect immunity and inflammation. toll-like receptor recognition and signalling was elucidated and showed, how bacterial "barcodes" are read and interpreted in order to trigger an adapted immune response (aderem and smith, 2004) . in order to address some of the challenging objectives of -omics-driven nutritional research, we have addressed (a) the effect of early antibiotic administration on the maturation of intestinal tissues, (b) protein discovery in human milk, (c) the effects of polyunsaturated fatty acids on gene expression and lipid profile in the liver, and (d) biomarkers for intestinal stress. (a) antibiotics and gut maturation: the effects of early administration of antibiotics on intestinal maturation were assessed at the gene expression level in a rat model. (b) human milk: rapid enrichment and iterative, consolidated identification of immunologically relevant milk proteins was achieved through the employment of restricted-access media and a tailored proteomic strategy (labéta et al., 2000; lebouder et al., 2003; panchaud et al., 2005) . (c) fatty acids and liver transcriptome/lipidome: epidemiological studies have correlated higher intakes of poly-unsaturated fatty acids (pufas) with lower incidence of chronic metabolic disease. the molecular mechanisms regulated by pufa consumption were examined assaying the liver transcriptome and lipid metabolome of mice fed a control and a pufa-enriched diet (mutch et al., 2005) . (d) gut stress markers: as a first step, we catalogued protein expression along the jejunum, ileum and colon of the rat intestine and found gut segment-specific proteins (marvin-guy et al., 2005) . the innovative combination of a neonatal separation model with proteomic analysis allowed us to study, whether early life psychological stress may impact the adult gut neuromuscular protein expression and the approach revealed specific protein biomarkers. omics for engineering lactic acid bacteria willem m. de vos wageningen center for food sciences, wageningen university, the netherlands. e-mail: willem.devos@wur.nl. url: http://www.wcfs.nl/ lactic acid bacteria (lab) are high at-rich gram-positive bacteria that have a well-established record in industrial food fermentations where they contribute to conservation, flavour and texture. in addition, several lab are used as food-grade hosts for the production of enzymes, peptides or metabolites. finally, lab are exploited in functional foods that contribute to the health and well-being of the consumer. a variety of metabolic engineering approaches have allowed for the improvement of many attributes of lab. these approaches have been facilitated by the possibility of uncoupling of growth and metabolite production in lab, the wealth of genetic tools that allow modulation of gene expression in a dynamic range, and the determination of several complete lab genomes (de vos et al., 2005) . we have developed lactobacillus plantarum as a paradigm for lab engineering by experimental and modelling approaches, the application of functional and comparative genomics, and the implementation of other post-genomics avenues (kleerebezem et al., 2003; smid et al., 2005; de vos et al., 2004) . examples of optimizing the production of vitamins and other cofactors, the impact of these engineering approaches on the global transcription and metabolite profiles, and determining the l. plantarum activity in the human host will be discussed. solanum tuberosum (potato) is the fourth major crop worldwide and used for food, feed and biotechnological applications. to fully realize the biosynthetic potential for production of starch, protein and metabolites, we conducted an extensive quantitative profiling of the expressed genes of mature potato tuber. a total of 58,322 sage (serial analysis of gene expression) tags of 19 nt representing 22,235 different tags were analyzed. the 695 tags seen 10 or more times were assigned a tentative function by comparison to homologous genes. contrary to the transcript profile of rice seedlings (gibbings et al., 2003) the storage organ of potato is not dominated by transcripts encoding storage proteins. transcripts for four types of protease inhibitors, a metallothionein and a lipoxygenase were more prominent than patatin isoforms. the lactic acid bacterium lactococcus lactis is used extensively in the production of fermented milk products. during cheese production the bacterium experiences many changes in its immediate environment, as a result of its own reactions. the most severe change is the accumulation of lactic acid, which changes the ph of the medium until growth is totally inhibited. we have focused upon a survey of these dairy related stress responses, as a means of constructing more robust strains. when l. lactis starter cultures are produced in rich media, they will experience an initial period with purine limitation after being added to the milk substrate, a stress condition that in several studies have been found to induce cross resistance towards a number of other stresses. we have analyzed both general purine nucleotide (atp and gtp) and specific gtp limitation in chemi-cally defined medium, using both proteomics and transcriptomics. the differential expression analyses were performed with a custom designed dna microarray of pcr amplified probes. the two stress conditions resulted in very different stress responses, both at the transcriptomic and proteomics level. from a new study on the temporal expression pattern of l. lactis during growth in milk, we present preliminary data showing differential expression of genes and proteins of the purine stress stimulon as well as other stress stimulons. cell physiology of the yeast saccharomyces cerevisiae glucose repression mutants ∆snf1, ∆snf4 and ∆snf1∆snf4 was studied in batch and glucose limited chemostat cultivations. detailed physiological studies were performed on cells grown in batch using glucose, galactose, or glucose-galactose mixture as a carbon source. during growth on glucose-galactose mixtures it was shown that after glucose was consumed, galactose consumption remained repressed for about 15 h in ∆snf1 or ∆snf4 mutants, and for more then 40 h in ∆snf1∆snf4 mutant, whereas it only lasted 6 h in wild-type cells. the global transcriptional response in the glucose repression mutants was studied using chemostat cultures. s. cerevisiae wild type and the mutants were grown in glucose limited aerobic chemostats at a dilution rate of 0.1 h −1 . biological triplicates were performed for each strain. to identify transcriptional responses of the glucose repression mutants, statistical tests, clustering method and a model-driven analysis method were used. the global transcription data analysis experiments showed that genes involved in hexose transport, carbohydrates metabolism, respiration, and signal transduction were differently expressed in ∆snf1 and ∆snf4 mutants comparing to wild type cells. combination of gene expression data and gene-scale metabolic model indicated changes in the metabolic sub-networks among studied glucose repression mutants. genomics technologies have recently been introduced into food and nutrition science for identifying targets of molecular actions of nutrients as well as non-nutrient components of foods. changes in the transcriptome, proteome and metabolome have been determined for assessing the molecular actions of zinc as an essential micronutrient and of flavonoids in processes such as colon carcinogenesis and atherosclerosis. zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. zinc deficiency effects in human cells and an animal model (rats) were analyzed by microarrays and showed that a low intracellular zinc concentration caused major alterations in the steady-state mrna levels of several hundred target genes-dependent on the tissues studied including liver, brain, muscle, intestine and kidney. proteome analysis from the same samples by 2d-page followed by peptide mass fingerprinting via maldi-tof-ms identified similarly a large set of proteins with altered expression level but allowed a common theme of action to be identified. although pleiotropic in first view, the obtained pattern of zinc-affected genes/proteins may represent a reference for defining the zinc regulon in mammalian cells. flavonoids occurring in large number in plant species are considered protective agents in a variety of processes including inflammation and cancer development. we have studied the effects of around 80 selected flavonoids in a screening program and identified for compounds such as flavon, genistein or quercetin by genomics technologies their putative mode of action in colon cancer models and endothelial cells. as part of a collaborative effort employing human endothelial cells and blood mononuclear cells from a human intervention trial with soy isoflavones (genistein/daidzein) the effects of the flavonoids on the stress-response to oxidized ldl and homocystein was analyzed. a set of markers of anti-inflammatory and anti-apoptotic activity was identified for genistein and daidzein and cell biological studies confirmed that both compounds prevented programmed cells death in stressed endothelial cells. in the food processing industry, unwanted presence of extremely heat-resistant bacterial endospores creates major problems due to their capability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. screening of spoilage isolates using genomic typing techniques to visualise putative genome-based biomarkers allowed us to classify strains according to the degree of thermal resistance of their spores. in addition, we showed that sporulation in the presence of ingredients rich in calcium ions promotes thermal resistance of developing spores and correlates with the expression of specific (marker) genes (see oomes and brul, 2004) . finally, the molecular program that forms the basis of spore germination has been assessed using genome-wide expression analysis. noticeably genes involved in dna-repair were transiently expressed in germinating wild-type spores. also, surprisingly, it was found that spores contain significant levels of ribosomal and messenger rnas. degradation of these rna molecules upon spore thermal injury was found to be characteristic for their thermal resistance and predictive for their subsequent outgrowth behaviour. this finding is currently being patented. the information on spore presence, predictions of their thermal resistance and process survival chances, is used to structure a process management system to facilitate optimal food quality assurance and allow for real time analysis in case of the need for quality control. the information on spore presence, predictions of their thermal resistance and process survival chances is now being integrated. this is used to formulate the conditions for a process management system with state of the art food production quality assurance, which allows for real time analysis in case of the need for quality control. the interplay of the pectinase spectrum of aspergillus niger as revealed by dna microarray studies elena martens, jac benen, johan van den berg, peter schaap fungal genomics group, laboratory of microbiology, wageningen university, dreijenlaan 2, 6703 ha wageningen, the netherlands. e-mail: elena.martens@wur.nl (e. martens) the saprobic fungus aspergillus niger is an efficient producer of extracellular enzymes many of which show carbohydrate modifying activities. these enzymes have gras status and therefore are widely used in the food and feed industry. after determination of the genomic sequence of a. niger by dsm, it was estimated that only a fraction of the potential of secreted enzymes is currently characterised. database mining using the proprietary genome sequence has resulted in the identification of more then 80 genes encoding enzymes involved in the depolymerisation of the back bone and the site chains of the complex polysaccharide pectin. additional enzymatic activities required to remove methyl and acetyl esters, present in pectin were also observed. by using dna microarrays we have sought to gain insight into the complex regulation of all the genes involved in pectin degradation. a. niger was cultivated on sugar beet pectin and on the monomeric constituents of pectin, viz. galacturonic acid, rhamnose and xylose. subsequently the corresponding transcriptomes were analysed. we will report on our findings concerning the regulation of the expression of the genes involved in the degradation of pectin and its main constituent-galacturonic acid and the consequences for the interplay of the encoded (novel) enzymes. since reactive oxygen species (ros) are formed in all living organisms a wide range of antioxidative enzyme systems are present to keep the system in balance. when an animal is slaughtered the cellular anti-oxidative capability is reduced, resulting in an accumulation of ros followed by an increased oxidation of dna, lipids and proteins. generally lipid oxidation is a well-known problem, causing increased rancidity during prolonged storage, of especially fatty fish. the implications of protein oxidation are, however, more unclear also in respect to quality decay of fish. protein oxidation causes a wide variety of amino acids modifications, where of the most studied is carbonylation of proline, argenine, lysine or threonine. these carbonyl groups can be labelled with 2,4-dinitro-phenylhydrazine. combining two-dimensional gel electrophoresis with immunoblotting enables the detection of carbonyl groups for each single protein. the results presented here, reveal that both during frozen storage and tainting of rainbow trout protein oxidation/carbonylation increases, furthermore there is an increase in oxidation/carbonylation for distinct proteins. anne-marie neeteson european forum of farm animal breeders, benedendorpsweg 98, 6862 wl oosterbeek, the netherlands society is concerned about food, animal welfare, food safety, new technologies, scientists and industry. these elements are all present in genomics for farm animals. therefore, it is important to build awareness in scientists and industry, start a dialogue with stakeholders and society, and to be transparent in a pro-active way. this paper will address the issues at stake for scientists and industry, when it comes to genomics and animal health. it will combine the results of imperical, ethical and sociological efforts in three eu funded projects. (c) the proper use of genomics in relation to infectious diseases in production animals, and the role of the scientist in the development in new technologies in this field, are being addressed in european animal disease genomics network of excellence (ead-gene, http://www.eadgene.org/). some observations are that: (1) genomics does not concern changing the gene. however, acceptability of any discovery dealing with living beings and edible products, is not obvious just like that! animals have a symbolic and emotional load. (2) genes are still related to eugenics, in the mind of people. genes as such cause reluctance, but it is seen as positive if the use of medication can be reduced, and if animals will be better resistant to disease. (3) consumers are in favour of consumer education, compulsory labelling and the imposition of minimum standards. the inclination to pay more for foods produced according to desired standards relates closely to income level. (4) animal welfare is the major issue citizens mention as a concern. the focus of breeding organisations on productivity should be counterbalanced by serious attention to the animal's needs in order to avoid unnecessary negative impact on the welfare of the animals. (5) when technical specialists and lay people communicate, they tend to use different languages: they use the same words, but with rather different interpretations. so transparency of breeding practices and clear definitions of terminology will be essential for effective communication among all stakeholders. (6) food safety and human health are the major concern for most people, when it comes to making a choice. during the latest decades research within the field of animal genomics has in general been following the same strategies as those used within the field of human genomics, although with much less resources. the porcine genome has been characterized intensively through the development of linkage maps, comparative maps and physical maps. until a few years ago it had not been anticipated that it would be possible to embark on whole genome sequencing of animals genomes. however, because of technological developments and much lower costs for sequencing, several animal genomes have now been assembled/are on the way to being assembled. the initial step towards sequencing the porcine genome was taken by the sino-danish pig genome project. the efforts within this project have now generated approximately 3.84 million genomic shotgun sequences and 700.000 expressed sequence tags (ests). the shotgun sequences have been included in a three-species alignment to make an initial evolutionary analysis. the results show that pig is much closer to human than mouse is. the ests represent 5 -end sequences from a total of 98 non-normalized cdna libraries. based on assembly and annotation of the ests the structure of the porcine transcriptome has been analysed. the relevance of assembling the porcine genomic sequence is justified both from the perspective of sustainable animal breeding and from the fact that the porcine model is an important research platform because of the anatomical, physiological, biochemical and metabolically similarities with man. examples of functional genomic studies both aimed at sustainable animal breeding and aimed at exploiting the pig as a model for medical studies will be discussed. genomics refers to global, systematic and high throughput approaches that allow collecting large amount of data and thus offer new possibilities for analysis and understanding biological processes. we will present some new knowledge related to reproduction in farm animals resulting from three different strategies. (1) functional analysis of gene and protein expression: the transcriptome and the proteome analysis allowed to identify new genes and proteins whose expression is associated with processes of ovarian follicular growth and atresia as well as oocyte maturation in bovine and porcine species, maturation of spermatozoa in the different compartments of epididymis. farm animals produce food as cost effectively as possible, however this may have negative side effects for their health and welfare. trade off processes between production on one hand and reproduction and health on the other hand play a crucial role. the principles of selective breeding for the best of naturally occurring variation has proven to be able to balance an increased level of production and quality of life for the animal. every year, the economic value of the genetic gain achieved by the breeders and carried over to the producers is 1.5% of the economic value of eu farm animal production. consequently, a conservative estimate of the gain from animal breeding is, every year d 1.2 billion in europe. recent developments, such as the sequencing of the genomes of the human, chicken and cow, together with high throughput laboratory techniques, means that there are new opportunities to enhance quality of life. the goal of this paper is to give an overview of the options offered by genomics for enhanced quality of life with focus on identifying relevant gene variants and technologies for large scale tracking and tracing. selective breeding for the best of naturally occurring variation remains the same as in traditional systems, but by pinpointing the relevant gene variants along genomics it is possible to identify directly the animals best selected for high production without comprising health and welfare. the combination of full genome sequences, software tools, study of functional physiological processes cost-effective high-throughput snp genotyping and comparative mapping have the (proven) potential to identify relevant gene variants, e.g. pork color, boar taint, general disease resistance. functional mutations have direct option of application in breeding programs. unfortunately this is not the case for genetic markers due to cost of genotyping and inconsistent phenotypic effects. new technologies for snp genotyping are cost effective and enable large scale genotyping (1.000 of animals/day). a selection of the best technology and strategic use of these opportunities enable tracking and tracing. the application of this technology offers new opportunities for quality of life, both for animal and humans. background: studies have shown that prebiotic and probiotic consumption alters the gastrointestinal flora, modulates the immune system, inhibits genotoxicity and has a protective effect on colon carcinogenesis. however, the effect of synbiotic consumption on these parameters in subjects at risk of colon cancer has not until now been investigated. aim: to determine if a synbiotic (prebiotic and probiotics together) modulates cancer risk biomarkers in human subjects at risk of colon cancer. methods: a 12-week randomised, double blind, placebo controlled, ethically approved trial of a food supplement containing lactobacillus gg, bifidobacterium bb-12 and raftilose synergy 1 (prebiotic) was performed in 37 colon cancer subjects who had undergone 'curative resection'. faecal and blood samples were obtained before (t1, week 0) midway through (t2, 6 weeks) and following intervention (t3, 12 weeks). rectal biopsies were obtained at t1 and t3. the effect of synbiotic consumption on the faecal flora was assessed using standard plate count techniques. genotoxic damage was measured in single cells derived from biopsies using the comet assay. fw was prepared by diluting faeces 1:1 in dmem, ultracentrifugation and sterile filtration. the genotoxic (comet assay) and cytotoxic potential (almar blue assay) of fw was determined. peripheral blood mononuclear cells were isolated from blood and cytokine production in vitro assayed by elisa. natural killer cell cytotoxic activity and the phagocytic and respiratory burst activity of monocytes and granulocytes in whole blood were determined by flow cytometry. results: in the synbiotic group faecal numbers of bifidobacteria significantly increased (p < 0.001) and lactobacilli increased although not significantly (p = 0.0674) while coliforms decreased (p < 0.05). enterococci, clostridium perfringens and bacteroides were unaffected. in the placebo group bifidobacteria decreased (p < 0.001), the other bacterial groups were unaffected. in biopsies genotoxic damage was increased in the placebo group at t3 versus t1 (p = 0.0301) but was unchanged in the synbiotic group. the genotoxic and cytotoxic potential of fw was unaltered. synbiotic consumption significantly increased (p < 0.05), ifn-␥ production by pbmcs but il-2, il-10, il-12, and tnf-␣ production was unaffected. natural killer cell, phagocytic and respiratory burst activities were unaltered. conclusion: synbiotic consumption did not have a strong immunomodulatory effect on the systemic immune system in this study, nor did it influence the genotoxic and cytotoxic potential of fw. however, synbiotic consumption altered the composition of the gut flora to a more beneficial composition as well as protecting against genotoxic damage in vivo, suggesting a protective effect of synbiotics against colon carcinogenesis. emmaårsköld, malin svensson, halfdan grage, peter rådström, ed w.j. van niel applied microbiology, lund institute of technology, lund university, p.o. box 124, sweden lactobacillus reuteri is used today in a variety of dairy products as a probiotic bacterium. several lactic acid bacteria have the ability to produce different kinds of exopolysaccharides (eps), which have the potential to be used as an alternative biothickener. however, the yield of eps is too low to be profitable in the food industry. to optimise the environmental conditions for eps formation a l. reuteri strain was chosen for a factorial design study. the factors used in this experiment were temperature (30-43 • c), ph (4.5-6.5) and sucrose concentration (50-150 g/l); for each factor three different values were chosen. the strain was grown in batch mode using a semi-defined medium at constant ph and sucrose as the carbon source. the results obtained with 27 fermentations revealed that the highest eps formation was found at 37 • c, ph 4.5 and 100 g/l of sucrose. sucrose did not further affect the eps formation above a concentration of 100 g/l. temperature and ph were significant for the eps formation, but only temperature was significant for growth. a central composite design study was chosen for further optimization of the ph and sucrose concentration for maximum eps formation. also the gene expression of the sucrase enzyme responsible for eps formation was investigated using qrt-pcr. the data were used to develop a model for growth and eps formation. dendritic cells (dc) play a pivotal immune regulatory role in the th1, th2 and treg cell balance. dc are present in the gut mucosa and may thus be target for modulation by gut microbes. here, we screened a large panel of human gut-derived lactobacillus and bifidobacterium spp. for dc polarizing capacity: bone marrow-derived murine dc were exposed to lethally irradiated bacteria and cytokine and dc surface markers were analyzed. substantial differences were found among strains in their capacity to induce proinflammatory cytokines, while the differences for anti-inflammatory cytokines were less pronounced. bifidobacteria were weak il-12, il-23 and tnf-␣inducers, while both strong and weak cytokine-inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12, il-23 and tnf-␣ production induced by otherwise strong cytokine-inducing strains, while il-10 production remained unaffected. those lactobacilli with greatest capacity to induce il-12 were also most effective in up-regulating surface markers. surface marker up-regulation was however reduced in the presence of weak il-12-inducing strains. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. cell surface-associated glycolytic enzymes from lactobacillus plantarum 299v mediate adhesion to human epithelial cells and extracellular matrix proteins s.m. madsen, j. glenting, a. vrang, p. ravn, h.k. riemann, h. israelsen, m.r. nørrelykke, a.m. hansen, m. antonsson, s. ahrné, h.c. beck bioneer a/s, hørsholm dk-2970, denmark among the main selection criteria of lactic acid bacteria for probiotic use, the ability to adhere to intestinal epithelial cells, mucus, or extracellular matrix proteins is considered important. using a proteom based approach we identified a group of novel surface proteins that are non-covalently bound to the cell wall of the probi-otic bacterium lactobacillus plantarum 299v. surface proteins were extracted and analysed by gel electrophoreses followed by mass spectrometry analysis. the surface proteins included glycolytic enzymes like, e.g glyceraldehyde 3-phosphate dehydrogenase, which usually is a typical intracellular enzyme. a collection of lactobacillus species was screened and the phenomenon of surface-associated glycolytic enzymes was found in many of the analyzed species. this is to our knowledge the first example of surface-associated glycolytic enzymes in probiotic bacteria. however, in pathogenic bacteria these enzymes are well known and their surface localization is involved in adhesion to human epithelial cells and invasion. we suspect that the presence of these enzymes on the surface of probiotic bacteria could prevent adhesion of pathogenic bacteria and possibly also involve other probiotic activities such as immune modulation. binding studies showed that the surface-associated glycolytic enzymes of lb. plantarum 229v were able to bind to caco-2 intestinal cells and extracellular matrix proteins like fibronectin. scientific and industrial interest on exopolysaccharides (eps) synthesized by microorganisms and on their chemico-physical properties has been quickly growing in the last years. many strains of lactobacilli produce eps allowing them to adhere to human mucosae and therefore to have probiotic effects such as the stimulation of immune response and even antitumoral activity of these molecules has been claimed. futhermore prebiotic actions of eps beneficially affect the human host health improving the properties of indigenous microflora. in this research we have studied two novel interesting lactobacilli strains: lactobacillus plantarum dsmz 12028 and a particular human isolated strain of lactobacillus crispatus that have probiotic potentialities and are good eps and l(+)-lactic acid producers. the aim of this work has been to characterize these strains and their metabolites (eps, organic acid and bacteriocins), to state them as probiotics and to study their adhering ability on human cells. we have studied the physiology of l. plantarum and l. crispatus in shaking flasks as well as their optimal fermentation conditions to obtain high cell density cultures suitable for use as starters in the food industry and eventually for probiotic preparations. fermentation experiments have been performed in a bioreactor equipped with microfiltration (mf) modules using a semidefined medium and various carbohydrates in different culture conditions (aeration, temperature). the kinetic of eps production has been followed and according to results both strains have shown a growth-related production ranging from 200 to 400 mg/l. in vitro studies concerning the ability of these strains to adhere to human mucosae are in progress as well as the structural characterization of the exopolisaccharides. previous studies have shown that compression coating improves the storage stability of freeze-dried lactobacillus acidophilus, although this stability is related to the degree of cell injury, which in turn is related to the compression pressure used. compression coating has also been found to improve the survival of freeze dried l. acidophilus during exposure to simulated gastric fluid (sgf). the aim of the present work is to create a compression coated l. acidophilus formulation, with targeted release at the terminal ileum and beginning of the colon in the human gastrointestinal tract. dissolution studies were performed using a phosphate buffer with a ph of 2 and 6.8, to simulate gastric fluid and intestinal fluid (sif), respectively. cell viability was monitored using multi-parameter flow cytometry (mpfc), together with traditional cfu/ml counts. mpfc was used to identify live, dead and stressed cell populations, using the fluorescent stains propidium iodide (pi), 3,3 -dihexylocarbocyanine iodide (dioc 6 (3)) and to-pro-3. results show that an enteric coating material, eudragit l100-55, is both suitable for compression coating, and enhancing the survival of cells when exposed to sif. pectin usp 100 has also been shown to promote targeted release of the cells. the opium poppy papaver somniferum contains more than 80 tetrahydrobenzylisoquinoline-derived alkaloids. it is the source of the narcotic analgesics codeine and morphine, which accumulate in specialized internal secretory cells called laticifers. in the aerial parts of the plant, the laticifer cells are anastomosed, forming an articulated network. laticifers are found associated with the vascular bundle in all plant parts. the morphinan alkaloids morphine, codeine and thebaine are found both in roots and in aerial plant parts and specifically accumulate in vesicles within laticifers. the benzo[c]phenanthridine alkaloids sanguinarine and 10-hydroxysanguinarine are found in root tissue. the syntheses of sanguinarine and of the tetrahydrobenzylisoquinoline latex alkaloid laudanine are completely understood at the enzyme level. nearly all enzymes of morphine biosynthesis have also been described. in more recent years, cdnas encoding enzymes of alkaloid biosynthesis in p. somniferum have been isolated and characterized. the cell-specific localization of several of the enzymes of morphine, sanguinarine and laudanine biosynthesis has also been described. with knowledge of many of the gene of alkaloid formation and their sites of expression, the metabolic engineering of p. somniferum for tailored alkaloid profiles is now being undertaken. an agrobacterium tumefaciens-based transformation and a regeneration protocol have recently been developed specifically for narcotic tasmanian cultivars. the various cdnas encoding genes of alkaloid biosynthesis in p. somniferum are being systematically reintroduced into the plant to achieve engineered plants with altered alkaloid profiles. the first results have now been obtained with sense and antisense genes stably expressed in a regenerated tasmanian cultivar. the ultimate goal of exploiting the genes of alkaloid biosynthesis is to produce transgenic medicinal plants of specific alkaloid content that would facilitate commercial production and improve our understanding of the factors that regulate biosynthesis as well as provide experimental systems with which to investigate the ecological role of alkaloids in planta. integrated transcript and metabolite profiling for gene discovery in plant natural product pathways richard a. dixon, lahoucine achnine, bettina deavours, mohammed farag, marina naoumkina, lloyd w. sumner plant biology division, samuel roberts noble foundation, ardmore, ok 73401, usa the rich diversity of chemical structures found in the plant kingdom arises in large part from a limited number of basic chemical scaffolds (e.g. terpene, polyketide) that are modified by a limited number of chemical substitution types (hydroxylation, glycosylation, acylation, prenylation, o-methylation, etc.) . much of the diversity is brought about by the substrate-and/or regio-specificities of the substitution enzymes. in contrast to the large collections of gene sequence and transcript level data available on-line, little detailed information exists on the plant (secondary) metabolome. promiscuity of substrate specificity in vitro may complicate attempts to assign functions to genes of secondary metabolism accessible to researchers through various cdna library collections. using the isoflavonoid and triterpene pathways in medicago species as examples, we describe how integrated metabolite and transcript profiling approaches can aid functional genomics, help explain metabolic regulation, and provide tools for assessing the impacts of genetic modifications in plant secondary metabolism. focused and non-targeted approaches were used to assess the impact associated with introduction of new high flux pathways in arabidopsis thaliana by genetic engineering. transgenic a. thaliana plants expressing the entire biosynthetic pathway for the tyrosine derived cyanogenic glucoside dhurrin as accomplished by insertion of cyp79a1, cyp71e1, and ugt85b1 from sorghum bicolor accumulated 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome and metabolome. in a similar manner, plants expressing only cyp79a1 accumulated 3% dry-weight of the novel tyrosine derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. in contrast, insertion of cyp79a1 plus cyp71e1 resulted in stunted plants, transcriptome alterations, accumulation of numerous new glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae specific uv protec-tants sinapoyl glucose and sinapoyl malate as well as kaempferol glucosides. the accumulation of new glucosides in the plants expressing cyp79a1 and cyp71e1, was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify novel xenobiotics. the pleiotrophic effects observed in plants expressing sorghum cyp79a1 and cyp71e1 were complemented by retransformation with s. bicolor ugt85b1. accordingly, insertion of high flux pathways directing synthesis and intracellular storage of high amounts of natural products is achievable in transgenic plants with marginal inadvertent effects. arabidopsis thaliana-distinct function in gene transcription dynamics? jeppe madura larsen, brian stougaard vad, søren mølgaard, kell andersen, mads n. davidsen, klaus d. grasser department of life sciences, aalborg university, 9000 aalborg, denmark in recent years it has been shown that introns to some extent can regulate expression in the eukaryotic cell. insertion of introns in the 5 utr in arabidopsis genes has shown to increase gene expression at both rna and protein level. in order to investigate if 5 utr introns have distinct characteristics, we analyse these in the well annotated arabidopsis thaliana genome published by the arabidopsis genome initiative. 12,898 loci annotated with full-length cdnas were analysed and 1989 loci (15.4%) containing 5 utr introns were isolated. we studied if the genes containing these introns showed different patterns in alternative splicing and gene function (gene ontology classification) compared to the remaining genes not containing this intron type. 1802 of the isolated loci (90.6%) contained only one 5 utr intron and these were used for further analysis, where it was investigated if the 5 utr introns had a characteristic size distribution. genes containing transcripts with 5 utr introns were more subjected to alternative splicing (9.63% versus 2.39%) and had a tendency to be more involved in cell regulatory functions compared to genes without this intron type. it was also found, that 5 utr introns was characteristically size-distributed. we identified thee predominant sizes of approximate 110, 270 and 390 bp compared to only one for orf introns. this suggests widespread multiple splicing events in 5 utr introns. the results presented here suggest that 5 utr introns have distinct characteristics and function in gene transcription dynamics. cytochrome p450 monooxygenases appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites in microbial, animal and plant cells. in particular, cytochrome p450 scc catalyzes the conversion of cholesterol into pregnenolone-the precursor of all steroid hormones in mammalian steroidogenic tissues. cytochromes p450 are also involved in the biosynthesis of different plant steroid derivates that play important role in regulation of plant growth and development. therein, investigation of possible influence of cytochrome p450 scc expression on plant regulatory system is of a great interest. this report devoted to the investigation of transgenic tobacco plants, which have been generated by the transformation with recombinant plasmid pgbp450f constitutively expressing cyp11a1 cdna of the bovine cytochrome p450 scc . the transgenic state of the plants was confirmed by southern blot analysis. transgenic plants are phenotypically different from the control ones. in particular, they obtained a substantially higher growth rate and are larger than wild type plants. we have demonstrated that incubation of fragments of the transgenic plants leaves in [ 14 c]-labeled cholesterol containing medium results in formation of the radioactively labeled product with chromatographic mobility corresponding to pregnenolone. the presence of this metabolite in the steroid fraction of lipid extracts obtained from the transgenic plants leaves was confirmed by gas chromatography mass spectrometry (gc-ms) method. the data obtained indicate that cytochrome p450 scc synthesized in transgenic plants displays its specific catalytic activity. biotechnological production of glucose isomerase enzyme with streptomyces olivochromogenes for production of fructose syrup from hydrol m. hashemiravan, a. sadat barikani department of food science and technology, azad university (pishva, varamin unit), institute of food science and agriculture, tehran, iran the use of glucose isomerase for isomerization and production of fructose syrup was performed by selected industrial strain of streptomyces olivochromogenes ptcc 1457. growth of microorganism and production of enzyme in different culture media was studied, and effects of different parameters such as phosphate and aeration was evaluated. the growth of microorganism at 28 • c, caused a production of high amount of enzyme. the production of enzyme was considered in two culture media (a and b). medium (a) was selected for the higher production amount of enzyme. the highest amount of enzyme production was seen in medium a, which was 36.4 giu/ml, after 80 h. the use of baffles in culture flasks, increased the amount of enzyme production, four times more. the production of enzyme was increased, 1.25 times more, in phosphate deficient medium. the cells containing enzyme (intra cellular glucose isomerase) was separated by centrifuge, and extraction and release of enzyme was performed by ultra sonication, that is a physical-mechanical method. this method released about 89.9% of total intracellular enzyme. the best length of time for sonication was found to be 4 min. experiments showed that optimum ph and temperature of the enzyme were 7.5-8 and 80 • c, respectively. the highest activity of the enzyme was observed at ph 5.8 for up 120 min. at the time of isomerization reaction the existence of magnesium ions showed to be necessary and omission of this ion cause a decrease of enzyme activity in isomerization process, but this effect was not necessary for the enzyme activity results showed that treatment of glucose syrup at temperatures of 40, 60 and 80 • c, by the enzyme, caused 48%, 50% and 52% of isomerization, respectively. efficiency increase of high acetic acid production with the use of acetobactereace iranian native strains mutation m. hashemiravan 1 , a. alirezasadat barikani 2 : 1 department of food science and technology, azad university (pishva, varamin unit) institute of food science and agriculture, iran; 2 young iranian researchrer's club, tehran, iran the first step in the present research is the isolation of acetobactereace native strains from fruits (such as grape or apple) and fresh vinegar. this separation has been done with the use of effective isolation methods and optimized mediums. ten strains were isolated effectively, the bio chemicals test were performed, each of them were detected, classified and nominated with a special code. two methods have been used for mutation: (a) mutation with the ultraviolet radiation; (b) mutation with nitrous acid. in the first method the microbial cells were treated with us radiation for different periods of 15, 30, 45 and 60 s, and the effect of the uv mutation was assessed. as a result, the period of 45 s was determined as the optimum mutation periods. in the second method, the microbial cells were first washed with the acetate buffer 0.2 m with the ph of 4.5, then 10 ml nitrous acid 0.07 m was added and was mixed for 2-10 min. finally the sampling was done in the periods of 2, 4, 6, 8 and 10 min and was transferred to a plate containing the medium of ethanol-phenol red-agar. the two methods have been compared with each other. each method has its own advantages and disadvantages. the mutation pathway in method (b) is more stable and conducted, while mutation with the uv radiation method, change the position of thiamine-cytosine bases absolutely randomly. finally, the best mutant site was inoculumed with the medium containing alcohol 16%, acetozyme gz 0.6 g/l, acetozyme d 1 g/l, acetic acid 1.5% and 1 l double distilled water. the acetic acid was produced in the rate of 16 g/100 ml. also the mutant strains were detected with scanning electron microscope (sem) and interesting photographs were taken from mutant cell. the performed experiments were planned with the use of taguchi statistical method (qualitek 4). this research has been performed in the irost as the thesis of ph.d. in food biotechnology industry. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red 646 and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed 2d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with 0.6% (w/v) epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than 75 consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph 8.1) or chloride (ph 8.5) as leading ion and -amino-caproic acid (ph 8.9) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic 2danalysis. genotypical differences affecting the response of pisum sativum to differing boron/iron applications e.e. hakki, u. zeynep, m. hamurcu, a. tamkoc, m.b. babaoglu, s. gezgin department of field crops, faculty of agriculture, selcuk university, kampus, konya 42079, turkey. e-mail: eehakki@selcuk.edu.tr (e.e. hakki) boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plantation areas of turkey. both defficiency and toxicity problems exist in a total of about 50% of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant aquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f9 plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. increase in sulfite production by accelerating sulfate uptake in brewing yeast t. fujimura, y. kodama, y. nakao, n. nakamura, w. miki institute for advanced technology, suntory ltd., mishima-gun, osaka 618-8503, japan sulfite plays a role as an antioxidant, which stabilizes beer flavor. therefore, it is important to control the sulfite concentration during fermentation. sulfite is produced as an intermediate in the sulfate assimilation pathway in yeast. we have already reported that over-expression of a lager yeast-specific ssu1 gene, encoding a sulfite efflux pump, leads to increase of sulfite production (fujimura et al., 2003) . in the present work, we have clarified that there are two types of sul2 genes (scsul2 and non-scsul2) each encoding a high affinity sulfate permease in lager brewing yeast. eighty percent and 86% identity are found by comparing the dna sequences and the deduced amino acid sequences, respectively. a comparative functional analysis of the two genes has been performed aimed at achieving further increases in sulfite production by accelerating sulfate uptake. over-expression of scsul2 and non-scsul2 has been achieved by transformation of lager brewing yeast, saccharomyces pastorianus. experiments have been done with and without expression of non-scssu1. the resultant transformants have been evaluated by fermentation tests in wort. over-expression of either scsul2 or non-scsul2 failed to show significant effect on sulfite formation. a combination of over-expression of non-scsul2 and non-scssu1 resulted in two-fold higher sulfite production compared with overexpression of only non-scssu1 and four-fold higher compared with the parental strain. these results suggest that the non-sc-gene types significantly contribute to sulfite production in lager brewing yeast. , t., et al., 2003. functional phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyse the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. possible applications of phytases have been suggested in animal nutrition to increase mineral bioavaliability and to decrease phosphate pollution in area of intensive life stock management and in human health. zea mays is one of the cereals that contain high amount of phytate as the major phosphate storage compound. over 200 bacteria were isolated and screened for phytases from the halosphere, rhizosphere and endophyte of malaysian maize plantation. the phytase activity of the isolates was screened by a modification of the ammonium molybdate method. the highest extracellular phytase activity was detected from bacteria that isolated from the endophyte of the maize root. in this paper, results for 24 isolates chosen for media, temperature and ph optimization will be presented. production of plant proteinase from jack fruit seeds (artocarpus integrifolis) and its influence on rheological and sensory characteristics of low fat yogurt el-sayed el-tanboly dairy sciences department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, 3.9 (t1), 7.8 (t2) and 11.7 (t3) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period (15 days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t3 showed more less firm after 15 days of storage being 20.17 g/100 g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t2 and t3 of 433 and 479 mpa s, respectively, compared with control of 299 mpa s at 15 days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t2 gained the highest scores (85 points) followed by the control (81.5 points) after 15 days of storage, while yogurt of t3 showed a low scoring being 75. from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of 7.8 units of plant proteinases/ml milk. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions 77 g/l from white distilled lees and 45.6 g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. toasted wine was traditionally produced in galicia, northwest of spain. nowadays this technique is being recovered. grapes after harvesting are air dried in order to concentrate sugars, acids and flavor compounds. raisings are pressed to obtain a must with high sugars concentrations. two different grape wines were prepared concentrating the sugars up to 37 and 57 brix, respectively. in order to get a better knowledge of the problems involved, synthetic media simulating the grape musts were prepared. theses musts were used to optimize the initial sugar concentration, the amount of nutrients required, the optimum temperature to carry out the fermentation and the influence of the type and amount of yeast. under the best conditions some fermentations with grape must were carried out to produce wines with intense aroma and flavor notes and high residual sugar concentrations. in this studies, bacillus sp. e1 strain was isolated from koreanstyle fermented soybean paste and it was producing the biological response modifier (brm). the brm activated the b cell selectively. it was identified the bacillus licheniformis e1. the brm was purified by ion-exchange chromatography and gel filtration. chemical properties of brm: molecular weight of brm was estimated to be about 1,594,000 da. sugar content of brm was 33.0% (w/w) and glucosamine (35.1 mol%) was the high level. protein content of brm was 4.3% (w/w) and serine (17.2 mol%) was the high level. infra-red absorption spectrum was showed the characterization of glycoprotein. biological properties of brm: the brm which isolated from fermented soybean paste was similar to that of bacillus licheniformis e1 by immuno-fluorescence assay. we confirmed that the brm was capsular substance of b. licheniformis e1. potato nitrogen concentrate (pnc) is a highly viscous liquid with high complex nitrogen content produced from the protein-fraction in potato starch extraction. the concentrated extract is rich in minerals and ␣-amino nitrogen. although ␣-amylase nowadays is mainly produced exploiting bacillus production systems there is still considerable demand for fungal ␣-amylase from aspergillus oryzae origin. the aim of the experiments to be reported here was to investigate, if pnc can replace commonly used complex nitrogen sources in the production of fungal ␣-amylase. the following data have been measured in pnc pretreated by diluting to 1/2 and clarifying by centrifugation. total-n: 8.4 g n/l; ␣-amino-n: 2.8% (w/v) (as glycine); soluble protein (bradford): 51.2 mg/l (as bsa); total carbohydrates: 110.0 g/l; reducing sugars: 5.6 g/l; dry weight: 57.3% (w/w). in the following experiments nitrogen sources were replaced on the basis of their ␣-amino nitrogen content. the carbon source for all experiments was maize starch. the formation of ␣-amylase by a. oryzae atcc 1011 in shake flasks -using pnc (centrifuged or not), yeast extract, malt extract, casein hydrolysate or meat extract -was compared to "standard" cultivation with corn steep liquor. the experiments showed only small differences in ␣-amylase titers using complex nitrogen sources. no remarkable differences were observed in the resulting biomass. in general no differences in enzyme productivity and biomass formation could be seen after 50 h of incubation. especially the bench top bioreactor experiments indicated an optimal fermentation time of about 100 h. cultivations of a. oryzae atcc 1011 were carried out in bench top bioreactors. comparing cultivations in a medium with pnc as the sole complex nitrogen source to one containing csl as such no significant differences both in the formation and amount of ␣-amylase and the fungal growth were observed. thus pnc might be able to replace complex nitrogen sources such as csl or even the more expensive yeast extract and casein hydrolysate in fungal amylase production systems. 1.19) is involved in the metabolism of inositol and catalyzes the conversion of d-glucuronic acid to l-gulonic acid with nadph as a cosubstrate posterior to the oxidation of inositol to glucuronic acid by the enzyme inositol oxygenase. although the yeast sporobolomyces oryzicola (nakase and suzuki, 1986) is not able to grow on inositol as the sole carbon source, intracellular glucuronate reductase can be found in cells grown in a medium containing d-glucuronic acid. the enzyme could be a useful tool in the design of a specific quantitative assay for glucuronic acid, e.g. in so called energy drinks. the organism was grown in media containing either glucose and glucuronic acid or only glucuronic acid and difco yeast nitrogen base. whereas growth on both media was similar in shake flask culture, hardly any growth in either medium was observed in bench top bioreactors. the influence of dissolved oxygen tension was investigated and the relevant data will be shown. the formation of intracellular glucuronate reductase activity by sp. oryzicola is inducible by media containing glucuronic acid. no activity is found in cells grown in a medium containing only glucose as the carbon source. besides the activity against d-glucuronic acid, activities against 5-ketogluconate and -at very low levelsagainst galacturonic acid and the lactone of glucuronic acid were detected. the enzyme activity is stable up to 35 • c. the ph has relatively low influence on the activity against glucuronate, whereas the reduction rate of 5-ketogluconic acid is optimal at ph 7.0-7.5 with significantly lower values at ph 6.0 and 8.0, respectively. data on the kinetics of the conversion of both glucuronate and 5-ketogluconate will be shown. nakase, t., suzuki, m., 1986. j. gen. appl. microbiol. 32, 149-155 . the multiple nutritional and functional impacts of food fermentation on human health have been widely accepted (reddy and pierson, 1994; hugenholtz et al., 2002) . however, the related role of the involved microorganisms to the nutritional effect from the fermented food is still not well defined and the mechanisms involved are still largely unknown. the present study was to investigate iron bioavailability in carrot juice fermented by two selected lab strains, l. pentosus fsc1 and ln. mesenteroides fsc2. after digestion by gi enzymes, the juice was supplied to fully differentiated caco-2 cells to study iron uptake and transepithelial transport by caco-2 cells from the digested juice. our data revealed strain specified changes in iron bioavailability in carrot juice fermented by these two strains. after in vitro digestion with pepsin and pancreatic-bile enzymes, the best yield of soluble iron was from ln. mesenteroides fsc2 fermented juice. surprisingly, the l. pentosus fsc1 fermented juice yielded about five times higher uptake iron as compared to fresh juice, while ln. mesenteroides fsc2 fermented juice was not significantly different from the fresh juice. interestingly, the transepithelial transferred iron across the cell line was however better from ln. mesenteroides fsc2 fermented juice than from l. pentosus fsc1 fermented juice. to summarise, our study showed that level of soluble iron after in vitro digestion does not necessary indicate iron absorption, especially in the case of lab fermented food. data on improved iron uptake from l. pentosus fsc1 fermented juice indicated exiting of promoter(s) for iron absorption in such juice that is not related to the production of organic acids and lowering ph effect. peng zhang, herve vanderschuren, martin stupak, wilhelm gruissem institute of plant sciences, 8092 zurich, the tropical root crop cassava (manihot esculenta crantz) is a major source of food for approximately 1 billion people worldwide. in sub-saharan africa, more than 200 million people rely on cassava as their major source of dietary energy. in many parts of africa and latin america, cassava leaves are a vegetable source for daily uptake. cassava is grown mostly by poor farmers under marginal environmental conditions and in areas where few other crops can sustain competitive yields. the crop is therefore fundamental for subsistence farming and food security, but it is also very susceptible to stresses common in the areas and conditions where it grows. in many parts of africa, reliable cassava production is strongly impacted by infections with the african cassava mosaic geminiviruses (cmgs), a rapidly spreading disease that causes large yield losses. in the coastal areas of east africa, cassava production now is threatened by another devastating disease, cassava brown streak disease (cbsd). cassava plants are also frequently attacked by many pests, such as cassava hornworm and stemborers. several reports also indicate that greater leaf longevity, especially under drought conditions, could be important for increasing yields and/or the stability of production in cassava, as well as improve the access to an important nutrient source. conventional breeding efforts have attempted to address the constraint to cassava production, but with limited success. the new tools of biotechnology can change this situation by offering new approaches to the challenges of cassava. these new technologies have the potential to make cassava much more productive, a better source of nutrients, and profitable to grow, hence, greatly contributing on the sustainable development of tropical agriculture. recently we have developed biotech cassava with value-add traits, including resistance to cassava mosaic virus, prolonged leaf life and insect resistance. new strategies are also explored to increase protein content of cassava storage roots. we are currently undertaking pilot studies with two teams of leading scientists and experts for projects to test acmv-resistant transgenic cassava lines in africa and lines with extended leaf retention at ciat, colombia under field conditions. this development of substantially equivalent improved transgenic cassava lines is part of a larger study to analyze the need, effectiveness and biosafety of biotech cassava for agricultural production. the goal of the pilot studies will be the development and coordination of a broader project that produces important and novel scientific results, valuable information on the need and impact of biotechnology at the subsistence farming level, and a sound scientific basis for the development of guidelines for biosafety assessments and release of transgenic organisms into the environment and agricultural production in africa and latin american countries. this study was conducted to reveal the effects of different pretreatments on obtaining haploid plants by using the anther culture in pepper capsicum annum l. cultivars demre sivrisi and sirena. buds were collected at uninucleate microspore stage. anthers collected from buds were cultured in ms medium containing different hormones and hormone concentrations. experiment results revealed that when sirena anthers were pre-treated cold at +4 • c for 24 h and kept in darkness at 25 • c for a period of 1 week gave good results. in the case of demre sivrisi anthers were pre-treated cold at +4 • c for 48 h and kept in darkness at 25 • c for a period of 1 week gave good results. on the other hand no cold pretreatment to anthers resulted with low embryo formation. similar results were also observed on the anthers kept at 35 • c for 1 week as callus was produced in some petri dishes but no regeneration was observed. as a conclusion, since no cold pretreatment to anthers resulted with low embryo formation it is possible to say that cold pretreatment should be applied to anthers in pepper another culture studies. the yeast d. hansenii ufv-170 was tested in this work in batch experiments in synthetic media at constant initial substrate concentration (100 g l −1 ) under variable oxygenation conditions. to get additional information on its fermentative metabolism, a stoichiometric network was proposed on the basis of the general knowledge available in the literature on xylose metabolism in pentose-fermenting yeasts and the specificities of xr and xdh activities in d. hansenii and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. it can be stressed that under strongly oxygen-limited conditions xylitol production was negligible, whereas under semi-aerobic conditions maximum xylitol production (p max = 76.6 g l −1 ) and yield (y p/s = 0.73 g g −1 ) were obtained. a progressive decrease in these parameters was observed under fully aerobic conditions, suggesting that xylitol-producing yeasts require limited oxygen conditions, which is species-dependent. the proposed model, which utilizes the experimental specific rates of substrate consumption and product formations, allows estimating the main bioenergetic parameters. besides, it proved to be an effective tool to investigate different metabolic situations and showed how they can influence the flux distribution of the carbon source and the bioenergetics of this biosystem. the effect of disinfectants on fungi anne svendsen, pernille skouboe bioneer a/s, hørsholm dk-2970, denmark prevention of mould spoilage of foods can only be carried out successfully, if the species, which are actually spoiling the food product, are known. a very limited number of fungal species has been associated with the spoilage of each food category. proper disinfection of production facilities is very important to avoid mould spoilage. resistance of moulds to disinfectant treatments are known and different species have shown different response to the same disinfectant. to obtain proper disinfection it is important to know the resistance of the spoilage fungi against different disinfectants. in this study the effect of disinfectants on the spoilage fungi of cheese, rye bread, liver paté and fruit juice was investigated. in collaboration with five food companies the dominating species responsible for spoilage of each food product were isolated and used for testing. commercial disinfectants and disinfectants "under development" were tested. tests were performed in suspension and on surfaces, the methods used were modified after en 1650 and en 13697. considerable variability in fungicidal effect among the species was observed. some disinfectants were ineffective at low temperature. some disinfectants showed different effect in suspension and on surfaces, resulting in an effective kill in suspension and almost no effect on surface. the identification of effective disinfectants in the food industry includes: (1) testing against the specific spoiling species of the food product, (2) testing on surface, not only in suspension, (3) test parameters adapted to the food manufacturing plant. intensified research efforts in recent years confirm the major importance of the microbial flora in the gastro-intestinal tract for human health. ingestion of prebiotic oligosaccharides increases the number of the desirable bacteria like bifidobacteria and lactobacilli in the colon. we are looking at beta-galactosidases from lacto-bacillus spp. for the production of galacto-oligosaccharides (gos) because we speculate that the enzymes of probiotics will form gos with high prebiotic potential. in this present study, purified betagalactosidases of selected lactobacillus strains were used for the production of gos from lactose. different enzyme reactor set-ups, both discontinuous and continuous, were tested and compared. temperatures up to 37 • c and ph values between 6 and 6.5 were required for satisfactory enzyme stability during the process. enzyme source, substrate concentration and the level of substrate conversion were found to be critical process parameters for gos yields and composition. yields of up to 40% (w/w) of total sugars were achieved when the initial lactose concentration was 200 g/l. capillary electrophoresis (ce) and hplc with pulsed amperometric detection were the analytical tools for investigating the influence of reactor type, enzyme source and conversion level on gos composition. the prebiotics market is increasing rapidly and is expected to more than double until 2010 to about 180 million d world-wide. therefore, the development of enzymatic processes on an industrial scale is a high priority goal of our research. starter addition does not always succeed in improving standardisation and quality of the complex sensory properties of traditional fermented foods. in many cases the added strains do not grow as well as the environmental strains present in the production plant. here, a method of geometric simplification (by dichotomy) of a complex ecosystem found on a raw milk livarot (82 strains) was tested on cheese curd. by a limited number of cultures, successively, 40 out of 82, 20 out of 40 and 10 out of 20 strains were selected on the basis of two criteria (i) respect of the taxonomic proportion, (ii) generation by the daughter ecosystems of an odour close to the one of the mother ecosystem. finally a sub-ecosystem of 10 strains gave an odour similar to the one of the more complex mixture. the use of molecular methods (pcr-sscp) permitted to follow the main species growing. mother and daughter ecosystems were characterized by sensory analysis and gc-ms. probably because of an important redundancy of the strain functions, the method was very efficient. this method may permit to improve a lot the set up of mixture of strains and species used in fermented food industry. effect of the dilution rate on the exopolysaccharide production by bifidobacterium longum atcc 15707 c. shene, m. rubilar, s. bravo universidad de la frontera, chemical engineering, av. francisco salazar 01145, casilla 54-d temuco, chile exopolysaccharides (eps) producing lactic acid bacteria are used in dairy industry (cheese and yogurt) due to the rheological properties that these compounds confer to the products. preliminary results also suggest the use of eps as health-promoting (anti-tumor and immunostimulatory actions) ingredients. bifidobacteria are grampositive bacteria natural inhabitants of the gut of warm-blooded animals and man. a number of investigations have shown that bifidobacteria promote host health mainly because of the reduction proliferation of some pathogenic bacteria through acid synthesis. in this work results obtained in the experiments carried out to test the capability of b. longum atcc 15707 to synthesize eps are presented. continuous culture fermentations were carried out at dilution rates between 0.04 and 0.44 h −1 . composition of the culture media was that of the mrs broth. biomass concentration presents higher values (2.9-3.2 g l −1 ) at dilution rates between 0.1 and 0.2 h −1 . biomass growing at these rates is difficult to pellet and adheres to the fermentor walls behavior that was not observed at other growth conditions. eps from cultures grown at these rates were preparated and fractionated. authors wish to thank the chilean conicyt for the economical assistance given through the project fondecyt 1050602. high pressure-low temperature (hplt) inactivation processes were performed on bacillus subtilis vegetative cells at various conditions. at atmospheric pressure, lowering the temperature to as low as −45 • c was found to have minor anti-microbial effects. upon application of high pressure various phase transitions occurred in the microbial suspensions under study. after pressure treatment at 150-450 mpa, cells were plated under optimal conditions to assess cell viability. treatments at 250-450 mpa and −25 • c were the most effective in inactivation. in these cases, ice i-iii solid-solid phase transition was observed. in addition, we hypothesised that intracellular thawing (solid-liquid phase transition) had already occurred while the extracellular surrounding was undergoing solid-solid phase transition. this double effect is suggested to be key in mediating the observed large drop in viability. we speculate that more cells survived after treatment at −45 • c compared to the same treatment at −25 • c because both the extra-and intracellular surrounding remained fully frozen. at −45 • c a solid-solid phase transition was observed when pressure was higher than 350 mpa. a metastable state of ice i was observed at 250 mpa treatment. results from the current study will be presented (see also shen et al., ifset in press) . the data call for a mechanistic evaluation of the effects of hplt as an anti-microbial treatment. such data are currently being gathered and will be used in defining optimal hplt process conditions for the food industry. the influence of saccharomyces cerevisiae, kloeckera apiculata and candida pulcherrima mixed cultures on the selected alcohols formation during model fermentation pawel satora, tadeusz tuszynski department of fermentation technology and technical microbiology, food technology faculty, agricultural university, cracow, poland. e-mail: psatora@ar.krakow.pl (p. satora) for the study five yeast species were chosen, isolated from successive stages of plum fruits spontaneous fermentation: from the beginning (candida pulcherrima, kloeckera apiculata, saccharomyces cerevisiae w4), middle (s. cerevisiae w54) and final fermentation (s. cerevisiae k1). to characterize the potential influence of yeast mixed cultures on the selected alcohols formation, wick-erham synthetic medium (10% glucose) was fermented by mixed cultures of two and three yeast species. after distillation, ethanol, propanol, isobutanol, isoamyl alcohols, hexanol and 2-phenylethanol were determined using gas chromatography. findings were compared with the results obtained after monoculture fermentations. the use of mixed cultures resulted in increasing of glucose utilization rate, ethanol and fusel alcohols formation (except propanol) and decreasing of methanol synthesis. the samples fermented using two yeast species characterized higher (about 10%) amount of volatile compounds in relation to monocultures. it takes note of especially high level of ethanol (av. 44.3 g/dm 3 ), methanol (16.7 mg/dm 3 ) and isoamyl alcohols (47.6 mg/dm 3 ). the positive feature of triple cultures using was limitation of methanol and fusel alcohols synthesis that was accompanied by relatively high ethyl alcohol production (av. 41.5 g/dm 3 ). the consumption of sugar syrup becomes increasingly significant in industrial processes due to economic advantages and the easy of use. the production of sucrose syrup using enzymatic hydrolysis represents the safest alternative, once the reaction does not produce any toxic or undesirable substance. this work consists on the production of sugar syrup by immobilized inulinase from kluyveromyces marxianus, with two alternatives process: (a) syrup enriched with fructooligosaccharides or solely with glucose and fructose. the process is comprised by the following stages: production and purification of the enzyme in optimized conditions, immobilization of the enzyme in solid support and the conversion of sucrose in a fixed bed bioreactor with the immobilized enzyme. the final composition of the product can be a mixture of glucose, fructose, sucrose and fructooligosaccharides or a mixture of fructose and glucose, according to the operational conditions. the bioreactor can be operated continually for approximately 5 months with the same biocatalyst. the product from this process is ideal for applications in the food products such as sweet, candies, chocolates, yogurts, etc. besides, the prebiotics properties of the fructooligosaccharides, is a beneficial stimulant of the intestinal flora, which gives to the product a functional property. studies on plant microbial interactions using azotobacter sp. as bio-inoculants towards soil fertility baljeet singh saharan faculty of biotechnology, jcdm college of engineering, sirsa 125055, india. e-mail: baljeet.saharan@gmx.de, baljeet br@yahoo.co.uk (b.s. saharan) high nitrogen fixing, phytohormone producing isolates of azotobacter, azospirillum, acetobacter and pseudomonas were used as inoculants on wheat and cotton with varying doses of nitrogen under field conditions. bio-inoculants were selected on the basis of yield, dry weight and survival rate of bacteria under field conditions. seeds of wheat variety wh 711 were treated with different biofertilizers using nitrogen level of 90, 120 and 150 kg ha −1 and one level of p, i.e. 60 kg ha −1 in field along with control. under field conditions, maximum yield was obtained with azotobacter chroococcum e 12 at 90 (2506 ± 0.04 kg ha −1 ) as well as 120 kg ha −1 (2817 ± 0.07 kg ha −1 ) followed by a. chroococcum ht 57 (2482 ± 0.16 kg ha −1 ) and avk 51 (2474 ± 0.37 kg ha −1 ). whereas, with 120 kg ha −1 highest yield was observed with mac 27 (2833 ± 2.59 kg ha −1 ) followed by e12 (2817 ± 0.91 kg ha −1 ) and avk 51 (2804 ± 0.16 kg ha −1 ). maximum height at 90 kg ha −1 was observed with mac 27 inoculation (71.1 ± 5.24 cm) followed by avk 51 (70.8 ± 4.70 cm) and ht 57 (70.3 ± 1.76 cm). various chosen strains were tested with desi (hd 123) and american cotton (h 1098) under similar pot and field conditions as for wheat in the following season. plant height and yield were determined at the time of harvesting whereas survival rate was monitored at various intervals of time. survival rate of inoculated bacteria was determined after 30, 80, and 135 days. highest survival rate was observed in mac 68 ((3.34 ± 2.56) × 10 6 ), which decreased after 80 and 135 days, respectively. (3.38 ± 1.48) × 10 5 , (1.53 ± 0.92) × 10 5 with mac 68 and (2.97 ± 2.01) × 10 5 and (1.26 ± 3.01) × 10 5 with ht 54, respectively. maximum boll weight was with avk 51 (76.2 ± 2.34 g boll wt. plant −1 ) followed by pseudomonas (71.3 ± 1.77 g), ac 18 (61.5 ± 1.73 g) and ala27 (61.4 ± 2.79 g) boll no. plant −1 was maximum with ala 27 and avk 51 (46 ± 2.59 plant −1 ) followed by pseudomonas (34 ± 0.07 plant −1 ). maximum height and dry matter was obtained with pseudomonas (179.7 ± 1.97 cm) and avk521 (146.7 ± 3.49 cm) with variety hd 123 under field conditions. net saving of 20% nitrogen was observed using a. chroococcum (e 12 and avk 51) bioinoculants for wheat and cotton, respectively. to characterize the antioxidative properties of tempeh-fermented food prepared from vicia faba (l. kontu) with the use of rhizopus oligosporus, the sulfhydryl groups content and surface aromatic hydrophobicity of albumins were investigated. the results obtained for tempeh albumins were compared with raw vicia faba and bovine serum albumin (bsa). these results indicate that tempeh fermentation increased antioxidative activity of albumins. the measurements of antioxidative activity were carried out with the use of 1,1-diphenyl-2-picrylhydrazyl (dpph) and 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid (abts). the albumins of faba bean-tempeh have possessed much higher activity for scavenging free radicals as measured with the dpph and abts (49.4% and 41.1%) than raw seeds (27.9% and 23.2%) and bsa (5.8% and 13.4%), respectively. it has been also found that tempeh fermentation process increased 2.5 times sulfhydryl groups content (43.2 m/mg of albumins) as compared to raw seeds (17.5 m/mg of albumins). the tempeh albumins have possessed lower surface aromatic hydrophobicity than raw seeds (352.3 fi and 692.1 fi, respectively). orange peel characterization and generation of fermentable sugars solutions for the biotechnological production of food additives b. rivas 1 , j.m. domínguez 1 , p. torre 2 , j.c. parajó 1 : 1 department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, 32004 ourense, spain; 2 department of chemical and process engineering, genoa university, via opera pia 15, 16145 genoa, italy. e-mail: brivas@uvigo.es (b. rivas) the citrus processing industry generates in mediterranean area around 3 millions tonnes of orange peel as byproduct from the extraction of citrus juices in industrial plants. in order to avoid ecological problems and provide an extra profit, this residue was studied in order to generate a suitable substrate for the fermentation process oriented to the production of food additives. orange peels were characterized and the data collected allowed quantifying a 97% of this waste. soluble sugars (21.2%), cellulose (17.0%) and pectin (42.5%) were identified as more important fractions. this material was submitted to two hydrolysis techniques, prehydrolysis (with diluted sulfuric acid) and autohydrolysis (with water) under different experimental conditions. autohydrolysis was selected as the most appropriate technique for the production of suitable fermentation media. finally, the liquors obtained at 130 • c and liquid:solid ratio of 8 g/g, containing 38.2 g/l of sugars, without additional nutrients, were employed to citric acid production by aspergilus niger cect 2090 (atcc 9142, nrrl 599) . the influence of the addition of calcium carbonate and methanol were studied. under the best conditions an effective conversion of sugars into citric acid was attained, showing the viability of the production of fermentable solutions from this industrial waste. today there is an increasing interest in using high gravity fermentation in brewing. high-gravity fermentation involves production of beer wort of up to 18 • p or even higher and results in beer that has more consistent product quality. the main aim of this study is an increased understanding of how brewer's yeast respond to the various stress factors imposed during high gravity beer fermentation and the consequences these stress factors have on the gene regulation and its consequences on the metabolite levels (both intra-and extracellular). higher attenuation of the wort will be achieved by two different techniques: by the addition of highly fermentable adjuncts such as sucrose or glucose syrups and by mashing with addition of microbial enzymes such as pullulanases and glucoamylases. in the first part of the study model fermentation conditions are established, where the sugar uptake and product formation can be studied in details. characterization of the carbohydrate profile is analyzed by hplc. as flavour changes may occur at higher gravities, it is important to study changes in formation of secondary metabolites, especially esters. transcriptome and metabolome analysis will be used to establish how the stressful conditions prevailing under high gravity fermentations may influence the secondary metabolism in saccharomyces cerevisiae. furthermore, analytical aroma characterization of final beer will be studied by spme and gc-ms. detailed analysis of the effect of different stress factors on the cellular response using dna arrays and metabolite profiling will be carried out. dna arrays will be employed to evaluate if specific metabolic pathways are up-regulated or down-regulated as a consequences of the stress factors. naringin, a bitter compound that occurs in citrus fruit juices, may be converted to a nonbitter form by enzyme hydrolysis. the enzymatic complex naringinase was produced in aspergillus niger cect2088 cultures with naringin as inducer (pérez-mateos et al., 2004) . crude extracts from a. niger and purified naringinase from penicillium decumbens were immobilized into a polymeric matrix of polyvinyl alcohol (pva) hydrogel cryostructured in liquid nitrogen. the operating stability of the pva-naringinase beads was tested using synthetic citric juice (gray and olson, 1981) . immobilized enzymes reduced 40% the naringin content at 20 • c and ph 3.2. furthermore, immobilized preparations from aspergillus and penicillium could be re-used through six cycles (144 h) remaining 70% and 38% catalytic efficiency, respectively. financial support from "ministerio de ciencia y tecnología" and feder (no. agl2003-08006/ali). gray and olson, 1981 . j agric. food chem. 29, 1298 -1301 . pérez-mateos, et al., 2004 (1), 230. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box 6121, campinas, cep 13083-862 são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was to identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during 3, 5 and 7 days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the medium was composed by, cwb:cr were mixed with distilled water and transferred into 250 ml capacity erlenmeyers flasks and auto-claved at 120 • c for 20 min. the medium was then inoculated with spores (5.0 × 10 7 ) and the flaks were incubated at 32 • c. tannase was assayed according to the methodology of mondal et al. (2001) . according to the statist analyses, the optimum conditions to produce tannase was the range of temperature (29-34 • c); tannic acid (8.5-14%); residues percent (coffe: wheat bran) (50:50) and 5 days fermentation time. the enzyme production increased 8.6 times more enzyme production than that was obtained before this optimization. yeast and lactic acid bacteria are two major microbial groups of the most fermented products. a large variety of fermented foods and beverage are made by the activities of both yeast and lactic acid bacteria, simultaneously or successively. during the spontaneous mixed fermentation of lactic acid bacteria and yeast population, it is extremely difficult to control microbial species due to the complexity of the microorganism involved. therefore, we have compared the antimicrobial activity of chitosan against two lactic strains, lactobacillus plantarum and lb. brevis, and yeast strains, saccharomyces cerevisiae to investigate the possible use of non toxic biopolymer chitosan for selective control in mixed culture. the lactobacilli were more sensitive to the inhibitory activity of chitosan than s. cerevisiae. the results suggest the possible use of low molecularweight-chitosan for the control of food fermentation in which both groups of organisms frequently occur together. the effect of vegetable oils on astaxanthin production of phaffia rhodozyma and xanthophyllomyces dendrorhous csaba vágvölgyi, gyöngyi lukács, miklós takó, árpád csernetics, tamás papp department of microbiology, faculty of sciences, university of szeged, p.o. box 533, astaxanthin (3,3 -dihydroxy-␤,␤-carotene-4,4 -dione) is one of the most important carotenoid product. it is used primarily as food colorant and animal feed additive. their effective antioxidant properties linked to a preventive action on various types of cancer and an enhancement of the immune response could lead to expanded commercial applications. among the natural microbial source available, the closely related red pigmented yeasts phaffia rhodozyma and xanthophyllomyces dendrorhous are of great biotechnological interest. these yeasts have desirable properties as biological sources of pigment, including rapid metabolism and producing high cell densities in fermentor, but the commercial production of astaxanthin is limited by the relatively low content in wild-type strains. the purpose of this study was to determine whether the different vegetable oils had an effect on the carotenoid production in p. rhodozyma. effects of media supplemented with corn germ oil, wheat germ oil, sesame-seed oil, palm oil, pumpkin-seed oil, coconut grease, olive oil (extra virgin), olive oil (sanza), sunflower-seed oil and cottonseed oil were tested. studies were performed on both a phaffia and a xanthophyllomyces strain. yeast was grown in yeast-pepton-glucose liquid medium complemented with the appropriate vegetable oil in different concentrations (0.5-2, v/v, %) . after four days the total carotenoid production was determined spectrophotometrically, and it was referred to dry cell mass. palm oil increased significantly the carotenoid production of the phaffia strain, while a similar effect on the xanthophyllomyces strain could be observed with coconut grease. composition of carotenoid compounds in the strains was determined by thin layer chromatography. lutein is considered a nutraceutic compound that has developed an increasing interest since it is one of the two carotenoids that are located in the macula of the human eye. its consumption is associated with the prevention of age related macular disease (amd). industrially, lutein may be produced using a saponification step of a mixture of lutein diesters that are previously extracted with hexane from natural sources. our proposal is to improve the process by catalyzing the same reaction using microbial lipases during the extraction step with hexane. additionally, the use of supercritical fluids represents an extension of enzymology in non-conventional media with process and environmental advantages. this work was developed using extracts from marigold flower (tagetes erecta) in hexane and supercritical carbon dioxide (sc-co 2 ) where the lutein esters were hydrolyzed by two commercial lipases: lipase b from candida antarctica (novozym 435) and lipase from mucor miehei (lipozyme rm 1m). in particular, we focused our interest in the role of water in the system. interestingly our results show an inverse dependence of the initial reaction rate with respect to the initial water activity (awi) for both lipases, a phenomena that seems to be related to the partition of substrates and products in the solid support)and the hexane phase as a function of water. when sc-co 2 was used as solvent an increase in the consumption rate of lutein diesters occurred, reaching conversions of 70% in 24 h. for hexane, the same conversion was reached after 160 h. this result suggests a significant effect of the media on the reaction that can be related to shifts in the partition of compounds that bring the substrates in closer contact with the enzyme. this work also demonstrates that lutein hydrolysis seems to be another potential application of commercial immobilized lipases in the food/nutraceutical market. the enzyme of interest in this work is ␤-galactosidase from lactobacillus sp. (ec 3.2.1.23). ␤-galactosidases catalyze the hydrolysis and transgalactosylation of ␤-d-galactopyranosides (such as lactose). an attractive biocatalytic application is found in the transgalactosylaction potential of these enzymes which is based on the catalytic mechanism of ␤-galactosidases. the products of transgalactosylation, galacto-oligosaccharides, are non-digestible carbohydrates which meet the criteria of 'prebiotics' and therefore have attracted increasing attention. to produce these 'prebiotic' galactooligosaccharides, an inexpensive and efficient process is desired. immobilization of the enzyme ␤-galactosidase on an insoluble support is an attractive tool to make the process of lactose conversion more economical because the enzyme can be recovered and reused during continuous operation. in this present study, we aimed at immobilizing ␤-galactosidase from lactobacillus sp. by covalent linkages on two solid supports which are commonly used for protein immobilization: chitosan and eupergit c. the protein-binding capacity, the immobilization yield, ph and temperature dependency of activity and stability, and the kinetic parameters of immobilized enzymes were studied. higher activity retention of the immobilized enzymes over a broader ph range and at higher temperatures compared to those of the free enzyme was observed. the immobilized enzymes were evaluated in terms of transgalactosylation activity and stability for a to introduce of foreign genes for the important crop plants such as rice, we need a reproducible efficient procedure for regeneration of the calli through somatic embryogenesis. for this intention, we established the best callus induction medium for tarom mahalli and deilamani cultivars and created the method that the regeneration frequency was reached to 48%. calli were induced from scutellar tissues of mature seeds on ms medium supplemented with three level of 2,4-d (2, 2.5 and 3 mg l −1 ) and n6 medium supplemented with five level of 2,4-d (1.5, 2, 2.5, 3 and 3.5 mg l −1 ). for deilamani cultivar the best medium was n6 with 1.5 mg l −1 2,4-d and for tarom mahalli the same medium with 2 mg l −1 were the best. in subculture media, sucrose was used instead of maltose. for regeneration analysis of plantlets, we used two-factorial experiment in base of crd; one factor was regeneration media with six levels (ms medium supplemented with five amount of kinetin and: naa (mg l −1 ) [(6:2), (4:2), (2:0.5), (3:1), (2:1), respectively] and 0.2 mg l −1 2,4-d and 2 mg l −1 bap). other factor was dehydration process with three levels (without dehydration, dehydration with two layers of filter paper for 30 min [prior to transfer to the regeneration medium], and third factor was factor of 2 with substitution of sucrose with maltose [after 2 weeks; 2 sucrose:1 maltose]). we conclude that maltose due to changing in osmolarity proceeding can elevate the regeneration frequency to 48%. therefore, type of carbon source is critical in callus induction and regeneration. márová ivana, hrdličková jana, kubešová jitka, kočí radka, vidláková tereza faculty of chemistry, brno university of technology, purkyňova 118, 612 00 brno, carotenoids are the most widespread natural pigments with important biological activities and applications mainly in food and feed industry. at present many ways including genetic engineering are developed to reach higher production of naturally formed carotenoids using microbial producers. in this work cloning and expression of crt gene cluster from pectobacterium carotovorum in recipient bacterial strain e. coli dh5␣ as well as in yeast strain s. cerevisiae was tested. plasmid vector phsg298 with inserted crt genes was used for transformation of chemically competent e. coli dh5␣ cells, while in s. cerevisiae shuttle vector paur135 was used. transformants were selected based on resistance to antibiotics, formation of orange-coloured transformant colonies, analysis of recombinant plasmid size and lc/ms analysis of carotenoids produced by recombinant cells. the yield of individual carotenoids (lutein, beta-carotene, lycopene) obtained from various bacterial transformants was several fold higher than in natural producer (lutein: 0.2-1.2 g/g of d.w., beta-carotene: 0.1-1.4 mg/g of d.w.). the highest yield obtained in transformed strain was 63.5 g/g of lutein and 15.4 g/g of beta-carotene. the yield of biomass and carotenoids in. transgenic s. cerevisiae was comparable to some industrial red yeast strains (4.5 mg of total carotenoids + 32 mg ergosterol/l; 36 g/l of biomass). so, transgenic yeasts could be suitable for large scale production of carotenoids and/or enriched biomass, while transgenic bacterial producers are perspective above al for high production of rare carotenoids as lutein or lycopene using transformation by specific genes of crt gene cluster. this work was supported by the project msm 0021630501 of the czech ministry of education, youth and sports. two forms of grape seeds, whole and powdered forms, were heated at four different temperatures-50, 100, 150 and 200 • c. after heating, grape seeds were extracted with 70% ethanol (0.1 g grape seed/10 ml of 70% ethanol), and total phenol contents (tpc), radical scavenging activity (rsa) and reducing power of the extracts were determined. thermal treatment of grape seed increased the antioxidant activity of extracts. the maximum tpc and rsa of whole grape seed extract (wgse) were achieved when the seeds were heat-treated at 150 • c for 40 min, while that of powdered grape seed extract (pgse) were at 100 • c for 10 min, and were greater than that of the non-treated control. according to the gc-ms analysis, several low-molecular-weight phenolic compounds were newly formed in the wgse heated at 150 • c for 40 min. these results indicated that antioxidant activity of gse was affected by heating conditions (temperature and time) and physical conditions of grape seeds at the time of heat treatments. analysis of the unexpected phenotypic consequences associated with plant transformation jonathan latham, allison wilson, ricarda steinbrecher econexus, 6, canon frome court, ledbury hr8 2td, uk. e-mail: jrlatham@gn.apc.org (j. latham) transgenic plants often exhibit unexpected phenotypes. such phenotypes could arise from pleiotropic effects associated with the transgene, or they could arise from other sources. a recent econexus report underlined the potential for the process of plant transformation to result in genetic damage to the transformed plant (genome scrambling-myth or reality? transformation-induced mutations in transgenic crop plants: http://www.econexus.info/). the report showed that mutations arising at the site of transgene insertion are often substantial, frequently resulting in loss or rearrangement of chromosomal dna and insertion of multiple superfluous dna fragments. unintended mutations were also documented at other locations in the plant genome. such transformation-induced mutations could provide an explanation for unexpected phenotypes in transgenic plants. we decided to survey regulatory documents and the scientific literature for instances of unexpected phenotypic consequences arising in transgenic plants. this poster documents the preliminary results of our survey. it is intended to assess the range and frequency of unexpected consequences and to examine whether there is sufficient data available to determine their origin. it is our belief that investigating the origin of these unexpected phenotypes should be a principal aim of biosafety research. biotechnological production of metabolites such as carotenoids could be of high interest because of their antioxidative and antimutagenic activities in human body. production of these metabolites by microbial cells is dependent on cultivation conditions. so, presence of exogenous stress in cultivation environment could stimulate biosynthetic pathways of desired metabolites. two non-conventional yeast strains, rhodotorula glutinis and sporidiobolus salmonicolor, were chosen for study of carotenoid production useful in feed industry. hydrogen peroxide, sodium chloride and/or their combinations were used as exogenous stimulators of carotenoid pathway. presence of exogenous stress led to important overproduction of pigments as well as of supplementary studied substances (ergosterol, glycerol). higher adaptability of yeast cells was observed not only in cultivations with one type of stress. combination of stress factors in cultivation media induced significant increase of pigment formation. moreover, under controlled conditions in laboratory fermentor s. salmonicolor produced about eight-fold amount of ␤-carotene (230 g/g) in medium with 2% nacl and 5 mm h 2 o 2 than in control sample. similar result was observed in r. glutinis cultivated in presence of 2% nacl in inoculation medium only (200 g/g of ␤-carotene). the use of stressed biomass of red yeasts in feed industry could have positive effect not only in animal and fish feeds because of high content of physiologically active substances, but it could influence nutritional value and organoleptic properties of final products for human nutrition. this work was supported by the project msm 0021630501 of czech ministry of education. the culture ph significantly affects mycelial growth and morphology, exopolysaccharide (eps) formation, and their molecular properties during submerged cultures of a medicinal mushroom ganoderma lucidum. when the culture ph shifts from 3 to 6, mycelial growth (12.5 g/l) and eps production (4.7 g/l) were favorable compared with other ph-control strategies. the mycelial morphology was also significantly varied upon culture ph: a feather-like pellets were found when the ph was controlled shifting from 6 to 3 at day 4, which was regarded as undesirable morphological form for eps production. compositional analyses revealed that the ratios and chemical compositions of the eps formed in bottom or top fractions of ethanolic precipitates were significantly different upon culture ph. the molecular characteristics of the eps were further investigated using a size exclusion chromatography/multi-angle laser light scattering (sec/malls) system. plant ␤-n-acetyl-hexosaminidase (hex) (ec 3.2.1.52) is reported to have diverse physiological roles like fruit ripening, degradation of reserved glycoproteins in germinating seed and chitin-elicited lignification. in this paper we report the purification and characterization of hex from korean ginseng roots. after extraction with citrate-phosphate buffer, hex was purified to homogeneity using ion exchanger chromatography, hydrophobic interaction chromatography and gel filtration. its molecular weight was determined using gel filtration and mass spectrometer. enzymatic parameters were studied with 4-methyl-umbelliferyl-n-acetylglucosaminide as substrate. the effect of heat stress and weak organic acids on escherichia coli and a comparison of its recovery by the plate count method and flow cytometry monica s. talsania due to the importance of microbiology for human health, methods have been developed to enumerate viable bacteria. dilution plating is seen to be the 'gold standard' for proof of a cells viability. however, the success of this method relies on post sampling growth, which is limited by our ability to grow cells in the laboratory. additionally, stressed or sub-lethally damaged cells, remain undetected. single cell measurements can provide rapid detailed physiological information, and the assessment of population heterogeneity. this work compared the recovery of stressed e. coli as measured by the number of cfu/ml and by multi-parameter flow cytometric analysis. weak organic acids and high temperature-short time processing (htst) were used to stress the cells both methods commonly used during food preservation. it was shown here that flow cytometry is a powerful tool for the enumeration and detailed analysis of any non-culturable microbial population, which is important because cytotoxic compounds and heat stresses used in food preservation often have a growth inhibiting effect but not necessarily a lethal one. paul g. kovalenko molecular biology & genetics nasu, zabolotnogo str. 150, 031473 kyiv, ukraine the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of 6 weeks. mean transformation frequency ranged from 27% (for 8196 up to 31% (for 15,834). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba 9402 tl-dna and the 35s gus gene showed an average of more than 35%. these obtained root cultures were additionally elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g. uralensis were obtained by infection of a. rhizogenes 8196 have produced gl at an yield of 4.5% dry weight on the period of culture as a 30 days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels (3.42 g/l) of the total flavonoids production have been identificated on the strains which transformed by lba 9402. this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. ayse gul nasircillar akdeniz university, biology, akdeniz univ. faculty of art-science, biological department, 07058 antalya, turkey mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-d) or 1 mg/l naphthalenacetic acid (naa). the developed calli and regenerated plants were maintained on 2,4-d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + 2,4-d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. the glycolytic enzyme triosephosphate isomerase (tpi), which catalyses the interconversion of the triosephosphates dihydroxyacetone phosphate (dhap) and glyceraldehyde-3-phosphate (gap), was studied for its control on glycolysis and mixed acid production in lactococcus lactis il1403. we constructed a number of l. lactis strains in which the tpi activity was modulated from 3% to 217% of the wild-type level. the enzyme was found to be present in high excess with 3% tpi activity supporting 30% of the wildtype glycolytic flux, and with 24% of the wildtype tpi activity the glycolytic flux was essentially unchanged. measurements of the upstream metabolites glucose-6-phosphate (g6p), fructose-1,6bisphosphate (fbp) and dhap were essentially unchanged for tpi activities from 24% to 217%, and only in the strain with 3% tpi activity we observed a significant increase in the intracellular dhap concentration. homolactic product formation was preserved throughout the interval of tpi activity studied, though a small increase in the amount of acetate and formate production was observed in the strain expressing tpi at the lowest level (3% tpi activity). the finding of an increased mixed acid pattern under intracellular conditions with a high dhap concentration is in contrast to earlier data from literature, which indicated that the triosephosphates play an important role in regulation of pyruvate metabolism in l. lactis with a negative effect on the mixed acid flux. we have recently shown that alcohols induce the adhesion of l. monocytogenes at low temperatures, presumably accompanied by enhanced exopolysaccharide (eps) production. however, little is known about the mechanisms involved in the formation of biofilm and eps by l. monocytogenes. in the present project, we show that deletion of selected regulatory and up-regulated genes did not abolish attachment, though the degree of alcohol-induction in some cases was affected. we are applying bioinformatics to search for homologues in l. monocytogenes of known eps genes from various gram positive bacteria. this has revealed candidate genes involved in the synthesis of eps, such as genes encoding glycosyltransferases. moreover, we are at present performing dna microarray analysis for the egde strain grown at 10 • c in the presence of 2.5% isopropanol. this data should, combined with the bioinformatic results, give us a good indication of the genes involved in alcohol-induced surface attachment. repetitive-pcr (rep-pcr) was applied in research on non-starter lactic acid bacteria (nslab) in cheese. we first showed that strains previously differentiated by pulsed field gel electrophoresis (pfge) also could be differentiated by rep-pcr. this was partially due to slight changes in the pcr conditions that allowed reproducible amplification of 7-9 kb bands. more than 20 bands were obtained for most strains. a clear differentiation was also obtained between lactobacillus paracasei, lactobacillus plantarum, lactobacillus curvatus and lactobacillus danicus (a new species found in danish and estonian cheeses and traditional starter cultures). we found that this technique is highly reproducible, e.g. identical profiles in three different pcr-machines, two different dna isolation procedures, and different trained personnel. we applied the developed rep-pcr technique to confirm that survivors after heat treatment, were the actual strains introduced and not due to post-pasteurization contamination. we also showed that when we added a cocktail combination of five strains as protecting cultures to cheese, two to three members of this cocktail was dominating the cheese nslab microflora. in control cheeses without the cocktail in most cases other strains dominated, but in a few cases we were able to show cross-contamination between cheese vats. these data indicate that the rep-pcr will be useful to follow development of adjunct cultures as well as provide a reproducible subspecies (e.g. strain) differentiation. rep-pcr is a much quicker and less labour requiring procedure than pfge, and is apparently a much more reproducible technique than what has been seen for rapd. dynamic modeling of lactococcus lactis metabolism and its dynamic behavior for lactate secretion and regulatory characteristics jinwon lee, ui sub jung, hye won lee department of chemical and biomolecular engineering, sogang university, seoul, south korea, 121-741. e-mail: jinwonlee@sogang.ac.kr (j. lee) dynamic metabolic model for lactococcus lactis has been developed in order to analyze a time-dependent behavior of lactate secretion mechanism and probe its regulatory roles. the model was used to compare and analyze the lactate metabolism through in silico simulation and in vitro experimental measurements most of all pyruvate branch point seems to play a major role in producing lactate, and the results of metabolic control coefficient analysis recommend to increase lactate dehydrogenase activity and to decrease nadh oxidase activity. for obtaining more realistic data, we have added some measured flux data including some intermediate metabolites. by combining the simulation results and experimental measurements, we could establish more reliable and robust systematic lactate secretion model. in addition, an efficient parameter estimation method was used to test the exactness of the reported kinetic parameters. what to choose -the fast or the detailed -strategy to get informative profiles of secondary metabolite produced by fungi in culture. chemo-diversity and lead discovery calls for high throughput techniques, but do we need columns will direct infusion esi-ms (dims) do the job. the latter may give matrix effects and lacks resolution resulting in loss of information, while lc-ms analysis takes time and challenge the data processing. results from nano-esi dims and lc-ms analyses of the same extracts important penicillium species are compared. these results illustrate advantages and problems using these techniques for rapid profiling of fungal secondary metabolites, reviling that matrix effects in dims do not seriously hampers detection of important metabolites while the specificity and certainty, for e.g. de-replication is much higher in lc-ms. phenotypic classification of fungi is essential in food biotechnology ulf thrane center for microbial biotechnology, biocentrum-dtu, søltofts plads 221, technical university of denmark, dk-2800 kgs. lyngby, denmark. e-mail: ut@biocentrum.dtu.dk fungi are of great importance in food and food production. the intended use of fungi as cell factories for production of food ingredients is an upcoming issue in food biotechnology; however, this brings up a possible contamination with mycotoxins as a major issue. a reliable identification of the producer strains is crucial as a correct identification at species level following an updated taxonomy is the key to information on functional characters, e.g. useful metabo-lites and potential mycotoxins, growth conditions, resistance, etc. unfortunately, many mycological reports do not specify the taxonomy used or do not pay sufficient attention to taxonomical systems based on classification by functional characters-in contrast they are using a nucleotide sequence based phylogeny, which conveys little -if anything -about function of the organism. this situation is a major challenge for biotechnologists and mycologists in the years to come and will be highlighted by illustrative examples. the commercial interest in functional foods containing sufficient amounts of living probiotics is paralleled by the increasing scientific attention to the beneficial effect in the digestive tract. a daily intake of viable cells is proposed to ensure probiotic effect on consumer's health. one of the approaches which seems to be feasible to enhance probiotic viability and stability is to improve the fermentation conditions. during batch fermentation the viability of lactobacillus gasseri 5714 decreases after reaching a maximal value apparently indicating cell death. in this work, the apparent loss of viability can be avoided during fed-batch fermentation. a three-fold increase in viability is obtained when nutrient concentration was controlled compared with the viability reached in batch cultures. as a consequence, higher biomass concentration and lower specific lactic acid production were obtained. a mathematical model was developed to simulate and describe the effect of nutrient limitation on growth, viability, glucose consumption and lactic acid production. contribution to the metabolic adaptation to food restriction in rabbits (preliminary results) s. van harten 1 , s. borges 2 , p. cravo 2 , l.a. cardoso 1 : 1 instituto de investigação científica tropical, cvz, lisboa, portugal; 2 instituto de higiene e medicina tropical, lisboa, portugal. e-mail: svharten@gmail.com (s. van harten) in order to understand metabolic differences between two breeds of rabbits (halop ab and oryctolagus cuniculus algirus) during food restriction, the activities and expression of key enzymes and hormones of the rabbit were studied. animals from each breed were divided in two groups (ad libitum and restricted), revealing the results a similar difference in glycemic levels between fed and underfed rabbits, with a restriction of 50% of ad libitum feeding in the wild animals (decrease of 23% lw) and 16% of that ingestion in the halop breed (decrease of 32% lw). the activities of glutamine synthetase and glutaminase show a higher reduction of these enzymes in the wild animals superior to that of the halop breed, compromising, in this way, the ammonium detoxification and the entry of residual carbonated groups of the protein catabolism into the krebs cycle. in the latter animals, a rapid mobilization capacity of triacylglycerols (tga) appears to exist, with a rapid catabolism of fatty acids leading to their oxidation. the wild breeds' results reveal a rise of circulating tga, reflecting difficulties in the lipolysis and mobilization of nefa for oxidation. in these underfed animals, phosphoenolpyruvate and pyruvate suffered a large increase and oxaloacetate a decrease. the halop breed revealed results that indicate a diminution of glycolisis, being glucoses' energy substituted by carbonated chains of lipolysis and protein catabolism. hormone results showed a higher decrease in insulin, t3 and igf-1 in the underfed halop animals. in order to confirm the biochemical results, relative quantification of enzyme expression was studied by real time-pcr. since the introduction of genetically modified (gm) crops in 1996, the area under their cultivation has globally increased from 1.7 million hectares in 1996 to 67.7 in 2003. the number of countries adopting gm crops also rose from one country, the usa, in 1996 to 20 in 2003. despite numerous successes public opinion still questions the ecological, moral, ethical considerations and issues concerning altering the natural state of the organisms. in this study, a survey of food shoppers' knowledge, attitudes and perceptions of gm foods was carried out in food outlets in nairobi. the food outlets were determined by simple random sampling. using systematic sampling, shoppers were interviewed at targeted imported food products. focus group discussions were also conducted with farmers at city markets. the survey reflected views of a systematic sample of 387 shoppers in seven food outlets between november and december of 2003. it revealed knowledge at 20%, with positive attitudes and good perceptions towards gm foods (χ 2 = 42.873, d.f. 9, p < 0.001). seventy nine percent of shoppers were willing to buy and consume gm foods (χ 2 = 61.321, d.f. 2, p < 0.001). cross-tabulation of shopper's position on various issues raised in the survey showed a strong correlation between the respondents' respective knowledge, attitudes, and perceptions (r = 0.84). nineteen percent of food sampled tested positive for gms. poisson statistics were used to calculate the number of sample sequences. the statistical tools were obtained from spss version 11.5. the results of this study will be of great interest in determining the use and adoption of gm crops in kenya. it will also guide the development of national foreign food policy on gm foods. the technology should be embraced as soon as it is acceptable to alleviate, drought, famine and hunger estimated to be affecting 3.3 million kenyans today, mostly children. consumers and gm foods: the case of turkeyözlenözgen 1 , mustafa yildiz 2 : 1 department of family and consumer sciences, school of home economics, university of ankara, ankara 06130, turkey; 2 department of field crops, faculty of agriculture, university of ankara, 06110 ankara, turkey the future development of food biotechnology depends on consumer acceptance. scientists are aware that consumer attitudes will have a crucial impact on the process of the food biotechnology. because, food is one of the central features in human life. consumers' attitudes and trusts in the institutions will determine how gene technology will be used in food sector, in the future. recently, research concerned with consumer aspects of gm foods accelerated. but in turkey, the literature that deals with this subject is very limited and sparse. therefore, this research was carried out on the turkish consumers with the purpose of analyzing the consumers' awareness, assessments about benefits-risks, market place and labelling, and trusts in institutions, towards gm foods. this study was based on interviews with consumers who have recently purchased from major malls, during shopping hours. of the four major malls, voluntary male and female consumers were included in the research if they had main or secondary responsibility for household shopping. the questionnaire form was applied to subjects through face-to-face individual interview. the data were analyzed by using statistical methods according to explanatory variables, including age, gender and educational level. findings indicated that consumers' awareness and views about gm foods were connected to selected demographic characteristics. the results of this study can be important for consumer educators, marketing managers and policy makers. benefit-risk perceptions and moral beliefs of turkish consumers towards transgenic productsözlenözgen 1 , haluk emiroglu 2 , mustafa yildiz 3 , ayşe sezen taş 1 : 1 department of family and consumer sciences, school of home economics, university of ankara, 06130 ankara, turkey; 2 faculty of law, university of bilkent, 06590 ankara, turkey; 3 department of field crops, faculty of agriculture, university of ankara, 06110 ankara, turkey the use of biotechnology in production has generated considerable debate involving the benefits-risks and moral beliefs associated with its use. consumer acceptance of genetically modified product is a critical factor will affect the future of this technology. this study was planned and conducted to determine the relationship between product/process related benefit perceptions, product/process related risk percentions and moral beliefs of consumers towards transgenic products. a total of 400 university educated consumers, consisting of 200 males and 200 females, employed at the ministries selected by random sampling method in ankara, were included into study. interview techniques were used in the gathering the research materials. the interview instrument had been prepared considering previous research and literature. answers given to sentences typed likert were scored, used "varimax analysis technique" for validity. in order to test the reliability of questionnaire were calculated "cronbach alpha" as inner consistency coefficient. the t-test were performed for determining the differences dependent on gender and age variables between product related benefit perceptions, process related benefit perceptions, product related risk perceptions, process related risk perceptions and moral beliefs of consumers. the examination of relationships between product/process related benefit perceptions, product/process related risk perceptions and moral beliefs of consumers was made by correlation analysis technique. it is thought that the results of this study are important both for scientists and social scientists. the application of the dna recombinant technology for food production is generating a great debate in our society with the participation of scientists trying to explain the way of obtaining these new foods and which are their implications; environmentalist groups and anti-biotechnology associations that systematically are against to the application of this technology; legislatives bodies and the public in general, represented by consumers' organizations that expresses their right to be informed. considering that university students will be the future professionals and consumers their opinion on this topic will be decisive in its success or failure, this research is aimed to performed a global and comparative study of the agrobiotechnology perception by students from different areas of knowledge and studies. this study was carried out during the academic years 2000-2004, being analyzed a total of 1516 valid surveys. the designed questionnaire included 30 questions relatives to: evaluation of the own knowledge and interest on the topic; evaluation of the information sources mainly used by university students to obtain nutritional information; the opinion about gm food labelling; risks/benefits perception; purchasing intention and support of biotechnology. results obtained showed that 37% of the university students interviewed have a clear positive perception of biotechnology, mainly the students of the health sciences area. these students understand the scientific terminology and they use the university as the main source of information. they support the development of the biotechnology and they consider that in a future it will report them benefits. other group (11%) has a clear negative perception, they are mainly students from law and art history. they do not understand the scientific terminology, they consider that biotechnology will cause them risks, and as a consequence they don't have intention of buying these foods. the technology of the dna recombinant can be also used to introduce in the plants genome the gene that codes a protein of interest for their use as antigen. the application of agrobiotechnology has allowed the development of a new generation of vaccines that try to reduce or to eliminate the inconveniences of the classic ones. for the new vaccines design, the detailed knowledge of the biology of the pathogen is considered. with this knowledge the genes implied in virulence can be inactivated or modified selectively. the term "edible vaccines" it is usually applies to the use of edible parts of the plants (tubers, fruits, leaves, etc.) genetically modified with the purpose to produce specific components (antigens) of a pathogen (virus, bacteria, etc.) against which is wanted to protect a person or animal. however, oral is not the best vaccination route since the quantity of antigen for an efficient immunization it is usually high, being also needed, the co administration of an adjuvant that stimulates the immune answer. on the other hand, it is also important to highlight that the levels of antigen accumulation in transgenic plants is usually lower than the necessary ones. another problem is the irregular accumulation of the antigen in the different parts of the plants, thus difficult the appropriate control of the doses. tannin is polyphenolic component having some antioxidant properties and exists in many plants and fruits. in pomegranate juice this component causes turbidity and haze. during fruit juice clarification by conventional gelatin method, all poly phenolic substances which are responsible for antioxidant activity are removed and as a result the quality of the product is reduced. in the present study tannase enzyme (tannin acyl hydrolase; ec 3.1.1.20) was used to decompose tannin to gallic acid and glucose and as the result the amount of turbidity of the juice is decreased, however, the antioxidant properties remain unchanged since tannin is not decomposed and not separated in the juice as it occurs in the gelatin method. the amount of gallic acid in pomegranate juice samples before and after addition of tannase was measured using hplc tests and the optimum temperature, the enzyme and juice contact time, ph, and solvent concentration for clarification of pomegranate juice were obtained as 45 • c, ph = 5.5, 2 h and 50 mm citrate buffer, respectively. the potential benefits of enzymatic clarification of pomegranate juice, that is preservation of antioxidant activity and hence increasing the quality of the fruit product, in comparison to that of conventional clarification method by gelatin introduce a new technique in turbidity and haze removed in tannin containing fruit juices. the objectives of this study are to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against escherichia coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. in lactococcus lactis the enzymes phosphofructokinase (pfk), pyruvate kinase (pk) and lactate dehydrogenase (ldh) are uniquely encoded in the las operon. we have applied metabolic control analysis to study the role of this organisation. earlier work showed that ldh at wildtype level has zero control on glycolysis and growth rate but high negative control on formate production (c j formate ldh = −1.3). we find that pfk and pk have zero control on glycolysis and growth rate at the wildtype enzyme level but both enzymes exert strong positive control on the glycolytic flux at reduced activities. pk has high positive control on formate (c j formate pk = 0.9 − 1.1) and acetate production (c jacetate pk = 0.8 − 1.0), whereas pfk has no control on these fluxes. decreased expression of the entire las operon resulted in a strong decrease in growth rate and the glycolytic flux. increased las expression resulted in a slight decrease in the glycolytic flux. at the wildtype level the control was close to zero on both glycolysis and the pyruvate branches. the sum of control coefficients for the three enzymes individually was comparable to the control coefficient found for the entire operon at the wildtype level; the strong positive control by pk almost cancels out the negative control by ldh on formate production. the analysis suggests that co-regulation of pfk and pk provides a very efficient way to regulate glycolysis, and co-regulating pk and ldh allows the cells to maintain homolactic fermentation during regulation of glycolysis around wildtype level. bovine chymosin is used extensively in cheese production because of its specificity and low proteolytic activity. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. we isolated and characterized the prochymosin cdna from the abomasum of milk-fed kid goats. this cdna predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. the caprine preprochymosin has 99% and 94% identity with the corresponding lamb and calf sequences. the cdna fragment encoding prochymosin was fused in frame to the killer toxin signal sequence in a constitutive vector, and to the ␣-factor signal sequence-flag in an inducible expression vector. kluyveromyces lactis pm3-5c, k. lactis sel1, characterized by a "supersecreting" phenotype, and saccharomyces cerevisiae bj3505 were transformed with the recombinant plasmids. activated culture supernatants of yeast transformants showed milk-clotting activity. the flag-prochymosin fusion was purified from bj3505 culture supernatants by affinity chromatography. after activation at acid ph, proteolytic activity assayed toward casein fractions showed that the recombinant caprine chymosin specifically hydrolyzed -casein. the recombinant caprine enzyme could be an alternative milk coagulant in cheese making. lipid accumulation in schizochytrium g13/2s was studied under batch and continuous culture. different glucose and glutamate source concentrations were supplemented in a defined medium. during batch cultivation, lipid accumulation occurred towards the end of the growth phase but ceased when cell proliferation stopped. under continuous culture, as dilution rate decreased from 0.08 to 0.02 h −1 , both cell dry weight and total fatty acid content (tfa) of the cell increased. with a constant dilution rate of 0.04 h −1 , nitrogen limitation induced lipid synthesis (28% tfa) as described for other lipid-accumulating organisms. however, with carbon-limited conditions, some lipid accumulation was still possible, the tfa being 22%. finally, the batch and continuous culture methods are compared for docosahexaenoic acid (22:6, n − 6) production. the objectives of this study is to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against e. coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. nitrite-oxidizing bacteria catalyze an essential step of nitrogen elimination in biological wastewater treatment. recently, novel and yet uncultured nitrite-oxidizing nitrospira-like bacteria were found to be abundant in municipal and industrial wastewater treatment systems where they outcompete nitrobacter, which has long been considered as the organism responsible for nitrite oxidation in bioreactors. despite the importance of nitrospira-like bacteria for wastewater treatment and for nitrogen fluxes in natural ecosystems, little is known about their ecophysiology and interactions with other organisms. cultivation-independent molecular techniques were applied to investigate the diversity, distribution, and physiological and genetic features of nitrospira-like bacteria in nitrifying activated sludge and biofilm. a surprisingly high diversity of these organisms was found to exist in these engineered and in natural habitats. moreover, significant physiological differences could be identified among various phylogenetic sublineages in the genus nitrospira. quantitative co-localization analyses performed by novel image analysis software revealed that these metabolic features are reflected by the spatial organization of nitrifiers living in biofilm and activated sludge flocs. based on an environmental genomics approach the genome of a nitrospira-like bacterium found in activated sludge is being analyzed. results obtained so far point at unexpected physiological capabilities of this organism, and allow us to propose that nitrospira-like bacteria may also play roles in the bioremediation of (per)chlorate and chlorite. the activated sludge process is the most common way to remove organic matter, nitrogen and phosphorus from wastewater by microbiologically means. knowledge about the microorganisms involved is fundamental for optimisation of existing plants and development of new plants and process designs. many of the bacteria believed to be involved in nitrification, denitrification, biological phosphorusremoval, and removal of organic matter in full scale plants are now identified by use of molecular methods. recent developments in experimental approaches have allowed the study of the ecophysiology of these uncultured and potentially important bacteria, thus providing a better understanding of their function in full-scale activated sludge ecosystems. relatively few dominant species in each functional group (e.g. denitrifiers and polyphosphate accumulating organisms) seems to be present. some species appear to be very specialized regarding nutrient requirements while others are more versatile. a new method for mercury remediation from industrial wastewater based on the enzymatic reduction of mercury by live mercury resistant bacteria immobilized on the pumice particles has been developed in gbf, germany, and implemented in the industrial scale (unknown). the experience gained during operation of this instalation led to the idea, that the process of bioremediation may be integrated in one bioreactor with the sorption of mercury from wastewater, by immobilization of the bacteria directly on the activated carbon. for this it was necessary to define several significant parameters of the activated carbon used and the sorption process itself. the paper presents results of the equilibrium and kinetics investigations of the process of mercury sorption from aqueous solutions onto seven different types of activated carbon. the effective diffusion coefficients in the particles were obtained from the transient-state experiments and the sorption isotherms, saturation capacity of the sorbents and its dependence on the temperature and ph were identified. then the hydrodynamic and sorption characteristics of the activated carbon bed in a laboratory-scale fixed-bed bioreactor were investigated in different process conditions (mercury concentration, volumetric flow rate, temperature, ph). the results (effective capacity of the bed, dispersion and diffusion coefficients, mass transfer coefficient) enable implementation of this bioreactor for modified, integrated process of mercury bioremediation from industrial wastewaters. research supported by the grant kbn 4 t09c 013 25. bacterial cr(vi) reductases convert the very mobile toxic cr(vi) to the less toxic and less mobile cr(iii). the ability to reduce cr(vi) was studied on cell extracts of ochrobactrum tritici strain 5bvl1 and microbacterium sp. strain 3a. both microorganisms were isolated from the same sample of chromium-contaminated sludge, taken from a wastewater treatment plant. while in the first case activity was found to be associated with the intracellular soluble extract, in the second case it was a process occurring extracellularly. cr(vi) reduction by the intracellular soluble extracts of strain 5bvl1 required the presence of nadh or nadph as electron-donor, while the extracellular fraction of strain 3a only used nadph. several studies were made on strain 5bvl1 intracellular soluble extracts. a k m of 26.11 m cr(vi) and a v max of 5.75 ± 0.13 nmol cr(vi) min −1 mg −1 protein were estimated from the lineaweaver-burk plot and michaelis-menten non-linear regression. the temperature and the ph optima for cr(vi) reduction were 37.5 • c and 5.0, respectively. hyperthermus butylicus is an anaerobic hyperthermophilic crenarchaeon, isolated from the solfataric sea floor off sáo migel island, azores (zillig et al., 1990) . h. butylicus grows at up to 108 • c (optimally between 95 and 106 • c) at ph 7. it can utilize peptides, polysaccharides, and other substrates, as carbon sources to produce acetate, butyrate, and n-butanol. the capability to produce enzymes (e.g. hydrolases, dna and rna polymerases, etc.) that can tolerate and function at temperatures 20 • c higher than most other thermophilic archaea, renders h. butylicus of particular interest to the biotechnology industry. the complete genome sequence of h. butylicus was determined and it contains 1,667,186 bp on a single circular chromosome. 1695 protein encoding genes were identified which use a high level of uug and gug start codons. many of these were assigned functions on the basis of sequence comparisons. our analyses revealed some unusual metabolic properties in h. butylicus. several sugar transporters were identified, although the set of genes required for glycolysis is incomplete. moreover, genes encoding enzymes converting glucose to trioses are absent and no genes encoding enzymes of the pentose phosphate cycle or the kdpg pathway were detected. the h. butylicus genome encodes many proteases and peptidases although the lon proteases, encoded in all other archaeal genomes, are absent. although it was reported that h. butylicus does not utilize free amino acids in the media, genes for amino acid transporters were identified, and several proteins involved in di-or oligo-peptide transport are encoded. genes encoding signal peptidases are absent. we will summarize gene products of special biotechnological interest. reference zillig et al., 1990. j. bact. 172, 3959-3965. 2 hot genomics: insights in the thermophilic lifestyle of thermus thermophilus from its complete genome holger brüggemann 1,2 , anke henne 1 , gerhard gottschalk 1 : 1 göttingen genomics laboratory, institute of microbiology and genetics, university of göttingen, germany; 2 institut pasteur, unité de génomique des microorganismes pathogènes, paris, france thermus thermophilus is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment. recently completed genome sequences of two strains, hb27 and hb8, provide a solid foundation for investigating many aspects of thermophilic lifestyle; these range from molecular stability determinants to key elements of organismic physiology. in addition, the species has considerable biotechnological potential; many thermostable proteins isolated from members of the genus thermus are indispensable in research and in industrial applications. the closely related genera thermus and deinoccoocus belong to a distinct branch of bacteria called the deinococcus-thermus group. genome comparison of t. thermophilus and d. radiodurans, a mesophilic organism, which exhibits high resistant to radiation, oxidative stress, and desiccation, is of particular interest for the identification and exploration of thermophilic determinants. a large number of orthologs with a high degree of sequence identity are shared between the two species. this opens the opportunity for comparative studies of conformational and chemical thermostability of proteins, as well as for the identification of specific traits for each organism, explaining their unique physiological properties and their intriguing differences in stress tolerance. although strains hb27 and hb8 share a highly conserved chromosome, striking differences can be found between their megaplasmids, which encode a huge proportion of genes not found in the genome of d. radiodurans. possible contributions made by the megaplasmids to a thermophilic lifestyle will be discussed. microorganisms that can live in high temperatures, extreme ph and high salt concentration are called extromophiles. extromophilic microorganisms have extended our knowledge and understanding of fundamental questions such as the origin of life. the ability to grow in extreme conditions and to produce stable proteins makes extremopliles very attractive for the researchers and also for the industry. extremozymes from extremophiles have a great economic potential in many industrial processes, including agricultural, chemical and pharmaceutical applications. concurrent development of protein engineering will increase the application of enzymes from extremophiles in industry. turkey has vast and various ecologi-cal areas, and so it has a broad microbial diversity. based on the extremophilles which defined in the scope of this project, halophilic microorganisms produced industrially important proteins were isolated from ç amaltı saltern area in izmir, turkey. in this work, growth of isolates at different temperature, salinity and ph values were investigated to determine the effects of various growth conditions. eight isolates grow at ph between 6.50 and 8.50 and two isolates at 6.50-7.50. they grow at temperature between 37 and 55 • c and salt concentration between 3% and 25%. the results of some phenotypic characters showed that they are gram (−) and oxidase,ürease, dnase and nitrate reduction are (−), and catalase (+). they used d(+) glucose, maltose, lactose, sucrose, l(+) arabinose, d(+) mannose, glycerol and four of isolates used d(+) xylose as a carbon source. the isolates resistant to erythromycine, ampicilin sulbactam, cefoxitin, penicillin, bacitracin, novabiocin, amikacin and sensitive to ceftazidine, ciprofloxacin, amoxycillin/clavulanic acid, imipenem, chloromphenicol, ceftazidime/clavulanic acid, aztreonam, cefepime, cefotaxime, cefoperazone amoxicillin. this project was supported by tubitak through project tbag 2321-103t069. the technology of producing renewable energy sources such as ethanol, methane and hydrogen from biomass holds the potential of creating in-house energy resources while lowering the emission of greenhouse gasses as demanded by the kyoto protocol. recently, goals were defined for the european union determining that 5.75% of the transportation fuel has to come from biofuels in year 2010. a large-scale implementation of biofuels into the transportation sector will demand that lignocellulosic biomass, which is found in a surplus throughout the world is used as the raw material for the production process. the presentation will include a comprehensive description of the special bio-refinery concept developed in denmark for production of biofuels and other valuable products from straw. the concept includes several innovative steps such as a pre-treatment method using wet oxidation, on-site production of enzymes and a continuous fermentation process using a genetic modified thermophilic bacterium. by co-producing several biofuels in the plant optimal use of the biomass has been assured and the price of for instance of bioethanol is getting close to conventional oil-based fuels. optimizing each step in the bio-refinery, while having the full integration in mind, will be the way to make an economical viable biofuel production. in the presentation we will present our road map for achieving this goal in the nearest future. replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. one such fuel, which has been found well suited, is ethanol. it may be produced from various lignocellulosic materials, such as forest and agricultural residues, which are fairly inexpensive. to compete with gasoline the production cost must be substantially lowered. ethanol production from lignocellulose comprises the following main steps: hydrolysis of hemicellulose, hydrolysis of cellulose, fermentation, separation of lignin, recovery and concentration of ethanol and wastewater handling. the enzymatic hydrolysis and fermentation can either be run separately (shf) or combined into a simultaneous saccharification and fermentation (ssf). the latter has been shown to result in higher ethanol yields than shf. some of the most important factors to reduce the cost are: efficient utilisation of the raw material by high ethanol yields, high productivity, high ethanol concentration in the feed to distillation and process integration in order to reduce capital cost and energy demand. in the last years we have performed several studies on the hydrolysis and fermentation of various forest and agricultural residues in a mini-pilot to improve the overall yield of ethanol and to reduce the energy demand and production cost. steam pretreatment, with small addition of acid catalyst, has resulted in sugar yields close to 90% of the theoretical for various types of raw materials, e.g. spruce, salix and corn stover. the ssf has been developed and optimized to give high yield of ethanol. for spruce an ethanol yield of about 80% of theoretical based on the composition of the raw material has so far been obtained using a two-stage steam-pretreatment of so 2 impregnated raw material followed by ssf. improvements of the ssf step, in the form of high dry matter content, recirculation of process streams and adapted yeast have resulted in ethanol concentrations around 45 g/l leading to substantial reduction in energy demand and production cost. these improvements have been assessed by techno-economic evaluation to determine the effect on the ethanol production cost. the process has been further optimised by process integration to further reduce the energy demand. the ethanol production cost was estimated to be around 0.38-0.46 euro/l ethanol assuming a yearly capacity of 200 000 tonnes raw material (dry matter). production of bioethanol from spent grain, a by-product of beer production sho shindo, tadanori tachibana, akita research institute of food and brewing, akita-city, akita 010-1623, japan. e-mail: shindo@arif.pref.akita.jp (s. shindo) the breweries generate one million tons of spent grain every year, and about 20% of the spent grain is recycled in japan. therefore, it is environmentally and economically significant to consider the production of ethyl alcohol as biomass energy using the spent grain from the breweries industry. ethyl alcohol production from spent grain with immobilized yeast cells was investigated. spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. when 1 kg of wet spent grain was treated under the 30 kg/cm 2 pressure for 1 min using a 5 l steam explosion reactor, 60 g of total sugar was obtained from the liquefied spent grain. furthermore, 1.3% (w/v) of glucose, 0.4% (w/v) of xylose, and 0.1% (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. ethyl alcohol production was carried out by immobilized sacchromyces cereviseae and immobilized yamadazyma stipitis simultaneously from liquefied spent grain. both yeast cells were immobilized on the glass beads carrier. xylose and arabinose were consumed after glucose was consumed completely during ethyl alcohol production. 5.8% (v/v) ethyl alcohol was produced from liquefied spent grain that was adjusted 17% of initial sugar concentration after 2 days. the vegetable oils constitute a resource of renewable potential for the production of fuels, becoming a viable alternative when compared to the diesel from petroleum. among the vegetable oils, the extracted oil of the castor plant seeds is a promising alternative source because it is constituted mainly of the ricinoleic acid (12-hydroxy-9octadecenoic) that represents 90% of the total constitution of the oil approximately. the biodiesel obtained from castor oil can be defined chemically as being a mixture of methyl esters or ethyl esters of carboxylic acids synthesized by transesterification reaction of the existent triglyceride and an alcohol of little chain through the use of alkaline or enzymatic catalysts. in this work, we described the results found in castor oil with different degrees of purity. initially, it was made a rheological characterization followed by structural characterization (rmn 13c, rmn 1h and infrared) and thermal characterization (dtg, dta and dsc) of the crude and refined castor oil. it has been also measured the hydroxyl tenor, acidity index, saponification index and iodine index in different oils. later, these results were used to evaluate possible differences in the quality of the biodiesel (ethyl esters) produced in the enzymatic alcoholysis of the castor oil catalyzed by lipases (novozym 435, liposyme rm im and lipozyme tl im). the degree of substitution of castor oil derivative was performed by titration with 0.1n hcl and confirmed by tlc analysis and the results showed conversion rates about 90%. has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip3) and inositol tetrakisphosphate (ip4) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip6 is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia1 was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia1 converted ip6 into ip5 (myoinositol 1,2,3,5,6-pentakisphosphates) and another isomer, which is yet to be elucidated. in a denitrifying pilot plant reactor, a new obligately anaerobic ammonium oxidation (anammox) process with great potential for nitrogen removal for high strength wastewater was discovered. after transfer of the complex microbial community to a laboratory sbr system, a highly enriched population, dominated by a single anaerobic chemolithoautotrophic bacterium related to the planctomycetes was obtained. the bacterium was purified via percoll centrifugation and characterized as 'candidatus brocadia anammoxidans'. survey of different wastewater treatment plants using anammox specific 16s rrna gene primers and anammox specific oligonucleotide probes revealed the presence of at least four other anammox bacteria, tentatively named 'candidatus kuenenia stuttgartiensis', 'candidatus brocadia fulgida', 'candidatus scalindua wagneri' and 'candidatus scalindua brodae'. a close relative of the two scalindua species, 'candidatus scalindua sorokinii' was found to be responsible for about 50% of the nitrogen conversion in the anoxic zone of the black sea and in the benguela upwelling system along the namibian coast, making anammox an important player in the global nitrogen cycle. electron microscopic studies of all five anammox bacteria showed that several prokaryotic membrane-bounded compartments are present inside the cytoplasm, which are surrounded by unique ladderane lipids. hydroxylamine oxidoreductase, a key anammox enzyme, was present exclusively inside one of these compartments, named the 'anammoxosome'. unique peptides fragments of the purified hao were used to locate the hao gene in genome assembly of 'candidatus kuenenia stuttgartiensis'. the implementation of the anammox process in the treatment of wastewater with high ammonium concentrations was started at the treatment plant in rotterdam, the netherlands, where it is combined with the partial nitrification process sharon. the estimated price for nitrogen removal with partial nitrification and anammox is about 0.75 euro/kg n. gas lift reactors could sustain the highest anammox capacity at 8.9 kg n removed/m 3 reactor per day. an alternative configuration of anammox is the oxygen-limited canon process in which aerobic ammonium-oxidizing bacteria protect anammox bacteria from oxygen and produce the necessary nitrite. maximum nitrogen removal with canon in gas lift reactors was 1.5 kg n/m 3 reactor per day. using several different conditions and parameters, the competition and co-existence of aerobic and anaerobic ammonium-oxidizing bacteria were modeled. in addition to ammonia, urea was also converted after a 2-week adaptation in the canon system. recently it was shown that anammox bacteria can use organic acids as additional energy source. murray moo-young, wa anderson department of chemical engineering, university of waterloo, waterloo, ont., canada n2l 3g1 bioreactors are central to the bioremediation of contaminated environments of water, air or soil. in all three areas of application, bioreactor design is critical to the development of new or improved processes. this overview focuses on the physical limitations of bioreactors caused by biological requirements. the information is based on our own research findings. the need for more applicationsoriented bioremediation research becomes apparent. for technoeconomic reasons, the airlift type has often been the bioreactor of choice for most bioremediations. however, lack of adequate understanding of the quantitative effects of operating conditions on its performance has been an ongoing concern. these effects have been characterized for engineering implementation. to enhance productivity, innovative pretreatment techniques of the polluted sources have also been developed using photocatalytic and chemical oxidation methods. case studies on petrochemical-contaminated water and soil reveal significant enhancement potentials. other studies on microbial biofilters for air bioremediation indicate that the active mass of the biological consortia is not sufficiently understood for rational design. analysis and retrofit design of wastewater treatment facilities using process simulation tools demetri petrides, alexandros koulouris, intelligen, inc., scotch plains, nj 07076, usa. email: dpetrides@intelligen.com (d. petrides) process simulators have been used in the petroleum and chemical industries for over four decades to facilitate the design of new processes and optimize the performance of existing ones. similar benefits can be derived from the use of such tools in the environmental arena, particularly in the field of physical and biological treatment of municipal and industrial wastewater. specifically, process simulators can be used to evaluate and improve options for: (1) more efficient removal of nutrients (e.g., organic nitrogen and phosphorous) that cause eutrofication, (2) estimation and control of volatile organic compound (voc) emissions from open tanks, and (3) more efficient removal and control of hazardous compounds. the potential benefits will be illustrated with cases studies involving both municipal and industrial wastewater facilities. the microbial reduction of metals has showed recent interest as these transformations can play crucial roles in the cycling of both inorganic and organic species in a range of environments and, if harnessed, may offer the basis for a wide range of innovative biotechnological processes. under certain conditions, however, microbial metal reduction can also mobilise toxic metals with potentially calamitous effects on human health. some effluents present heavy metals as soluble compounds, several microorganisms have the capacity to precipitate these metals as insoluble compounds, and this fact allows the collection and separation of these metallic precipitates from contaminated medium. sulfate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulfate as an electron acceptor and generate hydrogen sulfide. hydrogen sulfide reacts with heavy metal ions to form insoluble metal sulfides that can be easily separated from a solution. the purpose of this work was study the capacity of desulfovibrio sp. cultures to reduce mixtures of the heavy metals in presence or not of petroleum. for it the experimental design 2 k (k = 5) was carried out. the five studied factors were cr, cu, mn, zn and petroleum. the study was carry out with desulfovibrio sp. batch studies were performed in 50 ml sealed bottles with different concentrations (cr(iii)-10 ppm, cu(ii)-5 ppm, mn(ii)-10 ppm, zn(ii)-15 ppm) of metal sulfate and 2 g l −1 of petroleum. during batch incubation the dissolved concentration of metal studied in supernatant were decreased to undetectable levels for zn (70-100%), however with cu (40-60%), mn (40-70%) and cr (50-80%). the development of continuous process with sulfatereducing bacteria seems to be a suitable alternative to reduce metals in solution from contaminated media such as industrial or mine effluents. after these preliminary results, some experiments in course are focused to study that purpose. reduction of odour emissions from livestock buildings using a bioscrubbing system morten øgendahl, nawaf abu-khalaf, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, dk-5230 odense m, denmark. e-mail: tvede@bmb.sdu.dk (m. øgendahl) a bioscrubbing system for reducing odour emissions from livestock buildings is presented. the bioscrubbing system consists of two separate units; an absorption column and a water purification module. the absorption column is mounted in the ventilations stacks in the livestock buildings absorbing odorants in the effluent air flow. the odorants are absorbed in a spray of droplets formed by a grid of high pressure nozzles in the inlet of the absorption column. the spray of droplets is extracted from the air flow and pumped to a centrally located water purification module, an inverse three phase fluidised bed bioreactor, where the bio-degradation of the absorbed odorants occurs. the bioreactor features a split sparging system for maximum mixing and aeration. the cleaned water is recirculated to the absorption column. an electronic tongue will quantify key odorants in the bioreactor. the absorption column is designed to be retrofitted into existing livestock building ventilation systems. the water purification module is constructed in standard size units simplifying scaling to match the requirements of individual applications. the total bioreactor volume is increased by increasing the number of standard bioreactors. this work describes a "light off" toxicity bioassay sensor based on whole cell genetically modified bioluminescent bacteria. the biosensor was constructed by mating between the environmentally isolated phenol-degrading acinetobacter sp. strain df4 and the plasmid putk2 that is an inc p␤ plasmid with the bioluminescence genes luxcdabe inserted into a genetic region involved in plasmid replication and transfer. subsequently, the bioreporter designated df4/putk2 and used to investigate phenolics toxicity. among examined phenolics, pentachlorophenol, catechol and nitrophenol recorded the fastest effect on the bioluminescence of bioreporter df4/putk2 over incubation period of 350 min. the effect of various concentrations of phenol and its derivatives either in an individual, duple or triple mixture forms on the bioluminescence response of the constructed bioreporter df4/putk2 were also examined. significant reduction of the bioluminescence was observed whenever a mixture contained pentachlorophenol, catechol and nitrophenol, respectively. to develop a system appropriate to commercialize, the constructed bioreporter df4/putk2 was subjected for immobilization in microtiter plates using several entrapment gels. after a selection of materials was tried, lb/agar was chosen as the most suitable candidate material. characterization of key odour compounds in an air wet scrubber is presented. the key odour compounds represent five chemical groups, i.e. sulphide, alcohol, volatile fatty acids (vfas), phenol and indole. direct aqueous injection (dai) and solid phase extraction (spe) methods were used before injection of key odorants into the gas chromatography-flame ionisation detection (gc-fid). the dai and spe methods were efficient in the identification of odour compounds in the wet scrubber. the spe method had a high recovery and can be more effective in the identification of compounds at low initial concentration. however, dai showed a better linearity and a lower limit of detection (lod) than the spe method. the dai method was the method of choice for characterization, as it is cheaper, easier to handle and highly applicable. at least two odorants, phenol and 1-butanol, were quantified successfully using the dai method. their lod was less than their odour detection limit in the wet scrubber. dai method can be used as a reference measurement method for any further analytical application, e.g. electronic tongue. recent developments in biotechnology enabled the widespread use of microbial enzymes in textile, detergent, food and dairy industries and also in various environmental applications. microorganisms which live at extremes of temperature, ph and salinity, produce extremozymes that offer many exciting opportunities for their use in clean production. in this study, microorganisms were isolated from camaltı saltern area ini̇zmir, turkey. effect of medium salinity on the growth of these microorganisms was determined. seven out of 10 isolates required salt for growth. the salinity ranges at which growth was detected were: 5-25% for two isolates, 6-25% for one isolate, 7-25% for two isolates and 8-25% for one isolate. the isolates were also screened for their capability of producing industrially important enzymes such as amylase, protease, lipase, xylanase and cellulose which are widely used not only in textile, detergent, food and dairy industries but also in various environmental applications. all of the isolates were found to be producers of both amylase and xylanase enzymes at varying salinity array within 5-30% salt concentration range at ph 7.0. extracellular protease activity was detected in the medium of all isolates grown at 5, 10, 15, 20 and 25% salinity at both ph 7.0 (optimum growth ph) and ph 9.0. out of 10 isolates, 9, 10 and 9 were found to produce cellulase enzyme when the salt concentrations were 5, 10 and 15%, respectively. at 20% salt concentration, only one isolate was found to be cellulase enzyme producer. none of the isolates were found to produce lipase enzyme at 5-30% salt concentration range. this project was supported by tubitak through project tbag 2321-103t069. chemical engineering department, middle east technical university, ankara 06531, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) glass and ceramic tiles are very widely used industrial materials. in most cases, periodical cleaning is required to maintain their optical properties such as transparency and visual aspects. because of the ever-growing demand for healthy living, there is a keen interest in materials capable of killing harmful microorganisms. the application of these tiles in care facilities to reduce the spread of infections, in public and residental places to improve hygienic conditions are of general interest. in this study the aim is developing methods to apply thin film coatings on glass tiles to make them anti-bacterial by utilizing photocatalysis and investigating their anti-bacterial properties. semiconductors because of their reasonable band gap energies find great attraction through this purpose. the photocatalytic property of semiconductors are used in this process. oxidising radicals are formed on the coated surfaces and these radicals attacks the organic pollutants and bacteria on contact with the surface. titanium dioxide (tio 2 ) coated surfaces are considered to be very effective against organic and inorganic materials, as well as against bacteria. in the experimental procedure coating solution is prepared by sol-gel technique. after pretreatment of surfaces, the coating solution is applied on the surfaces by dip-coating method. after appropriate thermal treatments, to achieve thin, dense and strong coatings, indicator microorganism is directly applied on the coated surfaces and illuminated under solar simulater light source. finally, the number of surviving microorganisms are determined. in this study, the effects of titanium dioxide (tio 2 ), tin oxide (sno 2 ) solutions and metal doping to these coating solutions on anti-bacterial function were investigated. as a result of this study, the number of escherichia coli that is used as indicator microorganism, on tio 2 and sno 2 coated glasses with respect to the control glass reduced by 80-85% and 40-45%, respectively. doping with metals increased the activity of the coatings, hence the number of surviving microorganisms decreased. activity of a methanogenic ecosystem during the primary contact with a solid support s. michaud, n. bernet, p. buffière, j.p. delgenès inra-lbe, avenue des etangs, f-11100 narbonne, france in this paper, the biological activity during the first initial contact between a methanogenic sludge and a solid support was investigated in batch experiments, at different solid concentrations, using two different granular solid materials and with glucose as the main organic substrate. in all cases, the introduction of a solid material in a methanogenic suspended biomass induced a response of the anaerobic microorganisms, after a lag phase during which biological activity was not detected. this lag phase could be the consequence of a physical stress induced by the first contact between microbial cells and the solid surface. this lag phase was not observed when the biomass used originated from a biofilm reactor, i.e. using a biomass previously exposed to a solid material. a change in the metabolism of organic matter from catabolism and methane production toward production of other compounds could be observed, characterised by a sharp decrease of the methane yield in the anaerobic system. analyses of the gas and liquid phases did not show the production of any new gaseous or soluble compound as the biological end product of this activity. this suggests the production of non-soluble compounds by an anabolic pathway, which could indicate the initiation of biofilm formation. this metabolic activity was shown to be directly correlated to the ratio between the solid surface introduced and the microorganism concentration in the anaerobic culture (m 2 g vs −1 ). from kinetic observations, it could be observed that acetogenic methanogenesis recovered more rapidly than syntrophic propionate and butyrate degradation. evaluating microbial diversity of hydrocarbon degrading bacteria cleantis braithwaite, howard rosser, tawfiq al-ibrahim, hussain, al-bandi research and development center, saudi aramco, dhahran, saudi arabia the analysis of microbial diversity with molecular methods is central to isolating and identifying new and potential biocatalysts resources for research and industry. the ability to degrade hydrocarbon components of petroleum is widespread among bacteria, and is an effective method for remediation of a variety of ecosystems. due to the high carbon content of oil and the low levels of other nutrients essential for microbial growth, treatment of oil with phosphorus and nitrogen is generally required to enhance the growth of hydrocarbon-degrading bacteria and to stimulate oil sludge degradation. in this research study, three types of oily sludges from a gas plant, refinery, and terminal facilities were treated with nutrients. to assess the microbial diversity, both biolog culture method and culture independent polymerase chain reaction (pcr,) denaturing gradient gel electrophoresis (dgge) methods were used. nutrient addition significantly improved oil sludge degradation. we identified and characterized several hydrocarbon degrading bacterial strains that have the ability to convert petroleum. these bacteria included representatives both gram positive and gram-negative genera. there were slight difference in the quantity and type of hydrocarbon degrading bacteria found in the three sites. this is the first molecular analysis of hydrocarbon degrading microbial population in saudi arabian operations. mussel adhesive proteins, including the 20-plus variants of foot protein type 3 (fp-3), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. here we report the novel production of a recombinant mytilus galloprovincialis foot protein type 3 variant a (mgfp-3a) fused with a hexahistidine affinity ligand in escherichia coli, and its ∼99% purification with affinity chromatography. recombinant mgfp-3a showed a superior purification yield and better apparent solubility compared to those of the previously reported recombinant m. galloprovincialis foot protein type 5 (mgfp-5). the adsorption abilities and adhesion forces of purified recombinant mgfp-3a were compared with those of cell-tak (a commercial mussel extract adhesive) and mgfp-5 using qcm analysis and modified afm, respectively. these assays showed that the adhesive ability of recombinant mgfp-3a was comparable to that of cell-tak but lower than that of recombinant mgfp-5. collectively, these results indicate that recombinant mgfp-3a may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments. cresol, a monomethylated phenol, is an aromatic compound. the environmental protection agency (epa) has determined the carcinogenic potential of cresol. various options are being examined for the degradation of cresol because of their unavoidable large scale production and toxicity. many aromatic hydrocarbons can be used as electron donors aerobically by species of pseudomonas, thus leading to the ring cleavage of these compounds. in the present study, pseudomonas strains were isolated from activated sludge collected from sewage treatment plant. it was repeatedly transferred onto nutrient agar plate to check the purity of the culture. the organism was grown aerobically in an inorganic medium with p-cresol as the solitary carbon source. pseudomonas was confirmed by the expression of green pigment, gram staining and biochemical tests including koh, catalase, nitrate reduction and carbohydrate fermentation reaction. inoculation status was used to determine the rate of degradation of p-cresol. the effect of temperature on p-cresol degradation was studied. moreover, the effect of different concentrations of the aromatic compounds on pseudomonas as well as substrate variability was also documented. phenolic intermediates were estimated colorimetrically using 4-aminoantipyrene, folin-lowry method, uv spectrophotometry and hplc. the results indicated that pseudomonas could degrade up to 300 mg/l of p-cresol within 10 h. pseudomonas sp. exhibited good metabolic versatility and degraded other aromatic compounds including m-cresol and p-hydroxybenzoic acid. we conclude that this strain of pseudomonas has excellent potential for bioaugmenting the degradation of p-cresol-containing waste water treatment units. a considerable amount of waste cooking oil is produced by the restaurant industry worldwide. this poses a significant environmental and economic problem, since high oil and grease concentrations in the sewage system could lead to pipes occlusion and decreased efficiency in water treatment operation plants. therefore, sending these wastes to recycling companies or hazardous waste processors is usually required. yarrowia lipolytica, a well-known lipase producer, requires the presence of lipidic compounds (i.e. vegetable oils) to boost enzyme biosynthesis. in this work, the suitability of waste cooking oil as lipase inducer in submerged cultures of this yeast has been assessed. if successful, this procedure could allow both the degradation of an abundant waste and its valorisation as a raw material for the production of a high added value product. the microorganism was grown in a liquid medium to which various amounts of waste cooking oil were added. biodegradation degrees up to 80% (measured as decrease in cod) were obtained after 3 days of treatment. also, initial glucose concentration in the basal medium seemed to influence the efficiency of the process. on the other hand, addition of waste oil led to a significant increase in lipase production (more than two-fold), compared to oil-free cultures. moreover, chain-length specificity of the produced enzymes was significantly different: high activity towards medium chain length esters was found, which hinted to the occurrence of both lipases and esterases. biodesulfurization: a documental review j. ferrer, simon bolivar university, environmental engineering lab. caracas, venezuela a documental review about larger interest aspects in biodesulfurization technique is showed. especifically, the investigation is related to general framework and the justification of this technique, degradatives pathways elucidated up to now, involved microorganism, important elements in development of bacterial desulfurization and progress areas, and future tendency. in situ bioremediation of a p-nitrophenol contaminated site and assessment of its community structure debarati paul, gunjan pandey, sumeet labana, rakesh k. jain institute of microbial technology, sector 39a, chandigarh 160036, india. e-mail: rkj@imtech.res.in (r.k. jain) biodegradation of p-nitrophenol (pnp), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. bioremediation studies were carried out in pnp-spiked soil in small plots under natural field conditions using arthrobacter protophormiae rkj100. role of carrier material was examined by immobilizing the bacteria on corncob powder prior to adding them to soil. these studies demonstrated successful removal of pnp by immobilized cells that were able to deplete pnp completely in 5 days, whereas free cells were able to deplete 75% pnp in the same time period. monitoring the fate of released bacteria revealed fairly stable population of the cells when they were immobilized on corncob powder throughout the period of study. on the other hand, there was a decrease of 2.7 log units in colony forming units of free cells at the end of the study (30 days). bacterial community structure and diversity was also studied for the pesticidecontaminated site wherein the effect of addition of an exogenous strain on the existing soil community structure and on soil functionality was determined using molecular techniques. as revealed by restriction fragment length polymorphism (rflp) studies 45 different phylotypes could be identified on the basis of similar banding patterns. sequencing of representative clones of each phylopyte showed that the community structure of the pesticide-contaminated soil mainly constituted of proteobacteria and actinomycetes. terminal fragment length polymorphism (t-rflp) analysis showed only subtle changes in community structure during the process of bioremediation. bacteriocins encompass an array of structurally different molecules produced by a number of phylogenetically distinct bacterial groups and trigger the killing of the same or closely related species. the recombined escherichia coli strain harboring a bacterocin coding region of xanthomonas campestris pv glycines 8ra was disrupted to obtain cell homogenate. peptidic xanthomonas bacteriocins (pxb) were separated by lowering ph and adding salt. the resulting pxb's were partially purified using ion exchangers, gel filtration. two final active fractions, a and b, were obtained with a yield of 0.005% and 500-1000-fold purification. the activity of pxb was stable at the ph ranging from 7.0 to 10.0. andreja kresal, vanja kokol, vera golob textile department, university of maribor, 2000, slovenia wastewater from textile dyeing industries is characterized by high chemical and biological oxygen demands (cod and bod) and intense color due to the extensive use of synthetic dyes. as dyes of complex aromatic structures are resistant to removal by the typical microbial population and may be toxic to the microorganisms present in the treatment plants, discharge of the wastewater to the treatment plants may lead to its failure. beside, direct discharge of these effluents into municipal wastewater plants and/or environment may cause the formation of toxic carcinogenic and/or unhealthy breakdown products. different chemical and physical methods (adsorption, coagulation-flocculation, oxidation, filtration and electrochemical treatments) for color removal have been proposed, but due capital costs and slow operating speed as well as huge amounts of sludge creation there is still a great need to develop an economic and effective method. the use of lignin degrading white-rot fungi and their enzymes (laccase, lignin peroxidase, manganese peroxidase) has attracted increasing scientific attention due their ability to oxidative degrade a wide range of recalcitrant organic compounds. in the contribution, the decolorization efficacy of different commercial textile reactive dyes (anthraquinone, azo, triphenylmethane) will be investigated after the treatment by laccase from trametes versicolor. in order to examine the reuse of enzymatically decolorized liquors, the ecological suitability and the toxicity of the degradation products after different time of enzyme exposure will be studied. this work was carried out within the scope of research project e! 3100 cawab. influence of heavy metals on growth and extracellular enzyme production of a trichoderma harzianum strain with biocontrol potential l. hatvani 1 , l. kredics 2 , a. szekeres 1 , z. antal 2 , l. manczinger 1 , a. nagy 3 , c. vágvölgyi 1 : 1 department of microbiology, university of szeged, p.o. box 533, h-6701 szeged, hungary; 2 hungarian academy of sciences, university of szeged, microbiological research group, hungary; 3 pilze-nagy ltd. kecskemét, p.o. box 407, hungary. e-mail: kredics@bio.u-szeged.hu (l. kredics) trichoderma species are common soil inhabiting asexual filamentous fungi with teleomorphs belonging to the hypocreales order of the ascomycota division. besides the industrial and clinical importance of the genus, certain strains have been found to cause great losses in mushroom cultivation while other strains are well known to possess high antagonistic activity against several plant pathogenic fungi and therefore used as biocontrol agents. important mechanisms of antagonism include competition and mycoparasitism, which -among others -can be related to the fast growth of trichoderma strains and the production of several extracellular enzymes. the influence of certain, soil-occurring heavy metals on mycelial growth and the secretion of extracellular enzymes involved in competition and mycoparasitism was examined in this study regarding an effective, potential biocontrol isolate of trichoderma harzianum. the metal ions zinc, manganese, copper, iron, lead and mercury were applied at the concentrations of 8, 16, 24, 32, 40, 60 and 80 m, and dry mycelial weight as well as the activities of extracellular ß-glycosidase, cellobiohydrolase, trypsinand chymotrypsin-like protease and n-acetyl-glucosaminidase enzymes were determined. it was found that mercury totally blocked mycelial growth, while other metal ions exerted a much lower influence on growth. the presence of heavy metals did not have a significant effect on the activity of the examined extracellular enzymes with the exception of trypsin-like protease, which showed a four-to six-fold rise in activity in the presence of certain sublethal concentrations of copper. based on these results, our further aim is to develop copper-resistant derivatives by mutagenesis from trichoderma strains with biocontrol potential. since proteases play an important role in mycoparasitism, these strains could be applied within the frames of integrated pest management in combination with copper-containing fungicides, resulting in an enhanced level of crop protection even with reduced amounts of fungicides. this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. the significance of biocontrol agents (bcas) is that some of them possess good antagonistic abilities against plant pathogenic fungi. a significant number of the most prominent fungi for the purposes of agricultural application belong to the genus trichoderma. in previous studies, in vitro assays on agar plates were reported as the generally used method for the evaluation of antagonistic abilities, as the results of these assays are well transferable to the practical application. the aim of the present study was to develop an accurate, image analysis-based method for the evaluation of the biocontrol characters of bcas. randomly selected trichoderma isolates were tested against fusarium culmorum. in the currently developed method, the areas of the fungal colonies were calculated on petri dishes by measuring the occupied surface of the medium on digital images. the inhibition effect was recorded as the value of biocontrol index (bci), which was calculated from the ratio of the area of the trichoderma colony and the total area occupied by the colonies of trichoderma and the plant pathogen. the proposed method was tested for numerous parameters, and the results revealed that bci proves to be capable for the accurate measurement and scale of the biocontrol abilities of fungal isolates. this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. the effect of advanced oxidation processes and recirculation on biodegradation of leachates from aerobic landfills liliana krzystek, anna zieleniewska-jastrzębska, stanisław ledakowicz department of bioprocess engineering, technical university of lodz, 90-924 lodz, poland modern landfills are built and operated in a way which allows us to treat them as a special type of bioreactor. simulation of municipal waste biodegradation in lysimeters provides knowledge on basic processes that take place in an aerated landfill. the aim of aeration is to stabilise mainly biodegradable and nitrogen containing components and to reduce methanogenic potential. stabilised leachates from old landfills contain big quantities of refractive carbon compounds that cannot be removed by biological methods. in such case most advantageous is to apply advanced oxidation processes (aops). the objective of this study is an experimental simulation of a landfill aerobic stabilisation and the impact of aops and recirculation of leachate on the reduction of organic load. the performance of the processes was monitored by the reduction in time of basic indices of organic load (bod 5 , cod, toc, vfa, tkn, n-nh 4 + ) and changes in biogas composition. the simulation of aerobic landfill processes was carried out in lysimeters with a fixed bed of household solid waste stabilised during 8 months in anaerobic conditions. leachates taken from the lysimeters were recirculated and subjected to advanced oxidation processes, i.e. ozonation and uv radiation with the addition of h 2 o 2 . experimental studies showed that the aerobic waste stabilisation was a very quick process. during a month the bed was stabilised, reaching a significant reduction of organic load indices. aeration of the lysimeters caused a quick reduction of mainly degradable organic substance (in terms of bod 5 ) and n-nh 4 + and vfa. the reduction of methanogenic potential of the landfill was even faster. the composition of gas at the outlet from the lysimeter changed and after one day already its content was similar to atmospheric air. a more frequent recirculation of leachates enhanced greatly the aerobic biodegradation. it was found that application of advanced oxidation processes (especially ozonation) contributed to a growing reduction of the organic load in the leachates from aerated lysimeters. the application of leachate ozonation resulted in a very high degree of reduction of organic compounds (up to 77%). the objective of the experimental study was to assess the effect of temperature on the extent of aerobic batch biodegradation of potato stillage with a mixed culture of bacteria of the genus bacillus. the experiments were performed at 20, 30, 35, 40, 45, 50, 55, 60, 63 and 65 • c, at ph 7, in five l l working volume stirred tank reactor (str) (biostat ® b, b. braun biotech international). the duration of the process was 120 h. initial cod of the stillage amounted to 51.9 g o 2 /l, the main carbon sources being reducing substances (18.7 g/l), organic acids (determined as their sum) (12.2 g/l) and glycerol (3 g/l). at 65 • c, no cod reduction or biomass increment was found to occur. at the other investigated temperatures, the reduction in cod measured after suspended solids (ss) separation varied from 77.6% (55 • c) to 89.1% (35 • c). without ss separation, cod reduction ranged between 55.6% (20 • c) and 75.1% (35 • c). this indicates that, in terms of the extent of cod reduction, the optimal process temperature was 35 • c and that there was a local optimum at about 63 • c. according to the temperature applied, the content of reducing substances decreased by 84.3-96%, that the organic acids by 91.7-99.6%, and that of glycerol by 91.5-96%. the experiments also produced the following two findings: (1) the rise in temperature brought about a decrease of biomass concentration in the str (measured as ss and bacterial number), and (2) temperature was a factor affecting the demand for ammonia nitrogen (n-nh 4 ), which was the highest at 20 and 60 • c. the high n-nh 4 demand observed both over the higher and lover ranges of the investigated temperature should be attributed to the release of n-nh 4 and to the large amounts of the biomass produced, respectively. the results obtained imply that the extent of potato stillage biodegradation with a mixed bacterial culture was high over a wide range of the investigated temperature. polychlorinated compounds such as tetrachloroethylene (pce) have become serious environmental pollutants. considerable attention has been paid to these organochlorine compounds. this paper describes the molecular analysis of dechlorinating gene in halorespirating bacterium and efficient bioremediation process. an anaerobic bacterium, that dechlorinates pce to tce, was isolated and identified as a species of the genus desulfitobacterium. a novel pce reductive dehalogenase (prda) gene from the desulfitobacterium sp. strain kbc-1 was identified. these prd genes, including membrane anchor protein, were classified as a novel type of pce reductive dehalogenase (approximately 40% homology with the general pce dehalogenase). according to the substrate utility of this strain kbc-1 and phylogenetic analysis of prda, the type of this microorganism may be expected to play the role of a primary degrader of pce in the environment. high efficient bioremediation process so called the restricted aeration system which means microaerobic/aerobic reciprocal bioremediation process was developed. strong modifications take place, as ammonia production with a subsequent rise of the ph value and a rapid heat evolution leading to temperatures of up to 70 • c. little is known about the microbial community in the toscano cigar fermentation and its development as fermentation proceeds. the aim of this study is to investigate the microbial community composition, its dynamic and its influence on the toscano cigar production process. our results show that the fermentation could be divided into three different phases: initially yeasts are the predominant microorganisms while bacterial growth is partially inhibited; the middle phase is characterized by exponential growth of bacteria while yeasts disappear. in the final phase the microorganism population is mostly represented by sporigen microbial species. the occurrence of yeasts in the first phase could be attributed to their ability to grow at low temperature and low ph levels. the bacterial population flourishes after the yeast cells have reached a stationary phase and probably grows on residual nutrients and autolysing yeast cells. yeasts and bacteria involved in the fermentation process were isolated and characterized. the microbial community was investigated by a combination of phenotypic and molecular approaches. the phenotypic characterization was based on both colony and cell morphology. the isolates were then identified by rrna genes sequence analysis. finally, in order to clarify the role of the identified microorganisms in the production process, a preliminary biochemical characterization was carried out. biosensors have undergone rapid development over the last few years; in particular, in environmental field many biosensors using microorganisms and purified enzymes as biological component, were recently studied. benzene is present everywhere with high levels in the cities and sometimes, in petroleum processing plants. it is classified as carcinogenic compound of first class able to cause leukaemia. because the evaluation of benzene requires complex instruments and quite long analysis times, it is required to study alternative systems for benzene detection simple, fast and highly sensitive, such as biosensors. from pseudomonas putida mst, strain previously isolated in our laboratory and able to degrade benzene, we isolated genes encoding for benzene 1,2-dioxygenase and cis-1,2-dihydrodiolbenzene dehydrogenase to use in the development of two different hydrocarbon biosensors based on microorganisms and on purified enzymes. the genes isolated were cloned in pvlt33 and we developed three microbial systems carrying: (1) benzene dioxygenase, (2) dihydrodiol dehydrogenase and (3) benzene dioxygenase-dehydrogenase modified by pcr to obtain enzymes with histidine tag. the cloning was planned to construct recombinant strains able to overproduce the enzymes; the enzymatic activities will be evaluated both using whole cells and purified enzymes. study of operation condition of biofilter using fibril-form matrix for odor gas removal don-hee park, chonnam national university, this research was performed for developing of biological treatment process of odor gas such as mek, h 2 s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over 93% was obtained by biofilm formation. at 400 ppm of inlet odor gas concentration and 10 s of retention time, the removal efficiency was 76% and 93% in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over 97% at the operational conditions above 15 s of retention time. ozonated water is produced using an ozone generator in a container filled with cold water. it is useful for sanitizing the surfaces of various products for which heat or chemical treatment is inappropriate, such as fresh food products. in this study, we investigated the antimicrobial effects of ozonated water and electrolyzed ozonated water against escherichia coli, s. aureus, bacillus subtilis and yeast, saccharomyces cerevisiae for practical use in sanitizing various products. the results demonstrated that the electrolyzed ozone water was effective for the reduction of microbial population at relatively low concentration of ozone. also, the electrolyzed and the ozonated water showed synergistic antimicrobial effects. many xenobiotics can react spontaneously with thiol moieties of glutathione (gsh), forming gsh-conjugates, or via glutathione s-transferases (gst). these enzymes participate in detoxification of potentially harmful compounds from endo or xenobiotic origin. using saccharomyces cerevisiae as experimental model, we observed that cells mutated in the gtt1 or gtt2 genes showed twice as much cadmium absorption than the control strain. we proposed that the formation of the cadmium-glutathione complex is dependent on those transferases, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. the addition of glutathione monoethyl ester (gme), a drug that mimics glutathione (gsh), to gtt1∆ cells restored the levels of metal absorption to those of the control strain. however, with respect to gtt2∆ cells, addition of gme did not alter the capacity of removing cadmium from the medium. taken together, these results suggest that gtt1p and gtt2p play different roles in the mechanism of cadmium detoxification. by analyzing the toxic effects of this metal, we verified that gtt2∆ and gsh1∆ cells showed, respectively higher and lower tolerance to cadmium stress than control cells, suggesting that although gsh plays a relevant role in cell protection, formation of the gsh-cd 2+ conjugate is deleterious to the mechanism of defense. furthermore, analyzing the harmful effects of other xenobiotic, menadione (2-methyl-1,4-napthoquinone), we have also observed that gtt1p and gtt2p isoforms play distinct functions in the process of cell protection as well as in drug remove, since both strains showed lethal phenotypes after direct exposure to 20 mm menadione. however, after adaptive treatments (mild-heat or exposure to a lower menadione concentration), cells acquired tolerance to menadione stress, although the gtt2∆ mutant had still shown a higher sensitivity against drug toxicity. by analyzing the malondialdehyde (mda) produced in response to menadione, we observed that gtt2∆ cells exhibited increased levels of lipid peroxidation, indicating that, during menadione exposure, gsh-conjugates are formed by the same transferase isoform, gtt2p, involved in cadmium stress. financial support: stint (sweden), cnpq and faperj (brazil). polycyclic aromatic hydrocarbons (pahs) are ubiquitous and persistent throughout the environment. they are generally distributed from both natural and industrial sources. many pahs can have a detrimental effect on the flora and fauna of affected habitats through uptake and accumulation in food chains, and in some instances, they induce serious health problems and/or genetic defects in humans. many research efforts have been expended to find a suitable method for remediation of soil and water environments contaminated with pahs. amongst them, the use of ligninolytic fungi is particularly suitable for the development of such processes, since they produce extracellular lignin-degrading enzymes (mnp, lip, laccase, . . .) which degrade a wide range of organic pollutants. coriolopsis rigida has been reported to produce extracellular laccase as the sole ligninolytic enzyme. this makes this fungus particularly suitable for the study of xenobiotics degradation by laccase. the purpose of this research was to obtain high laccase activities by c. rigida in solid state cultures and to determine their ability to degrade anthracene (typical pah). both in vivo and in vitro assays were performed. the former led to 60-80% degradation in 3 days depending on the culture conditions, whereas the latter showed a degradation percentage above 90% in 2 days when low mediator concentration (hbt) was added to the reaction mixture. focus will be given on pressure-driven membrane bioreactors, gastransfer membrane bioreactors and the novel ion exchange membrane bioreactor (iemb). the latter concept has been developed and currently studied by our group. this process, based on integration of donnan dialysis with bioconversion of one or more target pollutants to harmless products, has been modeled and experimentally verified for the removal of various charged inorganic pollutants such as nitrate, perchlorate and bromate by mixed microbial cultures under anoxic conditions. tests of up to 3 months showed a very good operational stability. the essential role of the microbial membraneattached biofilm, which develops naturally in this type of systems, will be also demonstrated and discussed. poly(lactic acid) (pla), which is one of biodegradable plastics, is depolymerized by hydrolysis and releases soluble monomer or oligomer of lactic acid. many bacteria can use the monomer and oligomer as an energy source or a carbon source. in this study, we applied pla to an electron donor for denitrification process of the previously developed bioreactor, which could remove ammonia from wastewater by simultaneously carrying out two biological processes, aerobic nitrification and anaerobic denitrification. a bench-scale bioreactor was constructed with a gel-plate containing pure-cultured cells of nitrosomonas europaea and paracoccus denitrificans and a pla-plate. the pla-plate was prepared by mixing three kinds of plas with different molecular weight and tricalcium phosphate to keep the constant release of the electron donor for a long term. batch treatment experiment with the bioreactor was repeated with an artificial wastewater containing ammonia for 100 days. the bioreactor could remove nitrogen from the artificial wastewater at nitrogenremoval rate of approximately 4 g n/day per square meter of gel-plate surface during the experiment period without an additional electron donor. the performance was equivalent to that obtained with our bioreactor using ethanol as electron donor for denitrification. the bioreactor using pla dose not need an additional pump for serving an electron donor (e.g., ethanol) and a hollow space for serving. therefore, the concept using solid electron donors like a pla would be effective our bioreactor to compact and simplify, and would be possible to develop a portable or disposable bioreactor. leucosporidium antarcticum as a source of enzymes for biotechnology arkadiusz wojtasik 1,2 , marianna turkiewicz 2 , jaroslaw dziadek 1 , pawel parniewski 1 : 1 centre for medical biology pas, 106 lodowa street, 93-232 lodz, poland; 2 faculty of biotechnology and food sciences technical university of lodz, stefanowskiego 4/10 street, 90-924 lodz, poland. e-mail: awojtasik@cbm.pan.pl (a. wojtasik) leucosporidium antarcticum is a psychrophilic yeast able to growth at low temperature. these microorganisms live in antarctic marine waters and are endemic to that cold environment. furthermore, l. antarcticum is also isolated from the digestive tract of antarctic krill euphausia superba. enzymes isolated from coldadapted microorganisms such as l. antarcticum having a specific activity at low temperatures ranging from 0 to 30 • c are considered for utilization at biotechnological applications such as bioremediation, production of polyunsaturated fatty acids of dietary significance and might be a source of industrially useful enzymatic proteins. the main goal of this study was to construct a cdna library of l. antarcticum. the partial cdna library was obtained and some of the clones were analysed. the sequencing analyses allowed us to find an approximately 450 base pair nucleotide sequence which displayed a very high homology to disulfide bond chaperone belonging to the hsp33 family from psychrobacter sp. high similarity of that heat shock protein was found on an amino acid sequence level and was reaching nearly 85%. the main object of our further research is to clone hsp33 family protein gene and to obtain its expression in a mezophilic host strain. also, further clones will be analysed to find other interesting genes encoding the psychrophilic proteins. this work was partially funded by the kbn grant i29/205/05. out of 9000 plant species found in the flora of turkey, about 3000 are endemic. beautiful flowering (geophytes) bulbous plants form an important part of this rich biodiversity. besides use as ornamental plants, these have great potential in perfume and pharmaceutical industry. genera of fritillaria, ornithogalum, muscari, bellevalia, tulipa, galanthus, sternbergia, crocus, arum and biarum have important and critically endangered species with high export potential that enters into this group. most of these are endangered and their collection from wild and export has been banned to conserve them. large scale production and conservation of these species could also be achieved by in vitro techniques. therefore bulb scale and immature embryo explants of sternbergia candida, s. fischeriana, muscari muscarimi, fritillaria imperialis and f. persica were cultured on different nutrient media supplemented with various concentrations of plant growth regulators using different culture applications. large numbers of bulblets were produced (over 100 bulblets/explants) from single immature embryos on nutrient media in most species tested after 12 months of culture initiation. regenerated bulblets were kept at 5 • c for 5 weeks and then transplanted to soil successfully. to our knowledge the present study is the first report for in vitro bulblet production from immature embryos of geophytes. the procedure described here provides a prolific bulblet production system that may form the basis of bioreactor culture and conservation of endemic and endangered geophytes. the commercial use of organofluorine compounds in industrial, pharmaceutical and pest-control applications has dramatically increased over the past few years, resulting in the introduction of numerous new organic compounds into the environment. organofluorine compounds are chemically very stable and are assumed to be resistant to biological degradation. given the chemical inertness of fluorinated organics, their bioactivity, and their potential for accumulation in the environment, it is important to understand their environmental fate and the mechanisms by which they might be degraded. examples of the biodegradation of fluorinated compounds in literature are scarce, being fluorobenzoic acids the most commonly reported. information on the cleavage of carbon-fluoride bonds in synthetic compounds is limited to fluoroacetate dehydrogenase. in this project we try to obtain more insight in the defluorination mechanisms by investigating the diversity of degradation routes for these compounds in several soil bacteria by making use of modern genetic tools. a gram-positive strain capable of aerobic biodegradation of 4-fluorophenol (4-fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. batch cultures were set up and substrate consumption, accumulation of intermediates and product formation were monitored. the consortium was able to use 4-fp up to concentrations of 448.4 mg l −1 and was able to utilize a range of other organic compounds. stoichiometric release of fluoride ions was measured in batch cultures suggesting that there is no formation of dead-end products during 4-fp metabolism. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene 1 , nazif kolankaya 2 : 1 kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; 2 hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth (1% soytone, 4% d-glucose and 0.5% cellulose pulp). maximal extracellular ligninase production was detected after 7 days (7 nkat). the optimum biobleaching conditions are 30 • c and ph 4.8, with 10 days. in this condition p. versicolor decreased the kapa number from 38.55 to 19.42 and increased brightness from 28 to 32.7 in 10-day treatment. boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plan-tation areas of turkey. both defficiency and toxicity problems exist in a total of about 50% of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant acquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f9 plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. butachlor is one of the selective systemic herbicide toxins that are act by inhibition of protein synthesis. this toxin is used exclusively in the rice, barely, cotton and wheat farmlands. butachlor is belonging to chloroacetanilide herbicide group, which are consisting of butachlor, alachlor, acetochlor, metolachlor and poropachlor. in the view of bioenvironmental, butachlor is degraded in the soil by microbial activity. its stability is about 6-10 weeks. it is converted to the water-soluble derivatives in soil or water, with a slow evolution of carbon dioxide. because of butachlor is one of the herbicide toxin, it is inhibitor factor against growth of bacteria and microorganisms. microorganisms can be continuing their activities in the limited concentration of butachlor. therefore treatment of industrial wastewater consist of concentrated butachlor by the biological treatment is impossible and it is necessary chemical or physico-chemical treatment are used. in this research, biological treatment methods are used. in the biological treatment, an activated sludge system with volume of 6.5 l is used. in this method, butachlor with concentration of 5 mg/l are treated. removal percent of butachlor for concentration of 2.5 and 3 mg/l are calculated to 41.20% and 41.67%, respectively. removal percent of cod is also calculated to 86%. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized purification and downstream process of xylitol obtained biotechnologically from hemicellulosic hydrolyzate of corncobs b. rivas 1 , p. torre 2 , j.m. domínguez 1 , j.c. parajó 1 , a. converti 2 : 1 department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, 32004 ourense, spain; 2 department of chemical and process engineering, genoa university, via opera pia 15, 16145 genoa, italy. e-mail: brivas@uvigo.es (b. rivas) biotechnological production of xylitol from lignocellulosic materials has been widely studied in the recent years with promising results that confirming the possible industrial application of this technology. xylitol purification from fermented broth is the limiting stage of this process. previous works suggest crystallization procedures in order to recovery xylitol from fermented synthetic solutions. the complexity of fermented hydrolyzate not allows direct crystallization. in this work, corncobs hydrolyzate obtained with autohydrolysis-posthydrolysis techniques, detoxificated with activated charcoal and concentrated was fermented to xylitol by d. hansenii. the fermented broth composition was 64.9% of xylitol (68 g/l), 13.3% of other sugars and 21.8% of other compounds that interferes in the crystallization process (as dry matter of the liquor). the fermented media was submitted to an absorption process with activated charcoal and concentrated until a xylitol concentration of 340 g/l. the liquor was then submitted to a second step of precipitation with ethanol, the best results achieved in this study were obtained with an ethanol/liquor ratio of 4. in these conditions this treatment allows to remove a 56.9% of the impurities. the resulting solution was evaporated and crystallized containing 60% of ethanol and a xylitol concentration of 443 g/l. crystallization was performed at t = 5 • c with slightly agitation. after 36 h were separated xylitol crystals with a recovery yield of 13% and a purity degree of 90%. numerous publications have documented that only a minor number of the indigenous prokaryotic organisms found in complex environments such as the human intestine, biogas reactors, and soil are known, and probably only a fraction of this diversity can be accessed using traditional culturing techniques. some of the reasons for this are the lack of knowledge of specific growth conditions, specific nutrients, and obligate coculture requirements. also growth on a solid surface directly exposed to the atmosphere puts a very strong selective pressure on single cells supposed to develop into visible colonies. therefore, the knowledge of these microorganisms is scarce and generally limited to the 16s rrna genes that have been extracted from different environments and cloned for phylogenetic analyses. an obvious approach to circumvent these problems was the development of techniques based upon micromanipulation for isolation of single cells from complex mixtures. continuous development of modern microscopes in combination with the precision of a servo-powered micromanipulator and the development of the modern microscopic micro injectors used in ivf techniques has further aided the manipulation of single cells. this technique, however, does not solve the problems of the non-culturable cells, and other approaches are needed to gain more information about these organisms. an approach to the non-culturable cells could be genomic analysis of isolated single cells without preceding cultivation. this pcr-based technique is widely used for genetic analysis of human cells, but due to the small amounts of dna present in prokaryotic cells it has so far not been possible to produce identifiable amounts of dna from single cell amplification using conventional polymerases. a promising alternative used for amplification of small amounts of dna is the f29 dna polymerase operating under isothermic conditions. applying random hexamer primers, this polymerase carries out a multiple displacement amplification (mda) of high molecular weight dna template. in this study we demonstrate the successful application of mda for selective amplification of genomic dna from a single prokaryotic cell. the yield was >20 mg of amplified genomic dna corresponding to about a 5 billion-fold amplification from a single cell. the technique was used to approach a large group of non-thermophilic archaea found in agricultural soil. our results show that combining mda with fluorescent in situ hybridization and cell isolation by capillary micromanipulation enables an unprecedented ability to investigate new species without cultivation. also this combination of techniques opens for studies of genetic heterogeneity within populations and processes such as horizontal gene transfer. precipitation of zn 2+ , cu 2+ and pb 2+ at bench scale using biogenic hydrogen sulphide produced from the utilization of volatile fatty acids by sulphate reducing bacteria maria teresa alvarez 1,2 , carla crespo 2 , bo mattiasson 1 : 1 department of biotechnology, center for chemistry and chemical engineering, lund university, p.o. box 124, s-22100 lund, sweden; 2 instituto de investigaciones fármaco bioquímicas, universidad mayor de san andrés, la paz, bolivia biological production of hydrogen sulphide (h 2 s) from sulphate using sulphate reducing bacteria (srb) is popular within environmental biotechnology. srb require absence of oxygen, presence of nutrients required for growth and oxidizable organic substrates (to supply hydrogen atoms for reduction of sulphate). many organic wastes have been used as electron donors for the sulphate-reducers in the treatment of acid mine drainage (amd) including straw, hay, sawdust, peat, spent mushroom compost and whey, however, other wastes such as municipal organic waste can be used. the aim of this work was to study the possibility of using srb for the treatment of amd at bench-scale. this process involved three stages: the volatile fatty acid (vfa) production by hydrolytic bacteria from the degradation of vegetables and fruits, the production of h 2 s through the utilization of the produced vfas by sulphate reducing bacteria and the precipitation of metals by using the biologically produced h 2 s. the substrates used for vfa production consisted of tomato, papaya, apple and banana. the h 2 s produced from the degradation of vfas was utilised for the precipitation of an artificial effluent simulating the heavy metal concentrations of a mine located at bolivian andean region, containing approximately 9 mg/l of zn +2 , 8 mg/l of cu +2 and 4 mg/l of pb +2 . the maximum concentration of hydrogen sulphide obtained was approximately 17 mm. removal efficiencies of 97%, 98% and 100% for zinc, cooper, and lead, respectively, were achieved in the present work. at 30 • c during 14 days. bacterial population was determined by counting in a neubauer chamber with optical microscope. sulfate concentration was measured by turbidity method and metal concentrations in the filtered supernatant were measured by icp-aes. the first part of study consists of determine the maximum concentration of each metal at which d. vulgaris and desulfovibrio sp. grow in similar way than control culture (without metal). both cultures tolerate: cr(iii) 15 ppm, ni(ii) 8.5 ppm, zn(ii) 20 ppm. the maximum precipitation percentages were approximately: 25% (15 ppm cr(iii)), 96% (8.5 ppm ni(ii)) and 99% (for d. vulgaris-10 ppm zn(ii) and desulfovibrio sp.-15 ppm de zn(ii)). time to reach the highest precipitation was minor for mixed culture (desulfovibrio sp.) y all the cases. the next part was focused to study the precipitation percentage when metals are present in combination in the same metal levels (cr(iii)-ni(ii), cr(iii)-zn(ii), ni(ii)-zn(ii) and cr(iii)-ni(ii)-zn(ii)). the combination of metals does not affect significantly the bacterial growth and precipitation percentage of metals. this fact supposes an importance advantage so metals are commonly found together in the environment. future experiments are focused in development of this process in continuous operation mode. biosolubilisation and depolymerisation of coal has potential to produce a clean energy source or high value organic products from low rank coals such as lignite or sub-bituminous coal. these complex soluble phenolic compounds are of value as starting materials for biotransformation to value-added compounds such as antioxidants and flavourants. the bioprocess is carried out at ambient temperature and pressure and is perceived to be environmental benign. in the evaluation of coal solubilisation an important quantity for the assessment of process feasibility is the yield, i.e. the determination of the mass of product obtained per unit mass of coal solubilised. to date, results for coal biosolubilisation reported in the literature are qualitative or at best semi-quantitative, indicating trends with operating variables. the process kinetics has not been determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. in this paper, the use of an indirect method for the estimation of the growth and metabolism of fungal biomass by measuring co 2 evolution and o 2 consumption using an off-gas analyser is reported in the study of fungal coal solubilisation. coal determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. biosolubilisation was carried out in a stirred tank slurry bioreactor with working volume of 1.0 l. complete suspension of the coal particles of 650-800 m mean diameter was achieved at an agitation rate of 560 rpm. growth yield coefficients based on coal and oxygen as well as maintenance coefficients were calculated from growth of the fungus under the same conditions using a non-coal carbon source such as glucose. these data were used to determine the stoichiometric coefficients for biomass growth, enabling the biomass production rate to be quantified in terms of co 2 production rate and o 2 consumption rate. a dna-chip platform for parallel detection of microorganisms related to biofilm in industrial systems and drinking water systems pernille skouboe, dorte lauritsen, kim holmstrøm bioneer a/s, kogle allé 2, dk-2970 hørsholm, denmark. e-mail: psk@bioneer.dk (p. skouboe) an oligonucleotide microarray for simultaneous detection and identification of pathogenic bacteria related to technical water systems as well as drinking water has been developed. the approach is based on the use of a tandem hybridization technique with two ribosomal 16s rdna-pcr products, 1000 bp and 500 bp long, generated from two consensus pcr reactions using conserved ribosomal primers end-labeled with cy3 and cy5, respectively. the tandem hybridization technique implies an internally quality control for discrimination between target and non-target signals. the current prototype of the dna-chip platform includes 20 oligonucleotide probes representing 11 different genera (and subgroups of species), e.g. legionella, mycobacterium, aeromonas, campylobacter, vibrio and enterococcus. the platform has been used for detection and identification of species from pure cultures, and initial experiments with water samples from industrial systems have been performed. the potential as well as the limitations of using a dna-chip based detection format in its present form will be documented. particularly, its potential application as a rapid method for initial screening of environmental or food samples will be addresses. the aim is to reduce and optimize the number of samples required for traditional microbiological identification tests. in the course of a project for the development of a novel kind of a mycotoxin inactivating feed additive, the aim of this study was to isolate and characterize microorganisms with the specific ability to enzymatically break down and detoxify fumonisins, a group of structurally related fungal toxins, with fumonisin b 1 (fb 1 ) being the most abundant and -with respect to toxicology -also the most important representative of this group. these toxins are produced as secondary metabolites by some fusarium species such as fusarium verticillioides and f. proliferatum and are naturally occurring contaminants of cereal grains worldwide. they are found especially in maize and maize based products, and are known to be hazardous to human as well as to animal health. a natural feed additive, based on microorganisms and/or enzymes, should ensure the detoxification of fumonisins during feed uptake and digestion via microbial or enzymatic break down of these compounds, by that protecting the animal from the harmful effects of these mycotoxins. besides an extensive screening of microbial strains derived from strain collections, various different natural habitats were investigated for the presence of fb 1 degrading microbial activity, such as intestinal contents of pigs, soil samples, and naturally fumonisin contaminated maize. while testing of nearly 150 organisms from strain collections did not show positive results, fumonisin transforming activity could be detected in one soil sample and a number of maize samples. trials in order to isolate the respective fumonisin degrading microorganisms resulted in a number of strains, whose fb 1 degrading activity could be proven. the most promising bacterial and yeast strains were further characterized with regard to a general taxonomic description, and to different aspects of their toxin degradation behaviour. approaching a more relevant in vivo situation, fb 1 degradation trials in food-and feed-stuffs were conducted. further on, the applicability of the respective organisms as stabilized lyophilisates was investigated. arsenic is one of the most important global environmental pollutants and the toxicological effects are related to its chemical form and oxidation state. arsenite [as(iii)] is reported to be on average 100 times more toxic than arsenate[as(iv)]. this work shows the ability of one strain of the species ochrobactrum tritici to grow in presence of several metals including arsenite, arsenate, selenite, selenate, tellurite and antimonite. its arsenite mic was determined as 50 mm, whereas for arsenate, this bacterium could resist to concentrations upper than 200 mm. we report the identification of two loci involved in high-level arsenic resistance. sequencing of the first locus identified four complete genes in the following order: arsr, arsd, arsa, arsb. the second locus containing genes for arsenic resistance was also characterized. each sequence has been compared with nucleotide and protein databank (blast programs) and significant homology with known orfs coding for arsenic resistance has been found. it is also possible that the phenomenon of high-level arsenic resistance in o. tritici could evolve other genes or loci. the ability of the ␣-proteobacterium o. tritici to tolerate high levels of arsenic in addition to other oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. this work is based on the mathematical modeling of kinetics of a thermophilic bacteria cultivation system. the cultivation proceeded by way of batch and continuous on the synthetic medium with the main carbon source-lactose. this medium simulated an industrial waste approximately. mixed thermophilic aerobic bacteria popula-tion, applied to the wastewater treatment (sludge v&k bystrice pod hostynem), was used to the inoculation. the cultivation system consisted of the laboratory fermentor biostat b (b. braun biotech) with working volume 2 l and the control unit connected with a computer. it is possible the temperature, ph, aeration, stirring and foaming regulation. physical and chemical cultivation conditions were optimized. the chemical oxygen demand (cod), generally expressive the impurity level, was selected as the main parameter for the cultivations run classification. but into the mathematical model also the kinetics of biomass growth, lactose consumption, production of choice metabolites (acetate, lactate, succinate) and dissolved oxygen concentration was included. the modeling was located to two head distinguishable growth phases of the microorganisms. an optimization and identification of mathematical model parameters was practised in the software language psi/c. the difference between simulated curves and experimental data is not statistically significant on the relevancy level 0.05 (f-test). it took place the cod degradation at 74.0% with the average yield coefficient y chsk/x 3.8 g cod/g biomass in the batch process with the air aeration only. there is a better way to the cod elimination (>90.0%)-the aeration of air enriched by the pure oxygen. in experiments with the continuous system was obtained the 69.0% cod decrease after the steady state stabilization. this work was supported by project msm 0021630501. fluorescence in situ hybridization (fish) of whole cells using oligonucleotide probes was applied to study the influence of low temperature and temperature reduction on the bacterial community of biofilm reactors for the removal of chlorophenols (cps). two packed bed reactors were set up for degradation of a mixture of 2-cp, 4-cp, 2,4-dicp, and 2,4,6-tricp as sole source of carbon and energy at 14 • c (ra) and 24 • c (rb) and were inoculated with bacterial consortia adapted to these respective initial temperatures. the performance of the reactors was studied under different conditions of pollutant loading, aeration rate, and hydraulic retention times over 7 months. total chlorophenol removal capacities of 1240 and 1420 mg l −1 day −1 were achieved in the bioreactors ra and rb, respectively, under a total pollutant load of 1440 mg l −1 day −1 . the population of ␤-proteobacteria was the major bacterial community of the biofilm (35-47%) followed by the ␥-proteobacteria (12-6.5%). two bacteria with the ability to mineralize 50 mg chlorophenols l −1 were isolated from the bioreactors and characterized as ralstonia basilensis and alcaligenes sp., both belonging to ␤-proteobacteria. decreasing the temperature by 10 • c (in two steps of 5 • c each) resulted in an increase in the population of ␥-proteobacteria and a decrease in the population of ␤-proteobacteria in both reactors. application of genus specific probes showed an increase in the pseudomonas population from 25% of the ␥-proteobacteria at 14 • c to 59% at 4 • c. the pollutant removal capacity decreased to 548 and 833 mg l −1 day −1 in ra (4 • c) and rb (14 • c), respectively. the ␣and ␦-proteobacteria, cytophaga-flavobacteria and actinobacteria the survey was carried out in urban and rural areas of two cities (ankara and isparta). this paper is only analysing urban people by excluding villagers. urban sample is consisted of 400 urban consumers and 200 professionals. professionals were selected amongst pharmacists, doctors, agricultural engineering's and industrialists that is thought are affective in the process of developing new technologies and also the development of biotechnology in the society. basic data was gathered by a questionnaire including both structured and open-ended questions besides deep interviewed. workforce development for life sciences-the scottish experience carol booth scottish entreprise, uk the presentation will look at, the background and definition of workforce development for scottish enterprise, examine information available to support scottish enterprises economic intervention and where and when to intervene. conclusions emerging from the evidence base will be used to outline scottish enterprises approach to workforce development and look at which actions might be required to address identified issues. moving on to reasons for integrating workforce development into business development and how the life sciences cluster team at scottish enterprise, stakeholders and partners in scotland have approached their current and future contributions to workforce development for life sciences using a variety of projects. the national institute for bioprocessing research and training (nibrt) in ireland is a proposal that will be a state-of-the-art training, research and pilot plant service facility that brings together institutions with complementary expertise and state-of-the-art research technology, and industry partners. these include university college dublin, trinity college dublin, institute of technology, sligo and dublin city university. nibrt is an innovative collaboration between academic institutions at the forefront of biotechnology, cell biology, engineering and pharmacy and industry. for training, two separate training labs, for upstream and downstream training, in addition to 5 research labs are planted. it will also include a state-of-the-art pilot plant fermentation facility for fermentation optimisation, fermentation scale-up, product separation and purification, regulatory aspects and automation. by aligning with industrial demands, the new institute will tailor its training programmes while remaining on the cutting edge of biotechnology research and technologies. the fermentation facility will offer hands-on training workshops and educational modules for outside researchers and companies. these workshops cover the fundamentals of small-scale fermenta-tion, scale-up considerations, and fermentor design and set-up. the training and educational philosophy underpinning the nibrt will focus on the needs of industry with an emphasis on providing training for accreditation of existing industry staff and prepare technicians and graduates for the technical, business, regulatory and professional aspects of the industry. the strategy is to provide specialised modules in nibrt in support of courses established in the higher education institutions, which will provide the certificates, diplomas and degrees. modules will be offered for all categories of students and will be given credits respected by other third level institutions in ireland. the role of professional graduate degrees in meeting current and future biotechnology industry workforce needs a. stephen dahms san diego state university, usa the presentation will review the status of new graduate training models designed to meet the unique needs of the biotechnology industry as it transitions to commercialization. emphasis will be on professional master's degree programs in biotechnology and their various versions, with a focus on operational and funding strategies and industry acceptance. discussion will also centre upon the creation and operation of industry-validated, specialized and highly targeted professional masters degrees in various refined aspects of the drug development process, including regulatory affairs, biomedical quality systems, clinical affairs, management of drug development, management of reimbursement affairs, bioinformatics, etc. the eurodoctorate in biotechnology, new combined mba/phd combined degrees in the molecular life sciences, the u.s. professional doctorate in chemistry and the proposed u.s. professional doctorate in biotechnology will be also discussed. data will also be presented on the current workforce and the industry's projected needs. genetic studies show that mankind is a rapidly expanded population of closely related individuals with very similar disease sensitivity. bad nutrition and infections dominate among the main health problems in the world. apart from malnutrition, the overeating habits of the developed world are now creating problems in the developing world as well. infectious diseases are also a global problem since new contagious agents like hiv, sars and avian flew do not recognize borders. thus, the global responsibilities of modern health care are obvious. many research scientists from and in developing countries find it nearly impossible to use their talents for the benefit of their own countries. some struggle to develop research and education programmes with poor facilities, some leave science completely, and others migrate to more developed countries. the talents of such people are either being wasted or lost completely to their home countries just at the time they are most needed to combat the great humanitarian challenges of hunger, illness and lack of knowledge. europe must strengthen programmes which allow third world scientists to work to their full potential in their home countries or regions. yang beijing genomics institute, chine academy of sciences, beijing, china europe has all the reasons to be proud of being the cradle of modern science and of its achievements and resources in life sciences. as a model of having solved many of the problems that many other counties are now facing, europe is expected by the whole world to make its further contribution to a better future of mankind and to play a more important role in the international community of life sciences. tropical diseases and public health basilio valladares director of the university institute of tropical diseases and, public health, university of la laguna, 38200 la laguna, tenerife, spain. e-mail: bvallada@ull.es the presentation will look at the main research interests of the university institute of tropical diseases. these are the following: 1. immunology and molecular biology of parasites. we express and purify recombinant proteins from leishmania sp. which have been shown to act as immunomodulators and protect against disease such as l25, hsp70, hsp83. the study of acanthamoeba pathogenic factors has also resulted in the isolation and silencing of extracellular proteins related to their pathogenecity, which has a great potential in the development of novel chemotherapeutics. 2. diagnosis of parasites. the immunological diagnosis of leishmaniasis has been one of the main research interests in our laboratory for several years. as a result, we have identified peptides which could be used to develop kits for the immunological diagnosis of leishmaniasis such as hsp70 c-end, l25 n-end, etc. we have also developed some dna based methods for the identification of acanthamoeba species from biological and environmental sources. 3. water quality. biological parameters. our water research group has the expertise to identify and characterise bacterial, viral and parasitic indicators of faecal contamination in diverse water sources including tap water, rivers, reservoirs, sea, etc. this research area has been developed in collaboration with the local sewage treatment plant and reservoir managing authorities. currently, we are establishing a conjoined project with the center for disease control and prevention (cdc) in atlanta, usa for the identification of water-borne emerging pathogens. 4. development and formulation of chemotherapeutic antiparasitic agents. in this field, we evaluate the leishmanicidal activity both in vivo and in vitro of natural and synthetic drugs and synthetic peptides. in a later stage, the drugs which have shown the highest antiparasitic activity have been subjected to cytotoxicity assays and their molecular targets dissected. some of the drugs tested in the last few years have been submitted to patent due to their outstanding activity. finally, in order to allow the commercialization of these drugs, both in vivo and in vitro assays are being carried out to predict their chemical stability and degradation pathways. this will be followed by the use of liofilization and controlled crystallisation strategies for the development of efficient and safe treatments. 5. human and population genetics. tachykinins and their receptors in different tissues and groups of patients and their association with molecular polymorphisms is another one of our research interests. the knowledge of ligand and receptor sequences and their similarities will allow the rational development of drugs with specific activity against these receptors. furthermore, we are also interested in the interspecific variation along the evolutionary scale of these markers. 6. nitrate assimilation group. research in our group is focused on understanding nitrate assimilation in the yeast hansenula polymorpha. several biotechnological companies use this yeast to produce heterologous proteins (hepatitis b vaccine). genetic manipulation techniques for h. polymorpha are available in our laboratory. head of the department of biotechnology, technological institute of canary islands, pozo izquierdo 35119 santa lucía, las palmas, spain single cell analysis by flow cytometry has proved to be a tool to perform simultaneous and rapid measurements related to cell morphology and physiological state. previous studies showed the possibility of quantifying neutral and polar lipids spectrofluorometrically using a lipid specific fluorescent dye, nile red (nr), however the existence of inter and intraspecific variations in the fluorescent response had not been clearly established. in this work, two strains of marine microalgae: crypthecodinium cohnii and tetraselmis suecica, characterized both by high contents of polyunsaturated long chain fatty acids (dha and epa, respectively) and an hypersaline microalgae: dunaliella salina, characterized by a high ␤-carotene production, were grown under different conditions and collected at different growth phases to be used for in vivo lipid quantification with nr by flow cytometry. our results showed a high correlation between the mean fluorescence signal of nr stained cells and the neutral and polar lipid content measured by gravimetry for each strain. in this respect, these data make feasible the development of a rapid method for lipid quantification in monoalgal cultures. however, differences in the dye uptake related to specificity were detected. in this communication we also assess the possibility of use this cytometric technique to select microalgal strains with high lipid and polyunsaturated long chain fatty acids content from mixed samples. performance of such technique would be a good alternative to the time-consuming traditional screening protocols based on gravimetry and gas chromatography and would optimise the search of new commercial strains of microalgae. claverie-martín head of research unit, biomedical research institute, hospital universitario, de n.s. de candelaria, santa cruz de tenerife, spain our group is involved in the cloning and production of proteins of interest to the food and pharmaceutical industry. we have recently expressed in yeast the cdna that encodes the precursor of caprine chymosin. chymosin is the enzyme responsible for the coagulation of milk in the abomasum of unweaned calves. this enzyme is secreted by gastric mucosa cells as an inactive precursor, known as prochymosin. in the acidic conditions of the lumen, prochymosin is converted into the active form by autocatalytic cleavage of the n-terminal prosequence. chymosin is used extensively in cheese production because it cleaves -casein in a specific manner with low proteolytic activity. several biotechnology companies are producing the bovine recombinant enzyme for commercial use in the process of cheese making. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. it is well known that the activity of these types of extracts varies depending on the age of the animal and the type of food ingested. these difficulties should be overcome using a recombinant caprine chymosin. the caprine mrna used for the synthesis of the cdna was obtained from the abomasum of milkfed kid goats. the cdna fragment encoding the mature portion of caprine prochymosin was fused in frame to a signal sequence in yeast expression vectors. culture supernatants of yeast cells transformed with the recombinant plasmids showed milk-clotting activity after activation at acid ph. proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed -casein (patent 200402025). the recombinant caprine chymosin could be an alternative milk coagulant in cheese making. work is underway to optimise the expression of the new recombinant prochymosin for further purification and characterization. smart molecules for health victor martín head of research, university institute of bio-organics "antonio gonzález", avda. astrofísico francisco sánchez 2, 38206 la laguna, tenerife, spain the instituto universitario de bio-orgánica "antonio gonzález" (iubo) is a multidisciplinary research centre that belongs to the university of la laguna. the iubo is located at the town of la laguna, inscribed on unesco's world heritage list in 1999, and former capital of the canary island of tenerife. the geographical location in addition to its mild climate has made the canary islands to posses a variety of ecosystems with unique plants and animals. the institute was started up in the 1960s with the need to study the natural products and secondary metabolites produced by those marine and terrestrial organisms, thus providing a new source of bioactive products. the main research lines that are being developed at iubo are summarised in the following paragraphs: anticancer agents from natural sources: several natural products and their semisynthetic derivatives are produced at the iubo in diverse joint projects for the development of new antitumour drugs with novel mechanisms of action. as an example we can mention natural products from the mevalonic, shikimic or polyketide pathways. some products have recently shown in vitro reversion of the resistance in multidrug resistant (mdr) tumour cell lines. genetic engineering: in vitro cultures of the plants atropa baetica, maytenus amazonica and m. macrocarpa are developed in order to manipulate their biosynthetic pathways and induce the production in large scale of the secondary metabolites for diverse applications, including arthritis, rheumatism, and back pain. marine organisms and toxins: dinoflagellates are marine organisms responsible for the red tides and food poisoning episodes. among others, okadaic acid and yessotoxin are the most common toxins present in european shellfish. the isolation of these products is best done from the microorganism cultures, since they are present in very low amounts in the natural sources. at the iubo we develop culture systems to provide us with amounts of toxins large enough to perform biological, metabolic and bioactive studies. insecticide and repellents: natural products are being isolated for their use against plagues, specially those affecting agriculture. these projects are run in collaboration with a number of public institutions and agrochemical companies throughout europe and latin america. fine chemicals and pharmachemicals: our institute possesses large expertise in the field of organic synthesis devoted to the synthesis of medicinal substances, with special focus on asymmetric processes. of particular interest is the development of new methodologies for the total synthesis of biologically active substances like polyether toxins, (un)natural amino acids, sphingosine analogs, alkaloids, etc. with an annual source of 100s of new compounds, the fine chemicals and medicinal chemistry branch at iubo have recently started and anticancer screening program in collaboration with the biomedical research unit at the hospital universitario n.s. de la candelaria. the program is committed to the discovery of novel drugs for application in cancer treatment. the outcome of this project in its first year has been outstanding, leading to the finding of several leads that form the basis for current and future projects. institute of canary islands, biotechnological department, playa de pozo izquierdo s/n, 35119 santa lucía, gran canaria, spain the presentation will look at the main research interests of the biotechnological department of the technological institute of canary islands. these are the following: nutrition and feeding in aquaculture: we conduct studies on digestion, absorption, transport, and utilization of the different nutrients applying also different techniques such as histology, enzymology, genetic or immunology among others. aquaculture feeding is the main important cost in fish farms, being higher that the prize of fries, personnel cost or energetic costs. thus, studies conducted on the improvement of diet formulation and the use of different dietary ingredients is one of our main research lines (such as vegetable oils and meals to be used as alternative to fish meal and oil, or carotenoid sources to improve fish colour). not only the use of the different ingredient is studied, but also the effect of these ingredients on fish health, flesh quality, flesh healthy aspects related with human consumption, are being studied. different formulae are being developed and patents of different diets are being obtained. studies on nutritional requirements are also conducted allowing us to patent different formulae in larvae studies, developing microdiets to substitute the high-cost processes associated with live prey in larval nutrition. development of immunostimulants, anti-stress diets, immune techniques to be applied as bio-indicators of fish health and welfare, as well as use of dietary ingredients derived from bio-reactors are other research lines in our group. genetics: genetic techniques applied to aquaculture are being an important tool. microsatellites are being used to determine genealogy of the fish, allowing to decreases important problems in aquaculture such as fish deformities. genetic techniques such as micro-arrays and gene expression are being applied to obtain indicators of stress and health in fish. these technologies also permit to make different selective breeding programs, increasing the accuracy to estimate genetic parameters and evaluating brood-stock. furthermore, this technology allows to obtain a procedure to increase the productivity and quality of fish hatcheries. new species for aquaculture. development of new culture techniques: the diversification of species cultured is one of the main objectives of european aquaculture, since nowadays only four marine species are commercially produced: gilthead sea bream. european sea bass, turbot and salmon. we have been developed rearing techniques for new species such as red porgy or canarian abalone and also we are conducting studies on different new species, such as different sparids species, octopus or yellowtail. new rearing technology: the election of adequate systems for fish growth for each species and site of production and the localization of more appropriate sites for farms, using gis technology are also of special importance in our research team. besides, new larval rearing techniques such as semi-extensive hatchery (mesocoms) were development and nowadays is being used to increase fry quality (survival, no-deformities, better growth). this technology is being exported to other countries in order to offer new technology easy to manage to be implanted in developing countries. alejandro cañeque project officer, canary islands special zone, ministry of finance, c/leon y castillo 431 -4 a planta, edificio urbis, 35007 las palmas, spain. e-mail: acaneque@zec.org the canary islands special zone is the newest tax instrument within the canary islands economic and fiscal regime (ref). it offers a reduced tax rate of between 1 and 5% of corporate income tax for companies setting up a business. the companies must cover the following minimum requirements: create employment and make a minimum investment. the zec offers other tax advantages such as the exemption from paying transfer tax and stamp duty and the canary islands general indirect tax (igic). this tax scheme particularly fosters the biotechnology and pharmaceutical sectors. references fahnert references bechor the antimicrobial drugs high-level production of human collagen prolyl 4-hydroxylase in sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates microbial processes and products press. biotech. bioeng. 218. van hijum microbial xylitol production from corn cobs using candida magnoliae the role of bacillus methanolicus citrate synthase gene, city, in regulatiing the secretion of glutamate in lysine-secreting mutants plasmid-dependent methylotrophy in thermotolerant bacillus methanolicus 1,5-anhydrod-fructose; a versatile chiral building block: biochemistry and chemistry detailed dissection of a new mechanism for glycoside cleavage: the ␣-1,4-glucan lyase ␣-1,4-glucan lyase, a new class of starch and glycogen degrading enzyme ␣-1,4-glucan lyase, a new starch processing enzyme for production of 1,5-anhydro-d-fructose efficient purification, characterization and partial amino acid sequencing of two ␣-1,4-glucan lyases from fungi ␣-1,4-glucan lyases producing 1,5-anhydro-d-fructose from starch and glycogen have sequence similarity to alpha-glucosidases enzymatic description of the anhydrofructose pathway of glycogen degradation i. identification and purification of anhydrofructose dehydratase, ascopyrone tautomerase and ␣-1,4-glucan lyase in the fungus anthracobia melaloma a systems approach to dissecting immunity and inflammation integrative biological analysis of the apoe*3-leiden transgenic mouse innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble cd14 soluble forms of toll-like receptor (tlr)2 capable of modulating tlr2 signaling are present in human plasma and breast milk proteomics of the rat gut: analysis of the myenteric plexuslongitudinal muscle preparation an integrative metabolism approach identified stearoyl-coa desaturase as a target for an arachidonate-enriched diet the challenges of modeling mammalian biocomplexity rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by mudpit technology post-genomics of lactic acid and other food-grade bacteria to discover gut functionality genetics, metabolism and application of lactic acid bacteria. fems microbiol complete genome sequence of lactobacillus plantarum wcfs1 functional ingredient production: application of global and metabolic models metabolic engineering of lactic acid bacteria for the production of nutraceuticals reduction in antinutritional and toxic components in plant foods by fermentation the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no 2001k120240-41). the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no. 2001k120240-40) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aç ikel at gazi university. the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no 2001k120240-40) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aç ikel at gazi university. mrs. lynnette fernandez, johanna mäkeläinen, m.sc. and olli rämö, m.sc. are gratefully acknowledged for their help. this work was supported by the health science council, the academy of finland and the tekes-neobio program. supported by the mec of spain (ppq2002-04361-c04-02) and the canary islands government. jmp thanks icic for a postdoctoral fellowship. frpc thanks cajacanarias for a fpi fellowship. tm thanks the spanish mcyt-fse for a ramón y cajal contract. this work was supported by the korean systems biology research grant from the ministry of science and technology, lg chem chair professorship, ibm sur program and bk21 program. this study was supported by ankara university biotechnology institute (project no: 89). this work was partially supported by mec and feder (bio2004-00439), and fundación séneca carm (00842/pi/01). this work was supported by grants f037663 of the hungarian scientific research fund and grant omfb-00219/2002 of the hungarian ministry of education. keto sugars have long been implicated as attractive intermediates or substrates for further chemical or enzymatic reactions, to generate a number of synthetic sugar derivatives and fine chemicals. the quinone-dependent pyranose dehydrogenase (pdh) purified of the basidiomycete fungus agaricus meleagris catalyzes with high specificity the oxidation of the c-3 of glycosidically bound d-glucose, whereas in contrast it oxidizes simultaneously the c-2 and c-3 of free d-glucose. considering the broad substrate tolerance, pdh provides a new convenient tool for high yield production of 3-ketothis work was partially financed by the scientific and technological research council (cicyt, spain), grant ren2001-3224, by the iii pla de recerca de catalunya (generalitat de catalunya), grant 2001sgr-00143, and by the generalitat de catalunya to the "centre de referència en biotecnologia" (cerba). this work was supported by mega a.s. (czech republic) (www.mega.cz) and following vega grants: 1/2391/05 and 1/2390/05. this work was partially supported by mec and feder (bio2004-00439), and fundación séneca carm (00842/pi/01). financed by a marie curie re-integration grant merg-ct-2004-006378 and consejería de educación y ciencia de la junta de andalucía. this project is part of the collaborative research centre sfb 578 "development of biotechnological processes by integrating genetic and engineering methods", which is supported by the german research foundation dfg. this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658 and d48537) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb00103/2005). this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658 and d48537) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb00103/2005). this research was supported in part by grants from the hungarian scientific research fund (otka t37471, f46658, d48537) and gvop-3.1.1.-2004-05-0471. the authors thank dr. hamid narjiss (director of morocco inra) for the instruction to identify nadorcott mandarin by molecular markers and helpful discussions regarding this paper. this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). this work has been financed by xunta de galicia (pgidt03pxib30103pr). sevgil sadettin, gönül dönmez department of biology, faculty of science, ankara university 06100 beşevler, ankara, turkey this study was supported by ankara university biotechnology institute. demet ç etin 1 , sedat dönmez 2 , gönül dönmez 1 : 1 department of biology, faculty of science, ankara university, 06100 beşevler, ankara, turkey; 2 department of food engineering, faculty of engineering, ankara university, 06110 dışkapı, ankara, turkey sulfate-reducing bacteria (srb) that could grow on modified postgate c medium (pc) containing chromium(vi) were isolated from industrial wastewater and their chromium(vi) reduction capacities were investigated as a function of changes in the initial ph values, chromium, sulfate, nacl concentrations and carbon source. the optimum ph value at 50 mg l −1 initial chromium(vi) concentrations was determined as 8. chromium(vi) reduction by srb was investigated at 22.7-98.4 mg l −1 initial chromium(vi) concentrations. at the end of the experiments, the mixed cultures of srb were found to reduce more than 99% of the initial chromium(vi) levels which ranged from 22.7 to 74.9 mg l −1 within 2-6 days period. the effects of initial 0-9.0 g l −1 concentrations of sulfate and 0-6% (w/v) concentrations of nacl to chromium reduction were showed that, the lowest concentrations of sulfate and nacl, were the best for chromium reduction in the pc media including 50 mg l −1 chromium(vi). when the 25% whey was used as carbon source in the pc medium, 99.6% of the 65.9 mg l −1 initial chromium(vi) concentration was reduced within this study was supported by ankara university biotechnology institute. this study was carried out as a part of the project for analyzing and controlling the mechanism of biodegrading and processing entrusted by the new energy and industrial technology development organization (nedo). this work has been financed by xunta de galicia (pgidt03pxib30103pr). lead ions are considered a high pollutant of different waters. in our previous work were selected seven potential sorbent-strains rhodotorula mucilaginosa 1776, rh. aurantiaca 1195, rhodotorula sp. 4, williopsis californica 248, candida krusei 61t, cryptococcus sp. wt of lead ions. it was determined their stability to high concentration (up to 750 mg/l) of lead ions in medium. the ph changes and yeast physiologies of growth were studied in medium with these heavy metal ions. the influences of environmental factors such as ph of solution, age of microbial culture, biosorbent concentration in suspension, alive or living state of biosorbent, time of contact on sorption were investigated. the levels of maximal sorption ability and biomass affinity to heavy metal ions were established by experimentally received sorption isotherms with mathematical modeling of biosorption process separately for everyone researched yeast strain. the sorption isotherms obtained in these experiments for non-living yeast biomass showed that the maximal sorption capacity was 225 and 195 × 10 −6 mol (g sorbent) −1 for rh. aurantiaca 1195 and s. cerevisiae 1968, respectively. in the case of living biomass, the highthis work has been financed by the spanish ministry of science and technology and european feder (project ctm2004-01539). the authors wish to thank dra. m.j. martínez (cib, csic, madrid, spain) for providing coriolopsis rigida. this work was supported principally by embo and the mrc. fed-batch cultivation of haematococcus pluvialis under illumination with leds for production of astaxanthin abdolmajid lababpour, tomohisa katsuda, shigeo katoh department of molecular science and material engineering, kobe university, kobe, hyogo 657-8501, japan photosynthetic microalga haematococcus pluvialis is a most promising microorganism for production of astaxanthin, which has powerful antioxidant activity and is used both for human being as a food supplement and cosmetic; as well as animal farming such as salmon and poultry. the deficiency of nutrients in batch culture of h. pluvialis decreases the growth of cells and increases the induction of astaxanthin as an induction factor. therefore, it is impossible to reach to high cell concentrations in batch cultures. in previous experiments, the medium replacement increased the cell concentration, while accumulation of astaxanthin was not induced without other factors. in this work, the effects of fed-batch addition of culture medium on the cell growth and astaxanthin production in h. pluvialis cultures were studied. h. pluvialis was cultivated in 50 cm 3 culture medium containing sodium acetate, yeast extract, l-asparagines, mgcl 2 ·6h 2 o, feso 4 ·7h 2 o and cacl 2 ·2h 2 o (ph 6.8). light was supplied by panels of blue or red led lamps. the temperature was kept at 20 • c and the culture was mixed with a magnetic stirrer. in fed-batch cultures of h. pluvialis, the cell concentration and production of astaxanthin increased in comparison with those in batch culture. in addition, the operation in feb-batch manner is easier than medium replacement from industrial viewpoints for production of astaxanthin. simvastatin and similar compounds are wide used as antihypercholesterolemic agents. simvastatin is obtained by c-methylation of side chain of lovastatin. this process is not perfect and some unreacted lovastatin is present in the reaction mixture. simvastatin is separated from lovastatin using fungi clonostachys compactiuscula. using of living microbials has some disadvantages such as high cost, difficult product separation from the reaction mixture. we work on overcome these difficulties by separation and immobilization of enzyme that hydrolyses lovastatin ammonium salt in presence of simvastatin ammonium salt. our results of purification and immobilization lovastatin hydrolase will be presented. investigation of peptide antibiotics produced by trichoderma strains isolated from winter wheat rhizosphere a. szekeres 1 , l. kredics 2 , l. hatvani 1 , z. antal 2 , l. manczinger 1 , a. nagy 3 , c. vágvölgyi 1 : 1 department of microbiology, university of szeged, p.o. box 533, h-6701 szeged, hungry; 2 hungarian academy of sciences, university of szeged, microbiological research group, hungry; 3 pilze-nagy ltd. , kecskemét, p.o. box 407, species of the imperfect filamentous fungal genus trichoderma with teleomorphs belonging to the hypocreales order of the ascomycota division are of great economic importance as sources of enzymes, antibiotics, as plant growth promoters, decomposers of xenobiotics, and as commercial biofungicides. peptaibols and related peptaibiotics (prps) are secondary metabolites constituting a family of fungal peptide antibiotics which is constantly growing since alamethicin was isolated from cultures of trichoderma viride. these compounds are linear, amphipathic polypeptides composed of 5-20 amino acids and usually containing several non-proteinogenic amino acid residues, which are representing characteristic building blocks of the structure. one hundred and twenty trichoderma strains were isolated from roots of winter wheat grown in agricultural fields of southern hungary. the identity of species was examined based on morphological and molecular characters. the presence of prps-producing strains among the isolated trichoderma strains was detected by biological tests and the antibiotics were partially purified using a multistep chromatography procedure involving exclusion chromatography, adsorption chromatography and thin-layer chromatography. about 20% of the isolates proved to be able to produce prps. the antibacterial activity of the compounds was tested against staphylococcus aureus, bacillus subtilis, micrococcus luteus and escherichia coli, while the antifungal effect was recorded against fusarium oxysporum, f. culmorum, rhizoctonia solani and pythium debaryanum. ergezinger, m., bohnet, m., berensmeier, s., buchholz, k., 2005. integrierte enzymatische synthese und adsorption von isomaltose in einem mehrphasenbioreaktionsreaktor. cit 77, 167-171. glucansucrases from family 70 of glycoside-hydrolases are transglucosidases that produce ␣-glucans from sucrose, a very cheap substrate, without any use of nucleotide activated sugars. based on sequence analyses, these enzymes have been classified in two families, the family 70 and the family 13 of glycoside hydrolases. among the natural diversity existing in family 70 in which are found the glucansucrases produced by lactic acid bacteria, three enzymes have been selected for their distinctive specificities: dextransucrase from l. mesenteroides nrrl b-512f (dsr-s), which catalyses almost essentially the synthesis of ␣-1,6-linkages, alternansucrase from l. mesenteroides nrrl b-1355 (asr), which produces alternan polymer formed of ␣-1,6and ␣-1,3-alternated linkages and finally dextransucrase from l. mesenteroides nrrl b-1299 (dsr-e), which is responsible for the synthesis of a branched dextran composed of about 70% of ␣-1,6-linkages in the main chain and 30% of ␣-1,2-branched linkages. for all these enzymes, the natural polymerase activity can be shifted towards oligosaccharide production or gluco-conjugate syntheses by introducing acceptors in the reaction medium. a number of sugar acceptors have been successfully glucosylated with the view of developing new functional food products. acceptor glucosylation yield as well as acceptor reaction product structures were shown to be highly dependant on the enzyme specificity. consequently, using glucansucrases of distinctive specificities and varying the acceptors give access to a large variety of applications. amylosucrase, the sole glucansucrase found in family 13 of glycoside-hydrolases is also of great interest for functional food applications. this enzyme is able to synthesize highly resistant amylose from sucrose. again the reaction conditions can be used to modulate the yield and the size of amylose. the aim of our work is to further develop the applications of these enzymes via rational and combinatorial engineering. the most recent results obtained in this field will be discussed. novel food structure engineering concepts with enzymes johanna buchert vtt biotechnology, espoo, finland food structure is a very important quality attribute in food choice, since it affects not only the sensory perception of texture, but also release of flavour. enzymes offer specific means to engineer food structure by creating cross-links to food biopolymers, i.e. to proteins and/or carbohydrates. enzymatic cross-linking of food biopolymers can be exploited to create novel types of structures to foods without any need of added food ingredients. laccases and peroxidases can be used to crosslink ferulic acid containing carbohydrates, such as sugar beet pectin or arabinoxylan. proteins can be crosslinked by different oxidative or transferase type of enzymes. transglutaminases can crosslink protein via formation of isopeptide bond between glutamine and lysine residues. laccase and peroxidase can oxidize tyrosine residues to corresponding radicals, which in turn can further react with different groups in proteins. tyrosinases, on the other hand, oxidize tyrosine to a quinone, which can further react with aromatic ring, amine and thiol groups present in proteins. the biopolymer networks formed can be further engineered by combining adequate processing to the enzyme treatment. in this work the potential of enzymatic food structure engineering is reviewed. asparaginase-mediated reduction of acrylamide formation in baked, fried, and roasted products hanne vang hendriksen, beate kornbrust, steffen ernst, mary stringer, hans peter heldt-hansen, peter østergaard novozymes a/s, dk-2880 bagsvaerd, denmark. e-mail: hvhe@novozymes.com (h.v. hendriksen) in 2002, it was discovered that acrylamide is formed in several potato and grain-based foods (e.g. chips, french fries, toasted bread, biscuits, cereals) and in coffee, all of which have been prepared at high temperatures. the level of this potential carcinogen in the final food appears to range from 50 to 4000 ppb. later that year, the mechanism of acrylamide formation was unraveled, demonstrating that asparagine and reducing sugars are the precursors for acrylamide. this pointed to several potential enzymatic approaches to remove the root cause of the problem by degrading the precursors in situ. here, we demonstrate that asparaginase treatment leads to a more efficient reduction in acrylamide than alternative enzymatic treatments. asparaginase from aspergillus oryzae is used to reduce acrylamide formation significantly in laboratory models of a range of common food products. examples are french fries, biscuits, crisp bread, and fabricated chips. the sensory qualities appear to be constant. the implications for scaling up the processes for industrial food production are discussed. effect of cultivation conditions on folate content in yeast: exploring the potential of yeast as a bio-enrichment vehicle for folate in foods sofia hjortmo 1 , johan patring 2 , jelena jastrebova 2 , thomas andlid 1 : 1 department of chemical and biological engineering, chalmers university of technology, po box 5401, 402 29 gothenburg, sweden; 2 department of food science, swedish university of agricultural sciences, po box 7051, 750 07 uppsala, sweden. e-mail: sh@fsc.chalmers.se (s. hjortmo) over the past 10 years, the interest in health benefits of the b vitamin folate has increased considerably. a good folate status may hinder progression of several diseases such as neural tube defects and downs syndrome in foetus, as well as cancer, dementia, alzheimer's disease and cardiovascular disease in adults. it is however not easy to reach the recommended intake and new strategies have to be developed to increase the folate status. in this project we explore the use folate producing microorganisms for this purpose. many yeasts have the ability to synthesise folate de novo and can thus serve as a source for humans. folate enrichment in fermented foods could be much improved by using starter cultures better at producing folate compared to traditional strains. this is, e.g. applicable to bread making fb32 over-expression of isoprene biosynthetic enzymes in the ␤-carotene producer zygomycete mucor circinelloides tamás mucor circinelloides has been involved to study the carotene biosynthesis genesis of fungi. this fungus is more amenable to molecular techniques than the others traditionally used in carotenogenic studies (e.g. blakeslea trispora and phycomyces blakesleeanus). moreover, mucor has a great advantage: it is a dimorphic organism. this type of morphology is preferred by the fermentation industry because yeast-like growth allows the submerged culture, when usually higher biomass production can be achieved and cells can be more easily separated from the media. β-carotene is a terpenoid-type chemical compound likewise to sterols, quinones or chlorophylls. the production can be increased by improving the non-carotene specific terpenoid biosynthesis. this can be carried out by the overexpression of the genes responsible for the ratelimiting steps of these pathways. in this study, polyethylene glycol mediated transformations of m. circinelloides protoplasts were performed with autoreplicative expression vectors containing the known terpenoid genes of m. circinelloides (e.g. isoa encoding farnesyl pyrophosphate synthase and carg encoding geranylgeranyl pyrophosphate synthase). carotene production of the transformants and the wild-type strains were analysed by high-performance liquid chromatography (hplc). transformants harbouring plasmids with isoa or carg produce about 1.5 times more carotene than the recipient strain, while carotene production increased about two times in the co-transformants containing both type of plasmids. members of the genus rhizopus are important from biotechnological aspects in consequence of their effective extracellular enzyme, alcohol and organic acid production. moreover, rhizopus strains are used for fermentation of various foods, because they are capable of transforming soybeans into edible products. the high affinity iron permease (ftr1) contains both highly conservative and variable regions applicable for phylogenetic comparisons. the aim of this study was the comparative analysis of this gene of different rhizopus species in order to elaborate a simple and fast method to identify these fungi at a species and subspecies level. conserved regions of candida albicans and rhizopus oryzae ftr1 genes (fu et al., 2004) have been analysed to design degenerate primers for polymerase chain reaction. they were used to amplify the homologous regions from different strains of r. oryzae, r. microsporus, r. stolonifer and r. niveus. isolates of the similarly thermophilic rhizomucor miehei and r. pusillus, as well as a strain of m. rouxii were involved in the study as outgroups. deduced protein sequences were aligned and phylogenetic analysis was performed. surprisingly the r. oryzae isolates formed a group completely different with a significant distance from the r. microsporus isolate. r. niveus is currently not distinguished from r. stolonifer var. stolonifer because of morphological considerations. however, phylogeny of ftr1 gene sequences, in agreement with earlier results based on rapd data (vágvölgyi et al., 2004) , raise the need to handle r. niveus as a separate species. sequences and pcr primers useful for identification of all tested rhizopus strains were elaborated. potential application in a lactose conversion process. the prebiotics market is at high demand therefore the development of the process to produce 'prebiotic' galacto-oligosacharides efficiently and inexpensively is our particular interest. citrus, particularly mandarins and clementines, are among the most economically important fruit crops in morocco. besides morphological traits multiple molecular markers have been used for the caracterisation of citrus germplasm. the main aim of this study was to evaluate the moroccan mandarin germplasm and to identify specific polymorphisms among accessions sharing identical name. eighty mandarin and two sweet orange varieties were analyzed by dna markers. issr markers were amplified using 3 anchored primers and analysed by agarose gel electrophoresis. aflp markers analyses were performed using three primer combinations. the dice coefficient was used to estimate genetic similarities and the upgma algorithm was utilised to generate a phenogram depicting the genetic relationships among the acessions. the selection of 9 primers out of the 31 issr primers primarily assayed, allowed us to maximize the average number of amplified fragments analyzed per reaction (7.3), and the percentage of informative polymorphisms (49%). the three combinations of ecori/msei primers revealed 73 reliable aflp markers, 35 (47%) of witch were polymorphic. the range of fragment sizes varied from 100 to 650 bp. contrasting with the phenotypic diversity for agronomic and fruit quality traits, very low variability at the dna level has found among mandarins, which always showed a high (s > 0.81) coefficient of genetic similarity. the molecular marker analyses allowed the clarification of ambiguous denominations and the establishment of phenological relationships. the mandarin cultivars have been clustered into several different sub groups. this study allowed the identification of one issr marker, distinct and specific for the clementine sidi aissa and some aflp markers specific to maroc late and w. navel. many hybrids used in this study presented high coefficient of the similarity with one of their parents, such as siamelo and king of siam (s = 0.92), fortuna and clementine (s = 0.93), kara and king of siam (s = 0.92). this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). the new morocco mandarin variety nadorcott became very important in the international market because of high quality, good size, easy peeling and absence of seeds. also known as afourer or w. murcott, this variety was selected in 1981-1982 at the afourer experimental station, inra, located near to beni mellal city, as an original tree among several 18-year-old murcott honney (c. reticulata × c. sinensis) seedling trees. in order to shed additional light on the genetic origin of this variety, we have carried out isozyme and dna fingerprinting analyses. for better interpretation of the nadorcott molecular profiles, others mandarin cultivars, among which murcott honney, were also analyzed by molecular markers. three enzymatic systems (idh, pgm and pgi) permitted the discrimination between nadorcott and his female parent murcott honney. the molecular patterns displayed by these cultivars point out the sexual origin of nadorcott and discard the previously assumed hypothesis for its origin as a mutation of a nucellar zygote. the issr and rapd markers analyses allowed the identification of kinnow; du japon, vietnam; and swett lime as the genetically most closely related mandarins (s ∼ 0.95) to nadorcott. strong genetic similarity was also found with the clementine group, a possible male parent of nadorcott. the analysis by aflp markers confirmed the hybrid origin of nadorcott and the high genetic relatedness (s = 0.93) of this mandarin to its putative female parent murcott honey, and other cultivars as kinnow (s = 0.94) and clementine (s = 0.93). the possibility of an accurate molecular identification of nadorcott by specific molecular markers is of paramount importance for the protection and management of this original moroccan citrus variety. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions 77 g/l from white distilled lees and 45.6 g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. thermophilic cyanobacterial strains that could grow in the bg 11 media was isolated from hot springs and their reactive dye bioaccumulation was studied under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye accumulation. in the experiments performed with newly isolated synechocystis sp. and phormidium sp., the optimum ph values at about 25 mg l −1 initial reactive dye concentrations was determined as 8. lipases are extremely versatile enzymes, that catalyze both hydrolysis and synthesis reactions. they have a wide range of industrial applications, among which the manufacture of detergents, pharmaceuticals and fine chemicals are outstanding. the fungus rhizopus oryzae has been reported to synthesize a number of commercially interesting enzymes. in this work, its ability to produce extracellular lipases when grown in solid state culture has been assessed. cultures were carried out in erlenmeyer flasks, using a complex medium and several supports, both synthetic (nylon sponge) and natural (barley bran, ground walnut or peanut). the latter appeared to be more suitable for lipase production. since the best results were initially obtained with lipid-containing supports, barley bran cultures were supplemented with a vegetable oil, in an attempt to optimise lipase production and design an efficient procedure for reusing this agroindustrial waste. surprisingly, this strategy did not improve enzyme synthesis. however, when a surfactant (triton x-100) was added to the basal medium, a dramatic increase in extracellular activity was detected (up to 20-fold). the results agreed with those previously obtained in submerged cultures of r. oryzae, in which addition of olive oil did not increase lipase production, while the presence of triton x-100 had a remarkably beneficial effect. also, enzyme concentration in solid state cultures was up to two-fold that of the submerged ones. est maximal sorption capacity was found for yeasts cryptococcus sp. wt and rh. aurantiaca 1195 . these cultures also demonstrated the high sorption affinity, which makes them especially efficient biosorbents at low concentrations of lead ions. the high efficiency of lead elution was shown with 0.1n edta. isolation and identification of marine bacteria from deep-sea sediments els maas 1 , cara brosnahan 1 , vicky webb 1 , helen neil 2 , phil sutton 2 : 1 marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand; 2 oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand marine sediments were obtained using a piston corer with associated trigger core (0.5 m, 0.06 m diameter). cores were collected from depths of 270 to 3911 m, along norfolk ridge and across challenger plateau. sediments ranged from coarse carbonate sands in the north to sandy and silty hemipelagic mud with increasing depth and latitude. all samples sites underlie subtropical surface water masses associated with, and south of, the tasman front. sediment samples were aseptically taken from the triggers cores upon recovery. samples were stored in sterile tubes at 4 • c on board the vessel for 3-17days. the core samples were plated on several different agar types and incubated aerobically for 4 weeks at 16 • c. individual colonies were sub-cultured and purified using standard microbiological techniques. morphological and molecular taxonomy revealed that the bacteria isolated from the sediments were closely related to novosphingomonas, halomonas, stappia, glaciecola, pseudoalteromonas and leeuwenhoekiella. phylogenetic trees constructed using 16s rrna gene sequence data showed that two other isolates were unrelated to known genera. the bacterial isolates are currently being investigated for their biotechnological potential. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by 20% was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were 90 and 50%, respectively. while the color and aromatocity decreased by 60 and 95%, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was 0.045 h −1 while the monod constant based on the consumed tp and cod (mg/l) were 370 and 6900, respectively. the control of water pollution has become of increasing importance in recent years. the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g −1 ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph. the maximum adsorption at 50 ppm was 88.52% equal to 11.8 mg of dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of 8-12). the effect of contact time was studied at initial concentration (50 ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after 15 min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. flocculation of saccharomyces cerevisiae (diastaticus) ifo1958 was studied. cells of ifo 1958 did not flocculate even in the stationary phase without mg 2+ ("mg 2+ -deficient cells") although they began to flocculate strongly 18h after inoculation in the presence of mg 2+ ("complete cells"). cycloheximide completely inhibited induction of floc-forming ability of "mg 2+ -deficient cells". co-flocculation between "complete cells" and "mg 2+ -deficient cells" was investigated by chemical modification. treatment of "mg 2+ -deficient cells" by proteolytic enzymes did not affect the co-flocculation with "complete cells". photo-oxidation or mercaptoethanol-reduction of "mg 2+ -deficient cells" failed to weaken the co-flocculation with "complete cells" while treatment of "mg 2+ -deficient cells" by periodate brought about a significant loss of the co-flocculation. on the contrary, "complete cells" deflocculated by proteolysis or chemical modification of proteinaceous component failed to co-flocculate with "mg 2+ -deficient cells". these findings suggest that "mg 2+ -deficient cells" are non-flocculent because of lack of proteinaceous component essential for flocculation of cells of ifo 1958. the industrial toscano cigar production starts with the dark firecured kentucky tobacco fermentation process. during this phase the present work is a trial to study the portal serum factors which stimulate the cell proliferation of the schistosomules, aiming to find ways to block or inhibit their effects. our previous studies showed that portal serum of human and hamster (highly susceptible hosts) and a 1-50 kd fraction separated from human portal sera by ultrafiltration stimulate cell proliferation in immature schistosomules (20 days old) in vitro. for further identification of the portal serum factors in the range of 1-50 kd that stimulate cell proliferation, schistosomules were incubated in vitro in medium containing 10% fetal calf serum, 10% portal human serum or 10% peripheral human serum or their fractions separated by native electrophoresis followed by electroelution, incubations were performed in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. the results showed that human portal sera enhanced cell proliferation of schistosomules compared to the peripheral serum. this stimulatory effect was substantially reproduced by fraction separated from human portal serum with molecular weight 20.8 kd. these results may help in designing a drug or antibody therapy to block the stimulating effect of the portal serum fraction and subsequently to disturb the life cycle of the parasite at early stage of development. center of molecular biosciences, university of the ryukyus, okinawa 903-0213, japan. e-mail: naoya-s@comb.u-ryukyu.ac.jp (n. shinzato)oil strage tank sludge, mainly composed of water and solid hydrocarbons (waxes) needs to be treated when harvesting the stored oil. although the sludge treatment by microbial surfactant or microbial cracking are considered as the feasible method, microbial degradation of the waxes (ca. solid n-alkanes) have been reported in a very limited species such as acinetobacter. in addition, long-chain n-alkanes (so called paraffin waxes) are one of the major components of oil, and their resistant properties to biological attack hold up the recovery of oil-polluted environments. in this report, we have screened n-tetracosane (c24) degrading bacteria from soils in okinawan island, an unique sub-tropical area in japan, to know the bacterial diversity and their degrading mechanism.16srdna phylogenetic analysis of the isolates, totally ca 40, showed they were not only acinetobacter and pseudomonas, but also other proteobacteria (alcaligenes), actinomycetes (gordonia, nocardia, and leifsonia), bacillus, staphylococcus, and unidentified ones. they also grew not only the solid n-alkanes but also iso-alkanes, mid-chain n-alkanes as the sole carbon source. results for the biosurfactant production will also be shown. the properties of 188 environmental enterococci were studied. the strains were isolated mainly from surface and waste waters and several strains from sheep manure were also included. species identification was provided by combination of phenotypic (micronaut system, merlin) and molecular detection methods (automated its-pcr, ddl-pcr). several discrepancies were observed when comparing molecular and biochemical identification. six enterococcal species were overall identified; e. faecium and e. hirae were the most abundant ones, almost 80% of isolates belonged to these two species. the distribution of selected genes conferring virulence to enterococci (cyla, gele and esp) was investigated, the positive signal was obtained mainly for e. faecalis strains. the strains were also characterized for the possession of enterocin genes (enta, entb, entp, ent31, entl50ab) and high frequency of enterocins was observed. biosorption of three different dyes (reactive black 5, cibacron brilliant yellow, cibacron brilliant red) onto immobilized scenedesmus obliquus a microalga was investigated in a batch sys-tem. the immobilized alga exihibited the highest dye uptake capacity at the initial ph value of 2.0 for all dyes. the effect of temperature on equilibrium sorption capacity indicated that maximum was obtained at 25 • c for rb5, cby and cbr biosorption. the freundlich, and langmuir adsorption models were used for the mathematical description of the biosorption equilibrium and isotherm constants were evaluated. biocontrol properties of microbially-treated sugar beet wastes in presence of rock phosphate n. vassilev, i. nikolaeva, m. vassileva department of chemical engineering, faculty of sciences, university of granada, c/fuentenueva s/n, granada-18071, spain. e-mail: nbvass@yahoo.com (n. vassilev) the effect of soil application of sugar beet wastes (sb) treated with aspergillus niger in the presence of rock phosphate (rp) on the control of fusarium wilt of tomato were studied. two treatments and a control were used: inoculation with glomus intraradices (am), further inoculation with a. niger grown on sb + rp medium, and the control (c). application of the am fungus increased plant growth, p and n uptake and reduced disease caused by fusarium oxusporum f. sp. licopersici (fol) as compared to non-mycorrhizal control plants. soil amendment with sb + rp + a. niger resulted in 347% and 467% (versus c) higher plant shoot biomass in plant-soil experiments contaminated or not with fol, respectively. in this case, disease severity and number of fol cfu reached the lowest levels while soil phosphatase and beta-glucosidase activities increased compared to all other treatments. fol negatively affected plant root mycorrhization determined in the am treatment while the difference between the mycorrhization of plants grown in the presence and absence of f. oxysporum in sb + rp + a. niger-amended soil was insignificant (53% versus 59%, respectively). in conclusion, the fermentation mixture containing mineralized organic matter, partially solubilized rp, and a. niger biomass could be efficiently used not only in improving plant growth, nutrient uptake and properties of degraded and polluted soils, as previously reported (vassilev and vassileva, 2003) , but also in environmentally-mild management of fusarium wilt. vassilev, n., vassileva, m., 2003. appl. microbiol. biotechnol. 61, 435-440 . biological treatment processes allow for the effective elimination of charged inorganic micropollutants, e.g. a number of oxyanions, heavy metals, etc. from contaminated drinking water supplies. however, dedicated technologies have to be implemented in order to eliminate the target pollutants without changing the quality of treated water, avoiding its secondary pollution by cells, nutrients and metabolic by-products. some innovative technologies, which combine the use of membranes with the bioconversion of charged micropollutants in order to deal with the secondary water contamination problem, will be presented and critically compared. a special on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by 20% was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were 90 and 50%, respectively. while the color and aromaticity decreased by 60 and 95%, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was 0.045 h −1 while the monod constant based on the consumed tp and cod (mg/l) were 370 and 6900, respectively. advanced start-up strategy of an anaerobic three-phase turbulent bed reactor treating winery wastewaters r. cresson, h. carrère, n. bernet, j.p. delgenès laboratoire de biotechnologie de l'environnement, institut national de la recherche agronomique (inra), avenue des etangs, 11100 narbonne, france. e-mail: cresson@ensam.inra.fr (r. cresson)the objective of our study was to compare two start-up strategies for an anaerobic biofilm process, to create an effective biofilm and increase the organic loading rate (olr) as quickly as possible. two methanogenic three-phase biofilm reactors have been started, using the same operational parameters (solid hold-up ratio, gas velocity of 1 mm s −1 ), in order to test two different strategies:• maximal load strategy (reactor a): the olr is increased as long as the global amount of removed cod (biogas production) increased. • maximal removal strategy (reactor b): the olr is increased stepwise as soon as the cod removal rate reaches 80%.both reactors have been operated for 90 days, until a volumetric olr of 20 g cod l −1 j −1 , with more than 90% of carbon removal. the total amount of cod removed and methane produced were higher in reactor b (19.6 and 32.2%, respectively). in both reactors, the short hydraulic retention time (hrt) applied during all the experiment caused a rapid wash-out of planktonic bacteria and an exclusive use of the substrate by the attached micro-organisms, which accelerates the biofilm growth. the lag-phase was reduced to approximately 7 days. the reactor submitted to repetitive disturbance by the maximal removal strategy appeared to be more robust when confronted to perturbation like organic overload or nutritional deficiency. experiments have demonstrated capability and the efficiency of the aggressive strategy for controlling anaerobic bioreactor start-up. sustainability is the generally accepted paradigm for future industrial development. the re-integration of waste products into production processes is a major aspect of environmental sustainability. in this study the use of sugar cane molasses is being investigated for the production of bioplastics by mixed microbial cultures, with the added possibility of parallel biohydrogen production. polyhydroxyalkanoates (phas) are polyesters synthesized by bacteria and accumulated as granules in the cytoplasm. studies conducted by this group have shown that mixed microbial cultures subjected to dynamic feeding conditions may accumulate phas up to 80% cell dry weight, a value close to that obtained for pure cultures. volatile fatty acids are good substrates for the production of phas by mixed cultures. on the other hand, sugar molasses, with a very high sugar content (about 50% dry weight), can produce organic acids by fermentation. the two-stage process being implemented in this study includes a molasses fermentation step, in which the high sugar content of the molasses is converted into volatile fatty acids (vfas), and a pha production step, in which the vfas serve as the precursors to the formation of phas under dynamic feeding conditions. moreover, hydrogen can be produced by anaerobic bacteria from carbohydrate-rich substrates giving organic fermentation end products, h 2 and co 2 . to optimize the production of both high-value products, design of experiments (doe) is being used to elaborate a set of experiments to study the effect of ph, hydraulic retention time and organic loading on both the organic acids distribution (which will serve as precursors for pha production in the second step) and h 2 production in the acidogenic fermentation reactor (a 1 l cstr). preliminary results show that the effluent of the acidogenic reactor fed with 10 g/l total sugars and operated at ph 7 and d = 0.1 h −1 (composed mainly of acetate and propionate) can be successfully fed to a polymer-accumulating mixed culture. under these conditions, the h 2 production yield has been estimated at 3.9 mol h 2 /mol sucrose. vanillin is a flavour compound used in food industry, fragrances and pharmaceutical preparations, which is nowadays mainly produced by chemical synthesis. the increased demand of natural products for the food industry as well as the high cost of natural vanillin extracted from vanilla pods has recently stimulated the research for alternatives to produce this compound by a natural way. the microbial transformation of ferulic acid, a phenolic compounds from lignin degradation, is recognized as being the most interesting alternative to produce natural vanillin. the combined effects of initial ferulic acid concentration (s 0 ) and biomass concentration (x 0 ) on vanillin production by resting cells of escherichia coli strain were investigated using response surface methodology. e. coli jm109/pbb1 a recombinant strain producing key enzymes of ferulate catabolic pathway from p. fluorescens bf13 (feruloyl-coa synthetase and feruloyl-coa hydratase/aldolase) was utilized in this work. a 3 2 full-factorial design was employed for experimental design. the results shown a possible inhibition phenomena at a vanillin concentration of about 0.1 g l −1 leading to the accumulation in the fermentation media of secondary compounds like vanillic acid and vanillin alcohol. removal of dissolved nutrients from wastewater using a microalgae biofilter line christensen, suvina sooknandan, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, odense, a microalgae biofilter can be used for treatment of wastewater from landbased fish farms in order to remove excess amounts of dissolved nutrients such as nitrate, ammonium and phosphate. a bubble column bioreactor has been developed for cultivation and characterization of microalgae. this type of bioreactor is equipped with a control system that enables online determination of the photosynthetic quotient and optimization of light intensity. furthermore the bioreactor has a dualsparging system simultaneously allowing adequate mixing and high gas-liquid mass transfer coefficients. different species of microalgae have been cultivated in batch and fed batch cultures to characterize growth and ability to take up the different dissolved nutrients. the specific growth rate and substrate uptake rate have been determined to compare and select the algal species most suited for use in a biofilter. additionally the composition of lipid, protein and carbohydrates has been measured to determine the nutritional quality of the algae when used as animal feed. at present, biological nitrogen-removal is mostly carried out through several complicated steps. to simplify the present systems for nitrogen-removal, we have investigated a new nitrogenremoval bioreactor using packed gel envelopes capable of simultaneous nitrification and denitrification. the envelope consists of two plate polymeric gels with a spacer in between. ammonia oxidizer, nitrosomonas europaea and denitrifier, paracoccus denitrificans are co-immobilized in the plate gels. when the envelopes are exposed to wastewater containing ammonia, the immobilized n. europaea oxidizes ammonia to nitrite in the outer aerobic surfaces of envelopes. at the same time, as ethanol solution is injected into the internal anaerobic spaces of envelopes, the immobilized p. denitrificans reduces the nitrite to nitrogen gas using the ethanol solution as an electron donor for denitrification. in this way, the envelopes can remove ammonia from wastewater in a single step. we have already reported advantages of our bioreactor in laboratory-scale experiments. in this study, we show our large-scale bioreactor (water volume 1.8 m 3 ) could treat three kinds of wastewater derived from coal power plants. ammoniacontaining wastewater that occurred regularly in a coal power plant was continuously treated with the bioreactor using thirty envelopes for over a year. the bioreactor could remove more than 90% of total nitrogen at hydraulic retention time (hrt) of 24 h. at hrt of 4 h, the bioreactor accomplished a maximum rate (the transformation of nh 4 + to n 2 ) of 6.0 g n/day m 2 of the envelopes' surface. the performance was equivalent to that obtained in the laboratory-scale experiments. furthermore, our bioreactor showed similar nitrogen-removal performances when it treated nitrate-containing wastewater occurring regularly and condensed ammonia-containing wastewater occurring at irregular intervals in coal power plants. these results show that our bioreactor can treat various wastewater containing nitrogen in coal power plants. thus, our concept is effective to simplify the large-scale systems in coal power plants and the other plants. in order to establish an environmentally friendly process for the treatment of metal containing waste, in a portuguese refinery a process involving sulphur oxidizing acidophilic microbes is being considered. bioleaching of metal containing bottom ash, from fluidised bed incineration of sludge resulting from the refinery water treatment station, was performed using a sulphur oxidising acidophilic culture isolated from an acid pool resulting from the weathering of sulphur piles from the claus plant. this sample served as inoculum for liquid medium cultures with 1% sterile sulphur flowers as source of energy. application of monod kinetics to adapted culture growth of free cells presented a value of µ = 0.124 day −1 . yield of sulphur conversion to sulfate after 17 days was η = 78%. in the presence of bottom ash from the incineration of refinery sludges µ = 0.141 day −1 and the yield of sulphur conversion was η = 67.5%. a η fe = 90% removal of iron is obtained from the treated ash. x-ray fluorescence spectroscopy of the solid residue revealed a total removal of metals namely, v, cu, ni, zn and most of the fe after 15 days of bioleaching. the present of heavy metals in the environment is a serious problem. they are commonly present in effluents from mining and industrial activities. usually, chemical conventional methods are very expensive and they have limitations when heavy metals are in low concentrations. at the moment the interest increases for processes that involve microorganisms as alternative method. some effluents present heavy metal sulphates which are soluble compounds. sulphate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulphate as an electron acceptor and generate hydrogen sulphide. hydrogen sulphide reacts with heavy metal ions to form insoluble metal sulphides that can be easily separated from a solution. the purpose of this work was to evaluate the ability of srb to reduce cr(iii), ni(ii) and zn(ii) in artificial contaminated solution. desulfovibrio vulgaris and desulfovibrio sp. strains has been tested in this study. batch cultures was carried out in 50 ml sealed bottles with different concentrations of studied metals (1-20 mg/l), with 10% of inoculum bacterial and adapted to postgate's medium c. a gaseous nitrogen current was employed to purge oxygen and obtain anaerobic conditions. the assays were incubated statically represented very low portion (less than 4-5%) of the total bacterial community at all temperatures tested. schistosomules of schistosoma mansoni (20 days old) were incubated in rpmi 1640 medium containing 10% fetal calf serum, 10% hamster portal venous or 10% hamster peripheral venous serum (highly susceptible host) or 10% rat portal venous or 10% rat peripheral venous serum (poorly susceptible host) in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. also the rate of cell proliferation of s. mansoni were assessed in vivo in hamster to study the cell proliferation in the natural ontogeny of the organism. the rates of cell proliferation as expressed by brdu labeling indices (blis) were determined as a function of time of incubation by immunohistochemistry using monoclonal antibody to brdu. compared to schistosomules cultured in presence of rpmi plus 10% fetal calf serum, blis were increased by 41% in the presence of hamster portal, but not in peripheral serum. while in case of rat, no significant changes were observed in the blis in both portal and peripheral sera. the experiment was repeated using hamster portal and peripheral sera containing different schistosomal igg antibody titres. the results showed decreased values of blis compared to sera which did not contain the schistosomal antibody(ies). the in vivo results revealed that there was no cell proliferation of s. mansoni schistosomules (6 days old) in the lungs. cell proliferation was detected in schistosomules of 17 days old and the results revealed a significant decrement in the brdu labeling indices (blis) with the increase of the age of schistosomules in vivo. the results indicated that hamster portal venous serum (highly susceptible host) could have stimulating factor(s) for schistosomule cell proliferation which is not found in rat (poorly susceptible host) and the presence of antibody(ies) greatly inhibit the cell proliferation. this could be due to the blocking of some portal serum factors, which stimulate the cell proliferation by the antibody(ies). the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g −1 ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph, the maximum adsorption at 50 ppm was 88.52% equal to 11.8 mg dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of 8-12). the effect of contact time was studied at initial concentration (50 ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after 15 min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. compared to other european nations, the austrian population shows a low level of knowledge in biosciences and a strong denial of gene technology (1). the austrian non-profit organisation dialog<>gentechnik, a scientific society, organizes various activities to raise awareness for the "hot topics" in the life sciences. according to its principle of independence, all activities are funded publicly. projects on behalf of the austrian authorities and international cooperations demonstrate credibility and trust in dialog<>gentechnik (2). a few examples will be presented. dialogue with the public: on the occasion of the first anniversary that the gmo labelling rules became effective, the action "gene technology on my plate" is performed austrian wide in shopping centres. here, consumers are informed about health and labelling aspects of gm food. two days of open discussions were organized in the context of the austrian genome research program gen-au (3): topics were "gene diagnosis" (2002) and "genome research-what is in it for me?" (2004) . an open lab is currently set up in vienna to offer "hands on" experience in life sciences for everybody. motivating students: in the very successful gen-au summer school (3), high school students spend 3-4 weeks in the lab and work with scientists. the best documentations are awarded. in an innovative project, student groups (age 16-18) work on the topics stem cells and cloning and develop units of an elearning course which will be accessible for all austrian schools in the near future. engaging stakeholders: dialog<>gentechnik manages interdisciplinary wor king groups that develop leaflets, brochures and questionnaires on various aspects of gene diagnosis. four products are currently distributed to the public and to health services. (1) european commission. eurobarometer 55.2, december 2001; (2) www.dialog-gentechnik.at; (3) www.genau.at. the aim of this paper is to compare the attitudes of the urban consumers with professionals towards to new technologies, especially to biotechnology. it was tried to find out in which area, medical, agriculture or industry, people can accept biotechnological developments and in which area not accept. key: cord-015359-gf32a6f1 authors: nan title: b scientific sessions (ss) date: 2002 journal: eur radiol doi: 10.1007/s00330-002-0002-9 sha: doc_id: 15359 cord_uid: gf32a6f1 nan this icon indicates a pre-selected scientific paper to be judged by the subcommittee chairmen. the final winners of the best paper within one topic will be announced at the closing ceremony and presented with a diploma and their prize (€ 1500.-) in (a)symptomatic high-grade internal carotid artery (ica) stenosis, ultrasound and digital subtraction angiography (dsa) are the established techniques for grading ica stenosis; contrast-enhanced magnetic resonance angiography (cemra) is replacing dsa. aim of this study was to evaluate the diagnostic accuracy of cemra in studying the carotid arteries and to compare cemra with dsa findings. two mra protocols, differing in acquisition voxel size, were used to assess whether intravoxel dephasing effects could modify diagnostic accuracy of cemra. 34 patients were studied: 20 (group 1) underwent a cemra protocol with an acquisition voxel size of 1.07 mm 3 and 14 (group 2) with a voxel of 0.40 mm 3 . dsa was perfomed in all patients. morphology and degree of stenosis were evaluated, and a qualitative analysis comparing cemra and dsa findings was obtained. diagnostic accuracy and correlation with dsa results for ica stenosis were calculated in all patients and, separately, in the two groups. cemra diagnostic accuracy and agreement with dsa were respectively 94 % and k = 0.84; no significant difference was found among the two groups. qualitative analysis revealed a better correlation between cemra and dsa images in group 2 than in group 1. these data suggest that cemra could be a powerful tool for the non-invasive evaluation of atherosclerosis of neck vessels. stenosis overestimation of cemra is confirmed; reduction of voxel size, decreasing the dephasing intravoxel effect, allows to minimize it, with a better correlation of cemra and dsa findings; yet, no diagnostic gain is obtained in the patients' classification according to nascet criteria. mri perfusion changes in unilateral symptomatic internal carotid artery stenosis: evaluation of middle cerebral artery and border territories f. gaudiello, g. manenti, f. garaci, r. floris, a. bozzao, g. simonetti; rome/it background and purpose: to determine, with dynamic susceptibility contrast magnetic resonance imaging (dsc-mri), the hemodynamic changes occurring in patients with unilateral internal carotid artery stenosis (icas). methods: 15 patients with symptomatic unilateral icas (over 70 % by nascet criteria) as assessed by digital subtraction angiography and 15 age and sex matched control subjects underwent dsc-mri. on a separate workstation rcbv and rmtt were calculated on the basis of signal decay rate during the passage of gadolinium bolus through the sampled. middle cerebral artery (mca) and distal border territories (mca to posterior and anterior circulation) were evaluated in all patients. results: patients with unilateral icas did not showed statistically significant hemodynamic changes (rcbv and rmtt) between the two hemisphere both considering mca and border territories. comparing patients and controls statistically significant differences were found only for rmtt values in the border territories (increased, p = 0.005). conclusions: dsc-mri is a valuable tool for measurement of hemodynamic changes in the presence of carotid disease with hemodynamic impairment. based on our data no significant perfusion changes occurs between the two hemispheres in patients unilateral symptomatic icas. this indicates compensatory mechanisms between the two hemispheres in the cerebro-vascular reserve distribution. only rmtt values changes (increased) in the border territories (of both hemispheres) differentiate patients with icas from normal controls. correlation of doppler-and duplex ultrasonography based grading of unilateral internal carotid artery stenosis and blood volume flow quantification in brain supplying arteries using 2d cine phase-contrast mr imaging k.w. neff, a.k. kilian, s. meairs, a. schwartz, c. düber; mannheim/de purpose: correlation of unilateral internal carotid artery (ica) stenosis grading based on doppler-and duplex ultrasonography and the hemodynamic changes in brain supplying arteries. methods and materials: 72 patients with unilateral ica stenosis at the level of the bifurcation between 50 % and 98 % and 15 age-matched normal controls were examined using a 2d cine phase-contrast mr technique. blood volume flow was measured in both icas and the basilar artery (ba). the ultrasonography based grading of stenosis was compared to the changes in blood flow in the stenosed ica and to the changes in the contralateral ica and the ba. results: ica stenoses greater 70 % were hemodynamically relevant. with increasing stenosis, a decrease of blood volume flow in the ipsilateral ica was determined with a high linear correlation of r = 0.86. normal controls showed a blood volume flow in an ica of 256.8 ± 29.1 ml/min. in patients with 70 % stenosis a mean blood volume flow of 229.1 ± 32.2 ml/min; with 80 % stenosis 165.1 ± 52.3 ml/min and patients with 90 % stenosis a reduction of mean blood flow in the stenosed ica to 80.4 ± 25.1 ml/min. with decreasing blood flow in the stenotic ica an increasing blood volume flow in the contralateral ica was found. in patients with unilateral ica stenosis greater than 85 % a significant increase in ba blood flow was documented. the comparison of ultrasonography based grading of unilateral ica stenosis and blood volume flow determination in patients with hemodynamically relevant ica stenoses demonstrates a high correlation between increase of stenosis, decrease of ipsilateral blood flow and collateralization of brain supplying circulation. doppler ultrasound, multidetector ct and gd-mra in the assessment of carotid artery stenosis e. ferone, c. pistolese, s. fabiano, r. floris, a. spinelli, a. bozzao, g. simonetti; rome/it aim of the study was to prospectively compare gadolinium-enhanced magnetic resonance (mr) angiography, computed tomographic angiography with multidetector capabilities (md-cta) and doppler ultrasound for use in detecting atheromatous stenosis and plaque morphology at the carotid bifurcation. conventional angiography was available in all cases; surgical specimens were also available in many cases. methods and materials: 42 carotid arteries (in 21 patients) were analyzed by using md-ct, enhanced mr angiography, and dsa. the following features were analyzed: degree of stenosis on the basis of north american symptomatic carotid endarterectomy trial criteria, luminal surface and presence of ulcers. results: there was significant correlation between md-cta, enhanced mr angiography, and doppler for degree of stenosis. with enhanced mr angiography and ct angiography, degree of stenosis was underestimated in two of 44 cases (1 mild and 1 moderate). no case of overestimation with enhanced mra and md-cta was found. severe internal carotid artery stenoses were detected with high sensitivity and specificity: 100 % and 100 %, respectively, with ct angiography; 96 % and 100 %, respectively, with enhanced mr angiography. luminal surface irregularities were most frequently seen at md-cta as demonstrated by surgical specimens. conclusion: there was a significant correlation between md-cta, enhanced mr angiography and dsa in evaluation of carotid artery stenosis. cta was however slightly better than mra in detecting surface alterations. fucan, a natural polysaccharide from algal origin, inhibits intimal hyperplasia after stent implantation in normocholesterolemic rabbits we tested the hypothesis that fucan, a sulfated polysaccharide inhibitor of smcs proliferation in vitro, may reduce intimal hyperplasia in vivo. we performed a pharmacokinetic study on a rat model and a restenosis study on a rabbit model. a single dose of fluorescent fucan and heparin were administered to 10 rats and serum and urine were collected over time. serum and urine concentration and pharmacokinetic parameters were calculated. ten normocholesterolemic rabbits were treated with daily intramuscular injection of fucan (5 animals) or saline (5 animals) after stent implantation in both iliac arteries. fourteen days after injury animals were sacrified and histomorphometric analyses were performed. blood samples were systematically collected. results: fucan exhibited high plasma levels and a prolonged half-life (7 h 30 min) compared to heparin (1 h) after intravenous and intramuscular administration. fucan reduced intimal hyperplasia by approximately 60 % after stent implantation in rabbits (1.79 ± 0.4 vs 0.73 ± 0.2 mm 2 , p < 0.005). intimal hyperplasia inhibited was associated with a better longitudinal organization of cells layers. no toxic effect was noted in treated animals. blood samples showed no anticoagulant activity. conclusion: fucan, a natural polysaccharide of algal origin, reduces markedly intimal hyperplasia in a rabbit restenosis model without anticoagulant activity. methods: endovascular treatment was performed in 39 patients with 42 aneurysms presenting with an unruptured aneurysm either of the anterior (n = 31) or posterior circulation (n = 11). 8 patients had a previous sah from another aneurysm and 10 had neurological symptoms due to the aneurysm. in 21 patients the aneurysm was an incidental finding. aneurysm size was small in 18 (25 mm). occlusion rates was divided into complete (100 %), subtotal (95 -99 %) and incomplete (< 95 %). results: endovascular treatment was technically feasable in 38/42 aneurysms. complete or nearly complete occlusion was achieved in 34/38 aneurysms. in four aneurysms of the ica only incomplete obliteration could be achieved. procedural complications included one case of thrombembolic vessel occlusion with narrowing of the parent artery due to coil protrusion, resulting in anterior cerebral infarction. with the exception of one patient with a mild deficit, all had a good recovery according to the glasgow outcome scale, resulting in a morbidity of 2.4 % and a mortality of 0 %. conclusion: endovascular treatment in patients with unruptured aneurysms is an effective alternative with low morbidity and mortality and should be considered the first option for treatment. we designed three chains of calcium made from small cortical chicken bone fragments. before the fragments were fixed with glue onto tape, volume measurements using water displacement and mass measurements using a precise balance (mettlar h35ar) were performed. during open-heart surgery the chains then were sutured to the pig heart (n = 4) covering the coronary bed of each major epicardial artery as lad, cx, and rca in order to simulate real coronary motion. in each pig we performed a series of prospectively gated sequential and retrospectively gated half-scan and multisector helical scans (lightspeed plus, gems, milwaukee, wi) using a range of scanning parameters (ma, kv, slice thickness). we evaluated the agatston score as well as the volume and a mass score for each chain and each pig. results were compared to water displacement volume measurements and balance mass measurements. the cortical bone frag-purpose: electron beam computer tomography (ebct) and multislice computer tomography (msct) are used to evaluate the amount of sclerotic plaque in coronary arteries in clinical practice. to investigate the potential of both ct techniques to identify calcified coronary lesions, we compared the results of ebct and msct with intravascular ultrasound (ivus) and histology. methods: five human autopsy coronary arteries were fixed in a saline perfused bath with vessel side branches and sutures defined as landmarks: a total of 81 vessel cross sections (vcs) were randomly assigned to undergo all 4 investigative steps. ivus was performed with an automated pull back system (0.5 mm/s). vessels were scanned with ebct (imatron, 3 mm slice thickness, 3 mm increment) followed by msct (siemens plus 4 volume zoom, 4 × 1 mm spiral mode, 3 mm slice thickness purpose: to evaluate the reliability of ca-score measurements of identical multislice ct (msct)-derived raw data obtained by the traditional agatston method and a volume score using different software systems. methods and materials: 22 asymptomatic patients with known or suspected coronary heart disease were scanned on a multidetector ct system (somatom volume zoom, siemens) with retrospectively ecg-gated 4-slice spiral scanning. data was acquired with a 4 × 2.5 mm collimation. continuous volume data sets were reconstructed in the enddiastolic phase with 3 mm slice thickness. for each patient reconstructions without (3 mm non-incremental -ni) and with a 50 % overlap (1.5 mm incremental -i) were performed. two observers independently performed calcium-scoring using the agatston (as)-and volume-score (vs) on two software systems, the calcium-scoring (siemens) and the accuimage-scoring-system. replicated ca-scoring was performed by each observer. the data was categorized into score groups 0; 1 -10; 11 -100; 101 -400; > 400. results: intra-and interobserver reliability was 0.94 -1.0 for both scoring methods (as i/ni and vs i/ni). when ca-scoring measurements were repeated on different scoring software systems, the kappa statistics were found to be 0.75 for as i, 0.69 for as ni, 0.75 for vs i, and 0.75 for vs ni, resulting in different risk stratifications in up to 14 % of the patients. conclusion: intra-and interobserver agreement was excellent using the same software. the two different softwares do not yield the same calcium quantities. there were significant differences with regard to interobserver reliability, resulting in different risk stratifications. the interpretation of calcium quantities should acknowledge which type of software was used. thirteen patients with heterozygous fh (mean ± sd; age 17.9 ± 3.4 a) and 13 controls were studied. endothelium-dependent, flow-mediated dilation (fmd) and endothelium-independent, nitroglycerin-induced dilation (nmd) were assessed in the brachial artery using high-resolution ultrasound. in addition, fh patients were evaluated for coronary calcium using ecg triggered multidetector ct. results: fmd was significantly reduced in heterozygous fh patients compared to controls (10.7 ± 5.3 % vs. 17.3 ± 4.6 %; p = 0.002). in contrast, nmd values were similar in both study groups (17.7 ± 6.5 % vs. 21.0 ± 6.3 %; p = 0.22). on multiple stepwise regression analysis, a significant correlation was found between fmd and total cholesterol (r = −0.68; p = 0.0003) as well as between fmd and vessel size (r = −0.43; p = 0.01). calcifications of the aortic root or the major epicardial coronary arteries were not detected. conclusions: endothelial dysfunction is present in the brachial artery of heterozygous fh already in the second and third decade of life without evidence of calcification of the aortic root and the coronary artery tree and is independently related to total cholesterol. thus, altered vascular function precedes morphological evidence in the course of atherosclerosis. therefore at this early age endothelial function testing seems to be more appropriate to evaluate fh patients developing atherosclerosis and to assess the short-term effects of therapeutic interventions. the association between electron-beam ct detected coronary calcification and mri detected cerebral white matter lesions j.c. de groot 1 , r. vliegenthart 1 , j.c.m. witteman 2 , m.m.b. breteler 2 , a. hofman 2 , m. oudkerk 1 ; 1 groningen/nl, 2 rotterdam/nl purpose: coronary calcification is associated with angiographically detected coronary artery disease. cardiovascular risk factors are associated with cerebral white matter lesions (wml), which in turn play a role in cognitive decline. we investigated whether coronary calcification is associated with wml. materials and methods: participants were recruited from a population-based study of elderly patients aged 60 -90 years. during 1995 -96, cerebral mri (1.5 t) scans of 554 subjects (response 63 %) were obtained on which the severity of deep subcortical wml (scwml) and periventricular wml (pvwml) was estimated using a semi-quantitative scale. during 1998 -2000 thoracal electron-beam ct scans were obtained and coronary calcium scores (according to agatston) are available for 221 of these subjects. wml was dichotomised in severe (upper quintile) versus mild to moderate (other quintiles) and the calcium score was dichotomised in severe (upper tertile) versus mild to moderate (other tertiles) for additional analyses. results: mean age was 72.3 years (50 % women). mean severity of scwml was 1.2 (range 0 -15.9) and of pvwml 2.2 (range 0 -9). median calcium score was 125.3 (interquartile range 19.5 -501.4). subjects with higher calcium scores had more scwml (rho = 0.15, p = 0.02) and tended to have more pvwml (rho = 0.12, p = 0.08). when adjusted for age and sex, severe calcium scores were associated with the presence of severe scwml (or = 2.3, p = 0.05) but not with severe pvwml (or = 1.8, p = 0.19). conclusion: preliminary analyses show that coronary calcification is associated with cerebral scwml and as such may be indicative for a generalised vascular disease process. coronary calcification detected by electron-beam computed tomography is associated with mortality r. vliegenthart 1, 2 , h.-h.s. oei 2 , a. hofman 2 , j.c.m. witteman 2 , m. oudkerk 1 ; 1 groningen/nl, 2 rotterdam/nl purpose: coronary calcification is closely related to the amount of coronary atherosclerosis, but its predictive value for cardiovascular disease and mortality has not been well established. we studied the association between coronary calcification and mortality in the population-based rotterdam coronary calcification study. materials and methods: from 1997 -2000, rotterdam study participants were invited for electron-beam ct scanning to detect coronary calcification. calcifications were quantified according to agatston's method. calcium scores of 2013 scanned subjects (46 % men, mean age (standard deviation, sd), 71 (5.7) years) were used in the analyses. the rate ratios of mortality between categories of the calcium score were calculated. results: 40 % of men had a calcium score above 500, while 24 % had a calcium score above 1000. corresponding percentages for women were 17 and 4, respectively. during a mean follow-up period (sd) of 2.4 (0.7) years, 80 subjects died (51 men, 29 women). the age-adjusted relative risk for death was 3.6 (95 % confidence interval (ci), 1.4 -9.4) for men with a calcium score above 500 compared to men with a calcium score of 0 -100 (reference category). men with a calcium score above 1000 had a 4.5 times increased risk (95 % ci, 1.7 -11.9) compared to the reference category. in women, the relative risks were 2.3 (95 % ci, 1.0 -5.6) for calcium scores above 500, and 2.8 (95 % ci, 0.9 -8.3) for calcium scores above 1000, compared to the reference category. conclusion: the amount of coronary calcification detected by electron-beam ct is predictive for all-cause mortality. methods and materials: 20 consecutive patients (11 men and 9 women; mean age 64 years) with surgically treated mitral valve disease underwent preoperative contrast-enhanced, retrospectively ecg-gated mdct. two readers assessed visibility of the mitral valve annulus, mitral valve leaflets, tendinous cords, and papillary muscles using a four-point grading scale. abnormal findings of the mitral valve, such as thickening of the mitral valve leaflets, presence of mitral annulus calcification (mac) and calcification of the valvular leaflets, were compared to preoperative echocardiography and intraoperative findings. results: visibility of mitral valve annulus and mitral valve leaflets were good or excellent (grade 2 and 3) in 15 patients (75 %), and in 19 patients (95 %) for papillary muscles; visibility of tendinous cords was inferior. mdct yielded a 95 -100 % agreement with echocardiography and surgery with regard to thickening of mitral valve leaflets and presence of calcification of the mitral valve annulus and mitral valve leaflets. conclusion: retrospectively ecg-gated mdct allows good to excellent visualization of anatomical details of the mitral valve and its apparatus except for tendinous cords, and demonstrates high agreement between echocardiography and surgery with regard to the diagnosis of mitral valve abnormalities. with increasing experience a diagnostic niche may open up for preoperative assessment of mitral valve pathology using mdct, especially for patients for whom echocardiography is of limited use, e.g., for patients with suspected perivalvular abscess. conventional parathyroidectomy is a difficult operation and unsuccessful in 5 % to 10 % of cases. to improve the efficacy, preoperative (mibi) spect scintigraphy and intraoperative radio-guided localization of parathyroid adenomas with γ probe and a quick pth assay were used. methods: forty patients with the diagnosis of primery hyperparathyroidism were studied with (mibi) spect scintigraphy. we combined the preoperative administration with the intraoperative γ probe examination to identify the exact localization of parathyroid adenomas. the intact parathyroid hormone was measured by irma method (schering cis bio international). results: in all cases parathyroidectomy was performed successfully and the preoperative localization was always confirmed. after five minutes parathyroidectomy pth concentrations decreased more than 55 % compared with the highest preexcision value. at follow-up, serum calcium and pth levels were normal in all patients. in hyperparathyroidism, the combined use of preoperative imaging mibi-spect and intraoperative γ probe examinations improved the success rate of parathyroidectomy. the most helpful use of quick pth in patients is its ability to give the surgeon quantitative assurance that all the hyper functioning parathyroid tissue has been removed. 3d ultrasound technology for the calculation of thyroid volume and the evaluation of benign cystic, nodular disease v. osti, l. cova, l. solbiati, m. tonolini, d. della chiesa; busto arsizio/it purpose: to assess whether three dimensional sonography (3d-us) can permit reliable and easily calculated volumes of the thyroid and nodules. methods and materials: 24 patients, aged 33 -67, underwent 3d-us of the thyroid gland. ten patients had prior hemithyroid resection for multinodular disease and presented with goitre relapse. after examination, all had surgical resection. the surgical specimen volume at pathology was compared with the volumes calculated using 3d-us. the remaining 14 patients had either single or multiple, cystic nodules. following 3d-us, these nodules underwent percutaneous needle aspiration of the fluid content with measurement of the aspirated volume prior to ethanol injection. us examinations were performed using linear, 10 mhz probes (voluson 530d and 730d, kretztechnik, austria). volume acquisitions were automatically achieved by transverse scans, with the addition of coronal oscillations of the transducer crystals. subsequently, both automatic [n = 9] and/or manual [n = 15] 3d reconstructions were performed, including volume calculations, for either the entire gland [n = 10] or the nodules [n = 14] . results: comparison between surgical specimen volumes and in-vivo volumes calculated by 3d-us demonstrated a restricted margin of error of 6 % for automatic reconstruction, and 4 % for manual reconstruction. the quantitative assessment of the fluid aspirated from cystic nodules was identical to the 3d-us calculation (margin of error < 1 %). conclusions: 3d-us allows easy and accurate estimation of the volume of the thyroid gland and of nodular, cystic thyroid lesions. these measurements will undoubtedly provide both surgeons and interventional radiologists additional potentially useful information prior to procedures. role of color-doppler sonography in graves' disease diagnosis and follow-up after 131 i therapy v. summaria, a. costantini, v. rufini, m. garganese, g. maresca, s. speca; rome/it purpose: to assess the usefulness of cds in evaluating activity of graves' disease and the response to 131 i therapy. methods and materials: several cds patterns: thyroid volume (tv), parenchymal hypoechogenicity (ph), parenchymal vascularity (pv), peak systolic velocity (psv) in inferior thyroid arteries (ita), were evaluated in 51 patients with graves' disease divided in 4 groups: 13 pts at first diagnosis; 12 pts during antithyroid drug treatment; 11 euthyroid pts after withdrawl of therapy; 15 pts who relapsed into hyperthyroidism. all pts in the latter group underwent radioiodine therapy (rt) and cds was performed 1, 3, 6 months after rt. 13 healthy subject were included as control group. results: all 40 pts with hyperthyroidism showed an increase of tv, pv and psv in ita, compared with patients in remission and with control subjects. in pts within group 2, cds values were lower, but not significantly different from those in group 1. ph was found in 40/51 pts, without a statistically significant difference between pts with hyperthyroidism and pts in remission. in pts within group 4, a significant decrease (p < 0.1) of tv and psv mean values in ita was observed in 11/15; the others pts, with residual signs of hyperthyroidism underwent further rt. materials and methods: forty two patients with 57 nodules were prospectively enrolled in this study. the nodules were studied in a transverse plane with conventional high frequency b-mode imaging and with sono-ct (atl hdi 5000, l12-5 linear probe). the data were transferred on a pc (hdi lab). after selecting two comparable images in both modes, three parameters of quantification were measured using region of interest: the difference of contrast between the nodule and the adjacent thyroid (db), the slope of the transition between the nodule and the adjacent tissue (db/cm). the nodule heterogeneity was evaluated as the standard deviation of the mean signal intensity normalized with the mean signal intensity (linear units). the contrast between the nodule and the adjacent thyroid was systematically greater with sono-ct (mean 4.3 db). the transition slope was higher with sono-ct. the average difference between the sono-ct slope and the conventional b-mode slope was 5.2 db/cm. the heterogeneity index was superior in 83 % of cases with this modality. however, sono-ct imaging exhibited a blurring artifact when the probe was moved too fast, despite the use of a unique focal zone. conclusion: the objective evaluation using appropriate quantification technique showed a significant improvement in the visibility of thyroid nodules with both sono-ct when compared to conventional b-mode imaging when the linear probe was moved very slowly. pre-biopsy investigations included scintigraphy and thyroglobulin levels. then we examined the us characteristics of the nodule (low reflectivity, irregular margins, thick-irregular halo, intranodular blood flow pattern, hypervascularity, invasion of vessels and adjacent structures, vessel encasement). in 25 patients the nodule was solitary and not palpable and in 20 patients the disease was multinodular with a nodule having characteristics suspicious for malignancy. we performed us guided fna biopsy using 21 g needle. we used negative pressure on the syringe to obtain material through the needle. it is important to allow the pressure to equalize before removing the needle. the cytological specimen obtained was examined by cytopathologist. a post biopsy us was performed to exclude complications. the following results were obtained: follicular ca (n = 1), papillary ca (n = 1), undiferentiated ca (n = 1), hashimoto thyroiditis (n = 6), colloid and bening follicular cells (n = 7), blood (n = 5), nodular hyperplasia (n = 22), colloid and activated lymphocytes (n = 1), colloid and histiocytes (n = 1). conclusion: us guided fna is safe, fast and low cost method to determine the nature of thyroid nodules and exclude malignancy. papillary stenosis: differential diagnosis with mr-cholangiography using a cholecystokinetic drug m. di girolamo 1 , l. azzarri 2 , r. di nardo 1 , c. rende 1 , g. brughitta 2 , v. david 1 ; 1 rome/it, 2 grottaferrata/it purpose: to differentiate with mr-cholangiography (mrcp) inflammatory papillitis from functional papillary stenosis, using a cholecystokinetic drug. methods and materials: 17 patients with papillary stenosis diagnosed with ercp underwent mrcp before and 30 minutes after i.v. injection of ceruletide (takus, farmitalia, italy) . this drug stimulates the activity of the gallbladder and promotes the relaxation of oddi's sphincter. mrcp was performed by using 3-d non-breathholding, fat-suppressed turbo se sequence (tr: 3000 ms; te: 700 ms; n.ex.: 4; etl: 128; matrix: 128 × 256; acq. time: 5 min 12 s) on coronal planes with 0.5 t superconductive magnet (gyroscan t5 iii, philips medical system). the 3d coronal images were post-processed with mip algorithm. results: in 13 patients mrcp diagnosed fibrotic papillary stenosis while in 4 patients detected functional papillary stenosis that in 3 cases was confirmed by ercp with manometry. the diagnosis was based on the evaluation of the mbd calibre after drug administration: a partial reduction of the calibre is typical of functional papillary stenosis while the persistence of the same calibre allows the diagnosis of fibrotic papillary stenosis. mrcp could also exclude choledocholithiasis whose clinical symptoms can simulate papillary stenosis. conclusions: it is possible to differentiate with mrcp between fibrotic and functional papillary stenosis, without performing invasive procedures like ercp with manometry. this differential diagnosis is very important: in fact, fibrotic stenosis should be treated with sphinterectomy while functional papillary stenosis does not need any particular treatment. methods and materials: 52 patients (25 women, 27 men) with confirmed psc were examined with mri at 1.5 t comprising: t1-weighted spoiled gradient echo (sge), t2-weighted fat-suppressed turbo spin echo, haste, and serial postgadolinium t1-weighted sge sequences without and with fat-suppression. two radiologists reviewed retrospectively all images independently and in a consensus reading. the evaluations comprised: the imaging pattern of the liver parenchyma; the presence and grade of intrahepatic biliary ductal dilatation; the presence of areas of parenchymal atrophy or abnormal signal and/or gadolinium enhancement. results: a "diffuse pattern" of liver cirrhosis was observed in 17 of 52 patients (33 %). a "large macronodular pattern" (regenerative nodules ≥ 3 cm) was observed in 28 of 52 patients (54 %) with nodules showing low signal on t1-and t2-weighted images and low gadolinium enhancement. intrahepatic biliary ductal dilatation was observed in 44 of 52 patients (85 %) with a "general distribution" in 18 patients (35 %) and a "segmental distribution" in 26 patients (50 %). peripheral wedgeshaped areas of abnormal parenchyma showing atrophy were observed in 24 patients (46 %) and without atrophy in 18 patients (35 %). the signal intensity on t1-/t2-weighted images was usually low/high. gadolinium enhancement was typically late in areas with atrophy and early in areas without atrophy. conclusion: the spectrum of mri appearances of psc is diverse and comprises distinct patterns. large regenerative nodules are a frequent finding and may help to establish the diagnosis. for 17 months, 7 patients were diagnosed to have incomplete pancreas divisum on ercp and six of them underwent mrcp after secretin stimulation. the maximal diameter and length of pancreatic duct (ventral, dorsal, and main duct) and the angle between the main pancreatic duct (mpd) and the communicating channel (cc) were measured on ercp. morphologic changes of the pancreatic duct, the presence of santorinicele, the shape of cc, and associated bile duct anomaly were analyzed on ercp and mrcp. results: ercp showed dominant dorsal duct and foreshortened ventral duct with a small communication between the two ducts. the mean caliber of dorsal, ventral, and main duct was 3.8 mm, 4.2 mm, and 4.2 mm, respectively. the angle between the mpd and the cc was acute in 3 patients, right angle in 2, and obtuse in 2. the cc was classified into short (n = 5) and long (n = 2) types. the morphologic features of pancreaticobiliary duct included: santorinicele in 2, beaded appearance of pancreatic duct in 5, pancreaticoliths in 3, and bile duct anomaly in 1. mrcp successfully demonstrated the presence of the cc and associated morphologic features of the pancreaticobiliary duct in all 6 patients. purpose: since evaluation of bile ducts in mrcp can be complicated by other fluid filled structures as pancreatic or bile duct cysts superimposing the region of interest and the clinical relevance of a dilated bile duct regarding functional stenosis may be unclear we investigated the hepatobiliary contrast medium gd-bopta as a positive contrast enhancer for intra-and extrahepatic bile ducts in mri. material and methods: 34 patients were investigated on a 1.5 t scanner (magnetom vision, siemens, erlangen) using unenhanced t1 an t2 w breathhold sequences as well as 2d-and 3d-mrcp. to evaluate contrasted biliary structures 60 to 90 min after cm administration (gd-bopta, 0.05 µmol/kg bodyweight) a 3d gre sequence as well as t1w axial images were applied. results: in the majority of the patients (28/30) bile duct structures could be delineated by the mrcp sequence. however the identification of the common bile duct in the region of the pancreatic head was not possible in 6 patients. in 7 patients following pancreatitis or cholelithiasis a dilatation of the common bile duct was observed with unclear functional relevance, in 4 patients following resection of the pancreatic head the anastomosis of the common bile duct could not be displayed in detail in mrcp. ce mrcp was able to delineate the bile duct in 8 of the 10 unclear cases, functional relevance of dilatation could be evaluated in 4 of 7 patients. conclusion: in unclear cases gd-bopta can be helpful for a better delineation and functional evaluation of bile duct structures. follow-up of suspected pancreatic intraductal papillary mucinous tumours (ipmt) of collateral branches: preliminary results g.m. carbognin, a. guarise, e. dalla chiara, n. pagnotta, c. procacci; verona/it purpose: to verify the evolution of suspected collateral branches ipmt assessed as benign at the first imaging control. methods and materials: 14 patients with a presumptive diagnosis of benign branch duct ipmt formulated by ercp (#6) and/or mrcp (#10). diagnosis on the basis of the following criteria: (1) no previous pancreatitis; (2) one/multiple cystic lesion/ s, homogeneous liquid content, thin wall, no parietal proliferation; (3) maximum diameter of the lesion < 3 cm; (4) normal main pancreatic duct (mpd). the patients were submitted to mrcp follow-up every 6 months. two observers reviewed all the mrcp examinations. results: mean duration of the follow-up: 10.5 months (range: 3 -28 months). 12/14 (86 %) lesions with the requisite for benignity reported in literature, did not show any morpho-structural modification during the follow-up. in 1 case the lesion manifested rapid growth after 3 months, along with what seemed to be a small parietal nodule. at intervention, it was a retention cyst in a mild focal pancreatitis. in the other case, multiple apparently benign lesions were present, which demonstrated slight growth over 13 months of follow-up. nevertheless, the patient was submitted to strict mrcp monitoring, since a total pancreasectomy would have been warranted. conclusion: among those considered, lesions of different nature could be present. however, the evolution of all these lesion, when the sign of malignancy are absent, is so slow to justify the follow-up in the elderly, in the presence of high surgical risk or whenever the multiplicity of the lesions would require a largely demolitive intervention. results: mrt and mrcp were false positive in a patient with a benign stricture due to an inflammatory pseudotumor and false negative in two patients (suspected granulation tissue after prior surgery and contrast enhancement of the wall of the bile duct after interventional therapy). except for one false negative patient mrcp revealed stenosis of central bile ducts in all patients. in 15 patients a well demarcated mass was seen while 12 patients revealed circumferential tumor spread along the bile ducts. most lesions appeared hyperintense on t2-weighted images (n = 22) and hypointense on unenhanced t1-weighted images in all cases. contrast enhancement was either lower (n = 21) or higher (n = 7) than normal liver parenchyma. t2-weighted images and gadolinium-enhanced fat-suppressed t1weighted images showed best lesion conspicuity and tumor margins in 12 and 13 patients respectively. combined ercp, us and ct suggested hilar cholangiocarcinoma in 21/28 (75 %) patients. mrt with mrcp appears to be the most sensitive imaging modality for detection of hilar cholangiocarcinomas and may replace us, ct and ercp in the evaluation of suspected malignant bile duct obstruction. information supported by mrcp: consistency and repeatability t. gorycki, b. bobek-billewicz, m. studniarek, p. jagodzinski, k. grabska; gdansk/pl purpose: to compare decisions of radiologists watching mrcp images in order to find the strongest features on mip reconstructions which characterise biliary strictures. materials and methods: mrcp mip reconstructions (0.5 t gyroscan nt) from 80 cases of biliary stricture had undergone estimation by three independent observers who performed linear measurements on mrcp images concerning morphology of the biliary stricture including stricture length, proximal ductal dillatation and distance from stricture to hepatic ducts junction. last of the measurements has introduced information about stricture localisation along the biliary tree. measurements were compared in two stages: between two observers as well as counting their mean values against third observer. the analysis included change, consistency and repeatability values with relative repeatability coefficient (rrc). results: repeatability counted with rrc presented values from 3.7 % to 12.12 %, consistency did not exceed 1.52 mm when stricture length or proximal biliary dilatation were measured and 2.5 mm when stricture was localised along biliary duct. conclusion: mrcp mip reconstructions are valuable and easily understood images in radiologists' every day practice when morphology and localisation of biliary stricture is discussed. the most reliable conditions seem to be stricture length and stricture localisation. assessment of the role of translabial perineal ultrasonography (tpus) to evaluate urinary stress incontinence in females c. roy, d. pfleger sr., e. castel sr., h. lang sr., c. saussine sr.; strasbourg/fr purpose: assess the usefulness of translabial perineal ultrasonography (tpus) in the evaluation of the two mechanisms (hypermobility of bladder neck and intrinsic sphincter deficiency) of urinary stress incontinence (usi). method/materials: 52 women (mean age: 58.7) underwent us with translabial perineal approach (7.5 mhz transvaginal probe) located on the perineal floor, behind urethral meatus. examinations performed in sagittal and coronal, in decubitus and standing positions, at rest, and during pelvic floor contraction and straining. bladder was filled with 200 -300 ml of urine. morphology of the bladder neck (funneled) was evaluated to diagnose isd. to evaluate hbn, position and displacements of bladder neck was evaluated calculating the distance using inferior border of the symphysis pubis as reference according an orthogonal system, in sagittal plane. the diagnosis of hbn was based on a displacement up to 1.5 cm. correlation was made with clinical evaluation and urodynamic testing results. results: among 35 patients with the clinical diagnosis of hbn, tpus confirmed a displacement and depicted in 8 cases an additional isd in front of funneling of bladder neck at rest and straining. among 17 patients with diagnosis of isd; us confirmed an abnormal bladder neck. an association with bladder neck hypermobility was found in 10 cases of this group. for the diagnosis of cystocele (20 cases), correlations were excellent, but without additional findings for tpus. conclusions: tpus is easy to perform and no time consuming. it provides a perfect assessment of bladder neck during static and dynamic studies. it allows the diagnosis of associated mixed types of usi with a better classification of these disorders. dynamic fast mri of pelvic floor dysfunction in females using a t2-w turbo spin echo technique: value of knee flexion lateral position in comparison with conventional supine position c. roy, f. mayer sr., j. pfeiffer sr., e. castel sr., c. saussine sr.; strasbourg/fr purpose: evaluate organs displacements due to patient position by using a knee flexion lateral versus supine position and its role in the quantification of abnormalities. materials and method: 55 women symptomatic for pelvic floor descent and 27 asymptomatic were explored with dynamic sagittal mri (1.0 t) using a t2w tse sequence (tr/ete: 13950/100 ms) at rest, pelvic floor contraction, maximal straining and two series: supine and lateral position with knee flexion. distances from bladder base, cervix and anorectal junction perpendicular to the pubococcygeal line were measured in the sagittal plane. in symptomatic patients, correlations with clinical evaluation and colpocystorectography was performed. results: at rest and contraction, there was no significant difference. during straining; for asymptomatic patients mean descent differences of bladder base, cervix and anorectal junction between the two positions were 1.5 cm (± 0.5), 1.8 cm (± 1), 2 cm (± 0.9), respectively and for symptomatic patients 4.8 cm (± 1), 4.5 cm (± 0.8) and 6 cm (± 1), respectively. the lateral position revealed 5 rectoceles and 10 additional cystoceles with 4 enteroceles associated to rectal prolapse. the highest displacements in lateral position were better correlated with clinical findings. conclusion: dynamic mri in lateral position is easy to perform, is more reliable to assess pelvic floor prolaps and to detect associated defects of other compartments. role of translabial perineal ultrasonography in females after surgical treatment of urinary stress incontinence by tension vaginal tape (tvt): a prospective study c. roy, d. pfleger sr., e. castel sr., h. lang sr., c. saussine sr.; strasbourg/fr purpose: the efficiency of tvt unknown. to assess the morphologic and dynamic modifications of bladder neck in order to better understanding its action. method/materials: 41 females (mean age: 58.7) underwent translabial perineal us (7.5 mhz transvaginal probe located on the perineal floor behind urethral meatus) in sagittal, decubitus and standing positions, at rest, during pelvic floor contraction and straining, before and after surgery. bladder was filled with 200 -300 ml. position, morphology, and displacements of bladder neck were calculated using inferior border of the symphysis pubis as reference according to an orthogonal system. correlation was made with clinical findings and urodynamic results. results: tvt was always perfectly seen as linear hyperechoic structure; against lower third (31), middle lower third junction (8) and upper third (2) of urethra, respectively. before surgery, 15 cases had funneling of bladder neck in addition to hypermobile bladder base. after surgery, all bladder necks were closed, located at rest above the inferior portion of symphysis. in decubitus position, displacements before and after tvt were 30 -7 mm (m = 18.2 ± 1.2) and 45 -9 mm (m = 20.6 ± 0.5) at pelvic floor contraction and straining respectively. in standing position, they were 10 -5 mm (m = 7.2 ± 0.7) and 18 -3 mm (m = 8.2 ± 0.6) respectively. among 5 cases of dysuria, tvt was located against the middle (3) or upper part (2) with angulation. conclusions: tvt procedure limit excessive movement of bladder neck. it must be located against the lower third of urethra. us examination is reliable to detect malpositions. in 20 patients with symptomatic uterine fibroids cemra (symphony-siemens; 1.5 t) of the pelvic and uterine vessels combined with conventional mr imaging was performed. we used a breath-hold 3d flash cemra (tr/te 3.6/1.3, fa 25°) and injected gadolinium (0.1 mmol/kg b.w.) at a rate of 3.0 ml/s. clinical value of the arterial and venous cemra for the planning of the uae was evaluated. after the mr-imaging biphasic uae was performed. the selective and superselective dsa sequences were compared with a vessel by vessel technique with the cemra. results: all cemra were technically successful and all topographically relevant segmental/subsegmental arteries could be evaluated. the common iliac, external iliac, internal iliac and uterine artery could be identified in all cases. the vascular 3d information of mra provides additional information for the planning of the intervention and the superselective catheter placement. the arterial cemra phase allowed an exact demarcation of the hypervascular fibroids in 19 patients, thus defining the primary access site for the intervention. the venous phase did not provide an additional diagnostic information. the time for the interventions and the radiation could be reduced by using the diagnostic information of cemra in 18 of the patients. cemra combined with mri of the pelvis appears to be an important diagnostic tool for analysing the volume and location of the fibriod and planning of the vascular access. withdrawn by author b-0117 11:25 clinical stage i carcinoma of the uterine cervix: value of preoperative mr imaging in assessing parametrial invasion s. sironi; milan/it purpose: the purpose of this prospective study was to assess the efficacy of different mr imaging techniques in the evaluation of parametrial tumour invasion in patients with early stage cervical cancer. methods and materials: 73 consecutive patients, clinically considered to have stage 1b tumour (confined to the cervix), underwent mr imaging studies at 1 t, according to the following protocol: fast spin-echo (fse) t2-weighted, gadoliniumenhanced se tl-weighted, and fat-suppressed gadolinium-enhanced se tlweighted sequences. images obtained with each sequence were evaluated for parametrial invasion with the use of histopathological findings as the standard of reference. results: in the assessment of tumour infiltration into the parametrium, with fse t2-weighted images accuracy was 83 %, with se tl-weighted gadolinium-enhanced images was 65 %, and with se tl-weighted gadolinium-enhanced fat-suppressed images was 72 %. the difference between the accuracy rate achieved with fse t2-weighted images, and those obtained with the other two mr sequences was statistically significant (p < 0.05). the high negative predictive value (95 %) for the exclusion of parametrial tumour invasion was the principal contributor to the staging accuracy obtained with fse t2-weighted imaging. conclusion: unenhanced fse t2-weighted imaging is a reliable method in determining the degree of tumour invasion in patients with early stage cervical cancer. our data suggest that contrast-enhanced sequences, even with the use of fat suppression technique, have a limited value in the assessment of tumour extension. methods and materials: mri of the pelvis was performed 24 h after i.v infusion of uspio (2.6 mg fe/kg b.w.) in 9 patients (mean age 48 years) with gynecologic tumours (6 cervical, 2 endometrial carcinomas, 1 malignant mixed mullerian tumour) who were scheduled for surgery. axial t1w se, t2w fse (fatsat), fspgr, sagittal and oblique t2w fse sequences were acquired on a 1.5 t system. next to the primary tumour, lymph nodes were staged prospectively using newly established criteria and results correlated with histology. results: mri correctly diagnosed the primary tumour as stage i in 7 cases and stage ii in 1 case respectively. in 1 case preoperative diagnosis of stage iv led to a change in the therapeutic approach and chemotherapy was given. in this case, multiple malignant iliac nodes were demonstrated. no surgical proof was available. 58/62 of the lymph nodes detected on mri, in which a histological correlation was available, were correctly staged (accuracy 94 %). 1 node was false positive; 3 micrometastasis < 5 mm were missed. conclusion: according to our preliminary results, uspio-enhanced mri of the pelvis has the potential to become an important tool for preoperative assessment of uterine tumours. the prognostic significance of high-resolution ct morphology compared to a histological diagnosis in usual interstitial pneumonia (uip) and nonspecific interstitial pneumonia ( in idiopathic pulmonary fibrosis (ipf) both histological and ct features are known to predict outcome. the aim of this study was to compare the prognostic significance of the histological diagnosis (uip vs nsip) and ct findings. materials and methods: 53 biopsy proven cases of uip and nsip were diagnosed by consensus by two histopathologists. the hrcts were scored semiquantitatively by 4 observers with regard to the proportion of ground glass opacity to reticulation (1 = predominantly ground glass, 2 = mixed ground glass and reticulation, 3 = predominantly reticulation). in addition the exact proportion of ground glass opacity was recorded. results: 32/53 cases were classified as uip and of the 21/53 classified as nsip 5/21 were cellular and 16/21 fibrotic. at follow up (median 33 months) there were 22/53 deaths. both an increasing extent of ground glass opacity and a lower semiquantitative ct score were associated with a better outcome (p = 0.02, p = 0.01 respectively). the histological diagnosis of nsip was also associated with lower mortality (p = 0.05). on bivariate analysis hrct provided the more powerful prognostic information. conclusion: in terms of prognosis hrct features in ipf provide much information with little added value from the histological diagnosis. correlation between histologic features and ct morphology in idiopathic pulmonary fibrosis (ipf) s.m. ellis 1 , s.r. desai 1 , d.m. hansell 1 , a.g. nicholson 1 , t.v. colby 2 , r.m. du bois 1 , a.u. wells 1 ; 1 london/gb, 2 scottsdale, az/us purpose: there is increasing evidence that the profusion of fibroblastic foci seen at histology in ipf is predictive of mortality. the aim was to determine whether high-resolution ct appearances correlate with prognostically important histological features. the hrct scans of 34 histologically confirmed cases of ipf were scored by two observers according to the proportion of macro (≥ 4 mm) and microcystic (< 4 mm) honeycombing, fine reticulation and ground glass opacity. their relationships to histological scores of fibroblastic foci and macrophages were examined. results: there was a positive correlation between the proportion of combined hrct score of macro and microcystic honeycombing and the number of fibroblastic foci on histology (p = 0.04). a further positive correlation was found between the extent of ground glass opacity and the macrophage score (p < 0.01). there was no correlation between any histological feature and the extent of fine reticulation on hrct. purpose: to describe the radiological (thin section ct) findings correlated to activity and remission in anca associated pulmonary-renal small vessel vasculitis (svv) material and methods: we used retrospective analysis of 37 cts, 27 in disease activity (8 first manifestations, 19 relapses) 10 im remission of 17 patients with pulmorenal syndrome (9 wegener, 4 microscopic polyangiitis-mpa, 3 churg-strauss-syndrome, 1 idiopathic crescentic glomerulonephritis following the chapel hill classification) 7 women, 10 men, median 65.5 years (34 -84). the diagnosis was established by clinical presentation (cough, dyspnoea, hemoptysis and/or fever associated with significant hematuria or progressive renal failure), anca titers (11 c-anca, 5 p-anca, 1 anca negative) and renal biopsy in all patients. results: pulmonary involvement defined by pathological ct occured in all patients. nodules with (n = 4) or without (n = 2) cavitation, ground glass opacity (n = 5), air space consolidation with (n = 6) or without (n = 9) cavitation and interstitial septal thickening (n = 4) were detected. local interstitial infiltration and nodules in wgs, diffuse interstitial alveolar infiltration in mpa and css, neither lower versus upper lungfield involvement nor side preverence were evident. radiological course of pulmonary involvement was the best indicator for therapeutic success after immunosuppression, occured earlier compared to renal symptoms and was a stronger indicator in first manifestations then in relapses for disease activity. conclusion: thin section ct as the most sensitive radiological examination detects and classifies efficiently the extent of pulmonary involvement of svv, most likely air space consolidation and ground glass opacity, it proves to be the most efficient therapeutic monitor indicating remission. traction bronchioloectasis on hrct in simple pneumoconiosis d. ryu; kangnung/kr objective: traction bronchioloectasis and focal pericicatrical emphysema in p type pneumoconiosis have been well known in pathologic report. however, traction bronchioloectasis, earlier finding of p type pneumoconiosis has not been yet radiologically reported. therefore, we describe traction bronchioloectasis on hrct in p type pneumoconiosis and assess its incidence among small rounded opacities (p, q, r). we retrospectively analyzed the hrct scans of 11 patients that showed small rounded opacity less than 1 centimeter. the study group had a history of occupational exposure to dust and was comprised of all men, 47 -67 years old (mean age 60). type p, q, r rounded opacity consisted of 5, 2, 4 patients respectively. two radiologists assessed with consensus whether traction bronchioloectasis was present or not, and its incidence at three levels (aortic arch, carina, and between carina and diaphragm). the degree of traction bronchioloectasis was classified as follows: score 1: 1 -5 traction bronchioloectasis were visible at each scan level; score 2: 5 -10 traction bronchioloectasis; score 3: over 10 traction bronchioloectasis. the total score was the sum of three level score. result: all patients showed traction bronchioloectasis. the degree of traction bronchioloectasis decreased with increase of nodule size; p type 6.8; q type 4; r type 2.3. conclusion: traction bronchioloectasis is an early finding of pneumoconiosis with centrilobular dot or branching structure on hrct. such findings could be helpful to differentiate pneumoconiosis from other diseases showing centrilobular nodule. viewing efficiency of soft-copy reading of high-resolution ct of the lungs s.m. ellis, x. hu, l. dempere-marco, g.-z. yang, d.m. hansell; london/gb purpose: soft-copy ct reading is increasingly common and offers the radiologist a choice of many viewing formats. using eye-tracking data we have assessed the efficiency of reading high-resolution ct of the lungs comparing two fundamentally different viewing formats. the eye movements of 2 experienced radiologists viewing hrct's demonstrating diffuse lung disease (n = 6), airways disease (n = 6) or normal appearances (n = 4) displayed on a single monitor were recorded. the time taken to view each case, the number and duration of fixation points, the number of movements between sections and the proportion of these leading to fixation were calculated. the viewing formats used were 4 sections displayed simultaneously (2 × 2) and single sections displayed at full screen size (stack mode). sections in both formats were viewable by sequential scrolling. results: the number, duration of fixation points, and the number of movements between sections were greater in the 2 × 2 mode (p = 0.03, p = 0.05 and p < 0.001 respectively. however, the proportion of movements between sections leading to a fixation point was greater in stack mode (p < 0.001). there was no significant difference in the overall time taken on each case. the viewing efficiency of soft copy reading of lung hrct is greater with sections presented in a 2 × 2 format compared to a stack mode, despite the tendency towards non-fixation movement between sections in the 2 × 2 format. the group of most severe cases (parasitemia > 5 %, presence of complication) included 53 pts (19 %). radiographic pulmonary examination was performed in 200 pts. results: pulmonary radiographic changes were founded in 23 pts (43 %) with severe malaria. 21 pts (91 %) had solitary or multiple pulmonary infiltration. one patient had hospital acquired pulmonary infection during artificial ventilation, while noncardiac pulmonary oedema with fatal outcome was noted in one patient. the therapy in this group of patients was based on quinine/arthemeter parentally. in 40 pts (81 %) tetracycline, erythromycin or clindamycin was added as second antimalarial drug. all pts who suffered from pulmonary complications received also various antibiotics parenteral (ampicilin/ceftriaxon/cefotaxim/imipenem, ciprofloxacin, aminoglycoside, vankomcin) according to severity of pulmonary changes, bacteriological findings (blood, sputum) and up to date therapetutical recommendations. conclusion: severe malaria is often connected with pulmonary manifestations, which implies prolonged course of healing and even fatal outcome (noncardiac pulmonary oedema). the value of post-contrast flair in the detection of brain metastasis a. patsalides, j.a. butman, n. patronas; bethesda, md/us in this study we assessed the value of post-contrast flair in the detection of brain metastasis. brain mri studies were performed in 160 patients with melanoma and renal cell carcinoma. fluid attenuation inversion recovery (flair) and t1-weighted (t1-w) scans were obtained before and after contrast administration in each patient. each of these techniques was evaluated blindly by two radiologists who diagnosed the presence of metastatic tumors by consensus agreement. follow up studies were available in patients with positive studies and the temporal changes of the lesions were used to confirm the diagnosis. cases. at one month the mean serum creatinine level had decreased from 359 ± 138 mmol/l to 298 ± 87 mmol/l, blood pressure was reduced in all the patients from 160/82 ± 24/14 mmhg to 145/80 ± 16/10 mmhg and drug therapy was reduced from 3.3 ± 1.3 drugs to 2.4 ± 1.5 drugs. results were maintained at 3 months. conclusions: in cases of renal impairment, renal ptra/s using gadolinium chelates as radiographic contrast agents can be performed using standard digital subtraction angiography (dsa) equipment. gd-dota (0.3 mmol/kg) can be administered intra-arterially without any effect on renal function up to a total volume of 45 ml. carotid artery stent implantation with cerebral protection: multicenter experience of 320 procedures f. castriota 1 , b. reimers 2 , n. corvaja 3 , r. manetti 1 , c. cernetti 2 , c. di mario 3 , p. pascotto 2 , a. cremonesi 1 , a. colombo 3 ; 1 cotignola/it, 2 mirano/it, 3 milan/it background: distal embolization of debris during percutaneous carotid artery stenting may result in neurological deficit. methods/results: 320 consecutive procedures (308 patients) of elective carotid stent implantation with cerebral protection performed in 3 different centers were included in a prospective registry. cerebral protection was performed using filter devices (80.6 % of procedures), occlusive distal balloons (17.2 %), and endoluminal clamping of the common and external carotid artery (2.2 %). all lesions were > 70 % diameter stenoses (mean 82 ± 8 %). mean age of the patients was 67 ± 11 years, 83 % were males, and 58.7 % of patients had had a previous stroke or transient ischemic attack. in 313 procedures (97.8 %) it was possible to position a protection device. in 9 of 55 procedures using distal balloon protection, this was not tolerated by the patient (2.8 %). in 317 procedures (99.1 %) a stent was successfully placed. neurological complications during the procedure, in-hospital and during 30 days of follow-up, occurred in 6 patients (1.9 %). these were 1 major stroke (0.3 %; amaurosis of ipsilateral eye), 3 minor strokes (0.9 %), and 2 transient ischemic attacks (0.6 %). protection device related complications, all without neurological symptoms, occurred in 8 procedures (2.4 %). these were 7 distal dissections (3 required additional stents and 1 lead to occlusion of the internal carotid artery) and 1 filter entrapment requiring surgical removal. major adverse cardiac events during the 30 days of follow-up occurred in 2 patients (0.6 %). conclusions: routine use of cerebral protection during carotid artery stenting appears feasible. in this study the incidence of neurological complications was low. fresh dwi lesions after stenting of the internal carotid artery v. moeller; homburg a. d. saar/de purpose: our aim was to reveal the number of thrombembotic events after stenting of the carotid artery. methods: 38 patients underwent stenting of the carotid artery. afterwards patients were neurologically examined and underwent mr imaging. stenting was performed without protective devices. systemic anticoagulation was started three days in advance. in 10 cases predilatation of the stenotic vessel was performed. results: 40 stents were succesfully placed. 4 patients developed neurological deficits, 1 had a minor stroke, 3 a transient ischemic attack. retrospectively anticoagulation was found to be insufficient in one of these cases. 3 months after the intervention none of the patients had developed neurological deficits. diffusion weighted mr imaging detected fresh lesions in 16 patients. the medium degree of stenosis was 88.2 %, this being significantly higher than the 82.7 % among those without new lesions on dwi. furthermore the mean age of the first group was thromboembolic events -a challenge in interventional neuroradiology i.q. grunwald, v. möller, w. reith; homburg a. d. saar/de purpose: thromboembolic events are the most frequent complication in the endovascular treatment of intracranial aneurysms with gdcs. at the moment diffusion-weighted imaging is the most sensitive method for their early detection. our aim was to evaluate the number of ischemic events by using diffusion-weighted mri. method: before and within 48 hours of endovascular treatment with gdcs 13 patients with 15 asymptomatic aneurysms were studied with diffusion-weighted images. during positioning of the coils and for another 2 days the patients were heparinised. a neurological examination took place before and after coiling as well as on discharge. results: occlusion of the aneurysm was achieved in all cases. dwi showed small, fresh hyperintense areas in 6 cases. they were all located in the vascular territory of the aneurysm and have to be seen as intervention-related. none of the patients had clinical symptoms. occurance of thromboembolic events did not correlate with the number of coils used. conclusion: regarding the frequency of thromboembolic events, even though they are mostly silent, dwi monitoring should follow intervention. we assume that the thrombembolic lesions are due to embolic fragments that migrate from the aneurysm as well as due to ischemic events of other origin (vasospasm, arteriosclerosis). therefore we think that in the endovascular treatment of asymptomatic aneurysms systemic anticoagulation should be performed. purpose: for most of the pregnant women who underwent an obstetric flow study using color doppler imaging, we calculated low velocity values from the foetal middle cerebral artery in comparison to literature. the aim of this study was two-fold: to define the normal flow values of the foetal and maternal arterial systems and to compare the results with the literature. methods and materials: 124 healthy pregnant women of various gestational age who underwent a color doppler examination were studied after having an uncomplicated pregnancy and labour. pulsatility index (pi), resistance index (ri), peak systolic/end diastolic ratio (s/d ratio) values were calculated for the foetal aorta (fa), foetal middle cerebral artery (fmca), umblical artery on the maternal (mua) and foetal (fua) side and the maternal uterine artery (uta). the results were compared with the published literature. results: 20 to forty week pregnancies were divided into 10 different groups, each group represented a period of 2 weeks of gestational age. for each group, the studied parameters were defined and presented in a diagram. flowmetric values are helpful in defining the existing or potential abnormalities in the foetus. the published literature was used to compare patient flowmetric study results. in our study, we found significant differences for fmca and ua values compared with the published data. to study the relationship of uterine artery impedance measured as pulsatility index (pi) and resistance index (ri) on the outcome of in vitro fertilization -embryo transfer cycles. methods: 55 women in the age group between 25 -42 a were studied on the day of embryo transfer by performing tranvaginal color doppler sonography. uterine artery pulsatiliy index, resistance index and endometrial thickness were calculated. subsequently pregnancy rates were calculated. results: the pregnancy rate was 35 % in patients with a pulsatility index 2.45 ± 0.54 and resistance index 0.85 ± 0.04. in patients with a pulsatility index higher than 3.00 and resistance index higher than 0.91 the pregnancy rate decreased significantly to 15 %. purpose: in addition to histologic evaluation of the effect of antiangiogenic treatment with anti-vegf receptor antibodies, a functional investigation of tumour vascularisation was performed using contrast enhanced, intermittent power doppler sonography in tumour bearing nude mice. methods and materials: 10 thymus-aplastic nude mice with hacat-ras-tumours were examined weekly for 4 weeks after i.v.-bolus injection of levovist with intermittent power doppler sonography. this further developed method allowed statements about the quantification of blood flow and volume by varying the time delay between doppler imaging sequences to be made. besides other vascularisation parameters like time of arrival, slope of the curve, maximum and plateau phase of the colour pixel density (pcd) and mean colour value were measured. five mice were treated with a vegf-receptor antibody during the examination period. results: while without contrast agent nearly no blood flow was visible, the tumours showed clearly visible enhancment of doppler signal after injection of levovist. tumours without therapy showed an increase in pcd signals during the tumourgrowing phase. mice with antiangiogenic treatment showed a signal decrease in the first monitoring examination, even in growing tumours. there was a limitation of doppler signals to the periphery of the hacat-ras-tumours whilst untreated tumours showed a homogenous enhancement except in necrotic areas that were found in later phases. conclusion: contrast enhanced sonography enables vascularisation measurements of hacat-ras-tumours and the monitoring of antiangiogenic therapy. therapeutic effects could be visualized before a macroscopic change of tumour size was observed. furthermore intermittent doppler sonography enables objective perfusion parameters in ultrasound. we prospectively examined the va origin with cds using a 4 -8 mhz endovaginal transducer in 244 vas in 122 consecutive subjects referred for evaluation of carotid and vertebral arteries. we also evaluated va origins of the same subjects using a 5 -8 linear transducer and compared the visualization rates of both transducers. the criteria for visualization of va origin was identification of origin of an artery arising from the subclavian artery and entering the transverse foramen at the c6 level. angle-corrected peak systolic velocity (psv), end diastolic velocity (edv), and resistive index (ri) were determined. angiography was available for comparison in 40 vas of 20 subjects. results: two aplastic right vas of 2 subjects were excluded. the va origin was visualized in 241 (99.6 %) of 242 vas (120 right and 121 left vas) with the endovaginal transducer and 161 (66.5 %) of 242 vas (97 right and 64 left vas) with the linear transducer. psv, edv, and ri of 241 vas were 61.9 ± 29.6 cm/s, 15.3 ± 9.9 cm/s, and 0.75 ± 0.08 (mean ± sd), respectively. angiography revealed stenosis (> 50 %) of the va origin in 2 vas (psv > 150 cm/s at cds) and normal patency in 38 vas. in the only one of 242 vas missed at cds with the endovaginal transducer, the va was shown to arise directly from the aorta at angiography. conclusion: cds with an endovaginal transducer was a very useful and efficient method to visualize the va origin. doppler sonography of sympathetic vasomotor response in patients with diabetic foot syndrome a. hlawatsch 1 , j. bauer 1 , v. kuhl 1 , s. mink 2 , m. eicke 1 ; 1 mainz/de, 2 heilbronn/de purpose: neurophysiological assessment of the peripheral autonomic system is limited. continuous wave doppler sonography of the radial artery can be used to measure the sympathetic vasomotor response (svr). we studied 25 patients (mean age 63 a) with diabetic foot syndrome. the data from 4 mhz cw doppler sonography before and after sympathetic stimulation by coughing (leading to a temporary disappearance of diastolic flow) were compared with normal controls. correlation was made with nerve conduction studies and the extent of radiologically visible media sclerosis. results: under baseline conditions, the mean flow velocity was similar in diabetics and controls, but the resistance index (ri) was significantly higher in diabetics (1.1 vs. 0.8). after coughing no significant difference in the ri or the duration of the response was noticed between patients and controls, but the onset of the response was significantly delayed in the study group (2.1 vs. 1.5 s). no correlation was found with nerve conduction studies and radiologic results. conclusion: radial artery svr is an independent and sensitive marker for autonomic function. 14:00 -15:30 room i interventional radiology liver tumor ablation -metastases purpose: to evaluate in vitro and in vivo the use of mr-guided interstitial thermotherapy with rfitt in bipolar technique and to compare electrodes with active and non-active tips. material and methods: a newly designed internally cooled rf probe (∅ 3 mm) was used. both electrodes were 18 mm in length, the inserted insulator between both electrodes was 8 mm in length. for cooling, distilled water was used at flow rates between 40 and 130 ml/min and the power was between 40 and 70 w. three patients with a liver metastases were treated under local anesthesia. in vitro studies were done to compare electrodes with an active tip with electrodes with a nonactive tip. during mr measurements the rf generator was switched off. results: mri allowed a reliable visualization of the electrodes and the insulator in between. the experiments documented that a rf power of 40 w with an irrigation rate of 40 ml/min results in the largest possible area of coagulative necrosis with a diameter of 34 mm × 46 mm. the treatment of the patients was performed without any complications. the advantage of the active tip is that subcapsular lesions can also be treated using this bipolar rf technique with the active tip as the distal electrode. conclusion: mr-guided rfitt using a internally cooled, irrigated bipolar rf electrode results in a coagulative necrosis which is comparable to those of mr-guided laser induced thermotherapy (litt) . with the active tip as the distal electrode subcapsular lesions can also be treated with the bipolar system. to determine the value of multiplanar and 3d post-processing techniques for guidance of the ablative probe to an optimal position before radiofrequency treatment of liver lesions. methods and materials: 21 patients with malignant hepatic tumors (hcc n = 6, metastases n = 15) underwent radiofrequency ablation. following conventional ctguided (siemens volume zoom©, biopsy mode) positioning of the ablative device, its position was re-evaluated using multiplanar (mpr) and volume rendering 3dreformations. the position of the ablative device was characterized qualitatively based on the following scale: central -probe is localized within the central 20 % of the tumor; marginal -probe is lateralized towards the periphery of the tumor; outside -probe-position outside of the tumor. suboptimal positioning was corrected following initial ablative therapy to assure complete ablation of the tumor and imaging was repeated. results: multiplanar and 3d renderings proved superior over imaging in the axial plane in determining the position of an ablative device within liver tumors (p = 0.008). in 9 out of 21 patients the probe position was considered to be central on axial images whereas multiplanar and 3d renderings only revealed marginal probe localisation. axial, multiplanar and 3d renderings yielded identical results in the remaining 12 patients. comparing multiplanar and 3d reformative techniques, no significant difference was shown in determining the ablative device position. conclusion: mpr and 3d renderings aid in optimising the probe position for ablative therapy in a substantial number of patients. as a correct probe position is crucial for the success of an ablation procedure, these viewing techniques should be implemented without delay. clinical diagnosis was always underestimated at initial presentation. imaging modalities were requested for cerebral ischemic attack (2/3), cephalalgia, photophobia and tinnitus (1/3). initial imaging diagnosis was delayed (> 3 days) in all cases, as patients were referred for brain evaluation and carotid artery disease. follow up clinical and imaging examination demonstrated: a death (n = 1) serious brain damage (n = 1), complete recovery after antiobiotherapy and anticoagulotherapy (n = 1). conclusion: delayed diagnosis of septic thrombosis of the lateral sinus is probably related to its non-specific clinical symptoms. therefore, in patients with an history of diabetes immunodepression or of infection of the face or of the temporal bone, a careful examination of the skull base should be performed to determine the permability of the brain venous sinuses, the status of the mastoid cell pneumatization of the temporal and occipital bones and the normality of the related fatty spaces. objective: conventional or digital 2d dacrocystography and ct dacocystography is usually carried out after the catheterization of a lacrimal canaliculus. we tried to evaluate the quality of opacification on ct scans after simple instillation of contrast medium, without any catheterization. patients and methods: 39 patients (78 nasolacrimal ducts) were explored for lacrimal pathways obstruction by ct scans after instillation of diluted water soluble contrast medium instillation. a complementary ct acquisition after catheterization was performed when the first ones did not show any opacification. results: ct dacrocystography after instillation is a well tolerated and safe technique. in our study, it allowed detection all pathologic lacrimal canals; 7 normal lacrimal pathways (after catheterization) were not opacified after instillation (false positives). the sensitivity of the method was 100 %, its specificity 84 %. conclusion: ct dacrocystography after ocular instillation is an easy, physiologic and sensitive method to evaluate lacrimal obstruction. we propose it as the first step test, the catheterization being used only when there is total absence of opacification after instillation. results: inflamatory causes are responsible of lacrimal duct obstruction. we found no post-traumatic lesions, dacryolithiasis or nasolacrimal neoplasms. dcg evaluated lacrimal ducts and the nasal meatus. dct was performed only in patients with simple intubation or endoscopic dacryocystorinostomy to exclude all postsurgical complications of the surrounding nasolacrimal duct tissues or an inflammatory process of paranasal sinuses. discussion and conclusion: dcg and dct have almost the same sensitivity and specificity in a morphological study of nasolacrimal ducts. crystallin dosage is 0.04 to 0.2 msv in dcg and 1.8 to 2.6 msv in dct. dct with 3d reconstruction gives good spatial and anatomic details (orbital, etmoidal and nasal bone). dcg is always the gold standard in the imaging of the lacrimal ducts, but we propose dct to check possible post-surgical complications. purpose: poag is an ocular disease based on a progressive optic neuropathy, visual-field defects and elevated intraocular pressure. our purpose was to compare with color doppler the blood flow of the main orbital arteries of normal, atherosclerotic and glaucomatous subjects. methods and materials: we evaluated with color doppler (atl hdi 5000) the blood flow of ophthalmic artery (oa), central retinal artery (cra), nasal and temporal short posterior ciliary artery (spca). peak systolic velocity, end diastolic velocity and resistive index (ri) of each artery were considered. we examined 3 group of patients: 30 normal subjects (group a), 30 patients (> 50 years) with positive risk factors for atherosclerosis (smoking, hypertension) (group b) and 30 patients affected by poag (group c). inclusion criteria in group c were: iop > 21 mmhg, visus > 5/10, pseudoexfoliatio presence. results: all the arteries of group b showed higher ri than group a, with low end diastolic velocities. spca (0.87 ± 3.3) and cra (0.85 ± 2.6) of group c showed significantly higher ri than group a, but no significant ri differences were found in oa of group c (0.76 ± 4.2) and group a (0.77 ± 2.7). conclusion: normal, atherosclerotic and glaucomatous subjects can be distinguished by color doppler evaluation of the blood flow of the main orbital arteries. orbital color doppler can be useful in the diagnosis of poag and in the monitoring of these patients. which parameter should be used for rapid assessment of extraocular muscle enlargement in patients with graves' ophthalmopathy? z. szücs-farkas, j. toth, l. galuska, e. balazs, e.v. nagy; debrecen/hu purpose: to find the most accurate, easy-to-measure parameter that can be used as a substitute for extraocular muscle volume assessment in patients with graves' ophthalmopathy. subjects and methods: 70 orbits of 35 patients were examined in a conventional 1 t mr scanner and the rectus muscles evaluated. the diameter at the largest extent of the muscle belly, as well as the long and the short diameters and the cross sectional areas in a preselected coronal scan were measured for each muscle and compared with the corresponding muscle volume measured on contiguous t1w transverse slices. results: the measured coronal area correlated well with the volume of the superior (r = 0.694) and inferior (r = 0.783) recti. the largest transverse diameter showed strong correlation with the volume of the lateral (r = 0.868) and medial (r = 0.869) recti. for the latter muscle, the coronal area also exhibited a high correlation with the volume (r = 0.838). coronal cross-sectional areas can be well estimated by measuring both the short and long coronal muscle diameters (r values were between 0.914 and 0.966). p was less than 0.0001 for every above mentioned correlations. conclusions: in graves' ophthalmopathy, the volume of three of the rectus muscles seems to be well estimated by measuring their cross sectional area on a single coronal slice, while the largest transverse diameter of the lateral rectus is suitable for the same purpose. purpose: a prospective study was performed to determine the accuracy of unenhanced, enhanced and early delayed (15') enhanced ct densitometry in differentiating adenomas from non adenomas and to evaluate the usefulness of absolute and relative percentage of washout. methods and materials: 37 oncology patients with 44 undetermined adrenal masses were all studied by unenhanced, enhanced and 15 minutes delayed enhanced helical ct scans. attenuation values of all adrenal masses on each type of scan was measured by mean of a region of interest and then used to calculate absolute and relative percentage wash-out. proof of diagnosis was ct and/or mr follow-up (6 -36 months) in 31 patients; percutaneous ct guided biopsy in 5 patients; surgery in 1 patient. results: on unenhanced ct scans, with attenuation threshold of 18 hu, specificity and sensitivity for the diagnosis of adenoma were 100 % and 93 % respectively. on 15 minutes delayed ct scans, with an attenuation threshold of 38 hu, specificity and sensitivity for the diagnosis of adenoma were 100 % and 90 % respectively. moreover, all adrenal masses were correctly characterised as benign or malignant on delayed scans with a relative percentage washout of 35 % and an absolute percentage washout threshold of 50 %. conclusion: unenhanced and early delayed enhanced ct attenuation values can characterise an adrenal mass as a benign adenoma with high specificity and acceptable sensitivity. the percentage change in washout of contrast media can be a useful adjunct to absolute ct attenuation values in differentiation of adrenal adenomas and nonadenomas. ct adrenocortical carcinomas and pheochromocytomas: assessment of wash-out at delayed contrast-enhanced p.i. reittner 1 , m. korobkin 2 , p. wehrschütz 1 , k.w. preidler 1 , d.h. szolar 1 ; 1 graz/at, 2 ann arbor, mi/us purpose: to measure the changes in washout of contrast material on contrast medium-enhanced computed tomographic (ct) scans in patients with adrenocortical carcinomas and pheochromocytomas. materials and methods: fifteen patients with proven adrenocortical carcinomas and 17 patients with pheochromocytomas underwent helical ct. unenhanced ct was followed by enhanced ct at 60 seconds and 10 minutes. 121 adrenal masses (74 adenomas and 47 metastases, respectively) in 108 patients served as reference data. results: the adrenocortical carcinomas and pheochromocytomas enhanced significantly lesser than the adenomas at 60 seconds (p < 0.001). at 10 minutes, both the absolute and relative percentage loss of enhancement were significantly greater for the adenomas than for the adrenocortical carcinomas and pheochromocytomas (p < 0.001), respectively. delayed-enhanced ct at 10 minutes (sensitivity, 92 %; specificity, 95 %) was more accurate for differentiation of adenomas and adrenocortical carcinomas and pheochromocytomas than unenhanced ct (sensitivity, 82 %; specificity, 95 %) conclusion: adrenocortical carcinomas and pheochromocytomas exhibit similar wash-out than do adrenal metastases, but significantly lesser than do adrenal adenomas. the percentage change in washout of contrast material is a useful adjunct to absolute ct attenuation values in differentiation of adrenal adenomas and adrenocortical carcinomas and pheochromocytomas. incidence of malignancy in complex cystic renal masses (bosniak category iii): should biopsy precede surgery? m.g. harisinghani, k. jhaveri, d.a. gervais, p.f. hahn, j. varghese, p.r. mueller; boston, ma/us purpose: we sought to determine the incidence of malignancy and to assess a possible role for image guided biopsy of this category of renal masses materials & methods: of the 397 renal biopsies performed at our institution between 1991 and 2000; a total of 28 patients with 28 category iii lesions, were identified for analysis. there were 18 men and 10 women with age range from 40 to 70. the incidence of malignancy, based on surgical pathology or imaging follow up, and percentage of lesions proceeding to surgery, among these 28 lesions, was determined. the surgical results were correlated to the biopsy findings results: of the 28 biopsied category iii lesions, 17 were malignant (16 renal cell carcinoma, 1 lymphoma) and 11 were benign (4 hemorrhagic cyst, 1 oncocytoma, 3 inflammatory cysts, 2 adenomas, 1 focal glomerulosclerosis). seventeen of the 28 (60.7 %) lesions (16 renal cell carcinoma, 1 inflammatory cyst) had surgical resection following the biopsy. all lesions proceeding to surgery had pathological diagnosis identical to the pecutaneous image guided biopsy results. non-surgical patients had radiological follow up for minimum 1 year conclusion: the incidence of malignancy in bosniak category iii is 60.7 % (95 % confidence interval). presurgical renal biopsy and radiological follow up is useful in identifying non-malignant lesions in this category, thus avoiding unnecessary surgery in up to 39 % of patients method and materials: prospective analysis of 25 patients with histopathologicaly proved rcc included enhanced mdct studies in arterial and corticomedullary phase (detector configuration: 4 × 1 mm, rt 0.5). we compared 3 reconstruction protocols (slice width/reconstruction interval: 1.25/0.7 mm, 3/1.5 mm, 5/2.5 mm). in addition, multiplanar reconstructions (mpr) in 5, 3 and 1.25 mm coronal planes were assessed. finally, low-dose ct urography (delay 10 min) with mpr reconstructions was performed in the excretory phase. image data of all reconstructions were analysed for the size of the lesion, tnm staging, morphological, and enhancement characteristics by two radiologists. histopathological data were used to determine the efficacy of the different ct reconstruction protocols. results: different reconstruction protocols (5, 3 and 1.25 mm) have no influence on the evaluation of tumour size and t-staging. however, assessment of lymph node size was improved in 12 % of the patients by using thinner slice width and 1.25 mm mpr reconstruction. vertical tumour size and v. cava infiltration was more precisely assessed by evaluation of mpr-than axial reconstructions. in all patients ct-urography demonstrated sufficient opacification of the collecting system. conclusion: axial reconstruction protocols are equally suited for the t-staging of rcc. a mdct protocol using additionally 1.25 mm mpr reconstructions and lowdose ct urography appears effective as a "one-stop" protocol for a proper staging of rcc and eliminates further investigations such as iv urography. b-0178 14:40 0.59 (95 % ci: 0.22 -0.95) for the n-stage. the average tumour size was 5.2 cm. all accessory arteries and veins were correctly described. the renal pelvis was opacified in 94 %, the proximal ureter in 89 %, and the distal ureter in 77 %. distant metastases were found in 2 cases and 3 patients showed tumour extending into the renal vein. conclusion: isotropic multislice ct of the kidneys with a modified injection protocol provides good correlation with pathological tumour stage, allowing cta and ct-urography calculation from the same dataset and, thus, may represent a universal imaging technique in renal cancer staging. histopathological correlated, staging of renal carcinomas by multislice ct and high performance mri p.j. hallscheidt, s.o. schoenberg, g. riedasch; heidelberg/de aim: to evaluate the accuracy of multislice-computed tomography (ct) and magnetic resonance imaging (mri) in staging renal carcinoma and planning of nephron sparing surgery. material and methods: in a prospective study 58 renal carcinomas were preoperatively examined for tumour staging by multislice ct and mr imaging and correlated with histopathological staging. triphasic ct imaging was performed with a siemens volume zoom with a reconstructed slice thickness of 1 mm. 3d and coronal reconstructions were used to improve planning of the surgery. in mr imaging (siemens vision) additional to the transversal t1-weighted ge sequence with and without gdtpa, and a transversal t2 weighted respiration triggered tse, a coronal t1-weighted ge sequence with gdtpa and fat saturation were acquired. in addition a multi phase 3d angio after gdtpa injection was performed. results: in early stage renal carcinoma ct and mr imaging provide similar staging accuracies. in all cases multi slice ct allowed us to identify all renal masses, especially in multifocal renal cell carcinomas. accessory arteries could be identified in all cases, too. multiphase mri allows detection and differentiation of renal masses, especially in caval infiltration. conclusion: multislice ct and 3d reconstruction integrate essential information from angiography, venography, urography and 2d ct in a single imaging modality. in multislice ct all tumours in multifocal renal cell carcinoma were detected. in advanced renal carcinoma mri was superior to ct imaging, especially in diagnosing tumour thrombus. consequently the extent of tumour thrombus may be assessed by mri which therefore may replace conventional cavography. small renal masses: value of contrast-enhanced colour doppler imaging a. klauser, g. janetschek, g. helweg, r. peschel, l. pallwein, d. zur nedden, f. frauscher; innsbruck/at purpose: we assessed the value of a microbubble based ultrasound (us) contrast agent for blood vessel enhancement in colour doppler imaging (cdi) of small renal masses. methods and materials: 51 patients with "indeterminate" small renal masses (< 3 cm in diameter) underwent prospective cdi before and after intravenous administration of the contrast agent levovist® (schering ag, berlin, germany). the degree of tumour vascularity was subjectively graded from 0 to iv (indicating an increasing vessel count) and the peak systolic velocity (psv) was measured. cdi findings were compared with those obtained at histopathological examination. results: intra-and/or peri-tumour vessels were detected in 26 lesions (51 %) by unenhanced cdi and in 48 lesions (94 %) by enhanced cdi. the detection of vascularity was increased by contrast administration (p = 0.006, mcnemar test). higher grades of tumour vascularity (grade iii and iv) were found more often in malignant renal masses (p < 0.01). psvs higher than 80 cm/s were found only in malignant lesions. based upon receiver operating characteristic analysis, enhanced cdi (az = 0.789) is more accurate than unenhanced cdi (az = 0.576) for differentiating benign from malignant renal masses (p < 0.004). conclusions: enhanced cdi is superior to unenhanced cdi in the detection of tumour vascularity, and in the discrimination between benign and malignant small renal masses. assessment of renal masses with contrast enhanced sonography using pulse inversion imaging and fso69 j.-m. correas 1 , m. claudon 2 , a. lesavre 1 , a. méjean 1 , l. bridal 1 , o. hélénon 1 ; 1 paris/fr, 2 nancy/fr purpose: to evaluate the efficacy of contrast-enhanced sonography of renal masses using a fso69 (optison®, mallinckrodt, usa), with quantification of the signal intensity. materials and methods: 24 renal masses were studied with pulse inversion imaging (pii) at baseline and following a bolus injection of fso69 (1, 2, 3 and 4 ml, randomised dose, 6 patients per dosage group, atl hdi5000, c5-2 probe). cineloops were transferred to a pc for quantification with hdi lab. the cortical and solid mass enhancement was calculated as the difference between the signal intensity of a region of interest located upon the normal cortex and the mass before and after injection (db). the final diagnosis was obtained by ct, mri and/or surgery (adenocarcinomas 16, complex cysts 5; hamartomas 3). results: cortical enhancement was correlated with the dose (r = 0.98) and was consistently greater when the mechanical index was lower than 0.4. the detection of normal and atypical cysts was improved and was correlated with ct and mri features. renal mass delineation was improved except when the enhanced mass signals matched with the normal surrounding cortex (2 cases). the visibility of the necrosis within the lesion was comparable to the ct and mr appearance. the peak enhancement was: 9.6 db ± 4.0 db for the cancers, 5.8 db ± 3.5 db for the hamartomas and 0.8 db ± 0.5 db for the complex renal cysts. conclusion: pii of renal masses following fso69 injection improved the detection and the characterization of renal masses, particularly in small tumours and complex cysts. purpose: combined inhibition of the synthesis of nitric oxide and prostacycline predisposes rats to renal injury from radiographic contrast media (rcm). the reliability of these pharmacological manipulations was investigated. methods and materials: adult male sd rats were injected with l-name (10 mg/kg), indomethacin (10 mg/kg) and rcm (or normal saline) 15 minutes apart. serum creatinine (cr) was measured prior to and post these pharmacological insults. results: a significant increase in serum cr (from 54.66 ± 8.39 to 171.96 ± 24.49 µmol/l and from 80.95 ± 6.73 to 204.76 ± 16.73 µmol/l, n = 5/group) was observed 24 hours after injection of 6 ml and 8 ml of high osmolar rcm diatrizoate respectively. the increase in serum cr recovered spontaneously 7 days after the injection. no significant change in renal function was observed in the control group receiving 8 ml/kg of normal saline (n = 5) or after injection of 4 ml of diatrizoate (n = 5). the increase in serum cr observed with 6 ml of diatrizoate was significantly higher (p < 0.01) in comparison to the rise induced by equivolume of the low osmolar iopromide (serum cr was 68.47 ± 8.39 µmol/l pre contrast and 143.59 ± 32.03 µmol/l 24 hours post contrast, n = 5). the calcium channel blocker diltiazem (10 mg/kg i.p., 30 min prior to contrast injection) prevented the rise in serum cr observed with 6 ml of diatrizoate. the protective effect was less with lower doses of diltiazem. conclusion: the used animal model is reliable and capable of reproducing previously established observations. the protective effect of a calcium channel blocker has also been shown. purpose: aim of the study was to evaluate the efficacy of gadobutrol as contrast agent for diagnostic chest and abdominal ct compared to iodinated contrast media in a porcine animal model. methods: in 7 pigs spiral cts of the chest and abdomen were performed using 2 ml/kg bw gadobutrol (gadovist, schering, germany) given by single intravenous injection (siemens somatom plus 4; slice: 5 mm, table feed: 7.5 mm, reconstruction increment: 5 mm). one week later the same animals were examined under the same protocol using iopromide (ultravist 300; schering, germany; 2 ml/kg bw). in 3 additional animals serial cts were performed using gadobutrol on day 1 and iopromide on day 7 to detect contrast media kinetics, peak enhancement and time enhancement-product in important vascular regions and parenchymal organs. peak enhancement (net increase compared with native baselines) were measured in houndsfield units (hu) in defined regions of interest. additionally, diagnostic quality and accuracy of the cts were evaluated on a two-step scale by three independent blinded investigators. results: in vivo, the mean peak enhancement 0, 5, 10, 30, 60 and 120 seconds in the abdominal aorta after injection of 2 ml/kg bw gadobutrol and iopromide was 40, 200, 224, 261, 118, 95 hu and 41, 232, 298, 152, 143 , 123 hu respectively. all cts using gadobutrol were classified as diagnostic with excellent differentiation of vascular and parenchymal structures. conclusion: computed tomography with 1 mol gadobutrol resulted in an excellent contrast peak enhancement. computed tomography using gadobutrol is feasible and a possible alternative to iodinated contrast material. use of artificial neural networks in predicting adverse drug reactions in patients receiving contrast media e. grossi 1 , m. buscema 2 , a. seeberg 3 ; 1 milan/it, 2 rome/it, 3 constance/it neural networks (anns) are mathematical algorithms tools are able to determine the existence of subtle correlations between series of data and a particular outcome, and once "trained", can predict output data on the basis of the input data. although neural networks have been applied in various areas of medical research, they have not been previously applied to the prediction of adverse events to contrast media. materials and methods: anns (provided by semeion research centre, rome, italy) were used to predict adverse events occurring in over 600 patients receiving iomeprol 300 (300 mg i/ml) within a large observational clinical study performed by bracco-byk gulden in 6 german radiological centres. the database consisted of 76 independent variables obtained from the case report forms. during an optimising process based either on linearity criteria or specific evolutionary algorithms, just 9 independent variables resulted as best predictors of the prognostic target. the sample size was divided into two random subsamples: the training one (the only one containing the dependent variable) and the testing one. in the testing phase the overall prediction accuracy ranged from 91.6 to 95.6 %. these values were reproducible along ten consecutive experiments with independent networks of the same type trained in different random subsamples. these results indicate that neural-network analysis can be used to predict adverse effects of contrast media within a given class. mr imaging of atherosclerotic plaque with new ultrasmall particles of iron oxide (7228) compared to sinerem® in hyperlipidemic rabbits c.u. herborn 1 , t.c. lauenstein 1 , f.m. vogt 1 , c. corot 2 , j.f. debatin 1 , s.g. rühm 1 ; 1 essen/de, 2 aulnay-sous-bois/fr purpose: to evaluate a new uspio compound 7228, compared to sinerem® (both guerbet, france) as a marker of macrophage activity in atherosclerotic plaques. materials and methods: experiments were conducted on 4 heritable hyperlipidemic rabbits aged 4 -6 months. imaging was performed on a whole body scanner using the transmit/receive head coil. after initial 3d mr angiography of the thoracic aorta with conventional paramagnetic contrast agent (gd-dota), animals were injected once with either 7228 (n = 2) or sinerem® (n = 2) at equal doses. 3d mr angiography was repeated daily over 5 days. on day 6 the rabbits were euthanized for histopathologic evaluation of the aorta. results: 3d mras with gd-dota revealed no abnormal findings in any of the animals (n = 4). luminal signal intensity at different days was comparable between both compounds with no significant differences regarding the time-evolution of t2* effects. susceptibility effects within the aortic wall became evident on day 2 in both groups reaching the maximum after 4 days. these changes were more pronounced in the 7228 group without amounting to a statistical significance. histopathology proved slightly increased fe uptake in macrophages of atherosclerotic plaques of the two rabbits injected with 7228 compared to those who received sinerem®. conclusion: both uspio agents cause susceptibility effects within atherosclerotic plaques detectable by 3d gre sequences. since phagocytosis of 7228 seems to be superior to sinerem® it can be assumed that lower doses of this compound are suited to allow better visualization of active plaque in atherosclerotic disease. a.r. rudisch 1 , c. kremser 1 , w. judmaier 1 , h. zunterer 1 , j. griebel 2 , a. de vries 1 , w.r. jaschke 1 ; 1 innsbruck/at, 2 neuherberg/de aim: this study aimed to compare perfusion-index-values (pi) of gadolinum-dtpa (gd-dtpa) obtained in malignant tumors with pi's of normal tissue (muscle). material and method: perfusion data were obtained in histological proven primary rectal carcinoma and in left sided gluteus maximus muscles (n = 23). perfusion data were obtained by magnetic resonance imaging (mri) measurements using a specially ultrafast t1-mapping sequence in a 1.5 t whole body scanner. t1-maps were acquired in intervals of 14 s or 120 s before, during and after constant rate infusion of gd-dtpa. in order to investigate the relevance of spatial heterogeneities of microcirculation in tumors and muscles, relative frequency distributions of pi's were computed with equal class intervals of 0.021 ml/min/g. not only the mean pi but also the relative fraction of pi £ 0.126 ml/min/g between tumor tissue (0.093 ml/min/g ± 0.021; 89.1 %; ±12.1) and muscle tissue (0.037 ml/min/g ± 0.012; 99.8 %; ±0.6) was statistically significantly different (both p < 0.001, mann-whitney-u-test). discussion: our results can offer additional information about higher nurtrients supply, e.g. chemotherapeutic agents, to tumor tissue than to muscle tissue caused by high pi's and a higher relative fraction of high intratumoral pi's. the presentation was supported in part by grants from schering, germany and austria. can necrosis avid-mr-imaging with metalloporphyrins differentiate between stunned and infarcted myocardium: results of an experimental study in dogs p. reimer 1 , j. bankert 1 , c. bremer 2 , k.-u. jürgens 2 , t. filler 2 , t. weber 2 , b. tombach 2 ; 1 karlsruhe/de, 2 münster/de purpose: to investigate whether stunned myocardium may be differentiated from infarcted myocardium by metalloporphyrin-enhanced-mri. materials and methods: 10 open-chest anesthetized dogs underwent reversible (15 min; n = 5) and permanent (n = 5) pneumatic occlusion of the left anterior artery (lad) to induce myocardial stunning and myocardial infarction, respectively. mri was performed at 1.5 t obtaining ecg-triggered short and long axis t1-w tse at baseline and every 30 min following injection of gadophrin-2 (50 µmol/kg bw) up to 6 hours. postmortem, needle biopsies were taken from the lad-perfused and a remote area. ex vivo, mri (t1-w tse) and ttc staining were performed. results: gadophrin-2 enhancement was slightly higher in stunned myocardium than in remote regions but significantly higher than in infarcted areas when measured in the long axis. all regions showed highest signal intensity 30 min following injection followed by a decrease over time. conclusion: gadophrin-2 enhanced mri may serve as an additional tool besides functional measurements such as tagging or perfusion to differentiate stunned from infarcted myocardium. assessment of angiogenic tumor burden by susceptibility mri using long circulating iron oxide nanoparticles c. bremer 1 , m. mustafa 1 , a. bogdanov jr. 2 , v. ntziachristos 2 , a. petrovsky 2 , r. weissleder 2 ; 1 münster/de, 2 boston, ma/us purpose: to evaluate iron-oxide enhanced mri for the assessment of the angiogenic burden of various tumors methods and materials: a variety of tumors (9l-gliosarcoma, du4475-breast cancer, ht1080-fibrosarcoma and eoma-hemangioendothelioma) with various angiogenic potential were implanted into nude mice. tumors were imaged at 1.5 t using a pd-w ge sequence before and after i.v. injection of magnetic iron oxides nanoparticles (mion). ∆-r2* maps were calculated for all tumors and the relative tumoral blood volume was determined by roi-analysis. tumors were stained for cd31 for quantification of the microvessel density (mvd). moreover, vegf expression was analyzed for each cell line by western blotting and elisa. a subset of animals was injected with a tc-labeld intravascular tracer for measuring the blood volume distribution in vivo. results: blood volume maps generated by mri revealed a significantly higher tumoral blood for eoma-(6.6 ± 0.9 %) and ht1080-(5.5 ± 0.8 %) compared to du4475-(3.1 ± 0.4 %) and 9l-tumors (2.1 ± 0.3 %). mvd correlated well with the mr-data with eoma-and ht1080-tumors revealing a higher mvd (150 ± 13 and 81 ± 5 counts per field) compared to du4475-and 9l-tumors (43 ± 3 and 39 ± 2 counts per field). moreover, mri data closely correlated with tc-blood volume maps and the vegf expression patterns conclusions: mri with long circulating iron oxides can be utilized to assess tumoral angiogenesis. since iron oxide based particles are widely available, this technique can be readily adapted for clinical use. 1999) . a 3-month clinical follow-up was obtained in all patients who were not anticoagulated. results: for a longer mean z-axis coverage (group 1: 152 mm; group 2: 110 mm; p < 0.001), the mean duration of data acquisition was shorter with msct (group 1: 17 s; group 2: 21 s; p < 0.0001). examinations devoid of respiratory and cardiac motion artifacts were more frequent in group 1 than in group 2 (p < 0.001). in the absence of significant difference in the quality of vascular enhancement, the porportion of examinations interpretable down to the subsegmental arteries was higher in group 1 (57.5 %) than in group 2 (13 %) (p < 0.0001). the benefits of msct were more marked for patients with underlying respiratory disease and did not lead to a higher detection rate of peripheral pe. the negative predictive value of ssct and msct were 100 % and 99 %. conclusions: improvement in image quality on msct scans accounts for the improved diagnostic accuracy of scta, especially for patients with impaired respiratory function. incidental detection of pulmonary emboli on routine multislice ct of the chest c. ciccotosto, m.l. storto, f. guido, a. di credico, a. guidotti, l. bonomo; chieti/it purpose: to determine the prevalence of pulmonary embolism (pe) incidentally detected on routine multislice ct (msct) scans of the chest and to assess the influence of window width and level on pe detection. methods and materials: between january 1 and august 31, 2001, 542 routine contrast-enhanced ct scans of the chest were performed in 485 patients using a msct scanner (4 × 1 mm or 4 × 2.5 mm collimation; 5 mm slice width). ct angiographic studies performed for suspected pe or evaluation of the thoracic aorta were excluded. ct scans were retrospectively reviewed by 2 chest radiologists for the presence and site of pulmonary emboli using a cine-view mode on a dedicated workstation and 2 different widow settings: w = 400 hu, l = 40 hu (standard) and w = 600 -700 hu, l = 100 -150 hu (wide). results: unsuspected pe was present in 21/485 (4.3 %) patients, with an inpatient prevalence of 4.7 % (18/386) and outpatient prevalence of 3 % (3/99). most patients (15/21; 71.4 %) with unsuspected pe had cancer. the proximal extent of pe involved the main pulmonary artery in 5 patients, a lobar artery in 10, a segmental or subsegmental artery in 6. use of a wide window setting allowed detection of pe in 2 more patients than did the standard one. unsuspected pe was found in as many as 4.3 % of patients undergoing a routine study of the chest with msct, with a higher prevalence among patients with malignancy. the use of a wide window setting is recommended when interpreting routine msct of the chest in order to improve unsuspected pe detection. low kvp settings improve contrast enhancement and reduce radiation exposure in spiral ct of pulmonary emboli c. weidekamm, m. prokop, c.j. herold; vienna/at purpose: higher contrast of iodinated contrast material at low kvp settings may be used to compensate for increased image noise at lower exposure dose. we evaluated this concept for spiral ct of patients with suspected acute pulmonary embolism. we compared a standard protocol using 140 kvp, 175 mas (ctdi vol = 12.4 mgy) to a protocol using 100 kvp and 125 mas (ctdi vol = 3.8 mgy). we evaluated two groups of 25 consecutive patients, for whom identical scan parameters (3 mm collimation, 5 mm feed, 2 mm increment) and contrast injection protocols were used (140 ml, 3 ml/s, 20 s start delay). we measured the enhancement of the pulmonary artery in an roi at the level of the pulmonary trunk. for the enhancement of small arteries, we measured the maximum ct number in peripheral pulmonary arteries at the level of the aortic arch and at the lung bases. we determined the percentage of segmental and subsegmental arteries with sufficient quality for pe evaluation. to test the influence of observer experience on the accuracy of ct venography (ctv) for diagnosis of acute deep thrombosis (dvt) and to identify potential sources of misinterpretation. methods and materials: 64 patients with clinical suspect of pulmonary embolism (pe) underwent combined ct pulmonary angiography and ctv, using a multislice ct scanner. ctv was performed in the caudo-cranial direction, 3 minutes after administration of 140 ml of contrast material (300 mg i/ml). scan parameters were: 4 × 2.5 mm collimation, 5 mm slice width, and 5 mm reconstruction increment. ctvs were analyzed independently and in a blinded fashion for the presence of acute dvt by three readers: (a) an experienced radiologist in body ct, but without experience with ct imaging of pe; (b) a fellow in ct; (c) a third-year resident without any formal experience with ct imaging of pe. ct scans were scored on a 4-point confidence scale and different causes of interpretation errors were analyzed. the gold standard was lower extremity sonography. results: interobserver agreement was moderately good (k = 0.60; 95 % confidence interval), whereas interobserver disagreement occurred in 5 (9 %) cases. the two observers with ct experience were more accurate than the less experienced one (p < 0.02) who made a higher number of false-positive diagnoses. the most frequent causes of misinterpretation were chronic thrombosis and arterial thrombosis. no clear differences were found among the 3 readers for the error type. conclusion: ctv in addition to ctpa is a relatively accurate method for evaluation of dvt. ct diagnosis of acute dvt improves with general ct experience. conclusions: a pre-therapeutic hr-ct should always be done before medical treatment by epoprostenol. if poorly defined nodular opacities, septal lines, pericardial effusion, pleural effusion, and adenopathies are found, the pre-therapeutic strategy should be discussed again, in order to prevent patients from the risk of a paradoxical aggravation under medical treatment by epoprostenol. purpose: surgical repair of ventral hernias has been changed during the last years. this is due to new biomaterials as expanded polytetrafluoroethylene soft tissue patch (eptfe), which lets a transabdominal preperitoneal technique. this laparoscopic hernioplasty has a lower morbidity and recurrence rate, whereas it needs an accurate follow-up to assess complications which require readmission. in this prospective study we considered eight patients who underwent a laparoscopic repair of ventral hernia with an eptfe mesh between march 2000 and june 2000 at our institution. in all cases ct of the abdomen was performed after 30 days to evaluate the position of the mesh which is visualised as an higher density bandlike structure. results: in one patient after 30 days ct scan showed a fluid collection; one patient became syntomatic after 50 days and a new ct scan was performed with iv contrast medium which showed an infected fluid collection whith enhancing. conclusion: ct scan is a useful technique to assess post-operative complication of laparoscopic repair of ventral hernias, such as sieromas, abscess, hematomas, bowel obstructions and recurrences and is a great advantage to an accurate follow-up evaluation. methods: patients admitted with acute abdominal pain for which no immediate surgical intervention or ct was indicated, were randomised to "early ct" (within 24 hours of admission) or to follow "standard practice". diagnoses and confidences (on a 5-point scale) were documented by surgeons on admission and again after 24 hours. the admission, 24 hour, and final diagnoses (established at surgery and/or 6 months follow-up) were assessed by two reviewers for changes (to more serious, or less serious conditions, or "no change"). results: 55 patients were randomised to "early ct" and 63 to "standard practice". early ct reduced the length of hospital stay by 1.1 days, but this reduction did not reach statistical significance (p = 0.17). only 50 % (59/118) of admission diagnoses proved to be correct on follow-up, improving to 75 % (89/118) after 24 hours (both independent of the randomisation arm). although early ct was not associated with a significant change in diagnostic confidence at 24 hours, comparison with the final diagnosis revealed that early ct missed significantly fewer serious diagnoses (p = 0.006). there were 7 in-patient deaths, all in the "standard practice" arm. conclusions: ct undertaken early during an admission for acute abdominal pain may have favourable effects on length of hospital stay and mortality, and it is able to identify unforeseen diagnoses and complications, particularly those of a potentially serious nature. objective: to correlate isolated blunt spleen, and liver injury in children with presence and amount of free-fluid on ct scan. methods: 134 paediatric patients (range 3 months to 17 years) with isolated solid organ injury (liver or spleen) was confirmed with enhanced ct scan and graded according to the asst guidelines. the presence, location and amount of free-fluid was recorded. free-fluid was quantified as 0 = no fluid, 1 = small amount, 2 = moderate, 3 = large for each of the 3 anatomic areas. results: 134 paediatric patients with an isolated spleen (n = 66) or liver (n = 68) injury from blunt trauma were identified. free-fluid was noted in 101 injuries overall (75 %), more commonly with spleen (82 %) than liver (69 %) injuries. as injury grade increased, so did the frequency of free-fluid (grade 1 50 % to grade 5 100 %) and the total volume of free-fluid in the three quadrants (grade 1 0.75 to grade 5 5.6). the total volume of free-fluid was greater for splenic injuries (3.1) than liver injuries (1.7). the pelvis was the most common location for free-fluid (liver 53 %, spleen 71 %) and had the greatest mean amount of free-fluid (liver 0.9, spleen 1.5) of the 3 anatomic areas evaluated. (1) there is a direct correlation between the severity of the isolated injury and the frequency and amount of associated free-fluid. (2) the pelvis was the most common location to detect free-fluid and had the greatest estimated volume. whole body spiral ct in trauma patients -part i: assessment of cervical, thoracic and abdominal soft tissue and organ injuries t. albrecht, j. von schlippenbach, k.-j. wolf; berlin/de purpose: to assess the accuracy of standardized "whole body" spiral ct in the initial work-up of cervical, thoracic and abdominal soft tissue and organ injuries in trauma patients. method: 46 patients with potentially life-threatening injuries underwent a spiral ct of the neck (unenhanced; 3/5/3), the chest, abdomen and pelvis (150 ml contrast agent, 5/7.5/5) immediately after resuscitation. the ct findings were compared with the final diagnoses at discharge or death. in 22 patients abdominal sonography and ct were also compared. the final diagnoses were: cervical haematoma (n = 1), haemothorax (n = 15), pneumothorax (n = 11), pulmonary contusion (n = 12), mediastinal haematoma (n = 4), aortic dissection (n = 1), retroperitoneal haematoma (n = 6), renal haematoma/laceration (n = 4), hepatic haematoma (n = 3), splenic haematoma/rupture (n = 3), mesenteric laceration (n = 1), and isolated intraperitoneal haemorrhage (n = 2). spiral ct showed all these injuries with the exception of a delayed splenic rupture. in the 22 patients with sonography available, ultrasound missed 2 hepatic, 1 splenic and 1 renal injury. furthermore sonography produced two false positive findings: 1 haemopericardium and 1 renal contusion. conclusion: the initial standardized "whole body" ct used provided fast, comprehensive and accurate diagnosis of cervical, thoracic and abdominal soft tissue injuries in trauma patients. it is superior to sonography in assessing abdominal injuries. it should be preferred over a targeted approach with selective ct of certain body areas according to sonography results and the clinical situation since it is time efficient and often reveals unsuspected injuries. part ii: b-0898 (ss 1810a) b a c d e f 152 single-phase helical ct protocol in the evaluation of patients with suspicion of pancreatic carcinoma m. imbriaco, l. camera, m. romano, p. mainenti, e. soscia, a. puzziello, m. salvatore; naples/it purpose: to evaluate the diagnostic accuracy of single-phase (sp) helical ct in the detection and assessment of resectability of patients with suspicion of pancreatic carcinoma (pc). methods and materials: 78 (mean age: 62 ± 11 years) patients with a suspicious pc were studied. unenhanced scan was followed by one set of scans (5 mm beam collimation, 3 mm reconstruction intervals, pitch 1.0, 120 kv, 220 -240 ma) in the caudo-cranial direction, from the inferior hepatic margin to the diaphragm, with a scan delay of 50 s after i.v. contrast administration. results: a final histopathological diagnosis based on surgical findings was obtained in 57 patients, in the remaining 21 fine needle aspiration biopsy followed by 2 years clinical follow up showed no evidence of malignancy. final diagnosis was pc in 52 cases and chronic pancreatitis in 26. of the 52 tumors, 8 patients had surgically resectable disease and 44 were unresectable. sp helical ct showed a diagnostic accuracy for assessment of tumor detection of 93 % with sensitivity and specificity of 94 % and 92 % and positive and negative predictive value of 96 % and 89 %, respectively. the overall accuracy of sp helical ct to determine resectability or unresectability of pc was 92 %. conclusion: sp helical ct is an effective technique for the detection and assessment of resectability of patients with suspicion of pc. due to the lower radiation burden and to the lower cost a sp of acquisition in a caudo-cranial fashion might be considered the protocol of choice when evaluating patients with suspicion of pc. secretin assisted ct of the pancreas: is it of benefit for pancreatic tumour staging or diagnosis? s.m. lyon, t. fotheringham, p. o'sullivan, m.f. given, m.j. lee; dublin/ie purpose: to determine the effect of secretin assisted ct on contrast material delivery to the pancreas. methods: 31 patients (mean age 70; range 47 -88) were enrolled. triple phase helical ct of the pancreas was performed on successive days. unenhanced and enhanced ct in the pancreatic phase and pv phase was performed without (day 1)and with (day 2) secretin (100 iu) given at t = 0 s (n = 10), t = 60 s (n = 5), t = 120 s (n = 5), t = 180 s (n = 4), t = 240 s (n = 4), t = 300 s (n = 3). percent enhancement of the pancreas and pv system was calculated using roi's obtained from studies with and without secretin. two radiologists scored the images using a five point scale for: pancreas/tumour contrast, pv, smv and duodenal mucosal enhancement. results: overall mean pancreatic enhancement was 125 % without secretin and 132 % with secretin. no significant difference in pancreatic enhancement was noted when secretin was given at t = 0, t = 60 s, and t = 300 s. however, significantly increased pancreatic enhancement was noted (secretin vs no secretin) at t = 120 s (12 %), t = 180 s (66 %) and t = 240 s (30 %) (p < 0.05). pv/smv enhancement was significantly increased in the pancreatic phase in all secretin studies (p < 0.05). qualitatively scores for pancreas/tumour contrast, pv/smv and duodenal mucosal enhancement were all increased on secretin studies. conclusion: secretin administration increases pancreatic enhancement which aids tumour identification. early enhancement of duodenal mucosa and pv/smv is helpful for staging. for optimal results secretin should be given 2 -4 minutes before iv contrast administration. further patients are being recruited and study completion is estimated for november 2001. high resolution multislice spiral ct in pancreatic cancer: preoperative detection and assessment of resectability f. fraioli, c. catalano, f. pediconi, a. laghi, a. napoli, s. vagnarelli, m. danti, r. passariello; rome/it purpose: to prospectively evaluate multislice ct (msct) in the preoperative diagnosis and assessment of resectability in patients with clinical suspicion of pancreatic carcinoma. material and methods: 26 patients referred for suspected pancreatic carcinoma underwent msct. immediately prior to the examination, patients were given 300 -500 ml of tap water; i.v. scopolamine (20 mg/ml) was administered to reduce intestinal peristalsis. unenhanced volume was first acquired, followed by the postcontrast acquisitions, during arterial and portal venous phases. the post-contrast volumes were acquired afeter injection of 140 ml of non ionic c.a. at 4 ml/s. images were evaluated using a real time interactive 3d approach by two observers, in terms of lesion identification and conspicuity, infiltration of peripancreatic fat, vascular involvement of major peripancreatic arterial and venous vessels, adenopathies and liver metastases. results: pancreatic carcinomas were identified in 22 of the 26 referred patients; in 4 cases no abnormalities were detected. tumors were located in the head in 13 cases, the isthmus in 3 cases and in the tail in 6 cases. peripancreatic spread was seen in 12 cases. vascular involvement was identified in 10 cases. adenopathies were present in 14 cases, while liver metastases in 6 patients. 3d real time interaction with multiplanar reformations appeared particularly useful in the evaluation of vascular involvement and above all in the detection of liver metastases. conclusion: high resolution msct with multiplanar reformations is a very accurate technique in the identification and staging of patients with suspected pancreatic carcinoma. to develop, test, and evaluate an automatic brain contour segmentation technique for multispectral mri data of the human brain based on radial basis functions (rbf) neural networks. methods and materials: 17 healthy male volunteers (aged 23 -32 years) were examined employing a standardized mri sequence protocol (t1w mp-rage, t2w/ pdw se, ir, anatomically correct image registration) on a 1.5 t system. automatic brain tissue classification was performed by a 3-layer feed-forward rbf neural network: image data were transformed to a 33-dimensional feature space with 3 spatial and 30 gray level coordinates of each voxel and its neighborhood. for comparison, manual interactive brain contour tracing was performed by two neuroradiologists on each data set. five data sets served as training data. the procedure was tested on the remaining 12 data sets. contingency tables were calculated w.r.t. results of human interactive contour tracing and compared to interobserver variability of manual segmentation. results: computation time for automatic brain segmentation was 31 ± 2 min on a sun ultrasparc workstation. mri acquisition time was 27.5 min for the sequence protocol. manual processing time for interactive contour tracing of the training data was 110 ± 14 min. the ratio of voxels classified differently in manual and automatic segmentation was 2.5 ± 1.2 %. this is comparable with the interobserver variability for manual brain segmentation (2.4 ± 1.0 %). conclusion: automatic tissue classification by rbf neural networks is a powerful method for brain segmentation from extracerebral anatomical structures. although comparable w.r.t. segmentation quality, the rbf approach is considerably less time-consuming than manual contour tracing by human operators. purpose: for medical applications in the field of computer assisted surgery it is essential to complement human visual systems. in order to develop new ultrasound based minimally invasive therapy systems in the head and neck region we combine both mr and 3d ultrasound data. materials and methods: mr and ultrasound datasets from the head and neck region were prealigned manually and filtered using adaptive filtering techniques. the prealigned data were matched for the first time using our biomechanical model based on a linear elasticity model recently developed for nonrigid registration of mr datasets of the brain. in order to reduce the computational effort this model uses as input parameters a sparse deformation field estimated from the images as well as predefined homogeneous tissue parameters. results: mr acquisition technique leads to rigid nondeformed datasets. ultrasound techniques offer resolution capacity of higher quality specially for soft tissue diagnostics and therapy. but ultrasound volume datasets show distorted and deformed anatomical structures. in order to prealign the data all images need to be resampled using both global affine transformation and interpolation. after being carefully prealigned an edge enhancement was carried out using adaptive filter techniques. it is shown in some cases of the neck region, that it is possible to match both data types using a linear elasticity model. the resulting images show clearly that the method can be used with slightly deformed ultrasound data. up to now the model is limited to high quality image data with a highly optimized data acquisition. using mri and ct data with the nasa virtual glovebox; a simulation system for life science experiments aboard the iss s. wildermuth 1 , j. smith 2 , c. bruyns 2 , n. teodorovic 1 , r. boyle 2 , p.r. hilfiker 1 ; 1 zürich/ch, 2 moffett field, ca/us purpose: the international space station will provide an orbiting research facility for addressing fundamental questions on the long-term effects of microgravity on living systems. many of these life science experiments will require the use of the space station glovebox facility, a contained reach-in environment where astronauts will handle animals and collect biological samples. to aid in this endeavor, virtual environment technologies are being developed to assist astronauts in training and performing complex experiments in the space station glovebox. this "virtual glovebox" (vgx) is designed to integrate ultra-high resolution imaging technology and force-feedback devices with highfidelity graphics and real-time computer simulation engines to provide a realistic immersive environment. here, we describe the prototype vgx system and its initial simulation environment using ct and mri datas. material, methods, and results: the virtual glovebox was designed and developed for three main applications: engineering evaluation of operational efficiencies for glovebox equipment, experimental design for on-orbit life animal research and simulation for training astronauts to perform biological experiments in space. different animal models are built based multidetector-ct (mdct) and mri datasets. we present high resolution mdct data to reconstruct anatomy of small animals and represent them as deformable objects within a linear mass-spring simulation system. the vgx combines elements of aerospace flight simulator technologies with new imaging techniques (high resolution ct and mri datasets) and medical simulation systems to provide a versatile, state-of-the-art virtual environment for training astronauts in biology research tasks. development of the function of the high speed virtualized bronchoscopy system and it's medical significance h. natori 1 , m. mori 1 , h. takabatake 1 , k. mori 2 , j. toriwaki 2 ; 1 sapporo/jp, 2 nagoya/jp purpose: we previously developed the virtualized bronchoscopy system which enables us to do intrabronchial high speed free navigation with special functions for clinical application and education in the medical school. we report the medical significance of the developed system. materials and methods: chest helical ct data were stored with dicom format. on the onyx2 (silicon graphics inc), bronchial air space was extracted and three dimensionally reconstructed with surface rendering. this bronchial tree was used a c d e f 154 as the virtual environment of this system. external view of the bronchial tree with the view point marker and its trace, ct image at the view point, intrabronchial view for navigation were displayed on the crt. the system had the anatomical database of the bronchial tree for automatic display of the bronchial name and for the bronchial name quiz. the system had interactive artificial cough as a sonic alert against wall irritation by the virtualized bronchoscope. results: even faster speed than real bronchoscopy was obtained for navigation conducted by the mouse. the system enables navigation into the peripheral bronchus for example b1bi. navigation into the peripheral intrabronchial space was possible beyond the narrowing portion. the system displayed proper bronchial names. its accuracy was dependent on the spatial resolution of thin slice ct data. conclusion: a high speed virtualized bronchoscopy system with special feature based on the ct data was developed. this system is useful for anatomical education for students, training to get their bronchoscopic skill for residents, and for preliminary examination simulator of real bronchoscopy. concept of a ct based orthopaedic simulation enviroment for movement analysis and osteotomy planning t.c. mamisch 1 , j. kordelle 2 , p. everett 3 , m. das 3 , f.a. jolesz 3 , r. kikinis 3 , r.m.m. seibel 1 ; 1 mülheim a. d. ruhr/de, 2 gießen/de, 3 boston, ma/us purpose: the goal of the presented study was to investigate the relation between the proximal femur and the orientation of the epiphysis, to determine if the acetabular development is influenced by the femoral neck orientation, and to simulate reorientating osteotomy. material/methods: three-dimensional reconstructed models based on ct data sets of 22 patients scfe were reviewed. measurement of the hip joint geometry was performed by using a newly developed interactive software to determine projected angles. a phantom scan was performed to validate the accuracy of the tool. a computer program for simulation of movement based on cartilage filtering techniques and osteotomy developed by the authors, served for study execution. results: we found a significant positive correlation between the femur version and the version of the femoral epiphysis. furthermore, there was a highly significant positive interdependence between the shaft-neck-angle and the shaft-epiphysis-angle. there was no correlation between the epiphyseal orientation and the anteversion and inclination of the acetabulum. conclusion: the study of the hip geometry demonstrates a significant correlation between the femoral orientation and the epiphyseal alignment. in response to these results, we suggest that there is an interrelation between the development of the proximal femur and the epiphyseal growth plate, wich influences the planning of reorientating osteotomy purpose: in addition to tumour size, the therapeutic effect after neo-adjuvant chemotherapy is histopathologically described by regressive changes. the aim of this study was to evaluate whether regressive changes affect the accuracy of preoperative mri tumour measurement. in an ongoing study initial and preoperative mri was performed in 31 patients with advanced breast cancer. beside t-1w flash 3d pre-and post contrast mri, a fast turboflash sequence was used for the dynamic examination. the time-intensity curves were parameterised by a pharmacokinetic two compartment model and colour-mapped. in consideration of the conventional mri, tumour diameters were measured using the colour-mapped images and compared with the histologically determined tumour size. regarding sclerosis, cytopathic effects and invasive residuals the histological regression was classified between 0 and 4. results: in 12 cases without regressive changes (grade 0), the correlation (spearman rank test) between mri and histopathological tumour measurement was 0.88 (p < 0.0002). in 17 patients with regressive changes grade 1 -3 the correlation was 0.48 (p < 0.0538), without any tendency for systematic over-or underestimation (p = 0.32). compared to tumours without regressive changes, those with grade 1 -3 were associated with a distinct decrease of pharmacokinetic parameters (amplitude and distribution constant rate) derived from time-intensity curves (p < 0.037 resp. p < 0.034). in two cases without residual tumour (grade 4), a complete response was observed by mri. conclusion: histological regression of breast tumours after neo-adjuvant chemotherapy leads to an inaccuracy of preoperative mri tumour measurement. in addition to the lost tumour continuity the decrease of contrast uptake complicates the demarcation of residual tumour. (axial t1 3d ffe, axial and sagittal t2* ffe, axial and sagittal t2 tse) . two experienced radiologists analysed the lymph nodes regarding size, morphology, and signal intensity before and after sinerem®. the results were compared with histopathologic findings. results: a total of 123 lymph nodes were detected equally by pre and post-sinerem® mri. 200 lymph nodes were diagnosed by histology. a node-by-node correlation between mri findings and histology could be achieved in 93 lymph nodes. 10/93 lymph nodes were true positive, 75/93 true negative, 3/93 false negative, 5/93 false positive. sinerem®-enhanced mri revealed a node-by-node sensitivity, specificity, and accuracy of 77 %, 94 %, and 91 %, respectively. however, in the overall lymph node assessment for each patient sinerem®-enhanced mri did not miss any lymph node metastases. conclusion: sinerem®-enhanced mri is an accurate method in the assessment of lymph node metastases in patients with breast carcinomas and is superior to plain mri. in our institute we performed 297 breast mri examinations for different clinical queries. we used a 3d-dynamic contrast-enhanced fspgr sequence and a 1.5 t mr unit. before the examination patients underwent a careful clinical-anamnestic evaluation; in fertile women mri was carried out between 7 th and 22 th day of menstrual cycle; previous mrx and/or us examinations were reviewed; patients were recommended for the highest compliance in order to reduce motion and breathing artefacts. results: among 106 cases with an histological equivalent we observed 20 false results. the 6 false negative cases were performed before the 10 th day of menstrual cycle (2 in situ carcinomas, 2 small fibroadenomas, 2 sclerotic adenosis). false positive results were 14: 6 due to a hormonal therapy recently suspended or still active, 4 due to the presence of the so-called ubos (unidentified bright breast objects), 4 in which examination was performed after the 18 th day of menstrual cycle. in literature breast mri diagnostic accuracy varies from 65 and 90 %. this large variability is mostly due to pitfalls in cases selection and examination timing. we recommend an optimal examination timing (10 th -16 th day of the menstrual cycle); a 6 months interval after hormone or radiotherapy. purpose: this study was designed to investigate the relationship between greater tuberosity irregularities, subacromial space, age and rotator cuff tears in the subacromial impingement syndrome. methods and materials: sonographic examination of 67 symtomatic shoulders (age range of patients from 33 to 79 years) were performed. the rotator cuff, greater tuberosities and subacromial space were evaluated. full and partial thickness rotator cuff were given equal significance. supraspinatus degeneration was graded according to the neer classification. the sonographic finding of tears was confirmed surgically. a student's t test, and logistic regression analysis were used to analyze the data. results: sonography showed the greater tuberosity to be irregular in 78 % of 47 shoulders with a rotator cuff tear and in 75 % of 20 shoulders with supraspinatus degeneration. the thickness of supraspinatus outlet ranged from 8.5 mm to 2 mm in shoulders with rotator cuff tear and from 9 mm to 3.7 mm in shoulders with supraspinatus degeneration. subacromial space: with tihckness < 6 mm was found in 70 % of rotator cuff tears and a thickness > 6 mm was found in 60 % with supraspinatus degeneration. statistical significance was detected (p < 0.001) for the association of greater tuberosity irregularity and rotator cuff tear and for the association of subacromial space thickness < 6 mm and rotator cuff tear. patients and methods: the study population comprises 109 randomly selected patients suffering from rheumatoid arthritis. a radiograph of the left hand was acquired for each patient and subjected to a new automated method for estimating bone mineral density (bmd) from a plain hand radiograph using the pronosco x-posure system™. this system digitizes a radiograph with a scanner and derives subsequently a bmd, a metacarpal index (mci) and a porosity index (pi) from automated radiogrammetrical measurements of the three middle metacarpal bones. the severity of the decease was graded by two radiologists using the larsen score. a third radiologist reviewed the incongruent scored cases. results: the x-posure system was capable of providing a bmd estimate in all 109 cases. mean value of bmd decreased from 0.55 ± 0.08 (score 1)g/cm 2 to 0.44 ± 0.11 (score 5) g/cm 2 . mean mci-values decreased from 0.44 ± 0.08 to 0.33 ± 0.10. no significant change of pi was observed. correlation of bmd, mci, pi and severity score was -0.44, p < 0.05; -0.40, p < 0.05 and -0.07, n.s., respectively. conclusion: the new densitometric system based on radiogrammetry seems to be able to detect differences of bone mineralization depending on the severity of rheumatoid arthritis. with suitable normative data, the new technology may be able to reduce additional radiation exposure in the future, especially in patients receiving a radiograph of the hand for e.g. diagnostic purposes. and normal subjects (n = 48) with equal mean age and sex ratios underwent electron-beam ct of the thoracic spine. images were acquired using the single slice mode (slice thickness 1.5 mm, scan time 0.1 s, matrix number 512 × 512, field of view 34 cm). for bone density analysis, cortical, transitional, and medullary zones were semi-automatically segmented on four consecutive images of the vertebral bodies. averaged histograms were acquired according to zones and proportions of five density intervals (a: 55, d: > 130, e: -1024-1024) were measured by custom-made software. firstly, the feasibility of this method was evaluated by comparing normal and osteoporotic groups. secondly, verification was performed by measuring the "coefficient of variance". thirdly, the feasibility was compared with quantitative ct using intervals c and e in medullary zone, since the interval e in medullary zone was used on quantitative ct. results: in the medullary zone, intervals a-d showed significant differences between osteoporotic and normal subjects, while analysis of whole density measurements (interval e) could not reveal a significant difference between both groups. intra-and inter-observer errors of intervals c-e were lower than 5 % in all zones. in the normal subject group, inter-subject variability of interval c was exclusively lower than 5 %. therefore, interval c in all zones became the region for the histographic bone density analysis method. conclusion: bone density analysis using histographic intervals was feasible to diagnose osteoporosis and superior to quantitative ct. purpose: micro-ct may offer a worthwhile opportunity to analyse so called patterned injuries in bone. in the field of forensic science, it has not been possible until now to non-destructively document such damages to bone. materials and methods: based on a real murder case, porcine pelvic bones were experimentally stabbed with multiple knives. afterwards these bone samples were examined with a micro-ct system developed at the imp erlangen. this cone beam scanner can achieve an isotropic resolution from 10 to 100 µm for sample diameters from 1 to 40 mm. resolution in the specific bone samples is 30 to 75 µm depending on the sample size. so far analysis has been performed by visual inspection of double oblique slices of the reconstructed volume to optimally display the plane cut by the knife using impact view (vamp gmbh, erlangen, germany). additionally the stabbing wounds were quantitatively evaluated by measuring distances and angles. results: the micro-ct datasets of the injured bone samples were used to obtain those 2d slices that optimally showed the stabbing wounds inside. based on the measured distances and angles it was easily possible to uniquely identify the size and shape of the injury-causing knife in straight stabs. in the field of forensic pathology micro-ct provides a new and advantageous tool for the non-destructive examination and analysis of patterned tool marks in bones. by using the micro-ct technology new horizons are opened for matching a possible injury-causing instrument against the patterned lesion in the bone. a bone defect (diameter 6 mm) was generated in the calvaria of 12 adult rats in general anaesthesia. this defect was filled with a threedimensional extracellular matrix gel inoculated with osteoblasts, marked with lipophilic tracer components, latter to proof viability. implants were covered by gore-tex patches. the cells phenotype was determined by antibody staining. ms-ct examinations were performed 24 hours, 2, 4, 6, 8 and 10 weeks after implantation in short anaesthesia using a siemens somatom volume zoom unit (siemens, erlangen, germany). acquisition parameters were: collimation 2 × 0.5 mm, tube current 40 to 80 mas at 80 to 120 kv, reconstructed slice thickness/increment 0.5/ 0.3 mm. thin sagittal multiplanar reformations (slice thickness 0.5 mm, overlapping 0.5 mm) out of coronar acquisition were performed. the rats were sacrificed, the implants histologically examined, and correlated with ct findings with respect of defect size and ossification. results: in all cases there was an absolute correlation between ct-findings and histological findings concerning the size of the defect and the presence of mineralised bone matrix. conclusion: ms-ct is a very sensitive and specific diagnostic tool for this and similar studies. with respect to refinement and reduction of animal experiments following the directive 86/609/eec of the council of the european union ms-ct allows tremendous reduction of the number of animals to be sacrificed (in this study two-thirds). 3d-navigation with an infrared-based camera system for biopsies using spiral-ct datasets -initial preclinical results c.r. krestan, s. grampp, p.l. peloschek, m. pretterklieber, c. czerny, h. imhof; vienna/at purpose: to assess the accuracy and feasibility of biopsies with a novel 3d-navigation system (ct-sightline: philips medical system, best, netherlands) which is based on spiral-ct datasets. methods: this navigation system uses an infrared based camera, which can track the biopsy needle and the patient position by infrared light reflected from mounted passive spheres. the spiral ct dataset (philips, secura tomoscan 7000) gained during the procedure can be linked with the position of the instruments and the patient position. we used needle-marked water melons for biopsy purposes. after scanning the object, target and entry point are located by the physician. the accuracy of the simulated biopsies with 14 and 16 g standard coaxial biopsy systems was determined by a target scan. the table position of the target scan and the gantry tilt are given by the navigation system. results: we performed 18 biopsies (targeting of needle-tip) using water melons. the mean distance between the biopsy needle tip and the target needle tip was 5.3 ± 3.6 mm (range: 0.8 -9.2 mm). the average time for the procedure after set-up of the navigation-system was 15 minutes on average. conclusions: our preliminary results suggest that this navigation system allows accurate targeting of biopsy needles at defined regions of interests within a reasonable procedure time. the advantage of this system is the potentially safer and more accurate positioning of the biopsy needle at different targets. further studies and clinical evaluation will determine the value of this system in performing minimally invasive procedures. to explore the novel application of a methodology known engineering as pq diagrams to characterise the evolution of ischaemic stroke. methods: five acute stroke patients were imaged with diffusion tensor imaging (dti) at presentation, 1 week and 3 months on a 3 t magnet using a previously described dti data acquisition scheme. each dataset was then processed on a voxel-by-voxel basis, and each tensor was decomposed on into its p (isotropic) and q (anisotropic) components. when p and q are taken as axes of a cartesian plane, the evolution of tissue (e.g. due to pathology) can be visualized as characteristic trajectories or signatures in this plane. in this study, p, q, and the standard indexes relative anisotropy (ra) and fractional anisotropy (fa), were computed for the lesion (l) and contralateral control (c) regions, for each patient at each time point. results: all patients showed the same characteristic trajectories consisting of: an acute (pl < pc, ql < qc), subacute (pl ≈ pc, ql < pc), and chronic (pl > pc, ql < qc) stages. percentage acute changes in q, ra and fa for the five studies were: ∆q = 51 %, 69 %, 50 %, 51 %, 0 %. ∆ra = 3 %, 44 %, 14 %, −6 %, −110 %. ∆fa = 2 %, 35 %, 14 %, −5 %, −89 %. conclusion: ra and fa are less sensitive than p and q to detect diffusion changes associated with early ischaemia. in particular, q seems to be the most sensitive parameter to detect early loss of anisotropy in tissue; pq diagrams provide a powerful analytical and visualization tool to investigate the evolution of stroke. advances in diffusion imaging using sensitivity encoding (sense) r. bammer 1 , f. fazekas 2 , m. auer 2 , s.l. keeling 2 , m. augustin 2 , r.w. prokesch 1 , m.e. moseley 1 ; 1 stanford, ca/us, 2 graz/at purpose: diffusion-weighted single-shot epi (sshepi) is currently one of the most important tools for the diagnostic assessment of stroke patients, however it suffers from well known artifacts. sensitivity encoding (sense) was employed to greatly enhance the quality of diffusion-weighted echo-planar imaging (epi) in stroke imaging and for diffusion tensor imaging (dti). methods and materials: eight healthy volunteers and a consecutive series of stroke patients (n = 8) were examined with diffusion-weighted sense-sshepi using different reduction factors (1.0 < r < 3.0). moreover, in eight volunteers the potential of sense to improve dti was investigated. dti scans were carried out with regular (42 × 128; r = 2.0) and high resolution acquisition matrices (42 × 256; r = 2.0). to further improve the image quality, new reconstruction and co-registration algorithms were applied. results: derived maps of the trace of the diffusion tensor and of fractional anisotropy were of high quality. measured direction-dependent diffusion-coefficients and isotropic diffusion values were comparable to previous findings but showed less fluctuation. overall, the geometric distortions were substantially removed and the resolution enhancement was remarkable. all patient examinations were diagnostic and of better quality than conventional sshepi. the efficient use of thrombolysis requires robust algorithms for patient selection in acute stroke. intracranial vessel status and properties of brain tissue perfusion are predictive of cerebral ischemia. we present first clinical results using a multimodal multislice ct concept. material and methods: sixteen patients (age 34 to 90 years) with suspected cerebral ischemia and onset of symptoms within 6 hours (mean 2.6 h) were included in this prospective study. we performed noncontrast ct (ncct) followed by perfusion ct (pct) and ct angiography (cta). at the level of basal ganglia pct data were acquired using a 4 × 5 mm collimation after injection of 40 ml of cm (flow 8 ml/s). two consecutive slices of 10 mm slice thickness were reconstructed. cerebral perfusion maps (cbf, cbv, ttp) were calculated using ctp software. the size of the ultimate cerebral infarction was measured by follow-up ct or mri. results: in nine patients significant perfusion deficits were noted in one (n = 1) or both (n = 8) slices. four patients with normal pct showed small supra-or infratentorial infarctions outside the section levels. no false-negative pct results were seen. cta revealed major vessel occlusion in five patients. ncct showed early signs of infarction only in three individuals. conclusion: pct improves the sensitivity for the detection of cerebral ischemia in the section levels compared to ncct and can estimate the ultimate size of infarction. cta might localize the underlying pathomorphology. the presented multimodal ct concept is an accurate and reliable method for the assessment of acute stroke. the preliminary study of ct perfusion imaging in tias k. li, j. lu, x. du; beijing/cn purpose: to evaluate the application of ct perfusion imaging on transient ischemic attacks (tias). methods: conventional ct and ct perfusion imaging were performed on a group of tias and control group of normal adults. cerebral blood flow, cerebral blood volume, and time to peak enhancement were measured within specific regions of the brain on ct perfusion imaging. quantitative analysis was used for these images and comparative analysis was performed on these two groups to evaluate the differences on perfusion images between tia patients and control group. results: a gradient of perfusion between the gray matter and the white matter was shown on the ct perfusion images of normal adults and tia patients. persisting abnormal perfusion changes of decreased cerebral blood flow were found on about 86 % ct perfusion images of tia patients. abnormalities were detected on time-to-peak images of all tia patients with diffuse or regional prolonged time to peak enhancement. conclusions: perfusion ct can provide valuable hemodynamic information and depict abnormalities of perfusion in the patients with tias. prognostic value of admission perfusion ct in acute stroke patients: comparison with admission mr m. wintermark, j.-p. thiran, p. maeder, s. binaghi, p. schnyder, r. meuli; lausanne/ch purpose: comparison between perfusion-ct and dwi-/pwi-mr in acute stroke patients at the time of their emergency evaluation methods and material: 13 acute stroke patients underwent perfusion-ct and dwi-/pwi-mr on admission. 9 were treated with thrombolytic agents. the size of infarct and ischemic lesion (infarct + penumbra) on the admission perfusion-ct was compared to that of the mr abnormalities on the adc map and on the relative cerebral blood volume (rcbv), cerebral blood flow (rcbf), time-to-peak (rttp) and mean transit time (rmtt) maps extracted from pwi studies. the most significant correlations were found between the infarct size on the admission perfusion-ct and the abnormality size on the admission adc map (r = 0.971; p < 0.001) and between the size of the ischemic lesion (infarct + penumbra) on the admission perfusion-ct and the abnormality size on the rmtt map calculated from admission pwi-mr (r = 0.930; p < 0.001). information provided by both imaging techniques about cerebral infarct and total ischemia (infarct + penumbra) are similar, with slopes of 0.910 and 0.925, respectively. conclusion: perfusion-ct and dwi-/pwi-mr are equivalent in the identification of cerebral penumbra in acute stroke patients and in their selection for thrombolytic therapy. perfusion-ct takes advantage from its availability and from its feasibility in acute stroke patients, as part of their admission imaging survey. a novel ct brain perfusion algorithm for improved measurement of low flow regions: clinical results a.j. cook ii. 1 , s. pohlman 2 , s. lin 2 , p.c. johnson 2 , a. cook 1 ; 1 ravenna, oh/us, 2 highland heights, oh/us purpose: accurate quantitative measurements of ct perfusion studies will make a significant impact on patient care. low flow regions, associated with minimal contrast enhancement and poor signal to noise ratios, are susceptible to difficulties using traditional perfusion calculations. we propose a novel signal enhancement algorithm for analysis of low flow regions. methods: 30 ct brain perfusion cases were acquired. a low flow improvement technique, which works by clustering similar tissue types, was applied to each case. gray matter and white matter regions were manually segmented. for quantitative measurements, perfusion values and ratios were calculated for each region before and after the low signal correction. for qualitative evaluations, four radiologists reviewed the corrected and uncorrected images. the corrected images were found to be visually superior to the uncorrected images, particularly in terms of reduced noise and improved distinction between gray and white matter. perfusion values for the corrected images (gray matter: 75.9 ml/100 g/min, white matter: 29.1 ml/100 g/min) were closer to physiological norms than uncorrected images (gray matter: 84.0 ml/100 g/min, white matter: 46.7 ml/100 g/min). the gray/white matter ratio, typically reported in the literature to be approximately 3:1, improved from 1.9:1 for the uncorrected images to 2.8:1 for the corrected images. regions with no blood flow, such as the ventricles, were also more accurately depicted on the corrected images. conclusion: initial results using our approach for low signal enhancement is very promising and could potentially provide more accurate quantitative measurements using ct perfusion. to evaluate the effect of dose reduction on diagnostic performance, using a digital chest imaging system in which amorphous selenium serves as the x-ray detector. material and methods: 247 patients were examined with the selenium system. three sets of images were made in each patient: one set with a standard x-ray dose, one set with 55 % of the standard dose, and one set with 35 % of the standard dose. all 741 images were read by two radiologists. the diagnostic value of each set of images for detection of pulmonary, mediastinal and pleural pathology was analyzed with receiver operating characteristic (roc) methodology, using computed tomography (ct) as the reference standard. we also assessed the effect of the sex, height and weight of the patients on the diagnostic performance of the readers. a c d e f 166 conclusion: we found no statistically significant difference between the radiologists' performance in detecting abnormalities with standard x-ray dose images, and the performance with images made with 55 % and 35 % of the standard dose. sex, height and the weight of patients had no influence on diagnostic performance. withdrawn by author b-0259 10:40 flat panel x-ray detector: reduced radiation exposure for the detection of simulated interstitial lung disease, nodules and catheters u. rapp-bernhardt, t.m. bernhardt; münster/de purpose: evaluation of a flat panel detector (fd) using amorphous silicon with respect to detection of simulated interstitial lung disease, nodules and catheters and reduction of radiation exposure compared with an asymmetric screen-film system. materials and methods: an experimental model was used to determine the detection of reticular, micronodular, linear, ground glass patterns, nodules and catheters which were superimposed over an anthropomorphic chest phantom. 19200 observation fields were evaluated using the fd with the simulated speed of 400, 600 and 1600 and compared with the asymmetric screen-film system with the speed of 400. five radiologists performed receiver operating characteristics (roc) for a five-level confidence scale. the areas under the roc-curves showed no statistically significant differences between the two detector systems at the same speed with respect to the detection of all simulated lesions (p > 0.05). the fd with a digital speed of 800 yielded in decreased diagnostic performance compared with the asymmetric screen-film system, however, these results were not statistically significant (p > 0.05). using the fd with a digital speed of 1600 lead to statistically inferior results in the detection of catheters and nodules over obscured lung (mediastinum) as well as micronodular lesion over lucent lung (p < 0.05). conclusion: these results suggest that a dose reduction up to the digital speed of 800 is possible for fd. image quality of computed radiography and flat panel detector radiography: evaluation of simulated subtle lung abnormalities h. tagashira, k. arakawa, m. yoshimoto, j. ikezoe; shigenobu/jp purpose: to evaluate the image quality of computed chest radiography (cr) and flat panel detector radiography (fpd) for diagnosing subtle lung abnormalities. materials and methods: we studied the differences of observer performance among computed radiography (new type computed radiography (fcr 5501d, pixel size 100 µm, dual side (front and back) read out by laser beam of imaging plate) and flat panel detector radiography (indirect type with screen, canon cxdi-11, pixel size 160 µm). simulated nodule, ground-glass opacity and reticular shadow were made of acrylic resin, sand ground down and softened gauze with urographin (contrast material), respectively. a simulated abnormality was placed on the back of the thorax of each volunteer (n = 50) in order to overlap the right or left lung parenchyma and each volunteer was examined with three different modalities with the same exposure condition. so, total of 200 unilateral lungs were obtained, and of these 200 unilateral lungs, 100 lungs had one of the simulated abnormalities, and the remaining 100 lungs were normal. five chest radiologists evaluated these 200 unilateral lungs with roc analysis with continuous confident scale. the area under the roc curve (az) was used to evaluate the results. results: for all simulated abnormalities, dual read-out type cr (fcr 5501d) showed the best result (az = 0.820), followed by fpd (az = 0.780). however, in case of reticular simulated abnormalities, fpd showed the best result, followed by dual read-out type cr. conclusion: dual read-out type cr and fpd showed extremely high image quality. first experiences with a detector-based dual energy system for thorax radiography j. ricke, f. fischbach, u. teichgraeber, t. freund, r. felix; berlin/de purpose: to assess the influence of dose on the image quality of subtracted soft tissue and bone images generated by a dual energy system based on a flat panel detector. materials and methods: 88 patients were randomized in 2 groups. one group received a dual energy examination at a xqi revolution (ge medical systems, usa) with an intended approximative speed pair of 400/1000 for high and low energy shot, the other group with an intended approximative speed pair of 200/ 500. for data analysis, subgroups were specified according to additional dose measurements at the detector. image quality indicators were noise, residual bone structures, motion artefacts of pulmonary vessels, heart and aorta, display of retrocardiac ribs or other bone structures, display of lung apex or retrocardiac lung. image review was performed blinded by two experienced radiologists in consensus applying a rating score of 1 to 5. linear regression and chi square tests were performed for statistical analysis. results: overall image impression was rated good with no significant improvement of image quality with increasing dose. however, a trend for decreased noise in soft tissue and bone images was noted with higher dosage as well as increasing residual bone structures. conclusion: increased dose did not improve image quality significantly. dual energy thorax imaging at a flat panel detector proved potential as a future routine application. digital bedside chest radiography with and without antiscatter grid: impact on image quality and display of low contrast details m. uffmann, k.s. exter, i.m. nöbauer-huhmann, c. balassy, j. sailer, c.j. herold, c. schaefer-prokop; vienna/at purpose: use of an antiscatter grid also for bedside chest radiographs improves image quality however at the expense of increased dose requirements. image processing in digital radiography offers the option for optimising detail contrast. purpose of the following study was a) to test whether contrast enhancing processing compensates for increased scatter radiation if no grid is used and b) whether further dose reduction is feasible. materials and methods: routine chest films with storage phosphor plates (st vn, fuji) were performed on 24 icu patients on 3 consecutive days using an antiscatter grid and an acquisition dose approximating 250 speed system, without grid and an optimised image processing, and without grid and additional dose reduction of one third. all images were overlaid by a 9 element matrix with small fragments of various catheter types (n = 120). 4 readers evaluated the hardcopies for the presence of catheter fragments and diagnostic image quality. results: areas under the roc curve for the detection of catheter fragments were statistically equivalent for all images irrespective of the use of a grid or reduction of acquisition dose (0.92, 0.93 and 0.93, resp.). visualisation of lung parenchyma and monitoring lines was rated comparable for all 3 image types. visualisation of mediastinal and retrocardiac areas, however, was rated significantly poorer in images obtained without grid and reduced dose. conclusion: digital bedside chest radiographs can be obtained without an antiscatter grid by using optimised image processing. however, further dose reduction results in significant loss of image quality in high absorption areas. detection of simulated chest lesions using soft-copy reading: comparison of an amorphous silicon flat-panel detector system and a storage phosphor system j. goo, j.-g. im, j. kim, m. chung, j. seo, h. kim; seoul/kr purpose: to compare observer performance using soft-copy images produced by an amorphous silicon flat-panel detector system and a storage phosphor system for the detection of simulated chest lesions. materials and methods: to test the diagnostic performance of these two systems, we used four types of simulated lesions (nodules, micronodules, lines, and reticular opacities) that were superimposed over an anthropomorphic chest phantom. digital chest radiographs were acquired by amorphous silicon flat-panel detector radiography (3k matrix, 12 bits) and by storage phosphor radiography (4k matrix, 10 bits). six board-certified radiologists evaluated soft-copy images on a high-resolution video monitor (2560 × 2048 × 8 bit). a total of 14400 observations were analyzed in terms of receiver operating characteristics (roc). results: averaged performance in terms of the detection of nodules was significantly better (p < 0.05) on the flat-panel detector system than on the storage phosphor system (area below roc curve [az] values: 0.93 ± 0.015 and 0.85 ± 0.033). for micronodules, lines, and reticular opacities, no significant detection differences in averaged performance were found between the flat-panel detector and storage phosphor system (az values: micronodules, 0.86 ± 0.020 and 0.76 ± 0.049; lines, 0.85 ± 0.031 and 0.75 ± 0.051; reticular opacities, 0.96 ± 0.015 and 0.92 ± 0.022). in the evaluation of soft-copy images, the amorphous silicon detector system appears to be superior to the storage phosphor system for the detection of pulmonary nodules. the total realistic examination time in multi-slice-ct is 5 min 28 s faster than in single-slice-ct. due to shorter scanning time a reengineering of preparation and post-processing phase enables to increase the number examinations per day with multi-slice ct. process simulation showed to be superior to cpm/pert due to higher flexibility and ability to consider cycle overlap and determine cycle times. evaluation of a new patient transfer board (ptb) for diagnostic er-/icupatient-management in diagnostic ct t. schroeder, s. ruchholtz, s.g. rühm, s. heistrüvers, h. kuehl, j.f. debatin; essen/de purpose: multislice ct has vastly reduced scan times. rather than data collection, patient transfer and positioning has thus become the rate limiting step particularly for polytraumatized er-and icu-patients. to speed transfer to and positioning onto the ct table, we developed a patient transfer board (ptb) enabling the "en-bloc"-transfer of patient and life-support equipment. the ptb was assessed on 20 acute trauma-patients and 50 icu-patients. patients were placed on the ptb directly in the er/icu before transfer to the ct room. transfer-times between patient arrival in the er and completing of the radiological diagnostic procedures (er-patients) and the in-roomtimes in the ct (er-and icu-patients) were measured by stop-watch and compared to those determined in matched populations (40 acute trauma, 100 icu) examined without the ptb. results: the ptb enabled the "en-bloc transfer of er-/icu-patients and support equipment". the handling of the ptb was easy, and intuitive. the ptb had no adverse effect on image quality. transit-times between arrival of trauma patients in the er and completing the radiological diagnostic procedures including ct were reduced from 39 to 31 minutes (213 %, p < 0.05). the in-room-times in the ct (er-and icu-patients) were reduced from 14 to 9 minutes (36 4 %, p < 0.05). the ptb was well accepted by the medical staff. conclusion: the ptb is a simple device capable of vastly shortening patient transfer and positioning times onto ct-tables. the ptb accelerates the diagnostic management of er-and icu-patients and hence increases ct patient throughput. iliocaval thrombi were simulated using clotted bovine blood. four experimental set-ups were performed with 10 interventions each. thrombus particles and distribution were measured in the effluent. mechanical thrombectomy (mt) was performed using the ptd alone. secondly, a newly developed vena cava filter was inserted before and removed immediately after the intervention without manipulation within the filter. in a third procedure, the filter was filled with thrombus and closed without any fragmentation. finally, the filter was completely filled with thrombus material and mt was performed within the filter using the ptd. results: running the ptd in the flow circuit led to a maceration of 67.9 % of clots into particles below 500 µm. in the second set-up, additional placement of the filter safely prevented embolization of particles above 500 µm. closing the filled filter within the flow circuit macerated 75.2 %, while additional mt within the occluded filter led to a dissolution of 90.4 % of the initial clot weight. the ptd proved as an effective and safe device for clot fragmentation in this experimental set-up. the use of a cava filter is mandatory to prevent embolization of thrombus fragments. even large clot burdens can be further macerated easily by the ptd within the filter basket before removal. clinical evaluation of these two devices in iliocaval thrombosis is promising. cost effectiveness assessment of port devices chemotherapy implant in 200 m0 breast cancer patients: radiological versus surgical placement evaluation p.y.r. marcy sr. 1 , c. bailet 1 , n. magne 1 , e. chamorey 1 , j.c. machiavello 1 , j.c. gallard 2 ; 1 nice/fr, 2 caen/fr purpose: to report the feasibility and cost effectiveness of two venous chemotherapy port implantation techniques in 200 m0-breast cancer patients. material and methods: radiological venous arm port (r) and surgical subclavian (s) implantation techniques were retrospectively evaluated in an homogeneous set of 200 m0 breast cancer patients treated with adjuvant/neoadjuvant chemotherapy. mean age was 55.7 a [55.5 a, n = 100 (r); 55.9 a, n = 100 (s)] and the f/m sex ratio was 1.0. initial feasibility was evaluated for both techniques. procedure related direct costs and outcome were respectively evaluated. results: initial technical failures rates (r/s) were 4 % and 9 %. mean implant duration time was 168/222 days, the overall complication rate (r/s) was 9 %/13 % (chi2 test p = 0.5) (0.24 -0.4/1000 patient-days). mean implant duration time without any complication or death was 193 d vs 233 d. median number of chemotherapy courses was 6 (r = s). 6 %/7 % of the devices had to be removed prematurely. complications included device-related sepsis (n = 2 vs 5), skin dehiscence (n = 3), deep venous thrombosis (n = 1 vs 4), catheter occlusion (n = 1 vs 0) and fissuration migration (n = 1.1 vs 0.0) of the catheter. direct costs (r/s) were respectively 230.8 vs 219.1 • . conclusion: both techniques are successful and safe, with a 5 % higher relative cost for r placement. both are indicated for breast cancer adjuvant chemotherapy. b a c d e f 168 balloon angioplasty and stenting of subclavian and brachiocephalic benign venous obstruction b. guadagni, m. cariati, g. cittadini, g. de caro, c. ferro; genova/it purpose: report our clinical experience of the treatment with balloon angioplasty and/or stent placement of subclavian or brachiocephalic benign venous stenosis or obstruction in patients undergoing hemodialysis. materials and methods: among 27 patients with subclavian (n = 13) or brachiocephalic (n = 15) venous stenosis or obstructions, 10 were treated with pta alone and 17 with pta and stent placement. technical success and primary and assisted primary patency rates were calculated. results: technical success was 100 %. the ten patients treated with pta alone had good immediate results. primary patency rates at 6 months, 1 year and 2 years were respectively 90 %, 80 % and 80 %. two patients required additional procedures with repeated pta and stent placement (wallstent) with an assisted patency rate of 100 % at 2 years. 24 wallstents were inserted immediately after failed angioplasty in 17 patients because of early restenosis and unsuitablility for angioplasty. primary patency rates at 6 months, 1 year and 2 years were respectively 76 % (13/17), 61 % (8/13) and 70 % (7/10). eight patients required multiple reinterventions for restenosis or thrombosis with additional angioplasty and/or stent placement. assisted patency rates at 6 months, 1 year and 2 years were respectively 100 % (17/17), 92 % (12/13) and 80 % (8/10). conclusion: endovascular treatment of subclavian and brachiocephalic benign venous stenosis or obstructions with pta alone and/or stent placement can provide prolonged use of a hemodialysis access site. close clinical surveillance and multiple reinterventions are usually necessary to maintain stent patency. follow-up was available in 19 patients over 20 months. in this period 11/19 patients (57.9 %) had another treatment: pta of intra stent stenosis in 23 cases; stenting of restenosis after pta in 4 cases; positioning of a second stent in 2 cases. results: in total we performed 62 procedures, with a full technical success in 59 (95.1 %), 2 (3.2 %) were technically unsuccessful and 1 complication ocurred (1.7 %): rupture of the treated vein. primary patency rates were 82 % at 3 months, 53 % at 6, 17.6 % at 12 and 5.8 % at 24 months. secondary patency rates were 100 % at 3 months, 55.5 % at 6, 33.3 % at 12 and 22.2 % at 24 months. conclusion: interventional procedures allow treatment of stenoses with a high grade of technical success but the high incidence of restenosis requires continuous monitoring and frequent re-intervention to maintain the functionality of the hemodialysis vascular access. mechanical thrombectomy to preserve vital venous access in patients with subclavian vein thrombosis p.m. vos, h.j. baarslag, j.a. reekers; amsterdam/nl purpose: deep venous thrombosis is one of the most important catheter related complications in patients with central venous lines. preservation of venous access may be vital for these patients. feasibility of percutaneous mechanical thrombectomy of subclavian vein thrombosis in patients with central venous lines was evaluated. materials and methods: 7 patients (3 male; 4 female) with a mean age of 50 years were included. all patients had central venous catheters in the thrombosed subclavian vein. catheters were placed for chemotherapy 5, nutrition 1 and dialysis 1. before the procedure venography was performed to evaluate the level and extent of the thrombosis. if there was extensive thrombosis with extension in the brachial veins, patients were not included. mechanical thrombosuction was performed using a 6 or 7 french hydrolyser catheter (cordis j&j, 700 psi, 5 ml/s). prior to the procedure 5000 u of heparin was given intravenously. all patients were treated with anti-coagulants after treatment. re-establishing flow with or without wall adherent thrombus was defined as a clinical success. results: in 5 cases, percutaneous mechanical thrombectomy was successful. in these 5 patients the central venous line could be preserved. in 2 patients re-canalization of the subclavian vein thrombosis could not be established due to wellorganized (longstanding) thrombus clots. in one patient with a clinical success, reocclusion occurred 2 months after the procedure. conclusions: percutaneous mechanical thrombectomy of subclavian vein thrombosis in patients with central venous lines is feasible if the thrombosis is acute and not too extensive. factors that influence the results of percutaneous transluminal angioplasty for hemodialysis access dysfunction k. maeda, a. furukawa, m. yamasaki, m. onishi, k. furuichi, t. nagata, s. aoki, m. takahashi, k. murata; otsu shiga/jp purpose: to evaluate factors that influence the initial success rate and long-term results of percutaneous transluminal angioplasty (pta) for hemodialysis access dysfunction. methods and materials: a total of 81 pta procedures were performed in hemodialysis shunts with stenosis (61 procedures) or occlusion (20 procedures) in 47 patients between 1997 and 2001. initial success rates were compared between patients with stenosis and occlusion. in cases where initial success was obtained, cumulative rates of shunt-patency (crp) were evaluated to assess whether the following three factors may influence the long-term results; length of stenosis (≤ 3.5 cm vs > 3.5 cm), length of patent period of the primary shunt (≤ 12 months vs > 12 months) and diameter of the balloon used for angioplasty (≤ 4 mm vs > 4 mm). results: overall initial success rate was 87.7 % (stenosis: 98.4 % vs occlusion: 55.0 %). overall crp at 3, 6, and 12 months were 81.5 %, 54.9 %, and 44.4 %, respectively. crp was significantly higher in patients with shorter stenosis compared to patients with longer stenosis (93.8 %, 72.1 %, and 63.1 % vs 70.8 %, 37.1 %, and 20.6 %, respectively, p < 0.01). crp was significantly higher in patients with longer patent period of the primary shunt compared to patients with a shorter period (83.7 %, 67.8 %, and 63.8 % vs 81.4 %, 36.9 %, 21.5 %, respectively, p < 0.01). conclusion: pta is technically highly successful in patients with shunt stenosis (not occlusion) and a better prognosis is expected in patients with shorter stenosis and patients with a longer patent period of the primary shunt. long-term follow-up of vena cava filters: clinical and radiological findings s.c.a. herber 1 , t. knodel 1 , j. schneider 1 , c. düber 2 , m. thelen 1 , m.b. pitton 1 ; 1 mainz/de, 2 mannheim/de purpose: to evaluate the clinical efficacy, mechanical stability and safety of vena cava filters (vcf) in patients for prophylaxis of pulmonary embolism. material and methods: retrospective analysis of 80 patients undergoing vcf for thrombosis (dvt) in 9/80 and pulmonary embolism (pe) in 71/80 patients. 3 filtertypes were inserted (73 lgm; 8 antheor; 1 cook tulip) using a femoral approach in 66/80 and a jugular approach in 14/80 patients. follow up included ultrasound, conventional x-ray and ct-scans. results: no periinterventional complications occured. 71/80 patients were anticoagulated (51/80 marcumar, 20/80 heparin). mean follow-up was 40.9 months (range 14 -113 months). 54 patients are still alive. mean survival was 28 months (range 8 -40 months). 26 patients died (tumour progression n = 16, others n = 10). there was no evidence of further pe, but 3 dvt recurred during long-term follow-up. in 68/80 cases correct filter deployment was achieved including a suprarenal position (n = 3). technical problems with filter deployment occured in 12 of 80: incomplete opening (n = 8), non-opening of filters (n = 2), filters tilted > 15° (n = 2). follow up demonstrated significant filter migration in 28/80 with a mean migration of 19 mm (range 7 -48 mm), 7/28 within 30 days and 20/28 showed late filter dislocations (> 12 months). fractures of filter struts occured in 4 cases (2 lgm, 2 antheor) and perforation of struts through the venous wall occured in 2 (antheor). conclusion: vcf has a low periprocedural complication rate but significant migration, some filter fractures and strut perforations were seen. due to the low incidence of re-thrombosis under anticoaglation, vcf is restricted to very selected cases. patients and methods: 117 patients underwent percutaneous implantation of a snf from 1993 through to 1999. patient reports were retrospectively analysed for complications during and after implantation and deep venous thrombosis and pe before and after implantation. helical-ct with contrast media and plain abdominal radiography were performed on 35 patients, helical-ct alone on 2 patients. we checked the position and configuration of the snf and looked for perforation of the filter legs through the wall of the inferior vena cava (ivc). the ivc and deep pelvic veins were analysed for patency. results: during implantation 10/117 (9 %) patients had minor complications, major complications were reported in 0.9 % (1/117). pulmonary re-embolism was documented in 9/117 (7.7 %) patients. there was no significant increase in thrombosis of the deep pelvic veins or the ivc after implantation. 1/35 (2.9 %) examined patients showed a single strut fracture of the snf. tilting more than 15° was seen in 7/37 (19 %) patients. dislocation of the snf more than 10 mm occurred in 1/35 (2.9 %) patients and perforation through the wall of the ivc in all 37 patients. we found no occlusion of the ivc. the snf is easy and safe to implant. in our investigation, complication rates during and after implantation were low. the snf effectively prevents pe. (june 1999 to september 2001 . the estimated period of protection against pulmonary embolism was less than 15 days. before insertion, color duplex sonography was performed in all patients. all filters were implanted and retrieved through a femoral approach. the position and condition of the filters was assessed immediately and 4 days after insertion. results: there were no placement complications in any patient. no clinical manifestations of pulmonary embolism occurred during the filtration period or during removal. implantation time was 7 to 12 days (mean 9 days). there were no filter thrombosis at the time of retrieval in 8 patients. one patient required reinsertion of a gunther temporary vena cava filter 8 days after the first insertion because thrombus was captured. in two patients, permanent filtration was subsequently requested, in one due to an ongoing contraindication to anticoagulation whilst in the other due to sizable clot captured in the gunther temporary filter. one other patient died of causes unrelated to the procedure. there were no complications of retrieval. no damage was detected at the insertion site. clinical and imaging follow up of the patients did not demonstrate any pathology. conclusions: gunther temporary vena cava filters are easy and safe to use and effective in protecting against pulmonary embolism in high risk patients. tips creation using self-expandable stent-graft covered with eptfe (viatorr): first experience and short-term follow-up v. chovanec, a. krajina, m. lojik, j. raupach, p. hulek; hradec kralove/cz purpose: to evaluate technical success of tips dedicated stent-graft implantation and short-term patency. materials and methods: between february and september 2001, 13 patients (7 female, 6 male) aged 16 -76 years with end stage liver disease underwent tips procedure. liver cirrhosis was due to alcohol abuse (6/13), hepatitis (2/13), liver fibrosis (1/13), unknown cause (3/13) and mixed etiology (1/13; patient with hepatitis c and wilson disease). the shunt was created using eptfe covered selfexpandable nitinol stent-graft (viatorr, w.l. gore), which was placed from the portal vein to the ostium of the hepatic vein. the 2 cm non-covered part of the stentgraft was placed into the portal vein branch which remained patent. tips procedure was done using a standard technique and with antibiotic prophylaxis. the tips patency was assessed by doppler us at discharge and then at 1 and every 3 months. the follow-up period ranged from 1 to 7 months (mean 4 months). results: all tips were technically successful with a significant decrease in the portosystemic gradient (from 18.4 mmhg to 7.9 mmhg) without clinical complications and all were fully patent. in one patient (7.7 %) two stent-grafts had to be results: due to technical failures 2 patients were excluded from the evaluation. in the remaining 100 patients the exam rendered diagnostic image quality from the carotid arteries to the tibial vessels (3000 segments). apart from the clinically suspected pvd, additional clinical relevant disease was found in 21 %. 16 patients had a renal artery narrowing, in 13 patients a carotid arterial stenosis was detected, and 2 patients showed an aaa. conclusion: noninvasiveness, three-dimensionality, extended coverage and high contrast conspicuity are the characteristics of the applied whole body 3d mra approach allowing for a quick, risk-free, and comprehensive evaluation of the arterial system in patients with atherosclerosis. the impact of cardiovascular and colorectal disease processes can be diminished if recognized and treated at an early stage or pre-stage. inherent noninvasiveness and lack of harmful side effects make mri ideal for preventive imaging. the goal of this study was to assess the impact of a screening mr examination, encompassing depiction of the brain, the arterial system, the heart and the colon. materials and methods: 28 healthy subjects (mean age 53.3 a, range: 39 -76 a) were evaluated. using a high performance mr system (siemens sonata) equipped with a rolling table platform (angiosurf), mri of the brain (t1w, t2w and tof sequences), whole-body mr-angiography (five contiguous 3d gre acquisitions), cardiac mri (truefisp cine and ir-turbo flash) and eventually mr-colonography (3d gre imaging following administration of a water enema) were performed. paramagnetic contrast was administered i.v. prior to mr-angiography and for mrcolonography. all examinations were evaluated by two experienced radiologists. results: the compound mr examination was well tolerated by all 28 patients. mean examination time amounted to 61 (± 6) minutes. a total of 14 unsuspected pathologies were detected: colorectal polyps (4), cerebral aneurysms (1), aortic aneurysm (2), stenosis of renal arteries (1), stenosis of femoral arteries (2), reduced myocardial ef (2) and mitral regurgitation (2). conclusion: multi-organ screening with mri within a short time is possible. a considerable number of disease pre-conditions requiring subsequent treatment were identified. further work will be required to determine the true value of such an approach. the use of diluted contrast media in equipment with co2 software t. moreno sánchez, e. lopez jimenez; huelva/es objective: to determine the cost-effectiveness and efficiency of angiography of the abdominal aorta and lower extremities using diluted contrast media and co2 postprocessing software for obtaining diagnostic images. subjects and methods: forty patients with peripheral vascular disease were evaluated by co2 software-digital substraction angiography with diluted iodinated contrast material (ioversol 240, diluted at 30 %). results: diluted contrast was well tolerated by all patients with no complaints despite the severity of the vascular disease. good image quality was achieved for the aorta and iliofemoral arteries in all cases. the popliteal artery and distal tree images were of good quality in 35 patients. for the remaining five, it was necessary to use pure contrast along with the co2 software. conclusion: contrast diluted angiography with co2 software can be used as an alternative to pure iodinated contrast material for obtaining arteriograms of the abdominal aorta and lower limbs. the diagnostic quality is equivalent to that obtained with conventional methods. the lower dose of contrast reduces the risk of adverse reactions, increases the cost-effectiveness of the procedure and is more comfortable for the patients. the use of the co2 software produces good quality diagnostic arteriographic images with more comfort for patients, less secondary effects and less cost. the time for completing the study is similar to the conventional one and less time consuming than co2 lower extremities angiography. material and methods: cduv was performed using manual injection and digital substraction angiography in 40 consecutive patients (77 upper limbs). images were read on a 1k high brillance digital workstation. the international anatomic nomenclature was used to divide the superficial venous network into 16 segments per limb. two senior radiologists independently assessed the patency of all segments according to the following grading: absent or non-opacified (grade 1), poor quality (grade 2), small calibre (grade 3), good quality (grade 4). in the case of discrepancy, a consensus was reached by reviewing the images. the kappa-test was used to assess the inter-observer correlation in each of the 16 different venous segments. results: overall, grade 1 was seen in 24 %, grade 2 in 13 %, grade 3 in 12 % and grade 4 in 51 %. mean κ was 0.72 (p < 0.05) for the right side and 0.71 (p < 0.05) for the left side. the worst correlation (k = 0.55) was obtained for veins at the elbow level. conclusion: cduv allows satisfactory imaging of the upper limb venous network with good agreement between the observers. special attention must be paid to obtain good images at the elbow level. intraarterial digital subtraction angiography with carbon dioxide and nonionic gadodiamide in incomplete renal failure h.-p. dinkel, h. hoppe, i. baumgartner; berne/ch purpose: to evaluate the benefit of gadolinium for intraarterial use in diagnostic and therapeutic angiography in patients with incomplete renal failure. methods: 17 patients with planned peripheral or renal vascular interventions (1 iliac, 10 femoral, 3 renal, 3 transplanted kidneys) and renal insufficiency underwent digital subtraction angiography with intraarterial administration of gadolinium (1 gadolinium-dtpa, 16 gadodiamide). gadolinium was used selectively after peripheral catheter placement, usually as an adjunct to carbon dioxide (co2) if the quality of co2-angiography (n = 14) was insufficient to assess the distal run-off vessels or did not sufficiently characterize stenotic lesions. in 16 of 17 cases gadolinium yielded good or satisfactory results that were superior to those of co2-angiography in each case. this applied to the calf and femoropopliteal region in patients with femoropopliteal occlusions, but at times also to the aorto-iliac vessels and the renal arteries. mean serum creatinine level after angiography (295 µmol/l) was not significantly different from the initial level (279 µmol/l), p > 0.37. conclusion: gadolinium is a viable alternative contrast agent in digital subtraction angiography and percutaneous transluminal angioplasty (pta). it enhances the diagnostic evaluation of stenoses and run-off vessel. non-ionic gadodiamide is preferable to hyperosmolar gadolinium-dtpa. for economical reasons and since the maximal applicable dose of gadolinium is currently restricted to 0.4 mmol/kg bodyweight (40 -80 ml) its combined use with co2-angiography is recommended. evaluation of the aorto-iliac and renal arteries: intraindividual comparison of 3d mr angiography and mdct angiography j.k. willmann, p.r. hilfiker, t. pfammatter, j.e. roos, b. marincek, d. weishaupt; zürich/ch purpose: to compare contrast-enhanced 3d mr angiography (mra) and multidetector row spiral ct angiography (cta) in the assessment of the aorto-iliac and renal arteries, using digital subtraction angiography (dsa) as the standard of reference. methods and materials: dsa, mra and cta were performed in 39 consecutive patients with suspected occlusive arterial disease. two readers assessed all mra and cta data sets independently; a third reader evaluated the dsa images. all reviewers were unaware of the results of the other imaging modalities. for data analysis, the arterial system was divided into 16 segments, and a 4-point grading system was applied to assess arterial luminal stenosis. time for post-processing and reading times of mra and cta data sets were noted. results: for readers 1/2 sensitivities of cta were 80 %/83 % and specificities 96 %/96 %; mra had sensitivities of 91 %/92 % and specificities of 97 %/96 % for detection of hemodynamically significant stenosis (> 50 %). interobserver agreements for both mra and cta were good (kappa = 0.74 and 0.67). intermodality agreement between cta and mra for readers 1/2 were 0.73/0.70. post-processing of cta data sets was significantly more time consuming (mean 15 minutes) compared to post-processing of mra data sets (mean 4 minutes) (p < 0.001). similarly, reading time for evaluation of cta was longer than reading time of mra (4 versus 2 minutes). conclusion: both mra and cta demonstrate a similar diagnostic performance compared to dsa in the assessment of occlusive disease of the aorto-iliac and renal arteries. due to shorter post-processing and reading times, mra seems to be more suited to clinical routine. correlation of spatial resolution in multislice ct and angiography in kidneys p.j. hallscheidt, c.c. cardenas, j. boese; heidelberg/de aim: the aim of this study was to evaluate the maximum spatial resolution of multislice ct in comparison to digital subtracted angiography. material and methods: 15 kidneys were catheterised with a 4 french straight catheter and under went mulitslice-ct in an early arterial phase with a reconstructed slice thickness of 0.2 mm. the data were evaluated in mip and 3d reconstruction and the resolution was compared to conventional dsa angiography, which was performed after the ct scan. results: in reconstruced multislice ct with isotropic voxels all segmental and subsegmental arteries could be delineated. the reconstructed ct data allowed similar spatial resolution to dsa, but additionally the segmental anatomy could be evaluated. discussion: reconstructed multislice ct gives similar spatial resolution, with delineation of subsegmental arterial branches, as angiography. but the 3d data allows additionally the delineation of the segmental anatomy which is essential for the planning of nephron sparing surgery. material and methods: 51 patients with a clinical or ultrasound suspicion of renal artery stenosis were examined with mscta after a bolus injection of 100 ml of non-ionic c.a. at 4 ml/s. in most cases the renal artery study was part of a run off examination for patients with peripheral arteriopathy. a fixed time delay of 25 to 28 (in presence of abdominal aortic aneurysm) seconds was used in all cases. images were interpreted by 3 blinded radiologists either on axial or reconstructed images. in all patients dsa was performed within 72 hours and considered the gold standard. sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) for the assessment of degree of stenosis and evaluation of supernumerary arteries were calculated. results: we evaluated 123 renal arteries. mscta provided good to excellent diagnostic quality in all cases; all 21 supernumerary arteries were correctly demonstrated. regarding the degree of stenosis the values of sensitivity, specificity, ppv and npv were respectively 100 %, 95 %, 100 % and 92.6 %. cta was superior to dsa in the delineation of calcification. in 4 cases with ostial calcified plaque the stenosis, moderate at dsa, was overestimated with mscta and considered severe. multislice spiral cta provides excellent arterial delineation in patients with suspected renal artery stenosis. the limited amount of contrast agent used lends itself to use in patients with partially impaired renal function. to evaluate the utility of mri for the assessment of angiodysplasia compared to conventional duplex us, venography and arteriography. methods and materials: 14 patients (9 male, 5 female; age range, 10 -64 years) with clinically diagnosed angiodyplastic abnormalities of the extremities were examined on a 1.5 t whole body scanner (magnetom sonata, siemens, germany). based on a localizer sequence axial/coronal t1-, heavily t2-weighted (stir) as well as axial t1-weighted sequences post contrast were acquired to determine the extent and type of the arteriovenous malformation. in addition dynamic contrastenhanced (0.2 mmol/kg gd-bopta, multihance, bracco, italy) 3d gradient echo sequences were collected. a board certificated radiologist as well as an angiologist analysed the mr data sets for lesion depiction, determination of extent and involvement of neighbouring structures as well as characterisation of the malformation. results were compared to findings from duplex us, conventional venography and intraarterial arteriography. results: all mr examination were of diagnostic quality. a total of 16 arteriovenous malformations (venous malformation: n = 11, arteriovenous fistulae (avf): n = 2, posttraumatic avf: n = 3) were depicted. the stir sequence was helpful in the determination of the extent of the vascular malformation which was often underestimated by 3d mra alone, whereas dynamic 3d mra was mandatory for the classification of the lesion. these findings correlated well with the combination of conventional venography (n = 11) and duplex-us (n = 16). conclusion: mri appears to be valuable in the assessment of angiodysplastic lesions of the extremities. the protocol should combine dynamic contrast-enhanced 3d gre-sequences with heavily t2-w (stir) sequences. material and methods: all procedures were done on a multislice-spiral-ct (siemens somatom plus 4vz) using a standardized ct-fluoroscopy technique. robot assisted punctures were executed using a remote-controlled robotic system (unitedrobotsystems, evolution1) with unrestricted flexibility of movement. interventions performed were either punctures of contrast-media filled capsules, placed in a soft-tissue-phantom, or interventions within the spinal column of human bodies, such as vertebroplasty or discography. evaluation parameters were accuracy and application of the system, access to the patient and procedure time for the intervention. results: using robotic assistance high accuracy was achieved for all interventions. each lesion within the soft tissue phantom was punctured precisely (20/20). interventions on the spine (10) and the intervertebral discs (8) were performed without significant deviation from the approach determined initially. though application of the system was simple, free access to the patient was often aggravated by the scanner and robotic design. a marked learning curve was observed, thus leading to a significant reduction in intervention time in the course of all procedures (40 %) conclusion: a robotic system may be useful for ct-interventions in the spine, significantly reducing the radiation dose to the radiologist. as examination time is still markedly increased and patient access remains difficult, the system configuration has to be further optimized for the clinical setting. purpose: percutaneous vertebroplasty is used to strengthen the pathologic vertebral body and reduce pain in some diseases involving the spine. the purpose of this study was to evaluate segment stability and clinical status in patients who received vertebroplasty after radiofrequency ablation of spinal tumors. methods and materials: 23 vertebroplasties with polymethylmethacrylate were performed in 14 patients who had been treated with radiofrequency ablation of inter-or paravertebral tumors in the thoracic and/or lumbar spine. vertebroplasty was indicated due to presence or imminent tumor related vertebral fractures and presence of imminent neurological deficits. operations were carried out under ct/ fluoroscopy guidance under local anesthesia and on an out-patient basis. at follow-up, preoperative mr images were compared with follow-up mr-images in addition to clinical examination. follow up ranged from 3 to 12 months. results: at follow-up, mr images of treated vertebral bodies showed no signs of sintering or refracture. clinical examination showed improved mobility of the spine in 13 patients (92.9 %). in none of these patients was the presence or progression of neurological deficits diagnosed. 1 patient (7.1 %) was restricted in motion due to a new tumor in another segment. no intra-or postoperative complications were reported. our results indicate that image-guided percutaneous vertebroplasty seems to be a safe and effective, minimally invasive method to stabilize pathologic vertebral bodies in patients who have received radiofrequency ablation of spinal tumors. purpose: frequent complications of osteoporosis are fractures, especially in the vertebral bodies of the thoracic and lumbar spine. the purpose of this study was to ask whether percutaneous ct-/fluoroscopy-guided vertebroplasty can cause pain reduction in patients with fractured osteoporotic vertebral bodies. methods and materials: 25 verebroplasties were performed on 17 patients with osteoporotic vertebral body fractures. patients suffered from superior or inferior endplate fractures, combined end plate fractures without loss of height and combined end plate fractures with loss of height. inclusion criteria were new osteoporotic vertebral body fractures with acute pain as well as older fractures with persistent pain. exclusion criteria were fractures causing a narrowing of the spinal canal and neurological deficits, tumors, cement allergy and haemorrhagic diathesis. the procedure was carried out on an outpatient basis under local anesthesia and was performed under combined ct/fluoroscopy-guidance. follow up examinations were carried out after an average of 2 days and after 3 months. pain was assessed with the help of a visual analogue scale. results: at the time of the first follow up, patients reported on an average relative pain reduction of 29.9 %. after 3 months the patients obtained an average of 33.4 % relative pain reduction according to the visual analogue scale. conclusion: ct/fluorscopy-guided percutaneous vertebroplasty of osteoporotic vertebral body fractures is a gentle, effective and minimally invasive method to reduce symptomatic pain in the affected region. it can be carried out on an outpatient basis under local anesthesia, which results in direct mobilization and rehabilitation. steroid injection in sciatica due to lumbar disk herniation: technical aspects and clinical results (80 patients) d. krause, f. cognet, m. dranssart, d. ben salem, s. chapuy, j. lerais, j. cercueil, j. couaillier; dijon/fr purpose: to assess the feasibility and efficiency of foraminal or intracanalar steroid injections in cases of sciatica due to disk herniation. materials and methods: between january 1999 and january 2001, 80 patients presenting with typical sciatica, resisting usual medical treatment (four weeks), underwent a prospective protocol of pain treatment. a percutaneous approach was performed using ct guidance under local anesthesia. in foraminal lateral herniation (25 %), the 22 g needle tip was positioned through the soft tissues. in intracanalar herniations (75 %), the needle tip was advanced directly into contact with the posterior part of the root, through the ligamentum flavum (homo-lateral or contra-lateral approach in case of a narrowing lumbar tendency). in all cases the steroid injection (dexamethasone acetate 20 mg) was rigorously epidural. the procedure was possible in all cases, without any complications or side effects. the analysis of the results was based on a multi-factorial analysis: analogue visual scale for pain intensity measurement; family g.p questioning; professional activity recovery; stopping medical treatment. the mean follow-up was more than nine months. good or excellent clinical results were observed in 70 % of cases, poor results (without surgical intervention) in 20 % of cases and failures in 10 %. conclusion: this less aggressive and non expensive technique, performed under very precise ct guidance, is particularly adapted to ambulatory patients with encouraging results. effect of ct-guided lumbar facet joint infiltration on lower back pain r. knapp, a. glück, j. lukasser; kufstein/at purpose: ct-guided lumbar facet joint infiltration is a valuable tool in treating lower back pain. the duration and quality of pain relief is evaluated. methods and materials: 60 patients suffering from lower back pain caused by osteoarthritis of the facet joints underwent ct-guided lumbar facet joint infiltration with bupivacain and hydrocortisone. only one facet at one lumbar level was treated. evaluation of pain relief and change in medical treatment over a period of one year was performed with a questionaire. results: 58 patients (97 %) felt a complete relief of pain after a mean time interval of 5.2 days after the infiltration. after one year 19 patients (32 %) remained free of pain. 16 patients (27 %) improved markedly or reported rarely any pain, whereas in 25 patients (42 %) the pain recurred to the same extent as before the infiltration. the pain free interval after infiltration was a mean of 110 days. before infiltration 55 of the patients (92 %) took one or more different oral analgesic drugs. one year after infiltration 28 patients (51 %) still needed analgesics. in 25 patients (45 %) who took two or more different drugs before treatment this was reduced to 5 patients (9 %) after one year. no adverse effects of the infiltration were seen during the study interval. conclusion: ct-guided lumbar facet joint infiltration is a save and effective treatment for patients with lower back pain caused by osteoarthritis of the facet joints. purpose: offical recommendations for obtaining informed consent for operative or ir procedures are that the patient is consented by the operator. the purpose of the study was to identify the proportion of european interventional radiologists who conform to these guidelines. materials and method: a questionnaire was designed and consisted of thirteen questions on current working practice and opinions on informed consent. the questionnaire was distributed to 786 european interventional radiologists who were members of irish or british societies of interventional radiologists and members of cirse. the anonymous replies were then entered into a database and analysed. results: 254 (32.3 %) questionnaires were returned. institutions were classified as academic (56.7 %), 40.5 % non-academic (40.5 %) and private (40.5 %). 83.5 % of responders do not use specific consent forms and 61 % have patient information leaflets for procedures. although 72.8 % of radiologists consent patients for some procedures, junior medical staff (house officers or senior house officers) consent patients in 58 % of replies. over half (56.3 %) did not feel that their current policy was adequate. comments from responders indicated that there is insufficient time for radiologists to consent all patients. suggestions to improve current local policies included radiology outpatient clinics and radiology nurses visiting patients on the wards or in outpatient clinics. conclusion: only 24.8 % of european interventional radiologists follow current guidelines for obtaining informed consent. the causes identified were resource limitations and in particular constraints on time. optimizing material management in interventional radiology departments methods and materials: after compilation of a comprehensive stock list of all disposable materials necessary for interventional radiology an abc-analysis was performed with focus on high quality and high volume materials (a-articles). based on ordering frequency, all a-articles were subjected to xyz analysis, which is based on predicting the probability of requirement of the article. on this basis capital binding costs were considered with an annual interest rate of 8 % assumed. results: current inventory control reveals an annual capital binding for all articles of $ 250000 mainly due to a-articles, which accounted alone for $ 200000. if articles are bought in a just-in-time (jit) mode instead of mid term, stock costs could be reduced by $ 130000, thus storage time is shortened and capital binding is reduced and $ 10000 can be saved. with a stock turnover of the remaining material of 9 months, which matches our experience, jit-buying of articles can lead to an additional cost reduction of $ 5000. altogether $ 15000 could be saved, which means a relative cost reduction in stock of 6 %. besides this, there are lower costs for renting space and expiration dates of articles can better be controlled. late improvement of regional myocardial wall motion in the area of infarction after acute ptca in a 6-month follow-up study using delayed contrast enhanced magnetic resonance imaging: is "bright" really "dead"? purpose: acute recanalization in myocardial infarctions (mi) has shown to be of benefit with respect to global myocardial function and prognosis. the aim of our follow-up study was to investigate late effects of acute coronary angioplasty (ptca) on regional wall motion after the subacute phase of mi. patients and methods: 17 patients underwent mr imaging at 1.5 t after acute ptca in the subacute phase of mi and a second time 6 months later. corresponding short axis slices encompassing the left ventricle (lv) were acquired with standard cine and delayed contrast-enhanced magnetic resonance imaging (cemri). target parameters were the percentual size of infarction (psi (%)) and percentual wall thickening (pwt (%)) in the infarct area (ia) and the remote area (ra). results: psi was similar in the subacute phase and at 6 month's follow-up (22.2 and 21.9 %, respectively; n.s.). pwt improved significantly in ia (21.9 and 37.9 %, p < 0.05) in contrast to ra (46.4 and 38.4 %; n.s.), whereas global myocardial function did not change significantly. this late improvement was only observed in transmural mi. patients with subendocardial myocardial infarction did not show any further improvement. conclusion: pwt improved in ia after acute ptca even after the subacute stage of transmural mi in this 6-month follow-up study. this phenomenon could be explained by a prolonged stunning in transmural infarctions and contradicts the hypothesis that "bright" in delayed cemri represents nonviable tissue in all cases. contrast-enhanced (ce) mr can clearly depict definitive necrotic areas after myocardial infarction: the high resolution of mri permits discrimination of subendocardial versus transmural infarction. follow-up with cine mri allows documentation of functional recovery of both subgroups. methods: 30 patients (26 males, mean age 56 years) underwent emergency ptca ± stent for ami. mr controls were done on day 1, 3, 7 and 28 after ami using a 1.5 t unit (siemens, sonata) with the following protocol: (1) multiple truefisp (tr 3.2 ms, te 1.6 ms) along the short axis to assess lv volumes and wall motion abnormalities; (2) 3d inversion recovery sequence (tr to rr, te 1.64 ms) 10 -15 min after administration of gd-dtpa to visualise infarcted myocardium ("late enhancement"). results: 12 patients showed transmural infarction in late enhancement. all of these patients had wall motion abnormalities in the same segments which persisted over the observation period of 28 days. 21 patients had myocardial segments with non-transmural enhancement in late enhancement. improvement of the wall motion abnormality was shown in 11/21 patients. the transmural group showed a decrease of ef (0 -7 %) and the non-transmural group an increase (0 -4 %). conclusions: transmural infarction with late enhancement has a worse functional outcome in the involved myocardial segment and the entire ventricle than nontransmural infarction. longer observation periods than 28 days may be required for monitoring the outcome of non-transmural infarction. b a c d e f 174 combined first-pass and delayed contrast enhanced mri in the assessment of myocardial viability after acute infarction l. natale 1 , a. meduri 1, 2 , a. lombardo 1 , a. giordano 1 , r.m. razmi 2 , p. marano 1 ; 1 rome/it, 2 birmingham, al/us purpose: to define the role of mri in the assessment of myocardial viability we compared mri, rest 99 tc sestamibi spect and dobutamine echocardiography (de). methods and materials: 18 consecutive patients with first ami (63.9 a, 16 anterior, 2 inferior, 8 primary ptca, 4 thrombolysis) underwent mri, spect and de within the first week after onset of symptoms. mri was performed with a 1.5 t scanner (ge signa horizon echospeed); short axis single slice first pass (iv 10 ml gd-dtpa, 3 ml/s) and multi-slice delayed t1 imaging were performed. using a 16 segments lv model, both segmental analysis and percent infarct area were analyzed. segments were classified as: (1) normal first-pass, absent or delayed hyperenhancement; (2) hypoenhancement at first-pass, delayed hyperenhancement; (3) hypoenhancement both at first-pass and delayed imaging. in delayed images, segments out of first-pass slice were classified at delayed imaging as normal, hyperenhanced and hypoenhanced. patterns 2 and 3 and patterns hyper-and hypo-were considered non viable. results: segmental analysis results were poor, particularly if compared with de (sensitivity 70 %, specificity 78 %); infarct area analysis (at least 2 viable segments) was better (vs de and spect respectively: sensitivity 76 % and 72 %, specificity 83 % and 88 %). conclusions: patterns 1 and 3 respectively identify viable and non viable tissue. pattern 2 or delayed hyperenhancement is less specific as 25 % viability was demonstrated at de and/or spect. first pass plus delayed imaging are superior to delayed imaging alone (sensitivity and specificity with de and spect respectively 76 % vs 72 %, 84 % vs 78 %, p > 0.05), but multislice first pass is mandatory. comparison of magnevist™ and gadophrin-3 for the assessment of acute and chronic myocardial infarction j. barkhausen 1 , w. ebert 2 , c. heyer 2 , j.f. debatin 1 , h.j. weinmann 2 ; 1 essen/de, 2 berlin/de purpose: to investigate whether the area of hyperenhancement using extracellular contrast agents is larger compared to the region demarcated by a necrosisspecific contrast agent. methods and materials: 15 rabbits underwent thoracotomy and permanent occlusion of a branch of the left coronary artery. two animals died prior to imaging, 8 animals were examined 48 hours following occlusion and 5 animals were imaged six weeks following induction of the infarction. all animals received 50 mmol/kg of a necrosis-specific contrast agent (gadophrin-3, schering, berlin/germany) 24 hours prior to the mr examination. continuous short axis views were collected on a 1.5 t mr scanner using an ecg-triggered ir-turboflash sequence. imaging was repeated 10 minutes after additional injection of 100 mmol/kg of magnevist (schering, berlin/germany). the area of hyperenhancement demarcated following gadophrin-3 injection was compared with hyperenhancement seen on gadophrin-3 plus magnevist enhanced images and with ttc staining. results: in animals with acute myocardial infarction hyperenhancement was detected in 27 slices. the mean difference in the size of hyperenhancement seen on the two different in-vivo mr scans was −1.8 ± 6.0 mm 2 (p > 0.05). both measurements showed excellent agreement with ttc-staining. chronic infarctions showed no enhancement following gadophrin-3 injection, whereas application of magnevist resulted in hyperenhancement. conclusion: in acute myocardial infarction the area of hyperenhancement following gd-dtpa application does not exceed the area of hyperenhancement seen on gadophrin-3 enhanced images. the combination of gadophrin-3 and magnevist can distinguish acute and chronic infarction because chronic myocardial infarctions do not enhance with gadophrin-3. multi-slice first pass myocardial perfusion imaging: first results comparing saturation recovery (sr) truefisp 2d und sr turboflash 2d pulse sequences s. miller 1 , m. fenchel 1 , o. simonetti 2 , u. kramer 1 , u. helber 1 , n.i. stauder 1 , j. finn 2 , c.d. claussen 1 ; 1 tübingen/de, 2 chicago, il/us purpose: to compare two different pulse sequence techniques for multi-slice first pass myocardial perfusion imaging. methods: using 1.5 t (magnetom sonata, siemens, erlangen) 14 patients and 6 volunteers were examined using both sr truefisp 2d and sr turboflash 2d pulse sequences. t1-weighting was enhanced applying a 90° saturation recovery preparation pulse. sequence parameters were tr 2.4 ms, te 1.2 ms for truefisp 2d and tr 1.8 ms, te 0.8 ms for turboflash imaging with a 280 -300 mm field of view and 80 × 128 matrix. contrast injection was standardized to 5 ml (5 ml/s flow rate) resulting in a dose of 0.025 -0.035 mmol gd-dtpa/kg per injection. data evaluation included signal intensity (si) over time analysis and determination of baseline and peak signal and contrast to noise ratios (s/n, c/n). an automated post-processing software (argus, siemens) was used to derive perfusion parameters simax, slope, time to peak and area under curve. results: with diastolic ecg-triggering 2 -4 slices/rr-interval were acquired. values of s/n baseline were comparable (truefisp 5.2 ± 2.4, turboflash 4.5 ± 2.4, p > 0.3), but superior results of s/n simax (9.2 ± 5.4 vs. 6.1 ± 2.6, p < 0.006) and c/n (4.0 ± 2.1 vs. 1.6 ± 1.1, p < 0.0001) could be obtained with truefisp. additionally, analyzing individual myocardial segments, absolute and dynamic ranges of slope and area under curve were 2 -9 fold higher (slope 3.8 ± 2.2, area under curve 4.5 ± 2.2, p < 0.0001) using sr truefisp compared to sr turboflash imaging. conclusion: superior results for multi-slice first pass perfusion imaging can be obtained using sr truefisp 2d imaging compared to sr turboflash 2d. myocardial perfusion assessment with dynamic p792 enhanced mri compared to 99 tc-sestamibi-spect: phase ii european multicenter trial j. bremerich 1 , m. friedrich 2 , b. wintersperger 3 , f. brunotte 4 , j. piek 5 , n. al saadi 2 , t. schindler 1 , s. friedrich 2 , m.f. reiser 3 ; 1 basle/ch, 2 berlin/de, 3 munich/de, 4 dijon/fr, 5 amsterdam/nl purpose: to compare mri enhanced with the new rapid clearance blood pool agent (rcbpa) p792 with the gold standard 99 tc-sestamibi-spect for the assessment of myocardial perfusion. materials and methods: by september 2001 40 patients with subacute myocardial infarction were included in 5 european centers. dynamic first pass and delayed equilibrium phase mr images were acquired after injection of 0.0065 or 0.013 mmol/kg p792 (guerbet, france) at injection rates of 2 ml/s or 4 ml/s. mr was conducted at 1.5 t with phased array coils. mr images were acquired in the short axis during first pass of p792 with a dynamic saturation recovery turboflash sequence (magnetom vision/symphony; siemens, germany or cvi; general electrics, usa). subsequently equilibrium phase inversion recovery mri were acquired 5 and 10 min after contrast injection to assess late enhancement. ∆si and si-upslope were calculated. results were compared with 99 tc-sestamibi spect as gold standard. results: p792 was well tolerated by all patients. with 0.013 mmol/kg infarction was readily detected on dynamic first pass mr images as hypointense areas. with 0.0065 mmol/kg infarction was not visible in all patients on dynamic images, but upslope and delta si were attenuated. location and extent correlated well with 99 tc-sestamibi-spect. infarction was also visible on inversion recovery mr images. the results from this study exhibit the potential interest of a new gadolinium based intravascular contrast agent in assessing qualitatively and quantitatively hypoperfused myocardium on p792 enhanced mr images during first pass and equilibrium phase. this study was supported by guerbet, france. quantitative first-pass myocardial perfusion mri in patients with coronary artery disease after successful revascularization v. gramovitch, v.e. sinitsyn, m. gordin, o. stukalova, e. noeva, s.k. ternovoy, e. chazov; moscow/ru purpose: to determine the potential value of quantitative myocardial perfusion mri in the assessment of successful coronary intervention. method and materials: we studied 9 pts (9 men, age 49 ± 10 years, weight 80 ± 9.7 kg) with 1 -3 vessel cad and normal left ventricular (lv) function before and after (2 nd day to 1 month) coronary intervention. all pts underwent either ptca (7/9, stent 6/7) or cabg (2/9). mri was performed using a 1.0 t siemens magnetom scanner with snapshot-flash sequence. perfusion was assessed by injecting the contrast agent gd-dtpa-bma (omniscan, nycomed amersham) via the antecubital vein before (1 st bolus 8 ml) and after dipyridamole (0.56 mg/kg, 2 nd gd bolus 12 ml). myocardial and blood si were converted to concentration of gd according to the in vitro calibration curve and fitted by one-compartment model by saturday b a c d e f 175 the use of a custom written program. eleven normal and 11 segments supplied by stenotic coronary arteries (³ 50 % diameter stenosis) with successful intervention afterwards (residual stenosis < 50 %) were included in the final analysis. results: myocardial blood flow was similar in normal and "stenotic" segments at base line (1.21 ± 0.49 and 1.28 ± 0.37 ml/min/g of tissue) but significantly lower in "stenotic" segments during hyperemia (1.44 ± 0.57 vs. 2.52 ± 0.94 ml/min/g, p < 0.05). coronary vasodilator reserve (the ratio of flow during hyperemia to flow at base line) was significantly lower in "stenotic" segments than in normal (1.2 ± 0.36 vs. 2.1 ± 0.75, p < 0.05) and normalized completely after successful revascularization (3.1 ± 1.45). conclusion: quantitative mr perfusion assessment may be useful for the follow-up of patients with cad after coronary intervention. myocardial perfusion in the human heart: quantitative assessment using a spin-labeling technique at 2 t f. fidler, c.m. wacker, p.m. jakob, w.r. bauer, a. haase; würzburg/de purpose: the aim of this study was to determine myocardial perfusion of the human heart using a spin-labeling technique without exogenous contrast agent. method and materials: an ecg-gated fast saturation recovery flash sequence was implemented using a 2 t scanner (bruker) with a homebuilt quadrature surface coil for signal reception. t1 measurements were performed after global and slice-selective saturation under resting conditions and adenosine-induced stress. exams were obtained breathing room air (air) and pure oxygen (oxy). 10 ecgtriggered enddiastolic midventricular short axis views were acquired in a single breathhold of about 15 s in 9 healthy volunteers. results: myocardial perfusion was calculated as p = 2.5 ± 0.7 ml/g/min (rest, air), 4.8 ± 1.1 ml/g/min (stress, air) and 1.6 ± 0.6 ml/g/min (rest, oxy). t1 of lv (rv) blood was 1700 ± 100 ms (1600 ± 120 ms) (air) and 1440 ± 45 ms (1560 ± 150 ms) (oxy). the presented spin-labeling method is a robust alternative for quantitative perfusion evaluation and allows repeated measurements without contrast agents. oxygen induced changes of t1 (myocardium and blood) are detectable and may therefore become important in patients with coronary artery disease. purpose: in small larynx tumors (t1, t2) the presence of spread is crucial in determining adequate surgical or laser-surgical treatment. therefore, the purpose of the study was to determine sensitivity and specificity of preoperative multi-slice helical ct (msct) for the infiltration of various glottic laryngeal structures. material/methods: 30 patients with suspected laryngeal cancer were investigated on a msct scanner (siemens plus 4 volume zoom) with 4 × 1 mm collimation, 0.5 seconds rotation time in a single breathhold. multiplanar reconstructions (mpr) in sagittal and coronal planes were reconstructed in all patients and rated in consensus. all findings regarding tumor spread into the glottic fat, crossing the anterior commissure, infiltration of the arytenoids and the extension along the vocal cords were compared to surgery and histology. the sensitivity and specificity of msct regarding the infiltration of the glottic region were 93 %, 85 %. regarding infiltration of the anterior commissure sensitivity and specificity were 85 %, 94 %, respectively; regarding the infiltration of the anterior part of the vocal cords 89 % and 92 %, and regarding the infiltration of the arytenoids 83 % and 96 %, respectively. sagittal mprs were especially helpful regarding the surfaces of the arytenoids and the anterior commissure, and coronal mprs were most helpful in the evaluation of the vocal cords. the detection of spread of supraglottic laryngeal tumors is crucial in determining best treatment, as the preoperative assessment decides to what extend surgical therapy can be offered. thus, the purpose of the study was to determine sensitivity and specificity of multislice helical ct (msct) for the identification of infiltration of supraglottic laryngeal structures. material/methods: investigations on a msct scanner were performed in 30 patients with suspected supraglottic laryngeal cancer, with two entire acquisitions; one in breathhold and one in phonation. multiplanar reconstruction's (mpr 3 mm thickness) in sagittal and coronal plane were reconstructed in all patients. the findings were separately evaluated regarding tumor spread into the pre-epiglottic fat, the anterior commissure, the laryngeal and the superficial side of the epiglottis and the piriform sinus, and were compared to surgery and histology. results: regarding infiltration of the piriform sinus sensitivity and specificity were 86 % and 96 %, regarding the infiltration of the laryngeal side and the superficial side of the epiglottis 100 % and 93 %, and regarding the anterior commissure 85 % and 94 %, respectively. the cumulative sensitivity and specificity of msct regarding the infiltration of the entire supraglottic region were 89 % and 95 %, respectively. while the mprs were felt to be of general benefit, sagittal mprs were especially helpful regarding both surfaces of the epiglottis, and coronal mprs were mot helpful in the evaluation of the piriforme sinuses. the extend of supraglottic laryngeal tumor spread can be depicted with high accuracy using msct. referred pain in oropharynx carcinoma: a clinical symptom finds its anatomical correlate in mri h.c. thoeny, k.t. beer, r.h. greiner, p. vock; berne/ch purpose: is there a correlation between mri and the prognostic symptom of referred pain to the ear in patients with oropharynx carcinoma? method/materials: irritation in the region innervated by the glossopharyngeal nerve can be transmitted as pain to the ear due to connections between the glossopharyngeal nerve and the tympanic nerve. in a prospective study 36 consecutive patients, median age 59 (range, 41 -76 years) with oropharynx carcinoma underwent mri before radical radiotherapy. 21 patients had referred pain to the ear, 15 did not. the protocol included axial pd/t2-weighted tse sequences, axial, coronal, and sagittal t1-weighted se sequences before and/or after contrast medium administration. examinations were performed on two 1.5 t mris (magnetom vision, siemens, or signa, ge). two independent, blinded radiologists analyzed the mri studies for alterations of anatomical structures (effacement, signal alteration, or contrast medium enhancement) of the oropharynx and adjacent regions. a twosided 2 test was used to compare variables. results: patients with referred pain to the ear showed significantly more alterations of the following structures innervated by the glossopharyngeal nerve: palatoglossus (p < 0.002), stylo-(p < 0.006) and palatopharyngeus (p < 0.004), and constrictor pharyngis (p < 0.04) muscles; hard palate (p < 0.005), tonsil (p < 0.002), pre-epiglottic space (p < 0.03), and posterior soft palate (p < 0.003). no difference could be observed for structures innervated by different cranial nerves. conclusions: alterations of structures innervated by the glosso-pharyngeal nerve showed a significant association with referred pain to the ear in patients with oropharynx carcinoma. the symptom of reflex-otalgia has, therefore, an anatomical correlate in mri. virtual laryngoscopy with multislice ct enables grading of upper airway stenosis h. hoppe, h.c. thöny, h.-p. dinkel, p. zbären, p. vock; berne/ch purpose: to compare the efficiency of noninvasive virtual laryngoscopy in depicting and grading upper airway stenosis with multislice ct versus fiberoptic laryngoscopy. methods and materials: multislice ct and fiberoptic laryngoscopy were used to examine 116 upper airway sections (supraglottis, glottis, subglottis, trachea) from 29 patients (n = 19 malignant upper airway pathology, n = 5 benign upper airway pathology, n = 5 control group without upper airway pathology). ct data were obtained on a toshiba asteion multislice ct in 4 × 1 mm collimation, pitch 5.5/4 and 1 mm reconstruction interval. postprocessing was performed using surface rena c d e f 176 dering and multiplanar reformats (mpr). ct images were independently interpreted by two radiologists. fiberoptic laryngoscopy was the gold standard of reference. results: virtual laryngoscopy and mpr accurately demonstrated upper airway stenosis caused by intraluminal tumor growth. there was a close correlation (r = 0.94) between virtual laryngoscopic and fiberoptic laryngoscopic grading of stenosis. virtual laryngoscopy was limited in the differentiation of mucus and appositional tissue folds from tumor, which was more reliable with mpr and axial ct slices. furthermore, subtle mucosal irregularity could not be defined. conclusion: virtual laryngosopy with multislice ct enables high-resolution endoluminal imaging of the upper airways with a short scan time despite thin collimation, but should be combined with axial ct-slices and mpr readings. virtual laryngoscopy was especially useful for evaluation of subglottic and tracheal stenosis, which may be difficult to assess with fiberoptic laryngoscopy. it is a reliable non-invasive method for the assessment of upper airway stenosis as a complement for fiberoptic laryngoscopy. purpose: to evaluate the potential of mr-guided laser-induced thermotherapy using a high power irrigated laser application system for the treatment of recurrent head and neck tumors. materials and methods: 27 recurrent tumors in the head and neck tumors (recurrent squamous cell carcinoma n = 24, recurrent pleomorphic adenoma n = 3) were treated using mr-guided laser-induced thermotherapy and a newly developed irrigated power application system which allows power settings 6 times higher than the conventional system. a total of 53 laser applications were performed using 37 laser applicators. mr thermometry was performed using a temperature sensitive t1-weighted gradient echo sequence for monitoring thermal induced changes in signal intensity. follow up studies were performed using plain and contrast enhanced mr. results: all procedures could be performed under local anesthesia. laser applicators with an active length of 2 or 3 cm were used with 24 or 36 w respectively (12 w/cm active length of the laser applicator). the application time was between 4 and 15 minutes. all lesions showed a very rapid heat distribution. we were able to induce coagulative necrosis in all patients. clinical relevant improvement of clinical symptoms was observed in 24 lesions. the treatment of 3 lesions resulted in no improvement of clinical symptoms. conclusion: mr-guided litt using a high power irrigated laser application system proved to be an reliable and effective method to treat recurrent tumors in the head and neck region. mr-thermometry allowed monitoring of laser induced temperature changes during litt. fat suppressed t1 sequences in the evaluation of "t" factor in carcinoma of the tongue s. cappabianca, a. barberi, l. pasqualetto, g. colella, r. grassi; naples/it purpose: comparison between se t1, dp and t2 sequences and t1 fat-suppression sequences in staging of lingual carcinoma methods: 47 patients with lingual carcinoma underwent mri exam using a 1 t superconducting scanner. in all cases se sequences dp-t2w were obtained; t1w and t1w with selective fat suppression before and after intravenous administration of gd-dtpa were also acquired. two different groups of radiologists evaluated independently the dp-t2 and t1 sequences, and the dp-t2 and t1-fs sequences, in order to define the extension of the neoplasm ("t" factor). mri findings were compared with pathological staining. results: dp-t2 and t1: tumour not detectable n = 3; t1 n = 4, t2 n = 6, t3 n = 20, t4 n = 12. dp-t2, t1 and t1-fs: tumour not detectable n = 2; t1 n = 5, t2 n = 6, t3 n = 23, t4 n = 9. surgery/pathology: tumour not detectable n = 0; t1 n = 8, t2 n = 7, t3 n = 20, t4 n = 10. dp-t2w and t1w sequences diagnostic accuracy 93.3 %; dp-t2w, t1w t1-fs sequences diagnostic accuracy 95.5 %. conclusion: our data suggests that in the local staging of lingual carcinoma the use of se-t1 sequences with selective fat suppression can improve diagnostic accuracy of mri in evaluation of tumour volume. withdrawn by author ultrasonographic detection of iatrogenic accessory nerve palsy p. kovacs, g. bodner, a. gardetto, h. piza-katzer, w.r. jaschke; innsbruck/at purpose: to report the ultrasonographic findings of 4 cases of iatrogenic accessory nerve palsy after lymph node biopsy and neck dissection and to demonstrate feasibility of ultrasonography (us) in detecting the accessory nerve in three cadaveric specimens. methods and materials: 4 patients (range 42 -65 years) presented with neck and shoulder pain after surgical intervention in the neck region (2 after lymph node biopsy, 2 after neck dissection). us was performed with a linear broad-band transducer (5 -12 mhz) working on a hdi 5000 (atl). in 3 fresh cadavers the accessory nerve was detected by means of us and marked with blue ink. following, the accessory nerve was exposed carefully. results: in the right lateral cervical region of two patients, who underwent lymph node biopsy, we found a hypoechoic mass, in which a tubular structure ended. we suspected a cut accessory nerve. in two patients with unilateral neck dissection us revealed a tubular structure ending in a hypoechoic scar measuring 3 cm in length. in all cases the trapezius muscle showed hyperechoic texture, suggestive of muscular atrophy. us findings were confirmed by electroneurographic testing and surgical nerve inspection. on us the accessory nerve appears a small tubular structure (diameter 0.7 mm) lying just under the superficial layer of cervical fascia. in all specimens the nerve was correctly marked by means of us showing blue stained accessory nerves after dissection. conclusions: the capability of high resolution us allows detection of the normal accessory nerve and of iatrogenic accessory nerve palsy. saturday materials and methods: to date, 5 small (< 3 cm) and 5 large (> 3 cm) solid renal tumours and 2 cystic complex renal masses (< 3 cm) were evaluated by ultrasound (us), color doppler (cd) and pihi. pihi was performed after levovist injection by high mi (1.0 -1.2) intermittent manual triggered stimulation every 10 -15 seconds, during vascular (20 -60 seconds) and late phase (60 -120 seconds). helical ct pattern and/or histologic findings were considered as the reference procedures. results: reference procedures classified 3 small solid tumours and 5 large solid tumours as renal adenocarcinomas, 2 small solid tumours as angyomiolipomas and 2 cystic complex renal masses as cystic papillary tumours. small solid renal adenocarcinomas were hypoechoic (n = 2) or hyperechoic (n = 1) on us with a basket arterial pattern on cd. large solid adenocarcinomas appeared inhomogeneous with no vascular pattern, while angiomyolipomas appeared hyperechoic with peripheral venous flows. cystic complex renal masses revealed thick septa and a peripheral solid portions on us with a basket arterial pattern on cd. on pihi small solid renal adenocarcinomas appeared hyperechoic on vascular phase and isoechoic (n = 2) or hypoechoic (n = 1) on late phase. large renal adenocarcinomas revealed increased conspicutiy of necrotic intratumoural zones and inhomogeneous enhancement. renal angiomyolipomas revealed dot-like enhancement. cystic renal adenocarcinomas revealed peripheral enhancement limited on septa or solid portion. during a 6-month period, variable renal masses including renal cell carcinoma (n = 10), transitional cell carcinoma (n = 2), acute pyelonephritis (n = 3), angiomyolipoma (n = 1), and traumatic renal contusion (n = 1) were evaluated with cha us (logiq 700 expert series; ge medical systems) using microbubble contrast agent. us images were obtained before contrast administration and with a bolus injection of 4 g of microbubble contrast agent (300 mg/ml of levovist; schering) in every 10 -15 s for 5 min. the contrast enhancement patterns of variable renal masses were assessed. result: 10 renal cell carcinomas showed more enhancement than adjacent renal parenchyma was seen between 16 to 57 s (mean, 30 s) and 51 to 252 s (mean, 82 s) after injection. the duration of enhancement was 13 to 208 s (mean, 80 s). one angiomyolipoma showed heterogeneous enhancement from 27 s to 84 s. all transitional cell carcinomas showed no definite enhancement. three acute pyelonephritis and one traumatic renal contusion showed focal perfusion defects that were not apparent on pre-contrast scan. conclusion: cha us with microbubble contrast agent is an effective us technique in the evaluation of both tumour vascularity and renal perfusion abnormality. withdrawn by author (5). all patients were followed-up clinically, biochemically and by ct both early (< 7 days) and at 6 monthly intervals. results: one patient demonstrated a minor internal injury to the psoas muscle. no significant rise in creatinine was noted post-procedurally in 7/8 patients (mean rise: 3.6 µmol/l; range −8.1 to 19.8 µmol/l). one patient with von hippel-lindau syndrome had 4 tumours treated in a single kidney. there has been a gradual post-treatment rise in creatinine, from 130 to 233 µmol/l. early post-procedural ct demonstrated complete tumour necrosis in 9/11 tumours. two tumours (3.0 and 5.5 cm) required additional ct-guided treatments of residual crescents of viable tumour. follow-up (mean 7.4 months, 82 patient months, range 1 -17 months) revealed no evidence of local or distant recurrence. conclusions: early experience suggests rfa is a safe, well-tolerated, and minimally invasive therapy for renal cell carcinoma. in the era of nephron-sparing surgery rfa may have a role in the management of small problematic rcc. drug-induced mr-pyelography in the evaluation of renal collecting system malformations, tumours, renal calculi and obstructed renal ureters m. di girolamo 1 , a. grossi 1 , a. roncacci 1 , r. di nardo 1 , l. azzarri 2 , v. david 1 ; 1 rome/it, 2 grottaferrata/it purpose: drug-induced mr-pyelography (dimrp) is a diagnostic technique for the evaluation of obstructed and non-obstructed renal collecting systems. a c d e f 178 method and materials: 10 normal volunteers and 135 patients underwent dimrp. the examination was performed with a 3-d non-breath-holding fat-suppressed turbose sequence (tr: 3000 ms; te: 700 ms; n.ex.: 6; etl: 128; acq.time: 9 min) on coronal planes. these acquisitions were post-processed with a mip algorithm. to obtain maximum filling of the collecting system, the diuresis was pharmacologically induced by administering i.v. 250 ml of saline solution together with 20 mg i.v. of furosemide. one mr acquisition was performed 10 minutes after diuresis induction. 5 normal volunteers and 115 patients had undergone ivu and ascending pyelography was performed in 15 cases. results: we always obtained an excellent anatomical evaluation of the renal collecting system that was considered comparable to that obtained with ivu. it was always possible to study the renal collecting system malformations, even in patients with known contraindication for i.v. administration of contrast media. renal calculi larger than 2 mm were always identified by the analysis of 3d coronal scans. dimrp is particularly important for the detection of ureteral stones. 24 patients had non-functioning kidneys on ivu: in 23 cases with obstructive uropathy the site of the obstruction was determined and using conventional mri, abdominal plain radiograph and urinary cytology, the cause was always determined. conclusions: dimrp is recommended for patients with contraindications for i.v. administration of contrast agents; in non-functioning kidneys is considered the best diagnostic imaging modality to perform after us, especially in case of ureteral obstruction. filling defect artefacts in magnetic resonance urography g. girish, w.k. chooi, s.k. morcos; sheffield/gb purpose: (1) assessing the prevalence of filling defect artefacts (fda) in magnetic resonance urography (mru). (2) presenting the characteristic features of fda, that can differentiate them from true filling defects (tfd). method/materials: mru's of 45 patients with neurogenic bladder dysfunction were reviewed to assess the prevalence of filling defects within the pelvicalycyal system (pcs) and ureter. heavily t2 weighted fast spin echo techniques with fat saturation were used. these included axial images 5 mm (thick)/2 mm (gap), slab images (75 mm thick) of retroperitoneum and volume coronal imaging of kidneys and retroperitoneum with 3d and maximum intensity projections. dilatation of pelvicalyceal system as well as features of filling defects (central, eccentric, complete) were graded. clinical course and plain films were reviewed to determine significance of filling defects. results: filling defects were present in 27 pts (60 %). prevalence of filling defect artefacts amounts to 22/45 (49 %) and that of true filling defects was 11 %. the following are the characteristic features of fda that differentiate them from true filling defects: (1) vast majority of fda occurred in axial t2 weighted images and very rarely in slab and maximum intensity projection images. (2) fda's were usually small, centrally placed (94 %) and noted mostly in pelvicalyceal system (41.8 %) and the upper third of the ureter (39.5 %). (3) true filling defects were bigger in size and seen in two or more image sequences. conclusions: (1) awareness of these artefacts will avoid misinterpretation and prevent further unnecessary investigations or interventions. (2) when in doubt, correlation is suggested with other mri sequences, plain abdomen radiograph and clinical presentation. purpose: differentiation of healthy kidneys from those with haemodynamically significant stenosis or those with renoparenchymal disease. methods and materials: in 79 kidneys with suspected renal artery stenosis (confirmed stenosis n = 38, no stenosis found n = 41) or parenchymal disease (n = 30) renal flow and perfusion measurements as well as mr angiography were performed. flow measurements were done using an ecg gated cine phase flash sequence, perfusion was measured using an arterial spin labeling fair sequence. contrast enhanced mr angiography was done with a fast 3d gre sequence in a single breath hold. data was compared to that from 31 healthy volunteer kidneys and correlated to serum creatinin levels. results: substantial differences in mean renal artery flow and perfusion were found in kidneys with renal artery stenosis or parenchymal disease compared to healthy kidneys. in addition perfusion measurements showed significant agreement to 99 tc renal scintigraphy. using the discriminant analysis we were able to achieve a specificity of 79 % and a sensitivity of 90 % in terms of separating healthy kidneys from those with either vascular, parenchymal or combined disease. on the contrary, renal volume measurements did not show any significant differences. the combination of mr angiography, flow measurements and mr perfusion measurements offers a comprehensive way for assessment of both renovascular and renoparenchymal disease. it offers a non invasive way to separate normal individuals from either those with haemodynamically significant stenosis or those with underlying renoparenchymal damage. functional imaging of the kidneys using pure o2 inhalation r.a. jones, n. grenier, m. ries, c.t.w. moonen; bordeaux/fr purpose: because of its paramagnetic effects, pure o2 reduces the t1 of lungs. our purpose was to investigate the intra-renal signal intensity changes induced by the dissolved molecular oxygen in blood. materials and methods: nine volunteers were imaged while breathing normal atmosphere and pure o2 alternatively with an inversion prepared, segmented, half fourier tse sequence for t1 measurement and a multiple echo gradient echo sequence for t2* measurement. roi were positioned on the cortex, the medulla, the liver and the spleen. subtraction and cross-correlation images were also obtained after dynamic t1w acquisitions during three successive phases (air-o2air) results: the only significant intrarenal change was the reduction of t1 within cortex while o2 breathing (882 ± 59 ms vs 829 ± 70 ms, p = 0.0001) and a reduction of t1 within the spleen. neither significant change of t1 within the medulla and the liver nor significant change of t2*, in all organs, were observed. the effect was not related to flow because no change was noted between selective and nonselective inversion pulses. the observed t1 change induced by pure o2 within the cortex is related to the high arterial blood volume without o2 consumption. the absence of change within the medulla is probably related to the high o2 consumption and shunting effect. is to distinguish it from other entities for prognostic and management purposes. it is the unusual cases of lch that often create a particular diagnostic dilemma. however, careful analysis of the radiographs including the site of involvement and behavior, coupled with a detailed demographic and clinical history, can be suggestive of the diagnosis. methods and materials: 542 consultation cases, including 730 skeletal sites, submitted to the armed forces institute of pathology, were analyzed for unusual manifestations of lch. five unusual manifestations were evaluated including cortical involvement, disease at birth, presence of fluid levels, circumferential "pencilling", and involvement of the mandibular inferior cortex. results: unusual demographics included 46 non-caucasian cases. unusual sites of involvement were the epiphysis (2), apophysis (1), cervical spine (13), posterior elements of the spine (8), soft tissues (2), cortical involvement (6), and atypical locations (sternum, middle and distal phalanx). unusual manifestations included crossing the physis (1), crossing calvarial sutures (6), fluid levels (2), erosion of the inferior mandibular cortex (1), transforming from sclerotic to lytic (2), brain invasion (1), circumferential "pencilling" (5), sequestrum in noncalvarial sites (2), unusual size of a lesion (1), and bone expansion (4). these unusual appearances may mimic other diseases including paget disease, fibrous dysplasia, ewing sarcoma, coxa vara, and metaphysitis. conclusion: recognition of the spectrum of unusual manifestations of lch is helpful in allowing distinction from other differential diagnoses. the imaging of 6 patients (4 female, 2 male; age range 13 -55 a, mean age 24.8 a) with biopsy-proved subperiosteal aneurysmal bone cyst was reviewed. radiologic studies inculuded radiographs (n = 6), ct (n = 2), and mr images (n = 6). evaluation included patient demographics, lesion location and size, radiographic features, and intrinsic characteristics on ct and mr images. review of histologic specimens was carried out by an experienced musculoskeletal pathologist. results: all lesions were located at the surface of long tubular bones (femur 3, tibia 2, humerus 1); three involved the diaphysis, two the dia-/metaphysis, and one exclusively the metaphysis. lesion size ranged from 2.5 to 6 cm in maximum diameter. radiographs and ct images always showed a superficial bone defect, which on radiographs demonstrated irregular margins in four cases. all lesions caused an interrupted periosteal reaction (shell 3, trabeculated shell 1, codman angle 2). mr images always showed a multicystic appearance with hypointense rim, contrast-enhancing cyst walls, and fluid levels. edema of adjacent soft tissues was present in all cases. conclusion: aneurysmal bone cysts in a subperiosteal location can demonstrate an aggressive radiographic appearance. mr imaging appears to be most valuable in differential diagnosis, since it can demonstrate typical morphological features of the underlying process. rib sonography: can we obtain more information than with conventional radiologic studies? s.h. paik, m. chung, y. yoon, j.-g. im, j. ahn; seoul/kr purpose: we performed this study to evaluate whether high-resolution ultrasonography can give more information concerning rib lesions than plain radiography or bone scintigraphy. method: we selected 20 patients with high uptake rib lesion on bone scintigraphy. plain radiography and rib ultrasonography were performed on these patients. ultrasonography was performed using linear 12.5 mhz transducer. we analyzed cortical disruption, mass, callus formation, hematoma, deformity and bone destruction by rib ultrasonography. we considered radiologic findings and clinical information in the diagnosis. purpose: innovative therapeutic protocols of musculoskeletal tumors include neoadjuvant and antiangiogenic approaches. functional tumor imaging including dynamic contrast enhanced (dce) mri and fdg-pet seem to be capable techniques to evaluate ongoing response during therapy. aim of this study is to assess the clinical utility of dce-mri in such cases. materials and methods: within a therapeutic phase i protocol, dce-mri was included as a surrogate marker to assess the tumor prior and during therapy. after neoadjuvant treatment, surgical resection was performed with histological correlation as well as detailed micro-array analysis. mri was performed on a 1.5 t clinical system using optimized fast 3d gradient echo sequences. standard gd-chelates were given at 0.1 mmol/kg bw and the studies post processed using a pc based environment facilitating quantification and visualization. results: dce-mri could be performed and quantified in all cases. the enhancement patterns observed revealed a wide variability with areas of high uptake and rapid washout correlating to active tumor areas and regions of contrast agent trapping seen in less vascularized, necrotic areas. areas with decreased enhancement correlate with response to therapy. in more than 1/3 of the cases, active tumor areas were only detected by functional imaging, not seen otherwise and confirmed histologically. conclusion: functional imaging is an essential tool to assess ongoing therapy in musculoskeletal tumors, it is robust and can be used as surrogate marker in therapeutic trials. whole body mr imaging with a rolling we assessed a single whole-body mri examination for detection and staging of metastases and compared the results with findings of nuclear bone scintigraphy and ct examination. materials and methods: 37 patients with known primary malignancies were included in this study. patients were examined on a mr system (siemens sonata) equipped with a rolling table platform (bodysurf) allowing the swift movement of the patient through the magnet bore as well as through a phased array surface coil. t1w 3d vibe data sets were collected in five stations following the intravenous application of paramagnetic contrast covering the body from skull to the knees. the chest and abdomen were imaged with t2 haste and t1 flash before contrast. mean examination time amounted to 15 (± 3) minutes. mri findings were compared to results obtained with skeletal bone scintigraphy and ct-scans of the abdomen and chest. results: all pulmonary and hepatic metastases > 6 mm detected by ct were identified by whole-body mri. skeletal scintigraphy detected bone metastases in nineteen patients. in eighteen patients mri confirmed these lesions. in one patients presenting with bone metastases in the ribs mri failed to reveal the osseous lesions, whereas in four other patients mri detected bone metastases missed by scintigraphy and eventually confirmed by biopsy. conclusion: vibe whole-body mri screening for metastases correlated well with ct and scintigraphy. use of the rolling table platform provides the basis for a comprehensive whole-body exam in merely 15 minutes. early therapeutic evaluation of bone metastases from breast cancer: histopathology, mr imaging and ct i. ciray, g.k.o. åström, h. lindman, h. nordgren, j. bergh, h.k. ahlström; uppsala/se purpose: evaluation of early therapeutic changes in breast cancer bone metastases using various parameters of mri, ct and histopathology. materials and methods: 20 metastatic bone lesions in 17 breast cancer patients were examined with mri and ct, and were subsequently biopsied under ct-guidance before initiation of chemotherapy and 6 courses later (median). the changes in size and signal intensity (si) on t1-weighted and long te inversion-recovery turbo-spin-echo (long te ir-tse) mr sequences and in density on ct were measured. the histopathological changes in the amount of tumor and fat cells, in the density of fibrosis and in the trabecular bone were evaluated. results: the amount of tumor cells was decreased in 16 (responding) and was increased in 4 lesions (progressive). si could not be measured in one responding lesion. there was no change in the tumor size and si on t1-weighted sequence in 8 of 15 responding lesions. there was an increase in si on long te ir-tse sequence of 11 of 15 responding lesions. two progressive lesions remained unchanged, one increased in size and the other in si on t1-weighted sequence with no alteration on long te ir-tse sequence. density increased in 12 of 16 responding lesions and in 3 of the 4 progressive lesions on ct images. conclusion: an increase in si in metastatic lesions on long te ir-tse sequence may indicate an early therapeutic response while a t1-weighted sequence is of limited value. an increased density on ct can be measured in both responding and progressive lesions. belgian soft tissue neoplasm registry (bstnr) j.l. gielen, p.m. parizel, a.m.a. de schepper; antwerp/be introduction and objectives: based on epidemiological data from the usa, the total number of new cases of malignant soft tissue tumors in belgium is estimated at 200 per year. this low incidence and the overmedicalization with numerous university hospitals and large general hospitals with mr-equipment available limits the number of cases seen in each hospital and per year and hampers the gaining of expertise in this field. to cope with these disadvantages, the department of radiology of the university hospital of antwerp in cooperation with all mr centers (47) in belgium created a belgian soft tissue neoplasm registry ("bstnr"), starting the registration on january 1, 2001. material and methods: internationally accepted standard procedures are used in the development of the electronic database in order to guarantee future and systematic availability of the data. a clinical and statistical indexing of biomedical imaging and image-related information based on the attributes of image acquisition procedures and on the diagnostic image obeservations is applied. in this way, an electronic image data selection by image related, acquisition related as well as disease related criteria will be possible. results: until now 330 cases of soft tissue tumor sent by the cooperating centers have been registered. the bstnr achieves 3 objectives: (1) to provide the referring radiologist with a second opinion about diagnosis and differential diagnosis within 48 hours; (2) to create a digital archive at the disposal of national or foreign researchers; (3) to obtain epidemiological information about the prevalence of stt in belgium. the vascularity in malignant lesions was significantly greater than in benign (benign: 0.96 m%cda, malignant: 5.88 m%cda, p < 0.001). vascular heterogeneity (sd%cda) showed similar results, albeit less significant (p < 0.01). there was a monotonic linear relationship between m%cda and sd%cda with increasing tumor grade. best separation was achieved between benign, intermediate and high grade (p < 0.001). there was a clear correlation between increasing tumor grade and higher vascularity (m%cda). separation between benign and low grade was possible, but less significant (p < 0.08). this correlates to histopathology, where vascular markers are low -similar to benign -for this group. conclusions: qpd shows the potential to differentiate benign from malignant softtissue tumors. it can measure increasing vascularity and vascular heterogeneity and reliably predict higher-grade malignancy. qpd therefore is a promising new non-invasive method both diagnostically and prognostically. after gd administration there was no significant enhancement of the nodules. our findings were then confirmed in all cases with the exception of one patient in whom arthroscopy did not find the lesion as it was located outside the synovial membrane. conclusion: mr provides a good and detailed evaluation of the different recesses of the knee and demonstrates high diagnostic accuracy in the evaluation of pvns enabling lesion characterization. to verify the benefits introduced by a new cr system for digital mammography, using a dual-side reader and transparent imaging plates. methods and materials: two kinds of breast phantom have been used to compare the performance of two different cr systems employed in digital mammography. they are the high resolution imaging plate hr-v coupled with the fcr 5000-r reading unit and the last fuji cr system including transparent ips and a dual-side reader. this detects also the laser-stimulated light propagating toward the back of the plate, which was previously removed from the hr-v plates. consequently, the detection efficiency and the signal-to-noise ratio are increased, resulting in a better image quality. phantom images have been taken for different values of entrance dose. the images produced by the two cr systems have been scored by three expert observers and the results compared in terms of both contrast-detail curves and detail visibility. the benefits of the new cr system are emphasized by the contrastdetail curves and the cirs phantom scoring. the new system permits a dose reduction of about 30 %. the spatial resolution is heavily better and exceeds 7 lp/mm for the new system, while is slightly lower than 5 lp/mm for the previous. the contrast-detail analysis shows a gain of 13 % in disk size and 6 % in contrast. saturday purpose: dry and conventional laser printers have become an equipment for producing images from digital mammography. purpose of our study was to compare image quality of a dry versus a conventional laser imager for digital mammography. materials and methods: two view mammograms of 80 patients with different breast composition (pattern i -iv) and mammograms of 44 patients with pathologic lesions were printed on a conventional wet laser imager (agfa scopixlr 5200) and a dry laser imager (kodak dryview laserimager 8600). all examinations were performed on a full field digital system (senographe 2000d, ge). two radiologists independently scored images side by side on the following aspects: delimitation of the skin, subcutis, musculature, glandular tissue, fat, and calcification. each comparative aspect was rated by using a five-level scale. in case of a breast lesion (masses n = 22, calcifications n = 22) lesions characteristics and resulting birads-classification were assessed. additionally, artefacts were noted. objective comparison was performed in accordance to instructions of the producers. results: a total of 248 digital mammograms were assessed. breast parenchyma of different composition and pathologic lesions were equally displayed by dry and conventional laser imagers. dry laser images showed significantly more artefacts at the film surface (73.6 % vs. 22.6 %, p < 0.001). however, these artefacts did not influence image interpretation. conclusion: compared to a conventional wet laser imager, a dry laser imager is capable of producing images of equal quality. the dry laser imager offers advantages with regard to handling, installation, maintenance, and environmental criteria. full-field digital mammography and patient dose reduction g. gennaro, c. di maggio, e. bellan; padova/it purpose: to estimate the dose reduction factor allowed by the full-field digital mammography. methods and materials: exposure parameters and breast characteristics have been collected from 300 screen-film (ge senographe dmr) and 300 digital mammograms (ge senographe 2000d) randomly distributed. from the track/filter combination, kv and mas values and breast thickness we estimated the entrance skin dose by using the ipem (institute of physics and engineering in medicine) data. results: data analysis shows a mean dose reduction of 32 % for the digital system. it is mainly due to the larger dynamic range of the digital detector, which permits the use of rh/rh combination in almost 60 % of considered cases without image quality degradation. in screen/film the mo track was needed in most cases, while the rh/rh choice was convenient only for 20 %. moreover, by analysing data as a function of breast thickness, the digital system shows an extra dose reduction for each track/filter combination due to its higher quantum efficiency compared with the screen/film system. conclusion: the digital system delivers an entrance skin dose 30 % lower than the screen-film system. a quality control phantom for full field digital mammography: first experience p. baldelli 1 , c. di maggio 2 , m. gambaccini 1 , g. gennaro 2 , d. giannopoulou 1 , a. taibi 1 ; 1 ferrara/it, 2 padova/it purpose: to verify the usefulness of a simple phantom to monitor some quality control parameters in full field digital mammography. methods and materials: a phantom has been developed for periodic quality control measurements on a full field digital mammography system ge senographe 2000d). the phantom allowed us to verify the detector linearity, uniformity, contrast and spatial resolution and the reproducibility and stability of both the detector and the automatic exposure control system (aop). the system stability was daily controlled by using four different exposure settings. the raw images have been used for data analysis. results have shown that the presented phantom is able to give information on the detector characteristics and can detect any problem related to the system stability. the collected data have verified that the detector response is linear with a high correlation degree, for both the mo/mo and rh/rh combinations. the non-uniformity was lower than a few percent. the detector response variability was lower than 1 % when manual exposure is employed while the fluctuation in the aop choices was less than 5 % for the three available settings (std, cnt and dose). the phantom used was found useful to check the key detector performance parameters and the system stability. it is fast and easy to use, as required for a quality control. full-field digital mammography -comparison of hardcopy versus softcopy reading s. obenauer, s. luftner-nagel, k.-p. hermann, u. fischer, e. grabbe; göttingen/de purpose: to evaluate the feasibility of using a mammography workstation for the interpretation of digital mammography images in comparison to the results of hardcopy reading. materials: mammograms performed on a digital full-field mammography system (senographe 2000d, ge) of 50 patients were evaluated as softcopies and hardcopies by 2 readers. 19 of the 50 patients had histologically proven carcinomas, the remainder showed no malignancies after a follow-up of 2 years. the softcopies were displayed on a 2.5 × 2k monitor, the hardcopies were printed on a laser printer (scopix, agfa). the mammograms were evaluated with respect to image quality, artefacts, presence of masses and microcalcifications. all detected lesions were categorized according to the bi-rads classification. the time for this evaluation was written down. post-processing in softcopy evaluation (windowing and levelling, zooming, inversion) and additional views like spot views in both methods were also timed. results: diagnostic accuracy of the classification of the malignant lesions according to the bi-rads categories showed no significant differences between soft-and hardcopy. more time was needed for the evaluation of softcopies than for hardcopies. additional views were recommended in hardcopy more often than in softcopy evaluation (26 versus 21 for reader 1, 20 versus 18 for reader 2). postprocessing was used in 9 cases by reader 1 and in all cases by reader 2. the evaluation of softcopies needs more time than those of hardcopies, especially when post-processing is performed. however, it seems that softcopy reading reduces the need of additional views without change of diagnostic accuracy. full-field digital mammography (ffdm): intrapatient comparison of direct magnification versus monitor zooming u. fischer, f. baum, s. obenauer, s. luftner-nagel, d. von heyden, e. grabbe; göttingen/de purpose: our goal was to compare digital magnification mammograms with images zoomed from the digital contact mammogram in patients with microcalcifications. materials and methods: 55 patients with 57 microcalcification clusters were evaluated with a ffdm system (senographe 2000d, ge). in addition to a digital contact mammogram, a digital magnification mammogram (factor 1.8) and two images zoomed from contact mammogram with magnification factors of 1.8 and 5 -8 were obtained in each patient. the image quality (1 = perfect to 5 = inadequate) and the characterisation of microcalcifications (bi-rads™ 2 -5) were evaluated by 4 readers. the results were compared to histopathologic findings in 35 patients (37 lesions) and follow-up in 20 patients. in patients with mammographic microcalcifications, monitor zooming of the digital contact mammogram is equivalent to direct magnification ffdm. therefore, monitor zooming allows a reduction of the radiation exposure and an optimization of the work-flow. b a c d e f 182 full-field digital mammography -comparison of different exposure protocols s. obenauer, k.-p. hermann, e. grabbe; göttingen/de purpose: to evaluate the potential of radiation dose reduction in full-field digital mammography (ffdm) compared to screen-film mammography (sfm). materials and methods: ffdm was performed by using an amorphous silicon detector with a cesium iodide scintillator layer (senographe 2000 d, ge). sfm was performed by using a state-of-the-art system (senographe dmr, ge) combined with a dedicated screen-film. an anthropomorphic breast phantom with superimposed egg shell fragments (50 -200 µm) was used to evaluate the detectability of microcalcifications. contact mammograms and magnification views (m = 1.8) performed with both the digital and the screen-film system were compared. images were exposed automatically (aec mode) by using a molybdenum/ molybdenum (mo/mo) anode-filter-combination, 28 kvp and 63 mas (protocol a, dose ~100 %). dose reduction in digital mammography was achieved by using protocol b with mo/rh and 31 kvp (dose ~60 %) and protocol c with rh/rh and 32 kvp (dose ~40 %). the detectability of microcalcifications was assessed by 3 well-experienced readers with a confidence level ranging from 1 to 5. a receiver operating characteristic (roc) analysis was performed. results: in protocol a the area under the roc-curve (az) for contact views performed by the screen-film system was 0.63 and for those performed with the ffdm system 0.68. for the conventional and digital magnification views az was 0.70 and 0.79, respectively. for the protocol b/c the az value reached 0.74/0.65 for the contact views, and 0.81/0.77 for magnification views. conclusions: compared to screen-film mammography the digital mammography system allows an equivalent detection rate of microcalcifications at a slightly lower radiation exposure. comparison between analogue and digital reading performance in screening mammography a.a.j. roelofs 1 , s. van woudenberg 1 , j.h.c.l. hendriks 1 , a. boedicker 2 , c.j.g. evertsz 2 , n. karssemeijer 1 ; 1 nijmegen/nl, 2 bremen/de purpose: to investigate the use of a digital reading station for screening mammography and to compare results with analogue reading. materials and methods: 500 cases, 125 screen-detected cancers, 125 interval cancers, and 250 non-cancers, were collected from the dutch breast cancer screening program. for each patient, mammograms from two prior screening rounds were collected in addition to diagnostic mammograms. diagnostic mammograms and pathology reports were used to establish ground truth. all mammograms were digitised and processed by a cad system (r2 imagechecker, los altos, ca). experienced screening radiologists participated in a study in which each of them read one film the two prior screening mammograms of all 500 cases in randomised order and blinded to the detection results. the readers were asked to annotate potential abnormalities, and estimate the likelihood of malignancy of each finding they reported. a subset of 100 cases with visible cancers on the prior mammograms and 175 non-cancers were randomly selected. this subset was reread on a dedicated workstation we are developing for screening mammography, by a subset of 5 radiologists 1.5 years after their first reading. the workstation was has two highresolution monitors (barco). results were evaluated using roc analysis and compared to the results obtained with analogue reading. results: no significant difference in screening performance between analogue and digital reading was found, provided cad was available for microcalcification detection. conclusion: using a dedicated reading station for mammography, soft-copy reading of digital mammograms in screening is possible without loss of quality. management through pacs and digital telemammography a. grueva, m. mahesh; baltimore, md/us purpose: digital telemammography (dtmg) is rapidly developing into a mature technology due to advances in digital image acquisition, database design and communications infrastructure. current film-screen mammography has a number of inherent limitations, which can be overcome effectively with dtmg. since acquisition display and storage can be optimized independently, screening sensitivity is increased. the initial goal was to provide dtmg in a screening environment with radiologist teleconsultation. technical issues in planning the establishment of dtmg screening infrastructure include network design, image compression, archiving, and special viewing station. displays, picture archiving and communications systems (pacs), image storage and processing architecture are examined, all at a concrete functional level of dtmg. a component-based approach to software development is described and is validated for each of the abstract system requirements for future breast screening computing environments and information management. research approach is telediagnosis, teleconsultation, and telemanagement. results: the implementation of this architecture is described and analysed from image and clinical data. workstations and comprehensive database development is the most difficult and expensive technology for teleimaging and information management. full-field dtmg with technical components, study protocols, and preliminary results of a study are analysed. conclusion: the study shows the dtmg system to be successful in fulfilling the goals, dtmg is also considered in the general context of an integrated regional health telematics network, emphasizing its role and its interaction with other information and networking services for decision treatment strategies. dtmg standard needs to be further evaluated for quality of telemammography and for cost effectiveness. material & methods: seventeen subjects affected by drug resistant cryptogenic partial epilepsy underwent long-term computer assisted video-eeg monitoring and dsc mri in the same day. mri data were compared with eeg findings, and accuracy of perfusional mri was analyzed considering the spiking rate and the type of epilepsy. results: dsc mri showed a relevant asymmetry in a region compared to the contralateral areas in 8 (47 %) out in 17 patients, suggesting the presence of a relative focal hyperperfusional pattern. this area corresponded to the epileptogenic focus or to the hemisphere involved. in the genesis of epileptic discharges in 7 (77.7 %) out of 9 patients showing a higher spiking rate (hsr) conversely, all patients with a low spiking rate (lsr group) did not show significant asymmetry indexes in perfusional pattern. moreover we found that dsc-mri seems to be very sensitive for detection of epileptogenic focus in patients affected by temporal lobe epilepsy (tle). in 5 (83.3 %) of 6 hsr-tle patients dsc-mri showed a relative hyperperfusion region concordant with eeg focus or hemisphere involved in the genesis of epileptic discharges. conclusion: perfusional mri is a noninvasive procedure that provides useful additional information to better localize the epileptogenic zone, when the interictal epileptiform activity is sufficiently elevated. saturday materials and methods: eleven patients (6 females, 5 males), affected by intractable epilepsy were included. brain lesions were: 4 mesial sclerosis, 3 brain tumors, 2 cortical gliosis, 1 encephalomalacia, 1 cortical displasia. mri studies were performed on a 1.5 t scanner. fmri volumes were acquired according to a block paradigm of 6 alternating (rest-task) phases, including 10 scans for each phase. bold contrast images were obtained by echo-planar fid sequences (fov = 250, tr = 0.8 ms, te = 54 ms, thickness 3 mm). high resolution mprage sequence, covering the whole head, served as basis for the integration of anatomical, meg and fmri data. magnetic field recordings were performed using a whole head meg system, equipped with 165 integrated squid magnetometers. the location of magnetic field source during intercritical activity identified by meg was found to be in close proximity to the lesion in 6 cases. a displacement of the source with respect to the lesion was found in two cases. in three cases meg was not able to reliably locate a source. functional markers (sensory areas identified by meg and motor areas identified by fmri) where in accordance with the known cortical organization. conclusion: these preliminary results are encouraging toward the use of morphological mri, fmri and meg in the identification of the functional areas within the affected region. quantitative 1 h mrs investigations of the regional and age dependences of metabolite content in the human brain v.a. rogozhyn, z.z. rozhkova; kiev/ua purpose: the aim of our work is a quantitative study of the age and regional dependences of cerebral metabolism using primary spectral parameters of main metabolites. materials and methods: 65 healthy volunteers (age range 25 -75) were examined by 1.5 t magnetom vision (siemens). spectra were obtained in two locations of each brain hemisphere: in the occiput (gray matter), and in the forehead (white matter). all spectra were recorded with the steam sequence: tr/te 1365/ 135; 1500/270, 20 ms; voi = 2 × 2 × 2 cm 3 . results: for the normal brains the peak areas of 1 h mrs signals from n-acetylaspartic acid (naa), total creatine (cr) and choline (cho) were obtained. we define the metabolite concentration as the ratio of the peak area to the sum of all the peak areas. we have found a "fine structure" of the average metabolite concentrations which depends on the relative values of peak areas. to describe this fine structure we introduce the triad t* = {a, b, c}, where a, b, and c are the unequal peak areas of the signals from naa, cr and cho, respectively. we believe that each of the three peak areas takes three values: 1, 2 and 3, to represent symbolically six possible spectral configurations: t = {1*, 2*, 1/4, 6*}. the triads 1* = {3, 2, 1} and 2* = {3, 1, 2}denote the most frequent configurations in the spectra. metabolic variations with age are especially noticeable in the left forehead for them. we have built spatial maps of the age dependences of metabolite content in the normal brain. our results serve as the background for the description of brain pathologies. mrs data for the quantitative description of the brain metabolism in patients with brain tumors v.a. rogozhyn, z.z. rozhkova; kiev/ua purpose: the aim of our study is to propose the quantitative indicators of metabolic changes in the human brain and brain pathologies. methods and materials: 72 patients with gliomas (of various grades of anaplasty) or meningiomas, together with 65 healthy volunteers were examined using a 1.5 t magnetom vision (siemens). all spectra were recorded in 4 brain locations with the steam sequence: tr/te 1365/135; 1500/270, 20 ms, voi = 2 × 2 × 2 cm 3 . the peak brain areas of 1 h mrs signals from n-acetylaspartic acid (naa), total creatine (cr) and choline (cho) were obtained for all subjects. we introduce the metabolite concentration as the ratio of the peak area to the sum of all the peak areas. we describe the metabolic state of the brain in each voi by the triad t* = {a, b, c}, where a, b and c are the unequal peak areas of the signals from naa, cr and cho, respectively. each of the areas takes three values: 1, 2 and 3, to give six symbolic spectral configurations: 1* = {3, 2, 1}, 2* = {3, 1, 2}, 3* = {2, 3, 1}, 4* = {1, 2, 3}, 5* = {2, 1, 3} and 6* = {1, 3, 2}. the frequency of appearing each of the triads in spectra depends on the brain state. the triads 1* and 2* denote the most frequent configurations in the spectra. and the triads 5* and 6* we observed only in the pathological brains: both in the tumor location and in the intact tissue. we give a quantitative classification of the brain state by creating spatial triad maps. this essentially amplifies mri examinations of patients with brain pathologies. proton mr spectroscopic imaging and fdg-pet in irradiated glial brain tumors h.-p.w. schlemmer 1 , m.p. lichy 1 , m. henze 1 , p. bachert 1 , s. sammet 1 , a.a. maudsley 2 , j. debus 1 , g. van kaick 1 ; 1 heidelberg/de, 2 san francisco, ca/us purpose: to assess the clinical utility of proton mr spectroscopic imaging ( 1 h mrsi) for evaluating suspicious brain lesions after radiotherapy of glial brain tumors. methods and materials: in this ongoing study 17 patients with irradiated gliomas (who grade 2, n = 12; grade 3, n = 4; grade 4, n = 1) were examined by 1 h mrsi and positron emission tomography with [ 18 f]-2-fluoro-2-deoxy-d-glucose (fdg-pet) to evaluate suspicious brain lesions. the mr protocol included multiplanar t2w turbo spin echo mri, 1 h mrsi (press, tr/te 1500/135 ms; spatial resolution 8.75 × 8.75 × 15.0 mm 3 ), and pre-and post contrast t1w-spin echo mri. relative signal intensity ratios of choline-containing compounds (cho), total creatine (cr), and n-acetyl-aspartate (naa) were determined both in lesions and in contralateral normal tissue using the program sitools. fdg-standard uptake values of the lesions were obtained from pet examinations and were related to normal gray (suvgr) and white matter (suvwm). lesions were classified as neoplastic (p) and non-neoplastic (np) based on mri follow-up or biopsy. results: significantly enhanced intensity ratios cho/cr and cho/naa were observed in p compared to np (p = 0.05 for both ratios) and compared to the contralateral normal tissue (p = 0.01 and p = 0.00003, respectively). in all patients cho/naa and cho/cr correlated with fdg-standard uptake values, e.g., cho/cr and suvwm: r = 0.58, p = 0.01. conclusion: parameters from 1 h mrsi are helpful for differentiating neoplastic from non-neoplastic lesions after brain tumor radiotherapy. moreover, the correlation with glucose metabolism indicates that mrs parameters may also indicate tumor grade. proton magnetic resonance spectroscopy ( 1 h mrs) of brain meningiomas at long echo time c. majós, j. alonso, c. arús, c. aguilera, m. serrallonga, j.j. acebes, j. cabiol, j. gili; barcelona/es purpose: to assess the utility of 1 h mrs in the categorization of meningioma with especial emphasis in their differentiation with respect to other brain tumors. methods and materials: single voxel "in vivo" 1 h mrs at an echo time of 136 ms was carried out in 37 patients with meningioma. a data-set of 93 spectroscopic exams of brain tumors acquired under identical conditions was used for comparison. this data-set included 15 low-grade astrocytomas, 14 anaplastic astrocytomas, 30 glioblastomas and 34 metastases. fitted area of seven resonances of interest (lipids at 1.30 ppm, lact at 1.35 ppm, ala at 1.45 ppm, nac at 2.02 ppm, glx at 2.35 ppm, cr at 3.03 ppm and cho at 3.20 ppm) was calculated. we searched for the resonances that better discriminated meningioma from every other tumor type. an empirical algorithm for bilateral discrimination between meningioma and the other tumors was produced on the basis of these findings. the performance of the algorithm was assessed by means of the leave-one-out method. results: high ala, low or absent lipids, high glx, low cr and high cho were found to be the most characteristic trends of meningioma. when applied in pair-wise discrimination with the other tumors, these findings produced 91 % successful classification outputs, 7 % unsuccessful and 2 % "unclassifiable". conclusion: single voxel 1 h mrs provides biochemical information from meningioma that can be satisfactorily applied in its categorization. according to our results, high ala, glx and cho, as well as low lipids and cr are characteristic spectroscopic signatures of meningioma. a c d e f 184 materials and methods: 22 patients who underwent supratentorial cortical/subcortical infarction were studied, 3 months after the onset of clinical symptoms of ischemic stroke the mr studies were performed on 1.5 t system. the results of spectra approximation (presented as metabolite ratios: naa/cr, naa/sum, chol/cr, chol/naa, lac/naa, lac/sum, lip/naa and lip/sum were subjected to statistical analysis. mr spectra were recorded from a tissue adjacent to area of infarction and normal appearing brain region: contra and ipsilateral internal capsule. spectra from stroke patients were compared with control group from 32 healthy volunteers recorded with the same techniques. results: the statistical analysis revealed significant differences between data obtained from the various regions in the patients who had undergone ischemic stroke and between the infarcted and control groups. proton mr spectroscopy detects changes in cerebral metabolites levels also in apparently normal regions. in contralateral brain regions, as well as in the internal capsule we have noticed significant reduction in choline, creatine and naa; we found correlation between clinical outcome and mri/mrs findings. conclusions: proton mrs is a very useful tool for evaluating major changes in cerebral metabolite levels in infarcted patients. our preliminary results of h mrs, mri and clinical data support the idea that metabolic leasions distant from the infarcted tissue can be responsible for clinical course and have predictive value. fingerprinting of the etiological agent in brain abscesses using proton mr spectroscopy m. garg jr., r.k. gupta, m. hussain, k.n. prasad, s. chawla, r. kumar, n. hussain; lucknow/in purpose: in vivo proton mr spectroscopy data was analyzed from 70 cases of proven brain abscess(s) to look for the possible metabolites that may act as marker for the specific group of bacteria. methods and materials: data from 70 patianets with proven brain abscess by pus aspiration, bacterial culture, and histopathology of the wall were analyzed to look for spectral pattern relationship with the bacterial culture. in vivo data was obtained using single voxel steam (te = 20 ms) and se (te = 135 ms) sequences. results: the in vivo spectra show the following 4 patterns: • abscesses in 4 patients showing only lipids (1.33 ppm, 0.9 ppm) and without amino acids (0.9 ppm) and showed m. tuberculosis on smear and culture. • abscesses in 16 patients showing succinate (2.4 ppm) and/or acetate (1.92 ppm), along with lipid/lactate (1.33 ppm) and amino acids (0.9 ppm) and showed anaerobic bacterial growth namely peptococci, peptostreptococci, and bacteroides. • abscesses in 20 patients showing only lipid + lactate (1.33 ppm) and amino acids (0.9 ppm). orgainsms seen on culture in these were either aerobes (pseudomonas, nocardia) or facultative anaerobes (staphylococcus, proteus, and escherichia coli). two patients with this pattern also showed anaerobic bacterial growth (peptostreptococci). • abscesses in the remaining 30 patients were found sterile on culture. these patients were on treatment with broad spectrum antibiotics at the time of spectroscopy and show lipid + lactate (1.33 ppm) and amino acids (0.9 ppm) only. conclusion: our data suggests that it may be possible to group the brain abscesses on the basis of metabolite pattern seen in vivo and may be of value in improved management of these cases. subclinical cerebellar neuropathy as a result of chemotherapy in proton magnetic resonance spectroscopy b. ciszkowska-lyson, l. krolicki, a. janowicz-zebrowska, a. teska, e. tacikowska, m. krzakowski, s. budrewicz; warsaw/pl purpose: of our study was to evaluate the central neurotoxicity of chemotherapy using mri and mrs, in patients with lung cancer who were treated with cisplatin and vinca alkaloids. we expected to find a reduction in n-acetylaspartate, as a result of the neurotoxicity of chemotherapy. methods: 31 patients aged 42 to 73 years underwent before and after chemotherapy: clinical examination; mri of the brain, mrs (press, tr 1500 ms, te 80 ms) voi of 8 ml localized in centrum semiovale (cs) and hemisphere of cerebellum. each patient's naa/cr and cho/cr ratios have been analyzed statistically. analysis was carried out separately for cs and cerebellum. none of the patients demonstrated clinical manifestation of cns neurotoxicity. mri did not reveal any abnormalities caused by chemotherapy. analysis of naa/cr and cho/cr ratios in cs did not show significant differences before and after chemotherapy. analysis of the cerebellar spectra showed significant decrease of naa/cr ratio (p < 0.05) and time dependent decrease of cho/cr ratio (p < 0.05) after chemotherapy. analysis of pearson's correlation showed very strong linear relationship between naa/cr and cho/cr ratios (p < 0.001), both at cs and cerebellum. conclusion: decreased naa/cr ratio can indicate the neuronal loss caused by chemotherapy. decreasing cho/cr ratio could be associated with damage of myelin. the mrs results suggest presence of subclinical selective cerebellar neuropathy caused by chemotherapy. the mrs revealed different reaction to chemotherapy at cs and cerebellum. these initial results indicate that proton mr spectroscopy is a potentially useful modality for detecting early stage of the cns neurotoxicity caused by chemotherapy. the liver and splenic parenchyma were surveyed and evaluated before and after embolization with plain helical ct, including volumetry of the liver and spleen. results: dsa examinations revealed a dilated splenic artery (n = 18) or gastroduodenal artery (n = 4) combined with slightly decreased perfusion of the hepatic arteries, while immediately after successful embolization normal perfusion of the hepatic arteries was noted. volumetric measurements before and after embolization showed no significant changes in liver parenchyma (6 % -10 %, mean, 6 %), and an alteration in spleen volume (11 % -27 %, mean, 16 %) in comparison with initial measurements. clinical follow-up examinations revealed a normalization of the previously elevated hepatic enzymes and a normalization of the liver function tests after successful embolization. complications were observed in 4 patients (infarction of the spleen). the preliminary results reveal that in liver transplant candidates with splenohepatic and gastroduodenal steal syndrome, successful embolization of the splenic artery results in a improvement in organ perfusion with normalization of function tests. were noted after 11 sessions (5.9 %). the number of sessions of tace, pancreatic location of primary tumour, extent of hepatic involvement and use of tace as a first line non-surgical treatment were significant for response rates in an univariate as well as a multivariate analysis. when survival from date of tace was considered, previous resection of primary and number of tace sessions were significant on univariate and multivariate analysis, while location of primary, histopathological grading and extent of hepatic involvement were significant only on univariate analysis. the digestive location of the primary tumor appears to be the strongest positive prognostic indicator for both response to tace and survival. use of at least 2 tace sessions per patient is recommended. poor prognostic indicators are presence of extra-hepatic lesions (nonresected primary and/or extra-hepatic metastases), poor histological differentiation of the tumor and more than 60 % involvement of liver. (2), kidney (2), spleen (1), brain (1). mostly there was an asymptomatic clinical course postprocedure. in six cases there were serious complications (3 × pneumonia, 1 × renal failure, 1 × cholecystitis) which resolved after two weeks. without evidence of displacement of lipiodol, there was one renal failure, one abscess of the liver and one case of hepatic encephalopathy. post-embolization syndrome (pes) with abdominal pain, nausea and vomiting, subfebrile temperature and pleural effusion was seen in 59 % (41/69) for a period of a few days. no rupture of an embolized hcc and no reflux embolization was observed. conclusions: unexpected displacement of embolisation material is not seldom, but mostly clinically inapparent. in cases with complications, symptomatic therapy is sufficient and successful. the frequent postembolization syndrome is transient and can be easily managed. transcatheter arterial chemoembolization (tace) as a first line treatment for liver metastasis from digestive neuroendocrine tumors a.j. roche, b.v. girish, v. kuoch, t. de baère, e. baudin, m. ducreux; villejuif/fr purpose: to report the outcome in patients who underwent tace as a first line non-surgical treatment methods and material: from jan. 1990 to dec. 2000 fourteen patients with progressive unresectable liver metastases from digestive neuroendocrine tumor were treated with tace (mean of 3.6 sessions) before any non-surgical treatment (somatostatin analogue, chemotherapy or interferon). liver involvement was less than 50 % in 11 patients. the size of the largest lesion ranged from 1.5 to 10 cm. the number of lesions was more than 10 in 8 patients. ten patients presented with carcinoid symptoms. tace was performed with doxorubicin emulsified in lipiodol® and gelatin sponge particles. results: a symptomatic response relating to flushes and/or diarrhea was complete in 7/10 cases and partial in 2/10. an objective morphologic response was noted in 12/14 cases. one patient developed transient renal failure and hypertension after the first tace. there was no procedure-related mortality in the series. the 5 and 10-year survival from diagnosis was respectively 83 % and 56 %. six patients were alive at the end of the study after 27 to 100 months from first tace and 38 to 142 months from diagnosis. three of them were successfully palliated for 55, 69 and 100 months with only tace as treatment (they received 9, 3 and 8 sessions respectively). the control rate of carcinoid syndrome with tace is comparable to somatostatin analogues. long term clinical, as well as morphological palliation, is possible in unresectable liver metastases from digestive neuroendocrine tumors with a few sessions of tace as early and exclusive treatment. material and methods: 220 tace in 54 consecutive patients with hcc. sequential tace (interval: 6 weeks) was performed using a suspension of 10 mg mitomycin c and 10 ml iodized oil (lipiodol). follow-up included pre-and post-interventional ct scans and laboratory measurements (liver-function parameters; afp). the number of lesions and tumour size were investigated using spiral ct. results: mean follow up was 17 months (range 6 to 50). 35 patients are still alive (mean survival 23 months, range 7 -50 months). 152 lesions were detected on the initial ct scans. size of individual lesions was < 20 mm (i), 21 -50 mm (ii), and > 50 mm (iii) in 42.8 %, 38.2 % and 19 %. over all median change of tumour size was -8.2 % (range -61 % to +82 %). median change depending on the initial lesion size was -7.7 % (i), -9.5 % (ii), and -3.8 % (iii). stable disease was noted in 33/54 patients. 10/54 patients had significant tumour regression and liver transplantation was successfully performed. tumour progression was noted in 14 patients with multilocular hcc and tumours > 50 mm. complications occurred in 3 patients, pancreatitis in one and dissection of the hepatic artery in two. there were no periinterventional deaths. conclusion: sequential tace in primary hcc is an effective therapy in patients with small tumours. because of tumour size control and avoidance of new tumour lesions, tace is a promising method to bridge the time to liver transplantation or to change patients into a transplantable condition. hybrid mri: transarterial chemoembolization of liver metastases using interactive mr guidance t.j. vogl, j.o. balzer, s. zangos; frankfurt a. main/de purpose: to evaluate the effectiveness of mr guided transar terial chemoembolization (tace) of liver metastases by employment of a hybrid mri system. in a newly built suite with fully interactive high field (1.5 t) mri and c-arm angiography units, elective tace was performed in 30 patients. an interactive table was positioned between the mri and angiographic unit, and allowed repositioning of the patient on a carbonfibre-plate within 20 seconds. arterial access was successfully achieved by employment of the dsa-unit and a mr compatible angiography catheter was superselectively positioned in the tumor feeding artery. the patient was then positioned in the mr unit and gd-enhanced dynamic mri (turboflash) via the catheter revealed the regional perfusion and verified the correct catheter position prior to tace. results: in 14/30 patients immediate embolization of the tumor bearing liver segments was performed. in 14 patients repositioning of the catheter and in 2 patients a change of vascular access was necessary. therapy monitoring using mri therefore resulted in the repositioning of the catheter tip in 53 % of the embolization maneouvers. dsa control immediately after tace revealed successful tace of the appropriate liver segments. conclusion: the presented hybrid system optimizes complex tace procedures in patients with liver metastasis. mri guidance enables the exact identification of the tumor bearing liver segments and correct catheter placement. uterine artery embolisation for symptomatic fibroids: results in 400 women w.j. walker, j.-p.j. pelage; guildford/gb purpose: to evaluate the mid-term efficacy, adverse events and complications of uterine artery embolisation (uae) in women with symptomatic fibroids. to assess reduction in uterine and dominant fibroid volumes using ultrasound imaging and magnetic resonance imaging (mri). methods and materials: four hundred women were treated between december 1996 and february 2001. indications for treatment were menorrhagia, menstrual pain, abdominal swelling or urinary frequency. imaging was performed before embolisation and at regular intervals thereafter. clinical evaluation was made at regular intervals after embolisation to assess patient outcome. results: bilateral uae was achieved in 395 women whereas 5 women had a unilateral procedure. with a mean clinical follow-up of 16.7 months, menstrual bleeding was improved in 84 % of women and menstrual pain was improved in 79 %. the mean changes in uterine and dominant fibroid volumes were 55 and 73 % using ultrasound (performed at an average of 9.7 months after embolisation) and 53 and 64 % with mri (performed at an average of 6.4 months). three (1 %) infective complications requiring emergency hysterectomy occured. 23 (6 %) patients had clinical failure or recurrence and nine (2 %) had a hysterectomy. 26 (7 %) of women had permanent amenorrhea after embolisation including 4 patients under the age of 45. thirteen pregnancies occurred. 97 % of women were pleased with the outcome. conclusion: uterine artery embolisation is associated with a high clinical success rate and good fibroid volume reduction. infective complications requiring hysterectomy and amenorrhea under the age of 45 are rare but major complications. embolization of bleeding arteries from pelvic trauma n.-e. klow 1 , a. riise 2 , m. brekke 1 , o. roise 1 ; 1 oslo/no, 2 fredrikstad/no purpose: to study the effects of arterial embolization after pelvic trauma in hemodynamically unstable patients. material and methods: from 1994 to 2000, 17 patients were included, three women and 14 men, mean age 35 years (from 16 to 72 years). all patients were admitted with trauma to the pelvis, but freqently multiorgan trauma was present. the pelvic fractures were bilateral in five, a sacral fracture was present in seven and diastasis of the symphysis pubis in seven. over the 24 hours immediately before embolization the need for whole blood transfusion was 18 ± 17 units (range 2 -64). results: all 17 patients were successfully embolized. fourteen had embolization of one internal iliac artery (iia), one both iia and a lumbar spinal artery, one a lumbar spinal artery, and one a superficial femoral artery branch. in 15 patients coils were used exclusively, in one gelfoam and in one coils and gelfoam were combined. the total need for blood transfusion after the embolization was 0.8 ± 1.0 units (range 0 -3). three patients died during the hospital stay, and no deaths were related to the procedure or rebleeding from the pelvic arteries. conclusion: embolization of arteries bleeding from traumatic injuries of the pelvis is a life saving procedure with a high procedural success rate. the procedure can be done on call, in hemodynamically unstable patients, and should probably be performed in an early stage of bleeding. intraarterial chemotherapy of malignant pelvic tumors under tourniquet occlusion of the femoral vessels p.d. niggemann 1 , s. murata 2 , h. tajima 2 , y. okajima 2 , k. ichikawa 2 , h. kawamata 2 , t. kumazaki 2 ; 1 aachen/de, 2 tokyo/jp purpose: to evaluate the possible benefit of intraarterial chemotherapy in malignant pelvic tumors under tourniquet occlusion of the femoral vessels. methods and materials: the difference in contrast enhancement of 55 regions in 23 malignant pelvic tumors was evaluated with and without tourniquet occlusion of the femoral vessels. in nine patients (group a) two ct scans of the pelvis, one and five minutes after starting an intraarterial contrast medium injection under tourniquet occlusion, were performed and then two ct scans after injection of the same dose without tourniquet occlusion were performed. in the remaining 13 patients (group b), the study was done in the opposite order. results: in group a, a mean change of the contrast enhancement of all tumors was 0.6 hounsfield units (hu) after one minute and −3 hu after five minutes. in group b, a mean change in contrast enhancement of 2.1 and 0.8 hu at one and five minutes respectively was observed. in patients with prostate tumor, the results were 5.2 and 3.9 hu respectively in patients from group a and 14.1 and 10.2 hu in patients from group b. the average of both groups together showed an increase of 8.9 and 6.5 hounsfield units respectively. conclusion: an increase in the contrast enhancement of malignant pelvic tumors due to tourniquet occlusion of the femoral vessels has been observed only in patients with prostate cancer after one and five minutes. therefore intraarterial chemotherapy under tourniquet occlusion of the femoral vessels only seems to yield a benefit in poorly vascularized tumors. purpose: to establish a high resolution mri (hr-mri) protocol for precise and repeated non-invasive analysis of selected atherosclerotic vessel segments in vivo and to compare the results with intravascular ultrasound (ivus). materials and methods: 7 patients with atherosclerotic disease of the superficial femoral artery were examined on a magnetom vision, siemens using axial t1-w, fat-saturated contrast enhanced t1-w, t2-w and 3d-tof sequences. contrast enhanced mra was used to display the detailed vasculature for precise orientation. maximum matrix size was 320 × 512, minimum voxel size 0.49 × 0.49 × 2.0 mm. ivus (3.5 f, 40 mhz) images were recorded with a standardized motorized pullback system (pullback speed 1.0 mm/s). parameters analysed were minimum and maximum luminal diameter and cross sectional lumen area. vessel wall calcification was determined by 900 steps. results: 123/123 segments were available for comparison between hr-mri and ivus. intra-and interobserver repeatability for exact assignment of vessel segments was high (r = 0.92, r = 0.87). there was a good correlation for luminal parameters (correlation coefficients ranging from 0.82 to 0.98 depending on the plaque burden). no correlation was found for cross sectional vessel area. calcification could be classified with a sensitivity of 91 %, a specificity of 93 %. accuracy was 93 %. conclusion: our hr-mri protocol is highly accurate for non-invasive assessment of atherosclerotic lesions in human femoral arteries. the results are comparable with ivus as known reference standard in vivo. purpose: to evaluate ultrasonography as a method of characterizing plaque surface configuration and plaque internal structure in comparison to in vitro angioscopy and pathological specimens. the carotid plaque specimens of 15 patients were examined by in vitro angioscopy and histopathological examination after carotid endarterectomy (cea) in a comparative study with preoperative ultrasonography. colorcoded ultrasonography was obtained with a 7.5 mhz linear transducer (logic 500, ge). transverse and longitudinal sections were recorded from the bifurcation area as well as from the proximal internal carotid artery. the plaque surface was characterized as smooth if the plaque surface did not show any disruptions. further, the echogenic structure was recorded by grey scale analysis. after cea the inner surface of the plaque was visualized by a 1.4 mm angioscope. the results were compared with histopathological findings. results: peak flow velocity (mean: 2.77 m/s, range 1.76 -4.24 m/s) demonstrated a > 60 % stenosis in all cases. the median of gray scale analysis (gsm) was 55 with a high correlation between both investigations (p < 0.01; r = 0.954). in comparison to pathological findings there was a correct detection of plaque surface in 86.6 %. pathological classification of plaque composition revealed no correlation to computer-assisted analysis (p = 3.85; r = -2.63). conclusion: detection of an ulcerated plaque surface by ultrasonography is difficult with a wider degree of variation when compared with direct visualisation of the surface and pathological examination. computer-assisted analysis of the morphology provides a high interobserver correlation, however, there was no correlation between the gsm and histological findings in this series. common carotid artery intima-media thickness (cca-imt) and carotid plaque echogenicity: correlation with cardiovascular risk factors in patients with acute ischemic stroke g. terzis, a. chrysanthopoulou, g. gioldasis, a. kalogeropoulos, c. paschalis, j.a. dimopoulos; patras/gr purpose: to correlate the cca-imt and the echogenicity of the carotid plaques with the main cardiovascular risk factors in patients with acute ischemic stroke. methods and materials: 535 consecutive patients (male 344, female 191, mean age 67.6 years) with acute ischemic stroke were studied with b-mode and color doppler ultrasonography during may 1999 to july 2001. cca-imt was evaluated and four types of plaques were defined by their echostructure. type i: uniformly echolucent, type ii: predominantly echolucent, type iii: predominantly echogenic and type iv: uniformly echogenic. cardioembolic and undetermined strokes were excluded. all patients were evaluated for a history of smoking, hypertension, diabetes or hypercholesterolemia. smoking, hypertension and diabetes were all significantly associated with an increased cca-imt (p < 0.01 for all subgroup comparisons). hypercholesterolemia had no significant effect (p = 0.67). smoking was associated with an increased cca-imt (p = 0.0032) independently of other risk factors. hypertension was significantly associated with diabetes (p = 0.0033) as a risk factor. smoking, hypertension and diabetes had no significant effect on the type of the plaque (p = 0.374, p = 0.607, and p = 0.636 respectively). hypercholesterolemia was significantly associated with type i plaques (p < 0.05). a trend for advanced echogenicity was observed among smokers (p >> 0.1). smoking, hypertension and diabetes but not hypercholesterolemia were significantly associated with an increased cca-imt in patients with acute ischemic stroke. smoking, hypertension and diabetes were not associated with a specific type of plaque, but hypercholesterolemia was clearly associated with type i plaques. high-resolution mr of carotid atheroma: a non-invasive tool for assessing plaque morphology and potential risk? j.h. gillard 1 , m.t. gaskarth 1 , m.j. graves 1 , r.a. coulden 1 , h. wilson 1 , n.m. antoun 1 , m. goddard 2 , p.j. kirkpatrick 1 ; 1 cambridge/gb, 2 papworth/gb purpose: whereas current imaging techniques provide excellent structural information including the quantification of vascular stenosis, they provide no information of the degree of functional plaque stability. risk of rupture is more closely related to plaque composition and macrophage content than size. thus angiographic appearances are an inaccurate predictor of risk; even large atherosclerotic lesions may not produce a significant stenosis at angiography. we aimed to use highresolution mr to evaluate plaque morphology in patients being evaluated for carotid endarterectomy. methods: blood-suppressed fast spin echo imaging was performed using a dedicated surface coil (t1, t2, proton density, and chemical shift selective fat suppression; spatial resolution 0.24 × 0.24. × 3 mm) in 29 patients with symptomatic carotid disease. gadolinium-enhanced elliptic centric mra was also performed and compared with conventional digitally subtracted angiography (dsa). histopathological examination was carried out on 4 specimens. results: 37 plaques were identified, 15 of which were eccentric, extending outside the limit of the "normal" vessel wall. there was no significant difference in the degree of stenosis (using nascet criteria) assessed with dsa and gd-enhanced mra (p = 0.11). high-resolution axial mr measurements, however, demonstrated more severe stenoses than was apparent from 4 projection dsa (mean difference 11 ± 13 %, p < 0.005). mr correctly identified plaque constituents (fibrin, lipid and focal hemorrhage). conclusion: high-resolution carotid mr appears to be a useful tool in evaluating plaque morphology and effectively assesses plaque volume and degree of arterial narrowing. it may be an effective tool in evaluating plaque risk and the effects of pharmaceutical interventions. in 18 patients with btm (mean age 25.8 ± 7.6 year, 55.5 % male, mean hemoglobin 10.2 ± 1 g×dl -1 , mean serum ferritin 2396 ± 1754 ng×ml -1 , 44 % on optimal chelation therapy) without cardiac disease or diabetes mellitus and 18 healthy control subjects, flow-mediated dilatation (fmd) was measured as a percentage change of the post-ischemic right brachial artery (ba) diameter. endothelial-independent, nitroglycerin-induced vasodilatation (nid) and cca imt were also assessed. the study groups were matched for age, gender, body surface area, blood pressure and smoking habits. serum cholesterol levels were lower in the patient group (102 ± 39 versus 178 ± 30 mg⋅dl −1 , p = 0.001). the brachial artery diameter was 4.07 ± 0.66 mm in patients and 4.06 ± 0.52 mm in healthy subjects. post-ischemic increase in the brachial artery blood flow was 555 ± 383 % (p < 0.01) for patients and 1072 ± 6.61 % for healthy subjects. fmd was 5.97 ± 2.3 %(p < 0.001) for patients and 10.44 ± 44 % for healthy subjects. nid was 14.86 ± 6.22 % for patients and 16.66 ± 4.23 % for healthy subjects. cca imt was 0.539 ± 0.078 mm for patients and 0.453 ± 0.072 mm for healthy subjects. fmd and thickness of the carotid arterial wall did not correlate with serum ferritin levels. conclusions: in patients with thalassemia major, endothelial function of conduit arteries is impaired and cca imt is increased. our findings suggest a proatherosclerotic milieu with reduced bioavailability of nitric oxide, probably due to the iron-induced, high oxidative stress in these patients. functional imaging of the aortic wall by ecg-gated multislice-ct: an ex vivo experiment m.-k. ganten 1 , j. boese 1 , d. leitermann 2 ; 1 heidelberg/de, 2 prague/cz purpose: ecg-gated multislice-ct (msct) allow imaging with increased temporal resolution. the aim of this study was to evaluate the possiblity of obtaining functional information determining aortic-wall-elasticity in an ex-vivo experiment. elasticity is a piece of clinically relevant information not yet widely accessible. methods & materials: 16 samples of porcine aortas were examined ex-vivo. to imitate physiologic circulation an artificial heart phantom emitting an ecg signal was used to perfuse the samples with pulsatile flow and varying pressures. aortic wall elasticity can be determined by dividing the relative change in vessel area by the corresponding pressure change. as a reference, the wall distension was measured using an optical system with high temporal and spatial accuracy. ecg-gated ct images were acquired with a standard ct-angiography protocol applying specially developed reconstruction algorithms. this allowed us to obtain images with improved temporal resolution of about 100 ms. the aortic cross-section was determined from the images by manual segmentation. the accuracy of the crosssectional changes measurements was evaluated by comparison between the optical and the ct data. results: determination of aortic wall elasticity was possible in all samples. elasticity, expressed as mean compliance, was 1.4 × 10 −5 /pa. precision of the manually determined values was in the order of 10 % compared to a precision of about 1 % for the reference optical method. cross-section changes obtained from the ct images were in line with the error bounds obtained from the optical measurements. conclusion: our study shows that aortic wall elasticity determination using ecggated ct is feasible. this allows functional information about the aortic wall to be obtained. patient studies are required to investigate the in-vivo applicability. detection of atherosclerotic plaque using gadofluorine enhanced magnetic resonance imaging j. barkhausen 1 , w. ebert 2 , c. heyer 2 , j.f. debatin 1 , h.j. weinmann 2 ; 1 essen/de, 2 berlin/de purpose: to visualize atherosclerotic plaques independent of luminal narrowing using t1-w contrast enhanced mri. methods and material: 5 watanabe heritable hyperlipidemic rabbits (9 -18 months) and 5 age-matched controls (white newseeland) underwent magnetic resonance imaging of the aortic arch using a 1.5 t mr system (magnetom symphony, siemens ag, erlangen, germany) before and 48 h after injection of 100 mmol gadofluorine (schering ag, berlin, germany). a haste sequence (tr 700 ms, te 60 ms) and a t1-weighted inversion recovery turboflash sequence (tr 300 ms, te 4 ms, ti 120 ms) were used for data acquisition. immediately following the mr examination the animals were sacrificed and the aorta was stained with sudan. ex vivo imaging of the stained aortic specimens was performed. additionally, the gadolinium concentration in plaques and normal aortic wall was measured by means of inductively coupled plasma atomic emission spectrometry. results: plain mr imaging revealed no plaques in the aortic arch in either animal group. enhancement occurred in the aortic wall of all whhl rabbits but not in the vessel wall of the control group. sudan staining demonstrated multiple plaques in the aortic arch of the whhl rabbits and using ex vivo imaging the area of hyperenhancement matched the area of plaques stained with sudan. the gadolium concentration was 51 ± 21 nmol/g for normal aortic wall in the control group and 531 ± 144 nmol/g for plaques. conclusions: gadofluorine enhances atherosclerotic plaques thereby permitting the detection of plaque independent of luminal narrowing. vessel wall mri of the descending aorta: comparison with transesophageal ultrasound n. abolmaali, m. langenfeld, c. schick, r. kraforst, v. schächinger, t.j. vogl; frankfurt a. main/de purpose: to evaluate the validity of high-resolution contrast-enhanced mri of the aortic wall and to prove comparability of plaque morphology detected by transesophageal ultrasound (teus). is there additional information in mri? methods and materials: we performed mri in five patients (median age: 62 years) in whom aortic wall thickening and plaques of varying morphologies were detected by teus. after sequence optimisation in cadaver studies, all mr-examinations were performed with a 1.5 t scanner (magnetom symphony quantum, siemens) with the cp-spine surface coil (supine position) using ecg-triggered t1weighted tse-sequences with dark-blood-preparation and fat-suppression before and after iv-administration of 0.1 mmol/kg bw gadolinium-dtpa (gd). slice thickness was 5 mm, in-plane-resolution using a 256 2 matrix was 0.5 × 0.5 mm 2 . the heart-frequency dependent acquisition time for 10 slices was 10 -14 minutes. roi-measurements of the aortic wall before and after contrast enhancement were performed and mri was correlated with the teus findings. results: thickened wall segments and plaques of the descending aorta detected by teus were visualized by mri. different morphologies with evidence of fatty deposits were detected. unaffected aortic wall revealed two layers and signal isointensity compared to striated musculature. after iv-administration of gd most parts of the aortic wall did not enhance (< 6 %) while wall thickening and plaques showed considerable enhancement (22 -59 %) solely of the inner layer of the aortic wall. conclusion: high-resolution mri of the aortic wall provides similar morphologic information as teus does. additionally the distinct enhancement of the inner aortic layer may correspond to an inflammatory process of the vessel wall. , metastases (n = 11, mainly colorectal), carcinoid (n = 5) and two benign liver tumours. 3 patients were excluded from follow-up. results: mean survival for all patients was 15.2 months, with an adjusted mean survival of 16 months for hccs and 15.2 months for metastases. there were three major and five minor post-procedural complications but no deaths. an average of 57 % of tumour was ablated as assessed by per-procedural thermal mapping, with an average of 49.4 % of tumour ablated assessed by pre and post ablation gadolinium-enhanced mris. average tumour size was unchanged after ablation. in patients with multiple liver tumours ablated tumours grew significantly less than untreated tumours over the same time period (108 % compared to 196 % growth over an average follow up period of 5.8 months). conclusions: mr guided laser thermal ablation of primary and secondary liver tumours is safe and feasible and produces a better survival in patients with hcc than would be expected in untreated patients, as well as a mean survival in patients with metastases at least equal to the longest median survival in untreated patients. percentage viable tumour was decreased by a mean of 49.4 % per lta session. contrast enhanced harmonic sonography (ceus) used in the interventional room (ir) during radiofrequency (rf) ablation treatments: how to save time and reduce patients' discomfort l. solbiati, t. ierace, m. tonolini, l. cova, v. osti, p. marelli; busto arsizio (va)/it purpose: to evaluate usefulness of ceus performed during rf ablation procedures to assess the therapeutic result prior to ending the treatment session. materials/methods: 61 patients with 1 -5 hcc in liver cirrhosis (42 patients) or 1 -4 metastases from colorectal (10), breast (3), gastric (2) and endocrine (4) cancers underwent single session percutaneous rf ablation with cool-tip electrodes, under general anesthesia, in the ir. before and 5 -10 minutes after ending each treatment session, continuous mode, low mechanical index, ceus with a second generation contrast agent (sonovue, bracco) was performed. a total of 86 lesions were examined. when, residual enhancement was detected within treated tumors, a second targeted electrode insertion was carried out. when complete tumor avascularity was demonstrated, general anesthesia was terminated. contrast-enhanced helical ct (cect) was performed in all cases 2 -15 days after ablation. results: in 67/86 (77.9 %) lesions no post-ablation residual enhancement was found with ceus. subsequent ct showed unablated portions of tumors in 8/67 (11.9 %) lesions (all larger than 5 cm) and complete necrosis in the remaining 59 saturday in 19/86 tumors in 17 patients, ceus showed either single or multiple intralesional areas of residual enhancement in arterial and/or portal phase, 1 -2.5 cm in size. all these likely viable portions were immediately targeted with 1 -2 electrode insertions, until no residual areas were demonstrated. on subsequent cect, only in 2/19 (10.5 %) of these tumors, 1 -1.5 cm residual areas were found requiring new rf treatment. to prove the hypothesis that t1w thermal mapping is reliable and achievable in mr guided laser tumour ablation. methods & materials: 110 mr guided laser thermal ablations (lta) of liver, kidney and uterine tumours were studied. after laser fibre placement, near real-time grey and colour scale thermal maps are produced. previous work showed t1 signal is inversely proportional to temperature below 55°c (the point of irreversible tissue necrosis). measurements included: (i) percentage (%) of cases in which the thermal map provided sufficient information to control the procedure (ii) ability of grey and colour scale maps to demonstrate size (centimeteres) and conspicuity (10 point scale) of thermal lesions (iii) factors causing thermal mapping failure. results: (i) thermal mapping was successful in 84 % uterine, 74 % hepatic, and 20 % renal ablations. (ii) for hepatic/uterine tumours, size and conspicuity of thermal lesions were significantly greater on the colour than grey scale mapping. for uterine ablations, mean lesion size was 3.1 cm (colour) and 2.5 cm (grey, p = 0.001, paired student's t-test), while mean conspicuity was 7.3 (colour) and 1.7 (grey, p = 0.001). for liver ablations mean lesion size was 3.1 cm (colour) and 1.7 cm (grey, p = 0.001) while conspicuity was 7.5 (colour) versus 3.7 (grey, p = 0.001). (iii) patient movement (n = 24), fibre charring (n = 2), magnetic field distortion & reconstruction errors (n = 2) caused mapping failure. in hepatic & uterine thermal maps the colour scale produced significantly greater sized lesions with significantly greater conspicuity than the grey scale. t1w signal thermal mapping was reliable and successfully achieved in 73.7 % of procedures. the most important challenge on the follow-up of radiofrequency treated hypovascular liver metastases: the differentiation of coagulative necrosis from viable tumor. is there a role for contrast enhanced sonography? l. solbiati, m. tonolini, l. cova, d. della chiesa, v. osti; busto arsizio/it purpose: to assess if contrast enhanced harmonic sonography (ceus) can be useful for the differentiation of radiofrequency-induced coagulative necrosis from viable hypovascular liver metastases. materials and methods: 29 metachronous colorectal liver metastases (mean size 2.7 cm) in 9 patients treated 2 -9 months beforehand with rf ablation and systemic or intraarterial chemotherapy underwent conventional us and continuous mode, low mechanical index, ceus with a second generation contrast agent (sonovue, bracco) due to suspicion of local recurrence. triphasic contrast-enhanced helical ct (in all cases) and fnab (10 cases) were performed to confirm recurrence. results: usind ceus in arterial and very early portal phases, 25/29 metastases showed intense, predominantly peripheral enhancement. in full portal phase, complete disappearance of the enhancement was observed, with homogeneously hypoechoic appearance. the same arterial ct demonstrated enhancement, although less evident than with ceus, in only 10 of these 25 metastases (40 %). us-guided fnab was performed in at least one metastasis for each patient, with diagnosis of viable adenocarcinoma cells. in the remaining 4/29 lesions no enhancement was found with either ceus or ct and fnab (in 2/4 nodules) did not yield viable tumoral cells. conclusions: differentiation of coagulative necrosis from unablated or recurring hypovascular tumor is a diagnostic challenge. ceus demonstrates marked arterial enhancement of the viable tumor portions, better and with higher sensitivity than with ct. this finding could represent a quick and valuable tool for post-ablation follow-up. such arterial enhancement (more intense than that observed before ablation) raises a suspicion of more active neovascularity in uncompletely ablated tumors there was no effect on survival by tumor size, number of tumor nodes or the grading. however, the survival of 50 % of the patients with a child c cirrhosis or with afp levels > 1000 ng was less than 6 months with nobody living longer than 36 months. in patients with child a cirrhosis and/or normal afp levels the mean survival was 14 and 17 months, resp. 10 % of these patients lived more than 60 months. conclusion: tace is an effective therapy that may prolong the patients survival in patients with non-resectable hcc. however, child class in cirrhotic patients and elevation of afp levels have to be considered as prognostic factors for a life extending tace therapy. purpose: to evaluate a neoadjuvant treatment protocol for large sized liver metastases performing repeated transarterial chemoembolization (tace) and litt (laser induced thermotherapy) in patients with unresectable liver tumours initially unsuited for litt material and methods: 519 patients with different malignant liver tumours were evaluated between january 1999 and september 2001. tace was performed with 50 mg/m 2 mitomycin c, 10 ml/m 2 lipiodol and microspheres. the tumour volume was measured by mr-imaging. lipiodol retention and perfusion of the tumours were evaluated by ct and angiography. after response to tace and reducing the tumor size 106 patients (16 patients with hcc, 59 patients with metastases of colorectal cancer, 17 patients with metastases of breast cancer and 14 patients with metastases of different primary cancer) could be treated with mr-guided litt 4 to 6 weeks post embolization. these patients received 2 to 6 (mean: 3.6) tace before litt. results: repeated tace enabled a significant reduction in tumour size and/or tumour perfusion in 106 patients, forming the basis for the performance of mrguided litt procedure for a complete ablation of the tumour. in 178 tumors lesions 203 laser interventions archived a complete tumour ablation. the initial tumour volume pre-litt presented with a mean of 30.1 ml, the resulting necroses was 73.4 ml. the local tumour recurrence rate was 7 % in the 6 month control, the rate of side effects 11 %. conclusion: repeated tace alteres the size and structure of primary unresectable liver metastases and expands the indication for mr-guided litt. results: tace treatment were performed successful in 99.9 %. only in 2 cases sondage of celiac trunk and performing tace was not possible. during the tace procedure in 12 cases an occlusion of the a. hepatica was evidenced with good collateral arteries, so that tace could performed. in one case perforation of a. hepatica propria resulted in a small subhepatic bleeding. no therapy associated mortality was observed. only minimal side effects like pain (53.8 %), nausea (30.8 %), vomiting (20.5 %), fever (43.6 %) and apokamnosis (82.1 %) were associated with the tace, which could be managed by oral medication easily. in 15 patients aszites was documented after tace treatment. in 6 patients intrahepatic abscess cavities were evidenced and treated with percutaneus drainages. conclusion: tace in liver tumors proved to be a safe oncologic treatment in an outpatient setting. side effects must be treated fast to reduce failure. extrahepatic recurrences after radiofrequency ablation treatment of hepatocellular carcinoma: spectrum of imaging findings m. tonolini, l. solbiati, t. ierace, v. kirn, p. marelli; busto arsizio/it purpose: to describe diagnostic aspects and particularly ct findings of extrahepatic relapses observed after treatment of hepatocellular carcinoma. materials and methods: during a six-year span, 226 patients (aged 32 -88 years) with chronic hepatitis or cirrhosis were diagnosed with hepatocellular carcinoma confined to the liver and treated percutaneously with radiofrequency (rf) ablation. a total of 313 treatment sessions were performed. post-therapeutic follow-up is based upon serum alpha-fetoprotein levels and ct examination. results: mean duration of follow-up was 17 months. after successful treatment, actuarial probability of neoplastic relapse is 30.7 % after 1 year and 58.5 % after 2 years. 88 patients had recurrence of hepatocellular carcinoma after a variable time interval (mean 7.3 months). extrahepatic neoplastic relapse was observed in 14 patients, half of these without active hepatic disease. distribution of extrahepatic sites of recurrence was as follows: abdominal lymph nodes (6 cases), bone (3), peritoneum (2), adrenal (2), lung (1). five patients (2.2 %) had a second primary extrahepatic neoplasm. conclusion: extrahepatic hepatocellular carcinoma is considered rare and occurring in advanced stages, but may represent a modality of post-treatment relapse. the distinctive hypervascularity of this tumor histology may be observed in adenopathy and adrenal metastases. second primary neoplasms should be considered in the differential diagnosis of lesions observed during follow-up. radio-frequency (rf) ablation of liver tumors: postprocedural appearances on multiphasic spiral ct p. cabassa, l. romanini, d. guidetti, f. simeone, a. maggi, l. grazioli; brescia/it purpose: to describe the spectrum of ct findings of liver malignancies treated with rf. materials and methods: 40 histologically proven malignant liver lesion (35 hcc, 5 methastasis, size range 1.6 -7.5 cm) in 37 patients (26 male, age range 44 -82 years) were treated with rf (using either internally-cooled electrode or le veen needle electrode). multiphasic spiral ct at 1, 4, 6, 12 months (and every 6 months after 1 year) was performed for follow-up. spiral ct included unenhanced and enhanced arterial and portal venous phase images. absence of contrast enhancment within the lesion was considered as complete ablation. the location (local intrahepatic, remote intrahepatic) and morphology of tumor recurrence were rewiewed. any other findings (perilesional hyperemia, thad etc) were recorded. results: complete necrosis was described in 31 lesions (77.5 %) at 1 month, partial necrosis in the remaining 9 lesions (22.5 %). local tumor recurrences (persistence) appeared in three patterns: nodular, halo or gross enlargement. 4/31 lesions (12.9 %) tumor-free at 1 month showed tumor persistence at 4 months. analysis of these cases showed that residual disease was probably masquerade by perilesional hyperemia, a common finding at 1 month. any lesion considered complete ablated at 6 month didn't show tumor recurrence in the following controls. 15 cases showed remote intrahepatic recurrence. pseudolesions as thad or a-v fistula were common findings expecially in early controls. the median follow-up was 13 months (range 6 -34 months). conclusion: knowledge of any ct findings after rf ablation is important but correlation with timing of follow-up is mandatory to avoid misdiagnosis. ct-guided aspiration and drainage of suppurative residual cavities after hydatidectomy a.i. ikramov, n.m. djouraeva; tashkent/uz purpose: to evaluate the efficacy of ct controlled intervention procedures in infected residual cavities after hydatidectomy. material and methods: ct-guided drainage was conducted on 62 patients with infected residual cavities after hydatidectomy. age varied between 14 and 76 years (mean 39.6). size of cavities ranged from 4.5 cm up to 12 cm. 76.3 % of cavities were located in the right lobe. in homogenous low density collections (+10 ± 18 hu) with thin walls an fna procedure was used to allow aspiration. in other cases (37) with high density collections (+25 ± 30 hu) we performed percutaneous drainage with 9 -18 f pigtail catheters. in 12 cases when cavity sizes were more than 10 cm we performed drainage via two catheters. results: in 93.5 % of patients ct-guided interventions allowed us to achieve a full recovery. repeated aspirations (from 2 up to 5) were conducted in 12 cases. one complication occurred (partial lung collapse). in 4 cases with ineffective drainage surgical conversion was needed. conclusion: ct-guided drainage procedures can be an effective method of treating suppurative residual cavities after hydatidectomy and may be an alternative to surgical treatment. in none of the patients was contrast material administered for mri. 90 patients also underwent cardiac catheterization within two months after mri examination. all patients were examined with percutaneous echocardiography. the results were compared to the anatomical situs and functional parameters. patients with abnormal coronary arteries were excluded from this study. results: in 70 of 90 patients (77 %) cardiac catheterisation and mri examinations provided results which led to the same clinical decisions. discordance between the two methods occurred in the assessment of pulmonary stenosis, in the evaluation of spongy myocardium, and straddling of mitral or tricuspid valves. overall there was no significant difference between mri and cardiac catheterisation in the pre-and postoperative evaluation of congenital heart disease (chd). conclusion: mri can be more beneficial in difficult patients especially with complex postoperative situs. cardiac catheterisation should only be performed when mri is questionable. percutaneous echocardiography still remains the first diagnostic tool in the evaluation of chd. saturday methods: 29 patients with an established echocardiographic diagnosis of tricuspid valve insufficiency grade ii or grade iii and planned combined reconstruction of tricuspid and mitral valve were prospectively included and examined with mri. sequences included continuous short axis truefisp cine sequences of the whole heart for volumetry of atria and ventricles, phase contrast (pc) through-plane flow measurement and cine sequences for assessment of valvular function. patients were randomized and treated either by devega annuloplasty or by carpentier ring implantation. in 12 patients, postoperative results were obtained 2 -3 months after surgery. results: 21 of 29 patients examined had an absolute arrythmia, thus impairing diagnostic quality especially for flow measurements. atrial volumetry in these cases showed no discernible contraction, and through plane flow measurement in the valvular plane showed no a-wave. in all patients, the degree of insufficiency was established with pc-flow correlated with 4 chamber view cine sequences and echocardiographic findings. in the follow-up-studies, no significant differences between the surgical treatment groups could be detected. conclusion: cardiac mri with pc-flow measurement and cine sequences seemed to be well suited for evaluation of atrial function and valvular morphology and function before combined valvular reconstruction. atrial volumetry is less useful than valvular flow measurement. cine sequences in the valvular plane allowed for sizing of the valvular ring. purpose: severe pulmonary regurgitation late after total correction for tetralogy of fallot leads to progressive right ventricular dilatation and an increased incidence of severe arrhythmias and sudden death. mri was used to assess the effect of pvr on rv function and pr. materials and methods: 26 adult patients who underwent pulmonary valve replacement in our institution between 1998 and 2001 were studied. mean age at initial repair was 5.5 ± 3.6 years and mean duration of follow-up was 30.0 ± 8.9 years. cardiac mri was performed 6.2 ± 3.7 months before and 7.7 ± 2.3 months after pvr. pulmonary regurgitation (pr), rv end-diastolic volume (rvedv), right ventricular end-systolic (rvesv) and rv ejection fraction (rvef) were measured. results: preoperative pr was 45 % (range from 25 to 64 %). after pvr, 20 out of 26 patients (77 %) showed no residual pr. rvedv decreased from 305 ml ± 87 ml to 210 ml ± 62 ml (p < 0.01) and rvesv decreased from 181 ± 67 ml to 121 ± 58 ml (p < 0.01 purpose: retrospectively ecg-gated 3d volume data from multislice spiral ct (msct) coronary angiography enables image reconstruction in diastolic and systolic phase of the cardiac cycle. thus, the objectives of our study were to determine lv ejection-fraction (lv-ef) from msct data set and to compare the results to cine magnetic resonace imaging (mri). materials and methods: 28 patients (60.6 ± 8.5 a) with coronary artery disease (cad) underwent msct coronary angiography (somatom volume zoom, siemens ag; 4 × 1 mm slice thickness, 120 kv, 300 ma; 140 ml nonionic cm, flow 3 ml/s). an additional cine mr study was performed at a 1.5 t mr unit (breath-hold flash 2d sequence, tr 80 ms, te 4.8 ms, flip angle 20°, slice thickness 6 mm). from msct data set retrospectively ecg-gated axial images were reconstructed in systolic and diastolic phase (acv reconstruction algorithm). enddiastolic and endsystolic lv volumes were determined from multiplanar reformations (slice thickness 6 mm) in short-axis image orientation (3d volumetry) to assess lv-ef. cine mri data was analyzed by using the implemented argus™ software in shortaxis image orientation. multiplanar reformations from msct data set allowed good delineation of endocardial and epicardial contours. systolic reformations showed slight motion artifacts in patients with a heart rate above 75 b.p.m. enddiastolic (r = 0.92), endsystolic (r = 0.90) lv-volumetry and lv-ef determination (r = 0.90) from msct data set demonstrated good correlation to cine mri. conclusion: in patients evaluated for cad, retrospectively ecg-gated 3d volume data set from msct angiography provides lv volumetric and functional data in good correlation to cine magnetic resonance imaging. after intravenous administration of contrast media (120 ml, 370 mg/ml iodine, flow 3 ml/s) scanning was initiated by bolus tracking and performed using a ecg-gated protocol at 40 mas and 120 kv, collimation of 4 × 4.0 mm, pitch of 1.2:4 and 0.5 s rotation time. the raw data was reconstructed with 50 % reconstruction pitch every 10 % of the r-r interval. images were transferred to external workstations for diagnostic interpretation of the chest (easy-vision, philips) as well as the cardiac data (alato-view, toshiba). studies were evaluated with regard to image quality. left ventricular function parameters were calculated and were compared with echocardiographic measurements using pearson's correlation coefficient and student's t-test for paired samples. results: diagnostic interpretation of the images was feasible in all patients without recognizable reduction in image quality. the radiation dose of the modified protocol did not exceed the exposure of a routine chest ct. retrospective reconstruction of diastolic and systolic cardiac images succeeded in all patients, only minor motion artifacts occurred in 9 cases due to cardiac arrhythmia. comparison between msct and echocardiographic measurements revealed no significant differences in the mean values of the function parameters (p < 0.05 in all cases) and a good correlation between both modalities: diastolic/systolic wall thickness r = 0.88/ 0.84; diastolic/systolic diameter r = 0.91/0.86 and ef r = 0.82. conclusion: using a modified protocol for routine chest scanning, msct allows a reliable, fast calculation of heart size and function without additional radiation exposure. b a c d e f 192 value and reproducibility of multidetector-row ct in the assessment of cardiac function c. herzog, a. mehtap, n. abolmaali, j.o. balzer, s. schaller, t.j. vogl; frankfurt a. main/de objective: to explore the value and accuracy of multislice-cardiac-ct in the assessment of functional cardiac parameters. materials and methods: 23 patients prospectively underwent multislice-ct (siemensplus4vz, germany) and invasive angiocardiography. using retrospective ecg-gating scanning parameters were 4 × 1 mm collimation, pitch 1.5 and 500 ms rotation time. based on a proportional reconstruction algorithm two datasets were obtained from the endsystolic, endiastolic phase respectively. effective slice thickness amounted to 2 mm, increment to 1 mm. measurements were performed in 10 mm thick slices cut orthogonal to the short axis of the heart. functional parameters determined were endiastolic (edv) and endsystolic ventricular volume (esv), segmental wall thickening and ejection fraction (ef). all measurements were performed using a mri-proved evaluation software (argus 2.3 wip, siemens, germany). invasive angiocardiography served as a reference measuring method using centerline analysis for functional evaluations. results: concerning evaluation of the ejection fraction, the wilcoxon test for matched pairs did not reveal significant differences between the two methods (tukey confidence interval: p = 0.95). however ca tended to overestimate ef by 4.5 %. the limits of agreement between ct and ca in ef were ±24.2 %. significantly higher differences between both methods were found for edv rather than for esv, again values being overestimated by ca. the best correlations for edv, esv and ef were obtained in patients with dcm. hypokinetic wall areas were detected with a sensitivity of 87.5 % (21/24), hyperkinetic regions with 92.6 % sensitivity (25/27). conclusion: multislice-cardiac-ct provides precise evaluation of functional parameters such as ejection fraction, wall thickening or endiastolic and endsystolic volume. to evaluate left ventricular volumes and systolic function using multirowdetector spiral computed tomography (mdct) with retrospective ecg gated biphasic data reconstruction in comparison to left ventriculography (lvg). methods and materials: 25 patients underwent routine mdct coronary angiography using retrospective ecg-gating. dedicated algorithms allowed for a temporal resolution of 250 ms. contrast enhancement was provided by administration of 120 ml of iopromid. to assess lv volumes and systolic cardiac function biphasic reconstruction at end-diastole (ed) and end-systole (es) was performed. data reconstruction timing was based on the ecg trace with placing the ed window at −100 ms before the r-wave and the es window to cover the t-wave. short axis views were reconstructed from ed and es data sets and the simpson rule used for calculation of ed volumes (edv), es volumes (esv), stroke volumes (sv) and ejection fractions (ef). data were compared to results of lvg. results: edv (r = 0.59) and sv (r = 0.56) showed good correlation with lvg, whereas esv (r = 0.88) and ef (r = 0.82) showed very good correlation. mdct significantly overestimated edv (md: 22 ± 36 ml; p = 0.0045) and esv (md: 39 ± 22 ml; p < 0.0001) whereas sv (md: −17 ± 27 ml; p = 0.008) and ef (md: − 17 ± 9 %; p < 0.0001) were significantly underestimated. mdct intraobserver variability ranged between 4 % and 6 %. conclusion: ecg orientated biphasic reconstruction of coronary mdct data sets allow for estimation of lv volumes and systolic function without the need for additional scanning. however, with current possible temporal resolution cardiac volumes are consistently higher in mdct and therefore sv and ef are underestimated in comparison to ventriculography. r. rienmüller, g. reiter, u. reiter, n. gagarina, a. ryabikin, b. schröttner; graz/at purpose: comparison of left ventricular volumes and muscle mass studied by electron beam tomography (ebt) and magnetic resonance (mr) evaluated by a geometry-based model as well as 3d methods in patients with suspected or known coronary heart disease. materials and methods: 40 patients were studied by ebt (ecg gated, multislice mode, 50 ms exposure time, intravenous contrast agent application) in long axis view and by mri (1.5 t field strength, 40 mt/m gradients, cine-truefisp) in 4 chamber view. enddiastolic (edv) and endsystolic (esv) volumes as well as left ventricular muscle masses (lvmm) were evaluated by a single-plane ellipsoid model. in addition mri short axis scans were performed in 23 patients to use simpson rule (argus software) to determine the above parameters. results: correlation between ebt and mri ellipsoid model is r = 0.87 (95 %-confidence interval is 0.76 to 0.93) for edv, r = 0.92 (0.85 to 0.96) for esv and r = 0.82 (0.68 to 0.90) for lvmm. correlation between ebt and mri simpson approach is r = 0.92 (0.81 to 0.96) for edv, r = 0.93 (0.83 to 0.97) for esv and r = 0.79 (0.55 to 0.91) for lvmm, respectively. relative standard deviations of the differences of single paired measurements varied in the range of 15 % to 25 %. conclusion: both ebt and mr methods may be used to determine left ventricular volumes and mass. the differences of standard deviations implicate the need to determine ranges of normal values for all evaluation approaches in clinical routine. background: several tumor-host biological factors seem to be valuable predictors for the prognosis of patients with squamous cell carcinoma (scc) of the head and neck, such as the peritumoral lymphocytic infiltration (pli) or a sharp tumor border. in particular, significant associations have been reported between the presence of pli and the absence of cervical adenopathy. preoperative biopsy specimens are often insufficient for an evaluation of these criteria. therefore, this study was performed to examine whether an elevation of peritumoral ct-values correlates with the presence of pli. moreover, correlations with present cervical adenopathy were obtained to check whether an elevation of peritumoral ct-values can be regarded valuable in predicting a prognosis. methods: in total, 45 patients with primarily resected scc were involved (pt1 = 8, pt2 = 13, pt3 = 9, pt4 = 15); 28 patients were pn-positive. all tumors were histopathologically analyzed regarding the presence of pli and a sharp or infiltrating tumor border. based on standardized ct examinations, repeated roi-based density measurements were obtained in vital parts of the tumor boundary. statistics were performed to find dependencies between pli, pattern of tumor invasion, cervical adenopathy, t classification, and ct densities. results: our results show that an elevated peritumoral ct-density is a highly specific and sensitive sign for the presence of pli. moreover, there were significant statistical correlations of present pli and elevated peritumoral ct-values with absent regional lymph node metastases. our results show that the evaluation of peritumoral ct-values appears to be a valuable prognostic co-factor, useful in the majority of patients (especially in patients without clinically detectable cervical metastases). clinical trial of the accuracy of a freehand and sensorless three-dimensional power doppler ultrasound system measuring the diameters, volumes and vascularity of malignant primaries of the neck m. keberle, m. jenett, d. hahn; würzburg/de purpose: the diameters, volumes, and vascularity of malignant primaries of the neck have substantial impact on staging and prognosis. the purpose of this study was to determine the accuracy of a novel freehand and sensorless three-dimensional power doppler ultrasound (3d pdus)technique in the assessment of these parameters. saturday method: 24 patients with squamous cell carcinomas of the neck underwent conventional ultrasound in b-and power doppler-mode (us), 3d pdus, and computed tomography (ct). diameters (sagittal, longitudinal, and transverse) and volumes of the tumors were correlated with hand-segmentated ct images, and tumor vascularity with us. results: 3d pdus and ct were highly correlated regarding diameters and volumes (correlation coefficients r = 0.98/p < 0.001 and r = 0.98/p < 0.001, respectively). 3d pdus and us highly correlated regarding vascularity (r = 0.92/p < 0.001). conclusion: although 3d pdus is based on a freehand and sensorless data acquisition, it appears to be as accurate as ct or us for measurements of diameters, volumes, and vascularity of neck malignancies. in spite of the excellent results it has to be noted that this technique requires a lot of training and its general use has to be employed with caution. prognostic significance of the revised tnm staging 1998 in patients with locally advanced nasopharyngeal carcinoma (ncp) a. kalogera-fountzila, c. kouskouras, n. fotiadis, e. vakali, g. sevas, c. christoforidis, l. papadopoulou, a.s. dimitriadis; thessaloniki/gr purpose: to evaluate the prognostic significance of specific anatomical structure involvement related to the revised tnm staging (1998) in patients with locally advanced npc. materials and methods: ct images of 132 patients, who were treated in our hospital with radiation or chemotherapy and radiation, were retrospectively reviewed. we analyzed, the ct scans performed prior and after the completion of treatment. results: there were 99 men and 33 women (median age 54 a). histology was undifferentiated carcinoma in 104 patients and scc in 28. the paranasopharyngeal space was found to be involved very commonly (98 %). degree 1 (d1) of paranasopharyngeal extension included 27 % of the patients, d2 39 %, and d3 32 %. the incidence of osteolysis was 33 % (clivus 20 %, foramen rotundum 5 %). cavernous sinus involvement was present in 15 % and carotid sheath in 38 %. retropharyngeal nodes were found in 42 % of the patients. t2b (65 %) and t4 (20 %) as well as n1 (29 %) and n2 (50 %) were more frequent. after the completion of treatment 81 patients demonstrated complete response. after a median follow up of 79 months 31 complete responders have relapsed. median time to progression for all patients was 37 months and the median survival 55 months. cox univariate analysis revealed, age, t1, t2, paranasopharyngeal extension and cavernous sinus involvement, as significant prognostic factors for survival. the revision of tnm staging, in our study, was useful in the prognosis of npc. the degree of extension of the tumor into the paranasopharyngeal space has to be considered in future tnm staging revisions. the prognostic value of mri in nasopharyngeal carcinoma s.s.a. lingawi, y.f. ragab, m. mansour; jeddah/sa purpose: to evaluate the role of mri in the prediction of nasopharyngeal carcinoma (npc) response to radiotherapy. material and methods: 52 npc patients had mri before and after radiotherapy to the local disease and to the nodal involvement. the mri assessed the size, site, signal intensity, enhancement pattern and extension of the regional disease in 25 anatomical sites. radiotherapy was delivered using x-rays of 4 -6 mv linac. purpose: head and neck tumors show considerable variation in perfusion pattern and biological behavior as well as response to chemoradiation. the high frequent acquisition of multislice ct data after a contrast medium bolus allows quantification of tumor perfusion. the aim of this study was to assess therapy-induced changes of tumor perfusion early after initiation of treatment. methods and materials: dynamic msct was performed in 22 patients with histologically proven head and neck cancer before and 3 weeks after initiation of chemoradiation. after bolus injection of 80 ml contrast medium, two 10 mm-sections through the largest tumor region were scanned for 40 s (siemens somatom volume zoom). the arterial input function was derived from the largest arterial vessel in the field of view. perfusion values were calculated using graphical analysis and displayed as parametric color coded images. standardized circular regions of interest were drawn on the area displaying highest perfusion. the tumor area was determined by the product of the maximum perpendicular diameters. results: at baseline, all tumor lesions were visible in parametric images by enhanced perfusion values (0.474 ml/min/ml ± 0.212), however the perfusion pattern was very inhomogeneous. the tumor area was 1007 mm 2 ± 736. three weeks after initiation of chemoradiation, tumor perfusion increased to 0.493 ml/min/ml ± 0.214, whereas tumor area decreased to 734 mm 2 ± 960. there was no correlation between perfusion and tumor size changes (r = 0.185). these results indicate that chemoradiation induces significant changes of tumor perfusion. standardized assessment is possible by perfusion msct. evaluation of radiation response with dynamic gadolinium-enhanced mri for head and neck cancer: correlation of hemodynamic changes with pathology n. tomura, k. omachi, k. kato, s. takahashi, i. sakuma, k. sato, j. watarai, m. sageshima; akita/jp purpose: to assess the diagnostic value of dynamic gadolinium-enhanced mr imaging in the evaluation of response to radiation therapy for head and neck cancer, and to correlate hemodynamic changes after radiation therapy with pathology. methods and materials: mr imaging was prospectively performed before and after radiation therapy in 26 patients with various head and neck cancers. a dynamic gd-enhanced study was performed using a fast spgr sequence. the maximum slope of increase (msi) on the time-intensity curve was displayed as a colorcoded image. ratios were obtained for peak time of enhancement (tpr) and intensity of maximum enhancement (mer), as well as the msi (msir) between the a c d e f 194 tumor and normal muscles. tpr, mer, and msir after therapy were compared with those before therapy. pathologic specimens after radiation therapy were acquired in 23 patients. histological grading of radiation changes was divided into grades i -iv. tpr, mer, and msir after therapy were compared with histological grades. results: a significant difference (p < 0.05) was observed in msir before and after therapy. although only 4 of 19 patients with grade ii (viable tumor cells present) or iii (only non-viable tumor cells) showed a msir < 2.5, all patients with grade iv (no tumor cells) showed a msir < 2.5. conclusion: msi quantitatively reflects the treatment response of radiation therapy for head and neck cancer. color-coded msi display is feasible for depicting hemodynamic changes following radiation therapy. scintigraphic prediction of resistance to radiation and chemotherapy in patients with head and neck tumors k. sato, n. tomura, o. watanabe, k. sasaki, j. watarai; akita/jp purpose: 99 tc-sestamibi (mibi) spect was compared with 201 tlcl spect for prediction of multidrug resistance and radioresistance in patients with head and neck tumors. materials and methods: eighteen patients (age range: 48 -76 a, mean 63 a) with tumors in the head and neck region (larynx in 6, oropharynx in 5, hypopharynx in 5, oral cavity in 1, maxillary sinus in 1) were evaluated with dual-isotope spect imaging using 99 tc-mibi (600 mbq) and 201 tlcl (111 mbq) at 15 (early) and 120 (delayed) minutes after the injection. quantitatively, tumor-to-background (t/b) ratio of tracer uptake was calculated in the early and delayed phases of each tracer, and retention index was calculated as follows: [(delayed ratio − early ratio)/early ratio] × 100 (%). using radiation and chemotherapy concurrently, the patients were classified into partial remission (pr) and no change (nc) groups. the relationship between therapeutic response and tracer uptake was analyzed. the detectability of resistance to radiation and chemotherapy was examined. results: the delayed ratio for 99 tc-mibi in the nc group was significantly lower (p < 0.05) than that in the pr group. there was no significant correlation between t/b ratio and tumor response using 201 tlcl. there were no significant differences in the early ratio and the retention index with respect to the tumor response in both 201 tlcl and 99 tc-mibi spect images. conclusion: 99 tc-mibi spect may be more effective than 201 tl spect for prediction of multidrug resistance and radioresistance in patients with head and neck tumors. purpose: to evaluate the need for doubling the effective mas in obese patients when performing low dose multi-row detector ct (ldmrdct) for the assessment of ureteric lithiasis. materials and methods: 106 consecutive patients with known body mass index (bmi), and clinical suspicion of renal colic were referred for a ldmrdct with the volume zoom (siemens) and 120 kv, 30 mas, 4 × 2.5 mm collimation, 0.5 s/rotation, pitch of 1.5. a second acquisition limited to the region with artefacts, using the same parameters but 60 mas, was obtained if necessary. slice thickness and increment were of 3 and 2 mm respectively. workstation based assessments of ureteric lithiasis were later performed by 3 radiologists who were unaware of the final results, and compared all clinical, surgical and radiological results available. effective dose was computer simulated and correlated with bmi. results: intra and interobserver agreements were greater than 0.88. the prevalence of ureteric lithiasis was 36 % and the accuracy of ldmrdct ranged from 95 to 98 %. effective doses for males and females were 1.2 and 1.9 msv respectively at 30 mas and 0.5 and 0.8 msv respectively in 20 patients with focused acquisitions at 60 mas. a bmi of 35 was found to be a threshold over which all patients needed 60 mas. conclusions: ldmrdct with 30 mas in patients with normal weight and with 60 mas in those with bmi greater than 35 is an accurate method for assessment of ureteric lithiasis, resulting in a dose similar to a 3 film ivu. mdct was used with 4 × 1.0 mm collimation, 100 mas, and 120 kv (ctdi = 11.4 mgy). we compared 3 reconstruction protocols (slicewidth/reconstruction interval: 1.5/0.7 mm, 3/1.5 mm, 5/2.5 mm). in addition, multiplanar reconstructions (mpr) in coronal plane were performed. 3 experienced readers analyzed each scan. presence, number, size of calculi and additional findings not associated with urinary stone disease were evaluated. results: mdct was positive for calculi in 107 cases independent of axial reconstruction protocols. coronal mpr's were helpful in evaluation of the precise stone location, although on 5 mm mpr's significantly less calculi were depicted. there was also no significant difference between performed protocols in evaluation of additional findings. conclusion: axial reconstruction protocols (1.5/0.7 mm, 3/1.5 mm, 5/2.5 mm) are equally suited in the diagnosis of urinary stone disease. coronal mpr's are helpful in the evaluation of precise stone location, however very small calculi may be missed on 5 mm coronal reconstructions. purpose: this study investigates the possibility of using combined blood oxygen level dependent (bold) imaging and diffusion weighted imaging to detect pathological changes in renal tissue induced by chronic renal hyper-filtration. the apparent diffusion coefficient (adc) and the t2*value within the four inner compartments of the kidneys of 17 rats with diabetes mellitus were compared with the results obtained from a control group of 16 rats. furthermore, the influence of the hyper-filtration on the blood-oxygen saturation was evaluated by comparing the t2*-values before and after the active tubular transport was reduced by injecting furosemide. results: all compartments of the diabetic kidney showed significantly (p < 0.05) lower t2*-values compared to the control group. in particular, the very low values in the outer stripe of the outer medulla (t2*-normal: 69.4 ± 10.9 ms; t2*-diabetic: 51.4 ± 13.9 ms) indicated either hypoxia due to hyper-filtration or renal blood volume changes. diffusion imaging of the same area showed significantly lower adc values (adc-normal: 1.45 ± 0.26; adc-oedema: 1.19 ± 0.25 [10 -9 m 2 /s]) that correlated with pathological findings on biopsy. conclusions: bold-contrast imaging appears to be able to depict tissue that is at risk of suffering from ischemia by revealing information about the balance between tubular workload and delivery of oxygen and may thus reflect a measure of the reserve capacity. the diffusion measurements reveal complimentary information -adc imaging is not sensitive to the current energy metabolism but reflects the pathological changes within the tissue. therefore adc-measurement might be a sensitive indicator of the severity of the induced ischemic lesions. virtual endoscopy of moderate or non dilated renal pelvis and calices with unenhanced 3d t2-weighted static fluid mr urography: a prospective study c. roy, c. vasilescu sr., l. solair sr., c. tuchmann sr., c. saussine sr., d. jacqmin sr.; strasbourg/fr purpose: to assess the accuracy and usefulness of virtual endoscopy (ve) by mr to study moderate dilated renal cavities. method/materials: 33 patients, having excretory tumours (n = 18), calculi (n = 11), and extrinsic compressions (n = 4) underwent mru (1 t philips) using t2 3d tse (tr/ete: 1800/500 ms, etl: 130, 80 slices, slice thickness: 0.6 mm, 256 × 256, fov: 240, ta: 4 min 21 s. ve were reconstructed using volume rendering and were interpreted (mip reconstructions, sources images) by two radiologists. correlation was made with conventional endoscopy/pathological findings. results: mean operator time was 20 min. endoluminal masses (up to 3 mm) were depicted by source images and ve, but missed by mip in 5 cases (2 tumours, 3 stones). morphologic assessment (smooth or irregular surface, position, number, surface extension) was more pertinent on ve than on sources images. blood clots (3 cases) were misdiagnosed as tumoural polyps. a smooth surface with abrupt margins was present for all stones and in 8 cases of tumoural process. an irregular surface was only present in tumoural processes. ve images did not allow evidence of intramural extension of the tumour. assessment of surface alone did not allow to differentiate between a calculus from a smooth polyp. conclusions: ve is easily feasible in moderate or non dilated renal cavities. it permits precise location and number of filling defects with delineation of the surface extension. it is not really time consuming and it seems to be a promising modality for optimal visualization of pelvo-caliceal pathology. in 2 patients the process progressed to abscess formation. retracted cortical scars in 9 patients reflected the healing phase. the ivu was normal in 16 of 18 patients with early lesions showing minimal delay of contrast excretion in the collecting system in 2 patients. conclusions: early cortico-medullary and nephrographic phase spiral cts are highly sensitive in detection of compromised parenchyma in the pyramids indicating papillary necrosis and thereby allow treatment at a time when the disease process is still reversible. methods/materials: we minimize patient dose by adapting the tube current to anatomy in two ways: a local projection angle dependent tube current modulation that minimizes the current for projections of low attenuation is combined with a global tube current control that adapts the tube current 360° mean level to the anatomic level. our method uses the projections currently scanned to "foresee" the behavior while scanning the next 180° segment. based on user-defined image quality parameters (selected by choosing the desired reconstruction kernel and the image noise standard deviation in hu) the tube current curve is computed. the approach has been tested in simulations and phantom measurements on a somatom volume zoom scanner (siemens, forchheim, germany). to further reduce the projection noise we have implemented a raw data-based maf approach. it locally smoothes the projection values of highest attenuation. maf is utilized as a preprocessing step during image reconstruction; the algorithms are implemented on the syngo explorer workstation (vamp, moehrendorf, germany). results: maf reduces image noise by up to 50 % and thereby allows for respective dose reduction. the combination of aec and maf allows to secure this reduction and to increase it further. conclusions/discussion: the combination of aec and maf allows to greatly reduce patient dose for a given image quality over complete anatomical ranges. the user simply specifies the desired image quality instead of setting the "mas-value" or other parameters. spiral imaging properties of a 16-slice ct-system t.g. flohr, k. stierstorfer, h. bruder, j. simon, s. schaller; forchheim/de purpose: the spiral imaging properties of a newly introduced 16-slice ct system (siemens, forchheim, germany) are evaluated. the 16-slice ct system provides a novel image reconstruction technique for multislice spiral ct (adaptive multiple plane reconstruction ampr) which takes into account the cone-angle of the measurement rays and allows for a free selection of the spiral pitch. for a given collimation (e.g. 16 × 0.75 mm), a variety of different spiral slice widths is available. we measured spiral slice sensitivity profiles, image noise in a centered 20 cm water phantom and dose (ctdi 100) as a function of the spiral pitch in the range 8 to 24. in our definition, the spiral pitch is the purpose: aim of our study was to establish a complete analytical description of noise and resolution in x-ray computed tomography (ct) and to predict trends for patient dose with respect to future developements in ct technology. we performed various simulations to validate the analytical predictions of pixel noise dependency on sampling distance in parallel beam geometry and the convolution filter. further, we measured image noise and high contrast resolution on a clinical ct scanner (siemens somatom volume zoom), thereby operating with all available scan modes. data sets were reconstructed with several convolution filters. results: noise variance at the center of a typical 20 cm water phantom varies with the inverse third power of the sampling distance. the tests with measured data showed that the convolution filter influences noise and resolution directly with variance depending linearly on the total area under the squared modulation transfer function (mtf). based on the measurements we were able to derive noise equivalent quanta for the ct scanner under examination. conclusion: due to the interdependence of noise and resolution, which is an intrinsic property of tomographic reconstruction from projections, it can be predicted that the dose necessary for constant noise level and unchanged object size will increase by a factor of x 3/2 when the resolution element is reduced by 1/x. this has direct implications for future ct systems that employ flat panel detectors with sampling distances down to 0.1 mm, thereby yielding a tenfold improved resolution compared to today's high-end ct scanners. method and materials: 50 patients with head and neck tumours were scanned using a multislice ct scanner (somatom volume zoom, siemens medical solutions, germany) with a high resolution protocol. reconstruction was performed using dedicated reconstruction software (impactir, vamp gmbh, germany) with the standard algorithm and the maf approach. we measured the noise for 6 anatomical regions at the level of the humeral heads. image quality, image noise and the diagnostic value comparing the standard reconstructions to the multidimensional adaptively filtered images was rated. results: the use of maf significantly improves image quality by reducing noise levels and by removing noise structures in the images. noise is reduced by 39 % on average, depending on the patient shape and the anatomical regions. the overall image quality is rated significantly better with maf in comparison to the standard reconstruction without any loss of image sharpness. especially the visualization of the cervicothoracic junction is drastically improved for all examinations, whereas the image quality of the upper neck is unchanged. the differentiation of anatomical and patholocical structures is enhanced in particular in the upper mediastinum and in the supraclavicular regions. the reduction of image noise, the improvement of image quality and the high diagnostic accuracy achieved with maf without any loss of image sharpness offers new perspectives to reduce patient dose. the reduction in patient exposure and image quality and noise were evaluated. to assess the noise, the standard deviation of the density of the trachea and the aorta were used. for the image quality a three point scale: worse -equivalentbetter. the mean dose reduction in the modulated scans was about 29 % (p < 0.001). in the area of the shoulder it was about 35 %, of the thorax and abdomen about 25 % and of the pelvis about 29 %. the noise of the images in the modulated scans was between 17 % (p = 0.01) at the level of the trachea bifurcation and 15 % (p = 0.06) at the level of the aorta bifurcation higher compared to the standard scans. with attenuation-based on-line modulation of tube current in 21 cases the diagnostic image quality was assessed as equivalent to the images using the standard protocoll. in 3 cases the image quality was worse. the use of attenuation-based online modulation of tube current allows a substantial dose reduction without a diagnostic relevant decrease in image quality. characterizing coronary artery motion using multi-slice ct m. vembar 1 , d.j. heuscher 1 , d.e. smith 1 , d.d. matthews 1 , s. chandra 1 , m. garcia 2 ; 1 highland heights, oh/us, 2 cleveland, oh/us purpose: 3d motion of proximal regions of the coronary arteries during the cardiac cycle were measured from image data obtained from multi-slice computed tomography to identify cardiac phases for optimal coronary imaging and to provide a realistic model for cardiac simulation. methods and materials: spiral ecg-gated contrast-enhanced 1 mm retrospectively reconstructed scans were performed on patients with heart rates ranging from 49 to 75 bpm using four-slice ct (marconi mx8000) at a temporal resolution of 250 ms. using landmarks along the proximal regions of the coronary arteries (left main and anterior descending (lm and lad), right coronary artery (rca) and left circumflex (lcx)), we obtained estimates of 3d position and velocity of these arteries during a cardiac cycle. identifying phases with minimum velocity, we correlated the image quality in these regions with the estimates obtained. results: motion characteristics varied depending on the artery, with the highest degree of motion being observed for rca. the points at which the lowest velocities occurred correlated well with the images in which the arteries could be best viewed. though more than one minimum was observed within a heart cycle for all arteries, the most consistent image quality was observed at 70 -85 % of the cardiac cycle. we were able to estimate the 3d motion of the three major coronary arteries. by identifying the phase in the cardiac cycle with lowest velocity, imaging of each coronary artery can be optimized. using these estimates in characterizing cardiac motion also provides realistic simulation models for future cardiac ct applications. and t2-weighted (haste) sequences in coronal (5 mm) and axial (4 mm) plane, followed by a coronal gadolinium-enhanced-acquisition with fat saturation (flash 2d in accordance to the recist criteria for evaluation of tumor response, target lesions were analyzed. t2 weighted, native and dynamic enhanced t1 mri were acquired. shape and character were described. size was taken as the clinical gold-standard for response criteria progression, stable disease or regress. 3 groups of patients were compared: response (n = 19); stable (n = 6); progression (n = 3). signal-to-noise (snr) measurements in t1 and t2 were obtained and correlated with size. results: preliminary results show that sti-571 changes morphological characters of the target lesions dramatically. in the response group decreasing lesions correlate with snr in dynamic t1 mri (r = 0.78; p < 0.01) but not in t2 (r = −0.13). in the progression group mri shows no significant correlation between size and dynamic t1 (r = 0.43) but in t2 images (r = 0.99; p < 0.01). these findings are concordant to the morphological change from solid to cystic or necrotic lesions. the information of mri in the early follow-up is helpful to individualise the management of patients in a timely way and to keep patients with increasing, but morphologic-changing tumor leasions in the study protocol. saturday material and methods: a prospective, randomized and double-blind trial was conducted in 50 patients randomly distributed into 2 groups: the control group (received only an oral solution) and a treated group (received the oral solution plus a subcutaneous injection of 20 mg of butylscopolamine 10 minutes before the mr examination). breath hold t1w gre images were obtained. quantitative image analysis was performed of the signal intensity of the liver and in background air anterior and lateral to the patient. a qualitative analysis of the subjective image quality was also done, and the adverse reactions were registered. results: the groups were homogeneous regarding age, sex and weight distribution. no significant differences in the signal intensity of the liver and in the incoherent noise measurements were found between both groups. gastrointestinal noise was statistically lower for the butylscopolamine group compared to the control group. there was also a statistical difference in the image quality between groups: optimal studies were only found in the butylscopolamine group. regading adverse events, there was non-significant differences between groups. to define the spiral ct findings in patients with superior mesenteric vein (smv) thrombosis and to compare these with surgical findings. material and method: abdominal ct examination of 12 patients with partial or total smv thrombosis were retrospectively reviewed by two radiologists. the level of the thrombus, patency of portal vein and intestinal findings were noted. these findings compared with the surgical findings in patients who were operated following smv thrombosis. results: in 10 of the 12 patients (83.3 %) there was complete thrombosis of smv and 2 patients (16.7 %) thrombus was partial and non-occlusive. in one patient with complete smv thrombosis, air was observed within smv and portal vein. portal vein was totally thrombosed in 8 patients (66.7 %), partially thrombosed in 2 patients (16.7 %) and patent in 2 patients (16.7 %). all 10 patients with complete thrombosis smv were operated and smv thrombosis was confirmed in these patients. small bowel resection was performed in 9 patients and sigmoid colon resection was performed in 1 patient. ischemic changes in small bowel could be detected preoperatively in 8 of the 9 patients who had small bowel resection. colonic wall thickening was observed in 5 patients including the patient with sigmoid resection. colonic ischemia was not confirmed by surgical findings in other 4 patients. conclusion: spiral ct can reliably demonstrate the presence, level and extent of the smv thrombus. spiral ct can also detect ischemic changes in the intestine and especially small bowel findings correlate well with the surgical findings. to determine whether ultrasound-guided delineation of the tumour bed after breast conserving surgery can improve the accuracy of conventional electron boost planning. methods and materials: 20 patients underwent post-operative radiotherapy plus electron boost following wide local excision for breast cancer, were selected at random. conventional boost planning was undertaken using clinical examination, surgical details and pre-operative imaging, and a standard boost field recorded. next ultrasound was used to delineate the tumour bed and an optimum boost field calculated using parameters including cavity size measured by ultrasound, 10 mm margin and 5 mm error margin. the standard and optimum fields were compared and potential areas of under and over-treatment of the standard field calculated. clinically acceptable values for these were set at 0 % of optimum boost area for under-treatment and 300 % of optimum area for over-treatment. the dmni visualisation and sampling management include galactography (ggr), mr and ultrasound ggr, fiber-ductendoscopy to investigate patients with intraductal abnormality. using ductal lavage (dl) and saline retrieval we tested material collected from breast ducts cells by methylationspecific pcr (msp) methylated alleles of cyclin d2, rar-, and twist genes detected in fluid from mammary ducts or endoscopically visualized dcis. northern blot analysis was performed for expression in epithelial cells. results: we were successful in intraductal cannulation, endoscopies and molecular testing, resulting in 96 % diagnostic accuracy. high-risk subjects were enrolled in two groups of a previous history of breast cancer and, a gail index score m 1.7 or brca 1/brca 2 positive status. of 502 eligible subjects suspicious/malignant cells were detected in 2 8 % (36/502). a comprehensive database created from imaging, interventions and molecular study protocols explored current pathology management. mr, ultrasonography dgr, and ductoscopy revealed papillomas but no high-grade intraductal lesions correlated with the lavage cytology. immunohistochemistry, histology grading, and the relevance of diagnostic certainty were considered. the dmni is feasible for early breast cancer conservation and prevention. evaluation of cellular samples for tumour biology and molecular imaging are the strongest predictors of the outcome. diagnosis, risk assessment, and prognosis emphasise the indicators for breast dcis, dmni and for survival rates. preoperative assessment of axillary lymph node involvement in breast cancer by ultrasound r. wenke, l. sanders, a. grosse, i. schreer; kiel/de purpose: to assess the sensitivity of ultrasonographic axillary lymph node detection and accuracy of different sonographic signs of malignancy in correlation with histopathological examination. material and methods: we examined 61 patients with preoperative histologically proven breast cancer. in 34 patients conventional axillary dissection was performed, 27 patients underwent excision of sentinel lymph node (s). clinical examination was performed independently. sonographic criteria included lymph node size, form, shape, presence or absence of hilar reflex, echogeneity, cortical rim thickening and vascularisation. results: ultrasonography detected 0 -7 lymph nodes (mean 2.66) were detected in level 1 and 0 -2 (mean 0.5) in level 2. histologically 2 -29 (mean 12.07) were detected in level 1 and 0 -9 (mean 3.01) in level 2. in 20 patients (31.74 %) axillary lymph node metastasis was detected histopathologically. sonographically suspicious lymph nodes were inferred in 23/61 (36.5 %) patients. in 4 cases (20 %) lymph node metastasis was missed, including one case of micrometastasis. clinical examination revealed only 9/20 (45 %) axillary metastases. sensitivity of ultrasonography was therefore 80 %, specificity 82.9 %, positive predictive value 69.5 % and negative predictive value 89.5 %. a satisfying prediction of axillary lymph node involvement can be achieved by means of ultrasonography. the lack of detection of micrometastases and of lymph nodes smaller than 5 mm is still a problem. the false positive rate (7) could possibly be decreased by an optimised combination of morphologic aspects. with a negative predictive value of 89.5 % ultrasonography can be a powerful tool in pre-selection for sln biopsy and the necessary follow-up of these patients. .5 g needle with appendant coil was used. after the procedure the distance from the coil artefact to the lesion was measured in two planes (sagittal, transverse). all lesions have been histologically proven by excisional biopsy 12 to 72 hours after the mr-guided localisation. results: localisation and excision of the lesion was successful in all cases. each mr-guided procedure lasted 45 -150 minutes (mean 66 min). 20 % (11/55) of the lesions were malignant. in most cases (75 %, 41/55) the coil was placed immediately adjacent to the lesion. the coil was placed up to 10 mm and 11 -15 mm (mean distance 1.8 mm, range 0 -15, sd 3.5) away from the lesion in 20 % (11/55) and 6 % (3/55) of cases respectively. conclusion: mr-guided localisation of only mr-detectable breast lesions with an embolisation coil system is easy to perform and results in a high precision. the coils are hocked into the tissue and detectable by specimen x-ray. the aim of our study was to investigate the feasibility and potential of cryosurgery in the therapy of breast cancer. materials and methods: 21 patients (60.5 ± 9.4 a) with histologically proven breast cancers underwent cryotherapy. after ultrasound-guided placement of the cryo probe in the tumour, two freeze/thaw cycles with a duration of 7 and 5 minutes respectively were carried out. the diameters of the occurring iceballs were measured sonographically. the patients were operated within 1 to 5 days later. the operation specimens were evaluated histologically. the maximum diameters of the iceballs were between 21 and 31.8 mm. the surface of the iceballs was well definable in ultrasound. 6 tumours with a diameter below 16 mm and one tumour with a diameter of 19 mm did not show rests of invasive cancer after treatment. 3 of them had dcis in the surroundings. subtotal necrosis was observed in 14 tumours which had a diameter of 16 mm or greater. conclusion: after these first cases cryotherapy seems to be promising in the treatment of small breast cancers. the low detection rate of surrounding dciscomponents in pre-interventional mri of breast cancer results in remaining dcis after cryotherapy. in larger breast cancers two or more cryo probes should be used to achieve larger iceballs. purpose: to examine the effectiveness of a potential minimal-invasive method for the elimination of breast tumours by magnetic heating. methods: a human adenocarcinoma (mx-1 cells) was implanted into 16 scid mice. after intratumoral application of iron oxides (7 ± 3 mg per 350 mm 3 tumour tissue; average particle diameter: 10 nm), mice were exposed to an ac magnetic field (4 min; amplitude: 6.5 ka/m, frequency: 400 khz). 4 controls did not receive an iron oxide application. temperatures at the tumour and rectum were monitored and quantified as total heat doses (thd = temperature × time). dna damages were investigated using the comet assay (percentage of dna in the tail). a calibration curve between temperature and dna damage was determined. results: intratumoural temperatures ranged from 59°c to 96°c (thd from 105 to 315°c × min), the percentage of the total cellular dna in the tail was of 67 ± 23 %. in controls, no significant temperature elevation was observed and only 8 ± 2 % of the dna was found in the comets tail. in vivo and in vitro experiments showed a temperature threshold for cell destruction of 55°c. conclusions: reliable tumour cell destruction is possible by magnetic heating. methods and materials: mr images of the eurospin-to4 phantom were obtained using standard 2d se, 2d tse, 2d-spoiled gre and 3d mpr sequence types and evaluation of spatial and high contrast resolution was performed. all images were transferred to a standard pc workstation utilizing a dicom protocol. these images were then converted to the four most commonly used image file formats on the web: tiff, jpeg, gif and png. a total number of 136 images using different file formats and compression ratios were evaluated. image quality a c d e f 206 (iq) was evaluated by consensus by three reviewers utilizing a 5 point grading scale (1: poor, 2: moderate, 3: good, 4: very good, 5: excellent). image size to image quality ratio's (is/iq) were also calculated for all images. results: minimal is/iq was obtained when working with low compression (20 -30 %) jpeg images and moderate to high compression (60 -80 %)png images. high compression jpeg images showed severe image distortion while maintaining acceptable spatial resolution. this problem was not encountered in png images. is/iq was quite satisfactory on gif images. tiff images were of excellent quality but with larger image sizes. the optimum compression levels were tested on routine brain and abdominal mr examinations with results comparable to phantom measurements. conclusion: the study determined the acceptable compression levels for jpeg and png mri images used for educational purposes on the web. the newly introduced png format offers high image quality with acceptable file sizes. • checking about 1000 homepages of international radiological institutes, • inquiries to eufora -a popular mailing list, • asking about 100 medical students and residents in radiology. the 100 favorite on-line teaching programs in radiology were evaluated by representatives of the target group -100 students and 10 residents in radiologyaccording to user friendliness, didactic, interactivity, content and layout. results: we created an on-line list (radlist) of categorized and evaluated on-line teaching files in radiology, ranked according to user friendliness, didactic, interactivity, content and layout. conclusion: radlist (http://www.radlist.uni-erlangen.de/) is a useful on-line tool for students and residents in radiology to find their way within thousands of internet teaching files in radiology. radlist will be updated regularly. open connectivity and interoperability of web based medical teaching file servers using xml-based web services t. schaaf, j. hohmann, k.-j. wolf; berlin/de purpose: support of automated access to dynamic webbased medical teachingfile-servers. the connectivity and system interaction should be realized with the help of consistent methods based on common standardized concepts and protocols. methods and materials: by using soap (simple object access protocol) as the fundamental information exchange protocol in cross-language and cross-platform communication, the herewith provided webservices offers a consistent application interface to dynamic generated data of our medical teaching-file-server. soap is an standardized (http://www.w3.org/tr/soap) and xml-based (extensible markup language; http://www.xml.org) protocol consisting a framework for describing the exchanged messages and their processing, rules for expressing instances of application-defined datatypes and definitions for representing remote procedure calls and remote responses. we have extend our multimedia teachingfile-server, built with no-cost common software components, with an php-based (php: hypertext preprocessor; http://www.php.net) soap-adaptor and could thereby offer the dynamic medical teaching-knowledge encapsulated in a medical webservice. since soap could be combined with existent common transport protocols such as http (hyper text transfer protocol), our webservice is seamless internet accessible. results: the php-based soap-adaptor handles the application-driven xml-defined messages addressing the server-stored procedures for accessing the medical teaching-file-data via the inter-/intranet. thus this application interface addresses the same database functions used by the beside existent human-computer-interface, our webservice provides the same programmed security issues. conclusion: the soap-ready extension of our multimedia dynamic webbased medical teaching-file-server supports the application layer interoperability through a consistent xml-based interface. thereby the provided medical knowledge could be easily accessed and integrated in decentralized, webbased knowledge-and workflow application frameworks in healthcare environments. building up a teaching system out of the radiological workflow a. schroeter 1, 2 , k. annacker 1, 2 , t. geisbe 1 , m. kroll 1, 2 , j. martin 1 , j. holstein 1 , d.h.w. groenemeyer 1 , h.g. lipinski 2 ; 1 bochum/de, 2 dortmund/de purpose: the aim is the development of a radiological teaching system, whose contents of the learning contexts are inferred directly from the radiological work routine. this system pulls dicom images, dicom presentation states and findings out of the ris and pacs to transfer them to an html learning text whitin an embedded dicom viewer. methods and materials: a radiologist will select a few images and send them to the teaching system together with the finding and presentation states. a learning text will then be extracted out of the finding. this text will be converted to an html page with an embedded java-based dicom viewer to visualize the medical images. within the html text, buttons will provide a context-referred view of the images via dicom presentation states. so all that radilogists will have to do, is to perhaps correct some text phrases or the layout. results: the result is a teaching system with its contents pulled out of the normal radiological workflow making it possible to transfer dicom images, presentation states and findings to interactive learning texts. dicom images can be freely manipulated and presentation states can be applied to achieve a context-referred view of medical images. conclusion: the efficiency of learning can be increased by being as close as possible to the real-life diagnostics and by making the images available for free manipulation. building up a learning case is quite easy because image data and views on these images already exist. webbased knowledge transfer in a radiology department for scientific and educational purposes j. hohmann, t. schaaf, k.-j. wolf; berlin/de purpose: to implement a basic system which allows: (1) scientific chats and interactive lectures, (2) live broadcast of scientific events, (3) access to teaching-files and examples in a medical image database. materials and methods: (1) the hyperchatsuite by fh software (www.fhsoftware.net) is freeware and allows common and private chat rooms as well. (2) for evaluation purposes of the live broadcasting the no-cost realsystem server basic (www.realnetworks.com) was installed which allows up to 25 concurrent users the access to the delivered data stream. realsytem servers supports up to 45 media types and different operating systems. (3) part of the teleteaching/-learning system is an access to teaching files and the medical database (introduced on ecr 2000). the medical database consists of open source components (mysql, www.mysql.com; apache, www.apache.org; php, www.php.net) and is in use since last year. results: (1) we conceptualised a lecture together with scientific groups in the usa regarding the principles of digital imaging. after performing initial testlectures this lecture will be held next term at our radiology departement. (2) the benjamin franklin contest 2001 was availaible as our first live broadcasted videostream of about 4 hours. (3) due to the flexible structure of the former medical image database the added functionalities with teaching files consisting of multi-media-data were easily achieved. conclusion: since the parts of the system fullfills our expectations in first trials we now have to get more experience during next term or year to find out about the advantages and disadvantages and to yield the necessary changes. current concepts in digital conference communication in radiology m.v. knopp, h. von tengg-kobligk, f.l. giesel, p.l. choyke; bethesda, md/us purpose: clinical conference of cases as well as group conference with clinician or research partners is one of the bases of radiological interaction in academia and hospitals. while pacs and all the advances in telecommunication have changed tremendously our work, these concepts have not been embraced sufficiently in conference communication. our group based in a major academic and research institution investigated the different capabilities available such as telephone (isdn) based systems picturetel, direct satellite links and web based approaches. the different systems were assessed using objective criteria such as speed, costs, image resolution etc. and subjective criteria using a questionnaire. results: many radiologists feel at unease using digital conferencing mostly due to lack of experience. after training, it was readily used and a code of conduct was helpful for training purposes. while image quality and speed is still superior with commercial systems, innovative uses of web based applications with concurrent b-0439 09:35 improving the workflow with central scheduling: introducing a "rad call center" p. gocke, a.p. bruckmann, j.f. debatin; essen/de purpose: to improve the workflow in a radiological department with central scheduling, by introducing a radiological call center (rcc) method/materials: with a separate module of our ris (radiological information system) we were able to introduce a central electronic scheduling. appointment schemes were created for all work places, time table-based or list-based. one central telephone number was assigned to the newly etablished radiological call center. rules were created for scheduling, and staff were trained by a telephone and marketing coach. results: with central electronic scheduling, the task of scheduling is removed from every workplace and focused on one center with specially trained staff. this center is an efficient tool offering flexible workload balancing. information about daily workload is available for every employee. especially helpful in workflow optimization was the introduction of 'virtual work places' were patients can be scheduled for urgent investigations and directed to the next available appointment. conclusions: central scheduling by a radiological call center improves workflow and enables a department to (re)direct patient flow and balance work load. inherent advantages were optimis ation of investigation strategies and avoidance of double/unnecessary investigations. acceptance of referrers is good (only one central telephone number!), especially when offering add-ons like a radiological advice service. work flow analysis using process simulation in a routine ultrasound division c. gillessen, u.k.m. teichgräber, j. ricke; berlin/de purpose: (i) to evaluate a routine ultrasound workflow by means of project graph technique (pgt) and process simulation (ps) and identify ways to redesign and improve it. (ii) to evaluate pgt and ps for significance and feasibility in workflow management. a workflow analysis of a routine ultrasound division was performed by: 1. observation and definition of work steps to perform an ultrasound examination. 2. time measurements of 500 activities using a software tool for parallel work step measurement. 3. designing a project graph. 4. pgt: calculation of operational measures. ps: generating operational measures by applying process simulation on network plan. 5. identification of reasons for delay. 6. process redesign and re-evaluation. results: average realistic examination time for abdominal ultrasound was 44 min 52 s. average simulated examination cycle time with 3 ultrasound devices, 3 examiners and 2 technicians was 10 min 42 s. by increasing personnel, average cycle time was reduced but average personnel slack time increased. major structural work flow deficits were not identified under given circumstances. replacing paper and film work associated activities with activities as expected with an electronic archive reduced total work effort by 16 %. major advantages of simulation over npt were the ability to consider cycle overlap and to calculate cycle times. conclusions: (i) under given circumstances, the observed work flow showed high efficiency. major effort reduction by the introduction of an electronic report and image archive is predicted. (ii) process simulation is superior to pgt in evaluating workflows and anticipation of the effect of variations. is speech recognition the best reporting method for any case? p. gocke, c. hogh, e.r. gizewski, j.f. debatin; essen/de purpose: to decide in which cases speech recognition is the best method for reporting, and in which cases other existing methods should be preferred method/materials: in our department, 'instant reporting' is performed by residents typing preliminary reports into a ris (radiological information system) based reporting module using a special ms winword 2000 environment. additionally, we have reporting stations equipped with a state-of-the-art speech recognition system (latest dragon naturally speaking professional with radiological dictionary). to compare both methods, the residents hand-stopped the time needed for generating the first, preliminary report for three different types of investigations: ct, skeleton and chest x-ray. results were discussed and tips were delivered for developing efficient auto-texts and macros. results: for ct, speech recognition was faster (4 min 49 s ± 1 min 44 s) than typing (5 min 10 s ± 1 min 32 s). for chest x-rays, there was no significant difference (speech recognition: 1 min 58 s ± 46 s, typing: 2 min 5 s ± 1 min 6 s). for skeleton x-rays, typing was faster (1 min 35 s ± 44 s) than speech recognition (2 min 4 s ± 58 s). conclusions: in many cases, speech recognition is an adequate technique for radiological reporting, but especially in cases of short and standardizable reports an autotext-and macro-based reporting technique is faster and more convenient. long-term experience with speech recognition of more than 240000 dictations t. ybinger, w. kumpan, f. karnel; vienna/at purpose: x-ray departments are increasingly under pressure to reduce costs and save time. this paper presents the experience of many years of using speech recognition to optime our services. the kaiser-franz-josef-spital in vienna is a 720-bed hospital which specializes in oncology and infectious diseases. a speech recognition system for producing reports was integrated into our ris early in 1998. since then the system has proved itself by completing more then 240000 dictations. we also investigated the efficiency increase achieved by using the system and its influence on our work processes. results: speech recognition is used to transcribe more than 98 % of our reports. we have been able to reduce the turnaround time for our reports by 35 % to an average of 8 hours. despite a high number of trainee doctors, 95 % of all reports are signed within 24 hours. our secretaries save approximately 40 % transcription time, and we have become more flexible in the use of typists. the average recognition rate is around 95 %, with some doctors clearly achieving even better results. however, of even greater importance to the workflow is an optimal integration into our ris. a c d e f 208 conclusion: speech recognition has fully established itself in our hospital and is routinely used for practically all dictated reports. this has considerably increased the productivity of our typists and clearly reduced the turnaround time of reports. we are convinced that speech recognition will soon make its entry as state-of-theart technology in numerous other institutes. when the bladder was filled of contrast-material-enhanced urine, the patient in supine position was asked to urinate. during the micturition t1-weighted spoiled 3d-gradient-echo acquisitions on sagittal plane were performed (acq.time: 12 s). these acquisitions were post-processed with mip algorithm. 15 patients performed retrograde and micturating conventional cystourethrography in the month preceding mri. one patient was unable to perform the exam because of the inability to urinate in the supine position. results: we always obtained perfect evaluation of the male urethra with voiding mr-cystourethrography. the visualization of the urethra with mip reconstructed images was considered comparable to that obtained with conventional cystourethrography. the analysis of 3d sagittal scans allowed a better evaluation of the urethral strictures in comparison with conventional cystourethrography. we detected 10 cases of bladder neck obstruction, 12 cases of urethral stricture and 3 case of benign prostatic hypertrophy conclusions: voiding mr-cystourethrography demonstrates the morphology of the bladder neck and urethra during the micturition and can a substitute for standard retrograde and micturating cystourethrogram, avoiding radiation exposure to the gonads. patients underwent combined mri and 3d-mr spectroscopic imaging (mrsi) of the prostate after up to (n = 16) or more than (n = 20) 16 weeks of ht. pretherapeutic psa levels (11.8 ± 10.1 vs. 14.2 ± 10.0 ng/ml) and gleason sums (6.2 ± 1.2 vs. 6.4 ± 1.0) did not differ significantly. posttherapeutic psa was dichotomised as being up to or more than 0.20 ng/ml. ht of more than 16 weeks was significantly associated with psa up to 0.20 ng/ml (15/20 vs. 3/16 patients, p = 0.002, chi square test) and loss of citrate from the peripheral zone of the prostate (15/20 vs. 5/16 patients, p = 0.022). psa exceeding 0.20 ng/ml was associated with prevalence of citrate (12/18 vs. 4/18, p = 0.020). citrate was detected at mrsi in 4 patients with psa under 0.20 ng/ml, including 3 of 4 patients on monotherapy with lhrh agonists for more than 16 weeks. in conclusion, this study finds an association between psa decrease and loss of citrate from the peripheral zone of the prostate in patients on ht for locally confined pca. there is a hint of dissociation of psa loss and detectable citrate in patients on longer-term lhrh agonist monotherapy that warrants more extensive analysis. longitudinal follow-up with combined mri and 3d-mr spectroscopic imaging of patients on hormone therapy for locally confined prostate cancer u.g. functional assessment in non radicular low back pain patients with an ultrasound-based 3d-topometry-system (zebris®) and radiographic motion analysis of the lumbar spine correlated with the results of a self administered health status questionaire a. petrovitch, s.o.r. pfleiderer, t.u. schreiber, w.a. kaiser; jena/de purpose: non-radicular low back pain (lbp) is a leading cause for compensation in industrialised countries with high socioeconomic impact. in the past it was mostly described by structural deficiencies, but not by disturbance of functional spinal segments or the instantaneous axis of rotation. methods: 45 out-patients (age 22 -85 a; 22 males, 23 females) with suspected vertebral instability were included. general health and orthopedic status was evaluated at admittance. health status was evaluated using the self-administered sf-36 questionnaire, which includes eight scales of functional health. a self administered pain score was evaluated using the visual pain analogue scale (vas). the motion of the lumbar spine was analysed in lateral and ap projections using radiographs and in all three main axis of rotation with the topometric motion analysis system. results: compared to norm-based population scores for europe and north america, values are reduced below one standard deviation in almost all items using the sf-36-questionaire. the items body pain and physical function are decreased by two standard deviations (sd) and role physical has a score reduced by more than three sd. where heavy pain was reported, the correlation between disturbed physical and mental scores was high (r = 0.8950; p < 0.001), but no correlation to radiographic spondylolisthesis could be observed. conversely, a good correlation between reduced physical score (sf-36), pain and disturbed instantaneous axis of rotation was observed in 3d-topometry (r = 0.6820, p < 0.01). conclusions: this study supports a new concept in functional assessments of non-radicular lbp using ultrasound-based 3d-motion analysis. spinal pm msct-studies of 45 consecutive patients, assessed for non-radicular, multisegmental cervical and/or lumbar pain syndromes by myelography and pm ms-ct were reviewed for additional information when compared to the myelographic findings alone by two neuroradiologists. all pm msct studies had been performed on a four-slice ct scanner using the following parameters: slice thickness of 1 mm, reconstruction increment of 0.7 mm, field-of-view 15 (cervical)/20 (lumbar) cm. subsequently data postprocessing to multi-and curviplanar reconstructions was performed. in addition, the radiation exposure was assessed by calculating the effective dose values. results: msct yielded additional diagnostic information, termed as clinically significant in 60 % of the studies. due to the resulting voxel size of about 0.4 × 0.4 × 0.8 mm multiplanar imaging of the spinal pathology in all planes with excellent image quality was possible. typical image artefacts at the cervico-thoracic junction as well as due to metallic implants were significantly reduced. the effective dose amounted to 2.8 msv (cervical spine) and 6.2 msv (lumbar spine). conclusion: pm spinal ms-ct is a useful tool in assessing multisegmental spinal pathology in patients in whom mr imaging is either contraindicated or not compatible with the clinical findings. in terms of radiation exposure, careful restriction of the scan area to the clinically relevant segments seems to be more important than the type of helical ct, i.e. single-vs. multi-slice. results: there were five women and one men, mean age of 66 years. a diabetes mellitus was present in half cases. mean delay between vertebral collapse and the infectious syndrome was 12 days. staphylococcus aureus, e coli, salmonella enteritidis, were the identified pathogens. bone biopsy isolated the pathogen in two third of cases, and was the sole positive sample in one third of cases. a thoracic vertebra was involved in three cases, a lumbar one in three cases. lysis of the spongious bone associated with a soft tissue mass thicker than 8 mm was observed in five cases. two patients died from consequences of the disease. conclusion: we want to highligh a seemingly unusual entity to avoid a radiological diagnostic pitfall. calcified cervical intervertebral disc in children -radiological findings v. jevtic; ljubljana/si purpose: to describe radiographic, ct and mri features in a relatively rare entity of unknown etiology, calcified intervertebral disc in children (cidc). material & methods: 12 patients (7 males, 5 females, mean age 9 years) with clinical signs of limited movements, stiff neck, torticollis and pain were radiologically investigated. the patients were imaged using radiography, functional radiographic examination, ct and mri. plain films, ct and mri were analysed in a qualitative fashion. results: radiography demonstrated a calcified nucleus pulposus which was flattened, oval or round. in 10 patients more than one disc space was calcified. additional radiographic findings included widening or narrowing of the disc space and moderate flattening of the vertebral bodies. in 6 cases functional abnormalities were revealed at the level of disc calcifications. ct clearly demonstrated hyperdense disc calcifications with posterior disc protrusion in 4 cases and extrusion into the epidural space in one patient. calcifications were shown as signal void areas by mri. there were also discrete signal intensity changes of bone marrow below the endplates. conclusion: cidc may be diagnosed using plain radiography, ct or mri. the advantage of ct examination is in exact demonstration of clinically important protrusion or extrusion of the calcified nucleus pulposus. sacroiliitis in children with spondylarthropathy: use of dynamic mr imaging to detect the therapeutic efficacy of intraarticular corticosteroid injection t. fischer, b. hamm, m. bollow; berlin/de purpose: our aim was to prospectively study the therapeutic efficacy of ct-guided intraarticular corticosteroid instillation of inflamed sacroiliac joints (sijs) in children with juvenile spondylarthropathy (spa) and to evaluate the role of mri as a procedure for establishing the indication and the therapeutic follow-up. method/materials: a total of 69 ct-guided corticosteriod injections of the sijs were performed in 42 children with inflammatory back pain (ibp): 27 bilateral, 15 unilateral. forty milligrams of a crystalline longacting corticoid was instilled in each inflamed joint. all 42 patients underwent continuous clinical follow-up at 10 week intervals after corticosteroid injection to a maximum of 18 months. intensity of back pain before and after the intervention was graded on an visual analogue scale from 0 (no pain) to 10 (very severe pain). dynamic contrast-enhanced (gd-dtpa, 0.1 mmol/kg body weight) mri with quantitative determination of contrast enhancement was performed in all patients before the intervention and 8 ± 4 months after therapy. results: 37 of the 42 study patients (88.1 %) showed a statistically significant abatement of subjective back pain from 8.4 ± 1.4 to 3.3 ± 2.2 (p < 0.05) at 1.5 ± 1.0 weeks after therapy, and this improvement lasted for 12 ± 6 months. the percentage contrast enhancement at dynamic mri showed a reduction from 110.5 ± 52.3 % before to 53.4 ± 38.1 % after intervention (p < 0.01). conclusions: dynamic mri proved to be a reliable non-invasive tool for the assessment of inflammatory activity of sacroiliitis and the response to the therapy in patients with juvenile spa. congenital lesions of the lumbar spine: ct depiction and significance d. passomenos, g. mantzikopoulos, g. giannikouris, i. staikidou, c. pikoulas, s. ispanopoulou, c. dayiada; athens/gr purpose: congenital lesions of the bony skeleton are not uncommonly detected incidentally by plain films. moreover, posterior elements due to their complex anatomy and orientation are poorly visualized on plain films. in acute trauma setting, in order to exclude a fracture, further investigation without the problems of superimposition is needed. we retrospectively evaluated the files of 873 patients examined to our diagnostic department over a period of 9 months. patients ranged in age from 12 to 44 a. most of them presented with a history of minor recent trauma or had a vague intermittent back pain. cases of spinal cord lesions in association with bone dysplasias were excluded from the study. plain films were mostly suggestive of congenital lesions but further investigation with ct to ascertain the diagnosis was requested. scans consisted of consecutive 3 mm cuts through the questioned area of the spine. obtained scans were imaged at softtissue and bone windows. sagittal and coronal reformatted images were also obtained. we could locate a total of 18 cases of unilateral spondylolysis, butterfly vertebra, posterior limbus vertebra, hypoplastic or absent facets associated with hypoplastic arch, spina bifida and pseudo-arthrosis of transverse process. the lesions were mostly located at l5 -s1 level. purpose: to describe characteristics on mr imaging of radiation osteitis of the pelvis in patients who had received radiation therapy for gynaecological tumours. material and methods: 9 women (mean age: 67.5 years) with gynaecological tumours who developed radiation osteitis where examined with radiography, computed tomography and magnetic resonance imaging (mri). on a 1.5 t system we used plain t1-and t2-weighted sequences and contrast enhanced t1-weighted a c d e f 212 sequences with and without fat saturation. mri was performed at different distances between the end of radiation therapy and developing pain. mr images where correlated with the results of clinical examinations. results: depending on the time from radiation therapy, radiation osteitis showed different signal intensities. the acquired images suggest that signal changes in t2 weighted images as well as the different enhancement behaviour of radiation osteitis might be dependent on the time from radiation therapy. best visualisation of regions with even low contrast enhancement was achieved with fat-saturated sequences. computed tomography showed increased density in the affected regions corresponding to osteosclerosis. in all cases at least one iliosacral joint was affected with different topographies in sacral and iliac bone. conclusion: mri is helpful in detecting and characterizing orn and eases the demarcation from secondary manifestations of the known malignancy. changes in signal intensity, based on histopatholocical tissue changes, might make a chronological classification possible. the purpose of this on going study is to evaluate the usefulness of whole body mri in the diagnosis of the inflammatory myopathy seen in polymyositis and to assess the disease extent in affected patients. materials and methods: eight patients with laboratory, muscle biopsy and electromyographic evidence of probable or definite polymyositis were referred for imaging. an additional patient was referred for mr to identify a potential site for muscle biopsy. 16 coronal turbo stir images were taken in each of three or four body regions (tr/te/ti; 2400/40/160 ms). acquisition time per region was 3 minutes 30 seconds, with a total scan time of approximately 30 minutes. results: mean patient age was 43.5 years (32 to 57 years). one patient with "probable" polymyositis by conventional criteria had a normal scan. one patient with polymyositis had inflammation in a myofascial distribution in the gluteal region. two patients had florid symmetrical myopathy involving proximal upper and lower limb girdle muscles, psoas, intercostal muscles and neck flexors and extensors. in addition, mild involvement of the calf muscles was also seen. three patients had more patchy asymmetrical muscle involvement, but also demonstrated involvement of psoas, intercostal and neck muscles. conclusion: whole body mri gives a much more extensive assessment of muscle involvement in polymyositis than the conventional mr protocols used to image these conditions. extensive muscle involvement of psoas, intercostal, neck and distal limb muscle groups were diagnosed. these muscle groups are not imaged on standard sequence protocols. the posterior half of the lateral tibial plateau and the lateral half of the medial femoral condyle were statistically more frequently involved than their corresponding halves (p < 0.0001 for both parameters). the bare area of the medial, but not of the lateral tibial plateau is more frequently involved than the meniscus-covered area (p < 0.0001). the meniscuscovered area of the lateral tibial plateau was more frequently involved than that of the medial tibial plateau (p < 0.0001). results: mri allowed a good identification of the sites of osteochondral cylinder remotion ("donor" sites) and of the treated lesion sites at the 5 month mri. small ferromagnetic particles artifacts due to surgical instruments were often present, with limitation of diagnostic value, in particular the "gap" regions between cylinders and health tissue were not individuated. only the oldest two patients showed mild inflammatory signs (intraarticular fluid collection, synovial hypertrophy) at the 5 and 10 month mri. no case of donor site degeneration was seen at the 5 and 10 month mri. in all cases, the sites of treated lesions showed regularity of subchondral bone and the fillling-repair of donor sites at the 10 month mri. evaluation of the chondral portion was very difficult due to artifacts. conclusions: mri allows a good evaluation of knee chondral implant healing, especially of the subchondral bone repair and seems to be a good diagnostic tool, avoiding invasive examination. results: based on the statistical analysis of the ultrasonographic measurements, diagrams and tables were created. the study of these data concluded that: • articular cartilage thickness is the same in both knees. • articular cartilage of the knee is thicker in boys. • cartilage thickness reduces with age. • the rate of diminution is higher during the first six years of age and lower after the age of six years. • there is negative linear correlation between cartilage thickness and somatometric parameters. • body height is the somatometric parameter that has the strongest correlation with articular cartilage thickness. conclusion: equations, diagrams and tables of normal articular cartilage thickness of femoral condyles in children, measured with ultrasonography, represent a precise method for cartilage growth estimation. these equations, diagrams and tables afford an objective way to compare normal articular cartilage thickness with the thickness noted in various pathologic conditions affecting articular cartilage in children. opportunities for the use of high frequency ultrasound (10 -13 mhz) in the diagnosis of degenerative-dystrophic changes of the knee joint n. lordkipanidze, d. tatishvili, m. lortkipanidze; tbilisi/ge the purpose of study was to define diagnostic criteria for degenerative-dystrophic damage to the knee joint according to the stage of disease using high frequency ultrasound. the investigations were performed on the "siemens sonoline elegra" ultrasound system using siescape panoramic imaging with a 7.5 -13 mhz frequency linear transducer. a group of 956 patients, 10 -75 years of age, with complaints of knee joint pain were included in this study. 738 patients were diagnosed as having knee joint degenerative-dystrophic changes. the early stage is characterized by a normal or decreased hyaline cartilage thickness of the femoral condyle. ultrasound studies detected increased echogenity of the cartilage surface, existence of freely floating 0.4 -0.6 mm crystals in the joint cavity and suprapatellar synovial bursa. the same type of crystals, in addition to insignificant synovitis, were found in sportsmen and dancers who exercised vigorously. in the case of difficulties in examining the lateral patellar and medial inferiorlongitudinal surface the existence of crystals was revealed during its maximum displacement from the condyles of the femur. in severe cases, examination revealed marginal joint osteophytes, margin skipping of cartilage surface, discontinuous contour of the femoral condylar bony surface, narrowing or complete disappearance of joint space and the existence of hypertrophic synovial membrane and villi and baker's cysts. as a result, pan zoom mode is used to detect joint crystals, to study in details the structure of hyaline cartilage, fluid and to identify their minimal changes, which helps to diagnose degenerative-dystrophic changes of the knee joint. multislice spiral ct (somatom plus 4 volume zoom, siemens, germany) examination was performed after standard oral colonoscopy preparation and colonic distension with room air. images were obtained using 2.5 mm slice collimation; 3.0 mm slice thickness; 1.0 mm reconstruction interval; 17.5 mm/s table speed; kvp, 140; and mas 10. supine and prone acquisitions were obtained in all patients. dose exposure was calculated. images were directly reviewed on a dedicated workstation by two experienced gastrointestinal radiologists using a software with volume-rendering capabilities (vitrea 2.2, vital images, usa). conventional colonoscopy was performed on the same day in all patients and represented the standard of reference. results: all colorectal cancers were correctly identified at ct colonography (9/9, sensitivity 100 %). ct colonography also detected 12 of 14 polyps (sensitivity, 85.7 %). both false-negative findings were represented by lesions smaller than 5 mm. dose exposure (ctdi) never exceeded 1.37 mgy for each scan. conclusion: although studies on larger series are certainly needed, our preliminary experience demonstrates that ultra-low-dose scanning is a feasible and accurate option for multislice ct colonography. this technique allows to scan the patient in both supine and prone positions with a radiation exposure lower than that of a double contrast barium enema, which is of paramount importance to introduce this imaging modality in screening programs. results: of 264 cancers diagnosed in this cohort, 260 were detected by ct colonography. 3 were missed in our early experience with single slice spiral ct. 2 were flat cancers < 5 mm thick. 1 was a polypoid tumour hidden by residue in a poorly prepared patient. this was obvious on a repeat study with adequate preparation. one recently missed cancer was in a co-existent acute diverticulitis and was described as suspicious for malignancy only. there were 18 false positive diagnoses of cancer. all but 2 were cases of diverticulitis or ibd. in only 2 cases was no lesion found at laparotomy or repeated colonoscopy. 2.5 % of patients failed to complete their preparation or did not attend for scanning. this was no different to other complex ct examinations. 7 % of studies were sub-optimal (poor preparation, no iv access, could not lie prone etc). only 1 patient could not be scanned. conclusion: ct colonography is remarkably robust with a sensitivity of 98.5 % and a specificity of 98.4 % for the detection of colorectal cancer. is 1 mm collimation (effective slice width 1.25 mm) essential for multislice ct colonography? a.r. gillams, v. munikrishnan, w.r. lees; london/gb purpose: whilst the finest collimation possible with adequate signal to noise may seem desirable, fine collimation results in increased radiation dose and generates very large data sets. this increases expense, slows down analysis and 3d reconstruction times. we studied the impact of 1.25, 2.5 and 5 mm slice width on polyp detection and cancer staging. methods and materials: 55 symptomatic patients, 22 male, referred for ct colography ± virtual colonoscopy underwent multislice ct following standard bowel prep, bowel paralysis and rectal air insufflation. scans were performed with 1 mm collimation, effective slice width 1.25 mm, pitch 5, 0.5 s rotation time following iv contrast. retrospective reconstructions were performed at 1.25 mm, 3 mm and 5 mm. 1.5 and 2.5 mm overlap was used for the 3 and 5 mm data sets respectively. scans were analysed for the presence of lesions and any cancers detected staged. all patients had colonoscopic correlation and where cancer was detected the resected specimen was also included in the analysis. results: there were 23 cancers and 25 polyps. finer sections aided separate detection of small nodes but this did not impact staging. overall there was no difference in cancer staging between the three data sets. there was no difference in the number of polyps detected on the 1.25 and 3 mm data sets. small polyps were smeared out on the 5 mm data sets. conclusion: 2.5 mm slice width appears to be adequate for multislice colography. previous studies have shown no beneficial effect of using iv glucagons as a spasmolytic for ct colonography. studies of barium enema examinations, however, have shown that buscopan may be a more potent spasmolytic in the colon compared with glucagon. the use of buscopan in ct colonography has not been tested to date. the aim of this study was to assess the effect of iv buscopan on colonic distention and polyp detection when used as a muscle relaxant in ct colonography methods: 70 patients undergoing ct colonography were randomized to receive iv buscopan or no muscle relaxant prior to air insufflation and scanning. patients were scanned in both supine and prone positions using a multislice helical ct acquisition. for the purposes of reporting, the colon was subdivided into 6 different segments, yielding a total of 12 colonic segments per patient. the degree of luminal distention of each segment was scored on a scale from 1 to 4. the accuracy of ct colonography for polyp detection was based on findings at subsequent conventional colonoscopy. results: there was no significant difference in the degree of colonic distention achieved between the group receiving iv buscopan and those who did not. the addition of prone scanning was found to be the single most important factor in ensuring adequate visualization of the entire colon. there is no evidence to support the routine use of iv buscopan in ct colonography. mr was performed on a 1.5 t scanner by using gadolinium as a rectal enema. the 3d data set was post-processed on a workstation to obtain vdc and ssd images. 2 radiologists with knowledge of the colonoscopic findings compared coronal and rotated views. they were compared by consensus in terms of the visualization of the mass lesions and normal colonic segments. technical visibility and interpretation accuracy of the colonic lesions were rated on a score. coronal vdc was regarded superior to ssd for assessment in 11 of 17 patients, equal to ssd in 5 and worse than ssd in 1 case. rotation of vdc and ssd improved the assessement of the colon. rotated vdc was regarded superior to ssd in 4, equal in 11 and worse than ssd in in 2 of 17 cases. all mass lesions above 10 mm were equally well depicted with the vdc mode compared to ssd conclusions: vdc and ssd are useful as a first step and possibly online analysis tools and could possibly allow for an earlier finishing of the mr exam in case of poitive findings. vdc appears to be superior to ssd in excluding colorectal mass lesions at the slice position with the visually largest lymph node extension in plain sequences, dynamic mri was performed using a turboflash starting withj a bolus injection of 0.1 mmol/kg b.w. gd-dtpa. results: in a retrospective evaluation, lymph node staging using the contrast enhanced endorectal and body-coil mri studies was based on the following criteria: stage n0 designated failure to identify nodal structures with a diameter larger than 3 mm in the imaging volume including the perirectal space and the pelvic structures, and stage n1 designated visualization of four or less nodes larger than 3 mm in diameter. in our series eleven patients (n = 11) had histopathologically proven lymph node stage n1; all other patients (n = 12) were stage n0. we were able to depict the enlarged and involved lymph nodes in eight of eleven patients with stage n1. we staged correctly nine of twelve patients with stage n0 (sensitivity of 85 % and specificity of 72 %). conclusions: high-resolution contrast endorectal mri was excellent for depicting perirectal nodes larger than 3 mm in diameter. further studies are necessary to assess the architecture, geometry, and contrast-enhancement characteristics of lymph nodes to improve higher specificity. to investigate systemic and regional lv functional parameters including myocardial wall thickening in patients with symptomatic coronary artery disease and cabg. methods/materials: on a 1.5 t magnetom vision (siemens) 40 patients with angiographically proven cad underwent prospective evaluation of ejection fraction (ef) and regional myocardial function in 320 myocardial segments by cine mri (flash-2d, tr = 11 ms, te = 4.8 ms, flip 25°) at rest. consensus reading by two observers was used for the analysis of myocardial function. a phase-contrast flash-2d sequence (pixel 0.98 × 0.98, venc 250 cm/s) was applied for flow measurements in the ascending aorta in order to derive functional parameters such as lv ejection time frame (ät) and cardiac index. patients were re-examined 6 months after surgery. results: clinical symptoms improved in 35 of 40 patients after cabg. in patients with significantly reduced ef (n = 10) an improvement from 38.4 ± 10.3 % to 49.8 ± 15.3 % (p < 0.05) was found postoperatively. after cabg surgery functional improvement was observed in 45 of 53 myocardial segments (x of y patients) with severe hypokinesia (p < 0.03); mean increase of cardiac index was 15 % (from 2.26 -0.5 l/min/m to 2.65 -0.41 l/min/m, p < 0.02), mean decrease of ∆t was 10 % in patients with functional myocardial recovery (p < 0.05). in general, clinical improvement can be found 6 months after cabg surgery and corresponds to improvement of systemic and regional lv function. patients with persistent symptoms still presented with pathological findings. therefore, mri can be used as a tool to follow-up symptomatic patients after cabg. the following effective doses (male/female) were calculated for the different calcium scoring modalities: sequential msct prospectively ecg-triggered (3 msv/3.7 msv), spiral msct retrospectively triggered (3 to 5.2 msv/3.6 to 6.2 msv), ebt (1.0 msv/1.4 msv). effective doses associated with ct coronary angiography were: spiral msct retrospectively triggered (6.7 to 10.9 msv/8.1 to 13 msv), ebt (1.5 msv/1.8 msv). highest organ doses in all examinations were found for the female breast followed by the lungs and the oesophagus. conclusion: up to a 7-fold higher radiation dose is obtained in msct compared with ebct in coronary artery examinations. the high radiation exposure inherent in cardiac msct warrants careful analysis of the underlying clinical indication. bolus optimisation in multi-slice ct of the coronary arteries and assessment of diagnostic accuracy in comparison with cardiac catheter t.f. jakobs, c.r. becker, r. brüning, a. knez, c. thilo, m.f. reiser; munich/de objective: to determine optimal contrast concentrations and injection rates for detecting coronary stenoses with multi-slice ct (msct) angiography. materials and methods: 60 patients, 4 groups of 15 patients, underwent msct (somatom volumezoom, siemens) with different contrast protocols: (a) 300 mg iodine/ml at 2.5 ml/s; (b) 300 mg/ml at 3.5 ml/s; (c) 400 mg/ml at 2.5 ml/s; (d) 400 mg/ml at 3.5 ml/s (byk gulden, konstanz, germany). assessment of location and degree of coronary stenoses was compared with cardiac catheter. results: the faster injection rates resulted in higher enhancement compared with the lower injection rates at both iodine concentrations (210.9 ± 34.2 hu (a) versus 366.6 ± 84.2 hu (d)). diagnoses of significant stenoses obtained by msct were confirmed by coronary angiography in 37 out of 43 patients (86 %). among those with false negative results there were 3 patients in whom msct failed to determine significant stenoses in the coronary arteries. the diagnostic findings as described in msct were not consistent with angiography with regard to localisation and degree of stenoses: in 42 % of cases, the degree of the most severe stenosis was identical in msct and cardiac catheter. conclusion: superior coronary enhancement was achieved with higher iodine concentrations and flow rates. however, msct with any contrast protocol was unable to achieve the diagnostic level of coronary angiography in assessing location and degree of significant coronary stenoses. therefore further improvement of spatial and temporal resolution in msct-technique is required. coronary artery by-pass grafts: evaluation by retrospectively ecg-gated multislice spiral ct r. marano, m. zimarino, m.l. storto, r.l. patea, n. maddestra, l. bonomo; chieti/it purpose: to assess the potential value of multislice spiral ct (msct) using retrospective ecg-gating in patients who had undergone coronary artery by-pass grafting (cabg). retrospectively ecg-gated msct was performed in 61 asymptomatic patients (144 grafts) 147 ± 86 months after cardiac surgery. scanning parameters were: 4 × 2.5 mm collimation, 3 mm slice width, and 1.5 mm reconstruction increment. images were reconstructed during end diastole with an absolute or relative delay before the next r-wave and volume rendered images were obtained to display the grafts. visualization of the proximal, middle and distal segments of each graft was assessed. the presence of artifacts was recorded. results: 59 left internal mammary artery (lima) to left anterior descending artery (lad) and 85 non-lima grafts were studied. 3 non-lima grafts were shown to be occluded. the entire lima to lad and non-lima grafts could be visualized free of artifacts in 39/59 (66.1 %) and 19/82 (23.2 %) cases, respectively (p < 0.001). the most frequent causes of incomplete visualization were the presence of surgical clips (21.3 %) and motion artifacts (35.5 %) impairing distal anastomosis evaluation. conclusion: ecg-gated msct is a promising imaging technique for non-invasive evaluation of cabg, allowing a complete assessment of lima to lad grafts. current limitations are artifacts from surgical clips and irregular heart rates. isotropic sub-millimeter volume scanning of the heart with ecg-gated multislice spiral ct: first experience t. we investigated the performance of a new multislice ct system with simultaneous acquisition of up to 16 slices and sub-mm collimation (siemens, forchheim, germany) for ecg-gated cardiac imaging. ecg gated spiral data were acquired at pitch 3.5 -4 (pitch -feed/rot divided by one collimated slice width), providing continuous image data with up to 110 ms temporal resolution. spatial resolution, image quality and artifacts were evaluated with a simulation study of an anthropomorphic heart phantom for 0.8 mm, 1.0 mm and 1.5 mm slice width. the results were confirmed by scans of coronary specimens. scan times and radiation exposure for coronary cta protocols were evaluated. first clinical results are presented. results: with the investigated ct system a 120 mm heart volume can be covered in less then 20 s with sub-mm slices. sub-mm slice width allows for improved assessment of non-calcified coronary wall changes, heavy calcifications and instent lumen. radiation exposure for coronary ctas with sub-mm slice width is about 7 msv (male), which can be considerably reduced by ecg-gated dose modulation. a 16-slice platform can cover the heart with ecg-gated spiral acquisition with sub-mm slices within short breath-hold times. increased scan speed and isotropic resolution with voxel size >> 0.5 mm allow for substantially improved coronary imaging. a c d e f 218 results: biexponential relaxation was more pronounced in "black hole" lesions and homogeneous enhancing plaques while dwm, nawm and hypointense lesions presented biexponential behavior with a lower frequency. non-enhancing isointense lesions and normal white matter didn't reveal any biexponentional behavior. linear regression between monoexponential t2 relaxation time and mtr measurements demonstrated excellent correlation for dwm, very good correlation for "black hole" lesions, good correlation for isointense lesions, moderate correlation for hypointense lesions and non-significant correlation for homogeneous enhancing plaques, nawm and nwm. conclusion: biexponential behavior is more evident on plaques with high degree of demyelination and homogeneous enhancing lesions when using conventional sequences with long first echo time. a strong correlation between mtr and t2 values in regions where either inflammation or demyelination is present was found while when both pathological conditions coexist this linear relation is destroyed. method and materials: mri of 24 pediatric patients affected with definite ms (13 male and 11 female; mean age 15) and 20 with adem (7 male and 13 female; mean age 12) were retrospectively evaluated to identify differences in the morphology, location and post-contrast behavior of the demyelinating lesions at the onset of the disease. clinical and laboratory data were not used. plain t2 and t1weighted sequences were always available, flair in only 30 % of more recent cases; i.v. contrast injection was done in 18 adem and 21 ms patients. results: lesions in adem patients were multiple in all but 1 case and in 16/19 cases were more then 3; 10/20 cases infratentorial region was involved; gray matter involvement was present in 10/20 and thalamus was the most common involved (6/20); corpus callosum was involved in 2/20 cases; contrast enhancement was present in 16/18 cases; a "lumpy-bumpy" effect was never remarkable. conclusion and discussion: differentiation of ms and adem has an high prognostic significance. clinical and laboratory data may often overlap between the two demyelinating disorders. even though single mri findings can be non specific, the global evaluation of each patient leads to a differential diagnosis in more then 90 % of cases. results: mri of the brain revealed no abnormality in the 7 patients who had lower plasma fa levels both in the last 5 years and on the day of mri (mean 474.0 and 510.9 mmol/l respectively). however, the 3 patients who had demyelination changes detected on mri had average plasma fa levels in the last 5 years and on the day of mri of 768.6 and 1026.0 mmol/l respectively. no difference of iq between both subgroups were observed. conclusion: the low number of patients within the study group precludes evaluation of the statistical significance of the detected differences, but a trend toward correlation between a degree of metabolic compensation of the disease and a presence of brain lesions can be observed; no correlation was detected between the lesions and a degree of mental development. intracranial tuberculosis: mri evaluation and 1 year follow up i. tsitouridis, m. emmanouilidou, s. chondromatidou, f. goutsaridou, s. stratilati, p. papapostolou, a. morichovitou; thessaloniki/gr purpose: to present our experience in the mri diagnosis and follow up of patients with intracranial tuberculosis. materials and methods: 27 patients with intracranial tuberculosis were evaluated by mri. the examinations were performed on a 1 t, siemens expert plus scanner, using conventional se t1wi and t2wi. all the patients were conservatively treated and underwent clinical and mri examinations every two months for a year. results: 14 patients revealed basal leptomeningeal dissemination and meningeal enhacement and 4 of them also had a tuberculous abscess in this area. 7 patients had disseminated parenchymal tuberculosis and 3 of them had also and meningeal enhancement. one case had tuberculomas and aspergillomas closed together and this patient had a biopsy in the left parietal lobe. the other 5 patients also revealed spinal subarachnoid dissemination. there was no correlation between the mri findings and the clinical status at the early stages of he disease. conclusion: we believe that mri alone or in combination with the other clinical data clearly can detect and characterize this group of patients. differentiation of tuberculous from non-tuberculous meningitis using magnetization transfer mr imaging: a prospective study p. kamra, r.k. gupta, s. pradhan, k.n. prasad, r. kumar, s. chawla, s. jha; lucknow/in purpose: infectious meningitis presents similar features on mr imaging regardless of etiology. the purpose of this study was to characterize meningitis of different etiology using mt imaging. methods and materials: one hundred patients with meningitis on post-contrast mt imaging -65 tuberculous, 9 with viral, 9 with fungal, and 17 with pyogenic meningitis -were studied. the visibility of the meninges on pre-contrast mt imaging in different etiologic groups was studied and percentage difference between the mean signal intensity (si) of the meninges and the mean si of the surrounding t2 normal brain parenchyma was calculated. the mt ratio was also calculated from the thickened meninges in tuberculous meningitis. the mt ratio could not be calculated in other etiologic groups due to difficulty in the placement of pixel due to thin nature of the meninges and their location in the cerebral sulci. results: thickened meninges appeared hyperintense relative to surrounding brain parenchyma in the cisterns on pre-contrast mt-se images in all 65 patients with tuberculous meningitis. meninges were not visible on pre-contrast mt images in the non-tuberculous group. the percentage difference in the mean si of the meninges and the surrounding brain parenchyma was significantly higher (p < 0.05) in the tuberculous group (21.21 % ± 1.98) compared to that in the non-tuberculous group (5.55 % ± 1.01 viral, 3.76 % ± 2.39 fungal, 6.18 % ± 2.18 pyogenic) and explains this difference in visibility. the visibility of meninges on pre-contrast mt-se imaging is specific for tuberculous meningitis, and helps in its differentiation from other non-tuberculous meningitis. purpose: septo-optic dysplasia is a variable condition, characterized by developmental anomalies of midline structures, the optical pathways, and the hypothalamus/pituitary system. while visual and hormonal disturbances are well known in these patients, neurological and mental development has not yet been related to the morphological findings. patients and methods: 22 children, 12 female, 10 male, aged between 0 -13 years at first presentation underwent mri (0.5 -1.5 t superconducting systems, t1w and t2w sequences in 3 section planes, slice thickness 1.5 -5 mm. in 19 patients behavioural abnormalities and school performance could be analyzed with age appropriate tests. results: mri abnoralities consisted of a completely or partially missing septum pellucidum in 14 cases, anomalies of other midline structures (corpus callosum, fornix, other commisures) in 12, 3/14 mr scans revealed hypoplastic optical structures. anomalies of the hypothalamius/pituitary system were seen in 10, hemisperic lesions (mainly schizencephalic clefts) or infratentorial abnormalities occured in 10, and pathological formation of the hippocampus in 7/15 scans. marked develthe purpose of this presentation is to report on the prevalence of cerebrovascular complications in children with aids and investigate whether the mode of hiv infection plays a role in the development of this complication. 508 children (ages, 4 months to 17 years) with aids were periodically evaluated with pre-contrast ct scans. further evaluation with mri was perfomed for patients with either focal neurologic deficits or ct findings other than diffuse atrophy. in five patients mr angiograms and in one conventional angiography were also performed. eleven children were found to have vascular lesions. only one had focal neurologic symptoms at the time of diagnosis. six children were found to have 25 aneurysms. a seventh child had a surgical clip at the site a previously treated anerysm. eight patients were found to have 27 infarctions. in four of the patients with infarctions, fysiform aneurysms of the cerebral arteries were also identified. nine of the 11 patients in our study were infected by transplacental route or during blood transfusion in prematurely born infants. in this group of patients the diagnosis of cerebrovascular disease was made earlier (mean, 8.2 years) compared to the two patients that were infected later in life (mean, 14.9 years). there is increased prevalence of cerebrovascular disease in children infected by hiv. the risk is greater if the exposure to the virus took place prior to the 40 th week of gestation. this finding suggests that the immature vessels of the fetuses or the prematurely born children are more vulnerable. a quantitative study of mr imaging in vascular dementia l. wang; beijing/cn purpose: to identify the neuroimaging determinants which could predict the occurrence of the vascular dementia (vad). the findings of cranial mri were compared in 30 vad patients and 30 stroke without dementia (swd) patients by means of quantitative measurement of some indexes. the indexes of measurement included the cerebral white matter lesion (wml) area, the cerebral infarct area, the ratio of ventricle-to-brain (vbr) and the ratio between the areas of the corpus callosum and supratentorial brain in the midsagittal plane. discriminant analysis was used to search for the indexes which could contribute significantly to distinguishing the two groups. results: small cerebral vessel disease and multi-infarct were two major basal diseases of vad in this series. the wml areas, the left cortical infarct and vbr were significantly higher and the corpus collsum areas was significantly lower in the vad group than the swd group. the indexes that could significantly discriminate the two groups was: callosal atrophy, ventricle-to-brain ratio, white matter lesions area, left cortical infarct area, left parietal infarct area, total cortical infarct area. conclusions: callosal atrophy, lateral ventricle enlargement and extensive wml are important predictors of incidence of dementia in the small vessel disease; however, left cortical infarct, especially left parietal infarct, is important predictor of incidence of dementia in the multi-infarct group. biological markers of alzheimer's disease: diagnostic imaging and oxidative stress d. lupoi, r. squitti, a. orlacchio; rome/it purpose: a bulk of evidence indicates that oxidative stress mediated by redox transition metals plays a central role in the neurodegeneration of alzheimer's disease (ad). iron and copper are strongly concentrated within neuritic plaques and represents the hallmark of the ad brain. recent studies indicate that peripheral markers of oxidative stress in ad patients could be informative about the pathophysiology of this brain condition and suggested that elevated copper in serum may represent a peripheral marker for ad. we report a pilot study examining the relation between copper and oxidative parameters in serum and the lesions present in the ad brain. we performed retrospective subjective qualitative and quantitative analysis of the mr brain images of 53 subjects affected by ad and 30 without ad (healthy control group). on imaging, we noted the degree of atrophy and amount vascular lesions in both patients and controls and correlated these with oxidative stress parameters. the preliminary results indicate that oxidative stress, with higher copper levels, are related to atrophy of temporal lobe. vascular lesions and global atrophy do not correlate with copper and peroxides in serum. our evidence sustains copper as peripheral marker in ad to help in the early diagnosis. to determine whether diffusion weighted mri (dwi) contributes to the differential diagnosis of patients with parkinson's disease (pd) and the parkinsonian variants of multiple system atrophy (msa-p) and progressive supranuclear palsy (psp) methods and materials: conventional dual-echo fast spin echo and dwi scans were obtained from 12 msa-p, 13 pd, and 10 psp patients matched for age and disease duration. dwi was performed using echoplanar imaging with diffusionsensitizing gradients switched in slice direction and three different b-values (30, 300, 1100). regional apparent diffusion coefficients (radc) were determined in different brain regions including basal ganglia and pons. results: using the kruskal wallis test and post hoc testing with the mann whitney u test, significant differences in radcs of the putamen (0.85 vs. 0.71 s/mm 2 ; p < 0.001) and the caudate nucleus (0.81 vs. 0.72 s/mm 2 ; p = 0.007) were detected between patients with msa-p and pd. between psp and pd patients, significant differences in radcs were revealed in the putamen (0.87 vs 0.71 s/mm 2 ; p = 0.008) and in the globus pallidus (0.72 vs 0.65 s/mm 2 ; p = 0.006). no significant differences in radcs were obtained between patients with msa-p and psp. conclusion: the significant higher radcs in patients with msa-p (putamen, caudate nucleus) and psp (putamen, globus pallidus) compared to patients with pd may reflect a more advanced alteration of the cns tissue integrity due to neuronal loss and gliosis leading to increased random movement of water molecules. metabolic impairment of the brain in patients with apallic syndrome s. mirzaei, c. stepan, p. knoll, h. köhn, h. binder; vienna/at purpose: a reliable assessment of prognosis in acute/persistent vegetative state following cerebral anoxia is mandatory for clinical decisions concerning extended intensive care procedures. we evaluated five patients with apallic syndrome with [ 18 f]-2-deoxy-d-glucose (fdg) positron-emission-tomography (pet). pet images of the head were performed using a siemens ecat-art scanner (cti, knoxville). two 137 cs point sources were applied for attenuation correction and osemalgorithm for iterative reconstruction. regional cerebral metabolism in 12 cortical and subcortical regions was determined and compared to a normal control group. the global cerebral metabolism of glucose was markedly reduced in all patients. so far, a follow-up pet scan of the brain in one patient without clinical amelioration showed further decrease of cerebral glucose metabolism. in accordance with limited reports in the literature these results suggest that the extent of metabolic impairment may play an important role in order to assess the probability of clinical recovery from severe anoxic brain injury. methods and materials: a pioneer instance of a fully digital radiology department has been implemented at shanghai first hospital since aug. 2000, which has been composed of a dicom compliance pacs and a chinese ris developed inhouse. two phases were implemented for whole setup procedures. results: in initial phase started in oct.99, ct, mri, rf, dsa and a film digitizer connected to a central archiving system which involved a 300 gbyte raid and a round about 3 tbyte dvd jukebox, simultaneously a chinese ris was installed to implement the computerized management for routine workflow; full implementation of digital department was completed with three new modalities, a dr, a digital mammography and a digital radiofluoroscopy installed in aug. 2000, which realized full electronically archiving of images and initiated a filmless procedure. central and distributed image store management and auto-routing procedure employed to reduce network traffic and improved processing response of the system. soft copy image diagnosis and the computerized management of imaging workflow elevate workflow efficiency and improve patients passthrough obviously. the chinese version ris, which was developed based on the workflow and routine manage model of typical radiology department of china, has successfully transferred traditional film-based management into a revolutionary efficient and reliable digital management. conclusion: phases approach is a perfect way to achieve the fully digital radiology department in china and a specifically ris would play an important bridge role in a complex medical information system environment. possible pitfalls in the digitalisation of the radiologic image archive p.m.a. van ooijen, m. oudkerk; groningen/nl purpose: with the increase in the amount of data produced at radiology departments, the importance of digitalisation of the radiologic image archive also increases. when planning a picture archiving and communiations system (pacs), an overview of the possible pitfalls is crucial to judge the quality of the implementation plan. we present such an overview based on our experience with the digitalisation of a radiology department in the netherlands. methods and materials: according to our experience, seven pitfalls are present when implementing a pacs: (1) system acceptation by the radiologist; (2) emphasis on storage instead of retrieval performance; (3) reduction of data transfer capacity; (4) undersized digital storage capacity; (5) unpredictable radiologic workflow; (6) functionality archive media; (7) pseudo dicom 3.0 solutions. results: for a successful pacs, all pitfalls described above have to be eliminated. to achieve this, the everything on-line (eol) principle was developed based on the ground rule that all images have to be available to the radiologist fast at every workstation at all times (emphasis on retrieval). the eol pacs is a, windowsnt™ based, full dicom solution with a large (last months to years) raid-5 on-line archive (fast, standardized, scalable) and a large dvd-r on-line backup storage (non-erasable, standardize, scalable), eliminating dependency on workflow management, pre-fetching or auto-routing and increasing the ease of acceptation by radiologists. conclusion: a large number of pitfalls are present when digitising a radiologic image archive but with careful planning and by putting certain demands on the manufacturer, these pitfalls can be eliminated. this new technique is based on a regular 15″ tft-flatscreen monitor equipped with a special filter system. this configuration allows the display of objects from 8 different angles with a shift of one degree in between. of these 8 perspectives only two reach the eyes of a viewer at one time. different viewers in front of the screen obtain different perspectives resulting in a stereoscopic view for all viewers in front of the monitor. to demonstrate 3d data sets, we use a 69″ plasma display equipped with the "stereo viewing" panel. the stereoscopic view is used for still images and for movies, e.g. avi files. the described system was evaluated over a period of 6 months. results: panel-based stereo viewing is possible. without the addition of cumbersome and costly glasses, the technique can be used to provide a 3d rendition of complex 3d mri-and ct-data sets for large audiences. the system proved easy to use and was widely accepted by referring physicians as a tool for rapid data assessment. a selection of cases will be presented in this presentation. conclusion: "stereo viewing" without glasses proved to be feasible and became widely accepted at our department, especially for demonstration purposes. accessing 2d purpose: accessing medical images everywhere and everytime on mobile hardware is very important for both routine diagnostic and research. therefore, we developed an application for mobile devices, which are increasingly present like pencils. methods and materials: java mobile-informations-device-profile (midp) was selected for development. for data-transfer the http network protocol is used. the image data is available in the dicom file standard. there is no need of special filepreparation before file access. stereoscopic images were generated with our previously developed medical interactive stereo-3d visualisation tool. results: java was used because of the growing range of java-enabled hardware like mobile phones, personal-digital-assistants and tablet-pcs. the mid-profile was created especially for limited devices. our reference application is running on 16 bit color palm-organizers. image files can be accessed through wireless or wired connections. once the data is retrieved from a server, the images can be viewed, edited and saved. it is also possible to list and edit the dicom tags and make additional comments. precalculated 3d-data (e.g. from ct-datasets) based on chromatek visualisation can be displayed stereoscopic on color devices. summary: the goals of a mobile implementation is not the replacement of medical desktop workstations, but enlargement. the implementation includes methods for intelligent interaction and reduction of drawbacks of limited devices. because of using distributed standards and the standalone implementation, the application can easily be integrated into medical systems. creating his-based quality mangement: the nottwil experience h. hawighorst, t. mayer; nottwil/ch the swiss paraplegic center was founded in 1990 and is a modern level i trauma center for patients with all type of spine injuries. in this presentation we will discuss our road to success and the difficulties lying ahead to fully integrate a knowledgebased quality management to our institution. pacs was installed in 1998 and is productive with his and ris as a knowledge based information system since the end of 1999. radiological images and reports are distributed within the hospital by a webserver based intranet technology. for management purposes the model of efqm (european foundation for quality management) was choosen at the end of 1999 and aims at total quality management. although the pacs sytem is running quite well there are still problems to be solved. in the efqm model "processes" are the essential element. we adopted from the efqm matrix an audit matrix with 90 quality steps. the result of the first quality selfassessment has shown that knowledge-based quality has to be transparent and understandable to employees and has to be "lived" and continuously "taught". to increase and assure widespread access and acceptance of quality management we bring intranet technology-based information together with continuous education of employee. however, trained people with skills in modern technology and quality mangement are warranted in this type of modern infrastructure. new approach to picture communication in an outpatient environment f.x.j. fruehwald, e. steiner, m. obermayer; st. pölten/at purpose: while pacs inside hospitals is a widely used technology nowadays, the situation is much different outside hospitals. a new approach offers picture communication to the medical community outside hospitals. while hospitals solutions are "islands" with little communication to the outside world, the new system supports communication between all providers of medical services materials and methods: application provided pacs allows storage of image data in a professionally organised server farm. patients are provided with a chipcard containing the name, a unique serial number and a pin. all doctors authorised by the patient can access the server and load down all images they want using secured lines. results: technological requirements and guaranty of privacy of medical data are described. the system has been implemented in a part of austria. some 50 physicians up to now take part in this project. only normal amateur pc equipment is needed on the physician's side, adsl is recommended for download of dicom images; for use of jpeg images isdn standard would be sufficient. global access of the server is possible. conclusion: a combination of application provided pacs and a plastic card with a chip can transfer all advantages of hospital pacs into the extra hospital world. as storage costs and costs for telephone lines go down it is to be expected that this internet based system will replace traditional image communication per hardcopy and mail shortly. purpose: magnetic resonance imaging (mri) is limited by artifacts and image distortion in vessels after stenting. with a rigid active magnetic resonance imaging stent (amris) the stent lumen can be illuminated without causing artifacts, however, the use is limited due to the need for surgical placement. a new balloonexpandable stent design enables catheter implantation of the amris. the purpose of this study was to evaluate the imaging properties of this stent in a rabbit model using mr angiography (mra) and flow measurements. the amris was expanded with a balloon catheter in the abdominal aorta of five rabbits. flow measurements and mra before and after injection of an iron-oxide-based blood pool contrast agent were performed at 1.5 t. signal-to-noise ratios (snr) were calculated within and outside the stent lumen. results: placement of the expandable stent was feasible in all animals. snr outside as compared to within the stent increased significantly (p < 0.05) from 5.0 to 23.2 for plain, from 19.5 to 30.7 for contrast-enhanced mra, and from 5.8 to 13.9 for magnitude images of the flow measurements. flow volume curves within and distal to the stent were comparable. conclusion: mr imaging after interventional placement of the expandable amris is capable of illuminating the inner stent lumen. thus, follow-up after stent placement is feasible and it appears to be a useful tool for clinical follow-up and basic research on vessel alteration after stent placement. we retrospectively studied 37 procedures of mechanical thrombectomy of ptfe dialysis access grafts with the at-ptd performed in our vascular department. 28 dialysis accesses in 26 patients were included (nine had a double procedure). the delay between thrombosis and percutaneous thrombectomy was less than 48 hours. duration of the procedure, immediate technical results, primary patency and complication rates were analysed. the mean duration of the procedure was 137 minutes. a venous stenosis was associated with thrombosis in 90 % of cases. the technical success was 89.2 %. 31 % of successfully declotted access grafts presented with early recurrent thrombosis less than 3 months after the procedure: 45.5 % of them presented with residual stenosis at the end of the initial procedure and 45.5 % with residual clots. in this group, the mean primary patency was 30.5 days. three major and 4 minor complications were observed. conclusion: thrombectomy of dialysis access graft with the at-ptd is a safe and effective technique. the mean patency rate is superior to pulse-spray and the complication rate is lower. moreover, the procedure seems to be faster to perform than other techniques. results: indications for pta were ulceration in 40 limbs, rest pain in 26 limbs, gangrene in 4 limbs and bilateral intermittent claudication in a patient requiring total knee replacements. 24 limbs underwent one procedure, 12 underwent two and 8 underwent three. technical success was achieved in 67/72 (93 %), partial success in 4/72 (5.6 %) with 1 failure to achieve any improvement (1.4 %). 30 day mortality was 1/40 (2.5 %) from bronchopneumonia 17 days post bkpta. mean/ median follow up was 24 months (range 0 -48). clinical improvement (ulcer healing, reduced or absent pain) was seen in 34 patients (77 %). expected major and minor amputations were 2 (9 %) and 3 (14 %). there were no major complications requiring further radiological/surgical interventions or increased hospital stay. there was 1 minor groin haematoma. conclusion: bkpta is a safe, worthwhile procedure in carefully selected patients, although repeat procedures were required in almost half of the limbs treated. following appropriate inflow procedures we consider pta as a first line treatment for patients with distal critical ischaemia. (3) after previous sfa angioplasty, underwent pta with adjuvant pdt using 60 mg/kg of the photosensitiser 5-aminolaevulinic acid and 635 nm light at 50 j/cm 2 to pta site. at 6 months of follow up all patients were asymptomatic with no restenosis and no arterial complications. because of previous restenosis/re-occlusion these patients would be at a high risk of this complication again. results: these patients have now been followed up for 26 ± 3 months. patients were reviewed clinically and were offered duplex examination. six of the 7 patients remained asymptomatic. one patient has had repeat angioplasty. a further patient had mild, unlimiting claudication. three of the 4 patients who had duplex showed no restenosis. the 4 th patient (the only currently symptomatic one) had a significant stenosis (psvr = 3.7). there were no arterial complications such as aneurysm formation or occlusion at the treated sites. conclusions: pdt as an adjunct to pta is a safe and feasible procedure and appears effective in the long term. we have now started a randomised control trial to assess its potential for the treatment of restenosis. purpose: to present statistical data from a registry including patients after angioplasty or stenting of atheromatous peripheral lesions (essentially renal and lower limb arteries). methods and materials: angioplasty with or without stenting of an atheromatous peripheral lesion was the criteria for inclusion: 729 patients were included (540 men, mean-age 59.9 years) between may 1998 and dec. 1999, in 10 centers. cardiovascular risk factors, symptoms and atheromatous localization were analyzed with a descriptive method; the population with renal disease was compared with the one with lower limb disease. results: smoking (78.5 %), hypertension (58.7 %), and hypercholesterolemia (50.9 %) were the most frequent risk-factors. men were more frequently smokers than women (92.7 % and 37.8 % respectively). lower limb arteriopathy was more frequent in men than in women (80.9 % and 44.4 % respectively). vasculo-renal disease was more frequent in women than in men (46 % and 20.4 % respectively). smoking was much more frequent in the group with lower limb arteriopathy (90.3 %) than in the group with vasculo-renal disease (53.3 %). conclusion: this registry gives access to statistical data about a population of atheromatous patients treated for peripheral localization. long-term follow-up will allow us to evaluate the changes in the profiles of the patients and the morbidity and mortality in this population. results: all lesions with a maximum diameter of 5 cm were treated with a safety margin of 5 -10 mm around the lesion. lesions with a close relationship to the liver capsule, the gall bladder and major vessels were treated. in patients treated with litt for liver metastases from breast cancer the mean survival was 3.6 years (95 % ci 3.0 -4.2 years, median 3.5 years) after the first litt treatment and 4.6 years after the diagnosis of metastases, which was treated with litt (95 % confidence interval 3.9 -5.9 years, median survival 4.5 years , partial necrosis in the remaining 8 lesions (23.5 %); two of these tumors were > 5 cm. patients had no major complications except one hemorrage that required transfusion but resolved spontaneously. transient pain, nausea and fever were common minor symptoms that resolved within 2 -3 days. the median follow-up was 12 months. conclusion: rf ablation with the le veen probe is a safe, well tolerated and effective procedure for the treatment of unresectable liver tumors. enhanced gray scale ultrasonography with levovist and c 3 -mode of hepatocellular carcinoma treated with interstitial laser photocoagulation l. tarantino 1 , a. giorgio 2 , g. de stefano 2 , f. esposito 2 ; 1 torre del greco/it, 2 naples/it purpose: to evaluate c 3 -mode enhanced ultrasonography of hepatocellular carcinoma (hcc) treated with interstitial laser photocoagulation (ilp). method/materials: 8 patients with a single hcc (2.0 -5.5 cm) treated with ilp underwent helical ct and were also studied with c 3 -mode™ (esaote biomedica, genoa, italy) and an i.v. microbubbles contrast agent (levovist, shering, berlin, germany). after injection of levovist (4 g; 300 mg/ml), c 3 -mode™ scans were recorded at the times 20 s, 40 s, 60 s, 2 min, and 3 min. the results of c 3 -mode™ were compared with ct. results: after injection of levovist, c 3 -mode™ showed homogeneous enhancement (hyperechogenicity) of liver parenchyma at 20 s, 40 s, 60 s, 2 min, and 3 min. in 6 patients, with complete necrosis of hcc at ct, the lesions did not show any intralesional enhancement during c 3 -mode™ examination (complete agreement with ct). in one nodule, with enhancing intratumoral areas suggestive of viable tumor at ct, c 3 -mode™ showed hyperechogenicity in the same corresponding areas (agreement with ct). in the remaining patient, ct showed complete necrosis of the nodule. howewer, enhanced spots along the inferomedial margin of the lesion suggested parenchymal infiltration left untreated by ilp. c 3 -mode™ did not show enhancement in that area and a biopsy did not show malignancy. no relapse has been observed after 8 months. follow-up of the patient is in progress. results: all patients tolerated the procedure well under local anesthesia. the total procedure time was 90 minutes. all complications observed were minor and no further treatment was necessary. online mr thermometry allowed exact visualization of the extension of laser-induced changes and their relationship to the neighboring anatomy. lesions up to 2 cm in diameter could be efficiently treated with a single laser application; larger lesions were treated with dual, triple and quadruple simultaneous applications. in 97.5 % we achieved complete necrosis of the tumor and a 5 mm safety margin, resulting in complete destruction of the tumor without local recurrence. mean survival after the first laser treatment was 3.4 years (95 % confidence interval (ci) 2.5 -4.2 years) and 4.4 years (95 % ci: 3.6 -5.2 years) after the time of diagnoses of the hcc. conclusion: in hepatic involvement with oligonodular hepatocellular carcinoma, litt appears to be an effective therapeutic procedure. results: before treatment, intratumoral arterial-phase enhancement followed by a hypoechoic appearance in the portal venous and delayed phases was demonstrated by contrast us in 38 (90 %) of 42 hccs. after rf, all the 32 (84 %) of the 38 hccs that were found to be necrotic at spiral ct, failed to enhance using contrast us. by contrast, in the six hccs with residual viable tumor at spiral ct, intratumoral areas of persistent enhancement -corresponding to the enhancing areas at spiral ct -were identified using contrast us. these six nodules were retreated with rf, targeting residual tumor with contrast us guidance. conclusion: contrast-enhanced us, performed using c3-mode imaging, shows promise in assessing the therapeutic effect of rf thermal ablation in hcc. purpose: cytokine-based gene therapy has been shown to be highly efficient in stimulating an immune response leading to tumor rejection in transplanted tumors. woodchucks infected with woodchuck-hepatitis virus (whv) develop orthotopic hepatocellular carcinomas (hcc). the purpose of this study was to evaluate the efficacy of mr-guided injection of a therapeutic adenovirus vector into hccs of woodchucks and to monitor the course of the tumor non-invasively using mri. for mr imaging five woodchucks with known hepatocellular carcinomas were anesthesized using ketamine and xylazine. the animals were examined using standardized sequences on a 1.5 t whole-body scanner. under mr-guidance either adil-12/b7.1 or a control adenovirus were injected into selected tumor nodules. the animals underwent axial mr examinations of the liver immediately as well as 2, 4, 7 and 10 weeks following application of the therapeutic agent. finally all animals were sacrificed and the liver was referred to histopathology. results: mri of all animals was feasible. injection of adil-12/b7.1 caused a reduction of the tumor nodule volume in all animals. in contrast, tumor areas injected with the control vector showed increased tumor size. histopathologic examination of liver sections revealed necrosis and inflammatory infiltration in tumor nodules injected with adil-12/b7.1 whereas typical features of neoplastic hepatic cells were found in areas injected with the control vector. conclusion: objective monitoring of gene therapy strategies in vivo by means of high resolution mr imaging in small animals is possible. purpose: paediatric diagnostic examinations in ireland are currently under investigation, such that recommendations may be made for greater optimisation of practice. methods and materials: a questionnaire survey established which irish hospitals perform paediatric examinations. dose measurement, using a combination of tld and dap monitoring, is being conducted in a sample of these. in line with european recommendations, diagnostic reference levels are being established for examinations with high effective dose. an evaluation panel is using image assessment criteria derived from those of the european commission to assign objective image quality scores. correlation of dose and image quality allows proposals for optimal techniques for paediatric examinations. results: there is a wide spectrum of paediatric practise nation-wide, in terms of caseload, equipment used, and technique applied. this has significant impact on the range of patient dose for similar investigations, and on the diagnostic quality of the images produced. comprehensive analysis of image quality is difficult, and in paediatrics, is complicated by the fact that essential image details depend heavily on the clinical situation. an absolute image quality measure such that the lowest achievable dose for an examination may be specified is being sought. the formulation of a non-biased, representative sample of hospitals is fundamental to the validity of any dosimetric survey, but particularly one concerned with the establishment of national reference doses. optimisation demands that image quality is evaluated in clinical radiographs -in paediatrics this requires consideration of the clinical context of the request. does an absolute image quality measure exist? purpose: to investigate in a controlled patient study the potential of online tube current modulation in subsecond multi-slice spiral ct (msct) examinations of children to reduce dose without loss in image quality. method/materials: dose can be reduced for non-circular patient cross-sections without an increase in noise if tube current is reduced at those angular positions where the patient diameter and, consequently, attenuation is small. we investigated a pre-release product version of an online control for the tube current integrated in a somatom vz with improved technical performance. we evaluated image quality, noise and dose reduction for examinations with online tube current modulation in 50 msct of the thorax and the abdomen. we evaluated mas for tube current modulation and compared to the mas in standard protocols. image quality was rated as very good, good and poor in a consensus by three radiologists. noise was assessed in comparison to a control group and by phantom measurements. results: dose was reduced typically by 20 to 37 %, depending on the patient geometry and anatomical region (thorax 25 to 29 %, abdomen 20 to 37 %). in general, no loss of image quality was observed. measured noise did not change significantly. in some cases the noise pattern was improved. online tube current modulation is now used as a standard in multi-slice spiral ct at our institution. conclusions: dose in multi-slice spiral ct examinations of children can be reduced substantially in routine examinations by online tube current modulation without a loss of image quality. purpose: malformations of the corpus callosum (cc) may occur in many different syndromes. various forms have been observed with different degrees of structural abnormality. the purpose of this study is to review characteristic signs of callosal abnormality and try to find a correlation with the degree of clinical disability. material and methods: mri examinations were performed in patients with developmental delay. imaging included t1 and t2-weighted sequences in all cases. all patients had a thorough neuropaediatric clinical examination. the mri images were evaluated regarding the degree of callosal hypoplasia, presence of a fornix and hippocampus, hydrocephalus and concomitant further abnormalities. results: seven different forms of malformation of the corpus callosum were encountered. in particular the development of the fornix and hippocampus differed significantly as did the amount of clinical disability. as a hippocampal commissure is a prerequisite of normal hippocampal development, the size of the temporal lobes depended on the degree of this midline abnormality. patients without hippocampi were all severely disabled. the clinical disability of the patients presented here differed significantly, which may in part be due to the different extent of the midline cerebral malformation. the relevance of the concomitant aplasia of the limbic system has not been addressed in detail before in the literature. purpose: to evaluate the role of ct and ct angiography in the diagnosis and treatment of pulmonary cystic and adenomatoid malformations in the newborn and young children. methods and materials: nine children aged between 2 days and 5 months with respiratory failure and cystic pulmonary malformations underwent conventional ct and ct angiography. lung abnormalities were present on all the chest radiographs prior to ct. the abnormalities had varying appearances; 2 resembled pneumonia, 3 appeared as a mass and 4 as cysts. the ct acquisition protocol included sequential scanning with a 5 mm scan thickness and scan interval, which was followed by high resolution ct in all cases and ct angiography in two. all ct studies were performed under general anaesthesia. the final diagnosis was based on the surgical data and morphologic examination of resected lung tissue in all patients. results: morphologic investigation revealed different types of cystic adenomatoid malformation in 6 cases and persistent interstitial emphysema in association with other hallmarks of bronchopulmonary dysplasia in 3 cases. the following types of congenital cystic adenomatoid malformation were found: type i (4), type ii (1), type iii (1), and the ct data was in close relationship to the morphologic findings. in 3 cases ct showed the presence solitary or multiply lung cysts. conclusion: ct is a useful diagnostic tool for precise estimation of the extent and differential diagnosis of cystic pulmonary malformations in the newborn and young child with respiratory failure. ct angiography can provide important diagnostic information in the preoperative investigation of pulmonary and mediastinal vessels in these cases. we analysed 97 aubmt (46 boys, age 11.5 a; 51 girls, age 11.2 a), 35 (36 %) died and 83 albmt (33 girls, age 12 a; 50 boys, age 16 a), 29 (34.9 %) died. at least one plain radiograph and one ct were obtained before albmt and aubmt respectively. results: only 13 ct examinations in clinically complicated cases were peformed after albmt. pathological findings were found in 6/83 (7.2 %). 3/6 (50 %) later died because of infections. inflammatory pneumonia occured in 3 cases, mycotic infection in 2 cases and pneumonitis after chemotherapy was found in one case. graft versus host disease was the cause of death in 3/97 cases. 655 ct examinations of patients were performed after aubmt, mostly to exclude lung metastases and to follow up mediastinal lymph nodes. in 4/7 patients with respiratory symptoms parenchymal changes were found. we diagnosed 1 pneumonitis after chemotherapy, inflamatory pneumonia in 2 cases and mycotic infection in 2 cases. two patients had clinical symptoms with a negative ct. only 1/7 (12.5 %) died due to respiratory complications. conclusion: different imaging strategies for aubmt and albmt were used. the therapy of pulmonary complications is more complex with albmt, but there are frequently diagnosed with plain films. ct is used more frequently after aubmt in which pulmonary infections are rare and less serious. ct has a decisive role in bmt patients. chrispin-norman-score and bhalla-score of patients with cystic fibrosis: results: cn-scores varied between 2 and 24 in rx and mr. the mean cn-score was 12.6 ± 6.4 in rx and 12.8 ± 6.2 in mr. the cn-score was higher in rx in 10 patients and higher in mr in 15 patients. in 30.6 % the difference between the two scores was 0, in 88.9 % the difference was smaller than or equaled 2. high correlation was found between scoring with rx and mr with minor restrictions in differentiation of nodules and rings on mr. bhalla-scoring was possible in mr-imaging. conclusion: cn-scoring and bhalla-scoring of cf is possible with fast low-field mr. since scoring differences between rx and mr are not higher than interobserver differences in rx scoring, further research is strongly suggested to reduce radiation exposure in the long term follow-up of patients with cf, especially in children with minor pulmonary involvement. true-fisp lung mri at 0.2 t in pediatric oncology patients presenting with fuo g. schultz, c. laub, w. kenn, a. trusen, j. kühl, d. hahn; würzburg/de purpose: to compare conventional chest x-ray and mri in pediatric patients with a diagnosed cancer suffering from fever of unknown origin (fuo). materials and methods: 13 patients (aged 3 -22 years, mean age 8 years) had 21 chest x-rays and underwent mri in a 0.2 t mri unit (siemens magnetom open) using a phased array surface coil. the images were acquired in coronal and sagittal planes. for the mr investigation a trufi-sequence (tr = 6 ms, te = 3 ms, matrix 256, slice thickness 20 -40 mm) was used with an acquisition time for 10 slices of 20 s. results: although breathhold-sequences were not possible in all cases, it was possible to evaluate all investigations. with regard to pulmonary infiltration coincident findings were achieved in 15/21 cases. 2 out of 6 infiltrations could only be detected on the mr images and the extent of infiltration was better evaluated in 4 cases. additional mri-findings such as sinusitis, pericardial effusion and hepatosplenomegaly were detected. conclusions: true-fisp lung mri was found to be more sensitive than the conventional chest radiograph. additional important clinical information was also obtained with mri. underwent duplex ultrasound and digital subtraction angiography, followed by percutaneous treatment. duplex us was performed via trans-lumbar approach: ri derived from the interlobular arteries was obtained. pre-procedural mean ± sd values of ri and referral cut off values were calculated. patients were classified as cured/improved or not improved according to blood pressure values and changes in antihypertensive drugs before and after treatment. results: pre-procedure, mean ± sd ri values were different (p = 0.001) for atherosclerotic (0.63 ± 0.11) and fibrodysplastic (0.56 ± 0.10) stenoses, with no difference between cured/improved and not improved patients (0.59 ± 0.10 vs. 0.64 ± 0.12); no difference (anova p = 0.5) was found for medical therapy. in cured/ improved atherosclerotic patients (41/58, 70 %), ri was < 0.65 in 28 (57 % sonovue enhanced us measurement of altered renal haemodynamics in patients with liver metastases e. leen, j. macquarrie, w.g. angerson, p. horgan; glasgow/gb purpose: to assess sonovue enhanced ultrasound in the measurement of renal haemodynamics in patients with liver metastases. materials and methods: 10 healthy volunteers and 10 patients with proven liver metastases were studied; non-linear ultrasound imaging (m.i: 0.15 -0.20) of each kidney was performed continuously in a sagittal plane before and after bolus intravenous injection of 2 ml contrast medium (sonovue, bracco, milan). there was a minimum of 10 minutes delay between the examinations of either kidney to allow for contrast clearance from the previous injection. digital data acquisition over a period of 60 seconds was subsequently recorded for quantification analysis. timeintensity curves were derived from regions of interests placed over the kidneys. the contrast arrival times (at), peak amplitude (pa), time to peak (tp) & gradient (g) of the steepest portion of the curves for each kidney were measured. results: there was no significant difference in all the parameters between the contra-lateral kidneys for both controls and patients with liver metastases. there was no significant difference in the pa, at and tp indices between controls and metastases. in contrast the gradient values were significantly raised in those patients with liver metastases (metastases vs controls: 1.59 ± 0.47 vs 0.40 ± 0.22, p < 0.01); there was clear separation of the gradient values between the two groups. to study diagnostic possibilities of 3d-vusa in evaluation of main renal vessels. materials and methods: 71 consecutive patients were referred to 3d-vusa for assessment of major renal vessels: aortic aneurysm (5 pts), renal tumors (16 pts), suspicion of ras (22 pts), suspicion of upj obstruction (28 pts). 3d-vusa was performed on sonoline elegra (siemens) version 6.0 with power doppler and a special 3d program. postprocessing was performed using volume rendering data to obtain angiogramm-like virtual images. verification was made by conventional angiography (51 pts) and surgery (20 pts). sunday b results: 3d-vusa revealed 17/22 unilateral accessory ra, 2/3 bilateral accessory ra (sensitivity 75 %, specificity 95 %). upj obstruction was diagnosed by 3d vusa in 12 cases and was confirmed by conventional angiography and operation in 11 cases (sensitivity 81 %, specificity 71 %). ras was revealed in 4/22 patients referred to 3d-us study (sensitivity 50 %, specificity 62 %). 3d-vusa diagnosed 3 infrarenal aortic aneurysms. in the cases of renal tumors 3d-vusa helped to delineate normal and abnormal renal vasculature, thus providing necessary information for nephron-sparing surgery. conclusion: 3d-vusa is a useful, noninvasive new promising method for evaluation of main renal vessels. we suggested it especially in children and young adults, in patients with renal failure, allergy to iodinated contrast agents, or fear of ionizing radiation or arterial catheterization. high resolution 3d mr-angiography in renal arteries using sense this was related by measurement to the umbilicus and asis. results: on ct, the median (interquartile range) of the distances between the umbilicus and the ab, from the asis to the ab and from the umbilicus to the iv were −9.0 (28.8), 48 (16) and −24.9 (32) mm respectively. the mean angle of the umbilicus from the ab was 21.6° (range 14 -34°). on us, the mean distance from the iea to the midline was 36.2 mm (10 -48) and from the iea to the asis was 100 mm (80 -140). the relationship between the level of the asis and ab/iv is more consistent than the position of the umbilicus. primary-port trocar insertion at an angle of 34° and secondary-port insertion 15 mm from the midline is recommended. renal cyst ablation using mixture of n-butyl cyanoacrylate and iodized oil in patients with autosomal dominant polycystic kidney disease: a preliminary report j. kim 1 , s. kim 1 , m. moon 1 , h. lee 1 , j. sim 2 , s. kim 1 , c. ahn 1 ; 1 seoul/kr, 2 koyang/kr purpose: to assess the feasibility and effectiveness of renal cyst ablation using n-butyl cynoacrylate (nbca) in patients with autosomal dominant polycystic kidney disease (adpkd). materials and methods: during the past 14 months, 50 renal cysts in 14 patients were treated with percutaneous needle aspiration and intracystic injection of 1:2 mixture of nbca and iodised oil. clinical follow-up was done in all 14 patients for the period of 1 -12 months. subjective symptoms, blood pressure, and serum creatinine level before and after the procedure were compared. ct follow-up was done in 31 cysts in eight patients for the period of 3 -12 months. the procedure was considered successful at follow-up ct when the diameter of the cyst decreased more than 50 % after the procedure as compared to that before the procedure. results: after nbca therapy, symptom was improved in 12 of 14 patients (86 %) and cyst was disappeared or decreased more than 50 % in diameter after the procedure in 25 of 31 cysts (81 %). there were no significant changes in blood pressure and serum creatinine level after the procedure. there was no significant complication related to the procedure in any of the patients. conclusion: percutaneous needle aspiration with intracystic injection of mixture of nbca and iodised oil appears to be feasible and may be an effective modality in ablating renal cysts in patients with adpkd. purpose: stimulated acoustic emission effect shows marked lesional difference in uptake of the microbubble levovist in its liver specific phase. benign lesions showed high uptake, malignancies appeared as defects. a multicenter trial using a novel non-linear mode (adi, acuson, mountain view) offering greater sensitivity and spatial resolution. methods and materials: 67 patients with focal liver lesions characterised either by biopsy or serial/multimodality imaging have been studied. diagnoses were: metastases n = 34, hepatocellular carcinoma (hcc) n = 10; haemangioma n = 8 and benign non-haemangiomatous lesions (bnhl) n = 15, comprising focal nodular hyperplasia fnh; n = 5, focal fatty sparing n = 4, focal fat 1, adenoma n = 3, regenerating nodule: rgn n = 2. adi imaging of the lesion was performed without interval scanning after a delay of 5 minutes from bolus injection of levovist (2.5 g). liver-lesional uptake differences (llud: as %) were compared using a visual analogue score. comparisons were made using non-parametric comparisons (kruskal-wallis anova; mann-whitney 2-column comparisons with bonferroni corrections). results: highly significant differences (p ≥ 50 %), while fat/fatty sparing, rgn and fnh showed minimal differences (< 10 %). hccs (mean liver-lesion difference 67 %, range 0 -100 %) and haemangiomas (average 45 %, range 7 -80 %) showed overlap features. intergroup comparisons showed significant differences between bnhl and all other lesion types and between metastases and haemangiomas. conclusion: this simple test shows marked differences between lesion types. it is a very useful test for fatty change, fnh and rgn. non-haemangiomatous lesions can be separated from metastases with particularly high accuracy. the present of high-uptake of levovist at 5 minutes is strong evidence for benignity. b a c d e f 230 contrast-enhancement patterns of hepatic lesions at c3-mode intermittent us imaging d. cioni, r.a. lencioni, f. donati, l. crocetti, m. perri, c. franchini, c. bartolozzi; pisa/it purpose: to describe contrast enhancement patterns of focal hepatic lesions at c3-mode intermittent us imaging. materials and methods: a series of 99 hepatic lesions in 66 patients were examined. us examination was performed by using c3-mode intermittent imaging (technos, esaote biomedica) with 1.4 mechanical index after bolus injection of 2.5 g levovist (schering ag) at the concentration of 400 mg/ml. us images were acquired in arterial (15 -25 s), portal in-flow (40 -55 s), full portal (70 -85 s), and delayed (180 -200 s) phases. findings at contrast us studies were correlated lesion-by-lesion with those at spiral ct or dynamic mri. results: four distinct enhancement patterns were observed: (1) rapid enhancement in arterial phase, followed by either isoechoic (5/5 focal nodular hyperplasia, 1/1 hepatocellular adenoma, 1/6 haemangioma) or hypoechoic appearance (38/38 hepatocellular carcinomas) in portal-venous and delayed phases; (2) peripheral globular enhancement in arterial phase, with centripetal fill-in in portal-venous and delayed phases (5/6 haemangiomas); (3) rimlike enhancement in arterial phase, followed by hypoechoic appearance in portal-venous and delayed phases (9/25 metastases); and (4) no enhancement in arterial phase followed by either isoechoic (13/13 macroregenerative nodules) or hypoechoic appeareance (16/25 metastases) in portal-venous and delayed phases. the enhancement patterns shown at contrast us correlated with spiral ct or dynamic mri findings in 80 of 88 lesions (90 %). conclusion: c3-mode intermittent us imaging is an accurate tool to evaluate the contrast enhancement patterns of focal hepatic lesions. findings correlate well with those at spiral ct or dynamic mri. clinical study of a 3d virtual sonographic system to be used for remote diagnosis d. jeanbourquin 1 , t. le bivic 1 , r. larrue 2 , p. pineau 2 , j.-c. provost 2 ; 1 clamart/fr, 2 paris/fr objectives: virtualprobe system (iôdp, paris, france) allows to acquire sonographic images simultaneously with spatial position of the probe, in order to rebuilt exact 3d sonographic volumes. the system allows to rescan through the 3d volume in order to perform a complete but asynchronous sonographic examination. one possible application would be military medicine, because the expert is not present at the time and on the site where diagnosis has to be done. the objective was to evaluate the ability of the system to be used for remote medical diagnosis. the examinations from 40 patients were done on non moving organs, mainly on cervical and abdominal areas. examinations using conventional method were performed and later on compared to those realized using virtualprobe. all examinations were done by qualified radiologists. the image quality of the 2 methods were scored from 1 to 5 (poor to very good). moreover, the ability of the system to allow proper diagnosis was recorded. results: there was no difference between methods in image quality. for virtualprobe, diagnosis was adjudicated as "possible" in 33 cases (82.5 %), "possible with some difficulties" in 5 cases (12.5 %) and "not possible" in 2 cases (5 %) due to technical problems (electromagnetic interferences and patient with polypnea leading to the impossibility to do acquisition). conclusion: virtualprobe system is a powerful tool that would provide a significant improvement in remote medical diagnosis using ultrasonography. differentiation of neoplastic portal vein thrombus from vascular thrombus in cirrhotic patients using harmonic sonography and second generation contrast agents m. tonolini, v. osti, l. solbiati, p. marelli, v. kirn; busto arsizio/it purpose: assessment of neoplastic nature of intraportal thrombus is of crucial importance for staging and therapeutic choice in patients with hepatocellular carcinoma (hcc). we sought to assess the utility of grey-scale harmonic sonography using second generation contrast agents for differentiating between neoplastic and bland portal venous thrombus. materials and methods: 85 cirrhotic patients with solitary or multifocal hcc were evaluated for potential rf ablation therapy with triphasic helical ct, b-mode and color doppler us. contrast enhanced ultrasonography (ceus) was performed using low mechanical index (0.1 -0.2) and a second-generation contrast agent (sonovue, bracco). liver parenchyma and main portal branches were examined over arterial, portal, and delayed phases. results: in 10 patients (11.8 %) conventional us and helical ct identified thrombus within the portal trunk or main branches. arterial enhancement was depicted by ct in 7/10 (70 %) cases suggesting malignant nature. unenhanced color doppler detected arterial-type flow signals within the thrombus in 3 of 10 cases (30 %). with ceus, 8/10 (80 %) thrombus showed rapid and inhomogeneous enhancement in the arterial phase (p < 0.05, compared to unenhanced us). in the portal phase, the enhancement of these 8 thrombi decreased in proportion to the respective hccs, as contrast filled the patent portions of the vessel. in the remaining 2 cases, no arterial enhancement was detected and no malignant cells were found by us-guided fine-needle aspiration biopsy. conclusions: our study suggests that continuous-mode, contrast-enhanced harmonic sonography may serve as an easy and highly reliable modality for the characterization of portal thrombus in cirrhotic patients. specificity of contrast-enhanced harmonic sonography for characterization of solid, incidentally detected focal liver lesions l. solbiati, m. tonolini, l. cova, d. della chiesa, v. kirn; busto arsizio/it purpose: to assess the use of contrast-enhanced harmonic sonography (ceus) in the differential diagnosis of incidentally detected focal liver abnormalities. methods and materials: 460 patients underwent abdominal us for clinical questions unrelated to the liver. none of the patients had chronic hepatitis/cirrhosis nor history of malignancy. solid focal lesions were found in 49 patients (8.75 %). lesions with characteristic features of hemangioma were excluded. low mechanical index, continuous-mode ceus was performed using a second-generation contrast agent (sonovue, bracco). final diagnosis was established by means of helical ct, dynamic mri and/or fnab. results: in 14/14 focal nodular hyperplasias (fnh) and 13/13 atypical hemangiomas the typical enhancement pattern was detected. in 6 patients presenting multiple hyperechoic lesions, ceus showed enhancement analogous to that of the surrounding liver in all vascular phases, suggesting the diagnosis of focal fatty changes, subsequently confirmed with ct and fnab. in four patients with poorly enhancing lesions, ceus did not provide diagnostic findings and fnab was necessary to diagnose fibrotic hemangioma (1 patient), multiple tubercolous granulomas (1) and disseminated metastases form unknown origin (2). conclusions: for the characterization of most incidentally detected and sonographically indeterminate benign lesions (fnh, atypical hemangioma, focal fatty change) ceus (through the assessment of the enhancement behaviour over the different vascular phases) showed specificity equal to that of helical ct. ceus will probably alleviate the need for further investigation with helical ct studies and aspiration biopsies. o. catalano 1 , a. nunziata 2 , f. sandomenico 1 , a. siani 1 ; 1 pozzuoli/it, 2 naples/it purpose: to evaluate if ce-pd is helpful in the differential diagnosis of focal hepatic lesions detected by grey-scale ultrasound. methods and materials: in a 1-year period 98 consecutive patients where found to have previously unknown, non-cystic lesions on ultrasound examination (51 subjects showed 1 lesion, 29 2 -5 lesions, and 18 > 5 lesions). these lesions were evaluated with levovist-enhanced pd and were fitted into 9 categories (hepatocellular carcinoma, cholangiocellular carcinoma, adenoma, focal nodular hyperplasia, angioma, metastasis, dysplasic nodule, abscess, other). diagnostic confidence level was graded subjectively from 1 (possible) to 4 (certain). categorisation and diagnostic confidence scoring were repeated after ce-pd study. eight cases without confirmation were excluded. results: addiction of ce-pd study resulted in a change of category for 5 patients (5.6 %), all with a single lesion; this modification proved correct in all of them. excluding 48 subjects with a maximal scoring (level 4), there were 16 patients with increased confidence after ce-pd study, 2 with decreased confidence, and 24 with unmodified confidence after ce-pd; score change was never greater than 1 level. in the two most commonly encountered issues, suspected nodule in chronic liver disease and angioma vs. hypovascular metastasis, ce-pd imaging was rarely useful. conclusion: ce-pd increased the characterisation accuracy of ultrasound only in selected cases while it is not cost-effective if employed routinely. these patients can usually be predicted on a clinical-sonographic basis allowing selective ce-pd imaging to be performed. purpose: to define and quantify acoustic parameters in order to characterize liver histopathologic findings in patients presenting with chronic hepatitis type c using a commercially available ultrasound system. materials and methods: 78 patients with a chronic hepatitis type c were prospectively enrolled. cineloops from the liver and the spleen were acquired using an atl hdi 5000 unit (atl-philips, wa, usa). raw data were transferred to a pc for quantification with hdi lab, a software that eliminates the effects of the nonlinear b-mode map. two parameters were calculated using a linear time gain compensation: the liver and spleen attenuation coefficients (respectively lac and sac), and the signals of a region-of-interest (roi) located at 6 cm depth (mean and sd, linear units). all results were correlated to the results of the liver biopsy (knodell and metavir score). results: lac was higher than the sac in all cases. lac and the normalized ac (lac − sac) was significantly higher in cases of cirrhosis (p < 0.01). the signal intensity from the roi was not correlated to the histological findings. the standard deviation increased in cases of cirrhosis due to the heterogeneity of the echostructure. conclusion: quantitative ultrasonography is feasible in routine practice and can improve the stadification of cirrhosis due to chronic hepatitis type c. evaluation of reformatted image quality using a new 3d imaging tool for ultrasound multiplanar reconstruction m.j. o'neill, s.i. lee, j.f. simeone, p. mattos, j. haase, p.r. mueller; boston, ma/us purpose: virtualprobe® (iôdp, paris) is ultrasound acquisition and post processing software that uses an electromagnetic field to acquire positional data, allowing retrospective multiplanar 3d rescanning through a volume of tissue. the aim of this study was to compare image quality of the 2d movie clips used for volume rendering and the 3d reformatted images to that of conventional 2d images. methods and materials: 30 patients referred for exams of the kidney (9) and abdomen (21) were enrolled. each patient had a conventional ultrasound exam, followed by the virtualprobe. the static images, 2d movie clips, and 3d reformatted images were then graded by 2 radiologists on a 5 point scale (1 = best quality and 5 = worst). the difference between either the 2d clips or 3d reformats and the static images for right and left (28) kidney (58) liver (21), pancreas (21), cbd (21), spleen (20), gallbladder (18), and bladder (9) , for the static images, 2d clips, and 3d reformats. there was no significant difference in image quality for 2d movie clips or 3d reformats when compared to standard images. ratings when compared to the static images yielded p values ranging from 0.4 to 1.0 and 0.1 to 1.0 for 2d clips and 3d reformats. there is no significant difference in image quality between static ultrasound images, 2d movie clips, and reformatted multiplanar reconstructions when using the virtualprobe® post processing software. pim 3 minutes after iv injection of levovist (2 g, 300 mg/ml). pim examinations were analysed in consensus by two radiologists who assessed the number of lesions, the conspicuity of lesions on a scale 1 -4, and the smallest lesion diameter in comparison with baseline us. ct studies were performed using a multidetector ct scanner with 130 cm 3 of a non ionic contrast agent (400 mg i/ml) at a flow rate of 4 ml/s. scans were acquired at 35 scan delay for the hepatic arterial phase, 70 s scan delay for the portal venous phase and 120 s for the delayed phase, with a collimation of 4 × 2.5 mm and a slice thickness of 3 mm. all ct scans were evaluated in consensus by two experienced radiologists blinded to the results of us analysis. results: pim improved the conspicuity and the detection of lesions in comparison to baseline us (43 % increase) and to msct (13 % increase). in comparison with msct, the number of lesions detected with pim increased from 60 to 69; the improved detection rate was limited to lesions less than 5 mm in diameter and to non-cirrhotic patients. conclusions: pulse inversion mode may depict more lesions than msct although patients with very inhomogeneous liver parenchyma can represent a limit. performance of a digital flat-panel detector system in the detection of subtle rib fractures: a comparison with a conventional screen-film system and a phosphor-storage system at different levels of exposure k. ludwig, c. schuelke, h. lenzen, t.m. bernhardt, s. diederich, d. wormanns, p. brinckmann, w.l. heindel; münster/de purpose: to compare a digital flat-panel detector in the detection of subtle rib fractures with conventional screen-film radiography and phosphor-storage radiography at different levels of exposure. method/materials: subtle fractures were created in 100 of 200 porcine rib specimens. specimens were mounted into a basin filled with water to obtain absorption and scatter radiation conditions comparable to a human chest. imaging was performed using a flat-panel detector system, screen-film system and phosphor-storage system. exposure settings comparable to clinical imaging were applied (77 kvp, s = 400). additional imaging was performed with both digital imaging modalities at lower exposure doses: with the flat-panel detector images were acquired at exposure doses corresponding to speed-class 800, 1600 and 6400, with the phosphorstorage system to 800 and 1600. for each image the presence/absence of a rib fracture was assessed independently by three radiologists according to a five level confidence scale. roc-analysis was performed for a total of 5200 observations (600 for each imaging modality/exposure level). multivariate analysis was used to compare aucs. introduction: new digital imaging techniques offer the ability to adjust the radiation dose required to answer the clinical question. irrespective of the type of imaging technique, the lowest dose compatible with the required image quality should be used. material and methods: a comparative study was performed with three units for storage phosphor and a flat detector unit. image quality was evaluated by a contrast detail radiography phantom (nuclear associates: digirad). this was completed by further studies using phantoms containing anatomical structures. clinical images and doses were also evaluated by 5 european centres for radiology and medical physics that co-operate within the dimond-3 ec project. results: the images of the digirad phantom demonstrate differences in the image quality of storage phospor systems, but flat panel detector technology showed far the highest image quality. for the flat panel detector radiation dose can be decreased significantly in comparison to storage phosphor systems. using flat panel detectors all examinations can be performed with a maximum of halve the dose compared to storage phosphor systems, operated at a 400 speed class. further dose reduction by a factor of 4 is possible for specific examinations. conclusion: dose mangement with new digital imaging techniques can decrease radiation dose to the patient significantly. in a broad range dose and image quality can be optimized and adapted to the clinical question. performance of a digital flat-panel detector system in the detection of simulated rheumatoid erosions: a comparison with a conventional screenfilm system, a mammography-system and a phosphor-storage system at different levels of exposure k. ludwig, a. henschel, h. lenzen, t.m. bernhardt, d. wormanns, w.l. heindel; münster/de purpose: to compare a flat-panel detector system in the detection of simulated rheumatoid erosions with a screen-film system, mammography system and phosphor-storage system at different levels of exposure. method/materials: 320 artificial lesions (diameter 0.5 -1.2 mm, depth 1.5 mm) simulating erosions were created in 640 predefined regions in the mcp-and pipjoints of 20 monkey paw specimens. specimens were enclosed in containers with water for absorption/scatter conditions comparable to a human hand;imaging was performed at 44 kvp using a digital flat-panel detector system with exposure-doses corresponding to speed-class (s) 25, 100, 200, 400, 800, 1600, 3200 system, a phosphor-storage-system at (s) 200, 400, 800, 1600, a speed-class 200 screenfilm system and a mammography system. three radiologists independently evaluated each region for the presence/absence of a lesion according to a five level confidence scale. roc-analysis was performed for 16.224 observations (1248 for each imaging modality/exposure dose). multivariate analysis was used to compare aucs. in erosive lesions the flat-panel system is superior to conventional screen-film radiography and phosphor-storage radiography at clinical exposure settings. it offers the same diagnostic performance with a lower exposure. reference levels during interventional cardiology procedures: the european dimond experience v. neofotistou; athens/gr purpose: to present the european dimond approach in defining reference levels (rls) for radiation doses delivered to patients during interventional cardiology (ic) procedures, namely coronary angiography (ca) and percutaneous transluminal coronary angioplasty (ptca). rls have been suggested by the council directive of the european community 97/43/euratom as a means of optimising diagnostic exposures. method: during the dimond (digital imaging: measures for optimising radiological information content and dose) european research program patients' doses during ic procedures were measured. this was done in terms of dose-area product (dap), fluoroscopy time and total number of radiographic exposures. values were obtained in three participating european hospitals, using previously checked x-ray equipment and according to a commonly agreed protocol. moreover, a dap trigger level has been defined in order to prevent skin injuries since ic techniques are known to be associated with high radiation doses and thus deterministic effects may be observed. results: rls for dap equals 60 gycm 2 and 100 gycm 2 , for fluoroscopy time 6.5 min and 20 min and for number of frames 1000 and 1400 for ca and ptca respectively. dap trigger level for cardiac procedures, which should alert the fluoroscopist for probable skin injury, equals 300 gycm 2 . the proposed values, which should be considered only as interim rls, take no account of the complexity of particular case or patient's physical characteristics dimond rls are being updated with patient doses measurements taken in seven european countries and with degrees of tolerances assigned to complexity of procedure as well as the patient's size. optimized spectrum in direct digital mammography w.j.h. veldkamp, n. karssemeijer, r.e. van engen; nijmegen/nl purpose: the objective is to find the optimum target-filter and kilovoltage for varying simulated breast thicknesses when using a flat panel full field digital mammography detector (ge senograph 2000d). we use the cdmam phantom that was developed at our institute. this phantom consists of a matrix of squared cells with dots of varying size and contrast. each cell contains two dots, one in the center and one in a scientific sessions sunday b a c d e f 233 randomly selected corner. a computer program was used for automatic readout of the phantom recordings. the program uses the ideal observer model for detecting the dots. image quality is expressed by the contrast detail curve relating object size and contrast at some fixed detection threshold. to cover a range of exposure conditions and breast thicknesses a total of 288 recordings were evaluated. the glandular dose was kept constant for each simulated breast thickness while spectrum parameters were varied. results: results showed that for 7 cm perspex the rh-rh target-filter combination gave superior image quality compared to mo-rh and mo-mo. for 3 and 5 cm perspex the different target filter combinations gave comparable image quality. the results suggest that in full field digital mammography a rh-rh target-filter combination can be used for the whole range of breast thicknesses. an investigation of the observer variability in reading a contrast detail test object in digital mammography p. . images were printed on high resolution printers. a cdmam contrast detail phantom 3.2 (nijmegen, the netherlands) was placed on top of 4 cm of polymethyl methacryllate. images were read by a group of experienced observers within the dimond iii ec project. optimal viewing conditions in respect of viewing box brightness and ambient light level were used. image scores were analysed using a computer program which incorporated the rules for interpreting images. results and discussion: contrast-detail curves and image quality factors could be calculated for the different observers and images. observer variability was compared with theoretical predictions and some readings from conventional mammography images. image quality factors were low, meaning that low contrast differences and small objects could be distinguished. the variability of the readings was large, but tolerances were similar in all observers. therefore, we propose to also acquire typical cdmam images from digital mammography systems as part of an acceptance testing programme. first clinical experience of a digital slot-scanning-area detector for mammography t. francke 1 , j. egerström 2 , m. eklund 2 , l. ericsson 2 , t. kristoffersson 2 , v. peskov 1 , j. rantanen 2 , s. sokolov 1 , p. svedenhag 2 , s. thunberg 2 , c. ullberg 2 , n. weber 2 ; 1 stockholm/se, 2 danderyd/se purpose: to compare image quality and dose for a novel digital slot-scanning area detector vs. conventional analogue film-screen images. the design of the detector, photon counting and small detector width of 50 mm, results in a very high image quality (high resolution and high contrast) at a low dose. the avalanche gaseous photon counting technique, allows for virtually scatter free and noise free images, resulting in very high signalto-noise ratio. this new detector has been mounted on a commercially available mammographic unit (mammomat 3000, siemens). the digital images taken with the instrument are compared to analogue film images, where the beam quality (kvp and anode/filter combination) as well as projections has been kept the same to allow a true comparison. results: a full field mammographic digital detector, 19 × 24 cm, based on a slotscanning-area technique with photon counting gaseous detectors has been evaluated. the performance of the detector has been studied and compared to analogue film-screen system by imaging phantoms, breast specimens and patients. the results are very promising, compared to conventional film-screen images, regarding image quality and a significant dose reduction. conclusions: noise-free photon counting is a promising method for digital x-ray imaging, improving image quality as well as significant reducing the glandular dose to the patient. first considerations regarding an acceptance protocol for full field digital mammography equipment: validation in a multicenter setting f. rogge 1 , a.-k. carton 1 , h. bosmans 1 , e. vano 2 , p. torbica 3 , a. crevecoeur 4 , y. palmers 5 , k. faulkner 6 , g. marchal 1 ; 1 leuven/be, 2 madrid/es, 3 innsbruck/at, 4 liège/be, 5 genk/be, 6 newcastle upon tyne/gb purpose: the introduction of full field digital mammography equipment requires the development of acceptance testing protocols. we have reviewed the current european protocol for quality control in (conventional) mammography within the dimond-3 ec project and we propose a similar series of tests for digital systems. methods and materials: following tests had to be redefined: the assessment of the focal spot size, different tests of the automatic exposure control and tests of the detector including the grid. image quality was evaluated via phantom exposures and dqe measurements. the protocols were tested on 3 digital senographe dmr systems (ge) and on 1 digital fuji detector. results and conclusion: all the tests included in the revised acceptance protocol may be completed in under 4 hours. the performance of the three systems was very stable. the doses for standard exposures were variable between the different sites. this has an impact on image quality. since the dose setting for a digital detector is a parameter of the x-ray system only (independent of any filmscreen combination) that can be freely adjusted in the different centers, we propose to use a quite strict guidance regarding the standard dose settings. a difficult practical problem is the transfer of data in order to perform some evaluations offline. further cooperation with the manufacturer may be needed to make this type of testing feasible for hospital medical physicists. interventional radiology purpose: percutaneous ct guided biopsy is an accurate and safe procedure to evaluate indeterminate adrenal masses in oncologic patients. we sought to assess the clinical outcome in patients with negative percutaneous needle biopsies of focal adrenal lesions materials and methods: retrospective analysis of 225 oncologic patients (f:m, 138:87; age range 33 -87 years, mean age 66 years) who had undergone ct guided fna biopsies of an adrenal mass over a 5-year period were reviewed. the incidence of negative (for tumor) biopsy results were identified and assessed for subsequent evaluation of the biopsied lesion in these patients results: of the 225 biopsies, 41 (18 %) were negative for neoplasm. the primary neoplasm in these 41 patients included 32 lung cancers, 1 bladder, 1 prostate, 5 breast and 2 renal malignancies with the size of the adrenal lesion ranging from 2.8 -5 cm. of these 41 biopsies negative for tumor; 10 were identified as adenomas and the rest showed benign adrenal cortical cells or hyperplasia on cytopathology and histopathology. an experienced pathologist interpreted all specimens as sufficient for diagnosis. repeat biopsies were obtained in 13/41 (31 %) patients; whereas 2/41 (5 %) had the adrenal glands analyzed on post mortem examination. none of these 15 repeat evaluations yielded tumor conclusions: a negative or normal pathology result in a ct guided percutaneous adrenal biopsy can be regarded as a true negative evaluation in oncologic patients with no necessity to repeat the biopsy. a c d e f 234 materials and methods: in 54 patients (32 liver tumors and 22 different abdominal tumors) mr-guided biopsies or punctures were performed with a low field mri (0.2 t, magnetom open, siemens). for guidance of the needles, t1-weighted flash sequences (tr/te 100/9; 70°) were performed in all patients and an additional fisp-rotated-keyhole-sequence (tr/te 18/8; 90°) was also used. after positioning of the needle tip in the tumor, 82 biopsy specimens were acquired with 14 -16 g cutting needles (somatex®) during the interventional procedures. the visibility of the needles, the tumors and the abdominal vessels were evaluated. results: all interventional treatments were performed without vascular or organ injury. adequate specimens for histological interpretation were obtained in 47 cases (87.0 %). in 3 patients (5.6 %) the biopsy result was non-specific and in 4 patients (7.4 %) the lesion was missed. t1-weighted flash images were useful for confirming needle-tip placement during the biopsies or punctures. the use of free slice orientation facilitated the interventional procedure. the organs, tumors and vessels were easily determined. the fisp sequence resulted in equal or inferior results. conclusion: mr-guided abdominal interventions can be performed with acceptable safety and accuracy with low field systems. lesion and needle visibility are significantly affected by user-defined parameters which must be considered during procedure planning. materials and methods: over 4.3 years, 1120 biopsies were carried out under ct-fluoroscopic control (philips aveu and toshiba aquilion) in the lung (33.6 %), mediastinum (6.6 %), abdomen (26.6 %), pelvis (7.5 %), retroperitoneum (11.8 %) and varia (14 %). in 867 cases, the obtained material was sent to pathology. 88 % (761) provided material for cytology, 48 % (413) for histology. a pathologist was on-site and decided, by means of a quick staining procedure, whether further tissue removal was necessary. the primary tumour was known in 403 cases. the number of punctures, unsuccessful biopsies, positive and negative results (classified according to biopsying examiner and location), differences between histology and cytology and complications were analysed. results: on average, 1.6 punctures were performed to complete the procedure. 2 tissue samples for cytology were obtained in 181 cases (21 %). the overall sensitivity for cytology and histology was 97 % and 96 %, respectively; 93 % and 97 % with known primary; 99 % and 95 % with unknown primary. in 83 (0.1 %) and 26 (0.03 %) of cases, no diagnostic material was obtained with cytology and histology, respectively, although the on-site pathologist assumed that sufficient material was present. benign lesions were found in 163 cases (18.8 %), 45 biopsies were unsuccessful (5.2 %). one less experienced examiner yielded only 61 % sensitivity. overall, complications occurred in 43 patients (5 %). conclusions: ct-fluoroscopy-guided biopsy represents a safe and reliable technique with a high sensitivity for malignancy and minimal difference between cytology and histology. the examiners experience plays a decisive role in the diagnostic outcome. the device used (pin point*) is integrated into a multisclice ct (mx8000, *marconi medical systems). it uses a laser assisted proprietary articulated arm technology and displays corresponding multiplanar ct images as the computer assisted arm is manipulated along the patient's body. the system includes an intuitive virtual planning tool that allows simulation of the best instrument path from the entry point to the target area. 50 biopsies of masses in 50 patients were carried out. all biopsies were obtained using an 18 or 20 gauge high speed tru-cut biopsy gun. masses biopsied included lung (25), lymph nodes (8), bone (10) and pelvis (7). an average of 2.5 passes was performed. results: using this system interventions could be confidently planned and safely carried out even in difficult or risky areas in all 50 cases. however, due to respiratory motion there are some limitations, particularly with lung lesions. mean proce-dure time was 8 min ± 10 min. in 95 % of the cases biopsies were diagnostic. after central lung biopsy 1 of 4 patients with pneumothorax required a chest drainage. 2 cases with haemoptysis resolved with conservative treatment. conclusion: the evaluated simulation and virtual planning system is safe, quick and convenient for ct-guided biopsies with some limitations due to respiratory motion. it may also become a useful tool for additional ct-guided therapeutic procedures. evaluation of age-related risk profiles for pneumothorax in ct-guided percutaneous thoracic biopsies b. rapprich 1 , t. werba 1 , r. tomczak 2 , n. rilinger 1 ; 1 offenbach a. main/de, 2 bad friedrichshall/de purpose: in studies of patients undergoing percutaneous thoracic biopsies, the risk for pneumothorax has varied from 2 -61 %. our aim was to develop a risk profile for pneumothorax based on the severity of pneumothoraces and in correlation with age and the presence of emphysema. methods and materials: we retrospectively investigated outcomes of 133 patients with ct-guided thoracic biopsies using an 18 g coaxial cutting-needle with a high-speed-automatic system (biopty bard). pneumothorax was graded as: i minimal, local; ii < 1 cm; iii > 1 cm; iv chest tube required. emphysema was graded as: i discrete; ii moderate; iii extensive. results: 97.7 % of the biopsies in 133 patients were successful. the diagnostic sensitivity was 94 % and the incidence of pneumothorax was 30.1 % (grade i: 17.3 %; ii: 5.3 %; iii: 3.8 %; iv: 3.8 %). for younger patients the risk of severe pneumothorax (grade iii or iv) increased linearly. the degree of emphysema did not correlate with the incidence of pneumothorax. conclusion: the pneumothorax risk for elderly patients decreases in transthoracal ct guided automated high-speed cutting-needle biopsies. the incidence of severe, clinically relevant pneumothoraces increases linearly in younger patients. the linear correlation is caused by morphological and functional linear changes due to the regular aging of the lung. no correlation with pathological emphysema was found. purpose: the aim of this study was to examine the feasibility of percutaneous drainage of abdominal fluid collections using a mri-compatible drainage system. material and methods: in 6 patients drainage procedures were performed with mri-compatible drainage systems (somatex®) for the interventional treatment of abdominal fluid collections). for all procedures a low field mr-system (0.2 t, siemens) was used. the punctures were controlled using t1-weighted flash sequences (tr/te 100/9; 70°) and a fisp-rotated-keyhole-sequence (tr/te 18/8; 90°). after positioning of the catheter in the fluid collection, the topographic details were controlled with intracavitary injection of an aqueous gadolinium solution (dilution 1:200). results: all drainage catheters were successfully placed into the fluid collections under mr-guidance. the catheter position was easily visualized with the t1weighted flash sequences. using the fisp sequences the visibility of the catheters was good or moderate and also useful for mr-guidance. no procedure-related complications occurred. the mean time needed for the drainage procedure was 30 min ± 15 min. the fluid collections were drained for a mean of 12 days. the topic of the study presented here is the evaluation of the feasibility and acceptance of repeated ultrasound-guided lymph node puncture for vaccination. we performed 548 ultrasound-guided punctures of inguinal lymph nodes in 58 patients with proven malignant melanoma and biopsy confirmed metastases of the disease. we delivered a suspension containing antigen pulsed dentritic cells, under ultrasound view, directly into the lymph node's paracortical area. to visualize the procedure we used a high-resolution computed ultrasound system (sequoia 512) with a 13 mhz linear array probe. the tip of a 20 g needle was placed into the paracortical area of the node, and application of the suspension was performed under ultrasound view. results: normal-sized, morphologically unsuspicious lymph nodes were found in every patient during every procedure. major side effects were not observed; we merely noticed fever in 1 patient, painful swelling of the punctured lymph node in 3 patients, and newly occurring vitiligo in 10 patients. the immunological results of our therapy are still under evaluation. conclusion: ultrasound-guided intranodal vaccination is a reliable and reproducible technique to deliver antigen-pulsed dentritic cells into peripheral lymph nodes. the method is well accepted by patients. it could lead to changes in the therapeutical options of both malignant and infectious diseases. slowly-heparin-releasing drainage catheter: in vivo and in vitro assessment of clog-resistant effect j. han, k. lee, b.-i. choi, c. yoon, y. byun, h. moon; seoul/kr purpose: clogging of catheter is frequently encountered during various drainage procedures. we evaluated the effectiveness of a novel clog-resistant drainage catheter. we have recently developed slowly-heparin-releasing catheter by coating standard drainage catheter with the blend of amphiphilic heparin-derivative (heparin-deoxycholic acid) and polyurethane. in vitro fibrin formation test was performed by dipping heparin-coated catheter and non-coated catheter (as control) into fresh plasma for 60 minutes, and the results were interpreted using scanning electron microscopy. for in vivo assessment, eight pairs of coated and control catheters (8 french) were inserted into the peritoneal cavity in normal new zealand white rabbits (n = 8). applying a basic principle in flow dynamics (resistance = pressure/flow rate), intra-catheter pressure was measured at the plateau (pmax) during saline infusion in constant rate (0.1 ml/s) and pmax was followed up for 14 days to estimate the changes in effective caliber. results: on in vitro assessment, no fibrin formation was observed in heparincoated catheter compared to strong reaction in control catheter. on in vivo assessment, decrease in mean effective caliber was 1.7 % vs. 7.5 % (coated vs. control) at 3 rd day (p = 0.14, paired t test), 4.8 % vs. 12.5 % at 7 th day (p = 0.059), 6.5 % vs. palermo/it purpose: hepatic artery stenosis and thrombosis are common complications in liver transplant patients. digital subtraction angiography (dsa) has served as the gold standard to make this diagnosis. more recently, three dimensional helical computed tomographic arteriography (3d cta) with maximum intensity projection and shaded surface display techniques has been compared with dsa. the purpose of this study is to determine if 3d cta with volume rendering technique is a useful and accurate tool in the detection of vascular complications after liver transplantation. methods: 35 consecutive liver transplant patients underwent 3d cta with volume rendering technique. the standard of reference was dsa for 20 patients and imaging and clinical follow-up for 15 patients. two blinded reviewers evaluated the axial and 3d cta images in consensus. results: 3d cta with volume rendering technique detected 10 hepatic artery stenoses, six hepatic artery thromboses, two hepatic artery pseudoaneurysms, two splenic artery aneurysms, two portal vein stenoses, and four redundant hepatic arteries. in one case ct detected a moderate hepatic artery stenosis, while conventional angiography showed a normal artery. the sensitivity of ct for detecting vascular lesions was 100 %, specificity 89 % (8 of 9), accuracy 95 % (19 of 20), positive predictive value of 92 % (11 of 12), and negative predictive value of 100 % (8 of 8). conclusions: 3d cta is a useful and accurate noninvasive technique for detection of vascular complications in liver transplant patients. evaluation of an "all-in-one" multidetector-ct protocol for potential living liver donors t. schroeder, m. malagó, s. heistrüvers, s. nadalin, j. stattaus, j.f. debatin, s.g. rühm; essen/de purpose: to evaluate an "all-in-one" multidetector-ct-approach for evaluation of potential living related liver donors. materials & methods: 21 consecutive potential living donors of the right lobe of the liver (mean age 32 years) underwent three-phase, dual-enhancement multidetector-ct imaging to delineate biliary, vascular and parenchymal morphology. for display of the biliary system the first ct-image set was collected 25 minutes following the infusion of 100 ml of a biliary contrast agent (biliscopin). subsequently ct (slice thickness/collimation 1 mm, pitch 4 mm, table-speed 12 mm/s) was performed for display of the arterial as well as the portal and hepatic venous systems, following automated injection of 140 ml of an iodinated contrast agent (imeron 350). analysis was based on source images and multiplanar reformats. the hepatic parenchyma was assessed for the presence of pathologies and volumetry was based on the venous data set. results: all potential donors tolerated the exam well. the 'in-room' time for the exam ranged between 7 and 12 minutes. one hemangioma and one adenoma were identified. total liver volumes ranged between 1040 and 1716 ml. the underlying biliary and vascular anatomy was displayed at least to the second intrahepatic branch in all cases. anatomic variations were observed involving the biliary system (n = 16), the arterial system (n = 13), the portal system (n = 5) and the venous system (n = 6). conclusion: the outlined approach proved efficient and robust. all relevant pre-op data for potential living related liver donation was collected in one single diagnostic step. ischemic-type biliary lesions following liver transplantation: evaluation with mr cholangiography p. boraschi, r. gigoni, l. urbani, e. neri, f. filipponi, c. bartolozzi, f. falaschi; pisa/it purpose: to assess the diagnostic value of mr cholangiography (mrc) when evaluating ischemic-type biliary lesions (itbl) in the follow-up of liver transplant patients. we retrospectively reviewed mr imaging and mrc of sixteen liver transplanted patients (13 men, 3 women) with ischemic changes of the biliary tree. the mr examinations were performed on a 1.5 t unit (signa, ge medical system) with high performance gradients. after the acquisition of axial t1w and t2w sequences, mrc involved a coronal, non breath-hold, respiratorytriggered, fat-suppressed, two-dimensional, thin-slab, heavily t2w fast spin-echo sequence, and a coronal breath-hold, thick-slab, single-shot t2w sequence. ten patients underwent either surgical reconstruction of the biliary system (n = 4) or liver retransplantation (n = 6); the pathologic specimens were employed as standard of reference. the final diagnosis was obtained through direct cholangiography in the remaining cases. without knowledge of the surgical, pathological and cholangiographic findings two experienced observers evaluated in conference the mr images to determine the presence of biliary tract abnormalities. results: mrc demonstrated strictures involving the hepatic bifurcation and extrahepatic pre-anastomotic bile duct with concomitant thickening of the biliary wall in 13 patients; sludge or stones were present in 7 out of these patients. in the other 3 cases with circumscript extrahepatic lesion, mrc clearly showed intraductal sludge and/or biliary tract dilation above the non-anastomotic stricture, but direct cholangiography was more precise in determining the length of the stricture and grading the stenosis. we retrospectively reviewed doppler us images of the hv in 113 consecutive patients who underwent lrlt. doppler us was performed between 1 and 25 times (mean, 5.2 times) during 1 -433 days after lrlt. nineteen patients who were excluded for analysis of hv stenosis from various causes (early postoperative death, etc). patients who had more than 10 mmhg of pressure gradient between the hv and the inferior vena cava during the procedure of hv stenting were considered to have significant hv stenosis. the control group included patients with no evidence of hv stenosis at least 3 months after lrlt. spectral doppler us findings between the two groups were compared. results: five patients (4.4 %) had significant hv stenosis, and three of them showed persistent monophasic waves on doppler us whereas two patients showed monophasic waves on most of us examinations and a biphasic or a triphasic waves on 6-and 9-day follow-up. in control group (n = 89), 52 patients with persistent tri-or biphasic waves and 35 with mixed monophasic wave with tri-or biphasic wave according to the follow-up period of us examinations, and 2 showed persistent monophasic waves. conclusion: persistent monophasic waves on doppler us in the hepatic vein is a suggestive, but not a specific finding to be significant hv stenosis after lrlt. persistent triphasic waves on doppler us can confidently exclude significant stenosis. 3d ct angiography using multidetector row helical ct in the preoperative assessment of arterial supply to the liver g. brancatelli 1, 2 , m.p. federle 1 , v. kapoor 1 , d.a. geller 1 , j.j. fung 1 ; 1 pittsburgh, pa/us, 2 palermo/it purpose: to evaluate the usefulness of ct angiography (cta) with 3d reconstruction to detect the arterial supply to the liver using a multidetector row helical ct. methods and materials: eighteen patients had cta. of these, 17 had surgical correlation, including 9 who also underwent conventional arteriography. the remaining patient had conventional arteriography only. images were reviewed in cine mode on a pacs station with 3d volume rendered images. axial ct sections and volume rendered images were prospectively reviewed by two blinded readers jointly. results: all studies were technically satisfactory. ct angiographic findings were confirmed at surgery in 17/17 (100 %) patients (classical arterial anatomy, n = 11; replaced left hepatic artery (lha) off the left gastric artery (lga), replaced right hepatic artery (rha) off the superior mesenteric artery (sma) and accessory lha to segment iv off the common hepatic artery (cha), n = 1; replaced lha off the lga, n = 1; replaced cha off the aorta, n = 1; replaced lha off the lga and replaced rha off the sma, n = 1; rha, lha and gastroduodenal artery off common hepatic as a trifurcation, n = 1; rha off the coeliac trunk, accessory lha to segment iv off the cha, n = 1). findings at cta and conventional arteriography were concordant in 8/9 patients. in the single discordant case cta depicted a rha off the celiac trunk and an accessory lha to segment iv off the cha, but did not identify a lha off the lga. conclusion: cta is valuable in detecting the arterial supply to the liver allowing for precise surgical plannning. there are a number of sonographic features of posterior tibial tendinosis (ptt). one of these is an increase in posterior tibial tendon calibre. the aim of this study is to compare the posterior tibial tendon calibre in patients with ptt against a group of normal subjects by ultrasound. methods: patients with ptt were referred by the orthopaedic department for assessment of the posterior tibial tendon. healthy asymtomatic volunteers were taken as normal subjects. the posterior tibial tendon was examined by high frequency ultrasound (at least 7.5 mhz, usually 10 -12 mhz). the cross-sectional area was measured at three points along each tendon whilst keeping the probe at a right angle to the course of the tendon as it runs behind and under the medial malleolus and then forward on to its insertion at the navicular bone. the reference points were behind the medial malleolus, below the medial maleolus and just proximal to the tendon insertion. results: over a four year period, 44 patients with uni-or bilateral ptt were examined. the tendon calibre was compared with measurements obtained from 100 normal subjects from which the mean and standard deviation values were determined. it was shown that the vast majority of those with symptomatic ptt had values greater than 2 standard deviations above the mean normal value at each reference level. conclusion: patients with posterior tibial tendinosis demonstrate an appreciably larger tendon calibre than normal subjects. this is a very helpful sign in the diagnosis of posterior tibial tendinosis. kinematic ankle mri with a low field mri system g. dazzi, e. silvestri, a. iozzelli, f. magnaguagno, g. garlaschi; genova/it purpose: to test the usefulness of kinematic ankle mri performed with low field mri equipment. methods/materials: from january 1999 until february 2000 we examined 150 ankles using a 0.2 t mr system (artoscan, esaote). 25/150 patients (n = 4 synovial anterior impingement, n = 6 osseous posterior impingement, n = 15 lateral ankle sprains) underwent kinematic evaluation. we obtained the adequate range of motion by moving the chair with the foot in a fixed position, so that the leg passes from a semiflexed to the fully extended position. in this way, the ankle moves from 90° dorsiflexed to 135° plantarflexed position. kinematic examinations were performed on sagittal and/or axial planes using hse t1w sequences. results: we didn not identify significative findings in all cases of synovial anterior impingement. on the other hand, kinematic exams allowed a better evaluation of all patients with osseous posterior impingement. we directly visualized peroneal tendon instability in 3/15 cases of lateral ankle sprains. conclusions: kinematic ankle mri, performed with a low field unit, is a simple and quick technique in the study of some ankle pathologies. in particular, it seems to be very useful to evaluate the biomechanical alterations of osseous posterior impingement and to demonstrate the presence of peroneal tendon instability which is often unrecognized and misdiagnosed. purpose: the aim of this work was to assess mri diagnostic accuracy in detecting pathological conditions which lead to tibio-talar joint (ttj) impingement, using arthroscopy as the gold-standard. method and materials: 21 patients (aged 19 -35 years) clinically complaining of ankle unsteadiness and swelling entered this study; 20 out of 21 patients referred to a history of ankle sprain. mri examination was performed with both a 1.5 t whole-body (tse t2-w and se t1-w sequences) and a 0.2 t dedicated system (se t1 and t2-w sequences). in 12 cases i.v contrast material was administered followed by se t1-w sequences with fat saturation pulse. all the patients underwent arthroscopy. results: mri examination revealed the presence of ttj synovial impingement in 9 patients and in only 4 of them the presence of contrast enhancement revealed acute inflammatory stage; arthroscopy always detected a thickened synovial lining. in 3 patients mri examination showed the presence of tibio-peroneal tendon injury associated with synovial hypertrophy ("meniscoid syndrome"); in 1 of these cases contrast enhancement was evident. in 6 patients mri examination documented the presence of a bony impingement due to talus and tibial osteophyte; in these cases arthroscopy confirmed mri diagnosis. three patients had a completely negative mri examination, one of them showing a meniscoid syndrome on arthroscopy. conclusion: because of a good correspondence with arthroscopic findings we may consider mri very accurate in the diagnosis of different pathological conditions causing ttj impingement. mri is also able to detect extra-capsular causes of impingement. forty patients affected by tibio-talar pain and swelling were submitted to plain films and to mri using dedicated and whole body mri units. in 21 of them history of a previous inversion ankle sprain was present. in five cases intra-articular injection of paramagnetic contrast media (5 -10 ml of contrast medium obtained from the dilution of 0.6 ml of gd-dtpa in 250 ml of saline) was performed. ct scanning was performed in nine cases. all patients underwent surgery. in 36 of the 40 cases arthroscopy confirmed the mri findings. 25 examples of chondral disease were discovered by mri with differing degrees of severity (6 low, 11 mild, 8 severe grade). in 3 of 6 low grade conditions, arthroscopy could not reveal the damage of the deepest aspect of the cartilage and subchondral drilling was performed on the basis of mri findings. in nine patients arthroscopy confirmed ct and mri evidence of osteochondritis dessicans. in the other 4 cases mri and arthroscopy could not reveal any pathological condition. conclusion: on the basis of our experience, mr has to be considered the gold standard in the study of ttj cartilage allowing evaluation of its full thickness and of the subchondral bone. subjects and methods: 63 patients with recurrent lateral derangement of the ankle were studied with ct and mri. ct was performed with coronal acquisitions using a conventional equipment. mri was performed with a superconductive scanner, and coronal and sagittal t1 and t2*w images. in 24 cases (selected group) no signs of alteration of both internal and external collateral ligaments were demonstrable. in these patient analysis of the angle between long axis of the tibia and major axis of subtalar joint (ts angle) was calculated and the appearance of the articular space of subtalar joint was also evaluated. analogous measurement was done in a control group of asymptomatic patients results: in 13 cases ts angle appeared acute; in 7 of them mri demonstrated partial synostosis/synchondrosis of the subtalar joint. in 7 cases ts angle appeared highly obtuse and no signs of synchondrosis/synostosis were identifiable. in 4 cases no signs of alteration of the subtalar joint were demonstrable both at ct and mri. to analyse the normal mr anatomy of the intermetatarsal space, with emphasis in the description of the intermetatarsal bursae and neurovascular bundles, using mr imaging, mr bursography, as well anatomic and histologic correlation in cadavers. high resolution mr imaging of 32 intermetatarsal spaces derived from fresh human cadaveric feet was performed. the 4 intermetatarsal bursae were injected, and mr imaging were performed. t1-weighted and fat saturated t1-weighted spin echo sequences were performed in the 3 orthogonal planes. the intermetatarsal space anatomy was analysed. histologic examinations were performed. the intermetatarsal spaces were located in the forefoot, between two metatarsal heads, below and above the deep transverse metatarsal ligament (dtml) that divided them in two levels. the superior levels contained the synovial bursa, the interosseous muscles and tendons, and the collateral ligament complexes of the metatarsophalangeal joints, while the inferior levels contained lumbrical muscles and neurovascular bundles. one case presented a morton's neuroma. mr bursography improved the visualization of the structures of the superior level. the bursae extended distal to the dtml in the second and third spaces close to the neurovascular bundles and did not extend beyond the dtml in the first and fourth spaces. objective: plantar fibromatosis is an uncommon benign condition manifested by proliferation of fibrous tissue within the plantar fascia. as opposed to plantar fasciitis which affects the calcaneal insertion, plantar fibromatosis affects the mid-to forefoot region of the plantar fascia. methods: over a four year period, 10 patients (8 females; 2 males, average age 54.8 years) with clinically suspected plantar fibromatosis were examined by ultrasound 13 mhz (siemens sonoline) or 12 -5 mhz (atl hdi 5000) linear array transducers. results: plantarfibromatosis was seen as a discrete fusiform-shaped nodular thickening of the plantar fascia removed from the calcaneal insertion. of the 12 patients examined, 8 (66 %) had unilateral disease while 4 (33 %) had bilateral disease. of the 16 affected feet, a single site of fibromatosis was present in 9 (56 %) feet with two or more sites in 7 (44 %) feet. of the 25 discrete foci of fibromatosis, 22 (88 %) were hypoechoic to the plantar fascia and 3 (12 %) were isoechoic. the fascial margin was well-defined in 12 (48 %) and ill-defined in 13 (52 %). 2 of the 25 lesions (8 %) showed mild internal vascularity. no correlation was found between the ultrasound appearances and the chronicity of patients symptoms. 15 (60 %) of lesions were located in the midfoot while 10 (40 %) were located at the forefoot. 17 (68 %) were located medially in the fascia while 8 (32 %) were located centrally. conclusion: this is the first reported series detailing the ultrasound appearances of plantar fibromatosis. while the ultrasound appearances are variable, they are still characteristic enough to allow a specific diagnosis in all cases. purpose: spontaneous involution of lumbar disc herniation in patients treated with conservative therapy is reported in up to 70 % of cases. the aim of our study was to evaluate any eventually predictive signs on mr imaging of disc herniation involution. methods and material: 65 patients, affected by 72 lumbar disc herniations, entered a perspective study. mri examinations were performed on 1.5 t magnet, using sagittal and axial t1w se sequences before and after contrast administration, and fse t2w ones on the same scan planes. patient age, sex and level and size of disk herniation, clinical onset interval, type of herniation, t2-w signal intensity and pattern of contrast enhancement had been registered. all the patients, conservatively treated, underwent clinical and mri follow-up after 6 months: disc herniation size and contrast-enhancement variations were re-evaluated. results: mri follow-up showed herniation regression in 66 % cases. extruded disk with high signal intensity on t2w sequences regressed in 83 % of cases. the same percentage of regression was registered in enhancing cases. free fragments disappeared in all cases. rest in bed for at least 15 days resulted to be a significant factor for a favorable evolution of acute disk herniation. no relationship was found with side, size, level. conclusion: mri features seems to have a significant meaning in assessing spontaneous tendency on herniation involution, thus furnishing additional prognostic value. the role or mri and mra in diagnoses of cervical spondilotic myelopathy v.p. marchuk 1 , a. chernenko 2 ; 1 vitebsk/by, 2 minsk/by purpose: there are three important pathophysiologic factors in the development of cervical spondylotic myelopathy (csm): static and dynamic mechanical spinal cord compression, spinal cord ischemia. this study was performed to analyze the role of combination mri and mra in patients with csm. materials and methods: 121 pts were examined on a 1.0 t picker vista unit -18 healthy subjects, 50 pts had signs of discogenic radiculopathy and neuroreflected disorders (i), 53 pts had signs of cervical spondylotic myelopathy (ii). mra was carried out in coronal projection, oriented perpendicular to the direction of blood flow of ra (tr/te 40/12.5, 256 matrix, 2dft tof, mast, overlap 0.5 mm). mr investigation included standard protocol. the difference in groups were mainly obtained in the evaluation of the number of radicular arteries branches (ra) -more than 3 branches were visualized (+) or were not visualized (-); and spine cord compression (c) -present (+) or absent (-). combination (ra+ c-) for healthy group displayed in 100 %, for i group in 52.8 %, ii group in 16.73 %. combination (ra-c+) for i group 11.1 %, for ii group 40.47 %. the spinal cord damage and csm arising from inadequate blood passage in ra branches and spine compression were increased twofold in cases of combination (ra− c+). the mr technique allows these complications to be revealed simultaneously and serves as a basis for appropriate treatment. material & methods: 20 patients who suffered from radiation myelopathy were examined by mr. patients underwent radiotherapy with total dose of 40 -44 gy and irradiation was performed in two series, with one month time interval between them. radiotherapy was performed in patients with bronchial carcinoma (14 patients) and malignant lymphoma (6 patients). examinations were performed on 1.5 t mr imager using a surface coil. the protocol included t1w and t2w sagittal images, t1w and t2w axial images and images after application of gd dtpa. results: clinical signs of radiation myelopathy appeared 6 -18 months after radiotherapy. symptoms included weakness of lower limbs and paresthesia. mr examination revealed abnormally decreased signal on t1w images, increased signal on t2w images, focal contrast enhancement and spinal cord enlargement. cord enlargement was detected in 19 patients while in 1 patient spinal cord atrophy was found. radiation-induced abnormal high signal intensity was presented in cervical and thoracal vertebral bodies due to fatty replacement of bone marrow with sharp boundary corresponding to the radiation field. conclusion: mr is easy, safe and valuable imaging method in diagnosis and differential diagnosis of radiation myelopathy. then we performed contrast-enhanced sagittal t1-weighted scans after i.v. administration of gadolinium-dtpa (magnevist, schering, germany). when we recognized these features with mri, the exam was completed with axial scans using the same sequences. all the patients underwent lumbar puncture within 2 days of the mr examination. results: in 10 patients mri was positive. we detected diffuse sheet-like infiltration of the arachnoid membrane or neoplastic coating of the conus and cauda equina to nodular deposition throughout the subarachnoid space. the most sensitive acquisition was the contrast-enhanced se t1-weighted sequence. only 7 patients out of 10 had a positive lumbar puncture. in the follow-up during therapy, mri was considered more accurate in the evaluation of the pathological evolution of leptomeningeal involvement by hematopoietic neoplasms. conclusions: in our limited experience, mri was more sensitive in the detection of spinal epidural and leptomeningeal involvement by hemopoietic tumors. in patients with strong clinical suspicion of this involvement and negative lumbar puncture, mri should be performed. purpose: knowledge of the exact site and localisation of a csf fistula is important for planning surgical procedures and to prevent further complications. the purpose of this prospective study was to establish and to evaluate gd-dtpa enhanced mr cisternography in the detection of rhinobasal csf fistulae in patients with suspected csf rhinorrhea. materials and methods: 10 patients with suspected csf rhinorrhea were examined. clinical diagnoses were: 7 severe head/brain trauma, 1 meningocele, 1 hydrocephalus and 1 chronic sinusitis. mr cisternography included the following investigation steps: acquisition of non enhanced fat suppressed t1-weighted se scans of the skull base and the paranasal sinuses, lumbar puncture with administration of 1 ml gd-dtpa solute with 4 ml nacl and performance of mr cisternography with the same fat suppressed t1-weighted sequences as used initially. in 5 patients gd-dtpa enhanced mr cisternography detected a csf fistulae (2 sphenoidal and 3 ethmoidal). while 4 of these depicted leaks were confirmed surgically and sealed. in one case the csf fistula closed spontaneously. in another case, csf leakage after severe head injury was highly suspected clinically, but ceased prior to mr cisternography, which was unable to detect the temporary fistula. in the remaining 4 patients with serous rhinorrhea mr cisternography did not provide any evidence for csf fistulae. intrathecal gd-dtpa injection was well tolerated. conclusions: mr cisternography after intrathecal administration of gd-dtpa is a safe, promising and minimally invasive method for detection of csf fistulae. this mr investigation provides excellent depiction of csf spaces and pinpoints csf fistulae. the description of the complex 3d shape of the ventricular system is an important aspect for the typisation of the ventriculomegaly. materials and methods: pd and t2w 3d datasets were used for the segmentation of ventricular system. using the volume and surface data's a size independent shape descriptor number (edginess) was created. edginess number of 8 atrophic, 10 alzheimer and 7 nph patient's was compared 44 normal subjects database. results: upon statistical analysis the data for the control patient group showed a significant difference from those for the alzheimer's group (p < 0.003) and for the nph group (p < 0.003), but no significant difference from the atrophy group. with respect to the atrophy group a significant difference was ascertained both from the alzheimer's group (p < 0.002) and from the nph group (p < 0.000). significant difference was also ascertained between the alzheimer's group and the nph group (p < 0.001). discriminate analysis enables 100 % reliability to be achieved in distinguishing the nph group. conclusion: size-independent shape description provides the opportunity for the objective monitoring of hydrocephalus cases, with particular respect to those occurring in developing children. it also enables ventricular shape changes developing in individual hydrocephalus cases to be differentiated. a substantial aspect the procedure creates a new possibility in differential diagnostics for the determination and type classification of every condition involving ventriculomegaly. aim of the study was to examine the effect of three different types of meal on the function of proximal stomach in alcoholic cirrhotic patients by ultrasound. the proximal stomach of 12 normal volunteers and 15 alcoholic cirrhotic patients were examined by ultrasound. each subject received on consecutive days 500 ml meat soup, or 500 ml of two different semisolid meals: yogurt with potato flakes or sour cream with potato flakes, the latter contained 12 % fat. the proximal sagittal area and the frontal diameter were measured fasting and 5, 10, 15, 20, 30, and 120 min. after a meal and my multiplication of these two parameters approximate volume (av) was calculated. relaxation of the proximal stomach was characterized by the ratio of fasting avf to the actual av at different time points. results: compared with healthy controls, av/avf was significantly decreased (p < 0.05) after liquid and semisolid meal in cirrhotic patients (2.9 ± 0.3 vs. 5.3 ± 0.7 after liquid and 3 ± 0.3; 6.5 ± 1.5 after semisolid meal), while difference of this value was not observed after semisolid fatty meal between cirrhotic and healthy patients. the abdominal ct scans of 42 patients were retrospectively reviewed by 3 radiologists. eighteen patients had surgically proven internal hernias (2 paraduodenal, 16 transmesenteric, case group) while 24 had no evidence of internal hernia at surgery (comparison group). images were reviewed in a random and blinded fashion. individual and group performance was evaluated by roc analysis, and inter-observer agreement was measured by cronbach's coefficient alpha. individual ct signs relevant as predictors of transmesenteric hernia were identified by logistic regression and ranked by their odds ratio and p-values. the 2 paraduodenal hernias were diagnosed by all 3 readers. ct signs of paradoudenal hernias include: a sac-like mass of small bowel, encapsulation of small bowel, mass effect on the posterior wall of the stomach, left displacement of the main mesenteric trunk and mesenteric vessel abnormalities. we found poor inter-observer agreement in diagnosing transmesenteric hernia in the case group, but good to excellent interobserver agreement for some of the ct findings predictors of transmesenteric hernia, with a cronbach's coefficient alpha value ³ 0.8. logistic regression analysis suggests that clustering of small bowel loops, en-gorgement, stretching and crowding of the mesenteric vessels, right displacement of the main mesenteric trunk, and small bowel obstruction manifested as bowel dilatation with a transition point are associated with transmesenteric hernia. conclusion: ct may allow a diagnosis of internal hernia, although this diagnosis remains difficult, especially of transmesenteric hernia. provoked gastro-intestinal bleeding with tissue plasminogen activator -a useful diagnostic tool in patients with occult lower gi bleeding j.m. ryan, s. dumbleton, t. smith; durham, nc/us purpose: the purpose of this study was to assess the efficacy and safety of provocative mesenteric angiography with tpa, heparin and tolazoline in patients with occult lower gastrointestinal bleeding. materials: 17 provocative bleeding studies were performed on 16 patients for occult lgi bleeding including 9 women and 7 men, aged 44 -79 years. all patients had previous negative endoscopic and negative angiographic studies. to provoke bleeding we used a combination of intravenous heparin (range 3000 -10000 units), intra-arterial tolazoline (range 25 -100 mg) and intra-arterial tpa (range 10 -50 mg, mean 20.3 mg). results: 17 studies were performed in 16 patients leading to bleeding in 6 patients (37.5 %). 2 vascular abnormalities were diagnosed which did not bleed during provocation -an abnormality was identified in 8 of 16 patients (50 %). there were no procedural complications encountered. of 6 patients in whom bleeding was successfully provoked, 4 bleeds occurred in the large bowel, and 2 in the small bowel. 3 patients had embolization at the time of provoked bleeding. 5 of these 6 patients required no further therapy for lower gastrointestinal bleeding. 10 patients (including 2 with vascular abnormality) did not bleed during the provoked study with tpa. of 8 patients with normal study 5 patients rebled during followup. conclusion: intraarterial provocative mesenteric angiography with heparin, vasodilator, and tpa identified the site of bleeding in 37.5 % of patients in our study group, and contributed to treatment in 50 %. this small study indicates that the procedure appears to be safe. monday b methods and materials: ct films of 10 cases with internal hernia including 4 cases with paraduodenal hernia, 3 cases with transmesenteric hernia, 2 cases with transomental hernia and 1 case with foramen of winslow hernia were retrospectively reviewed. surgical proof was obtained in all cases and strangulation was found in 5 of the cases. results: prevalent ct findings found in internal hernia were cluster of small-bowel loops, crowding of mesenteric vessels, engorgement of the mesenteric vessels, evidence of small bowel obstruction, and closed loop. saclike mass was a helpful ct finding to diagnose internal hernia having hernia sac. mesenteric edema, bowel wall thickening, and ascites were frequently observed in cases with strangulation. in paraduodenal hernia, herniated bowel loops showed a sac like mass and were found posterior to either ascending or descending mesocolon. in a subtype of paraduodenal hernia and foramen of winslow hernia, herniated loops were found in the lessor sac. in transmesenteric and transomental hernias, herniated bowel loops were found lying adjacent to the abdominal wall displacing the colon centrally. conclusions: ct may allow the diagnosis of internal hernia based on the shape and anatomical location of the herniated bowel loops. the presence of strangulation can be assessed by the ct features of the bowel wall and mesentery. cross sectional imaging findings of juxtapapillary duodenal diverticulum c. balci, a. akinci, e. akun, c. duran; istanbul/tr objective: the purpose of this study was to evaluate the ct and mr imaging features of juxtapapillary diverticulum, correlating with ercp, duodenoscopy. we retrospectively evaluated ct (n = 6) and/or mri (n = 11) examinations of 12 patients who were diagnosed juxtapapillary diverticulum on ercp (n = 10) or endoscopy (n = 2) examinations as correlative diagnostic examination. mri was performed in a 1.5 t scanner, ct was performed with helical technique using thin sections. ct (n = 5) and/or mri (n = 9) were primary imaging modalities in 10 patients. size, location of the diverticula and imaging findings of associated biliopancreatic disease were assessed. results: diverticula were missed initially in three patients on ct (n = 1) and/or on mri (n = 3). size of the diverticula ranged between 1 -3 cm. imaging findings of the juxtapapillary diverticulum was characterized as outpouching of the duodenal wall towards pancreatic head (n = 5). on 5 patients papillary relation to diverticular wall was evaluated on mri. on ct, layering of oral contrast with air level was characteristic imaging feature. on mri t2-weighted true fisp and haste techniques demonstrated the air fluid level with hyperintense fluid and signal void air level above. associated imaging findings as dilated cbd (n = 5), cholecystitis (n = 2) cholecystolithiasis (n = 2), chronic pancreatitis (n = 6) and were visualized on both imaging modalities. conclusion: with a better recognition of juxtapapillary diverticulum on ct and mri, initial cause of biliopancreatic disease symptoms in patients with juxtapapillary diverticulum can be depicted by these modalities. results of screening ultrasound diagnosis of gastric and colonic cancer s. pimanov, a. sikora, c. vergasova, n. lud; vitebsk/by purpose: the problem of colonic cancer (cc) and gastric cancer (gc) diagnosis remains a difficult issue. a number of screening programs for cc diagnosis based on faecal occult blood tests and endoscopy are well-known. the objective of this study was to determine the possibilities of screening ultrasound examination for gastric cancer and colonic cancer. patients and methods: 29467 patients aged 25 -87 with various diseases were examined for cc and gc diagnosis. screening ultrasound examination for cc and gc was performed in all the patients of the hospital and ambulance station who were examined by conventional abdominal sonography. cc and gc had not been suspected before the examination. revealing of concentric hypoechogenic thickening of the stomach and bowel wall with increased central reflection (i.e. non-specific "pseudokidney symptom") was defined as a pathological criterion. bowel and stomach sonography lasted for about 3 minutes. results: the ultrasound examination resulted in revealing 54 cases of cc and 18 cases of gc. almost all patients with tumours were operated and in all cases the diagnosis was confirmed histologically. according tnm classification most of these patients had t2 -t3 stages of cancer. false negative results were analyzed using regional cancer register. conclusion: it seems quite possible to diagnose actively colonic and gastric cancer by means of screening ultrasound examinations. the aim of our study was to develop an examination protocol for esophagus virtual endoscopy, that in association to axial ct images allowed to evaluate malignant stenosis and diverticuli. methods and materials: virtual endoscopy of the esophagus was performed in 29 patients with esophageal wall pathology. esophageal distention was obtained by oral administration of effervescent granules and intramuscolar injection of 20 mg of scopolamine butylbromide. in all patients, spiral ct volume zoom examination (120/80/1 mm/1 mm/8 mm/0.5 s kv/mas/slice coll./slice width/feed per rot/rot. time) of the thorax and upper abdomen was performed within a single breath-hold, before and after i.v. administration of contrast media. real time endoscopy images were reconstructed using volume-rendering techniques with a dedicated workstation and specific software (vitrea 2.2, vital images). conventional esophageal endoscopy was performed in all cases and an additional bronchoscopy was executed. results: 5 diverticuli and 24 endoluminal stenotic lesions were founded. most of them were localized in the middle or lower thoracic esophagus and were displaied as a discontinuity of esophageal lumen. 3d ct imaging associated to axial images depicts esophageal tumoral extension, other mediastinal air filled structures, the gastroesophageal junction and shows the presence of diverticuli complications. conclusions: our protocol achieves excellent distention of the lumen both proximal and distal to the stenosis and allows a correct differentiation between malignant stenosis and tumor located into diverticular task. this technique allows to obtain additional informations in the stenosis that do not permitt the passage of an endoscope and comparing to barium studies excludes the risk of aspiration of contrast agent. in 15 patients with positive egr, hp was negative, while both were negative in one patient. there was highly significant difference between the hp and egr findings (p < 0.01), meaning that statistically there was no dependence between hp and egr. in all 52 patients, hp finding did not correlate with index values (r = 0.181, p > 0.05). index of egr in patients with positive hp was c = 16.7 ± 11.5 %, while in those with negative hp were x = 25.3 ± 9.8 %. there were significant differences (p < 0.05) in the values of index in relation to the presence of hp, meaning that it was lower in the patients with positive hp finding. however, when only patients with pathologic values of egr were considered (egr > 10 %), there were no significant differences (p > 0.05) in the values of index in relation to the presence (n = 19, x = 27.7 ± 9.6 %) or absence (n = 15, x = 27.0 ± 7.15 %) of hp. conclusion: there was no relationship between the presence of hp and egr. birmingham, al/us, 2 rome/it introduction: cardiovascular mri (cmri)can accurately assess lv systolic function. diastolic function assessment involves complex methodologies such as phase contrast imaging or myocardial tagging. our purpose was to define diastolic dysfunction (dd) with a routine cmri approach. methods: using a 1.5 t system (ge signa cv/i; ge medical systems, milwaukee, wi), lv volumes and mass were calculated on cine short axis sequences in 19 patients, 8 with risk factors for dd (lvh, hypertension, coronary disease, etc) and 11 (control group) without risk factors. post-processing was performed with mass analysis package (medis, leiden, the netherlands) studied variables were ventricular relaxation rate, lv mass index, ef, and end diastolic volume index. results: diastolic relaxation rate was calculated from a linear regression line fitted to the rate of lv volume change and was normalized using the body mass index. ef and lv mass index were calculated. using binary linear regression analysis, diastolic relaxation rate predicted patients with risk factors for dd with a 2 of 9.8 (p < 0.005), whereas lv mass index predicted dd with a 2 of 5.6 (p < 0.05). ef and end diastolic volumes were not significantly different between groups. normalized diastolic relaxation rate distinguished the two groups with 79 % accuracy, cardiac mass index with 74 %, and the combination of the two with 95 %, with 2 of 18 (p < 0.0001). conclusions: diastolic relaxation rate and lv mass index can be used to evaluate lv diastolic function as a component of every cmri study, using a simple cine sequence, without additional time or complex techniques not widely available. clinical usefulness of high dose dobutamine stress mri for the detection and cardiac tagging for the detection of myocardial ischemia d.y. kuijpers, k.y. ho; groningen/nl purpose: to evaluate the clinical value of high dose dobutamine stress mri (dsmri) for the detection of myocardial ischaemia by wall motion analysis using myocardial tagging. patients and methods: a consecutive group of 159 patients with known or suspected coronary artery disease, who stopped their anti-anginal medication 4 days before the examination, were examined with a cine breathhold using a standard 16 segment short axis model. seven patients could not tolerate the examination. patients were examined at rest and during increasing doses of dobutamine stress at 6-minute intervals up to 40 µg/kg/min. cine images were acquired during breathholds at 3 short axis levels, including myocardial tagging. the data were evaluated for the presence of ischaemia, which was defined as a new wall motion abnormality (hypokinesia, akinesia, dyskinesia) in at least 2 segments at 2 different levels. mr images were interpreted during the examination by two 2 experienced investigators. two datasets were not interpretable. results: average examination-time was 52 minutes without major complications. 54 patients who showed signs of ischemia, were examined with coronary angiography within 2 weeks. 51 patients showed significant coronary artery disease of whom 49 needed revascularisation. three patients showed false-positive mri results, while sensitivity for 50 % luminal narrowing was 94 %. in the 86 patients without ischaemia on mri, the cardiovascular occurence-free survival rate was 98 % (average follow-up period was 10 months). conclusion: high dose dsmri using myocardial tagging is a safe and clinically useful method for the detection of myocardial ischaemia. aim of this study was to investigate the capability of mr imaging as a noninvasive tool to follow up ventricular and valvular function, perivalvular flow and myocardial remodeling in patients after avr. methods: on a 1.5 t mr system 15 patients with aortic stenosis and 20 healthy volunteers were examined. cardiac output (co), ejection fraction (ef), enddiastolic volume (edv) and lv ejection time frame as a parameter of contractility (∆tlv) were determined using a velocity encoded 2d flash technique (tr 24 ms, te 5 ms) and cine sequence (tr 100 ms, treff. 10 ms, te 4.8 ms, flip 25°, temporal resolution 50 ms). hemodynamic parameters and end-diastolic myocardial wall thickness (emwt) were determined 1 ± 0.5 days pre/7 ± 2 days and 10 ± 3 months after implantation of a st. jude medical prostheses. invasive measurements (fick principle/thermodilution) and echocardiography served as reference standard. intra-and interobserver variability of two observers were calculated for mr measurements. results: for co a good correlation with invasive measurements was found using phase velocity mapping (r = 0.66, p < 0.0007). lv parameters normalized late postoperatively (∆t = 158 ± 20 ms/98 ± 20 ms, edmt = 18 ± 3 mm/11 ± 3 mm, pre/ 10 ± 3 months surgery, p < 0.001). differences between normal volunteers and patients were detected on a high level of significance (normal ∆t = 102 ± 24 ms, emwt = 9 ± 3 mm, p < 0.0001). for mr flow measurements interobserver variability was 2.5 ± 2.7 %, intraobserver variability was 1.7 ± 1.6 %. in patients with suspected postoperative complications after avr cardiac mri should be applied prior to hospitalization and invasive diagnostic procedures. cardiac systolic rotation and contraction before and after valve replacement for aortic stenosis -a magnetic resonance myocardial tagging study j.j.w. sandstede 1 , t. johnson 1 , m. beer 1 , k. harre 1 , t. pabst 1 , w. kenn 1 , w. völker 1 , s. neubauer 2 , d. hahn 1 ; 1 würzburg/de, 2 oxford/gb purpose: aortic stenosis leads to derangement of cardiac function and contraction mode due to chronic pressure overload that is relieved after surgical valve replacement (svr). this study aimed to determine the changes in left ventricular (lv) systolic wall function before and after svr using mr tagging, compared to age-matched healthy volunteers. monday b a c d e f 249 materials and methods: 12 patients with aortic stenosis were examined with an ecg-triggered 2d tagging sequence at 1.5 t before and 12 months after svr. lv function and mass were determined by cine mri. cardiac rotation at the apical, mid-ventricular, and basal level and overall torsion were evaluated. 8 healthy volunteers within the same age group served as a control. results: before surgery, all patients showed a significant increase of apical rotation and overall lv torsion, basal rotation was not significantly different compared to volunteers. apical rotation and torsion were negatively correlated with lv mass and end-diastolic volume. one year after surgery, basal rotation was reduced compared to controls. in comparison to pre-operative values, apical rotation also decreased but was still elevated, and this resulted in a normalization of lv torsion. conclusion: pressure overload before surgery is associated with an increase of systolic lv wringing motion, possibly serving as a compensatory mechanism. this mechanism declines with increasing lv hypertrophy and dilatation. svr for aortic stenosis leads to normalization of lv torsion one year after surgery. patients and methods: global and regional function was determined by cine mri in 30 patients 3 weeks after anterior mi. at the same time, 31 p-spectra were obtained from infarcted as well as non-infarcted adjacent myocardium using a 3d-csi technique (voxel size 25 ml each). pcr/atp ratios and signal to noise ratios (snr) were determined using amares. 6 months after revascularization mri and mrs were repeated. gold standard for viability was recovery of regional function after revascularization. healthy volunteers served as control group. results: two groups of patients (15/15; wall motion recovery versus persisting wall motion abnormalities) were formed according to follow-up mri. at study entry patients with non-viable segments had significant reductions of pcr/atp-ratios in non-infarcted and infarcted myocardium due to a significant reduction of pcr snr, whereas patients with viable segments showed that only in the infarcted areas (p < 0.05 each). no significant differences were detected concerning morphologic or functional parameters between both groups. control examination 6 months after revascularization revealed significant increases of energy metabolism and cardiac function for patients with viable myocardium (p < 0.05). conclusions: restoration of regional oxygen supply by revascularization therapy is connected with improvement of metabolism and function in patients with viable segments, whereas irreversible damage of infarcted tissue is connected with continuous depression of energy metabolism. time course of 23 na signal intensity after myocardial infarction in humans j.j.w. sandstede 1 , h. hillenbrand 1 , m. beer 1 , t. pabst 1 , w. machann 1 , w. völker 1 , w.r. bauer 1 , s. neubauer 2 , d. hahn 1 ; 1 würzburg/de, 2 oxford/gb purpose: elevated sodium (na) content in myocardial infarction (mi) can be imaged by increased na signal intensity ( 23 na si). experimental studies described the temporal changes of myocardial na content post-myocardial infarction. aim of our study was to determine the time course of 23 na si after mi in humans. materials and methods: 13 patients were examined 14 ± 3 days and 3 months after mi, 7 patients underwent an additional examination 4 ± 1 days after mi. double angulated short axis 23 na images of the heart were obtained in prone position using a 23 na surface coil and an ecg-triggered 3d-flash-sequence with 32 acquisitions. wall motion abnormalities were detected by cine mri. 23 na si of mi was expressed as percentual increase compared with the entire circumference of noninfarcted myocardium. results: all patients showed an area of elevated 23 na signal intensity correlating with wall motion abnormalities. 23 na si remained elevated between day 4 and day 14 in the 7 patients that were examined twice in the early stage after mi. all patients revealed a decrease of 23 na si until 3 months after mi. conclusion: 23 na si is elevated in acute mi and throughout infarct healing but decreases until 3 months after mi. this is in concordance with experimental studies, these studies showed the highest na content at day 1 with a decrease until day 90. correlation of 23 na si and myocardial viability in humans has to be determined in future studies. p-sloop-mrs. 15 healthy volunteers were included in the study. results: compared to matched volunteers, a significant decrease of absolute pcr concentration was observed in patients with ms (p < 0.05), whereas atp concentrations showed no significant changes. functional analysis by mri depicted depressed left ventricular (lv) ef in 3 patients. for ms patients with mitoxantrone treatment, an association between cumulative dose and ef could be detected (r = 0.59; p = 0.0189). however, ef, lv esv and lv esv were not statistically significant between the two groups of patients. mrs detected no association for ratios or absolute values and cumulative mitoxantrone dosage. the reduction in cardiac high-energy phosphates as well as lv ef in some patients points to a subclinical involvement of the heart in ms. treatment with a putative cardiotoxic drug like mitoxantrone seems, however, not to aggravate a possible pre-existing heart disease within the examined cumulative dose range up to 100 mg/m 2 . absolute concentrations of cardiac high-energy metabolites -relation to clinical severity m. beer 1 , h. köstler 1 , j.j.w. sandstede 1 , k. harre 1 , s. neubauer 2 , d. hahn 1 ; 1 würzburg/de, 2 oxford/gb purpose: cardiac high-energy metabolism is impaired in failing human myocardium, and this may contribute to contractile dysfunction. current technical improvements of cardiac mr-spectroscopy (mrs) allow for absolute quantification. we tested the correlation between clinical severity and metabolite concentrations. patients and methods: patients with hypertrophic (aortic valve stenosis, avd) and dilated cardiomyopathy (dcm) were studied (10 each). patients were graded according to nyha; lv volumes, mass and ef were determined by cine mri. 31 pspectra were obtained using a 3d-csi technique. pcr/atp ratios and absolute values (mmol/kg) were determined using amares and sloop. healthy agematched volunteers served as control group. results: for nyha classes 0 (vol), ii (7 avd, 1 dcm) and iii (3 avd, 9 dcm), pcr concentrations were 8.48 ± 1.42, 6.78 ± 1.11, and 4.23 ± 1.01, atp levels 5.93 ± 0.85, 5.03 ± 0.81, and 3.58 ± 0.64; and pcr/atp ratios were 1.59 ± 0.28, 1.36 ± 0.22, and 1.21 ± 0.30. pcr absolute concentrations showed the highest, atp intermediate and pcr/atp ratios lowest correlations with clinical and functional variables. conclusions: absolute concentrations seem to be more representative of the extent of energetic derangement in heart failure than established pcr/atp ratios. material and methods: high resolution mr imaging was performed on a 1.5 t unit (symphony quantum, siemens) using t1-and stir t2-weighted sequences via a dedicated wrist coil (total acquisition time 8 min) a mean of 6.5 days after initial radiographs in 50 patients with clinical suspicion of wrist fractures and normal plain or indistinct radiographs. initial radiographs were evaluated independently by two senior radiologists without knowledge of the mri findings. the resulting change in therapeutic strategy was clinically evaluated. results: in 29/52 wrists mri findings resulted in a change of diagnosis. there were false positive diagnoses on plain radiographs in 23 cases (46 %) and false negative diagnoses in 6 cases. mri allowed to detect additional injuries of soft tissue in 19 cases (38 %). in 19/52 wrists the period of immobilization could be shortened or ended. in 11/52 it needed to be prolonged and in 3/52 a surgical intervention was necessary. in 19 wrists mri had no therapeutic consequence. correspondingly, after mri examination the period of being unable to work was reduced in 15/50, prolonged in 7/50 and was not changed in 28/50 patients. axial images were not helpful regarding diagnostic accuracy. conclusion: mri of the wrist is recommended on the day of trauma if there is clinical suspicion and normal plain radiographs. accurate diagnosis by mri examination on the day of trauma may reduce economic costs due to shortened immobilization time. mri evaluation of the wrist using dynamic studies m. mastantuono, e. bassetti, g. simonelli, l. di giorgio, r. passariello; rome/it purpose: in some painful syndromes of the wrist the conventional mri imaging is not satisfactorily for diagnosis. we describe a new dynamic mri method, that is accurate and easy to repeat, for the study of carpal instability and wrist pain. materials and methods: we performed a dynamic study of the carpus in 27 healthy volunteers and on 38 selected patients which we chose from a group of 232 previously studied by static mri wrist examination. we used a dedicated magnet (artoscan, e-scan 0.2 t) that has revealed itself to be particularly useful for dynamic acquisitions. we standardized a technique of image acquisitions at different degrees of flexion both on the coronal plane in ulnar-radial deviation and in sagittal for palmar-dorsi-flexion. we used se and ge sequences obtaining sagittal and coronal scans with a slice thickness of 2 -3 mm. results: the dynamic study is superior in the evaluation of carpal instability and in the assesment of capsular and ligamentous lesions if compared to standard mri examinations. the kinematic evaluation of the images acquired in the different positions of movement allowed to document the pathologic articular kinetics. conclusions: in our experience the mr kinematic study provided information that permitted differentiation of the possible mechanisms responsible for chronic wrist pain. kinematic mri is a simple and low time-consuming method of study that permits an accurate assessment of carpal instability, easily demonstrating articular biomechanical alterations that may be difficult to diagnose on static conventional mr examination. in 23 patients we assessed direct mr-arthrography in comparison to multiportal wrist arthroscopy for the evaluation of extrinsic and intrinsic ligaments, the tfcc. we used a 1.5 t mr system (magnetom). after fluoroscopic-guided direct wrist arthrography into the mid-carpal and radiocarpal joint with a solution of ultravist and magnevist (1:200) we applied following sequences: (1) t1 tse (tr 500 ms, te 20 ms) 3 mm slice-thickness, coronal and sagittal plane, matrix 512, fov 120 mm. (2) 3d flash (tr 24 ms, te 11 ms, flip angle 50°) 1.5 mm slice-thickness, coronal and sagittal plane, matrix 256, fov 120 mm. between 1 and 14 weeks after mr-arthrography, all patients underwent diagnostic multiportal arthroscopy. for the evaluation of the scapholunate ligament and the lunotriquetral ligament we subdivided lesions in partial defects and complete tears. the mr-sign of a partial-ligament defect was the passage of gadolinium-contrast agent between the mid-carpal joint and the radiocarpal joint, but with intact ligament structures palmar or volar to the defect visible. complete tfcc-defects, recognized by passage of contrast-agent into the distal ulnoradial joint, were classified according to palmer. results: for complete defects there was a high correlation between mr-arthrography and diagnostic multiportal arthroscopy (sen: 91.7 %, spez: 97.9 %, ppv: 78.6 %, npv: 96.2 %). for partial defects the correlation was moderate (75.0 %; 98.0 %; 85.7 %; 94.8 %). conclusion: for the evaluation of complete defects of the intrinsic and extrinsic ligaments, the tfcc mr-arthrography and multiportal arthrography have nearly the same diagnostic power. for evaluation of partial defects arthroscopy is superior to mr-arthrography. after undergoing arthrography with a mixture of 1:200 diluted contrast medium (gadolinium-dtpa: imeprol) injected radiocarpally and midcarpally under fluoroscopic control, 65 patient suffering from ulnar-sided wrist pain were examined with a 1.5 t mr scanner. using computer-aided "navigator"technique, the 3d data sets were converted into a virtual arthroscopy. all these patiens underwent a conventional arthroscopy within 24 hours. imaging results were compared with the virtual findings. results: in 57 of the 65 cases virtual arthroscopy could be performed adequately, whereas in 8/65 the procedure was not successful due to excessive narrowing of the ulnocarpal compartment or artefacts caused by patients movement. among to determine morphologic changes of the median nerve in median nerve entrapment syndrome after surgical decompression using high resolution ultrasonography and to correlate with the clinical outcome. materials and methods: 25 carpal tunnels of patients (18 women, 7 men, mean age 66.7 years) with documented median nerve entrapment were examined with high resolution sonography before and at least 3 months after surgical decompression. a subgroup of 10 subjects was scanned before and after 3 months following surgery. we evaluated nerve ap diameter, the flexor carpi retinaculum, postoperative scar tissue and correlation with clinical outcome. results: 3 or more months after decompression the mean nerve diameter in the proximal canal is reduced from 2.46 mm to 2.09 (p = 0.05), in the distal canal from 2.32 to 1.98 (p = 0.0005). there was no significant difference in the subgroup examined within 3 months after decompression and in the relation proximal to distal nerve diameter pre-and postoperatively. the flexor retinaculum could be depicted clearly in all scans. the existence and extent of scar tissue and reduction of nerve diameter were found unreliable parameters regarding outcome. conclusion: analysed at least 3 months after surgical decompression the ap diameter of the median nerve is significantly smaller compared to preoperative values. this diameter reduction, however, as well as existence and extent of scar tissue does not always correlate with clinical outcome. the relation between nerve diameter in the proximal and distal canal does not seem to be a helpful parameter in the diagnosis of median nerve entrapment. ultrasonography of hand joints in rheumatoid arthritis y. khodjibekova, t. zasteba; tashkent/uz purpose: the aim of this investigation was to study typical sonographic signs of hand joints lesion in rheumatoid arthritis. materials and methods: 67 patients aged 20 to 55 years (15 males, 52 females) with rheumatoid arthritis affecting hand joints were investigated. the control group comprised 10 healthy individuals. in all patients diagnosis was established by clinical, laboratory and radiographic examinations. results: soft tissues, bone contours, ligaments, joint-forming surfaces, joint space and synovium were visualized on hand joints sonograms. the first stage of the disease was characterized by soft tissue thickening, increase of ligaments echogenity, effusion and increase of joint space. at the second stage of the disease soft tissues thickness decrease, slight increase of their echogenity, periosteum thickening, joint-forming surfaces destruction, narrowing of joint space to a c d e f 252 0.1 -0.2 cm were revealed on sonograms. at the third stage of the disease soft tissues were not visualized, bone contours were well-defined and deformed, periosteum was thickened, joint-forming surfaces were indistinguishable. conclusion: sonography is effective method for the diagnosis of hand joints lesion in rheumatoid arthritis, particularly in its early stages. preliminary experience with "extended field of view" (efv) ultrasonography in musculoskeletal disorders e. santacroce, c. avanzino, g. dazzi, l. raimondi, e. silvestri; genova/it purpose: to evaluate the usefulness of efv ultrasonography in the study of musculoskeletal system. materials and methods: forty subjects with musculoskeletal disorders were submitted to an efv ultrasound examination after a routine us evaluation. 20 shoulders with anterior-superior impingement, 4 wrists with carpal tunnel syndrome, 2 wrists with de quervain's disease, 8 ankles with achilles tendonitis and 8 patients with post-traumatic muscle injuries were considered. an atl hdi 5000 system equipped with a broadband 7 -14 mhz transducer with efv software was used. results: the efv evaluation improved the muscular and tendon anatomy demonstration and correctly showed the pathologic process extension in all cases. the larger field of view of this technique allowed more precise anatomic definition of muscles, tendons and peripheral nerves but did not provide further diagnostic information. not easy images acquisition, evaluation of small structures and convex bone surfaces are the main limits to this technique. purpose: this study was performed to establish the value of breathhold mr imaging in the diagnosis of acute appendicitis. materials and methods: over a 14-month period 138 consecutive patients, clinically suspected of having appendicitis all underwent an us study and a subsequent mri. the mri images were obtained with breathhold coronal and axial fast spin-echo t1, t2, and t2 fat suppression sequences with 5 mm thick slices. mri images were evaluated prospectively. appendiceal diameter, signs of periappendicitis, abscesses, fecaliths, free fluid, mesenteric lymphnodes, paralytic ileus, or the existence of an alternative diagnosis was noted. also the total duration of the mri study was noted. mri findings were correlated with surgical pathology in 62 patients and clinical follow-up in 76 patients. the mri findings of 63 patients were interpreted as positive for appendicitis (62 true positive, 1 false positive). mri findings showed no signs of appendicitis in 76 (true negative in all) and showed an alternative diagnosis in 36. sensitivity and specificity for detecting acute appendicitis were 100 % and 98 %. the median total duration of the mri study was 15 min. conclusion: fast spin echo t1, t2 and t2-fat-suppression breathhold mri imaging for the evaluation of patients with suspected appendicitis achieved a high accuracy in the detection or exclusion of appendicitis or an alternative diagnosis. the use of short imaging sequences minimized the impact on the routine mri program. in selected cases mri is a reliable, safe and quick tool that can be implemented in a community hospital. helical ct diagnosis of closed-loop obstruction and strangulation: how and when m. scaglione, f. pinto, a. pinto, f. lassandro, n. renda, e. de lutio, s. romano, l. romano; naples/it purpose: definite confirmation or exclusion closed-loop obtruction (clo) is one of the most difficult tasks the radiologist has to face in the clinical practice. the aim of this presentation is to focus on the impact of helical ct in the diagnosis of clo and strangulation. materials and methods: 120 cases of surgical proven clo diagnosed by helical ct were retrospectively reviewed. helical ct scans were performed after administration iv contrast material (120 ml, 50 s scan delay, 2.5 -3 ml/s rate), with initial 10 × 10 mm collimation and repeated 5 × 5 mm over the region of interest. the state-of-art ct signs were considered for the diagnosis. results: serrated beaks with poor or no contrast enhancement of the bowel walls, ascites or engorgement of the mesenteric vasculature are ct signs allowed the diagnosis of clo complicated by strangulation in 26/120 cases. u or c-sharped of dilated loops, radial distribution of the mesenteric vessels, beaks and whirls suggested clo in 94/120 cases, but did not help differentiate from strangulation in 17 cases. conclusions: clo is a dynamic entity which may regress or need laparotomy depending on the time and degree of rotation of the incarcerated loops. ct is a reliable imaging modality able to differentiate clo from strangulation, which is rarely simple and obvious. detection of ischemic changes in the bowel walls and/or attached mesentery on ct scans imply strangulation highlighting the need for laparotomy; if only signs of clo are detected, the existence and/or development of strangulation cannot be predicted. results: a diagnosis of strangulation was made in 30 patients at surgery. in the patients whose ct findings were mesenteric haziness and decreased conspicuousness of the mesenteric vessels with normal enhancement of the affected small bowel, diagnosis of viable ischemia was made at surgery. ct findings predicting strangulation were poor contrast enhancement of the bowel wall, haziness of mesentery, diffuse engorgement of mesenteric vessels and ascites. (p < 0.05). among these findings, poor contrast enhancement of bowel wall was the most significant one. in the patients with small bowel obstruction, poor contrast enhancement of the bowel wall, haziness of mesentery, diffuse engorgement of mesenteric vessels and ascites were useful ct findings for detecting and predicting strangulation. purpose: the aim of the study was to define the role of high resolution ultrasound in establishing the imaging diagnosis of the right iliac fossa pain syndrome (rifps). the retrospective study has included 25 patients, which were admitted in the emergency room presenting the clinical picture of acute rifps. paraclinic investigations were: abdominal high resolution us, blood and urinary testes. we compared the diagnosis emitted ultrasonographically with the one established by the surgeons in the theatre following the intervention. results: ultrasound examination has showed: 10 patients with inflammatory changes suggesting acute appendicitis (with a thickening of the appendic's wall greater than 6 mm and free peri-cecal liquid collection; mild small bowel loops distension with reduced motility at the right iliac fossa in 4 patients; 1 patient with suggestive terminal ileitis and mesenteric adenitis; 7 women with gynecological abnormalities (4 with big right ovarian cyst and 3 with ectopic right pregnancy) and douglas free of fluid collections; 3 patient with ureteral impacted stone and dilatation of the right ureter. conclusions: ultrasound examination used in emergency rooms offers reliable differential diagnosis data between inflammatory ceco-appendicular disease and renal or gynecological disfunction. the high resolution us diagnosis has a high sensitivity and specificity concerning the gynecological abnormalities. the diagnosis of acute appendicitis requires a carreful and several "in dynamic" examination. the diagnosis of acute appendicitis is not in most of the cases exclusively done by us, it necessitates very often a correlation with the clinical examination and laboratory tests. helical computed tomography diagnosis of gastrointestinal perforation in the elderly patient a. pinto, f. pinto, m. scaglione, f. lassandro, e. de lutio di castelguidone, l. romano; naples/it purpose: to determine the diagnostic value of helical ct in a consecutive series of elderly patients referred with clinically suspected gastrointestinal perforation. methods and materials: our series includes 48 consecutive elderly patients (mean age: 69 years old) presenting with acute abdominal symptoms suggestive of gastrointestinal perforation. all the patients were prospectively submitted to abdominal ct evaluation. as revealed on helical ct, the presence of free air was considered diagnostic of gastrointestinal perforation. other ct findings considered indirect of perforation were: intraperitoneal free fluid, thickening of bowel wall, streaky density within the mesentery, the "dirty fat" sign, and focal collection of extraluminal fecal matter. results: at surgery, the following sites of perforation were found: duodenum (38.8 %); stomach (30.6 %); ileum (8.1 %); sigmoid colon (8.1 %); rectum (6.1 %); jejunum (4 %); appendix (2 %), and trasverse colon (2 %). in our series, helical ct demonstrated the presence of free air in 95.9 % of cases, intraperitoneal free fluid in 81.6 %, and thickening of bowel wall in 48.9 %. streaky density within the mesentery was found in 1 patient. conclusion: ct is a reliable and accurate imaging method for assessing gastrointestinal perforation by providing excellent contrast resolution to depict presence of even small amounts of free air in the abdomen. this is particularly helpful when elderly patients series are concerned. a c d e f 254 materials and methods: 63 female patients with disorders of defecation-and/or micturition were examined. video-fluoroscopy was performed in a sitting position. the urinary bladder was filled with water-soluble contrast media. rectum and vagina were contrasted with bariumsulfate. dynamic-mri was performed with a 1.0 t scanner (magnetom expert, siemens erlangen) using an t2 weighted gradient echo sequence ("true fisp"). patients were asked to present with full urinary bladder at the time of examination. rectum and vagina were filled with ultrasound-gel. results: in 28 patients we found a anterior rectocele with a anterior extent between 3 and 11 cm in video-fluoroscopy. mri failed to visualise rectoceles in 4 cases. in 9 cases rectoceles lead to a displacement of the urethra thus causing a voiding deficiency of the urinary bladder. these findings could be depicted better in mri. 12 patients presented a cystocele shown by both modalities. in another 4 cases a enterocele could be detected. conclusions: mri is inferior to video-fluoroscopy for the detection and the estimation of the degree of anterior rectoceles. however mri is superior in assessment of the severity of displacement of the urinary bladder and the urethra. the extent of pelvic descent could more accurately be assessed by mri. due to the capability of mri of direct visualisation of the pelvic contents it is superior in demonstration of enteroceles. in 1997, the gradual implementation of the nation-wide mammographic breast cancer screening in the netherlands was completed. since then, 625000 screening examinations are being performed annually in the agegroup 50 -75 years, at an interval of two years. the medical performance of the various disciplines in the regional screening centres is assessed by means of regular site visits. at these site visits, first, the overall screening outcomes are evaluated. subsequently, screening and diagnostic mammograms of interval and screen-detected st. ii cancer cases are reviewed. results: despite the small size of the country, notable regional variations e.g. in the relative frequency of small tumours (t1) are observed, ranging from 21 % to 35 % at a national average of 27 %. from a not blinded review of > 2000 interval cancers, we found that in about half of the previous screening mammograms in some way abnormal signs were notable. in one fourth of the cases, with a range from 17 % to 40 %, significant lesions were found, that should have been referred for further examination. conclusion: this large regional variation in outcomes prompted us to carry out the so-called national optimisation study. the objective is to learn in what aspects screening performance may improve as to the earlier detection of relevant lesions. these aspects include; (1) double reading; (2) contribution of additional craniocaudal views; and (3) recall criteria for further assessment. the role of individual mcs shape and cluster shape in the interpretation of mammograms i. leichter 1 , r. lederman 1 , p. bamberger 1 , b. novak 1 , s. fields 1 , s.s. buchbinder 2 ; 1 jerusalem/il, 2 bronx, ny/us purpose: to evaluate the diagnostic role of computer extracted features reflecting the cluster shape compared to features reflecting the shape of individual microcalcifications, identified mammographically. material and methods: 286 cases of clustered mc's with proven pathology were obtained from three university hospitals and reviewed by experienced mammographers. quantitative features characterizing the finding were extracted by a cad system following digitization at 42 µm resolution. the shape factor and number of neighbors, according to delaunay and gabriel methods, were com-puted for each mc together with the eccentricity of the cluster. while shape is related to the individual mcs, the average number of neighbors and eccentricity reflect the cluster geometry. stepwise discriminant analysis (sda) determined the significance of the extracted features in predicting malignancy. the performance of a classifier based on these features was evaluated by roc analysis. results: sda assigned variability of shape the highest predictive power followed by average number of neighbors (delaunay) and average shape but excluded cluster eccentricity and number of neighbors (gabriel) from the model. a classification scheme assigned the variability of shape a weighting factor, about 2.4 times greater than that assigned to the number of neighbors and average shape. a scheme based on these three features yielded a roc curve with an az of 0.88, indicating a ppv of 77 % for 95 % sensitivity. conclusion: the variability of shape of individual mcs was found to have a higher impact in differentiating benign from malignant clusters than the average shape or features characterizing the cluster geometry. the efficacy of the independent double reading of mammograms with consensus has been established, but the observer variability between the readers must be supervised to determine the homogeneity of radiological criteria. objective: the k index has been proposed as a method to calculate the interobserver agreement that was not due to hazard. we use the k index regarding the classification of mammographic findings in the assessment of mammographic categories (weighted k). status of nuclear medicine in breast cancer imaging, staging and therapy e. piperkova 1 , a. grueva 2 , s. sergieva 1 ; 1 sofia/bg, 2 baltimore, md/us purpose: accurate staging in breast cancer depicting and averaging primary lesions, multifocal and spread of the disease and lymph nodes imaging by nuclear medicine (nm) effects surgery and treatment plans. current status, diagnostic importance, priorities and limitations of spet and pet modalities are analysed using different tumourtropic agents. materials and methods: evaluation of primary lesions, detection of regional and systematic metastatic disease and the assessment of treatment response are discussed through spet using 99 tc-mibi or 99 tc-anticea and pet using fes or fdg for two patients' groups in four trials targeted categories. in addition localisation of "hot spots" and tumour margins in radiodense breasts are introduced. mrm showed a significantly higher sensitivity, but its specificity was significantly lower than smm. in the subgroup of lesions equal or greater than 0 mm (n = 97) the figure of the sensitivity was higher (93.2 %), than the overall sensitivity of smm. using receiver operating characteristic (roc) analysis, between xmm and the combination of xmm and mrm we found no significant improvement, but the combination of xmm and smm yielded a significantly higher diagnostic accuracy than xmm alone. conclusion: both mrm and smm have adjunct value to xmm. mrm is to be preferred when high sensitivity and spatial resolution is essential, while smm is recommended as a complementary method in lesions larger than 1 cm. tc-mibi. the one, measured in ipsilateral and/or contralateral area divided the count density, measured within a region of interest. this value represents coefficient of tracer uptake (in suv). differences between count densities in early and delayed images reflected the washout of the tracer from the tumour and thus sensitivity to chemotherapy. mr-mammography included assessment of t1 and t2 weighted images with the use of flash-mode and gd-enhancement. results: statistically significant correlation coefficients (t/nt) were observed for evaluated regions of increased uptake of radiopharmaceuticals in primary lesions: mibi > 1.67 ± 0.21, fdg > 1.69 ± 0.25. these data were concordant with mr findings of primary breast tumours. in all cases p < 0.001. regional lymph nodes involvement in scintigraphy was assumed in cases of axillary region uptake > 1.36 ± 0.29 for pet and > 1.33 ± 0.28. parasternal lymph nodes were only detected fdg-pet in 15 pts. washout coefficients calculated using 99 tc-mibi data stipulated for chemotherapy choice in 32 consecutive pts. conclusion: mri-mammography, mammoscintigraphy and fdg-pet correlate in assessment of primary bc. the best tools for the staging and adequate planning of radiation and/or chemotherapy are mammoscintigraphy and fdg-pet. contrast enhanced digital mammography: phantom experiment and first clinical results c. marx 1 , m. facius 1 , s. muller 2 , a. rick 2 , k. benali 2 , w.a. kaiser 1 ; 1 jena/de, 2 buc/fr purpose: we evaluated on phantoms the capability of a full field digital mammography (ffdm) system to show lesion contrast enhancement after intravenous injection of iodine. acquisitions on patients have begun to validate the potential of this new application as an alternative to contrast enhanced mri of the breast. method/materials: using appropriate phantoms, we simulated the iodine diffusion in vessels and lesions to evaluate the threshold of detection of contrast uptake on ffdm images (senographe 2000d, ge medical systems). the injections consisted of 1 -3 cm 3 of 2 ml hexabrix 160 diluted in 60 ml of water, and were washed out using water. sequences of 5 -7 images were acquired on ffdm (45 -49 kv, mo/cu) and were analysed to quantify contrast uptakes. clinical use of contrast enhanced digital mammography (cedm) requires the uptake contrast to be visible for iodine concentration between 0.12 mg/cm 2 and 1.8 mg/cm 2 . results: detection thresholds, with and without structured noise, were 0.50 mg/cm 2 and 0.25 mg/cm 2 respectively. breast thickness has a low impact on uptake detection. spatial and temporal analysis showed delayed marginal contrast uptake and slow increase of contrast in the background. initial results on patients have demonstrated contrast uptake in malignant lesions. the shape of the uptake curves is influenced by the thickness of the breast under compression (4 -5 dan). conclusions: given these initial results, contrast enhanced digital mammography may be a fast and less expensive alternative to mri for breast lesion characterisation. mri studies were performed at 1.5 t using a modified volumetric interpolated breathhold examination (vibe) sequence before and after administration of gadolinium. all images were evaluated prospectively regarding lesion detection and characterization. mri findings were correlated with final diagnoses. retrospective grading (scores 1 -4 for "poor" -"excellent") was performed for: (1) general image quality and presence of artifacts (rated "negligible" -"severe"); (2) imaging quality of detected pulmonary lesions (conspicuity and contrast on pre-and postgadolinium images) results: 23 solid pulmonary lesions, 25 infiltrates and segmental atelectases, and 1 cyst were detected and prospectively correctly diagnosed. sizes ranged from 0.3 -10 cm. the mean grading scores for overall image quality and presence of artifacts were 3.3 (sd 0.7) and 1.8 (sd 0.7), respectively. conspicuity and contrast of pulmonary lesions received mean scores between 3.0 and 3.8 (sd 0. we observed a lower peak intensity of emphysematous regions. in 22 subjects with distractedly hypo-perfusion a hyper-perfused region was detected and this occurrence was interpreted as pulmonary circulation re-distribution. discussion: the low specificity was due to three positive cases observed in the control group. these perfusion defects were very well defined and there was total agreement between the three radiologists. in our experience, mr perfusion might be an effective alternative to perfusion scintigraphy in patients with pulmonary emphysema. after an informed consensus, 20 non-smoker subjects, without any present or past pulmonary pathologies, were studied by means of an ultra-fast gradient echo sequence after a gd-dtpa intravenous administration. the acquisitions were performed in coronal and sagittal planes, in supine and prone position, in breath holding. an ulterior acquisition was performed in only five cases immediately after 2 -3 minutes of physical exercise. results: all the examinations, reviewed according to a visual score by three independent observers, resulted fairly interpretable. the i/t curves, obtained in postprocessing evaluation, have the same trend in all cases and are correlated to the lung physiological criteria: the positional changes of the examined subjects confirm the gravity dependence of perfusion; the study performed immediately after physical exercise showed a reduction in the perfusion of the whole lung due to a transitory massive shift of blood mass from visceral to muscular compartment. the extremely short acquisition and post-processing time coupled with a simple feasibility, the ability to detect physiological perfusional changes, and the minimal invasiveness indicates a potential clinical use of mr in the evaluation of pulmonary perfusion defects. tl myocardial scintigraphy. results: echocardiography was normal in 20 cases, including 2 patients with cardiac-symptomatic sarcoidosis. mr images were normal only in cardiac-asymptomatic stage i or lofgren syndrome patients (n = 4). multiple organ cardiac-asymptomatic sarcoidosis (n = 17) displayed similar mr abnormalities to those observed in patients with cardiac symptoms (n = 5). focal myocardial thickness and/or intramyocardial increased signal on t2-weighted and/or intramyocardial increased signal on gadolinium-dtpa-enhanced t1-weighted images, representing granulomatous inflammation, were correlated with segmental perfusion defects on the thallium scan. available mr follow-up in 11 patients showed regression of gadolinium-dtpa uptake after corticosteroid therapy while progression was recorded in 2 patients without treatment. conclusion: mr imaging enables valuable detection and localisation of cardiac sarcoidosis. the occurrence of subclinical lesions in multiple organ sarcoidosis may legitimise the use of mr as a screening method to identify early patients requiring careful review and treatment. detection of regional wall motion abnormalities: tissue doppler echocardiography in comparison with magnetic resonance imaging d.e. kivelitz, a.c. borges, g. baumann, b. hamm; berlin/de purpose: echocardiography when combined with spectral and color flow doppler is well established as a safe, non-invasive, and versatile diagnostic modality for evaluation of left ventricular global and regional function. tissue velocity imaging is a new technique that measures myocardial motion by tracking myocardial velocities with color doppler myocardial imaging principles. the purpose of this study was to compare this new tool with magnetic resonance imaging. methods and materials: 30 patients with wall motion abnormalities underwent echocardiography (harmonic mode-octave) in comparison with tissue tracking (vivedfive, ge, vingmed ultrasound, horten norway, equiped with the echopac software package) which allows real-time acquisition of color doppler images and on-line as well as off-line analysis of time-velocities-integrals (tracking). magnetic resonance imaging (magnetom vision, siemens, erlangen, germany) was performed using stripe tagging. for regional wall motion analysis we used the 16 segment model. results: in 29/30 patients we analysed quantitative data from tagging and tracking. the concordance was 85 % and the best results were in septal, posterior and apical regions. conclusion: tissue tracking, compared with magnetic resonance imaging, is a reliable method for on-line quantification of global and regional systolic function of the left ventricle. fast assessment of abnormal valve function using mri a. meduri 1, 2 , r.m. razmi 2 , v.k. rathi 2 , l. natale 1 , g.m. pohost 2 ; 1 rome/it, 2 birmingham, al/us background: valvular diseases can be rapidly assessed by fast cine (fc) mri. signal void is related to the te and determines the severity. a short te may impair assessment and detection of valvular lesions. we hypothesize that a lower receiver bandwidth, a longer te, increases sensitivity and accuracy. methods: a total of 30 pts and 59 valves were evaluated with the predefined bandwidth of 31.2 khz (te = 4.7 ± 0.2 ms) and with the narrower bandwidth of 15.6 khz (te of 7.1 ± 0.4 ms). length, area and maximum width of signal void associated with every diseased valve were calculated. each area was graded as mild, moderate or severe and compared with echocardiography. results: lowering the bandwidth significantly increases signal void area (117 ± 124 vs. 71 ± 78 pixel, p < 0.0001), length (21 ± 15 vs. 16 ± 12 p < 0.001) and the width (5.8 ± 5.2 vs. 4.4 ± 4.0, p < 0.001), graded as mild, moderate, or severe for each te. longer te increases the severity graded in 22/49 (44.9 %) valves with signal void and diagnosed one abnormal valve not detected with the shorter te. in the 23 cases compared with echocardiography, the longer te showed a significantly better correlation (contingency coefficient 0.708, r = 0.657). conclusion: lowering the bandwidth allows longer tes, giving better correlation with echocardiography. we conclude that a short te leads to underestimation of the severity of the valvular lesion. accordingly, a longer te should be used routinely in every comprehensive cardiac mri study. three-dimensional blood flow in the heart: evaluation with mri g. reiter, u. reiter, r. rienmüller; graz/at purpose: evaluate a method of 3-dimensional determination of the intercardiac flow for early recognition of flow based cardiac dysfunction using magnetic resonance imaging (mri). materials and methods: using flow quantification via phase contrast methods (tr = 33 ms, te = 6.5 ms, flip angle 30°, slice thickness 8 mm, pixel size 2.25 mm × 1.64 mm and matrix 140 × 256) of mri (1.5 t field strength, 40 mt/m gradients) we measured and reconstructed discretized time-dependent 3-dimensional velocity fields of blood flow within the different intracardiac regions. at any time of the cardiac cycle calculated velocity fields were projected onto a corresponding cine-truefisp image (e.g. 4-chamber view). the protocol was designed on healthy volunteers. results: as result of reconstruction and projection we obtained a time series of truefisp images where in-plane velocities were displayed by vectors proportional to the in-plane velocity and through-plane velocities were color encoded. images can be displayed picture by picture as well as in cine mode. the presented method appears robust and sensitive in the 3-dimensional evaluation of the normal blood flow and promising to observe changes as possible causes of ventricle wall dysfunction. benchmarking non-rigid registration techniques for the quantitative analysis of myocardial function in tagged-mr imaging c. petitjean 1 , n. rougon 1 , p. cluzel 2 , f. preteux 1 , p.a. grenier 2 ; 1 evry/fr, 2 paris/fr purpose: in the framework of quantitative analysis of myocardial function our purpose was to assess the relevance of non-rigid registration techniques for estimating dense displacement fields and functional parameters from tagged-mri sequences of the heart without performing tag extraction. the study was conducted on 2d tagged-mr sequences under short axis incidence using various gradient echo/tagging sequences including fisp/dante and bffe/spamm. pixel-based, non-rigid registration techniques which deliver dense displacement fields over the whole image domain by establishing pointwise correspondences without requiring preliminary segmentation of heart walls nor tagging pattern have been evaluated. state-of-the-art methods have been tested for major registration criteria: (i) "demon" algorithms using an optical flow constraint with prior elastic smoothing; (ii) fluid registration methods governed by deformation laws from fluid mechanics; (iii) informational techniques maximising entropic measurements between successive images. in each case deformation modelling has been addressed by comparing functional measurements derived from non-parametric and parametric motion estimates. using hierarchical solvers for handling large deformations has also been investigated. results: demon and mutual information maximisation techniques allow accurate derivation of dense maps of functional parameters including deformation tensor eigenelements, local contraction/dilation and circumferential/radial strains. moreover, informational approaches are insensitive to tag fade out. b-spline modelling offers a good trade-off between measurement accuracy and modelling compactness. multiresolution processing allows us to account for large deformations. conclusions: following this assessment stage, novel non-rigid registration techniques are have now been developed that incorporate local image geometry information to constrain and accelerate the matching process. evaluation of palliative procedures in functional singular ventricle with mri f. weiss, c.r. habermann, c. lilje, k. sasse, j. weil, g.b. adam; hamburg/de purpose: to determine the value of mri in the postoperative evaluation of singular ventricle compared to echocardiography and cardiac catheterisation. methods/material: 19 patients (range: 4 month to 16 years) with functional singular ventricle who had undergone palliative corrective operations. 5 patients were treated without separation of circulation to improve pulmonary perfusion with blalock-taussig-shunts. 13 patients had cavopulmonary shunts, 10 of them total cavopulmonary anastomosis, 3 only partial separation with bidirectional glenn-anastomosis. 1 patient was treated with a central shunt. the results were compared to percutaneous echocardiography, cardiac catheterisation and operation reports regarding postoperative morphologic changes. detectability of thombotic material in the low-flow-system was evaluated. conclusions: 3d-rotational angiography allows for a more exact depiction of anatomical details important in planning surgical and interventional therapy of intracranial aneurysms than dsa. ra also identified more aneurysms. b a c d e f 258 incidence of old intracranial microbleeds in patients with acute intracranial hemorrhage m. alemany, r. raininko, a. stenborg, a. terent; uppsala/se purpose: previous publications have demonstrated, using blood sensitive t2*weighted (w) sequences, the presence of incidental foci of signal loss which have been confirmed histopathologically to represent hemosiderin deposition from earlier bleeds. their clinical significance is still debated. our purpose was to evaluate the occurrence of old microbleeds in patients having acute intracranial hemorrhages and correlate these findings with clinical data. methods and material: we studied 26 patients (mean age 65 years) with acute intracranial hemorrhages. 23 patients had spontaneous bleeds, 1 appeared after subclavian dilatation and 2 after coronary and lung thrombolysis, respectively. axial spin echo t1-w and t2-w, flair and gradient echo t2*-w images were obtained using a 1.5 t system. results: besides the acute hemorrhages, evidence of prior bleeds were found in 13/26 patients (50 %). 18/26 patients were hypertensive and 8/26 were normotensive. 10 of the 18 hypertensive patients (56 %) and 3 of the 8 normotensive ones (37 %) had evidence of old bleeds. other clinical data were also investigated (diabetes, abuse of alcohol or tobacco, anticoagulant treatment, etc) but no clear correlation with intracranial microbleeds was found. only the t2*-w sequences were able to detect these lesions. our results support the hypothesis of a correlation between hypertension and old intracranial microbleeds. the microbleeds may be the sign of a microangiopathy that could give a increased tendency for intraparenchymal bleeding. the detection of old microbleeds is, thus, of diagnostic importance. they may also help to predict risk for spontaneous rebleeding or bleeding complications after anticoagulant therapy. multislice angio-ct in the evaluation of cerebral aneurysms m. wintermark, m. chalaron, p. maeder, a. uske, p. schnyder, r. meuli, s. binaghi; lausanne/ch purpose: evaluation of the accuracy of multislice angio-ct in the detection of cerebral aneurysms, by comparison with conventional cerebral angiography methods and material: 50 consecutive patients, who successively underwent multislice cerebral angio-ct (1.25 mm-thick slice reconstructed every 1 mm, pitch 0.75, 140 kvp, 200 ma, timing determined with a bolus test) and conventional cerebral angiography, were prospectively identified in our department between july 1999 and august 2001. both examinations were reviewed independantly by two neuroradiologists, who were blinded to their initial interpretation and who did not perform the cerebral angiography. angio-ct axial slices were reviewed, as well as 2d mip and 3d ssd reconstructions. angio-ct and angiography data analysis was performed separately, but according the same systematic interpretation strategy. results: 49 cerebral aneurysms were diagnosed. sensitivity, specificity and accuracy of multislice angio-ct in the detection of cerebral aneurysms were 95 %, 100 % and 96 %, respectively. the impact of aneurysm location was determined. the ability of multislice angio-ct in the characterization of cerebral aneurysms was evaluated: size (slope 1.138, r = 0.79, p < 0.001), orientation (accuracy 91.2 %), thrombosis, calcification, origin of arteries, apical teat. conclusion: multislice angio-ct of the willis circle is a worthy tool in the detection and characterization of cerebral aneurysms. its ability to detect aneurysm partial thrombosis is higher than that of conventional cerebral angiography. angio-ct allows for efficient planning of cerebral angiography and affords useful information in the determination of the therapeutical strategy. monday b a c d e f 259 methods and materials: in a blinded, prospective study 50 patients with acute subarachnoid haemorrhage underwent both cta and dsa. spiral ct with 3d reconstruction was performed. 120 ml of intravenous contrast was administered at 4 ml/s. collimation of 1 mm. with a pitch of 2, and a reconstruction index of 0.5 was used. there were 32 females and 18 males entered into the study. the cta images were separately reviewed by two consultant neuroradiologists, who were blinded to the dsa findings. the ct angiograms were assessed for aneurysm size and location, in addition to adjacent vascular anatomy. the results were compared with dsa. results: from the initial group of 50 patients with sah, no aneurysm was evident on either cta or dsa in eleven. of the remaining 39 patients, 51 aneurysms of varying types were diagnosed. cta accurately diagnosed 48 of these (sensitivity 94 %). of the 3 intracranial aneurysms missed on cta all of these were under 3 mm in size. specificity of cta is 98 %. conclusion: cta would appear to be a safe and relatively reliable alternative to dsa in the management of acutely ruptured intra-cranial aneurysms. other advantages include speed, cost and its non-invasive nature. in addition it may be of benefit in the follow-up of known aneurysms. in view of these results we have since modified our protocol to improve sensitivity further. purpose: to evaluate the blood flow dynamics in patients with arterio-venous malformations (avms) before and after radio-therapy treatment. patients and methods: two different mr imaging techniques were used in a total of 25 patients. a non enhanced dynamic mra based on a blood bolus tagging technique and a new contrast enhanced projection mra (fluoroscopic mra/ mrdsa) based on a fast flash sequence were used to assess the angioarchitecture of the malformation as well as the hemodynamics, mainly based on a calculated shunt-time between feeding arteries and draining veins. the conventional tof-mra and conventional dsa served as an internal standard. mrdsa was performed with a bolus injection of 0.1 mmol/kg bw of gadodiamide (omniscan®, nycomed-amersham) at an infusion rate of 3 cm 3 /s and a time resolution of 400 ms per projection. images were assessed in regard to vessel detection and demarcation as well as the hemodynamic aspects of the malformation. results: both techniques were able to assess the angioarchitecture of the avm in all patients. the arterial feeder, the avm nidus and the venous drainage pattern could be clearly delineated. the time resolution of the non enhanced tagging technique was substantial better enabling a more precise assessment of the hemodynamics. discussion: mrdsa and dynamic non enhanced mra are able to assess both the avm angioarchitecture and the avm hemodynamics non invasively. the techniques are easy to implement and can be used for treatment planning. hemodynamic changes due to therapy (embolisation or radiosurgery) can be monitored. the role of transcranial doppler sonography (tcd), and somatosensory evoked potentials (sep), in the detection of vasospasm after subarachnoid hemorrhage a. fatourou, c. constantoyannis, e. solomou, p.a. dimopoulos; patras/gr purpose: to evaluate the correlation between blood flow velocity (bfv) and vasospasm in patients suffering from subarachnoid hemorrhage (sh). to detect any relationship between conduction time of somatosensory evoked potentials and vasospasm in the same group of patients. to assess the simultaneous use of tcd and sep in the detection of vasospasm. materials/methods: in our study, we included 105 patients with subarachnoid hemmorhage (sh), diagnosed with ct. six of them also underwent a lumbar spine puncture (lsp). all of them had digital subtracted angiography (dsa), for aneurysm localisation. the follow-up was performed using tcd and sep. the diagnosis of sh, in 99 patients was made with ct, and in the rest of them (6 patients), was made with lsp. dsa revealed an aneurysm in 85 patients, whilst there were no remarkable findings in 17 patients. the latter were reviewed again and an aneurysm was detected in one more patient. vasospasm appeared in 27 patients, whose bfv in the middle cerebral artery was 160.8 cm/s, and central conduction time (cct), was 6.05 ms. the remaining 78 patients without vasospasm, had bfv 95.37 cm/s and cct 5.81 ms. conclusion: statistical analysis showed that the prediction of vasospasm, with the correlation of the two modalities tcd and sep, allowed a proper successful classification in 93.3 %. using two additional parameters, the fisher classification, and the classification hunt-hess, the percentage of proper successful classification did not change significantly. clinical and neuropsychological outcomes in 35 patients presenting with a ruptured anterior communicating aneurysm: mid term follow-up b. jean, k. martin, m. gely-nargeot, a. bonafé; montpellier/fr purpose: rupture of an anterior communicating aneurysm (acoma) is associated with substantial cognitive and psychopathological dysfunction, disability in psychosocial activities and daily living. we report the clinical and neuropsychological outcome of 35 patients treated by embolization of an acoma; subgroup of our base of 61 acom aneurysm. material and method: from september 1998 to may 2001 we treated 61 ruptured acoma by coil embolization. at admission age, aneurysm description, initial clinical wfns score, fisher grade were collected. at discharge occlusion rate, glasgow outcome scale (gos) and complications were reported. we followed patients at 6 months, and 1 year with clinical evaluation and an angiographic control. out of 61 patients, we submitted 35 patients to an extensive range of tests: comprehensive batteries of neuropsychological and psychopathological tests, daily living scales. cognitive outcome was evaluated using global rating scale (mattis), and assessment for attention, executive functions, explicit, implicit memory and psychiatric associated disorders. results: we treated 61 patients using gdc coils. technical and clinical results are provided. we report our technical complications and complications due to sah evolution (symptomatic vasospasm, acute hydrocephalus). 35 patients were submitted to the battery of neuropsychological tests. the patient's performances were characterized by neuropsychological deficits, especially impairments in learning and explicit long term memory, associated with functional frontal dysfunction. conclusion: aneurysm morphological characteristics and occlusion rate do not influence early clinical evolution in ruptured aneurysm. patients submitted to a battery of neuropsychological tests showed neuropsychological deficits and frontal dysfunction on mid term follow-up. purpose: although preoperative embolization of meningioma is a helpful procedure to minimize bleeding and to facilitate resection, some meningiomas showing lack of vascularity are not suitable for embolization. the dsa itself would therefore be an unnecessary procedure. the goal of our study was to predict poorly vascularized meningiomas using time resolved mra (tr-mra). materials and methods: tr-mras were performed on a 1.5 t whole-body unit (vision, siemens, erlangen, germany) in 18 patients with meningioma using a snapshot flash optimized for 2d projection (tr/te 4.2/1.5 ms, slab thickness 45 mm). a standard dose of gd-dtpa was injected with an automatic injector (volume 15 ml at 3 ml/s). coronally directed tr-mra was obtained using single slice, 3 frames/s during 34 seconds without time-interval. tr-mra, dsa and embolization were done at the same day when possible. signal intensity (si) of meningiomas in tr-mra was graded as 0 if no si is detected. faint si was graded as +, good si as ++, and dense si as +++. grades of si on tr-mra of meningiomas and possibility of embolization were analyzed using x 2 -test. results: in 18 meningiomas, 7 graded as +++, 5 as ++, 3 as +, and 3 as 0 in tr-mra. embolization was done in all graded as +++ and ++. however, embolization could not be performed on grade + and 0 due to lack of vascularity from external carotid artery (p = 0.000). conclusion: tr-mra is an useful study to avoid unnecessary dsa in cases of meningiomas with lack of vascularity which would not be candidates for embolization. purpose: traditionally estimation of pancreatic exocrine function has been invasive e.g. duodenal intubation or non-specific. secretin mrcp can be used to calculate exocrine flow rates. we compared 29 patients with normal pancreatic morphology and 28 patients with chronic pancreatitis. materials and methods: following baseline ssfse, 0.1 ml/kg iv secretin was administered and the acquisition repeated every 2 minutes for 7 minutes. the receiver gain was held constant. flow rate = change in total signal intensity over time divided by the signal intensity of a voxel containing 100 % water. breath-hold axial t1w images and spiral ct were also performed as part of their work-up. 29 patients had a normal pancreas on ct and ercp, 28 had chronic pancreatitis of whom 8 had had surgery, pancreatoduodenectomy in 4 and a puestow procedure in 4. of the remainder, 15 had severe, 3 moderate and 2 mild chronic pancreatitis. results: mean flow rates in the normal group were 8 ml/min ± 2.6 and for patients with severe chronic pancreatitis 5.8 ± 2.6. this difference was significant (student t test p < 0.05). within the chronic pancreatitis group there were 5 patients with flow rates < 3 ml/min all with longstanding chronic calcific pancreatitis. significantly different smrcp flow rates were seen in normals as compared to patients with severe chronic pancreatitis. very low flow rates were seen in patients with long standing chronic calcific pancreatitis. materials and methods: 46 patients with suspected pancreatic lesion were studied in preoperative evaluation, including mri with vibe -a fast t1w sequence tailored for dynamic 3d studies -with gadolinium dtpa dynamically, t1w fse and t2w haste. the mri findings (as described in the reports) were analysed, scored and compared to those from surgery and pad. results: mri detected 45/46 lesions (one papillary tumour was missed). 17/26 neoplastic tumours were correctly characterized as malignant (sensitivity 65 %; specificity 35 %). vascular encasement (of portal, superior mesenteric vein/artery, celiac trunk, hepatic artery and/or splenic vein/artery) was correctly described in 7/8 cases (sensitivity 87 %; specificity 94 %; ppv 100 %; npv 97 %). nodular metastases were correctly characterized in 1/7 (sensitivity 14 %; specificity 86 %; ppv 20 %). lymph node enlargement (> 10 mm) was metastatic in 1/5. 0/2 liver metastases were found. the remaining 20 non-tumoral lesions were histopathologically classified as 19 inflammatory pseudotumours and 1 papillary fibrosis. (1) pancreatic lesions were accurately detected with mri/vibe. (2) the method cannot differentiate benign from malignant lesion. enlargement of lymph nodes was not correlated to lymph node metastases. (3) mri/vibe is an efficient tool to predict resectability in patients with pancreatic tumour. vascular encasement was well evaluated. small liver metastases (≤ 5 mm) were not detected. methods and materials: all available ct and mr/mrcp studies of 29 patients who underwent surgical resection were considered. the pathological exams revealed 11 ipmt with hyperplasia/low-grade dysplasia, 10 ipmt with moderate/ borderline dysplasia, 4 ipmt with high grade dysplasia/carcinoma in situ, and 4 ipmt with invasive carcinoma. 2 observers retrospectively reviewed all the images, searching for signs indicative of either benignity or malignancy. results: considering the criteria reported in literature, after complete evaluation of the lesions the assessment of malignancy was made in 13/29 pts after consensus, being correct in 11/13 (84.6 %) cases. the 2 false positive cases, regarding lesion histologically classified as low-grade dysplasia, the wrong diagnosis was related to their dimension (> 4 cm). in 7 false negative errors were made, concerning lesions lacking any sign indicative for malignancy (moderate/high grade dysplasia: 5; carcinoma in situ: 2). these results yielded a sensitivity, specificity, and accuracy of 61 %, 81 %, and 69 %, respectively. conclusion: (1) the assessment of malignancy is trustworthy, and warrants surgical resection. (2) invasive carcinomas are always recognisable, thanks to the wall thickening, and parietal proliferations. . additionally, t1-w contrast enhanced (ce), triphasic "volume interpolated breathheld examination" (vibe) was performed. in a control group of 45 pts. vibe was replaced by ce 2d-gre. two observers analysed data with regard to image quality, tumor conspicuity (roc), diagnostic accuracy and interobserver variability. results: 15/45 pts in the study-and 18/45 in the control-group had pancreatic ca. vibe revealed the best image quality in the study-group (3.6 ± 0.5) outperforming 2d-gre (2.5 ± 0.8) (p < 0.001). haste, t2-tse and mrcp also performed less well: 2.2 -2.9 (p < 0.001). roc-analysis (1/2. observer) showed that vibe (0.76/ 0.76) obtained higher values than haste (0.74/0.67), t2-tse (0.69/0.73) or mrcp (0.62/0.77). 2d-gre performed signif. worse than vibe (0.69; p < 0.05). best diagnostic capability was obtained by combination of all sequences in the study-(0.88/0.91) and the control-group (0.72/0.78). hence, sens. and specif. was higher in the study-(73 %/87 %) than in the control-group (58 %/75 %) (p < 0.01). interobsever variability ranged from moderate 0.4 (haste), good 0.6 (vibe; 2d gre) to very good 0.85 (combination of all sequences). purpose: to evaluate the use of mr perfusion imaging to monitor organ function of pancreas and kidney grafts after combined transplantation. methods and materials: a coronal fgre sequence was used to monitor the signal intensity of transplanted pancreas and kidney grafts, of psoas muscle and aorta before and during the first 6 min after bolus injection of 0.1 mmol gd/kg bw. a perfusion index as the percentage of maximum signal intensity in the organ relative to the maximum signal intensity in the aorta was calculated. in a prospective study 33 patients underwent a total of 77 examinations. mean follow-up after transplantation was 98 days (range 7 -486). perfusion index was used to judge function as good, fair or poor. results were correlated to clinical course and laboratory data. results: absolute values of perfusion index correlated with clinical course of organ function. in addition a strong correlation was found between the intraindividual course of the perfusion index and the clinical course during repeated measurements. the perfusion index can be used to monitor organ function after combined pancreas and kidney transplantation and is especially important in follow-up examinations to assess the intraindividual course of organ function. results: mean number of ct examinations per patient was 3.68, maximum 27 (in those with severe pancreatitis). mean number of series was 1.1. mean effective dose was 4.1 mgy, maximum (performed with incremental ct and high exposition parameters) was calculated to result in 18.44 msv. mean dose lenght product was 292 mgycm. risk for radiation-induced cancer could be estimated to be 0.0085 % per average examination, cumulating to 0.23 % in a patient with 27 ct examinations. as a worst case scenario, assuming that the dose per examination would have been the maximum found in this survey (18.44 msv), the radiation risk would be 2.5 %. conclusion: average number of examinations, cumulative dose and estimated radiation risk resulting from ct imaging in pancreatitis patients is moderate, especially in regard to the often severe course of the disease. however especially in young patients, examination parameters should be chosen with care, and mri should be taken into consideration. multidetector ct of isodense pancreatic adenocarcinoma: more there than meets the eye r.w. prokesch 1 , l.c. chow 2 , c.f. beaulieu 2 , r. bammer 2 , r.b. jeffrey jr. 2 ; 1 vienna/at, 2 stanford, ca/us purpose: to assess the frequency of isodense pancreatic adenocarcinoma with multidetector ct (mdct) and determine the value of secondary signs which aid in their detection. materials and methods: 53 patients with pancreatic adenocarcinoma underwent contrast-enhanced, biphasic mutidetector ct with curved planar reformations (cpr). tumors were initially deemed isodense or hypodense on the basis of visual inspection and then confirmed by calculation of mean attenuation differences between normal pancreatic parenchyma and tumor during the pancreatic phase. indirect signs of pancreatic tumor were tabulated in cases where an isodense tumor was identified. results: out of the 53 patients, six (11.3 %) had isodense tumors with a mean tumor-pancreas contrast of 9.25 ± 11.3 hu during the pancreatic phase and 4.15 ± 8.5 hu during the portal venous phase. ancillary signs of a pancreatic tumor included an interrupted pancreatic duct (n = 5), dilated biliary and pancreatic ducts (n = 1), atrophic distal pancreatic parenchyma (n = 3), and mass effect/convex contour abnormality (n = 3). mean tumor-pancreas contrast for the remaining 47 cases was 74.76 ± 35.61 hu during pancreatic phase. conclusion: isodense pancreatic tumors, even studied with high-resolution mdct, represent a significant percentage of pancreatic carcinomas. with no visible tumor-pancreas contrast, indirect signs such as mass effect, atrophic distal parenchyma and an interrupted duct sign are important indicators for presence of tumor. cpr of the pancreatic duct are particularly helpful in delineating the obstructed pancreatic ductal system. multislice helical ct with 2d and 3d multiplanar reconstructions using minimum intensity projection for assessment of the pancreatic ducts m. zins, n. bouzar, l. fontanelle, s. lenoir, c. strauss, r. palau; paris/fr to evaluate imaging quality of 2d and 3d multiplanar reconstructions with minimum intensity projection (minip) in assessment of the pancreatic ducts using multislice ct. methods and materials: 35 consecutive patients with potential disorders of the pancreas were scanned using multislice ct. the ct parameters were: 40 s scan delay for the pancreatic phase and 75 s scan delay for the portal venous phase. pancreatic phase images were used for 2d and 3d reconstructions with the following parameters: 1.25 mm slice thickness; interval of reconstruction: 0.6 mm; the 2d and 3d data sets were evaluated in oblique axial and oblique coronal planes, using minip. qualitative assessment of the multiplanar reconstructions included: (1) visibility and size of the main pancreatic duct (mpd), (2) visibility of the major papilla, (3) visibility and size of the duct of santorini, and (4) visibility and size of the branch ducts. the mpd was entirely visualized in 33 patients (94 %) including 15 with non dilated mpd. the major papilla was visualized in all patients. a non-dilated duct of santorini was visualized in 9 patients; a dilated duct of santorini was visualized in three patients with intraductal papillary mucinous tumors of the pancreas (ipmtp). non dilated branch ducts were visualized in two patients and dilated branch ducts were visualized in 18 patients. purpose: to assess main pulmonary artery dimensions in normal subjects and in patients with marfan's syndrome. materials and methods: 50 marfan patients (mean age 33 (10) years, 34 men, 16 women) and 15 matched control subjects (mean age 28 (4) years, 9 men, 6 women) underwent cardiac magnetic resonance imaging (mri). pulmonary artery dimensions were obtained on axial spin echo images at two different levels: 1) the level of the pulmonary artery root and 2) the level of the pulmonary artery bifurcation. results: upper limits of normal (mean + 2 sd) at the pulmonary root and at the pulmonary artery bifurcation were 34.8 mm and 28.0 mm, respectively. pulmonary artery dilatation was demonstrated in 37 (74 %, root) and 38 (76 %, bifurcation) of the 50 marfan patients. there was a good correlation between pulmonary and aortic root diameter in non-operated marfan patients (r = 0.76). dimensions of pulmonary root were larger (38.4 mm, range 28.3 -50.7 mm) than dimensions at the pulmonary bifurcation (30.7 mm, range 21.4 -38.5 mm, p < 0.001). marfan a c d e f 262 patients with aortic root replacement (n = 35, root 39.7 mm, bifurcation 31.7 mm) had significantly larger pulmonary artery dimensions than non-operated marfan patients (n = 15, root 35.5 mm, bifurcation 28.5 mm, p < 0.01). conclusions: in the majority of marfan patients the main pulmonary artery, particularly the pulmonary root, was dilated. a good correlation between pulmonary and aortic root diameter was demonstrated. pulmonary artery dimensions were significantly larger in marfan patients with aortic root replacement than in nonoperated marfan patients. methods and materials: selective pulmonary dsa was performed in 91 consecutive patients with ctph. six bolus injections of non-ionic contrast media were used (pa-, oblique and lateral projections of both pulmonary arteries, iomeprol, 25 ml, 13 ml/s). hemodynamics were obtained using swan-ganz catheters and classified in three groups depending on systolic pulmonary pressure (pasyst): group i: < 30 mmhg, ii: 30 to ≤ 60, and iii: > 60 mmhg). results: pasyst was 21.4 ± 2.2 (group i, n = 7), 49.8 ± 8.5 (ii, n = 18), and 87.0 ± 18.9 mmhg (iii, n = 66). pulmonary vascular resistance index (pvri) was 238 ± 103 (i), 703 ± 364 (ii), 1587 ± 569 dyne⋅s⋅m 2 ⋅cm −5 (iii), mean cardiac index (ci) was 3.2 (i), 2.8 (ii), and 2.3 l⋅min −1 ⋅m −2 (iii). pcw pressure was normal in all three groups indicating normal left heart function. contrast bolus injection caused only slightly increased pa pressure (dpasyst: 1.1 ± 1.1 (i), 2.9 ± 2.3 (ii), and 3.7 ± 3.2 mmhg (iii)). after completion of angiography right atrial pressure and pasyst were moderately increased (dra: 1.8 (i), 2.6 (ii), 3.0 mmhg (iii), dpasyst to determine the value of mri in the diagnosis of pulmonary embolism (pe) in patients unable to sustain apnoea, e.g. in acute pe. methods: a real time (rt) steady state free precession (ssfp, truefisp) sequence was adapted for the examination of the thoracic vasculature. 21 consecutive patients with suspected pulmonary embolism were prospectively examined with mri using a rt-truefisp sequence and contrast enhanced mr-angiography (mra). the clinical stages of suspected pe were as follows: chronic 4, mild 7, severe 6, massive 4. results: 71 % of the mra-sequences were diagnostic, excluding all patients with massive and some with severe pe. all 21 rt-sequences were diagnostic. all 125 lobar arteries and 85 % of the segmental arteries could be evaluated, segments 4/5 being the most difficult. for the rt-sequences, no patient preparation, ecg or breathing commands were needed. pe was diagnosed in 67 % of the patients by rt-mr. in 6 patients with pe, mra detected 9 additional thrombotic segments. two diseases mimicking pulmonary embolism could be diagnosed. in all remaining patients with clinically suspected severe and massive pulmonary embolism, thrombotic material could be visualized in the pulmonary arteries. chronic and acute pulmonary embolism could be differentiated, and the effect of thrombolytic therapy on thrombus size could be visualized. conclusion: rt-mri allowed the examination of any patient regardless of his clinical condition in 3 min and required no preparation. therefore, the range of indications for mri in patients with suspected pulmonary embolism is extended. the well established mra technique has advantages in patients able to sustain apnoea. a differentiated approach to pulmonary embolism and deep venous thrombosis using multi-slice ct j.e. wildberger 1 , a.h. mahnken 1 , a.m. sinha 1 , p. haage 1 , s. schaller 2 , r.w. günther 1 ; 1 aachen/de, 2 forchheim/de purpose: to establish a protocol for multi-slice ct (msct) examinations for clinically suspected pulmonary embolism (pe) using pulmonary ct-angiography and indirect ct-phlebography (ctp). methods and materials: from february 2000 to july 2001, 161 patients (92 male, 69 female; age: m = 55.2 a ± 18.1 a) with suspected pe were examined on a msct (somatom volume zoom; siemens, germany). after intravenous injection of 120 cm 3 of contrast-medium, thin collimation chest ct was performed. ctp was added if pe was present or previous examinations and clinical signs suggested deep venous thrombosis (dvt). the latter was performed using a 4 × 5 mm protocol (slice thickness 7 mm, reconstruction increment 6 mm). venous phase scanning was completed at the level of the popliteal fossa 3 minutes after contrastmedium injection. results: 62 patients in our series suffered from pe. 47 of these had additional deep venous thrombosis (78.3 %). the latter was ruled out in 13 patients, one patient with pe did not receive this additional examination protocol due to their poor clinical condition, one subsegmental pe was initially not detected. of the 99 patients without pe, 47 also received indirect ctp. in 10 cases dvt was proven, which was already known from previous examinations in 8 patients. only in 2/47 patients (4.3 %) was a previously undiagnosed dvt found, despite exclusion of pe. the examination protocol presented is suitable for clinical use in patients with suspected pe and offers detailed examination of the venous system. if dvt is not likely, additional ctp is not recommended if pe has been ruled out by msct. methods: patients with severe chronic ctph were evaluated using multislice ct and selective pulmonary dsa for operative treatment planning. ct findings and angiographic findings were correlated at the level of major arteries, interlobar arteries, segmental and subsegmental arteries. diagnostic criteria included patency or occlusion, thromboembolic deposits, webs, bands, and anatomical allocation of the respective findings. results: in 14 consecutive patients with severe ctph, ct and dsa findings of 994 pulmonary vessel segments from major to subsegmental arteries were separately analysed. using multislice ct 30 segments were insufficiently detected (25 subsegments, 5 segments) compared to 50 segments in dsa (37 subsegments, 11 segments, and 2 major arteries). predominantly segment iii and less often segment i arteries were insufficiently detected due to the position of the pigtail catheter. concerning inconspicuous vessles versus occlusion or any thromboembolic alterations, the diagnostic concordance of both methods overall was 67 %, was 79.4 % for segmental arteries and 63.6 % for subsegmental arteries. concerning patency versus complete occlusion diagnostic concordance overall was 82.4 %, was 88.6 % for segmental arteries and 77.1 % for subsegmental arteries. dsa was superior for detection of peripheral thromboembolic alterations. ct was superior for anatomical allocation. conclusion: multislice ct is suitable for diagnosis of ctph on segmental and subsegmental level. however, for planning of pulmonary thrombendarterectomy both modalities, multislice ct and selective pulmonary dsa, are still necessary for exact anatomical allocation and precise visualisation of thromboembolic findings. epidemiologic, clinical and morphologic criteria as listed below. (advanced disease was defined as hepatic insufficiency leading to olt within the subsequent 2 years). results: common and characteristic findings were as follows: 81 % of patients were women with the onset of disease (diagnosis) in middle age (mean, 50.7 years, range, 26 to 71 years). the average time from diagnosis to liver transplantation was 6.1 years (range, 0.5 to 20 years). ct findings in advanced pbc often resembled those seen in other forms of cirrhosis with a small heterogeneous liver, varices and splenomegaly. the liver in less advanced disease was usually enlarged or normal in size, with a smooth contour, little atrophy and lace-like fibrosis and regenerative nodules in nearly a third. even patients with less advanced disease frequently had varices (62 %) and ascites (24 %). prominent lymphadenopathy was seen in 88 % of all cases. hepatocellular carcinoma was found in only 4 patients, 2 of whom also had chronic hepatitis c. only two patients had recurrence of pbc following olt conclusion: pbc is an important cause of liver failure with distinctive clinical and ct findings that may allow confident diagnosis and management. we reviewed retrospectively the imaging studies of five patients (4 women; aged 16 -25 years) with liver nodules associated with a spontaneous intrahepatic portosystemic venous shunt. color-doppler ultrasoud (us), helical-ct and arteriography were performed in all patients and mri in 4 patients. nodules location, number, size, density or signal, homogeneity and pattern of vascularition were evaluated. hepatic vascularization, the morphologic type of the shunt and portal hypertension features were also analyzed. these findings were correlated to pathological results (transcutaneous biopsy of the nodules (n = 3); transplantation (n = 2)). results: sonography and angiography demonstrated a portohepatic venous shunt (persistent arantius ductus (n = 1), congenital absence of portal vein (n = 2), left portohepatic venous shunt (n = 1), left portoatrial shunt (n = 1)). hepatic arterialisation were observed in segment where no portal flow was seen and where hepatic nodules were present. the number of nodules in each patients was at least 3 with a size ranging from 1 to 12 cm. most lesions were heterogeneous, hypoechoic on us, hypodense on ct, hyperintense on t1, slightly hyperintense on t2 and enhanced slightly and heterogeneously on arterial phase after contrast injection. histological results showed nodular hyperplasia in the liver suggestive of focal nodular hyperplasia in all cases and adenoma in one case. conclusion: these findings emphasised the hypothesis that nodular transformation of the liver is probably due to the lack of portal blood flow and hepatic arterialisation and is usually of benign type. high resolution multislice spiral ct in liver metastasis: comparison of a high resolution vs a low resolution protocol f. fraioli, c. catalano, a. laghi, f. pediconi, a. napoli, r. brillo, m. danti, r. passariello; rome/it purpose: to evaluate the sensitivity and specificity of multislice spiral ct (msct) in the assessment of patients with suspected liver metastasis and to compare two different acquisition protocols. material and methods: 50 patients referred for different neoplasms underwent msct. all patients underwent intraoperative ultrasound or, in case of diagnosis of unresectability in the first examination, a follow up ct at three months. pre (4 × 2.5 mm collimation) and post-contrast (4 × 1 mm collimation, 1 mm and 5 mm reconstruction interval) acquisitions, during arterial and portal venous phases were performed after i.v. administration of 140 ml of c.a. at 4 ml/s. two observers blindly evaluated either 5 mm axial images or 1 mm axial and relative mprs, in terms of presence and number of lesions. results: ct correctly showed 123 (92 %) of the 133 liver lesions shown at intraoperative ultrasound in 42 patients. ct correctly evaluated all lesions greater than 2 cm in size. regarding lesions smaller than 2 cm in size ct evaluated as cyst 4 lesions shown at intraoperative ultrasound as metastasys. real time interaction 3d data set with 1 mm axial images, allowed to identify 24 subcapsular lesions. sensitivity, specificity and accuracy in detection of smaller lesions smaller than 1 cm was 98 %, 94 % and 97 % using 1 mm axial images and 89 %, 93 % and 90 % using 5 mm protocol. conclusion: high resolution msct with 3d data is a very accurate technique in the assessment of patients with liver metastases. the difference of images secretory and non-secretory carcinoid tumours using standard ct and functional techniques j.b. cwikla, j.r. buscombe, a.j. watkinson, m.e. caplin, a.j.w. hilson; london/gb functional and anatomical imaging modalities can underestimate presence or extent of disseminated carcinoid and should be used together. aim of study was to assess if there is any difference in imaging pictures between secretory and nonsecretory carcinoid and toconsider both techniques. overall 50 patients, all with confirmed carcinoid. there were 31 patients with primary disease. half of the patients had secreting tumours with carcinoid syndrome. ct and 111 in octreotide study was performed in each case, using standard imaging protocol. in non-function tumours ct was able detect disease within liver in rate of 0.53. 111 in octreotide was positive in all patients. different sites within abdomen and chest in patients with non-secretory tumours ct and functional imaging detected with rate as follows: pancreas 0.57/0.71, paraaortic nodes 0.7/0.8, gut and/or mesentery 0.67/ 0.83. other site of tumour spread: chest, pelvis, spleen or bone using ct was 0.25 and 111 in octreotide 0.92. those patients with secretory tumours had results as follows: liver deposits using ct in 0.96, 111 in octreotide 0.91. in this group of patients other sites of tumour deposits within abdomen and chest ct and functional imaging detected with rate as follows: pancreas 0.25/1, paraaortic nodes 0.79/ 0.93, gut and/or mesentery involvement both 0.61. other site of tumour spread like chest, pelvis, spleen or bone involvement using ct was 0.41 and 111 in octreotide 0.82. these results suggest that any imaging modality is perfect to detect carcinoid deposits, additionally there is a difference with deposits distribution of carcinoid in patients with secretory and non-secretory tumours. lymphoma therapy monitoring by multislice perfusion-ct w. römer, l. muresan, r. repp, a. taubald, h. greess, w.a. kalender, w.a. bautz; erlangen/de purpose: the high frequent acquisition of multislice ct data after contrast medium bolus injection allows quantification of tumor perfusion. it was our goal to assess therapy induced changes of tumor perfusion early after initiation of lymphoma treatment using dedicated software to calculate parametric images. method: dynamic msct was performed in 19 patients with lymphoma before and 7 days after initiation of chemotherapy. after automatic bolus injection of 80 ml contrast medium (flow 8 ml/s), two 10 mm-sections through the largest tumor region were scanned for 40 s (siemens somatom volume zoom). the arterial input function was derived from the largest arterial vessel in the field of view. perfusion indices were calculated using graphical analysis and displayed as parametric color coded images. results: at baseline, all lymphoma lesions were visible in perfusion images by an enhanced perfusion index (0.474 ml/min/ml). seven days after initiation of chemotherapy, tumor perfusion decreased by 46 % in high grade nhl and hodgkin's lymphoma, whereas in two patients with low grade nhl the perfusion index increased by 55 %. in contrast, 2d-tumor size only decreased by 19 % and 11 %, resp. conclusion: these results indicate that chemotherapy-induced changes of tumor perfusion may be assessed by perfusion msct and documented in parametric images. in highly responsive tumors like high grade nhl and hodgkin's lymphoma, therapy induced perfusion changes occur within the first 7 days after therapy and precede morphologic changes. we hypothesize that outcome of therapy may be predicted as early as 7 days after treatment. the effect of intravenous secretin administration on hepatic enhancement during ct examinations of the abdomen s.m. lyon, t. fotheringham, p. o'sullivan, m.f. given, m.j. lee; dublin/ie purpose: intravenous secretin increases blood flow to the pancreas in animals. in an associated study we found that iv secretin caused significantly increased enhancement of the portal venous (pv) system. this study investigated the effect of secretin on hepatic enhancement. methods: 32 patients (mean age 70; range 47 -88) were enrolled. triple phase helical ct of the abdomen was performed on successive days so that each patient acted as their own control. all patients had intra-abdominal malignancy and the study was approved by the hospital ethics committee. unenhanced and enhanced ct in the arterial phase and pv phase was performed without (day 1)and with (day 2) secretin (100 iu) given at t = 0 s (n = 10), t = 60 s (n = 5), t = 120 s (n = 5), t = 180 s (n = 4), t = 240 s (n = 4), t = 300 s (n = 3). percent enhancement of the liver was calculated using roi's obtained from studies with and without secretin. results: overall mean hepatic enhancement in the portal venous phase was 109 % without secretin and 117 % with secretin. the relative increase in hepatic enhancement after secretin when compared to hepatic enhancement without secretin was 13 % at t = 0, 16.6 % at t = 60 s, 24.6 % at t = 120 s, 18.9 % at t = 180, 3.1 % at t = 240 and 19.5 % at t = 300 s. pv/smv enhancement was significantly increased in all secretin studies (p < 0.05) when compared with non-secretin studies. conclusion: secretin administration causes an increase in hepatic enhancement which may lead to better lesion conspicuity. the optimal timing for secretin administration and its effect on lesion-liver contrast differences will be determined as more patients are recruited. study completion is expected in november 2001. imaging findings in extraosseous multiple myeloma m. patlas, i. hadas-halpern, c. reinus, e. libson; jerusalem/il purpose: extraosseous manifestations are rare and are found in less than 5 % of patients with multiple myeloma. the purpose of this study is to illustrate the imaging features of extraosseous myeloma and to heighten the awareness of this phenomenon. we retrospectively reviewed the radiological files of 200 myeloma patients. patients in whom the extraosseous masses were in contiguity with bony involvement were excluded from the study. results: seven of the 200 (3.5 %)myeloma patients had extraosseous masses. there were 2 men and 5 women and the age range was 48 -82 years old (mean age 60 years). ct was available in five patients, mammography in three cases, and ultrasound in one case. biopsy revealed the diagnosis of plasmacytoma in all cases. the gamut of findings included breast masses (three patients), supraclavicular lymph nodes (1 patient), pancreas and stomach (1 patient), adrenal and pleura (1 patient), thyroid cartilage (1 patient). more than one site was present in 2 of the 7 patients (adrenal and pleura; pancreas and stomach). soft tissue plasmacytomas presented as relatively well-defined masses of various sizes, and could not be differentiated from other malignant or benign lesions on the basis of the imaging findings alone. conclusions: exraosseous myeloma is very uncommon in multiple myeloma. radiologists should be aware of this occurence so that extensive unnecessary interventions can be avoided when extraosseous sites of disease are encountered. quality indicators in radiology management: a methodology development study p.e. varga 1 , e. belicza 1 , e. sík 2 , g. nagy 3 ; 1 budapest/hu, 2 szekszárd/hu, 3 zalaegerszeg/hu purpose: quality indicators are widely used in healthcare. though imaging is involved in almost all diagnostic procedure, few quality indicators are accepted in radiology. method: repeated x-ray examination data were collected in 3 hospitals. the 3-month multicenter study aimed to develop an indicator as well as to test the indicator development method itself, using common data sheets. in the 886 repeated examinations, the influence of different reasons of repetition was analysed. results: 21 causes were identified. inappropriate exposition or positioning were leading causes, in 71.6 % of the cases in hospital 1, 72.6 % in hospital 2 and 22.0 in hospital 3. failure of the equipment was the second cause: 25.0 %, 7.9 % and 0.1 %, respectively. low rates in hospital 3 can be attributed to the newly equipped digital x-ray department. in other words: hospital 1 and 2 pay the price of the lack of investment with repeated examinations. day-section has a higher influence than workload: though the majority of the examinations were carried out during normal hours, 23.7 % of the repetitions occur on night duty. nearly half of the technicians involved found the data sheet too detailed and they proposed to modify his for automatic registration. as repeated examination means increased risk for the patient and the institution, economical consequences will be demonstrated. conclusion: repeated x-ray examination proved to be an appropriate quality indicator, as it is profession-specific, relevant and convenient for benchmarking. data collection is easy and economic data can be linked. quality management can be based on reason analysis. materials and methods: of 59 patients who were referred for percutaneous drainage of hepatic abscess between 1995 and 2001, cases with incorrect diagnosis or without pre-procedural contrast-enhanced ct were excluded, and 78 abscesses in 47 patients (29 -79 years, m:f = 35:12) were included. 24 patients had the predisposing factor such as hepatocholedocholithiasis (n = 7), the history of biliary (n = 6) or gastrointestinal (n = 5) malignancy, or others. findings in dual-phase helical ct (n = 34) or single-phase ct (n = 13) were analyzed concerning the enhancement pattern of the abscess wall and adjacent parenchyma, and the presence of the thrombosis of intrahepatic vasculature. the abscess wall appeared as single layer of hyperdense ring in 38 lesions (48 %) or as double target (inner hyperdense ring and outer hypodense ring) in 32 lesions (41 %). there was thrombosis in the hepatic vein (n = 20) and/or portal vein (n = 18) noted in 30 cases (64 %). in cases with dual-phase ct, regional difference in parenchymal attenuation was noted in 28 cases (82 %), showing wedge-shaped hyperdense area surrounding the absecss in arterial phase (n = 24) or hypodense area peripheral to the abscess in portal phase (n = 11). of those cases with regional difference in parenchymal attenuation, 21 cases (75 %) had venous thrombosis (p = 0.07, fisher's exact test). the abscess wall has characteristic enhancement pattern on dynamic ct. the enhancement pattern of adjacent parenchyma is more complex, and it may be associated with the venous thrombosis. the liver is the most commonly damaged organ in children following blunt abdominal trauma. a conservative non-operative approach is now the recognised standard of care. computerised tomography (ct) is the primary imaging modality of choice. this conservative approach can result in bile duct damage remaining undetected for several days. we have evaluated the pre-emptive use of tbida hepatoscintigraphy, to detect biliary leakage prior to the patient becoming symptomatic. at birmingham children's hospital, all patients who have a history of trauma and clinical suspicion of abdominal injury undergo an abdominal ct. in those cases where there was liver fracture greater than 4.0 cm or involving the portahepatitis, a tbida study was performed. 21 patients underwent an abdominal ct abdomen. 7 patients had a significant liver injury, all with fluid in the peritoneum and additionally had a tbida study. in 2 patients the tbida demonstrated activity within the peritoneal cavity. intraoperative cholangiograms confirmed the biliary leaks that were surgically stented. both patients were asymptomatic with no evidence of a biliary peritonitis. the tbida in a third patient demonstrated a biloma that on follow-up ultrasound has shown to be shown to be the site of a portovenous fistula. all patients showed good recovery and returned to a normal lifestyle. tbida hepatoscintigraphy, when used pre-emptively, can detect biliary damage prior to the development of clinical symptoms. this early detection improves surgical outcome and reduces hospital stay. the investigation also allows the detection of other potential intrahepatic complications. b a c d e f 266 acute pancreatitis in childhood l. laufer, o. kleiner, g. greenberg, z. cohen, y. hertzanu; beer sheva/il purpose: acute pancreatitis in children is rare. the aim of this study was to demonstrate the spectrum of imaging findings in relation to the aetiology. materials and methods: 46 children aged 1 -18 a between 1984 and the year 2000 were diagnosed at our hospital with acute pancreatitis. the diagnosis was established using clinical data, us and ct examinations. a history of trauma was present in 22 children, 20 cases were considered to be idiopathic. the other cases were secondary to biliary stones, thalassemia, steroid treatment and chemotherapy. results: there is a considerable difference between traumatic and nontraumatic pancreatitis. the imaging in most cases of idiopathic pancreatitis was normal or minimal changes were showed. the children with pancreatic injury demonstrated a large range of imaging findings. laceration or fracture (13 cases), local or diffuse pancreatic enlargement (16), nonhomogenous structure (20), and pseudocyst formation (6). the tail was involved twice as frequently as the head. the study showed a large difference between the 2 groups. the contribution of imaging in diagnosis of idiopathic cases was minimal and follow-up was unnecessary. the imaging of posttraumatic pancreatitis demonstrates a large spectrum of pathology in the early examination and follow-up studies. when can balloon dilatation of esophageal strictures in children be considered successful? j. lisý jr., j. snajdauf, m. vyhnánek, s. tuma, j. neuwirth; prague/cz purpose: healing of a stricture by scar formation after balloon dilatation results in narrowing of the eosophageal lumen and reccurrence of dysphagia. consequently repeated dilatations are usually necessary. the authors tried to establish the delay after dilatation when the procedure could be considered successful and further dilatation or surgery was not indicated. methods and materials: eosophageal strictures in 49 children were treated by 189 balloon dilatations in total. 20 children had an anastomotic stricture after surgery for eosophageal atresia, 12 had a reflux stricture, 6 a tight cuff after nissen fundoplication causing achalasia, 6 a corrosive stricture, 4 congenital stenosis and 1 a stricture after radiation. dilatations were considered successful, when absence of dysphagia lasted for at least 1 year after dilatation and the patient hadn't undergone surgery. results: one procedure completely treated the stricture in 11 children (22 %). none of them had surgery. repeated procedures were neccessary in 38 patients (78 %). the delay between dilatations in the case of repeated procedures ranged from 1 week to 3 years. dilatation avoided surgery in 27 cases (56 %). only 6 of these patients had more than a 6 month delay between procedures. the remaining 11 children (22 %) required surgery after repeated unsuccessful dilatations. however in none was there a need for repeat dilatation later than 6 months following a previous procedure. conclusion: the authors conclude that a delay of 6 months after dilatation is the key time for evaluation of success rate of eosophageal balloon dilatation. crohn's disease in paediatric patients: mr imaging of the small bowel using peg solution as an oral contrast medium a. laghi, i. carbone, i. baeli, r. iannaccone, p. paolantonio, f. iafrate, c. catalano, r. passariello; rome/it purpose: the aim of this study was to evaluate mr findings of the small bowel using peg solution as an oral contrast medium in paediatric patients with suspected crohn's disease. subjects and methods: twelve patients with clinical and laboratory findings indicating possible crohn's disease underwent mr study of the small bowel. after an overnight fast, immediately before mr examination, a fixed amount of 10 ml/kg of weight of peg solution was orally administered. no antispasmodic drug or other drugs were given. mr study was performed using haste (tr/te/acq.t.: inf/90 ms/ 18 s) and trufisp (tr/te/acq.t.: 4.8 ms/2.3 ms/14 s) sequences obtained sequentially for up to 20 minutes, in axial and coronal planes. t1 weighted flash sequences (tr/te/acq.t.: 140 ms/5.3 ms/16 s) were acquired before and after dynamic contrast medium administration (0.1 mmol/kg of gd-dtpa). image were analysed by consensus by two experienced gastrointestinal radiologists. results: seven patients presented positive findings for crohn's disease. mr findings were represented by: wall thickening of the terminal ileal loop, with stricture in three cases. following contrast medium administration wall enhancement was observed in all the cases. conclusion: mr imaging of the small bowel after oral administration of peg solution is a reliable, reproducible, and safe imaging modality for the evaluation of crohn's disease. colonic duplication: clinical presentation and imaging features m.-j.g.j.g. grandsaerd, m.p. hartkamp, c. boetes, p. rieu, c. buonomo, c.e. van die, j.g. blickman; nijmegen/nl purpose: to make the imager and clinician aware that colonic duplication may present at birth, associated with other congenital anomalies or late, as an isolated finding. in the latter instance, the duplication almost always features connecting lumens. material and methods: we reviewed the clinical and radiological features of 9 cases of colonic duplication over the last 10 years. there were 7 girls and 2 boys. their age at presentation was either newborn (6, all girls) or between 4 and 11 years old (2 boys, 1 girl). all had contrast enemas, and 3 underwent abdominal ct. results: those that presented at birth had multiple associated congenital anomalies, including partial and complete vater associations (5, all girls), duplication of the bladder in 2, uterus and vagina in 2, as well as situs inversus totalis in 1 girl. the older presenting children all had long-standing symptoms of constipation and distended abdomens. all duplications had connecting lumens. conclusion: duplication of the colon is a rare congenital anomaly. this report illustrates that there are 2 peaks of presentation: perinatally and later in childhood. furthermore, in the former group multiple associated congenital anomalies are present in all (or almost all) cases. as opposed to other gi duplications, these colonic duplications displayed connecting lumens. renal parenchymal volume assessment with 3d ultrasound in paediatric uroradiology g.a. fritz, m. riccabona, e. ring; graz/at purpose: to prospectively investigate the accuracy of renal volume assessment using three-dimensional ultrasound (3dus) compared to the results of 2dus, ct/ mri, or scintigraphy in neonates, infants and children. methods and materials: 60 patients (mean age 9.81 ± 8.1 years) underwentadditional to conventional us and other conventional imaging as appropriate -3dus of the kidney for anatomical and volumetrical assessment. 3dus was performed with a voluson (kretztechnik/ge, austria/usa) or an external 3d-system (echotech/ge, germany/usa). volume calculations were performed in 2dus applying the ellipsoid equation v = (p/6) × l × w × ap, for 3dus using the system specific volumetric software; in ct/mri volume was calculated by planimetric analysis, and for scintigraphy the renal volume was quantified as relative renal volume. volume measurement focused on renal parenchyma, a dilated renal collecting system was subtracted. results: in 55/60 patients at least one 3dus acquisition/kidney was of diagnostic quality. 3dus volume measurements were accurate compared to ct/mri (± 5 %). 2dus volume estimates showed a larger variation and difference (± 10 %), particularly in kidneys with dilated collecting systems or scars, volumes differed significantly. there was a good correlation of relative renal volume in 3dus compared to scintigraphy in patients without acute pyelonephritis or other perfusion alterations. conclusion: 3dus is applicable to the paediatric genito-urinary tract. 3dus is a more accurate method than 2dus for assessment of (relative) renal parenchymal volume, particularly valuable in patients with hydronephrosis. 3dus improves sonographic potential and can be considered a useful adjunct to conventional imaging. pediatric excretory mr urography a. borthne, c. pierre-jerome, k. gjesdal, t. storaas; oslo/no purpose: normal-sized ureters are difficult to visualise in neonates and small children with the haste-technique (hydrography). an additional contrast-enhanced t1-weighted fast gradient echo (fge)sequence was used for improvement of the image quality. methods and materials: an experimental study with 3 pigs was first performed to validate the excretion-technique. we then studied 34 patients (17 neonates and 17 children) with the same technique. after injection of gadolinium and 5 mg of frusemide, contiguous coronal fge images were acquired with: matrix 163 × 512, tr 2.6 ms, flip angle 60, fov 260 mm, slice thickness 3.3 mm, gap 1.6 mm, nsa 6. 2-and 3-dimensional reconstructions of the entire genitourinary system were performed, followed by quantitative and qualitative image analysis. in all neonates the excretion technique proved better than hydrography; 79 % of the ureters were completely visualised. in children 4 patients with obstruction/reduced kidney function were better seen with hydrography. all the others were better or equally visualised with the excretion technique: complete assessment of the ureters were achieved in 74 % of the children. the mean diameters of the ureters in neonates were: 3 mm (proximal), 4 mm (middle) and 3 mm (distal segment); in children: 4 mm, 6 mm and 4 mm respectively. conclusion: excretory mr urography is better than the non-enhanced hastetechnique (hydrography) for the assessment of the entire excretory system in neonates and children, except for those patients with marked obstruction or reduced kidney function. grade one vesicoureteral reflux -an undergrading? k. darge 1 , g. roessling 2 , j. , with the intravesical application of microbubbles containing the us contrast medium levovist, were performed successively during one examination session. any grade i reflux detected on vcug was compared to the reflux grade of the respective vus. furthermore, in an in-vitro set-up simulating the urinary tract the possibility of passive ascension of microbubbles in the ureter was examined using a uv-spectrometer. results: grade i reflux was identified on vcug in 22 patients comprising of 23 kidney-ureter-units [kuus] . in 7 kuus the reflux was also grade i on vus. in the remaining 16 kuus microbubbles were detected in the renal pelvises, the grades of vur being 2 and 3 in 12 and 4 kuus, respectively. prior to the administration of the us contrast medium dilated ureters were seen in 13 kuus. the renal pelvis was dilated only in 2 cases. in the in-vitro set-up using the same us contrast medium, despite the absence of counter-flow no microbubbles were detected at the site corresponding to the renal pelvis. conclusion: microbubbles detected in the renal pelvis can only have been actively propagated by the reflux pressure and do not ascend passively up the ureter. 70 % of the grade i refluxes diagnosed in the vcug are actually grade ii or higher. review of imaging before and after surgery for posterior urethral valves n.p. power, k. mchugh, i. gordon, d. wilcox, p. duffy, p. ransley; london/gb purpose: to compare the imaging appearances on both micturating cystourethrography (mcug)and ultrasound (u/s)before and after surgery for posterior urethral valves (puv). to determine the clinical effectiveness of routine postsurgical mcug. materials and methods: the imaging findings of 43 boys, of whom 7 had two operations for puv were reviewed. mcug was performed before and after each operation making 100 mcug in total. features evaluated included the appearances of the bladder, posterior and anterior urethra, the presence of vesico-ureteric reflux (vur) and the contemporaneous u/s results. age at surgery and creatinine results were also noted. results: age at surgery ranged from 6 days to 5 years 9 months. 50 % were less than a month at surgery. vur was detected in 40 % of patients; most of these were unchanged post-operatively. the commonest bladder abnormality was trabeculation (82 %); 75 % were unchanged on follow-up. 50 % of patients had a small volume bladder. when the posterior urethra was visualised it was invariably dilated. 52 % of patients had an apparently narrow anterior urethra initially; most improved on follow-up. the commonest u/s findings were bilateral hydroureteronephrosis (62 %)and bladder wall thickening (42 %), although 4 % of pre-operative u/s were normal. most patients had little change in u/s features. 78 % had a fall in creatinine post-operatively. conclusion: a small number of boys with puv may have normal u/s appearances. imaging appearances on both mcug and u/s are frequently unchanged after puv resection. as patient management depends on several factors including renal function and cystoscopy findings, we question the clinical effectiveness of routine post-operative mcug. the transbrachial approach for sclerotherapy in paediatric varicoceles p. agresti, s. pieri, l. de medici, g. fiocca, a. calisti; rome/it introduction: percutaneous sclerotherapy has gained wide acceptance in the treatment of male and female varicocele. we introduced the percutaneous approach into the paediatric population, because it is minimally invasive, uses local anesthesia, is minimally traumatic and less expensive than a surgical option. we report our 10 year experience with the transbrachial approach. materials and method: from 1991 to 2000 we have done 425 procedures. inclusion criteria were positives at physical, doppler and ultrasound examination for varicocele type i, grade iii, and ii only if symptomatic. phlebography was done under local anesthesia, with a transbrachial approach. we first try to enter the right spermatic vein and then work on the left side. sclerotheraphy was done with tetra-decilsolfato (trombovar). follow up with clinical examination was done at 1 -6 -12 months and 6 -12 months with colour-doppler ultrasound. results: median age was 14.7 years. symptoms were present in only 3 % of boys. there were 78/425 bilateral varicoceles, 15 on the right and the remainder on the left. there were good clinical results with sclerosis in 95 % of the treated boys. discussion: percutaneous sclerotherapy of varicoceles is a safe, effective and less expensive option than surgical therapy, especially in the bilateral form, with a low complication rate. methods: 20 patients with fractures of the mandible underwent mri (gradient echo sequences before and after contrast administration). continuity of the nerve was evaluated by visual inspection, signal intensity was measured by region of interest in 4 different locations. the nerve could be evaluated in all 20 cases. the continuity or the disruption of the nerve detected on mri was confirmed by the surgical findings in all patients. there was no difference in the increase in signal intensity after contrast enhancement between patients with and without disruption of the nerve. some patients with neurological deficit without disruption of the nerve, showed a decrease of signal intensity distal the fracture. all the other patients without neurological deficit had an increase of signal intensity from proximal to distal. conclusion: it is possible to detect the disruption of the nerve by mri. the reason why the signal intensity in some patients decreases seems to be unclear. as we used gradient echo sequences, which in particular show blood flow effects, it might be possible to measure a reduced blood circulation in the perineurium distal the fracture by regions of interest. beside prevention of neurological deficits, this might be another argument for urgent surgical treatment. bmd measurements in the jaw from dental ct images. verification of the method and first applications in dental implantology a. beer, p. homolka, a. gahleitner, m. tschabitscher, r. nowotny, h. bergmann; vienna/at purpose: in implant dentistry, both bone quantity and quality, are of vital importance for preoperative planning. while bone quantity is well defined, different approaches on measuring bone quality exist. usually quite crude grading schemes are applied, that may not provide a quantitative measure for local bone quality exactly at the intended implantation site. a c d e f 268 methods and materials: a calibration standard with known concentrations of hydroxyapatite is scanned simultaneously with the patient using an adapted dental-ct technique. conversion of hounsfield numbers to bmd values is accomplished using a linear relationship. bmd is evaluated locally using full ct image resolution on reformatted views to evaluate possible implant sites. results: in a cadaver mandible pilot study a strong correlation (r 2 > 0.85) between bmd and the insertion torque of dental implants is demonstrated. therefore, bmd may serve as an estimate of achievable primary implant stability and thus, as a measure of bone quality. first clinical applications are going to be presented. conclusion: since a correlation with mechanical bone properties and accepted measures of primary implant anchorage has clearly been established, preoperatively determined bmd values of the jaw bone could help to assess possible implant sites to optimize primary stability and long term prognosis of the implants by guiding the surgeon through the choice of best suited implant site or implant type and preparation technique. periapical dental findings evaluated with coronal sinus ct c.r. krestan, p.l. peloschek, c. czerny, s. grampp, a. gahleitner; vienna/at purpose: to evaluate the frequency of chronic peripical periodontitis (cap) in teeth in the upper jaw using coronal sinus ct. methods: we reviewed 95 coronal sinus ct examinations (ct-secura, philips medical systems, best, the netherlands) from non-selected clinical patients. sinus ct was performed with the patient in the prone position with a collimation of 3 mm from the ventral border of the frontal sinus to the posterior edge of the sphenoid sinus. reconstruction was done using a high-resolution bone algorhythm. the frequency and size of hypodense periapical lesions was determined by two radiologists in a consensus reading. frequency and severity of artifacts from restoration dentistry and disease of the maxillary sinus was recorded. results: in 4 out of 95 patients (4.2 %) periapical disease (cap) could be dignosed. in 40 of these 95 patients metal artifacts reduced the image quality of the periapical region. the regions 16 and 26 were most affected by artifacts. there was no statistically significant difference between the site of periapical disease and concomitant sinus disease. conclusion: in up to 4 percent of non-selected patients, referred for sinus imaging, we found periapical dental disease on coronal standard ct. although coronal sinus ct is not the method of choice for evaluating dental disease, it is worth to pay attention to periapical disease. atypical non-odontical chronic jaw pain: findings on panoramic radiographs and dental-ct-scans correlated with histology and microbiology p.l. peloschek, d. turhani, f. watzinger, m. püregger, i. sulzbacher, j. sailer, c. czerny; vienna/at purpose: the aim of this study was to compare radiographic, histological and microbiological findings of patients with atypical non-odontical pain of the jaw. patients and methods: from 1998 to 2001 20 out-patients with jaw pain of uncertain origin were prospectively analysed by panoramic radiographs and ct following open biopsy. the axial ct scans (philips ct secura; 120 kv, 70 ma, scan time 2 seconds, slice thickness 1 mm, table index 1 mm, high resolution filter) were reformatted using dental-ct software (philips easyvision, dental reformatting package). usually no contrast enhancing media were given. inclusion criteria were long-term patient history with pain and/or swelling located in a tooth-free region and unclear conventional and panoramic radiographs. exclusion criteria were previous fractures, osteoradionecrosis and dental residuals. in this cohort, laboratory parameters were inconclusive. the bicortical bone-biopsies were classified following histological and microbiological findings. radiographic findings were correlated with the results of the histological and microbiological examinations. results: 20 patients could be included. no significant correlation between radiological and microbiological or histological findings was found. histological diagnosis included unspecific post-inflammatory changes, active osteomyelitis and healthy specimen. conclusion: the use of conventional radiographs, panoramic radiographs and dental-ct in the diagnosis of chronical atypical non-odontical pain of the jaw should be re-thought. if panoramic radiographs give no appropriate information in finding a focus in chronic pain of a tooth-free region, dental-ct will not give further information. maybe dynamic mri will resolve this diagnostic dilemma. appropriate studies are in progress. does a dose reduction by a factor of 27 affect the diagnostic value of multirow detector helical ct of the sinuses? d. tack, j. widelec, j.-m. bailly, c. delcour; charleroi/be purpose: to compare multiplanar reformations (mpr) and volume rendering technique (vrt) as well as virtual endoscopic views (ve) obtained from helical multirowdetector computed tomographic (mrdct) respectively with low-dose and standard-dose acquisitions in patients with sinusitis. method/materials: 50 consecutive patients with headache referred for a standard dose brain ct and in whom a sinusitis was found or had to be excluded had a second acquisition with a low dose technique (volume zoom, siemens). following parameters were used for the standard dose acquisition: collimation 4 × 1 mm, rotation time: 0.75 s, 140 kv, 150 mas, pitch: 0.875, ctdiw: 57.2 mgy, reconstruction width and increment 1.25 and 0.8 mm; low-dose acquisition: collimation 4 × 1 mm, rotation time: 0.5 s, 120 kv, 10 mas, pitch: 2, ctdiw: 2.04 mgy. coronal, axial and sagital 2 mm thick mpr and selected vrt and ve views were obtained from both native image sets and analysed on workstations by 3 radiologists who had to state the normal or abnormal appearence of 16 different anatomic regions. the effective dose was computer simulated. results: in 785 of of 800 regions of interest, no difference was found by all 3 readers. in the remaining 15 regions, 2 of 3 readers stated that low-dose and standard dose images were equivalent. computer simulated effective dose were respectively for standard-dose and low-dose mrdct of 1.72 and 0.063 msv. purpose: due to the complex anatomy of the facial bones ct examinations of fractures in at least two planes are required for reliable diagnosis and operative planning. however, in some cases examination can be performed in the axial plane only. therefore high quality mprs from axial ct-datasets have to be obtained. aim of this study was to analyse the influence of collimation, reconstructed slice thickness/increment, reconstruction-kernel, tube current and ultra high resolution (uhr) and non-uhr acquisition on detectability of facial fractures. methods and materials: a cadaver head with artificial blunt facial trauma was examined using a siemens somatom volume zoom unit (siemens, erlangen, germany). acquisition parameters were: collimation 2 × 0.5 vs 4 × 1 vs 4 × 2.5 mm, tube current 120 vs 90 vs 60 mas, uhr and non-uhr, reconstructed slice thickness/increment 0.5/0.3 vs 1.0/0.6 vs 3/1.5 mm. out of these volume datasets coronar and sagittal mprs were obtained with the following reformation parameters: slice thickness 0.5 -3 mm, overlapping 0.5 -3 mm. 6 fracture locations were scored blinded by 5 experienced radiologists. inter-observer variability was determined with the κ-test. differences in fracture detection of the respective algorithms were assessed with wilcoxon-and friedman-tests to p < 0.05. results: detectability of fractures was higher in 2 × 0.5 mm collimation (p = 0.001 to p < 0.0005). uhr is superior to non-uhr (p = 0.001). 120 mas exceeded 90 and 60 mas (p < 0.0005) in uhr and non-uhr, respectively. thin mprs (0.5/ 0.5 mm, 1/0.5 mm) are superior to thick mprs (p = 0.023 to p < 0.0005), whereas there is no significant difference between 0.5/0.5 mm and 1/0.5 mm (p = 0.317). conclusion: our data reliably prove that for subtle fractures thin mprs (0.5/0.5 mm, 1/0.5 mm) should be obtained out of thin slices (0.5 mm) with 120 mas in uhr. multi slice ct with 3d reconstruction in the maxillofacial diseases f.m. drudi, s. padula, a. righi, f. trippa, p. ricci, f. cascone, r. passariello; rome/it purpose: aim of our study was to demonstrate the utility of 3d-reconstruction and virtual endoscopy in the planning of surgical intervention and evaluation of treatment in maxillofacial diseases. materials and methods: thirteen patients (mean age 42; range 22 -67 years) underwent multi-slice ct examination (slice thickness 1 mm; scan time 2.8 seconds; rotation time 0.4 seconds; feed rotation 4 mm) using intravenous contrast agent, if necessary. volumetric data sets were post-processed with vitrea 2 (vital images, milwakee) volume rendering software. results: in 7 patients who presented keratocyst, 3d-reconstruction permitted an exact definition of volume and site of the cyst. also the trigeminal mandibular nerve branch pathway was clearly depicted. after surgery 3d-reconstruction showed a complete "restitutio ad integrum" of the bone. in 3 patients, who presented floor-ofmouth carcinoma, 3d-reconstruction was performed before treatment to evaluate the dimensions of the mass and to exclude bone involvement. after treatment the examination was repeated to evaluate response to therapy. one patient who had undergone numerous operations for bilateral cleft leap, was studied to plan further surgery and to control the outcome. in 2 patients who presented maxillary sinus osteoma, 3d-reconstruction evidenced tumor origin. virtual endoscopy showed the exact position of the neoplasia in relation to the maxillary sinus floor and permitted postsurgical evaluation of the maxillary sinus floor. conclusions: multi-slice ct with 3d-reconstruction and virtual endoscopy are useful tools in the evaluation of maxillofacial diseases before and after therapy. facial hemangiomas as anexternal manifestation of the segmental angiodysplasia: mri and mra diagnosis v. panov, a. ivanov, m. inaneishvily, a. nadtotchi; moscow/ru purpose: diagnosis of facial soft tissues angiodysplasia (fad) is not hard because of its well-known clinical symptoms. ultrasonography with color doppler mapping, invasive selective and super selective x-ray angiography are usually used for the evaluation of tumor volume, its syntopy, morphological structure variants and identification of main supplying vessels. the main task of this presentation is to the show possibilities of noninvasive mr-imaging (mri) and mr-angiography (mra) in fad diagnosis. methods & materials: 38 patients with different forms of fad were examined. all mri and mra examinations were obtained on a 1 t mri system harmony (siemens, germany) and were compared with noninvasive ultrasound and invasive x-ray angiographic data. results: in 7 patients mri has found the following arterial abnormalities: different forms of unclosed willis ring (4 cases); significant middle cerebral arteries asymmetry (4 cases); lateral choroid artery displacement (1 case). in 5 patients vascular malformations were found: intracranial arterial malformations in 4 patients, meningeal venous malformations in 1 case. the disadvantages of mri were: the long time of examination, the necessity of sedation in young patients (younger than 5 -8 years) and in patients with claustrophobia. conclusion: use of noninvasive mra made it possible to establish that in some cases fad was an external manifestation of a segmental angiodysplasia which could be diagnosed only by the combination of noninvasive ultrasound and invasive x-ray angiography. mri and mra allow to obtain the same information about fad as traditional methods but has additional advantages in diagnosis of such lesions due to opportunities for intracranial arterial and venous visualization. from ct through *.stl to rism: a few real steps in the future of craniofacial surgery a. nadtotchi, v. roginskij, o. topol'nitskij, a. evseev; moscow/ru purpose: the evaluation of ct-datasets allows the possibility of creation of realsize facial skeleton individual stereolitographic plastic models (rism). methods & materials: 20 children with severe diseases and abnormalities of facial skeleton were examined by ct (conventional or spiral): 6 patients had mandibular tumors, 4 patients had mandibular congenital deformations and 5 patients had postoperative defects, and 5 children had complex craniofacial syndroms. after ct examination, the ct-dataset was transformed into the specially produced computerized stereolitographic files (*.stl). stl-files were send to the computer operated stereolitographic device that finaly created the facial skeleton rism. the rism was used not only for diagnosis but as an aid for operation planning in each individual case. using the rism it was easier for surgeons to define the access and operation techniques and to choose the optimal localisation of osteotomy lines. the rism was used as the base for preoperative manufacturing of exactly anatomical endoprotheses. this application of rism-technology is very progressive because: (1) it allows an increase in both the quality of preparation of endoprothesis and in their organospecificity; (2) it allows a decrease in the duration of operation of 1.5 -2.0 hours. where it was necessary to enlarge the mandible with compression-distraction apparatus, rism allowed definition of construction and optimal position of compression-distraction devices. conclusion: rism will be used not only as diagnostic model, but as important and powerful aid in the different branches of cranio-facial surgery. that is why we are sure that the system "ct -*.stl -rism" is a real step in the future of surgery. purpose: investigate the utility of ct cystography in the study of bladder tumors. method and materials: 10 patients with bladder tumours, ranging from solitary polypoid lesion to multiple endoluminal lesions with diffuse alterations of mucosal surface, were studied with ct cystography and the results compared with conventional cystoscopy. radiologists were blinded to the results of cystoscopy. ct cystography was performed with one helical acquisition of 3 mm slices at pitch 1.5, with images reconstructed each 1 mm. in all patients, the bladder was inflated with room air via a foley catheter. the data were then downloaded to a workstation and reviewed, both on axial 2d and with 3d-intraluminal navigation. results: tumour lesions (12) of the 8 patients having localized lesions were recognized on the ct data. this included one small lesion less than 5 mm. in the two patients having diffuse bladder involvement, ct cystography was able to detect the major polypoid lesions, but also identify a diffuse irregularity of the bladder inner surface corresponding to the spread of the lesions over the entire mucosal surface. ct axial data also predicted the extra-serosal spread of the disease by contiguity to the inner part of the anterior abdominal wall, which was confirmed by open surgery the day after ct cystography. urologists found the 3d images very similar to their own cystoscopy images. conclusions: recognition of bladder wall tumours is feasible by means of ct cystography. lesions equal or bigger than 5 mm were shown. b a c d e f 270 three-dimensional volume and surface rendered ultrasound of the urinary bladder: early experience g. helweg, a. klauser, l. pallwein, a.h. schuster, g. feuchtner, a. stenzl, d. zur nedden, f. frauscher; innsbruck/at purpose: we evaluated the feasibility of three-dimensional (3d) surface and volume rendered ultrasonography (us) for detection and differentiation of urinary bladder lesions. methods and materials: fourteen patients suspicious for bladder tumours on two-dimensional (2d) us underwent 3d imaging of the bladder prior to cystoscopy and transurethral bladder biopsy. the suspicious areas on 2d us were evaluated using the perspectivetm 3d us (acuson, moutainview, ca). tumour volume was calculated using the built-in software. documentation was done on video and printouts. the us findings of 2d and 3d us were compared with endoscopic findings and the biopsy results. results: eight of 14 patients (57 %) had biopsy proven bladder cancers. in addition to conventional 2d us, the mean time for the assessment of 3d data sets was 2.5 minutes (range: 2.0 -4.0 min). 3d us allowed a better visualization of the inner contour, corresponding to the mucosal layer of the bladder. 3d surface and volume rendered us data sets, enabled the correct diagnose of a benign lesion in 5 of 6 patients, and of cancer in 7 of 8 patients. this revealed an overall accuracy of 85 % for differentiating benign from malignant lesions. the tumour volume was overestimated ≥ 20 % in 4 of 8 patients with cancer compared to cystoscopy measurements. conclusions: 3d volume and surface rendered us improves the differentiation between benign and malignant lesions of the urinary bladder, compared with conventional 2d us. this technique requires only a short additional examination time and does not put any burden on the patient. use of flair sequences for detection and local staging of bladder tumours with mri m. di girolamo 1 , a. roncacci 1 , g. brughitta 2 , d. fini 2 , s. cavalaglio 2 , v. david 1 ; 1 rome/it, 2 grottaferrata/it purpose: to increase the accuracy of mri in the detection and local staging of bladder tumours by using flair sequences. method and materials: 32 patients with bladder tumours detected by us underwent mri using 0.5 and 1.5 t superconductive magnet. we performed se t1weighted (tr: 500 ms, te: 30 ms), tse t2-weighted (tr: 2500 ms, te: 120 ms) and flair sequences (tr: 6000 ms, te: 150 ms, ti: 2000 ms, n.ex.: 4; acq.time: 7 min 30 s) on axial scans. the contrast to lesion ratio was evaluated in all sequences. all the patients underwent cystoscopy with transurethral biopsy and 14 had subsequent cystectomy. results: in comparison with other sequences, flair sequence was more sensitive in the detection of bladder neoplasms. this sequence demonstrates the hyperintense signal of bladder neoplasms from the filled bladder lumen with no signal. the sensitivity in the identification of bladder neoplasms was 100 % with flair sequences, 89.6 % with tse t2-weighted sequences and 86.2 % with se t1-weighted sequences. that was due to the higher signal to lesion ratio of the flair sequences in comparison with the others. in fact on flair sequences the mean value of contrast to lesion ratio of bladder neoplasm was 33.1 while on se t1-weighted sequences and tse t2-weighted sequences was respectively 15.2 and 29.2. flair sequences allowed the detection of small papillomas (less than 2 mm). tse t2-weighted sequences were more sensitive in the study of bladder wall infiltration. conclusions: flair sequences were more sensitive in the detection of bladder neoplasms, due to their higher contrast to lesion ratio and can be very helpful in the visualization of small papillomas, especially when multifocal. purpose: evaluation of the diagnostic performance of virtual mr-cystoscopy for the assessment of bladder tumours. materials and methods: 35 patients with bladder tumours were examined with a 1.5 t scanner, using a t2-weighted 3d-tse-sequence (tr = 2911 ms, te = 500 ms) with a voxel size of 1.1 × 1.0 × 1.5 mm 3 . in 10 patients the new "sensitivity-encoding"-technique (sense) was used, which allows a higher spatial resolution without increasing scanning time. the bladder-wall was divided into 6 re-gions, which were analysed separately for the presence of tumours by 3 radiologists without knowledge of the tumour location. findings of conventional cystoscopy and operation served as standard of reference. results: 39 of 43 tumours were detected by virtual cystoscopy with 6 false positive results, resulting in an overall sensitivity of 90 % and a specificity of 95 %. all tumours larger than 1 cm (21/21, sensitivity 100 %) and 17 of 22 tumours smaller than 1 cm (sensitivity 79 %) were detected. the tumours missed had a size of 5 mm or smaller. on the average, the time needed for virtual-endoscopic reconstruction was 15 minutes. with the new sense-technique, spatial resolution could be improved without increasing scan-time. limitations of virtual cystoscopy are the detection of flat lesions and the differentiation between neoplastic and inflammatory lesions. conclusion: virtual cystoscopy is a promising, completely non-invasive tool for the diagnosis and follow-up of bladder tumours. by using the new sense-technique, the spatial resolution can be further improved. mr virtual cystoscopy detection and staging of bladder lesions before performing conventional cystoscopy v. panebianco, r. iannaccone, i. sansoni, a. laghi, c. catalano, m. ciccariello, p. paolantonio, r. passariello; rome/it purpose: to identify and to stage bladder lesions with mr-cystoscopy combining axial and virtual images. methods and materials: 31 patients with suspected or known diagnosis of bladder tumour underwent mr cystoscopy. we used natural contrast (urine) for bladder distention, monitoring bladder filling with 3dflash sequence with i.v. c.m. (optimal results after 20 min). mri examinations were performed on a 1.5 t siemens vision plus mr imager. we obtained a 2d fse sequence on axial planes. for evaluation of bladder wall, we performed a t1 weighted gre sequence, with and without fat suppression, pre and post gd-dtpa (0.0025 mmol/kg) i.v. injection. a 3dspgre sequence was obtained for monitoring bladder filling and for virtual endoscopy analysis, prior to furosemide (1 cm 3 ) administration. real time endoscopic views were reconstructed using volume-rendering reconstrution algorithm (vitrea 2.2, vital images). results: image quality was considered optimal in all cases. a total of 24 lesions were detected and confirmed at biopsy. in 4 cases no lesions were evident at mri and at conventional cystoscopy. all lesions were evident on morphological t1 and t2 weighted images, with size ranging of 4 -20 mm. mr virtual cystocopy allowed detection of the same lesions than conventional cystoscopy, providing comparable images. however, endoscopic images did not show intramural and extravescical extension of the tumour, whereas these findings were easily appreciated on morphological t1 and t2 weighted images. conclusions: mr virtual cystoscopy optimises detection and staging of the bladder pathology by combining axial t1 and t2 weighted and ve images. three-dimensional visualization of pelvic lymph nodes in staging and treatment of urinary bladder cancer using high-resolution mri a. welmers 1 , b. schrier 1 , h. huisman 1 , j.r. fielding 2 , l. o'donnell 2 , w.m.l.l. deserno 1 , r. kikinis 2 , j.g. blickman 1 , j.o. barentsz 1 ; 1 nijmegen/nl, 2 boston, ma/us purpose: in this study we evaluated the possibilities of 3d modeling in lymph node visualization for surgical guidance during lymph node dissection (lnd) in bladder cancer using high resolution mr data. methods and materials: mri was performed on a 1.5 t mri using the 3d t1-w mp-rage sequence (tr 11.4, te 4.4, 1.4 mm isometric voxel-size) with a pelvic phased-array coil. an iv uspio contrast agent was used to enhance the visibility of the lymph nodes. bladder, iliac vessels, ureters, obturatory nerves and lymph nodes were manually segmented from the mr data, using the software "3d-slicer". from these segmentations 3d models were created. in total 25 patients with histology proven urinary bladder cancer were evaluated. in all cases lnd was performed. five patients had enlarged metastatic lymph nodes, which were all segmented. during lnd the 3d models were used as a guiding tool for removing lymph nodes. results: in all patients a 3d model was created successfully, showing iliac vessels, ureters, obturatory nerves and lymph nodes. the 3d models allowed accurate localization of normal and metastatic nodes in relation to the surrounding structures, which enabled the surgeons to find and remove more lymph nodes than without this technique. conclusions: this technique simplifies visualization of lymph nodes. this increases urologist awareness of the exact location of lymph nodes, which results in a higher yield of nodes at node dissection. long fov whole body 3d mri using continuous table motion s.j. riederer, d.g. kruger, p.j. rossman, r.c. grimm; rochester, mn/us purpose: current methods for imaging an extended longitudinal field of view (fov) in mri either use multiple fixed stations or rapid 2d axial imaging similar to single slice, helical ct. the purpose of this work was to develop a method for 3d mr imaging of an arbitrarily long fov using continuous table motion. methods and materials: a method was developed in which data are collected as the table is moved continuously through the mr scanner gantry. after fourier transformation along the readout direction, every echo is assigned to hybrid (x, ky, kz) space, with longitudinal (x) location determined by table position at the time of the echo. as the table moves, all desired phase encodings are measured periodically. the method was tested in phantom, animal, and human studies. table velocity ranged from 0.8 to 3.0 cm/s. the longitudinal fov for readout ranged from 15 to 30 cm, and the total fov was as large as 170 cm. results: complete 3d image sets were formed of objects up to ten times longer than the fov of the acquisition. correction for position at sub-pixel precision is critical to suppress ghosting artifacts. contrast-enhanced studies in pigs demonstrate the ability to follow the contrast bolus in peripheral runoff studies. results in humans suggest the feasibility of whole body imaging. conclusions: 3d mr acquisition during continuous table motion has been demonstrated. it offers potentially reduced acquisition time vs. fixed station 3d methods and improved snr vs. rapid serial 2d axial acquisition. spatial resolution in t1-weighted conventional se and tse sequences: phantom measurements and in vivo results c. fellner, f.a. fellner, j.-c. georgi, w.a. kalender; erlangen/de purpose: assessment of spatial resolution in t1-weighted tse sequences with different echo train lengths (etls) compared with an se sequence using phantom measurements and examination of healthy volunteers. materials and methods: t1-weighted tse sequences with different etls (3, 5, 9) and an se sequence with identical pixel size (0.8 mm) were evaluated with a custom-built resolution phantom offering resolution patterns from 0.1 mm to 1.5 mm in steps of 0.1 mm and in 25 healthy volunteers focusing on the cranial nerves. the resolution in the phantom images was assessed visually, and the standard deviation in an roi containing the 0.8 mm stack was evaluated. image quality and delineation of cranial nerves (ii, iii, v) of the in vivo measurements were assessed by an experienced radiologist blinded to the sequence technique. statistical evaluation of the visual evaluation was done using wilcoxon's test (p < 0.05). results: the visual evaluation of phantom images yielded identical results for the se and the tse sequences with short etls (3, 5); for an etl of 9 the spatial resolution was deteriorated. the quantitative evaluation, however, showed a continuous decrease of the standard deviation with increasing etl. image quality of in vivo images and delineation of cranial nerves was significantly superior in se images compared with tse images -even if a very short etl was applied. conclusion: qualitative as well as quantitative evaluation of spatial resolution and anatomical details correspondingly revealed disadvantageous results for t1weighted tse sequences with increasing etl. this result was even more pronounced in the in vivo examinations. deep brain stimulation during interventional mri: safety issues r. girnus, v. hesselmann, k. luyken, b. krug, g. nimtz, k. lackner; cologne/de purpose: to investigate safety aspects of linear conductive devices in interventional mri. materials and methods: the temperatures occuring in long wires placed in a 1.5 t mri (philips, gyroscan intera) were measured. the temperature increases (δt) at the tip of the electrode were determined using a mr-compatible opticalfiber-temperature system. the end of the wire was surrounded by 3 cm 2 of a 0.9% nacl solution. the length and the position of the wire were tested as parameters influencing the hf-induced increases in temperature. furthermore the influence of the sequence was taken into account. result: the temperature increase of the wire aligned parallel to the z-direction and symmetric to the isocentrum showed a significant dependence on the length. a resonance length of about 2 m could be observed, with a ät of 30°c at the tip located in the saline solution during a tse-sequence (sar = 3.9 w/kg). by moving the wire out of the isocentrum in x or y direction, the heating effect exceeded 100°c at the electrode tip. the ät fell below 2°c, if the wire length was different c d e f 272 from 2 m, the part of the wire inside the scanner bore was minimized and the wire was located in the center of the bore parallel to the z-direction. in general the ät can be reduced by applying gradient echo sequences with low flipangels. conclusion: under controlled conditions mentioned above it seems feasible to put a wire or stimulation electrode into an mr without risk of burning. liver iron overload: comparison of two methods in measuring liver r2* values. correlation with serum ferritin concetration (sfc) and liver iron concentration (lic) t.g. maris 1 , o. papakonstantinou 1 , e. chryssou 1 , v. ladis 2 , s. kostaridou 2 , n. papanikolaou 1 , p.k. prassopoulos 1 , n. gourtsoyiannis 1 ; 1 iraklion/gr, 2 athens/gr purpose: to compare liver r2* (r2* = 1/t2*) values obtained by means of quantitative mri (r2*-qmri) using two regression analysis models and correlate them with lic and sfc. materials and methods: liver r2* values were calculated in 10 thalassaemic patients, and 10 normal subjects on an 1.5 t mri system using a breathhold multislice-double-echo spoiled gre sequence with initial parameters: tr/te1/te2 160/ 2.7/5.3 ms. the sequence was repeated four times, each time altering inter-echo time spacing. t2* calculated image maps were post-proccessingly reconstructed using four different fitting methods (a, b, c, d) . methods (a) and (b) were based on a simple linear regression model applied to image pixel data. methods (c) and (d) were based on a weighted linear regression model on which pixel data were weighted according to background image random noise figures. results: differences of mean r2* values between patients and normal subjects were considered extremely significant (t = 19.25, p < 0.0001). r2* values correlated much closer with lic levels (r = 0.89, p < 0.005) when using weighted linear regression models (c) and (d) than when using normal regression ones (a) and (b) (r = 0.85, p < 0.005). r2* were moderately correlated to sfc values regardless the use of the regression model (r = 0.6, p < 0.05). r2* values were linearly correlated with lic [r2 × (s −1 ) = 4.94 + 93.94 lic (mg/g) −1 , r = 0.89, p < 0.005] conclusion: r2* when calculated using weighted regression methods on 1.5 t systems seem to be a valuable means for the evaluation of liver iron overload when lic does not exceed 8 mg/g (liver dry weight). improved mri-monitoring during rf-hyperthermia h. reinl 1 , m. peller 1 , m. hagmann 2 , r. issels 1 , m.f. reiser 1 ; 1 munich/de, 2 salt lake city, ut/us purpose: in a mri-hyperthermia hybrid-system t1 relaxation-time changes are investigated for monitoring hyperthermia of deeply seated tumors. a water-bolus is needed for power transmission into the patient body. the signal of this bolus limits image quality by signal compression and artifacts. this can be avoided by using d2o which would result in additional technical effort and high costs. the purpose of our study is to improve image quality, spatial and temporal resolution by using a paramagnetic suspension of magnetite instead. material and methods: all experiments were done on the hybrid-system which consists of a 0.2 t open mr-system and a hyperthermia system with a mr-compatible phased array applicator and an integrated mr receive-coil. the tested paramagnetic suspension was a commercially available ferrofluid (msg w11, ferrofluidics corp.), normally used for material separation. a polyamidacryl gelphantom mimicking the human body was used for heating experiments. results: we found ferrofluid in a low concentration as a useful substitute. in the 0.2 t system a concentration of 0.25 % showed optimum results. below 0.2 % the bolus is still slightly visible and above 0.3 % the mr system is no longer tuneable. no artifacts are induced in this concentration-range. conclusion: by using the new bolus-filling an extinction of the bolus artifacts and a significant improvement in snr, spatial and temporal resolution are possible. this method of signal extinction in mri may be adapted to other experimental demands where mr-invisible fluids are necessary. stereological estimations of liver volume from mr images m. mazonakis, j. damilakis, t.g. maris, p.k. prassopoulos, n. gourtsoyiannis; iraklion/gr purpose: to investigate the possibility of generating stereological estimations of liver volume from magnetic resonance (mr) images. methods and materials: 38 consecutive patients underwent 1.5 t abdominal mr imaging. two radiologists evaluated the mr images and found that the liver size appeared normal in 27 and increased in 11 cases. liver volume was estimated using the cavalieri method of modern design stereology in combination with point counting. the effect of the separation distance between test points of the grid in the efficiency of stereological estimations was examined. a systematic sampling of mr sections was performed to find the minimum number of sections needed to provide acceptable volume estimations. results: for both subgroups of patients with normal and increased liver size, it was found that 100 -150 test points counted on 7 -8 systematically sampled mr sections may provide reliable liver volume estimations with a coefficient of error (ce) of less than 5 %. the mean time required for the stereological measurements was approximately 10 min. the mean liver volume for the patients with normal and increased liver size was found to be 1477.7 ± 230.7 and 1944.7 ± 196.1 cm 3 , respectively. the proposed volumetric technique may provide efficient liver volume estimations from mr images in patients presenting both normal and increased liver size. improvement of diagnostic accuracy of pet imaging using a high performance in-line pet-ct system: preliminary results t.f. hany, h.c. steinert, g.w. goerres, a. buck, g.k. von schulthess; zürich/ch purpose: we describe the first application and optimisation of a novel in-line system combining a high performance clinical positron emission tomograph (pet) scanner and a fast multi-slice helical computer tomograph (ct) scanner in a single machine (pet-ct) for tumour staging. methods: we examined 53 patients with diagnosed or highly suspected malignancy. non-contrast-enhanced ct data with 4 different tube currents (10, 40, 80 and 120 ma) were acquired, followed by pet acquisition. step-wise image analysis was performed using pet images alone compared to co-registered pet-ct with increasing ct energies (pet-ct10, … pet-ct120). clinical or pathological staging was used as standard of reference for lesion-by-lesion as well as on patient based analysis. results: the following accuracy ratios for classifying tumour lesions were calculated: pet alone 91 %, pet-ct10 97 %, pet-ct40 97 %, pet-ct80 98 %, pet-ct120 98 % and a significant difference was found between pet alone and co-registered images (chi square p < 0.01). conclusion: pet-ct fusion in a combined scanner using non-enhanced lowdose ct with 80 ma is significantly increasing diagnostic accuracy regarding lesion classification, and by using the ct scan for transmission correction will reduce the acquisition time by 30 % when compared to pet imaging alone. transmission scanning for attenuation correction in cardiac spect studies: patient effective dose and radiogenic risk k. perisinakis, j. damilakis, n. gourtsoyiannis; iraklion/gr purpose: to determine patient effective dose and associated radiogenic risk from the transmission scanning performed during cardiac spect myocardial perfusion studies to correct scintigraphic images for attenuation and scatter effects. materials and methods: transmission scans were obtained using an optima nx ge system, which involved an l-shaped dual headed gamma camera equipped with transmission scan hardware. two flat scan boxes each containing a collimated rod source of gadolinium 153 were mounted on the gantry opposite each detector. a rando anthropomorphic phantom was used to determine radiation dose from transmission scanning in 14 organs and tissues using 352 thermoluminescent dosemeters. for each projection the resulting effective dose was calculated using the icrp 60 tissue weighting factors. radiation risk was determined using age and sex related radiation induced fatal cancer risk factors. results: the effective dose corresponding to a typical 5 s acquisition per transmission scan was 620 psv. the organs receiving the highest amount of radiation dose is the lung and the oesophagus. the average lifetime risk for fatal malignancy is 4.9 × 10 −8 for us and 3.9 × 10 −8 for uk population. conclusion: present data allow the accurate estimation of patient effective dose and associated radiation detriment risk from myocardial perfusion spect studies. purpose: assessment of three different rectal agents (water, ultrasound gel and methylcellulose) for their suitability for colorectal imaging in multislice ct material and methods: 115 patients with colorectal diseases underwent msct with varying rectal contrast agents in a prospective study. images were assessed by two independent ct-experienced radiologists. ct scans were analyzed for the following criteria: contrast of wall versus lumen (w/l), contrast of pathological wall versus lumen (p/l), distension of anal verge, distension of colon and artefacts (ar). a rating scale of 1 (no) to 5 (severe) was used. interobserver variability was assessed by kappa statistics. results: mean rating values of methylcellulose were higher in contrast of wall/ lumen (4.66 ± 0.6), pathological wall/lumen (4.73 ± 0.5) and least rate of artefacts (4.13 ± 0.7) than of water (w/l 4.36 ± 0.8; p/l 4.36 ± 0.7; ar 3.76 ± 0.9) and ultrasound gel (w/l 2.4 ± 0.96; p/l 2.55 ± 1.01; ar 1.76 ± 0.9). the distension of the anal verge was better in using ultrasound gel (4.5 ± 0.6) and methylcellulose (4.35 ± 0.7) than in water (2.82 ± 1.1). with water the best distension of colon (4.1 ± 1.1) could be obtained, it was nearly equal to methylcellulose (3.82 ± 0.8), but remarkable superior to ultrasound gel (1.5 ± 0.6). air artefacts only appeared in ultrasoundgel. an excellent interobserver correlation was found for the contrast of pathological wall versus lumen of methylcellulose (k = 0.81). a high rate of agreement between the criteria of both radilogists in final diagnosis could be achieved. conclusion: rectal filling with methylcellulose significantly improves diagnostic confidence in colorectal examinations. ease of application and lack of use suggest to use as a clinical routine. ct differentiation of colorectal mucinous and nonmucinous carcinoma e. ko, h. ha; seoul/kr purpose: to evaluate the ct findings which can help differentiate mucinous from nonmucinous colorectal carcinoma. ct scans of 86 patients with pathologically proven mucinous colorectal carcinoma during a 10-year period, were analyzed. as a control group, 105 consecutive patients with pathologically proven nonmucinous colorectal carcinoma in a 3-month period were included. retrospective review of ct was performed by two radiologists in consensus who were blind to the pathologic results. ct scans were analysed with regard to the site and length of involved segment and types of morphological features, bowel wall thickening and contrast enhancement patterns, degree of contrast enhancement in solid portion of the tumor. also evaluated were the presence of calcification, obstruction, lymphadenophathy, pericolic infiltration, local tumor extension, and distant metastasis. in heterogeneous masses, the extent of hypoattenuated areas within tumor was graded into three. statistical analyses were performed by using student t-test and pearson's ÷ 2 -test. results: as compared with nonmucinous carcinoma, mucinous carcinoma showed more severe (p = 0.026) and more eccentric (p = 0.025) bowel wall thickening. heterogeneous contrast enhancement was more common in mucinous carcinoma (p < 0.001). significant difference was noted in the extent of hypoattenuated areas within tumor (p < 0.001). the solid portion of mucinous carcinoma showed hypoattenuation while nonmucinous carcinoma showed hyperattenuation (p = 0.001). although statistical values were not obtained, the presence of intratumoral calcification was more frequent in mucinous carcinoma (21 % vs 5). conclusion: ct is very useful in the differentiation of mucinous from nonmucinous colonic adenocarcinoma. usefulness of the hydrogen peroxide enhancement in the diagnosis of the anal and ano-vaginal fistulas i. sudol-szopinska, w. jakubowski, m. szczepkowski, d. sarti; warsaw/pl purpose of the study was to assess the usefulness of contrast-enhanced anal endosonography (aes) with hydrogen peroxide in the diagnosis of the anal fistulas. method and material: a bruel & kjaer scanner with a 7.0 mhz transducer was used. after visualization of the fistula tract in non-contrast aes, hydrogen peroxide was introduced into the fistula tract through the external opening in 22 patients with different types of anal fistulas. results: both contrast-and non-contrast aes revealed 13 transsphincteric, 3 intersphincteric, 2 suprasphincteric and 4 ano-vaginal fistulas. simple tracts were found in 16 cases and complex in 6 cases in non-contrast aes. contrast-enhanced aes revealed 19 simple and 3 complex fistulas. 15 internal openings visible in non-contrast aes were confirmed in contrast-enhanced aes in 6 cases, which additionally found 11 more internal openings. in all cases, surgery confirmed the diagnoses of the contrast-enhanced aes. conclusion: contrast-enhanced aes appears to be superior to non-contrast aes in preoperative assessment of the anal and ano-vaginal fistulas and in locating of the internal openings. contrast-enhanced multislice ct colonography in the diagnosis and staging of colorectal cancer i. carbone, a. laghi, r. iannaccone, i. baeli, r. ferrari, f. mangiapane, f. iafrate, c. catalano, r. passariello; rome/it purpose: to evaluate the role of ct colonography (ctc) in patients with suspected colorectal cancer. methods and materials: forty-eight patients (21 females and 27 males; age range 41 -78) underwent conventional colonoscopy (cc) and ctc for suspected colorectal mass. multislice spiral ct (somatom plus 4 volume zoom, siemens, germany) examination of the abdomen and pelvis was performed after routine bowel preparation and colonic distention with room air. patients were scanned in prone and supine positions using the following parameters: slice collimation, 2.5 mm; slice thickness, 3.0 mm; reconstruction interval, 1 mm; mas, 80 (prone) and 120 (supine). 130 ml of contrast medium were administered i.v. during the supine acquisition with a 60 s delay time. images were subsequently downloaded and analysed on a dedicated workstation. surgical specimens were used as the standard of reference against which the findings of cc and ctc were compared. results: cc failed to visualize the entire colon in 27 patients due to occlusive neoplasms. ctc allowed whole-colon evaluation in all the cases and correctly detected and staged 43 out of 48 colorectal cancers. ten synchronous colonic lesions (8 adenomatous polyps, 2 carcinomas) were identified in 10 different patients at ctc. liver metastases were detected in 10 patients. conclusion: contrast-enhanced multislice ctc is a valuable tool for the preoperative staging of crc. this imaging modality is very useful for presurgical planning due to whole-colon evaluation even in the presence of stenosing lesions, as well as optimal assessment of bowel wall invasion, lymphoadenopathies, and hepatic parenchyma. detection of colorectal lesions with ct colonography: comparison with conventional colonoscopy in 165 patients i. carbone, a. laghi, r. iannaccone, i. baeli, r. ferrari, s. trenna, c. catalano, r. passariello; rome/it purpose: to compare the performance of ct colonography with that of conventional colonoscopy (cc) in a blinded, prospective study in 165 patients with suspected colorectal lesions. methods: 165 patients, all referred for cc, underwent preliminary ct colonography. after standard oral colonoscopy preparation and colonic distension with room air, ct colonography was performed either with a single-slice (somatom plus 4; siemens, erlangen, germany) or multislice (somatom plus 4 volume zoom, siemens, germany) scanner. imaging parameters for the single-slice scanner were: 3.0 mm slice collimation; 6.0 mm/s table speed; 0.75 s tube rotation; 2.0 mm reconstruction interval; 512 × 512 matrix; 120 mas; 130 kvp; and scan time 35 -47 s. imaging parameters for the multislice scanner were: 1.0 mm slice collimation; 8.0 mm/s table speed; 0.5 s tube rotation; 1.0 mm reconstruction interval; 80 mas; 120 kvp; and scan time 25 -32 s. ct images of all suspected lesions were analyzed on a workstation and subsequently compared to cc findings. results: there were 30 colorectal cancers and 37 polyps identified at cc. ct colonography correctly detected all cancers, as well as 11 of 12 polyps of 10 mm in diameter or larger (sensitivity, 92 %); 14 of 17 polyps between 6 and 9 mm (sensitivity, 82 %); and 4 of 8 polyps of 5 mm or smaller (sensitivity, 50 %). the per-patient sensitivity and specificity were 92 % and 97 %, respectively. conclusion: ct colonography has a diagnostic sensitivity similar to that of cc for the detection of colorectal lesions larger than 6 mm in diameter. colorectal polyps: improvement of detection with multi-slice ct colonoscopy j. wessling, r. fischbach, d. domagk, e. neumann, s. schierhorn, w.l. heindel; münster/de purpose: to compare the performance of virtual and conventional colonoscopy for the detection of colorectal polyps using a multi-slice spiral ct scanner (msct). material and methods: 48 patients (20 women, 28 men, mean age 61.5 years) with clinical indication for conventional colonoscopy were prospectively studied using a msct (somatom volume zoom, siemens, forchheim). examination was performed after standard oral colonoscopy preparation and colonic distension with room air and i.v. buscopan. images were obtained in prone and supine position using a detector configuration of 4 × 1 mm, a feed of 5 mm/rotation at 140 mas and 120 kv. slice thickness and reconstruction increment were 3 and 1.5 mm, respectively. ct data were assessed by 2 blinded radiologists on a vitrea workstation (vital images, usa) using a software with multiplanar and volume-rendering capabilities. results: 33 patients had normal findings on conventional colonoscopy. a total of 30 polyps and 2 carcinoma were identified in 15 patients. msct-colonoscopy detected all carcinomas and 23 polyps (77 %). 3 of 3 polyps were 10 mm or more (100 %), 6 of 7 polyps were 6 to 9 mm (86 %) and 14 of 20 were smaller 6 mm (70 %). there were 13 false positive findings for polyps (10 were smaller 6 mm) and no false positive finding of cancer. conclusions: compared to single-slice-ct, multi-slice ct colonoscopy increases the rate of detection of small colorectal polyps in particular. however, false positve results still remain a problem. does contrast enhancement contribute to ct colography? a.r. gillams, v. munikrishnan, w.r. lees; london/gb purpose: controversy persists over the usefulness of iv contrast in ct colography. enhancement in small lesions should facilitate detection particularly when there is adjacent fluid. we measured the attenuation values of polyps and cancers both before and after iv contrast. materials and methods: forty-nine patients, 32 male, mean age 57 (42 -80) were studied. all patients had routine bowel preparation, iv smooth muscle relaxant and rectal air insufflation. supine and prone imaging was performed using a multi-slice ct, collimation 1 mm, effective slice width 1.25 mm and pitch 1.2. the second acquisition was performed 30 s after pump injection of 100 -150 ml of iv contrast injected at 5 ml/s. all lesions were confirmed on colonoscopy. the attenuation values of polyps and cancers were measured both before and after iv contrast. results: eighteen cancers were detected and 28 polyps. mean polyp size was 12 mm (3 -25 mm). both cancers and polyps demonstrated enhancement following iv contrast enhancement but was more marked in polyps. the mean attenuation value in the cancers prior to contrast was 53 ± 16 and following contrast 105 ± 26. the mean attenuation value in the polyps prior to contrast was 62 ± 11 and following contrast was 113 ± 26. the mean increase for cancers was 39 and for polyps 51. conclusion: contrast enhancement is seen in both polyps and cancers but is more marked in polyps. enhancement could be used where there is diagnostic doubt on the pre-contrast imaging. colour doppler visualization of hemorrhoidary arteries during transrectal us a. sias, v. alvino, f. lecca, g. mallarini; cagliari/it purpose: the scope of this work is to evaluate the usefulness of color doppler transrectal us in the visualization of hemorrhoidal artery in patients undergoing non invasive surgery for hemorrhoids. material and methods: we have examined 25 patients undergoing doppler-guided hemorrhoidal artery ligation for the treatment of hemorrhoids with modified morinaga technique. discussion: new surgical less-invasive techniques of treatment of hemorrhoids are being developed. one of this techniques involves the use of a modified proctoscope for artery ligation. colour doppler during transrectal us was used to evaluate the position of terminal arteries originating from the superior rectal artery. we were able to confirm the arrangement of these terminal arteries at 1, 3, 5, 7, 9, 11 as seen in the lithotomy position. this was noted first by meintjes in 2000. conclusion: colour doppler transrectal us allowed a clear visualization of hemorrhoidal arteries in the fixed position described by meintjes. our study was the first to show the reliability of this classification. although useful, well tolerated, quick and easy to perform, we do not recommend routine use of colour doppler transrectal us in all patients undergoing treatment for hemorrhoids with modified morinaha technique. this examination can be useful in those with recurrent secondary hemorrhage, as it can help avoiding further bleeding problems. results: volunteers: using the corrections the mean difference between the volume of the 3 he-mri and the pft measured -74 ml (r = 0.9). patients: after the corrections the mean difference measured 616 ml (r = 0.96). functioning lung grafts contributed 66 ± 6 %, their corresponding native ipf lungs 34 ± 6 % to total ventilated volume. conclusion: 3 he-mri of the lung offers a new approach to regional determination of ventilated lung volume. the volunteers show good correlation and high consistence of the absolute values between 3 he-mri and pft. the patients with sltx show good correlation and the possibility to measure the individual contribution of the graft in comparison to the native lung. he-mri (300 ml hyperpolarized 3 he gas; flash 2d, tr/te 11/4.2 ms, fa < 10°, slice thickness 10 mm, pixel size 4.2 × 2.7 mm, coronal) were performed. hrct was evaluated for 3 main lesions: airway disease (bronchial wall thickening, air trapping), emphysema, and fibrosis. 3 he mri was assessed for three main ventilation defects. functional compromise was scored on a 4-point scale independently for hrct and 3 he-mri by two readers and compared to forced expiratory volume in 1 s (fev1) and residual volume (rv). results: 63 ventilation defects were depicted in total: 70 % in both, 18 % only on 3 he-mri and 12 % only on hrct. mean hrct score was 2.7, mean 3 he-mri score 2.5, mean fev1 was 60 %. there was no relevant correlation between hrct and fev1 (r = 0.3), but between 3 he-mri and fev1 (r = 0.7), exhibiting significant differences between scores 2 and 3 (p < 0.05) as well as 3 and 4 (p < 0.05). there was no relevant correlation between hrct and rv (r = 0.1), but for 3 he-mri (r = 0.6). conclusion: in comparison to paired in-and expiratory hrct, 3 he-mri has a higher sensitivity in the detection of ventilation defects and correlates better with lft. thus, functional information is predicted better using 3 he-mri. no single lung function test index correlates well with the severity of emphysema. the aim was to develop a composite pulmonary function score that best reflects the ct quantification of emphysema. material and methods: the hrct scans of 97 cases of emphysema were scored objectively using a density mask. the following standard pulmonary function tests (pft): dlco, kco, fev1, total lung capacity (tlc), residual volume (rv) were recorded. stepwise regression was used to identify the combination of pfts that best fitted the hrct score results: significant negative correlations were found between the hrct emphysema scores and dlco, kco and fev1 (r 2 = 0.39, r 2 = 0.36 and r 2 = 0.42 respectively) and positive correlations with tlc and rv (r 2 = 0.29 and r 2 = 0.37 respectively). a composite score representing the best fit combination of pfts against the ct emphysema score was: 24. purpose: this study evaluated the relationships between high resolution computed tomography (hrct) morphologic quantification of bronchiectasis and clinical and lung function parameters. materials and methods: 60 chinese with steady state bronchiectasis underwent thoracic hrct scan and full lung function tests. exacerbation frequency/year and 24 h sputum volume were determined. extent of bronchiectasis, bronchial wall thickening, and presence of small airway abnormalities and mosaic attenuation were evaluated in each lobe, including lingula. relationships between lung function, sputum volume, clinical exacerbation and hrct parameters were analysed. results: exacerbation frequency was associated with bronchial wall thickening (r = 0.32, p = 0.03); 24 h sputum volume with bronchial wall thickening, small airway abnormalities (r = 0.30, 0.39, p < 0.05), and fev11, fev1/fvc and fef25 -75(r = -0.33, -0.29, -0.32; p < 0.05). extent of bronchiectasis, bronchial wall thickening and mosaic attenuation were respectively related to fev11 (r = -0.43 to -0.60 p < 0.001), fef25 -75(r = -0.38 to -0.57; p < 0.001), fvc (r = -0.36 to -0.46, p < 0.01), and fev11/fvc (r = -0.31 to -0.49, p < 0.01). after multiple regression bronchial wall thickening remained a significant determinant of airflow obstruction, while small airway abnormalities remained associated with 24 h sputum volume. women in general had milder disease than men, but showed more hrct-functional correlations. conclusion: this study has established a link between morphologic hcrt parameters and clinical activity, and emphasised the role of bwt in bronchiectasis. there are gender differences in bronchiectasis with respect to disease severity and sensitivity to hrct evaluation. morphology and lung function in healthy smoking and non-smoking men: a 5-year follow-up study j.d. vikgren, m. boijsen, b. bake, u. tylén; gothenburg/se purpose: there is demand for a reliable method for early detection of smoking induced disease. healthy smokers can present with or without normal lung function. having abnormal lung function, the smokers present with airway obstruction and/or emphysema. a possible way to elucidate early smoking induced changes, bronchiolitis, could be analysis of e/i quotient. material and methods: our study was a follow-up study. subjects were recruited from the randomised epidemiological study "men born 1933 in göteborg". hrct and lung function tests were performed with a five-year interval. smokers were subdivided according to presence of emphysema or not at the beginning of the study. computer calculation of e/i quotient was performed, i.e. quotient between cross-sectional area and mean attenuation values in inspiration and expiration. visual quantitation of emphysema and air trapping was evaluated by consensus. results: at follow-up there was a significant increase of the e/i quotient for mean attenuation for smokers with and without emphysema, but also for non-smokers. e/i quotient for cross-sectional area showed a significant decrease at follow-up c d e f 276 only for smokers without emphysema. there was a significant progression in emphysema for smokers. analysis of the relation to lung function parameters as fev 1.0, fvc, co-uptake and n2-test are in progress. conclusion: there is a progress of emphysema with continued smoking. an increased e/i quotient has been interpreted as obstruction, but the fact that the e/i quotient increases in smokers as well as non-smokers requires further analysis. mediastinal lymphadenopathy in left heart failure: correlation of ct abnormalities with clinical and echocardiographic findings v. chabbert 1 , g. canevet 1 , g. philippe 1 , p. otal 1 , v. delannoy 2 , f.g. joffre 1 , m. rémy-jardin 1 ; 1 toulouse/fr, 2 lille/fr purpose: to evaluate the frequency of mediastinal lymphadenopathy due to left heart failure on computed tomography (ct) scans in correlation with clinical and echocardiographic findings. materials and methods: 31 consecutive patients (mean age, 69 years) with left heart failure in a subacute phase were included in a prospective study between september 2000 and august 2001. ct examinations (hrct and spiral ct scans) and transthoracic echocardiography were performed within 24 hours after presentation. ct follow-up were obtained within 8 days after initiation of medical treatment. results: at presentation, dyspnea was graded as type iv (39 %), iii (39 %), ii (19 %) and i (3 %). mean ejection fraction was 39 %. enlarged mediastinal lymph nodes were seen in 42 % of cases, heterogeneous in 23 %, with a hazy perilymphadenopathy fat in 38.5 % of cases. all patients had pleuroparenchymal abnormalities due to left heart failure. peribronchovascular thickening, septal thickening, fissures thickening, ground-glass attenuation, condensations, micronodules and pleural effusions were present respectively in 20 %, 71 %, 71 %, 74 %, 16 %, 16 % and 74 % of cases. after treatment, dyspnea was graded as type iii (16 %), ii (61 %) and i (23 %). lymphadenopathy decreased in size in 38.5 %, was stable in 38.5 %, disappeared in 23 %, and weas heterogeneous in 20 %, fat abnormalities disappeared in all cases. pleuroparenchymal abnormalities decreased in 74 %, disappeared in 19.5 % and were stable in 6.5 %. we examined 23 consecutive patients (138 lung lobes) referred for the assessment of possible airways disease using multislice ct. the thorax was scanned contiguously at 1 mm slice thickness and this was reconstructed at 1 mm slice thickness (lung windows utilising high spatial frequency algorithm) in the axial (10 mm apart), sagittal (4 per lung) and coronal (6) plane. pedal wheel reconstructions were also performed. axial images were assessed by 2 chest radiologists with and without the help of mpr at two separate occasions. the presence of bronchiectasis, emphysema and bronchiolitis in each lobe was documented on a confidence scale of 0 -3 (0 no disease, 3 definite disease). result: there was no significant difference (p = 0.280) in the mean confidence in the diagnosis of airways disease with the help of mpr [observer a (1.98 axial, 2.01 mpr); observer b (2.88 axial, 2.71 mpr)]. in addition, weighted kappa showed no improvement in inter-observer agreement (kappa = 0.566 for axial; kappa = 0.530 for mpr). isolated cases wherein mpr was beneficial were noted, particularly in patients with bronchiectasis (3 patients). due to the small number, this did not reach statistical significance. conclusion: our results did not demonstrate a significant increase in confidence in the diagnosis of airways disease. mpr was beneficial in a few selected cases of bronchiectasis. methods and materials: 40 consecutive patients were randomized into 2 groups of 20 patients each. group 1 underwent ssct with a standard protocol for the study of the airways and group 2 underwent msct with the following protocol: 4 × 1 mm slice collimation, pitch 7 (1.75 m), 1 mm slice width, 0.8 mm reconstruction increment. two radiologists evaluated by consensus the axial images and vb reconstructions in order to assess the degree of visualization of the bronchial tree according to a three point scale (grade 0, 1 and 2). results: on axial images, msct yielded better results in the evaluation of the segmental bronchi of the middle lobe (32 bronchi rated as grade 2 by msct vs 16 by ssct; p < 0.05), of the anterior segmental bronchus of the left upper lobe (19 rated as grade 2 by msct vs 10 by ssct; p < 0.05) and of inferior segmental bronchus of the lingula (18 rated as grade 2 by msct vs 8 by ssct; p < 0.05). evaluation of lobar and segmental bronchi was always possible when vb was obtained from msct data sets whereas 53/280 bronchi were scored 0 when vb was obtained from ssct. subsegmental bronchi could be better evaluated with msct than ssct either on axial images or vb (p < 0.05). conclusion: msct with 1 mm collimation significantly improves the visualization of segmental and subsegmental bronchi and is particularly useful for oblique oriented bronchi. methods: over a one year period 96 patients had by pct. 10 patients were directly examined by pct on admission day on icu. for this purpose a special interventional suite was prepared directly on icu. in this suite radiological modalities like radiography, sonography and portable angiography can be performed under intensive care monitoring and therapy. ct examinations are even possible by using pct in this special suite. on day of admission patients underwent extensive evaluation using modalities of this special interventional suite. results: in two cases a bedside ct procedure was performed the others were performed in the intervention suite on icu. in 80 % of the cases a final diagnosis could be determined by ct examination. in 40 % ct findings led to follow up evaluations by pct. even 40 % of ct examinations led to direct therapeutic consequences such as surgical intervention. 60 % of the ct indications were assessed as necessary or essential by an experienced icu physician. half of the follow up examinations had also direct therapeutic consequences. monday b results: using the sense technique the temporal resolution and spatial coverage could be increased from 12 slices per 1.7 s up to 20 slices per 1.4 s. as a consequence of the increase in k-space velocity using sense the typical susceptibility artifacts of epi sequences at the skull base were almost negligible. the s/n is strongly influenced by the specific coil arrangement and the actual number of profiles in k-space. for the applied double loop coil and a sense factor of two the s/n equals that of the standard scan (head coil) in superficial temporal regions and is reduced by a factor of 0.65 to 0.8 in central and occipital regions. however, the increased temporal resolution compensated for the decrease in s/n and the calculated parameter maps were always of good diagnostic quality. with a dedicated coil the sense technique allows substantial improvement in perfusion imaging. diffusion weighted mr imaging in the early diagnosis of periventricular leukomalacia a. bozzao, f. garaci, s. marziali, r. floris, g. simonetti; rome/it -presented by f. fasoli; rome/it purpose: the use of mr diffusion weighted imaging in detecting hypoxic-ischemic encephalopathy (hie) in neonates is still controversial. moreover few reports concern pre-term infants with possible periventricular leukomalacia (pvl). we examined the ability of this technique to detect cerebral changes of acute pvl. methods: fifteen mr examinations were performed in eleven preterm infants. imaging comprised conventional and diffusion-weighted (dw) sequences. conventional, dw sequences and apparent diffusion coefficient (adc) maps as well as us obtained in the acute phase were compared. all the neonates underwent us follow-up 6 and 12 months after delivery, those with suspect pvl also underwent mri follow-up at two months and one year. qualitative and quantitative evaluations were performed to assess the presence of dwi changes compatible with pvl. results: dwi showed signal hyperintensity associated with decreased adc values in 3 subjects (27 %); in these patients conventional mri sequences were interpreted as normal and us (performed at the same time) as doubtful in 2 and compatible with pvl in one. mri and us follow-up confirmed severe damage in all these patients. in one neonate hemorrhages involving the germinative matrix were identified at mri and us without signs of pvl. follow-up mri and us confirmed the absence of pvl 3 months later. in eight neonates mri was considered normal. in these subjects us follow-up confirmed no signs of pvl. apart from the well known image artefacts time consuming reconstruction periods often inhibit continuing data acquisition, particularly if sequences are used with more than one acquisition or with built-in postprocessing capabilities to automatically generate adc-maps. by using these sequences as the last sequence in the whole examination prior to the start of the subsequent examination of the next patient this time delay can be reduced. the aim of this prospective study was to find out if there is any diagnostically significant difference between acquisition of epi-dwi images before and after intravenous application of gd-dtpa. in 203 patients a epi-dwi sequence was used (tr/te: 4000/100, 19 slices, 6 mm slice thickness, 230 fov) both as the first (before i.v. cm) and the last sequence after cm. the mr indications were ischemic stroke, encephalitis, multiple sclerosis and brain tumor with and without disturbed blood brain barrier. the mri images were rated by two blinded, neuroradiologically experienced radiologists. results: intravenous cm had no influence on the diagnostic information of the images. there were no significant signal differences even in lesions with pronounced disturbances of the blood brain barrier. conclusion: it is possible to use epi-dwi after cm application without loss of clinical information. the proposed approach allows to reduce examination times. brain perfusion studies with trans cranial colour doppler using ultrasound contrast media g. salvaggio, g. caruso, t.v. bartolotta, a. scisca, g. caputo, r. lagalla, a.e. cardinale; palermo/it purpose: to evaluate the intracranial micro circulation using trans-cranial color doppler (tccd) with a contrast medium (levovist). material and methods: 25 patients, (age range: 41 -73 years; mean age 64 years) affected by mellitus diabetes were selected. 20 healty volunteers (mean age: 63 years) acted as control. the examinations were performed using an atl hdi 5000 ultrasound unit provided with a phased array 3.25 mhz probe. mechanical index was calibrated to high value in order to obtain microbubbles rupture under ultrasound beam exposition. after positioning of an operator defined region of interest, intensity/time curves related to the parenchymal micro circulation were plotted, and the areas under the curves were calculated. results: in all controls, the areas under the curve showed a mean value greater than 0.05 that we considered as cut-off value of normal perfusion. on the other hand, the diabetic patients showed a lower mean value, to indicate a micro circulation disease. conclusion: thanks to software improvements and ever more effective algorithms, contrast-enhanced tccd is able to provide information on brain perfusion non invasively and at low cost. purpose: to present our initial clinical experience with a one-piece design 8-channel neuro-array coil for mri of the brain. methods and materials: mri examinations of the brain were performed in 5 normal volunteers using an 8-channel parallel acquisition technique (pat) optimized neuro-array head coil (mri devices corporation, wisconsin, usa), and a standard circularly-polarized (cp) volume head coil. examinations were performed on a 1.5 t superconducting magnet (sonata, siemens ag, germany). signal-to-noise ratios were calculated and compared for the two coils in various anatomical locations including peripheral (cortex, subcortical white matter, etc.) and central (e.g. corpus callosum, sella, brainstem, etc.) parts of the brain. results: the 8-channel neuro-array coil provided excellent high-resolution imaging with anatomical coverage of the entire head. in the peripheral portions of the brain, snr was 50 -100 % superior to that obtained with a standard cp volume head coil. in the deeper parts of the brain snr was at least equally good. the neuro-array coil is pat optimized and allows the use of sense/smash type sequences with flexible phase encoding. conclusion: the 8-channel neuro-array head coil provided excellent high-resolution mri of the entire brain. snr was markedly improved, especially in the peripheral parts of the brain. the pat optimized imaging capablities can be used to decrease imaging time, improve spatial resolution, or a combination of both. genetic approach to ct appearance of brain ischemic stroke l. cyrylowski, a. ciechanowicz, a. fabian, i. goracy; szczecin/pl purpose: there is evidence that an allelic variation in the angiotensin converting enzyme (ace) gene may confer an increased risk of cardiovascular disease. the aim of our study was to evaluate the relationship between the ace insertion/deletion (i/d) polymorphism and the prevalence of brain ischemic stroke as well as between the polymorphism and ct type of ischemic stroke. materials and methods: the ace i/d genotype was identified by polymerase chain reaction in 46 patients (26 males and 20 females) with brain ischemic stroke, and in 100 controls (50 males and 50 females). to assess the polymorphism frequencies, statistical analysis was performed. results: the d/d polymorphism frequency was significantly higher in the stroke group than in the controls (32.6 % vs. 13 %; p < 0.01). the i/i and i/d polymorphism frequencies were similar in both groups (26.1 % vs. 30 %, and 41.3 % vs. 57 %, respectively; p > 0.05). conclusion: a positive association between the ace d/d allele and ischemic stroke was found in our study. the ace d/d allele may be an independent risk factor for the development of cerebrovascular disease. . the motorized c-arm provides fluoroscopic images during a 190° orbital rotation computing a 119 mm data cube. from these 3d data sets mpr reconstructions were obtained. all images were evaluated by four independent readers for the detection and extend of fracture lines. all fractures were classified according to the müller ao-classification. to confirm the results, the specimens were finally surgically dissected. results: 93 % of the fractures were detected with cr, 97 % with iso-c-3d and 100 % with ct. differences between cr and ct were significant (p = 0.046), not significant between iso-c-3d and ct (p = 0.157). with cr 27 % of the fractures were correctly classified, 96 % with iso-c-3d and 100 % with ct. there was again no significant difference between ct and iso-c-3d (p = 0.066), but between ct and cr (p < 0.001). the preliminary results suggest a remarkable efficient detection of tibial joint fractures with the iso-c-3d. the evaluation of fractures with the iso-c-3d is better than with cr alone and comparable to that of ct-scans. even if image quality is definitely inferior to ct, iso-c-3d may be useful in planning operative reconstructions and evaluating surgical results in orthopaedic surgery of the limbs. dynamic ultrasonography in evaluation of muscular trauma a.k. nath, r. bouras; muscat/om purpose: role of dynamic ultrasonography in muscular trauma. methods and materials: 50 male football players (aged 20 to 30 years) presenting with clinical muscular trauma in the thigh and calf region were evaluated in this study. dynamic ultrasonography of both the affected and contralateral normal region, with toshiba power vision 6000 ultrasound equipment using 7.5 mhz phased array linear transducer, in sagittal, coronal and angulated axis was performed, both without contraction and with contraction of the muscles. needle aspiration of suspected hematomas was performed for diagnosis and treatment. all muscles tears and hematomas were studied and followed up after 72 hours, until complete healing. results: 46 of the total 50 patients had muscle tears and/or hematomos in thigh and calf region. 4 pateints had no abnormality. 32 patients had clear-cut muscle tears appearing as echogenic retracted portions surrounded by hematomas ranging from highly reflective mass to complete echo poor areas were observed on follow up. the remaining 14 patients had partial tears. healed tears appeared as highly reflective scar tissue. (1) ultrasonography is very useful in diagnosis, management, and followup of muscle tears and hematomas. (2) dynamic ultrasonography is essential for diagnosis of partial tears. ultrasound and color doppler imaging of thrombosis of gemellary veins in patients with "tennis leg" lesion m. dahmane 1 , c. martinoli 1 , s. bianchi 2 , f. zandrino 3 , f. monetti 1 , a. beghello 1 , s. paltenghi 1 , l.e. derchi 1 ; 1 genova/it, 2 geneve/ch, 3 alessandria/it purpose: to assess with ultrasonography (us) and color doppler imaging (cd) the association between rupture of distal myotendineous junction of the medial gastrocnemius ("tennis leg" lesion) and thrombosis of gemellary veins. materials and methods: 32 consecutive patients with suspected "tennis leg" lesion were prospectively examined with 12 -5 mhz us and cd to confirm the diagnosis and evaluate the possible association with thrombosis of gemellary veins. the us study had to be extended to the most proximal portion of the leg, to image the veins up to the popliteal; both compression evaluation and a cd exam were obtained to confirm patency. results: 30/32 patients had typical "tennis leg" lesions; 1 had a ruptured baker cyst; 1 had proximal rupture of gastrocnemius muscle. an associated thrombosis of the gemellary veins was detected in 6 cases (18 %); none had extension to the popliteal vein. conclusions: presence of associated thrombosis of the gemellary veins has to be considered in patients with "tennis leg" lesion, and the us examination technique has to be changed accordingly. in this study, we report our experience regarding the usefulness of mri in the evaluation of traumatic and microtraumatic bone pathology. we performed mri examinations in 4324 patients with knee pain and a negative plain film examination where a post traumatic or non traumatic pathology at the level of the joints (knee, ankle, wrist, elbow and hip) was suspected. mri examinations were performed with a low field dedicated unit for the study of limbs (e-scan and artoscan esaote) and in some cases with an high field mri unit (vision plus siemens). we used se t1 and turbo-t2 sequences, ge and stir. in some patients we administered i.v. contrast media. in 964 cases we demonstrated the presence of post traumatic bone alterations or bone alterations not related to trauma that were not visible on conventional radiological examinations. in 112 the bone alteration was the most important finding. in 42 patients, a follow-up mri after 1 month allowed correlation between the evolution of the clinical symptoms and the changes in the appearance of the bone injury. in 75 patients, it was very useful to correlate the changes in the bone appearance with the clinical outcome. conclusions: in our experience mri has been very important in the evaluation of traumatic, post traumatic or non traumatic bone pathology and for a correct therapeutic assessment of the patient. purpose: to compare bone bruise patterns identified at mri incurred by impaction, distraction and shear injuries correlated with microtome sections and histopathology. methods: freshly harvested sheep cadaveric limbs were imaged on a 1.5 t philips mri intera scanner following exposure to impaction, distraction and shear forces. each limb was imaged using se t1 and stir sequences. following imaging, the cadaveric limb was sectioned using a microtome and pattern of trabecular damage correlated with identified bone bruise at mri. results: impaction forces produce poorly marginated globular bone bruises with extensive concertina type trabecular damage. distraction forces produce localised bone bruises in a linear pattern perpendicular to the axis of distraction with localised trabecular disruption, attenuation and stretching. shear injuries produce linear bone bruises obliquely oriented with localised linear disruption of trabeculae on microtome sections. conclusion: bone bruise patterns are dictated by mechanism. mri appearances reflect the underlying extent of trabecular damage at histopathology. to determine the diagnostic accuracy of contrast-enhanced, three-dimensional panoramic-table-mr angiography in the assessment of pelvic and peripheral arteries compared with conventional digital subtraction angiography as the standard of reference. in 169 patients with suspected peripheral arterial vascular disease, both conventional digital subtraction angiography and three-dimensional mr angiography using an automatic tracking technique were performed. in a prospective blinded analysis, each vascular segment (29 segments per patient) was evaluated for the presence of obstructive stenosis, graded as normal (0 -10 %), mild (10 -50 %), severe (50 -99 %), or occlusion (100 %). results: obstructive lesions were identified and graded correctly with mr angiography. overall sensitivity and specificity for the detection of hemodynamically significant disease (severe stenosis) on panoramic mr angiographic images were 93 % and 97.7 %, and for the detection of occlusive disease were 95 % and 99.8 %, respectively. the diagnostic accuracy of contrast-enhanced, three-dimensional panoramicpurpose: with 3d-mr-angiography gaining more acceptance for assessing the arterial system, the amount of data produced requires new tools for visualization of the datasets. recently, a new volume rendering method, "stereo viewing", has become available. the purpose of this study was to assess the potential diagnostic gain by "stereo viewing" in comparison to combined mip and mpr for various vascular territories. over an 8-month period 3d-mra was performed in 40 patients. mras were obtained of the carotid, pulmonary, renal and pelvic arterial systems. imaging was performed on a 1.5 t mr scanner (sonata®, siemens). image sets were analyzed on a workstation (virtuoso®, siemens, germany), firstly based on a combination of mip and mprs, and a second time based on mip, mprs and "stereo viewing". the data sets were analyzed using a 5-point confidence scale ranging from 'stenosis/occlusion definitely present' to 'stenosis/occlusion definitely not present'. in addition to the level of confidence, the time required to reach the diagnosis was documented. results: the addition of "stereo viewing" led to a significant increase in diagnostic confidence regarding the presence/absence of stenosis/occlusion in all anatomic regions, evidenced by a greater area under the roc curve (p < 0.05). "stereo viewing" prolonged the analysis process by an average of 10 %. conclusion: "stereo viewing" is an effective and accurate tool for assessing complex 3d-mra datasets as it provides more diagnostic confidence compared to the combination of common post-processing techniques (mip + mpr). the added time needed for "stereo viewing" results in enhanced diagnostic confidence. purpose: image quality of dsa of the hands is still superior to ce-mra due to the lower spatial resolution of ce-mra which is limited by the passage time of the contrast bolus through the arteries. the aim of this study was to demonstrate that temporary blood flow interruption with an inflated blood pressure cuff during the arterial first-pass can significantly increase imaging time and spatial resolution. materials and methods: 8 volunteers and 3 raynaud patients were examined with tac-cemra on a 1.5 t mr machine (philips intera). a blood pressure cuff was placed around the upper arm. after timing (bolustrac), 5 ml iv. gadolinium-dota was administered and tac-cemra was performed. after cuff inflation at 200 mmhg during the arterial phase, a 3d-gradient echo imaging sequence (tr 4.7 ms, te1.6, flip 35°, matrix 512 × 1024, fov 300, 0.7 mm-partitions) was acquired. slopes of histograms were measured perpendicular to the radial and one arch artery and cnr calculated. results were compared to a classic ce-mra study in all volunteers. results: compared to the classic first-pass mra with pixel dimensions of 1.17 × 0.59 mm, the use of tac-cemra allowed a significant increase in spatial resolution with voxels down to 0.59 × 0.29 mm. this gain did not impede the cnr which was not statistically significantly different ( a c d e f 282 materials and methods: 4 patients underwent pta of a single distal sfa stenosis. lesion length was < 1 cm. hr-mri (magnetom vision, siemens) was performed within 16 hours after the intervention using axial t1-w, fat-saturated contrast enhanced t1-w, t2-w and 3d-tof sequences. maximum matrix size was 320 × 512, minimum voxel size 0.49 × 0.49 × 2.0 mm. contrast enhanced mr angiography was employed for assignment of exact matching sites. ivus (3.5 f, 40 mhz) images were recorded with a standardized motorized pullback system (pullback speed 1.0 mm/s). quantitative analysis for each segment included minimum and maximum luminal diameter and cross sectional lumen area. morphologic analysis included the absence or presence and extent of dissection. results: precise pre-and post-pta assignment of 16 segments was successfully carried out according to our protocol. the lumen increase measured 100 % to 400 % with a remarkable agreement between ivus and hr-mri. post pta correlation for cross sectional lumen area (r = 0.90) was better compared to pre pta analysis (r = 0.80). hr-mri detected 7/7 ivus proven dissections while digital subtraction angiography failed to demonstrate 4/7 dissections. conclusion: our preliminary findings suggest that hr-mri has a high potential for non-invasive in-vivo assessment of quantitative and morphologic changes in atherosclerotic sfa lesions following angioplasty. the method for quantitatively determining contrast medium (cm) concentration by measuring the t2*-effect (brady, 1990 ) was adapted to t2w liver-mri. the cm used was an indium-doted spio. three groups of study animals (liver cirrhosis, hepatitis, fatty liver) and a control group were investigated (15 rats/group). se and gre imaging was performed (n = 6/group, 7.5 µmol/kg bw); 3 rats received no cm and 3 animals each 15 and 25 µmol/kg. t2-relaxation time of all livers were measured ex vivo by relaxometry, and cm concentrations were determined. q-values representing the phagocytosed cm were computed from the pre-and postcontrast si on both the se and gre images. spearman's correlation coefficient between cm concentrations and t2-relaxation rates was significant in the control group at 0.506 (p < 0.05). in the 3 study groups, pearson's correlation coefficient (pcc) between cm and t2-relaxation rates was significant, ranging from 0.627 -0.717 (p < 0.05) -a positive correlation between cm and t2-relaxation rates in all groups. pcc between q-values and cm concentrations was significant at 0.767 (se) and 0.587 (gre) (p < 0.05) -positive correlation between the cm and q-values. the mann-whitney test yielded a significant difference in the qse values between the liver cirrhosis and fatty liver groups compared with controls (p < 0.05). regarding qgre values, a significant difference was identified only for the fatty liver group versus controls (p < 0.05). the animal experiments suggest that quantitative determination of phagocytic capacity as a functional parameter using contrast-enhanced mri has a potential for differentiating diffuse liver disease from healthy livers. results: in the detection of focal liver lesions, unenhanced plus dynamic plus late phase enhanced mri was more accurate than unenhanced plus dynamic and than unenhanced plus late phase enhanced examinations, which were more accurate than unenhanced imaging. the accuracy and observer confidence in the characterization of focal liver lesions were higher when dynamic examination was considered. methods and materials: 45 patients were prospectively investigated using a 1.5 t magnetom (siemens symphony; philips acs nt). the sequence protocol included t2w tse and t1w se/gre scans using unenhanced and spio-enhanced studies (endorem: 15 µmol/kg bw). diagnostic criteria unenhanced were: homogeneous isointensity/mild hyperintensity (t2w); homogeneous iso-/mild hypointensity (t1w); scar detection. the uptake of the spio-particels was calculated comparing contrast-enhanced with unenhanced scans. results: the standard of reference was histopathology providing surgery (liver resection) or needle biopsy. as a second option gadolinium-enhanced mri was accepted within a timeframe of 6 month. in the resection group sensitivity was 95.2 % with a specificity of 95.8 %. signal intensity was homogeneous in t1-and t2w unenhanced sequences and similar to surrounding parenchyma in 37 out of 44 cases (group 1). in eight cases the lesion was heterogeneous hyperintense in t2w and hypointense in t1w scans unenhanced (group 2). group 1 documented a significant signal intensity loss using spio-enhanced t2w sequences compared with unenhanced prtocols. for group 2 a statistical significant decreased uptake of spio-particles was documented. histopathology revealed high degree of fibrotic changes (group2). using spio-enhanced mri detection and delineation of central scar tissue was increased compared to unenhanced mri. conclusion: spio-enhanced mri is an effective imaging tool for the diagnosis of fnh nodules providing morphological and functional details sufficient for characterization. purpose: capillary and cavernous hemangiomas represent the most frequent benign lesions of the liver; atypical features due to complications may simulate different lesions; the rare occurrence of other vascular histotypes makes the differential diagnosis even harder. the aim of our study was to describe their imaging features and discuss their differential diagnosis. we reviewed ct, mr images in 152 patients. ct protocol included: 5 mm collimation, pitch 1.5 injection of iodinated contrast medium in the arterial, porto-venous and delayed phases. mr protocol included 5 -8 mm slice thickness, t1, t2 weighted sequences and post-gd evaluation. we evaluated the morpho-structural appearance and the contrast enhancement of the lesions. the comparison between different imaging modalities, the dynamic of contrast enhancement, the follow-up, the bioptic and surgical specimens allowed for the correct diagnosis in all the cases. results: we identified the typical patterns of capillary hemangioma (50), cavernous hemangioma (65), giant hemangioma (10). in 5 case we demonstrated intratumoral hemorrhage, in 7 cases prevalent jaline and/or stromal components, in 6 cases calcifications. we also found 2 hemangioendotheliomas (1 single, 1 multiple), 4 hemangiosarcomas (2 showing extravisceral growth, 3 multinodular, 1 single), 3 kaposi's sarcomas (2 showing peri-portal infiltration, 1 multinodular). conclusions: ct and mr demonstrate an elective role in the identification and often in the characterization of vascular tumors of the liver in adults. the presence of complications is also well depicted. mr gives important information regarding the structure of the lesions and in particular of the hemorrhagic components. methods: 12 children (5 m/7 f, mean age 14.3 ± 2.1 a) suffering from marfan's syndrome were consecutively chosen from the department of pediatric genetics. bone ultrasound attenuation (bua) and speed of sound (sos) were calculated by sahara (hologic, usa). the standard-deviation-scores (sds) for both parameters were calculated using regional normative data (3299 children). and porosity index (pi) were estimated automatically from radiogrammetric measurements and bone texture analysis of the three middle metacarpal bones. for statistical analysis each case was pair-matched to a sex-and age-related healthy control. the mean bmd was 0.53 ± 0.04 g/cm 2 in the marfan group versus 0.53 ± 0.08 g/cm 2 in the control group. only pi showed a significant difference: 1.99 ± 1.14 versus 5.14 ± 2.22 (p < 0.05). conclusion: bmd was very similar in our patients with marfan syndrome compared to the reference population. statistically significant differences were only observed for the porosity index. data in the literature concerning osteoporosis in marfan patients is ambiguous. our findings may indicate a specific textural disturbance in the bones of marfan patients which does not result in overall altered bone density. there are potential limitations due to specific anatomic properties of the metacarpal bones in marfan syndrome. radiogrammetric based bone densitometry seems to be of promising clinical value in pediatric patients receiving x-rays of the wrist and hand routinely for determination of skeletal maturity. this new technology might be able to give additional information and reduce radiation exposure. gender-specific standard-deviation-scores (sds) were calculated using age, height and weight matched regional normative data (3299 healthy children). follow-up was performed in 9 children during bisphosphonate-treatment over a period of 2 years. results: 20 patients had oi type i, 6 had oi type iv. age-matched bua values were < −2 sd in 9/20 patients, < −1 sd in 6/20 patients and normal in the remaining children with type 1. 13/20 patients had sos values below −2 sd, 4/20 below −1 sd and 3 were within the normal range. no bua and sos-values below −2 sd were observed in type 4; and 2/6 patients had values below −1 sd. 1/6 patients had sos values < −1 sd, the others were within the normal range. mean bone values differences were significant: −1.82 sd/−0.10 sd (bua) and −2.34 sd/0.02 sd (sos). there was a strong correlation of bua and sos (r = 0.90, p < 0.01). 4/9 children showed a significant increase of bua and sos (more than 1 sd), 1/9 a slight increase in values during follow-up treatment, 4 had no change. in all treated cases fracture rates were significantly reduced. conclusion: ultrasound based bone densitometry may be of clinical value in children suffering from oi. oi type i causes severe disturbances of bone mineralization in contrast to type 4. ultrasound based bone densitometry of the os calcis measured on asthmatic children using regional normative data a. (sos)). bua/sos correlation was significant (r = 0.62; p < 0.01). conclusion: bone densitometry of the os calcis using ultrasound appears promising in detecting disturbances of bone mineralisation in children treated with low dose topical steroids. sos was more sensitive in detection of bone mineralisation in comparison to bua. girls seemed to be osteopenic more often. in contrast to steroid intake, asthma severity does not correlate significantly with bone mineralisation disturbances. the use of mri in the evaluation of paediatric wrist trauma k. johnson, a. page, f. haigh; birmingham/gb traumatic wrist injury in children is common but detection of fractures and other injuries can be difficult. we have evaluated the role of mr imaging in the assessment of paediatric wrist trauma. 99 children with a history of wrist trauma in whom the there was either a discrepancy in the clinical assessment and the plain radiographic findings or normal radiographs and persisting symptoms underwent an mri examination of the wrist. all mri examinations were performed within 2 weeks of the initial trauma, 76 % within 6 days. 101 mri examinations were performed (2 children has repeat examinations). 34 (34 %) patients with normal plain radiographs had fractures detected on mr imaging (59 % in the carpus and 41 % in the radius and ulna). in 20 patients there was a discrepancy between the plain radiographic findings and the clinical assessment and in 12 cases (60 %) mri detected further occult fractures and soft tissue injuries. no patient with a negative mri examination has represented with a complication of the initial injury, indicating that mri has a 100 % negative predictive value for wrist injuries. mr examination of the wrist in children significantly alters management. mr imaging detects radiographically and clinically occult fractures and soft tissue injuries. the technique is well tolerated by children. methods and materials: 21 patients (age 3 -16 years; m = 11, f = 10) suffering from juvenile aseptic osteonecrosis (legg-calvé-perthes disease), underwent mri in a 0.5 t mr-scanner (gyroscan t5 nt, philips, eindhoven, the netherlands) using a fat-suppressed stir sequence, t2-weighted turbo spin-echo and t1-weighted spin-echo sequences. postcontrast t1-weighted sequences were performed in each patient after administration of 0.1 mmol gadolinium/kg bodyweight (magnevist, schering, berlin, germany). all examinations were retrospectively assessed by two paediatric radiologists, evaluating the signal patterns of the femoral head and the metaphysis in each sequence in consensus. the mr features and the diagnostic accuracy were analyzed for the different sequences and compared to conventional x-ray. monday b results: based on the fat-suppressed and the contrast enhanced t1-weighted images, six different signal patterns were differentiated within the femoral head as well as three different signal patterns being identified within the metaphysis. combinations of separate signal patterns within one thigh were a common finding, representing different stages of necrosis and reparation. for the differentiation of viable and necrotic osseous fragments, the administration of contrast material was mandatory. the combination of fat-suppressed and contrast enhanced t1weighted images allows distinct staging of juvenile aseptic necrosis. a combination of these imaging modalities permits an efficient surveillance of the legg-calvé-perthes disease. the purpose: saethre-chotzen syndrome is an autosomal dominant syndrome with craniostenosis and syndactylies. the purpose was to evaluate the variability of anomalies on hand and feet radiographs of patients with genetically proven saethre-chotzen syndrome or muenke-type coronal synostosis. we evaluated radiographs of the hands in 31 patients, 25 with saethre-chotzen syndrome related to twist gene mutations and 6 with muenke-type mutation. x-rays of the feet were available in 27 cases. the age range was between 1 month and 36 years. radiographs were evaluated by 2 radiologists in respect of morphological anomalies. results: we found a bone age delay in 10/27 patients, whose epiphyses were not fused. a brachyphalangy was noted in 22/31 patients, clinodactyly in 18/31. partial syndactyly occured in 16 cases, involving the soft tissues of the 2 nd web space and additionally the 3 rd in 3 cases. 7/31 patients presented with partial carpal fusion, affecting the trapezoid and the trapezium in 5. coned epiphyses were present in 16/16 patients. other anomalies included a duplicated distal phalanx of the hallux (n = 9), a triangular deformity of the epiphyses of the distal phalanx of the hallux (n = 10), partial syndactyly (n = 11) and brachyphalangy (n = 10). conclusion: saethre-chotzen syndrome and muenke-type mutation have a variety of morphological anomalies of the hand and foot in which skeletal anomalies such as brachyphalangy and syndactyly are non-specific signs. different patterns of anomalies between both are a duplicated distal end and triangular shaped epiphysis of the hallux, which were only detected in patients with saethre-chotzen syndrome. purpose: saethre-chotzen syndrome (scs) and muenke-type mutation fgfr3 pro250arg (mtm) are both complex syndromes with skeletal anomalies such as craniosynostosis, syndactylism and cervical spine abnormalities. in this study we analysed the variable cervical spine abnormalities identified on radiographs for specific features in patients with genetically proven scs and mtm. methods and materials: cervical spine radiographs of 20 patients (11 female, 9 male; mean age 6 a) were reviewed by to 2 radiologists with experience of skeletal dysplasias. the patient population included 17 patients with scs and 3 patients with mtm. the appearance of the vertebral bodies, the posterior elements including the neural arch and the atlanto-axial joint were assessed. x-rays of the hands and feet were available in 10 patients. results: fusion of vertebral bodies and posterior elements were noted in 3 patients (level c2/3, c3/4 and c5/6). an isolated fusion of posterior elements occurred in 8 patients (level c 1/2 (n = 1), c2/3 (n = 7), c 4/5 (n = 1), c5/6 (n = 2)). 5/10 patients with cervical spine fusion additionally showed carpal and/or tarsal fusion. in 7/20 patients an enlarged vertebral space between c 1/2 was found. a hypoplastic neural arch was detected in 5 patients and a long spinous process at c7 in a single case. the atlanto-axial joint space had a mean distance of 3.4 mm (range 2 -9 mm). conclusion: scs and mtm display a great variety of cervical anomalies, however the radiological signs are morphologically non-specific. however, cervical vertebral fusion in these patients is often associated with carpal and/or tarsal fusions. ultrasound and mri in children with pyomyositis a. trusen, m. beissert, g. schutz, b. chittka, d. hahn; würzburg/de purpose: pyomyositis is an acute bacterial infection of the skeletal muscles, most often caused by staph. aureus. the purpose of our study was to evaluate the role of ultrasound and mri in the diagnosis of pyomyositis. material and methods: 11 children with myositis were evaluated. the areas involved included the pelvic region (n = 4), the shoulder (n = 4), the thigh (n = 1), the lower leg (n = 1) and the elbow region (n = 1). all patients were examined by ultrasound, 9 of them had mri. symptoms and signs included swelling, pain and inflammation. results: in 7/11 the correct diagnosis was made by ultrasound. in 4 cases, 3 involving the pelvic region the ultrasound diagnosis was initially incorrect. in one child where the diagnosis of a tumour was made both on ultrasound and mri, the diagnosis of a proliferative myositis was subsequently made by histology after ultrasound guided biopsy. the extent of myositis was determined better with mri than ultrasound. in the extremities an abscess was detected by ultrasound, the accompaniing osteomyelitis could only be detected with mri. conclusion: in cases of suspected myositis ultrasound is the primary imaging modality, however, it has a lower sensitivity in the pelvic region. mri is necessary to exclude osteomyelitis and often to determine the exent of inflammation. a c d e f 286 materials and methods: 4 healthy volunteers and 7 patients with tongue tumors underwent videofluoroscopy and real-time mri before and after tumor-resection, using a t1-ffe sequence (tr = 3.2, te = 0.9, fa = 10, slice thickness 1.5 cm) with 6 images per second. images were acquired in the midsagittal plane. swallowing of diluted magnevist-enteral solution, phonation of test words and defined testmovements of the tongue were evaluated. results were analysed by 2 radiologists in comparison to videofluoroscopy as the standard of reference concerning overall quality of the depiction of tongue-movement and deglutition (mri better, equally good or worse), and depiction of the stages of swallowing (from 0 = not recognisable to 3 = very good depiction). results: in all cases, the important stages of swallowing could be successfully depicted with real-time mri. however, quality of videofluoroscopy was considered to be better in all subjects. discrete pathologies like laryngeal penetration could only be seen on videofluoroscopy. depiction of tongue-movement was better with real-time mri in 13/18 cases and equally good in 5/18 cases. this was due to the better soft tissue contrast of mri and the lacking of artefacts from teeth and metall implants. conclusion: real-time mri can successfully depict the normal physiology of swallowing. for the analysis of dysphagia, quality is inferior to videofluoroscopy, while for the analysis of tongue movement-disorders, real-time mri is superior to videofluoroscopy. examinations were performed on a 1.5 t scanner (philips nt intera), equipped with sensitivity-encoding mri (sense) imaging software. sense shortens acquisition time by reducing the number of phase encoding steps, using the coil sensitivity as an encoding effect. 5 volunteers and 5 patients suffering from post-operative dysphagia were examined using a sagittal dynamic t1w 2d ffe sequence with 8 frames/s (tr/te 2.2/1.1 ms, 30°, slice 15 mm, matrix 144 × 256, fov 230 mm, 124 ms/image, sense reduction factor 2). water and yogurt (spiked with gadolinium 1:50) with a bolus size of 3 -15 ml were used as oral contrast agents. oral and pharyngeal swallowing phases were assessed. results: oral and pharyngeal transit, laryngeal closure, epiglottic tilting and pe sphincter opening upon swallowing could be visualized in all volunteers. boluses of yogurt ≥ 5 ml were found most useful. in 4 patients, residue and intra-or postdeglutitive aspiration and in 1 patient nasal regurgitation could be seen realtime. conclusion: mr fluoroscopic visualization of swallowing is possible using realtime 2d ffe imaging with sense. our initial experience indicates that functional swallowing abnormalities such as aspiration can be assessed with real-time mri. the leading symptoms were globus pharyngis or dysphagia, which could not be verified by means of endoscopy or ent-examination. in 40 % of the patiens reflux disease was indentified as underlying disease be means of ph-monitoring. 80 % showed reflux-associated motility disorders in videofluoroscopy. 82 % of them showed a dysfunction of the upper esophageal sphincter, which could not be found by other diagnostic tool. due to the resultant pressure elevation they showed constant/inconstant zenker's diverticula of pharyngeal pouches of different degree. in 85 % of those patients motility disorders of the oesophageal body were detected 6 -7 % of them sufferd from a delayed cleaning function of the oesophageal tube. in the remaining collective hypermotile disturbances like segmental not propulsive contractions or reflux-associated non specific motility disorders could be verified. the results of the study will be demonstrated by digital and videofluoroscopic recordings. evaluation of patients with dysphagia by modified barium swallow examination u. coskun, m. cigiltepe; ankara/tr purpose: to determine the perceived radiologic abnormalities of dysphagia with modified barium swallow examination. materials and methods: 250 patients with dysphagia were undergone modified barium swallow examination. utilization of a thickener abled us to determine the deglutition skill in different consistencies in anteroposterior, and lateral planes. results: the perceived radiologic pathologies are as follows. oral phase: 35 % of the patiens with bolus formation whereas 42 % with bolus transfer problems. pharyngeal phase: 85 % of patients revealed pulling in pyriform sinuses, 55 % had ventriculer residue 73.5 % problems with basal tongue retraction, 13.5 % had abnormal epiglottic tilt, 27 % delayed elevation of the pharynx and hyoid elevation problems. premature spill is seen in 25 % of them with stasis at the valleculae or pyriform sinuses, 15 % had aspiration and 7 % of them had backflow reflux. functional swallow refleks was delayed in 65 %. oesphageal phase: 15 % had upper oesophageal pathologies. 7 % had lower esophageal pathologies and 5 % had diffuse peristaltism disorder. conclusion: in our centre each study is tailored to the clinical history, the patients ability to undergo the examination, and the initial flouroscopic findings. according to the findings intervention techniques were establihed. those included positioning, alteration of the food texture, oral motor range of motion exercise. according to the results aspiration and aspiration related pneumonia free status was achieved. mealtime and videofluoroscopic fluids features affecting deglutitive events s. de giorgi, a. carniato; treviso/it a four years experience in dysphagic patients suggested that assessment of deglutition tested with videofluoroscopic fluids, doesn't closely enough reproduce deglutition with mealtime fluids. with this technique in fact only poor and approximate indications can be given to swallowing therapist concerning food to be used for rehabilitation. the aim is to give clinicians more precise and objective information about deglutition assessment. density and viscosity of videofluoroscopic fluids were measured and compared by means of a precision balance and a viscosimeter. videofluoroscopic fluids were more dense and viscous than mealtime food. different abilities to manage boluses in relation to their density and viscosity during each stage of swallowing were examined as well. the analysis of a recurrent aspiration pneumonia is difficult in newborns or children. besides the special requests of radioprotection the diminuished capacity of collaboration of small children is a considerable problem. therefore we use a dynamic recording unit with cine-fluoroscopy and with a videorecording system. the test-bolus consists of jotrolan (isovist r), which is a iso-osmolar, non ionic hydrosoluble preparation with a reduced pneumotoxicity. the taste is sugarlike. the analysis of the study should be done with the pediatrician or the paediatric ent-specialist in order to integrate the clinical findings with the radiologic pattern. the differentiation between an oral and a pharyngeal morphologic or functional swallowing disorder is of special therapeutic interest. in the analysis of the swallowing disorder the individual situation of the study has to be considered, since a rejection might be misinterpreted as a swallowing disorder of the first, the volontary phase. functional disorders as a complete or an incomplete paralysis of the velum or the pharynx, dysfunctions of the upper esophageal sphincter and alterations of the sensomotoric system can be differentiated with a high precision. the sensitive diagnostic tool allows to find an individual treatment plan in accordance to the therapy of castillo-morales, voita, pörnbacher or bobart for the single infant. patient examples will be shown and discussed. we studied up to now more than 10000 patients of which nearly 3500 had a neurologic disorder. only an adequate pretherapeutic work-up of the pathomechanism of an aspiration allows a correct therapeutic approach. the aim of the study is to assess the efficiency of the close cooperation of swallowing therapists and diagnostic radiologists, specially trained in the analysis of the pathomechanism of swallowing. the differentiation of the "pre-", "intra-" and "postdeglutitive" aspiration, which means aspiration before, during and after the triggering of swallowing reflex, turned out to have a great impact in the differential therapy of aspiration. moreover on the basis of dynamic imaging other important pathomechanisms of aspiration could be detected, which were the bases of the application of new therapeutic manoevers. by means of videofluoroscopy we examined patients before and after therapy. in 80 % of them a sufficient swallowing rehabilitation without aspiration could be achieved. videoflouroscopy shortened the time of training-hospitalisation, because the therapist could immediately center on the relevant exercises for swallowing rehabilitation. conclusion: dynamic evaluation of the neurologic impaired patient makes rehabilitation more efficient and shortens hospitalisation-time and costs. flat panel x-ray detector radiography: comparison with storage phosphor radiographs and conventional radiographs p. boehm, p. homolka, s. grampp, c. czerny, a. ba-ssalamah, h. imhof; vienna/at purpose: to compare images obtained with a flat-panel x-ray detector based on amorphous silicon technology with conventional screen film radiographs and storage phosphor radiographs using different exposure parameters to evaluate dose reduction and image quality in skeletal radiology. a digital x-ray detector (trixell, siemens, erlangen, germany) based on cesium iodide and amorphous silicon technology was used. state of the art screen film radiographs and storage phosphor radiographs (adc system, agfa) were compared with digital detector images obtained at doses equivalent to those obtained with system speed of 200 (hand: speed 50, femur and tibia: speed 100). in vitro human cadaver specimen (femur, tibia, head, lumbar/thoracic spine, hand) were embedded in a pmma tube filled with water. a conventional and storage phosphor radiograph and digital detector radiograph were made of each specimen by varying the exposure parameters (kv, mas). the resulting 228 images were evaluated independently by 3 radiologists using a subjective ranking (grade 1 -3) rating exposure, contrast resolution, spatial resolution and soft tissue presentation. from each group the best image was chosen and separately evaluated for comparison. results: radiation doses for digital detector images were equivalent or below the other methods giving the same or better diagnostic performance. the diagnostic performance of digital detector radiographs compared with conventional radiographs and storage phosphor radiographs suggests that this technology will be a useful tool in diagnostic imaging. comparison of a flat panel digital detector to screen-film by x-ray images of the hand t. pollack, h. pauls, k. köhler, r. friedberg, u. günl, f. stösslein; dresden/de purpose: to evaluate the image quality of hand images obtained with a new flat panel digital detector to those obtained with an extremity screen-film system. methods and materials: hand images were obtained on 50 patients presenting for a rheumatologic baseline or follow-up exam. the patients were imaged such that one hand, selected randomly, was imaged with a digital flat panel table system (general electric, xr/d, milwaukee, wi) and the other with an extremity 100-speed screen-film (sf) system (agfa, curix ht 1.000g plus). the dr images were printed using a laser printer. all images were obtained using the same x-ray tube and generator, without a grid, 100 cm source-to-detector distance, 50 kv, and 2.5 -3.2 mas (dependent on the patient acquired with matched techniques). five radiologists scored pairs of images dr vs. sf using a 5-point scale (−2 clearly worse to +2 clearly better). observers were asked to score the image quality of eight anatomical criteria. results: all anatomical structures were scored equal or higher with the flat panel system compared to screen-film. comparing the digital flat panel to screen-film, dr was graded statistically superior: by 3 of 5 readers for cortical structures, by 3 readers for trabecular structures and by 4 readers for border structures, soft tissues and overall image quality. none of the observers rated the sf statistically superior to dr for any category. the results demonstrate that the digital flat panel system with a 200 µm pixel size produced equal or superior hand images with respect to all anatomical criteria. cost-effective data recording device for mobile c-arm which has only analogue output: optimizing ordinary software of video-capturing and real time mpeg compression with limited funds a. sada, w. ikeda, e. nakagaki, k. hisazumi, i. imai, k. nakanishi; osaka/jp purpose: in our department, a mobile c-arm is used for routine digital subtraction angiography (dsa) because of limited funds. the purpose of our study is to find an adequate data saving device for this mobile c-arm which was equipped with only analogue output. materials and methods: a philips bv 312 mobile c-arm was used as dsa. all dsa images were obtained at five images per second and imaging matrix was 752 × 582. this machine has no facility for a data transport system. imaging data were generated by a video cassette recorder as an analogue output. a personal computer (macintosh powerbook g4) and mobile c-arm were connected through the analogue-digital converting and real time mpeg-1 compressing system (the pixela). from july 2001 to september 18 2001, 20 cases of dsa were performed (abdominal aortogram: 8, celiac arteriogram: 15, sma: 15, renal arteriogram: 6, common iliac arteriogram: 9, the others: 75). the compressed image qualities were evaluated. the amount of data and cost were estimated. results: compression was about 3.7 %. average compressed data per one-series of dsa was 3.2 mb. average data per patient was 20.6 mb. therefore, about 32 patients' data can be saved on 640 mb mo disks and two mo disks will be required per year in our department. conclusion: with limited funds, our device (using ordinary computer and the real time mpeg compression software) was useful for saving the imaging data of dsa using a mobile c-arm system which has only analogue output. hodgkin's disease -radiological procedures for determination of clinical stage l. todoric; belgrade/yu purpose: to assess the value of us, ct and lymphography in determination of the clinical stage of the hodgkin's disease and to evaluate the reliability of the techniques in identifying the spread of disease into infradiaphragmatic lymph nodes. methods: 100 patients with histologically confirmed diagnosis were examined using all three methods before the onset of treatment. the clinical stage of the disease was based on us and ct findings. lymphography was performed as the last diagnostic procedure. after this final staging took place. we compared and statistically analyzed us, ct, and lymphographic findings as well as the change of clinical stage after lymphography. results: lymphography did not show high aortic lymph nodes. us and ct did not show the changes in small or normal size nodes especially in pelvic region. statistically significant differences in alteration of the clinical stage, before and after lymphography, has been demonstrated in all stages. conclusion: lymphography enables a more accurate evaluation of lymph nodes in the pelvic and aortic region in comparison with us and ct and the assessment of the clinical stage resulting in an optimal therapeutic approach to hodgkin's disease. a c d e f 288 methods and materials: five patients with chronic low back pain and two volunteers underwent routine magnetic resonance imaging (mri) protocol for the lumbar spine. a 3-plane localizer sequence was taken with and without a standard supportive cushion. the angle of lumbar lordosis was calculated by two 'blinded', experienced radiologists from consensus measurements of the angles at the l5/ s1 and l1/l2 levels using a ruler line tool on a workstation. results: observations in the seven subjects who underwent lumbar spine imaging (mri) with and without the supportive cushion showed no change in the degree of lordosis; the mean angle with the supportive cushion was 46.7° (sd. 17.5) and 48.2° (sd. 17.6) without. the mean of the difference between the measurements for each subject was −0.14 (sd. 4.05). conclusion: as the use of supportive cushions produces no practical change in the lumbar lordosis, the decision to employ them should be made entirely with respect to patient comfort. the military radiographer: a profession in constant evolution e. martinello, a. giardino, m. pari, c. turchetti, c. ottonello, s. panica; rome/it diagnostic imaging in military medicine can be divided into two branches: clinical (in homeland and in field hospitals) and forensic. the clinical branch is performed in all clinical hospitals, while the forensic branch is performed both in clinical hospitals and in dedicated medical forensic military centers. in the clinical branch, field hospital radiological activity (fhra)is very specific and interesting; it is performed in the homeland (great disasters or accidents) and abroad (military operations, as peace-keeping/enforcing). fhra is influenced by three important factors: mass (high spatial-temporal concentration of injured personnel), environment (e.g.: tropical countries: infectious or climatic changes or unusual diseases, and injurious agents, ballistical or thermal. a special modular rx unit (8 parashutable packages) that can be easily and rapidly assembled and disassembled, and a us unit with 3.5 and 7.5 mhz probe are available in our field hospitals. the modular rx unit is composed of a "c" arm, tv unit with fluoroscopy, radiographic facilities and the ability to print on us type film. in the near future dedicated ct units, digital radiology devices and dry film processing units shall also be available. methods and material: over a two year period, 95 patients ranging from 5 to 80 years with or without an intraspinal component of the lesion were subjected to fnac. all were performed with 20/22 to gauge needle. two or three passes were made. us guided biopsy was performed with 5 -7 mhz linear probe with a free hand technique. ct was performed using a lead wire grid for accurate location. results: of these 45 (67.16 %) were benign and 22 (32.87 %) were malignant. 6 patients underwent ct guided fnac as us guided was negative. the success rate of us guided fnac was 33/45 (73.3 %) and that of ct guided 34/46 (73.9 %). a conclusive diagnosis could not be reached in 18 patients due to insufficient material caused by necrosis, clot or blood. conclusion: image guided fnac is an effective and safe procedure for evaluation of spinal and para spinal lesions for effective patient management. to evaluate the value of mri as an adjunct to clinical and sonographic findings for the prenatal diagnosis of fetal malformation as well as for the assessment of maternal disorders during pregnancy. methods: 77 women with complicated pregnancies (fetal pathologies n = 71, maternal pathologies n = 6, mean age of gestation 26.82 ± 6.88 weeks) were referred for mri. imaging was performed in a 1.5 t system (gems) using t2w ssfse sequences. images were analysed with regard to fetal and maternal abnormalities. findings were correlated with sonography, postnatal clinical and imaging findings and/or autopsy. results: most of the fetuses (n = 37) had isolated pathologies of the cns. in 7 cases complex malformation syndromes were present. the remaining pathologies included pulmonary (n = 6), gastrointestinal (n = 2) and other (n = 20) pathologies. mri was equal or even superior to sonography for diagnosing cerebral malformation. due to the small size, the lack of real time examination and the missing flow information, the diagnostic confidence of mri was lower for pathologies of the heart, abdominal organs and extremities. maternal disorders included anomalies of the uterus (n = 2), myoma (n = 2), 1 cervical carcinoma and 1 borderline ovarian tumour. purpose: aim of this work was to compare the new 3d mr-histerosalpingography with the x-ray conventional one in the evaluation of female infertility. method and materials: 15 infertile female patients (aging 29 -43 years, mean 35) were studied with a 1.5 t magnet unit (ge horizon 8.5) using a t2 weighted fse sequences on axial and coronal plane and a 3d fast spgr sequence (3d mra sequence) on coronal plane after injection of a diluted gadolinium solution into the uterine cavity via a balloon catheter. all the data were compared with conventional x-ray hysterosalpingography. the tomographic images provided a complete evaluation of the uterus and ovaries. the 3d mr technique provided the evidence of the uterine cavity and the patency of the fallopian tube with a complete concordance with x ray histerosalpingography. however the accuracy in detecting the fallopian tube calibre was considered inferior with mri than x-ray by two expert radiologists. two fallopian tube stenosis were missed at 3d mr-histerosalpingography. conclusion: 3d mr histerosalpingography may represent a real promising technique in the non-invasive evaluation of the infertile female. the main advantage is related to the lack of ionising radiation in these young females. up to date, in our experience, the mr technique does not have the same accuracy as x-ray in detecting the fallopian tube. improvement in spatial resolution probably have to be considered. the purpose of this study was to establish mr pelvimetric reference values in a large study taking into account the mode of delivery. intra-and interobserver errors and intra-individual variability of mr measurements were assessed in a complementary volunteer study. methods and materials: mr pelvimetric measurements of 781 women (28.9 ± 5.2 a) were reviewed and correlated to obstetric history in order to establish normative values. 2 subgroups were identified: (1) spontaneous deliveries, and (2) cases undergoing caesarean section or vacuum extraction due to fetalpelvic disproportion. in addition, mr pelvimetry was performed 5 times in 10 female volunteers (34.7 ± 6.0 a) on a 1.5 t system using t1w fspgr sequences. all measurements were then performed twice by 5 different observers to assess intra-and interobserver errors and intra-individual variability. results: mean obstetric conjugate measured 116.6 ± 10.8 mm, interspinous distance 106.6 ± 9.5 mm, intertuberous distance 114.8 ± 12.3 mm, transverse diameter 125 ± 9.8 mm and sagittal outlet 112.8 ± 11.0 mm. there were significantly different measurements in the 2 subgroups (p < 0.001), with wider measurements in the spontaneous delivery group. in the volunteer study intra-, interobserver and intra-individual reliabilities were accurate for obstetric conjugate (0.94 -0.96), interspinous distance (0.92 -0.95) and transverse diameter (0.95 -0.98), however they were poorer for intertuberous distance (0.64 -0.87) and sagittal outlet (0.66 -0.85). the normative values established in this study stratified for mode of delivery are likely to influence obstetrical decision making in the future. intertuberous distance and sagittal outlet presented the largest variabilities. obstetric decisionmakers would be justified in approaching this parameter with caution. a single-shot echo-planar diffusion mr sequence with 2 b-values (0 -500 s/mm 2 ) in the 3 planes of space (x, y, z) was implemented on a 1.5 t magnet and systematically added to a routine liver protocol including t1-, t2-and gadolinium-enhanced t1-weighted sequences in 66 patients. 52 fll were evaluated in 43 patients separated in 5 groups: metastases (n = 9); hepatocellular carcinomas (hcc, n = 9); benign hepatocellular lesions (bhl) including focal nodular hyperplasias and adenomas (n = 15); hemangiomas (n = 7); and biliary cysts (n = 3). the adcs were measured on the normal and the cirrhotic liver and on the fll. the results were then compared between the groups of patients. the liver parenchyma and the fll were found to be isotropic. the adc (× 10 −3 mm 2 /s) of the cirrhotic liver (1.37 ± 0.52) was lower than that of the normal liver (1.83 ± 0.36, p < 0.05). signal intensities of the focal lesions were rated by two radiologists as follows: hypointense, isointense, hyperintense, and markedly hyperintense. lesion-to-liver contrast-to-noise ratio (c/n) was measured for a quantitative assessment. results: on ferumoxides-enhanced fse images, 92 % of cysts were 'markedly hyperintense' and most of the other lesions were 'hyperintense', and the mean c/n of cysts was significantly higher than that of other focal lesions. t2*-weighted gre images showed most lesions with similar hyperintensities and the mean c/n was not significantly different each other. t1-weighted in-phase images showed all fnhs and hemangiomas, 29 (69 %) hccs and 8 (20 %) metastases as 'hyperintense'. on t1-weighted out-of-phase gre images, all hccs and metastasis except one were iso-or hypointense, while all of the fnhs and hemangiomas were hyperintense. ring enhancement was more commonly seen on the out-ofphase images than on the in-phase images. conclusions: addition of t1-weighted in-phase and out-of-phase gre images is helpful for characterizing focal lesions in ferumoxides-enhanced mr imaging. was acquired before and immediately following i.v. injection of 0.1 mmol/kg of gadodiamide, during arterial and portal-venous phases. in-phase and opposedphase images were calculated at the end of the sequence; subtraction was performed for each series of images on the main console. results: images of diagnostic quality were obtained in all the cases. nine metastases, 3 hemangiomas and 9 cysts were detected, all showing low signal intensity on unsubtracted and subtracted images. increase in lesion/liver contrast was observed on subtracted images (p < 0.05). three hepato-cellular carcinomas previously treated with chemioembolization showed high signal on in-phase images and low signal in opposed-phase images; after subtraction bright signal was observed with increase in liver/lesion contrast (p < 0.05). focal fatty areas showed bright signal on subtracted images. in two patients with diffuse liver steatosis, three metastases were missed in both opposed-phase and subtracted images, but they were visible as low signal intensity nodules in in-phase images. in-phase and opposed-phase images with electronic subtraction may help in identifying focal liver steatosis and increase lesion/liver contrast in case of fat-containing lesions. which is the optimal phase for the detection of hypervascular liver lesions with multislice helical ct? comparison beetween early and late arterial phase r. basilico, m. ricciardi, a. di credico, e. cucci, l. bonomo; chieti/it purpose: to compare early and late arterial phase imaging with multislice ct in the detection and conspicuity evaluation of hypervascular liver lesions. materials and methods: 50 patients with known hypervascular liver lesions (20 hccs, 15 metastases, 10 haemangiomas, 5 fnhs) were evaluated with double arterial phase ct. ct examinations were performed with multislice ct scanner using 4 × 2.5 mm collimation and 3 mm slice thickness during administration of 130 cm 3 of contrast agent (400 mg i/ml) at 4 cm 3 /s. after a test bolus, early and late arterial phase images were obtained serially during a single breath hold in a cranio-caudal direction, with interscan delay of 4.0 seconds and mean scanning time of 10.5 seconds for each phase. each set of images were evaluated in consensus by two blinded, experienced radiologists. they determined lesion detection rates, recorded lesion size and scored the conspicuity of lesions on a scale 1 -4. the mean scanning delay for the early arterial phase was 21.5 seconds (range 13 -28 seconds), whereas the mean delay for the late arterial phase was 36 seconds (range 27.5 -42.5 seconds). the number of lesions detected was 118 with early phase and 242 with late phase (p < 0.01). late arterial phase resulted in 51 % increase in detection rate versus early arterial phase. lesion conspicuity and diameter significantly increased on late arterial phase. the detection of hypervascular liver lesions did not significantly improved with early and late arterial phase evaluated in combination. conclusions: late arterial phase imaging (15 seconds after the peak enhancement in the aorta) significantly improves the detection of hypervascular liver lesions. a c d e f 292 material and methods: 24 consecutive patients underwent mri of the liver using a 1.5 t system with 30 mt/m gradients with phased array bodycoil. imaging protocol includes t2w tse with and without fatsaturation and breathhold t1w flash. seven t1w 3d flash datasets with parallel imaging (vibe with sense; tr/te/ matrix/slice thick./acq.t.: 6.21 ms/3.16 ms/256 × 192/4 mm/14 s) was performed as dynamic contrast study during infusion of gd-dtpa i.v. additional postcontrast breathhold t1w flash was acquired. results: images of diagnostic quality were obtained in all cases. normal findings were observed in 6, ecchinococcus cyst in one, liver metastases in 3, hemangiomas in 4 and hepatocellular carcinoma in 12 cases. dynamic contrast studies gives important additional informations about the perfusion of focal hepatic lesions. with a slice thickness of 4 mm in vibe sequence even in small hemangiomas typical contrast behaviours are seen. hepatocellular carcinomas are detected best in vibe sequence in early arterial phase compared with other sequences. conclusion: the vibe sequence with sense technique covers the whole liver within one breathhold. thus dynamic contrast studies in a slice thickness of 4 mm are possible and important perfusion informations of liver lesions are available in a high spatial resolution. we think that this mr technique is a very promisable tool for liver diagnostic. the value of mri in the diagnosis of hepatic tuberculoma f.h. yan, m.s. zeng, k.r. zhou; shanghai/cn purpose: to analyse the mri findings of hepatic tuberculoma, to discuss the value of mri in the diagnosis and differencial diagnosis. methods and materials: ten cases with hepatic tuberculoma underwent mr se sequence and fmpspgr sequence dynamic contrast scanning. results: mri findings of total 12 lesions in 10 cases appeared as: (1)se sequence: all lesions were hypointense on t1wi, 10 lesions were inhomogeneous hypointense on t2wi (central hypointense and peripheral hyperintense in 8 lesions and punctual hyperintense in the center in 2 lesions). the other 2 lesions showed as hyperintense. (2) fmpspgr dynamic contrast scanning: 10 lesions had no enhancement and 2 lesions had slight peripheral enhancement on the arterial phase, all lesions had various patterns of enhancement on the portal venous and delayed phase scanning, mainly peripheral enhancement and internal septal enhancement. conclusion: mri could reflect the pathological changing of hepatic tuberculoma and be of great value in the diagnosis and differencial diagnosis. comparative study of ctap and mndpdp enhanced mri of the liver in the pre-operative evaluation of colorectal metastases e.j. van der jagt, t. kok, m.j.h. slooff; groningen/nl purpose: to compare the sensitivity of ctap (computer tomography during arterial portography) with mndpdp enhanced mri in assessing the exact number of liver metastases before liver surgery. material and methods: in 30 patients evaluated for surgical treatment of metastases of colorectal carcinoma, we performed ctap and mri with mndpdp contrast medium (teslascan). peroperative ultrasonography performed jointly by surgeon and radiologist served as the gold standard. ctap and mri were evaluated by two experienced radiologists blinded to the results of the operative findings and the result of the other technique. results: in 18 patients at operation 37 metastases were found, and 1 focal nodular hyperplasia. ctap correctly showed 31 (81.6 %) and mndpdp enhanced mri 24 (65 %)of these lesions. of the missed meastases in mri 7 were smaller than 7 mm and 10 smaller than 1 cm. two were missed because of technical failure, two of three by interpretative failure were found in retrospect. ctap showed 13 lesions thought to be metastases not proven by surgery, whereas mri only scored 3 false positive findings. in the non operated group 6 patients underwent petscanning showing a total of 15 metastases in the liver. 13 of these were shown by ctap and 14 by mri. when this group of patients is added to the operated group, sensitivity for ctap rises to 83 % and for mri to 70 %. conclusion: mndpdp-enhanced mri is less sensitive than ctap (70 % versus 83 % of proven lesions) but showes less false positive lesions. = 1) , sle (n = 1), idiopathic (n = 1)), cystic acinar transformation (n = 3), oncocytic endocrine tumor (n = 1) and isolated polycystic disease (n = 1). we reviewed retrospectively the preoperative investigations (computed tomography, cholangio-mri, us-endoscopy, pancreatography) and correlated them to the histological results. results: ct and mri showed isolated abnormalities of the pancreatic ducts in 7 patients involving the main pancreatic duct (mpd) (n = 3) and the branch duct dilatation (n = 5). the mpd was normal (n = 4), dilated (n = 3) or stenosed (n = 1). the cystic lesions involved the head and/or uncinate process (n = 4), the body and/or tail (n = 3). the location was diffuse (n = 3) or multifocal (n = 1). the number of cystic lesions varied from 3 to 20 or more and the size was less than 10 mm. a solid tumor was demonstrated in one patient. correlation with us-endoscopy was good. none of the 8 patients were found to have mucous extrusion through the papilla. pancreatography was normal in 1 case, showed a communication between the cystic lesions and mpd in 2 cases or a stenosis in one case. ductal biopsies performed in 5 cases (eus-guided n = 3; transpapillary n = 2) revealed epithelial proliferation in 2 patients and were non-informative in 3. the pancreatic resection consisted with a pancreatico-duodenectomy in 6 patients and total pancreatectomy in 2. to compare mr cholangiography to multislice ct cholangiography without biliary contrast agent in the assessment of patients with biliary obstruction. methods and materials: 25 patients with clinical or biochemical signs of biliary obstruction were studied. mr cholangiography was performed with a 1.5 t unit, using respiratory-triggered three-dimensional fast-spin-echo and haste sequences; source images, maximum intensity projection, and multiplanar reformatted images were evaluated. ct cholangiography was performed using a multislice scanner, without biliary contrast agent, with intravenous injection of iodinated contrast material; axial, minimum intensity projection, and multiplanar reformatted images were evaluated. ct and mr findings were compared to ercp in 17 patients, ptc in 6, intraoperative cholangiography in 2. six patients underwent surgery or fine needle aspiration. results: ercp, ptc, surgery, and pathology demonstrated: 10 pancreatic, 2 gallbladder, 2 klatskin's, and 1 ampullary carcinomas; 1 bile duct obstruction due to enlarged hilar lymph nodes; 7 patients with choledocholithiasis; 1 chronic pancreatitis; 1 patient with negative findings. regarding site of obstruction, agreement was observed among ct, mr cholangiography and conventional cholangiography in all cases. concerning the cause of obstruction, the correct diagnosis was made in 23 patients by both mr and ct cholangiography. in two patients with choledocolithiasis, ct cholangiography was judged to be negative, but stones were correctly identified by mr cholangiography. conclusion: in the assessment of patients with biliary obstruction, multislice ct cholangiography can be considered a useful diagnostic tool alternative to mr cholangiography, which is still the non-invasive standard of reference in the evaluation of the biliary tract. mdct-ca was performed as part of a comprehensive preoperative assessment in 30 potential living liver donors (16 women, 14 men; mean age 31 years). for display of the biliary system, mdct of the liver (slice thickness/ collimation 1 mm, pitch 6 mm, table speed 12 mm/s) was performed 25 ± 5 minutes following intravenous administration of meglumine iodipamide (biliscopin). subsequent mdct angiography was added to depict the topographic relationship between biliary and vascular structures. mdct findings were correlated with intraoperative findings (n = 14). results: mdct-ca was diagnostic in all 30 patients. 21 of 30 patients presented with variations in biliary anatomy. these include drainage of liver segment four into right hepatic duct (n = 9), additional right/left segmental ducts draining into the common hepatic duct (n = 8) or the left hepatic duct (n = 4) and trifurcation at the upper confluence (n = 3). biliary-vascular topography was well depicted in all cases. intraoperative assessment confirmed the preoperative mdct-ca findings in all 14 cases. conclusion: variations in biliary anatomy appear to be the rule rather than the exception. mdct-ca represents a non-invasive means for accurately assessing biliary morphology. a continuous helical data set of the heart was acquired in 50 patients (group 1) using the standard protocol with constant tube current, and in 50 patients (group 2) using an alternative protocol with reduced radiation exposure during the systolic phase. the standard deviations (sd) of predefined regions of interest (rois) were determined as a measure of image noise and were tested for significant differences. results: there was no significant difference between group 1 and group 2 in respect of image noise. radiation exposure with and without tube current modulation was 1.0 msv and 1.9 msv, respectively (p < 0.0001) for males and 1.4 msv and 2.5 msv, respectively (p < 0.0001) for females. thus there was a mean dose reduction of 48 % for males and 45 % for females, respectively. conclusion: ecg-controlled tube current modulation allows significant dose reduction when performing retrospectively ecg gated msct of the heart. purpose: to analyse myocardial contrast dynamics and to calculate myocardial perfusion parameters using ecg-triggered multirow-detector computed tomography (mdct). methods and materials: 9 patients with a suspicion of or known cad underwent retrospectively ecg-gated mdct angiography of the coronary arteries. prior to cta data acquisition a prospectively ecg-triggered transaxial dynamic scan (4 × 5 mm) over 35 heart beats was applied to analyze myocardial enhancement patterns with subsequent assessment of perfusion parameters. contrast enhancement was provided by administration of 40 ml of iopromid 300 at a speed of 8 ml/s followed by a 50 ml saline flush. sequential image data was analyzed to calculate maximum left ventricular (lv) and myocardial signal intensity (si) increase and for maximum si upslope. all data were normalized to the lv input function. in addition quantitative measurements were performed using a fermi-function. coronary angiography was available in all patients. the lv si increase showed an amplitude of 244 ± 62 hu (136 -324 hu) and an up-slope of 39.7 ± 4.6 (34.0 -45.6). the range of si increase in myocardium was 31 ± 10 hu. within myocardial regions the normalized si increase and upslope were 0.13 ± 0.07 and 0.065 ± 0.022, respectively. in regions of impaired blood supply si amplitudes and si upslopes tended to be lower. quantitative flow calculations revealed values close to those within normal myocardium (0.73 ± 0.20 ml/g/min). the use of mdct scanners the assessment of myocardial contrast dynamics is possible. however, ventricular coverage so far is insufficient. with optimized contrast enhancement protocols the reliability may be even better. so far, the relatively low myocardial enhancement leads to a low snr. a c d e f 296 method/materials: in 30 children (22 male, 8 female, mean age 9.1 years) with suspected aortic isthmus stenosis or re-stenosis mr imaging was obtained using a 1.5 t body scanner (magnetom vision, siemens). an optimized 3d ge sequence with double-slab excitation (tr 12.3 ms, te 5.5 ms, slab thickness 12.5 mm) was used in a sagittal plane. in 14 children an additional ce mra was performed using a standard 3d ge sequence with short tr/te (~6 ms/~1 ms) after intravenous administration of gadolinium (0.1 mmol/kg). source images and maximum intensity projections (mip) were analyzed, evaluating blood-tissue contrast as well as size and focal stenoses of the aortic arch. results: in 21 of 30 an aortic coarctation could be found. using the double-slab technique, in 87 % the image quality was high and there was a low sensitivity to flow and breathing motion. there was a good blood-tissue contrast without the use of contrast agent. using a combined analysis of source images and mip diagnostic accuracy could be improved. conclusion: mr imaging represents an excellent tool for non-invasive examination of the cardiovascular system of children. the double slab method allows recording of a large 3d data set in an adequate measurement time especially in small infants, which were not able to hold their breath, and it is well suited for 3d reconstruction. evaluation of the tais stent in short de novo coronary lesions i.v. pershukov 1 , t.a. batyraliev 1 , a.n. samko 2 , z.a. niyazova-karben 1 , y. pya 1 , y.n. belenkov 2 ; 1 gaziantep/tr, 2 moscow/ru purpose: the tais is a new balloon-expandable, stainless steel, tubular stent. this study was designed to assess the safety and efficacy of this novel coronary stent and by indirect comparison to indicate equivalence with other formal stent studies. methods and materials: patients with angina and a single short (< 15 mm) de novo lesion in a native coronary artery of ≥ 2.75 mm diameter were included. a total of 588 patients were allocated in 2 centers. most patients (59 %) had stable angina, and 34 % of lesions were type b2 -c. clinical data was collected at sixand nine-month follow-up. in the first 308 patients angiography was routinely performed at six months. the remaining 280 patients were merely followed up clinically. results: no stent deployment failure occurred, as well as acute or subacute stent thrombosis. the primary end-point of the study, the six-month mace-rate was 12.3 %, which is similar to the calculated 15 % mace-rate in comparable reference studies. secondary end-points included among others restenosis at six months in the first population. the target vessel diameter was 3.13 ± 0.51 mm. minimal lumen diameter pre/post procedure and at follow-up was 0.81 ± 0.33, 2.97 ± 0.44, 2.21 ± 0.77 mm, respectively. the binary restenosis rate was 15.5 %. conclusion: the coronary tais stent is safe and effective as a primary device for the treatment of native coronary artery lesions in patients with stable or unstable angina pectoris. clinical and angiographic results are in the statistical range of equivalence with comparable studies with other current stents. the aim of this study is to asses if the bmi affects the amount of pulmonary atelectasis developing after general anaesthesia, and its effect on atelectasis evolution. material and methods: two groups of volunteers aged from 27 to 66 years old were prospectively randomised. they all underwent surgical celioscopy. 20 patients with a bmi over 35 kg/m 2 underwent a gastroplasty and 10 patients representing the control group (bmi under 30 kg/m 2 ) underwent a cystectomy. before induction of general anaesthesia, three ct sections of 7 mm, were acquired in expiration at the level of interventricular septum. immediately after surgery and 24 hours later, three ct sections were acquired at the same level. with a threshold of −1000 to +100 hounsfield units, the surface of pulmonary parenchyma was manually extracted from the ct sections. the amount of pulmonary atelectasis was determined with a −100 to +100 hu threshold. results: a statistically significant difference was observed in comparing the group with a bmi over 35 kg/m 2 and the control group in the development of pulmonary atelectasis (p = 0.0021) and in its evolution (24 h p = 0.0008). the bmi affects the amount of pulmonary atelectasis developing after general anaesthesia and its evolution at 24 hours. lymphomatoid granulomatosis: pulmonary abnormalities and correlation with pathology a. patsalides, g. atac, u. hegde, w. wilson, n. patronas; bethesda, md/us lymphomatoid granulomatosis (lyg) represents an angiodestructive lymphoproliferative disorder. immunosuppression is a major risk factor for the development of lyg and the lungs represent the most commonly affected organ. the purpose of our presentation is to report the pattern of respiratory tract involvement in lyg and assess evolution of these lesions after treatment. 22 patients, six females and 16 males, ranging in age from 26 to 73 years (mean, 48 years) were enrolled on a study on the diagnosis and treatment of lyg. the diagnosis was confirmed by lung biopsy in all patients. ct imaging was performed in all patients upon entering the protocol and follow up scans were performed after the initial evaluation in order to assess the effect of treatment. pulmonary involvement was evident in all patients. masses were identified in 19, alveolar and/or interstitial infiltrates in 20, and lymphadenopathy in five patients. pleural effusion was present in six and pleural thickening in one patient. eight patients were found to have pulmonary cavities. pathology studies showed infiltration of the pulmonary parenchyma by lymphoid cells with prominent perivascular distribution. granulomas with various degrees of ischemia and necrosis were also present. on follow up studies, partial or complete resolution of the pulmonary lesions was noted after treatment. the pattern of pulmonary involvement in patients with lyg is consistent with the pathological findings. the imaging findings are not specific. pulmonary biopsy is therefore essential for the establishment of an accurate diagnosis. imaging studies however are useful for evaluating the response to treatment. method: 13 patients with histologically diagnosed neoplasms underwent initial assessment with clinical examination, pulmonary function tests and high resolution ct. pulmonary clearance studies were carried out using using a proprietary nebulizer (amertec, uk) and baselines were obtained contemporaneously with pre-treatment hrct. investigations were repeated immediately after treatment and at one and four months. results were documented independently and compared to determine the presence, severity and timing of changes. results: eight patients were able to complete the study; one died and four were withdrawn on medical grounds. clearance was unaffected by treatment in only one patient, the rest showing a marked increase. in five patients clearance increased immediately post-treatment and occurred in both irradiated and contralateral lung. only 3 of 8 patients showed demonstrated late changes on hrct; there was no corresponding change in the contralateral lung on hrct. there was no correlation between radiation dose and alteration in lung clearance. the pilot study confirms that 99 tc dtpa pulmonary clearance studies are a highly sensitive method of detecting radiation pneumonitis and presumed contralateral sympathetic pneumonitis, and confirms that pulmonary radiation has a systemic in addition to local effect. optimization of the acquisition protocol m. rémy-jardin, a. amara, p. campistron, i. mastora, v. delannoy, j. rémy; lille/fr purpose: to evaluate the accuracy of 3 mm thick reconstructed sections in the diagnosis of bronchiectasis with msct. materials and methods: 40 consecutive patients suspected of bronchiectasis underwent msct of the entire thorax with a 4 × 1 mm collimation. from each data set, two series of images were systematically reconstructed with a high-spatial frequency algorithm: 1 mm (group 1) and 3 mm (group 2) thick scans, at 10 mm intervals. three observers independently analyzed the presence of bronchiectasis and associated abnormalities on group 1 and group 2 lung images. results: no significant difference between group 1 and group 2 was found in: (a) the detection of bronchiectasis [group 1: n = 24 (60 %); group 2: n = 23 (57.5 %); (p = 0.08)]; (b) the evaluation of the extent of bronchiectasis [focal: group 1; n = 10 (25 %) and group 2; n = 7 (17.5 %); (p = 0.39) and diffuse: n = 16 (40 %) in both groups]; (c) the characterisation of bronchiectasis [cylindrical: group 1: n = 24 (60 %); group 2: n = 21 (53 %); p = 0.08; varicose: group 1: n = 5 (12.5 %); group 2: n = 6 (15 %); p = 0.56; cystic (group 1: n = 2 (5 %); group 2: n = 2 (5 %)]. apart from the identification of abnormal bronchial wall thickening (group 2: n = 35; 87.5 % vs group 1: n = 31; 77.5 %; p < 0.05), recognition of associated abnormalities did not differ between the two groups. conclusion: a comparable accuracy of 3 mm and 1 mm thick reconstructed scans in the detection and characterization of bronchiectasis allows the recommendation of a 4 × 2.5 mm collimation with reconstruction of 3 mm thick scans in the screening of bronchiectasis, reducing the radiation dose of msct examinations by 20 %. purpose: for the development, optimization and validation of inhalation therapies it is important to develop a model that covers individual anatomical data, application process, deposition and uptake of applied medication. using cfd, velocities and deposition within the individually segmented tracheo-bronchial tree were simulated. material and methods: multislice ct datasets (n = 20) of the lung were segmented using a hybrid segmentation algorithm to detect the tracheo-bronchial-tree. this algorithm consists of: (a) 3d seeding process, (b) 2d bronchus finder (fuzzy-logicsystem), (c) 2d module to identify peripheral bronchi (template-matching). the influence of three reconstruction kernels on the segmentation process was inves-tigated. the hybrid system was compared to a merely threshold-based segmentation tool by visual assessment performed by two radiologists. the segmented images were used to reconstruct the 3d geometry of the tracheo-bronchial airways. this is meshed using an unstructured tetrahedral mesh. flow analysis and particle tracking model is carried out using cfd to simulate in-and expiratory breath velocities. results: due to the combination of the 3-step algorithm it is possible to segment down to the 8 th generation of bronchi semiautomatically. depending on the kernel used the following sensitivity ranges were obtained: 5 th generation 80 -90 %, 6 th 70 -80 % and 7 th 50 -60 %. calculation of the velocity during in-and expiration revealed a ratio of up to 2.4 (peripheral bronchi/trachea). conclusion: combining individual anatomical data derived from radiological images and a comprehensive model of the respiratory tract enables the simulation of distribution and deposition of inhaled drugs. support: ec (ist-1999-14004: "cophit"), british council b-0863 09:40 ct bronchography: assessment and validation of two approaches for 3d reconstruction c.i. fetita 1 , f. preteux 1 , c. beigelman-aubry 2 , p.a. grenier 2 ; 1 evry/fr, 2 paris/fr purpose: to validate and compare two reconstruction techniques leading to the 3d ct bronchography, by means of a scoring system established with respect to a reference anatomical bronchial tree. the study was conducted on volumetric helical data sets obtained from 20 patients with various chronic airway diseases. acquisitions were performed during breath holding following a full inspiration without any contrast agent. axial images were reconstructed with 1.25 mm thickness and 0.6 mm intervals. for each patient, the bronchial tree was reconstructed using 2 different techniques. the first one consists in a slice-by-slice segmentation of airway lumens followed by a 3d reconstruction using a topological propagation and filtering. the second one is fully-3d and relies on a diffusive-aggregative markovian modeling, which takes advantage of the structural and topological features of the airways. in both cases, the bronchial tree was visualized in a ct bronchogram mode, using a semi-transparent volume rendering technique. the images were assessed independently by 2 radiologists using a scoring system established on the basis of a reference bronchial tree defined up to the subsegmental level. results: the bronchial tree was reconstructed up to the 6 th order by both techniques without any significant difference (96 % accuracy and 97 % robustness). the fully 3d technique running twice faster, proved in addition the capability to reconstruct smaller bronchi distal to the 6 th order. conclusion: 3d bronchography obtained from multislice ct is an accurate technique to reconstruct the airways up to the 6 th -7 th order of division. . the results were compared to 50 patients, which were examined with a standard protocol (120 mas: g120). all other parameters were kept constant. subjective image quality was rated using a three point scale. objective criteria, based on signal-tonoise measurements, were assessed. results: image quality was sufficient in all cases. subjective gradings of image quality, based on soft tissue window settings, were 1.1 for the 120 mas protocol, 1.1 (g+10), 1.1 (g±0), 1.3 (g−10), and 1.2 (g−20), for the body weight adapted protocols. objective criteria showed mean standard deviation values of 5.7 hu for the 120 mas protocol. for the dose reduced protocols, values were calculated as 7.6 hu (g+10), 7.9 hu (g±0), 8.7 hu (g−10) and finally 9.1 hu (g−20). best correlation for the whole subgroup was achieved for the −10 protocol, with nearly constant noise related to the body weight in all patients. conclusions: by deriving mas values from body weight estimation, an individually adapted protocol for chest ct can be recommended. with an adaptation of the tube current time product (x mas = m − 10, m … body weight in kg), a well balanced examination, even in soft-tissue window settings, can be performed. therefore, for other types of ct scanners, analogous protocols may be adapted. highland heights, oh/us purpose: current ct liver perfusion methods typically involve limited (2 cm or less) z-axis coverage with the results displayed in the form of axial cross-sectional perfusion maps. we implemented a full-organ liver perfusion technique and explored the usefulness of providing full-organ volume-rendered perfusion maps. methods: ten ct liver perfusion studies were acquired (technique) for patients with hepatic masses using a series of 5 consecutive acquisitions of the liver during one breathold after the injection of a bolus of contrast (4 -5 ml/s). images of each slice location were acquired every 8.5 seconds. liver perfusion maps were produced using previously published techniques and displayed in both axial and volume rendered modes. results: multislice-spiral acquisitions with a pitch of 1.5 and a 0.5 s rotation enabled full coverage of the liver, portal vein and spleen, which were used as inputs for the perfusion calculation. both the axial and volume-rendered perfusion maps depicted characteristic perfusion patterns of various types of hepatic tumors. small tumors were depicted as area of hyperperfusion, while advanced tumors exhibited a ring of hypervascular tissue surrounding a necrotic core. perfusion values of normal liver tissue corresponded to expected physiological ranges. further investigation is warranted to understand the sampling rate required for clinically relevant quantitative measurements. conclusion: multislice ct scanners may enable full organ assessments of liver perfusion. volume rendering of perfusion images allows potentially superior assessment of perfusion abnormalities and might be used to predict the effectiveness of chemo-and thermoablation therapy. (ratios) were determined using amares. additionally, absolute values of phosphorous metabolites were determined using sloop and an concentration of 2.5 mmol for atp as internal standard, including corrections for inhomogenous b1-fields, t1 relaxation. 10 healthy volunteers and 10 patients with liver chirrosis were included. results: amares allowed relative quantification (metabolite ratios) in all volunteers, however, only in 7/10 patients, due to low signal to noise ratios. using, sloop as postprocessing method, all acquired 31 p-spectra had sufficient signal to noise ratios for all analyzed metabolites. in healthy liver parenchyma, absolute concentrations for pme, pi and pme were 2.3 ± 0.8, 1.5 ± 0.6 and 15 ± 6.2 mmol respectively. for patients pme and pi increased to 6.2 ± 3.8 and 1.8 ± 0.7, pde showed no significant changes (15.2 ± 7.2). conclusion: using sloop, significant changes of energy metabolism, which is a parameter for hepatic vitality, can be observed in patients with chronic liver disease. compared to conventional postprocessing packages, sloop allows for a robust and quantitative assessment. methods and materials: ct scans of 408 patients with 311 malignant (79 primary and 232 metastatic) and 97 benign changes were retrospectively rewieved over a 5-year period. there were 181 women and 227 men. the study protocol consisted of 5 and 8 mm slices of the liver area. images were obtained before and after intravenous injection of non-ionic contrast agent (iopromidum 623.40 mg/ml) in a standard dosage 2 cm 3 /kg, as a bolus. original films were rewieved and sought in favour of capsular retraction by two independent radiologists. the number, location, size, density of the tumor and presence of capsular retraction were evaluated. the final diagnosis of focal changes was based on histopathological findings. results: twelve of 408 patients (prevalence 2.9 %) revealed retraction of liver capsule, which was associated with adjacent hepatic tumor. all 12 tumors were proven pathologically to be malignant (4 hepatocellular carcinoma, 3 colorectal metastases, 1 stomach cancer metastasis, 1 epithelioid hemangioendothelioma, 1 prostate cancer metastasis, 1 breast cancer metastasis and 1 carcinoid). the occurrence of capsular retraction adjacent to the liver tumor in our study was a rarely observed but a specific sign (specificity 100 %) of a malignant process. purpose: virtualprobe® (iôdp, paris, france) is ultrasound acquisition and post processing software that uses an electromagnetic field to acquire positional data, allowing retrospective multiplanar 3d rescanning through a volume of tissue. the aim of this study is to assess the accuracy of measurements made from the reformatted images of normal structures. methods and materials: 30 patients referred for exams of the kidney (9) and abdomen (21) were enrolled. each patient had a conventional ultrasound exam, followed by the virtualprobe®. the average of 3 measurements of sagittal right (30) and left kidney (28), sagittal bladder (9), sagittal spleen (20), cbd (21), and gb wall (18) were recorded. the correlation of the measurements between both methods was caclulated. analysis of variance for measurements of structures < 1 cm (cbd, gb wall) was performed to assess for the intra-observer variability. the correlation coefficients between methods were 0.84, 0.90, 0.96, and 0.83 for the right and left kidney, bladder, and spleen. the correlation coefficients were 0.62 and 0.23 for the cbd and the gb wall. the correlation coefficient for cbd and gb was 0.56. the variance of measurements for the cbd and gb was 0.22 cm for conventional and 0.24 cm for virtualprobe®. although it exists a statistical difference between both methods for these two structures (p = 0.023), the absolute value of the measurement difference (0.02 cm) was less than the intraobserver variability of the conventional data alone (s.d. = 0.09 cm). to evaluate the possible role of multislice ct (msct) in the preoperative evaluation of patients candidate to laparoscopic splenectomy. material and methods: 32 patients underwent msct with 0.5 seconds gantry rotation time. pre (4 × 2.5 mm collimation) and post-contrast (4 × 1 mm collimation) acquisitions, during arterial and portal venous phases were performed after i.v. administration of 140 ml of non ionic contrast agent at 4 ml/s, with a delay time of respectively 22 and 60 seconds. real time interaction with the post-contrast 3d data set was performed on a dedicated workstation to determine total spleen volume, and to assess arterial and venous vascular anatomy. for volumetric determination an hand-trace editing was used to calculate spleen mass to select candidates for laparoscopic procedure. results: at surgery mean volume was 998 cm 3 . calculation based on ct provided a mean error of 8 %. in none of the patients undergoing laparoscopic splenectomy, a conversion in laparotomy was needed. five patients underwent laparotomic splenectomy because of spleen volume greater than 1200 cm 3 . in all cases optimal vascular detail was achieved. conclusion: high resolution msct afforded complete parenchymal, vascular, and volumetric preoperative evaluation of potential laparoscopic splenectomy. volumetric determination provided accurate and reproducible information. changing the imaging paradigm in ultrasound: can 3d technology improve productivity, decrease sonographer work-load and maintain study quality? m.j. o'neill, s.i. lee, j.f. simeone, g.j. harris, p.j. whelan, p.r. mueller; boston, ma/us purpose: virtualprobe® (iôdp, paris, france) is ultrasound acquisition and post processing software that uses an electromagnetic field to acquire positional data, allowing retrospective multiplanar 3d rescanning through a volume of tissue. the aim of this study was to assess the workflow impact for both sonographers and radiologists as well as overall image quality when conventional ultrasound examination was converted to an off-line interpretation model. methods and materials: 38 patients referred for exams of the kidney (9), thyroid (8) and abdomen (21) were enrolled. each patient had a conventional ultrasound exam, followed by the virtualprobe®. the 3d data were blindly reviewed. the time for conventional vs. 3d performance/review was compared for sonographer and radiologist. the diagnostic findings between both methods were compared. radiologist desire to rescan a patient was recorded on an ascending 5 point scale. the average scanning and image review/rescanning time for the sonographer was 14.2, 17.5, and 15.6 min during the conventional and 3.9, 7.0, and 3.8 min during the 3d for renal, abdomen and thyroid. the average review/ interpretation time for the radiologist was 1.3, 1.5, 1.6 min for the conventional and 1.3, 2.7, 3.0 min for 3d for renal, abdomen and thyroid. the desire to rescan averaged 3.4/5 and 1.2/5 for conventional and virtualprobe. all findings on conventional scans were observed on 3d. 2 findings were observed during the 3d only. conclusion: virtualprobe® allows for a 3 fold increase in sonographer efficiency while minimally increasing radiologist time. overall study quality was not different between both methods. predictive ultrasound factors of an adequate biliary drainage through an endoprosthesis in proximal biliary obstructions z. spirchez, m. tantau, b. diaconu, o. anton; cluj-napoca/ro purpose: to study the ultrasound performance in the evaluation of an internal biliary drainage through an endoscopically inserted endoprosthesis in proximal biliary obstructions. materials and methods: 32 patients (14 males, 18 females, mean age 56, range 41 -82) with proximal biliary obstruction due to hilar tumors, treated by biliary stenting using plastic, 7 -12 f, stents were studied. the efficacy of the drainage was assessed clinically and by biochemical parameters. ultrasonographically the size of the right and left hepatic ducts (rhd/lhd) and the intrahepatic bile ducts were noted before and after stenting. the presence and degree of aerobilia and the position of the stent (the proximal end above or below the stenosis) were noted after stenting. of pancreatic structures and abnormalities were evaluated using a three point grading scheme. furthermore the differentiation of cystic versus solid tumors was compared. results: in the detection of pancreatic lesions there was no statistically significant difference between both modalities (overall sensitivity 67 % with thi and 64 % with bm, specificity 100 % with thi and 97 % with bm). the differentiation between simple cystic, complicated cystic or solid tumors, is improved with thi. the visibility of the pancreas was not sufficient in 23 % because of technical problems (eg. meteorism). in patients with adequate visualisation of the pancreas the pancreatic duct could not be sufficiently delineated in 38 % with thi and 59 % with bm. conclusions: image contrast and delineation of the pancreas as well as diagnostic accuracy are improved with thi in comparison to conventional sonography. characterization of focal liver lesions by pulse inversion harmonic imaging (pihi) with levovist in patients with cirrhosis b. forgács 1 , e. quaia 2 , m. bertolotto 2 , l. crocè 2 , l. dalla palma 2 ; 1 budapest/hu, 2 trieste/it purpose: the aim of this study was to determine capabilities of pulse inversion harmonic imaging (pihi) with levovist in characterization of focal liver lesions in cirrhotic liver. methods and materials: 39 liver focal lesions in 25 consecutive cirrhotic patients identified by baseline ultrasound (us), were evaluated by color doppler (cd) and pihi. pihi was performed 30 seconds (vascular phase) and 3 -5 minutes (late phase) after levovist injection. helical ct (hct) (n = 15) or surgical/bioptic histologic findings (n = 10) were considered as reference procedures. results: 30 lesions classified as hepatocellular carcinoma (hcc) by reference procedures appeared hypoechoic (n = 19), isoechoic (n = 5) or hyperechoic (n = 6) on baseline us, with basket pattern (n = 10), vessels within tumor (n = 6), peripheral (n = 4) or no vascular pattern (n = 10) on cd. on pihi they appeared hyperechoic (n = 26) or isoechoic (n = 4) on vascular phase and prevalently hypoechoic (n = 22) or isoechoic (n = 6) and rarely hyperechoic (n = 2) on late phase. four (4) lesions classified as regenerative nodules by reference procedures appeared hypoechoic on baseline us, with peripheral (n = 1) or no vascular pattern (n = 3) on cd. on pihi they appeared hypoechoic (n = 3) or isoechoic (n = 1) on vascular phase, remaining prevalently hypoechoic (n = 3) or isoechoic (n = 1) on late phase. five (5) lesions classified as hemangioma by reference procedures appeared hyperechoic (n = 4) or hypoechoic (n = 1) on baseline us with few flow signal on cd. on pihi they revealed progressive fill-in or dot-like enhancement during vascular and late phase. conclusions: pihi with levovist had identified some typical enhancement pattern in focal liver lesions in cirrhotic patients. conclusions: mri has low sensitivity to identify small lymph nodes. when visible, mri with sinerem has high sensitivity to identify metastatic lymph nodes; the overall low specificity was due to a learning curve in the first cases. when objective data are combined with subjective impression of the radiologist results are quite satisfactory. to assess the performance of ms-325, a gd-based and albumin-bound contrast agent, for the differentiation of normal and tumor-bearing (vx2) lymph nodes of rabbits with interstitial mr lymphography. materials and methods: experiments were conducted on 4 mature male white new zealand rabbits. fully anesthetized, the animals underwent mr examination twice: prior to and three weeks following implantation of vx2-tumor cells to the left flank. for each session 1.0 ml of undiluted ms-325 was injected subcutaneously into both dorsal foot pads. 3d mr imaging was performed on a 1.5 t whole body scanner prior to as well as 5, 10, and 15 minutes post injection of the compound. contrast medium uptake in the individual lymph nodes and the lymph node size were determined. finally all animals were euthanized and lymph nodes were analyzed by histopathology. results: interstitial mr lymphography with ms-325 visualized lymphatic structures as well as popliteal and inguinal lymph nodes in all animals. vx2 tumours were well differentiated from surrounding tissues in all rabbits. maximum enhancement values were present 10 minutes following contrast medium injection in both normal and tumour-invaded lymph nodes. roi measurements in normal lymph nodes revealed statistically significant higher cnr values compared to tumourbearing lymph nodes. vastly reduced contrast medium uptake in tumour invaded lymph nodes permitted easy detection thereof. the size of normal and tumor-invaded nodes was not significantly different. in addition to providing a means for display of the normal lymphatic system, ms-325-enhanced mr lymphography allows for depiction of direct tumour invasion in lymph nodes. interstitial mr lymphography in rabbits: assessment of inflammatory and normal lymph nodes by different gadolinium-based contrast agents (dota, p760, p792) c.u. herborn, t.c. lauenstein, f.m. vogt, m. goyen, j.f. debatin, s.g. rühm; essen/de purpose: to assess three different gadolinium-based contrast medium compounds in order to distinguish normal from reactive inflammatory lymph nodes by interstitial mr lymphography in rabbits. materials and methods: experiments were conducted on 9, fully anesthetized mature white new zealand rabbits. in each set of three rabbits 1.0 ml of undiluted gd-dota, p760, or p792 (guerbet, france) was injected subcutaneously into the dorsal foot pads. whereas gd-dota is an extracellular, commercially available contrast medium agent, the latter are new macromolecular compounds, undergoing pre-clinical testing. mr examinations were performed three times in each rabbit prior to as well as 2 and 14 days following induction of inflammatory lymph node reaction by the application of freund's complete adjuvant. imaging was performed on a 1.5 t mr system using conventional 3d sequences. contrast uptake in the individual lymph nodes as well as the lymph node sizes were determined. results: interstitial mr lymphography visualized popliteal and inguinal lymph nodes with all evaluated contrast agents. maximal enhancement occurred 10 minutes after contrast medium injection for all three tested compounds. inflammatory lymph nodes revealed statistically significant higher cnr values compared to normal nodes (p < 0.05). average cnr values for the macromolecular contrast medium agents (p760, p792) and the extracellular compound (gd-dota) were not statistically different. furthermore, the size of inflammatory and normal nodes was not significantly different. tuesday b a c d e f 307 conclusion: all three contrast medium agents appear well suited for interstitial mr lymphography to visualize popliteal and inguinal lymph nodes in rabbits. determination of contrast medium uptake can aid in differentiating reactive inflammatory from normal lymph nodes. initial experience with mr-lymphography and employment of iron oxide contrast material for the detection of metastatic lymph node involvement in rectal cancer w. purpose: to evaluate mr-lymphography by employment of iron oxide contrast material (sinerem) in the preoperative evaluation of lymph node (ln) metastases of rectal cancer. method/materials: mr-lymphography of the pelvis was performed using plain and enhanced mr images after 24 to 36 hours after intravenous administration of superparamagnetic iron oxides at a dosage of 2.6 mg fe/kg b.w. conventional t1and t2-weighted spin-echo sequences as well as t2-weighted gradient echo sequences were performed. a total of 8 patients were examined. mri findings were correlated to histopathology in every case. results: a total of 108 ln in 8 patients were histopathologically evaluated and the findings correlated with mri. 7 ln metastases were detected histopathologically. mri was able to detect 6 of the 7 metastatic ln. the size of the analysed ln was approx. 5 mm in most of the cases. all malignant ln presented with a size between 10 and 25 mm. mri missed one ln metastasis with a size of 6 mm. 3 ln metastasis had a size between 6 -10 mm, 2 ln metastasis were 16 -20 mm in size, and 1 ln metastasis had a max. diameter of 25 mm. conclusions: mr-lymphography with employment of the new iron oxide contrast material is a promissing imaging modality for the detection of ln metastasis. studies with a large study population need to further evaluate the potential of this imaging modality. optical imaging of lymph nodes -a new technique for lymphography p. wunderbaldinger 1 , c. bremer 2 , u. mahmood 3 , l. josephson 3 ; 1 vienna/at, 2 münster/de, 3 charlestown, ma/us purpose: to validate the use of near infrared fluorescence imaging (nirf) using enzyme sensitive optical probes for lymph node detection. material and methods: an optical contrast probe that is activated by cystein proteases was used to visualize lymph nodes by nirf reflectance imaging. in order to quantitate the uptake of the optical probe in lymphatic tissue the biodistribution was assessed using the 111 in labeled optical probe. sixteen balb-c mice were injected either intravenously (i.v., n = 10) or subcutaneously (s.c., n = 6) with the nirf-probe (2 µmol gy/animal) and imaged 24 hours after injection. signal intensities and target-to-background ratios of various lymph nodes were measured by manual regions of interest (rois). additional signal intensity measurements were performed of excised lymph nodes (n = 21) from i.v. injected mice (24 hours after injection) and compared to excised lymph nodes (n = 8) of non-injected mice. the probe employed in this study was lymphotropic with about 3 -4 % accumulation in lymph nodes (3.4 ± 0.8 %id/g). lymph nodes showed a high fluorescence signal throughout the body after i.v. injection (96 ± 7.8 au) and/or regionally after s.c. injection (141 ± 11.5 au). the signal intensities of lymph nodes was significantly higher after s.c. probe administration compared to iv administration (p < 0.01), as was the target-to-background ratio after s.c. administration (6.6 ± 0.81 vs 4.8 ± 0.67; p < 0.05). measurements of the excised lymph nodes (after i.v. injection) confirmed a significant increase in lymph node fluorescence signal (26 ± 7.6 au vs. 146 ± 10.9 au; p < 0.0001). conclusion: detection and visualization of lymph nodes is feasible by nirf imaging using enzymatic activatable optical probes. ultrasmall superparamagnetic iron oxide particles (uspio) in magnetic resonance imaging (mri) of primary malignant lymphoma: are there changes in signal intensity of enlarged lymph nodes after contrast application? g. michna 1 , j. kromeier 1 , j. laubenberger 1 , g. paul 2 , m. langer 1 ; 1 freiburg/de, 2 bonn/de purpose: the aim of this study was to assess the potential value of mri with uspio-particles (g534-70, sinerem®) for the nodal staging in primary malignant lymphoma. according to several studies normal tissue in liver, spleen, and lymph nodes shows an uptake of uspio particles due to phagocytic activity of res-cells. material and methods: ten patients with histologically proven untreated primary malignant lymphoma were studied. pre-and post contrast injections of sinerem® mr-scans were obtained from 9 patients. the relative signal intensities of 23 lymph nodes with a size of more than 10 millimeters were measured. we compared (incl. sd) the delta of mean values of pre and post contrast signal intensities in t1-w and t2-w images. results: the diameter of lymph nodes ranges from 13 mm to 105 mm. in t1-w images the delta of mean values of pre-and post contrast signal intensities varied between 0.70 and 1.62. post contrast images showed no significant change in signal intensity. in t2-w images the delta of mean values of pre-and post contrast signal intensities varied between 0.60 and 1.20. there was no significant change in signal intensity in post contrast images. conclusion: there were no significant changes in signal intensity in post contrast t1-w and t2-w images in primary malignant lymphoma. this finding suggests that in these enlarged lymph nodes no active res-cells are present to phagocytise the uspio agent due to the malignant involvement. ionic and non-ionic iodinated contrast media induce neutrophil apoptosis through a caspase-dependent pathway n.f. fanning, b.j. manning, h.p. redmond, j.g. buckley; cork/ie purpose: iodinated contrast media (icm) can induce apoptosis (programmed cell death) in renal and myocardial cells. following intravascular injection, circulating immune cells are exposed to high concentrations of icm. as neutrophils constitutively undergo apoptosis, we hypothesised that icm may adversely affect neutrophil survival. our aim was, therefore, to investigate the effect of icm on neutrophil apoptosis. materials and methods: neutrophils were isolated from healthy subjects and cultured in vitro with ionic (diatrizoate and ioxaglate) and non-ionic (iohexol and iotrolan) icm. neutrophil apoptosis was quantified by annexin v flow cytometry, and caspase dependence determined using the caspase inhibitor zvad-fmk (100 µmol). results: data presented as percentage apoptosis ± s.e.m. iso-iodine concentrations (20 mg/ml) of ionic (diatrizoate 69.6 ± 2.9; ioxaglate 58.9 ± 2.0) and non-ionic (iohexol 57.3 ± 2.9; iotrolan 57.1 ± 2.6) icm significantly induced neutrophil apoptosis over control levels (47.7 ± 1.4, n = 10). ionic icm had a more significant (p < 0.01) apoptotic effect than non-ionic icm (p < 0.05). furthermore, icm reversed the anti-apoptotic effect of lipopolysaccharide (1000 ng/ml) treated neutrophils (23.0 ± 3.5 to 61.2 ± 5.3; n = 4; p < 0.05). iohexol induction of apoptosis is caspase dependent as zvad-fmk reversed its pro-apoptotic effects to control levels (75.7 ± 2.7 to 53.7 ± 1.1; p < 0.05). these results clearly demonstrate that icm promote apoptosis in unactivated and activated neutrophils by a caspase-mediated mechanism. a recent report suggests that icm can increase the incidence of local and systemic septic complications in patients with mild acute pancreatitis (carmona-sanchez r.; arch surg, 2000; 135:1280-4) . icm, through induction of neutrophil apoptosis, could have significant deleterious effects on host immune defence and resolution of an inflammatory response. effect of intravenous injection of iopromide on regional renal blood flow and medullary oxygen tension in diabetic rats f. palm, p.-o. carlsson, p. hansell, a. fasching, o. hellberg, a. nygren, p. liss; uppsala/se purpose: hemodynamic disturbances with subsequent renal medullary ischemia have been suggested as key mechanism in the pathogenesis of contrast media (cm)-induced nephropathy. increased risk of cm-induced renal failure is seen during diabetes mellitus with nephropathy. the present study investigated the effect of an a c d e f 308 injection of the cm iopromide (600 mg i/kg) on regional renal blood flow (cortical and outer medulla) and oxygen tension (outer medulla) in streptozotozin induced four-or nine-week-diabetic wistar furth rats. material and methods: oxygen tension was measured with modified clark-type microelectrodes and blood flow with laser-doppler flow probes. results: administration of cm decreased the outer medullary oxygen tension in non-diabetic animals by ∼ 35 %. this response was absent in diabetic animals. renal outer medullary blood flow of non-diabetic animals increased after cmadministration and remained elevated (∼ 25 %). in contrast, no change in outer medullary blood flow occurred in the diabetic animals. glucose infused non-diabetic animals responded similarly in medullary oxygen tension to normoglycemic animals, but similarly to diabetic animals in medullary blood flow, following cminjection. conclusion: we conclude that diabetic animals have an altered response in renal outer medullary blood flow and oxygen tension to cm compared to normoglycemic animals. however, in contrast to corresponding control rats, outer medullary oxygen tension did not decrease in diabetic animals, which suggests other mechanisms than hemodynamic changes to explain the increased renal failure frequency in this risk group for cm-induced nephropathy. doppler intensitometry: quantitative comparison of two ultrasound contrast agents in humans j.-m. correas 1 , v. dhalluin-venier 1 , l. bridal 1 , p. burns 2 , o. hélénon 1 ; 1 paris/fr, 2 toronto, on/ca purpose: to quantify the doppler signal enhancement following the injection of two different ultrasound contrast agents (uscas) in humans, levovist® (schering sa, germany) and optison® (fso69, mallinckrodt, usa). materials: 24 patients received 2 bolus injection of optison® (1, 2, 3, and 4 ml, randomized dose) and 1 injection of levovist® (2.5 g, 5 ml, 400 mg/ml). the continuous doppler signals from the radial artery were digitized to calculate mean duration of the enhancement, peak enhancement and area under the time-intensity curve (auc). in 3 patients, the doppler signals could not be analyzed due to wrist movement artifacts. results: the duration of the enhancement was respectively (mean ± standard deviation) 255 ± 50 s for the levovist® and 307 ± 93, 304 ± 78, 301 ± 85 and 386 ± 97 s for each dose of optison® (1, 2, 3, and 4 ml). peak enhancement was respectively 17.8 ± 5.1 db for the levovist® and 20.4 ± 3.4, 21.6 ± 3.4, 21.9 ± 2.8 and 22.3 ± 2.2 db for each dose of optison®. auc was respectively (linear units) 5009 for the levovist® and 6311, 10765, 13522 and 15854 for each dose of optison®. a linear relationship was found between the dose of optison® and the enhancement (r > 0.97). levovist® enhancement exhibited a larger variability between patients. purpose: this study was designed to validate the reliability of the ao comprehensive classification using routine radiographs to predict the intra-operative findings. a second goal of this study was to assess interobserver variability of this classification. the radiographs or computed radiographs (cr) of the thoracic and lumbar spines of 32 patients (18 male, 14 female) from jan 2000 to march 2001, were reviewed retrospectively by four separate observers, two experienced staff and two residents without knowledge of the operative findings. each fracture was categorized according to the ao comprehensive spinal fracture classification into type a, type b or type c injuries based on agreed well defined radio-logic criteria. this was compared to the intra-operative findings which assessed the integrity of the posterior ligament complex and mobility of the injured segments. the concordance of the radiologic classification and the intra-operative findings was 75 % (24/32) for orthopedic spine surgeon, 62 % (19/32) for orthopedic resident, 54 % (17/32) for skeletal radiology staff, and 66 % (22/32) for radiology resident. the mean correct designation of the fracture classification was 64.5 %. there were 28 % (9/32) of fractures categorized as type a injuries by 50 % or more of the observers which proved to be type b injuries at surgery. conclusion: the use of plain films or cr to categorize fractures of the thoracolumbar spine according to the ao classification was associated with a designation of 28 % of unstable b injuries as stable type a injuries. there is moderate interobserver variability. with suspected spine injury were evaluated using rx and spiral ct performed in admitting area. all patients received standard thoracic ct (5 mm/pitch 1.5) and abdominal ct (8 mm/pitch 1.5) according to an established trauma protocol including sagittal mpr reconstructions of suspicious vertebral bodies. rx and ct were independently evaluated by two blinded radiologists for detectability of fracture signs and classified in three groups (g1 -g3): definitely fractured (g1); non conclusive result (g2); definitely no fracture (g3). all suspected fractures were confirmed by an additional high resolution ct (2 mm -3 mm/pitch 1.5) and clinical follow up. results: in 21 patients (37 %) of the 56 patients a spine fracture could be depicted. 2 fractures were missed in rx, none in ct. 98 % of the ct reports and 89 % of the rx reports were classified correctly true positive (g1) or true negative (g3). 8.4 % of rx-reports were non conclusive (g2), but only 1 % in ct. in comparison ct versus rx sensitivity was 1/0.88, specificity 0.98/0.92, positive predictive value 0.93/0.92 and negative predictive value 1/0.97 conclusion: using an established standard ct protocol for chest and abdomen examination with slice thickness of 5 mm and 8 mm respectively all relevant spine fractures can be detected. these results suggest that rx of thoracic and lumbar spine is not mandatory in the admitting area, when thoracic and abdominal ct is performed. correlation between ct and clinical findings in patients with spine injuries -analysis of 193 cases t. turek, m. sasiadek; wroclaw/pl purpose: to analyze the correlation between ct appearances of spine injuries and clinical findings and thus to establish the clinical significance of the finding at ct. methods and materials: ct studies have been performed in 193 patients after spine injury. in most of them plain films of the spine were performed prior to ct. 27 patients had also mr. head ct was performed in 25 cases and chest ct in 18 patients. ct findings (the presence of fractures and facet joints injuries, number of involved vertebrae and spinal columns, narrowing of spinal canal, instability features, intervertebral disc lesions, normal study, etc.) have been compared statistically with clinical symptoms (pain, spinal medulla injury, radicular symptoms, disturbance of consciousness, etc.). results: there was statistically significant correlation found between clinical signs of spinal medulla injury and several ct symptoms (instability, spinal canal narrowing, number of affected vertebrae and columns, facet joint lesions). in patients with pain and no severe neurological signs there was positive correlation with a normal ct study. radicular symptoms have been associated with intervertebral disc lesions and in chronic cases with degenerative changes of the spine. consciousness disturbances have been negatively correlated with spine fractures, while positively with normal ct appearance of the spine and traumatic changes on head ct. conclusion: ct symptoms of spine injury are highly correlated with severity of the clinical and neurological findings, except for consciousness disturbances which are associated with simultaneous head injury. thus, ct is very helpful in establishing prognosis and treatment planning. the purpose of this study is to evaluate the efficiency and safety of self-expandable stent insertion in patients with acute colonic malignant obstruction before elective surgical resection. between march 1999 and march 2001, 24 patients (15 males and 9 females, mean age 74, range 41 -93) were included. all the patients were scored asa iii. colonic cancers were located between the rectosigmoid and the middle part of the descending colon. all patients had plain abdominal radiography and ct scan to demonstrate the complete obstruction of the colon and to localise the stenosis. immediate technical success was obtained in 23 of 24 patients (96 %). the mean time between stent insertion and surgery was 6 days (3 to 9). in 2 cases (8 %), stents were misplaced and did not covered the lesion which required the insertion of an other stent. in 2 (8 %)other cases, the stent was initially well positioned but migrated secondarily which required the insertion of a second stent in one and a surgery in a other one. colonic perforation occurred in 3 (12 %) patients. these 3 patients underwent surgery, one of them developed an abscess which required percutaneous drainage under radiological guidance, other (8 %) patients underwent ileostomia because of a missprepared colon by stent dysfunction (obstruction). there was no more morbidity (21 %) and no death (0 %) at the moment. self-expandable stent insertion is an efficient minimally invasive procedure that allows for single-stage surgery. however, this technique is quite difficult to perform and may be associated with some complications. sites of obstruction were antro-duodenum (n = 18), postoperative anastomotic site (n = 19; 12 gastroenterostomy, six esophagojejunostomy, and one duodenojejunostomy). all stents were covered with polyurethane. the size of the stent ranged from 15 -20 mm in diameter and 7 -20 cm in length. with fluoroscopic guidance, covered self-expandable metallic stents were placed. results: stent placement was technically successful in all patients without or with the gastrostomy (n = 3) and balloon dilatation (n = 3). after stent placement, symptoms improved in all but one patient, who had another stenosis at the proximal jejunum. during the follow-up of 2 -73 (mean 13) weeks, stent migration occurred in four patients (10.8 %) 1 -41 days after the procedure. those patients were treated successfully by means of placing a second covered metallic stent (n = 3) or endoscopic reposition (n = 1). recurred obstructive symptoms due to tumor overgrowth (n = 2) and mechanical failure of stent (n = 2) were noted. one of tumor overgrowth and both of the mechanical failure of stent were treated by means of coaxial placement of a second covered stent with good clinical result. conclusion: fluoroscopy-guided placement of covered self-expandable metallic stents can be considered as the primary choice for the palliation of malignant obstructions in those patients. the laparoscopically placed adjustable gastric band (lap-band™) has been popularised as a minimally invasive, adjustable and completely reversible surgical technique for the management of morbid obesity. we report our experience with particular emphasis on the radiological aspects of follow-up and patient results in terms of short-term weight loss and complications. methods: between may 1998 and december 2000 50 patients were treated (8 male/42 female) with a mean age of 37 years (range 30 -48 years). the technique of fluoroscopy guided band inflation is illustrated with particular emphasis on radiological lessons learned. a new method of measurement of pouch volume and the passage within the band is described. results: mean number of radiology-guided inflations per patient was 2.6 (range 1 -7). the volume used for inflation was noted to be critical, with 0.5 ml making the difference between complete obstruction and optimal band inflation. the mean hospital stay was 2.8 days (range 2 -10 days). 4 % had gastric pouch formation and 4 % had nonfatal pulmonary embolism. there was no mortality. the follow-up was up to 30 months. the mean bmi decreased from the pre-operative level of 43 kg/m 2 (range 38 -55 kg/m 2 ) to 34.5 kg/m 2 . the lap band is an effective procedure in the treatment of morbid obesity. initial weight loss is comparable with other open bariatric procedures but with the added benefit of a shorter hospital stay, less pain and a low frequency of complications. fluoroscopy guidance is useful in puncturing the subcutaneous valve and mandatory in assessing optimal band inflation. radiological percutaneous gastrostomy with ballon retained catheters in malignant upper digestive tract obstruction h.-p. dinkel, j. triller; berne/ch purpose: to assess success and feasibility of radiological percutaneous gastrostomy in malignant pharyngeal or esophageal obstruction where endoscopic passage was impossible. methods: we report a prospective series of 30 consecutive patients with esophageal and/or head and neck tumors who required artificial enteral feeding by means of gastrostomy. in all patients endoscopic passage was not possible or extremely difficult. in order to avoid surgical gastrostomy we performed a radiological gastrostomy using the following technique. first, the stomach was punctured under fluoroscopic guidance and gastropexy using 3 -4 cope-type t-fasteners was performed. then a balloon-type gastrostomy catheter was inserted using a peel-away sheath. results: pg was technically successful in all cases. the gastrostomy remained for 5 to 26 weeks. complications occurred in 10 % (2 minor, 1 major). one patient developed a peristomal leakage due to bowel atony and incomplete voiding of the stomach managed by insertion of a jejunal nutrition catheter through the gastrostomy tract whereafter the leakage subsided an the gastrostomy tract healed. one patient had minor suture infection and delayed gastric voiding. in one patient the early catheter dislocation and peritonitis occurred due to erroneous food injection into the balloon port. conclusion: radiological gastrostomy by means of ballon-retained catheters is a feasible and relative safe procedure. most complications can be managed conservatively or interventionally. care should be taken to avoid erroneous injection into the balloon port, especially within the first week of pg in order to avoid tube dislocation. to compare the diagnostic accuracies of sonography, scintigraphy in diagnosing ileocecal crohn's disease (cd) and assessing its activity compared to enteroclysis and final diagnosis. method: a retrospective analysis of the studies of 54 patients (30 females, 24 males, ages of 18 -71) with verified cd (operation: 8, biopsy: 50 cases). located to the ileocecal region was made. all patients had enteroclysis, sonography (4 -8 mhz receiving frequency with native tissue harmonic imaging, and measurement of the flow in the superior mesenteric artery (sma)) and 45 of them had 99 tc agab immunoscintigraphy with spect. the results were compared. results: overall sensitivity of enteroclysis, sonography and immunoscintigraphy was 100 %, 89 % and 71 %. in early crohn disease (12 cases) the sensitivities were 100 %, 75 % and 40 % respectively. in detecting active disease (determined by the presence of ulcerations or fistulas in enteroclysis) immunoscintigraphy had 70 % sensitivity, 33 % specificity. increased sma flow had 76 % sensitivity; 100 % specificity, while wall thickening (> 3 mm) had 92 % sensitivity; 33 % specificity. using a combined sonographic criteria (presence of destroyed wall stratification or detection of fistulas or abscesses or increased sma flow) 96 % sensitivity and 100 % specificity is obtained. surgical indications (fistulas or abscesses) were detected in 6/8 cases by both enteroclysis and sonography. conclusion: although the sensitivity of sonography in early cd is not sufficient for detecting suspected cd, for follow up in advanced cases sonography could replace enteroclysis. new combined sonographic criteria gives excellent correlation with enteroclysis detected activity. sensitivity for detecting abscesses might compensate for missed fistulas. crohn disease: evaluation of small and large bowel with combined ct enteroclysis and ct colonography a. cieszanowski, k. wojciechowski, r. pacho, b. pruszynski; warsaw/pl purpose: to evaluate the new method -combined ct enteroclysis and ct colonography -for the assessment of whole bowel in patients with crohn's disease. method and materials: 20 patients suspected of having crohn's disease underwent ct colonography after i.v contrast material injection. in 10 of them 1500 ml of fluid was administered by nasoenteric tube positioned in the duodenojejunal region (ct enteroclysis) and in another 10 patients water was given orally (1500 -2000 ml), beginning 30 -40 minutes before the examination. intestinal and extraintestinal abnormalities were evaluated. small bowel distention was assessed in 4 grade scale (good = 4, poor = 1). results were compared to enteroclysis, colonoscopy and intraoperative findings. results: in 15 patients with confirmed crohn's disease ct correctly identified all involved small bowel (n = 17) and colonic (n = 8) segments, although underestimated their length in 5 cases (20 %). mesenteric involvement was seen in 10 of 15 patients with crohn's disease (67 %), lymphadenopathy in 14 (93 %), perianal disease in 6 (40 %), abscess and fistula in 1 patient (7 %). the distention of small bowel was significantly better on ct enteroclysis (mean score 3.3) than on ct performed after oral fluid administration (mean score 2.7). conclusion: both techniques enabled simultaneous evaluation of small and large bowel during single examination and allowed identification of all involved bowel segments, although, visualization of small bowel was significantly better with the use of ct enteroclysis than after oral administration of fluid. material and methods: 50 patients affected by cd were examinated at 1.5 t, using fat and non fat-suppressed t2 w haste sequences after administration of a negative superparamagnetic oral contrast agent and t1w flash sequences, pre and post gd-dtpa injection. all patients underwent endoscopy, us, barium studies (gold standard), 7 had surgery. all mri data were blindly evaluated by two radiologists. the degree of strictures, the presence of small bowel dilation, adhesions, sinus tracts, fistulas, abscesses, biliary, pancreatic and renal abnormalities were evaluated. results: mri correctly detected 95 % of strictures (100/105), 100 % of adhesions (47), 85 % of fistulas (18/21), 100 % of abscesses (5), hydronephrosis (2), biliary, renal and pancreatic stones (5). in 6/50 patients (12 %) mri showed complications undetected by the gold-standard modalities. conclusions: mri is a promising method to assess main complications of cd. differentiation of inflammatory bowel diseases by hydro-mri k. schunk, s. reiter, a. kern, s. schadmand-fischer, t. orth; mainz/de purpose: to assess hydro-mri regarding the differentiation of inflammatory bowel diseases. after an oral bowel opacification using 1000 ml of a 2.5 % mannitol solution and a rectal bowel opacification using 250 -500 ml of an 0.9 % saline solution, axial and coronal breath hold sequences ± gd-dtpa (haste ("half-fourier acquisition single-shot turbospinecho") and dynamic flash ("fast low angle shot")) were acquired in 27 patients with inflammatory bowel disease. by mri findings, crohn's disease (cd; n = 15) and ulcerative colitis (uc; n = 12) should be differentiated. results: cd and uc showed significant differences regarding the number of affected bowel segments (2.3 ± 1.5 vs. 1.2 ± 0.5; p = 0.01), the incidence of nodular bowel wall thickening (1/15 vs. 8/12; p = 0.002), and the incidence of a blurred demarcation of the inflamed bowel wall against the surrounding mesenterial fat tissue (9/15 vs. 1/12); p = 0.007). there were no significant differences regarding the contrast enhancement of the inflamed bowel wall (+37.7 ± 28.3 % vs. results: before treatment unenhanced pd showed circumscribed hypervascularity of affected bowel loops, consistent with acute disease relapse, in 13/18 patients (72.2 %); in 5 patients the typical acute stage hypervascularity was demonstrated only with ce-pd (p < 0.05). in seven patients with complete clinical recovery no residual enhancement of the affected loop was found at post-treatment follow-up by either unenhanced or ce-pd. of the patients with clinical persistence of acute disease, 6 (54.5 %) showed typical hypervascularity on conventional unenhanced pd, 4 (36.3 %) had hypervascularity documented only on ce-pd (p < 0.05) and 1 (9.2 %)had no enhancement detected even after contrast administration. ce-pd had higher sensitivity (90.8 %) compared to conventional pd and 100 % specificity. purpose: to assess feasibility, safety, and effectiveness of percutaneous imageguided radio-frequency (rf) thermal ablation of the lung. the research project includes an experimental animal study and two clinical phases. in the animal study, 12 rabbits will undergo in vivo rf ablation of lung tissue. rabbits will be sacrificed and histologic analysis of the specimens will be performed to assess cell viability. the clinical part of the project includes two phases: (1) 10 patients with resectable cancerous nodules will undergo ct-guided rf ablation and subsequent pulmonary resection and pathology examination of surgical specimens; (2) 30 patients with unresectable lung tumors will undergo ct-guided rf treatment; in these cases the outcome of rf ablation will be assessed by follow-up ct studies performed 1 month after the procedure a c d e f 314 and at 3-month intervals thereafter. rf ablations are performed by using a 150 w generator (rita medical systems) and a 15 gauge, 9 hook expandable electrodeneedle. results: to date, 8 new zealand white rabbits underwent in vivo fluoroscopyguided rf of lung tissue. two rabbits were sacrificed immediately, one after 24 hours, two after 3 days, two after 2 weeks, and one after 1 month. on gross examination, round, sharply demarcated thermal lesions measuring 21.4 ± 2.1 mm in diameter were observed in all cases. histopatology analyses confirmed coagulation necrosis with no intervening viable cell. conclusion: findings of the animal study showed that rf ablation of pulmonary rabbit parenchima can be safely and effectively performed via a percutaneous, transthoracic approach, thus prompting the start of clinical studies. b-0922 10:40 2-component fibrin clue injection for needle tract obliteration during ctguided percutaneous cutting biopsy of lung lesions m.c. freund 1 , c. riedl-huter 1 , k.m. unsinn 1 , m. hackl 2 , t. schmid 1 , w.r. jaschke 1 ; 1 innsbruck/at, 2 natters/at purpose: pneumothorax is the most common complication of percutaneous lung biopsy. in order to reduce the incidence for pneumothorax a commercially available 2-component fibrin glue (tissucol® duo quick) was injected in the puncture tract during the withdrawal of the coaxial sheath at the end of the examination. methods and materials: in total 120 consecutive adult patients underwent ctguided percutaneous biopsy for histological evaluation of an intrapulmonary lesion utilizing a 17 -18 g coaxial cutting biopsy system. all lesions were surrounded by aerated lung parenchyma and were located within 0.5 -12.0 cm distance (mean, 3.3 cm) from the pleural surface. lesion size measured 0.8 -10.0 cm (mean, 3.6 cm), in total 1 -8 biopsy passes per lesion (mean, 3.6) were performed, and in 57/120 patients a perilesional hemorrhage 0.7 -4.5 cm diameter (mean, 1.9 cm) was observed. patients were assigned to undergo biopsy either without (group i) or with (group ii) the use of a 2-component fibrin glue. all patients were observed after biopsy; after 2 -4 hours a spiral-ct of the thorax and after approximately 24 hours a chest radiography was performed for pneumothorax detection. the rate of ct-proven pneumothorax resp. for chest tube insertion in group i was 40 % resp. 4 % and in group ii 10 % resp. 2 %. conclusion: the injection of a 2-component fibrin glue in the puncture tract during withdrawal of the coaxial sheath significantly reduces the incidence for pneumothorax as well as the rate for chest tube insertion. parietal extrapleural saline for the reduction of pneumothorax in percutaneous lung biopsies (plb) t. petsas, i. tsota, c.p. kalogeropoulou, m. karamesini, n. samaras, d. dougenis, i. dimopoulos; patras/gr purpose: in this study we evaluate the efficacy of extrapleural injection of normal saline at the initial procedural phase of percutaneous lung biopsy in order to prevent pneumothorax. methods: 66 patients underwent plb under ct guidance, for diagnosis of lung lesions. the patients were randomized in either group a or b. in 33 patients (group a) normal saline was injected beneath the parietal pleura just before the procedure. in the remaining 33 patients (group b) no saline was used. all biopsies were conducted under ct using a 19 g -22 g coaxial needle system. in group a, after local anesthetic infusion, the needle was advanced in the subpleural fat and 20 -30 ml of saline were injected extrapleurally, in order to create a bulge of pleura into the lung parenchyma, adjacent to the lesion. all patients had ct scan 15 min after, and plain chest radiogram after 4 hours after the procedure in order to evaluate the presence of pneumothorax. results: the procedure was well tolerated in group a. pneumothorax developed in 3 patients (9.1 %) in group a. in group b, pneumothorax occured in 7 patients (21.2 %) and in one instance drainage was required (3.03 %). the technique prolonged the duration of the procedure 9 -17 min in group a. conclusion: our results suggest that extrapleural injection of saline reduces plb induced pneumothorax. it is a supplementary technique, which can be used in patients who are prone to develop pneumothorax. application of a new specially designed guide stylet set in chest biopsies t. petsas, c.p. kalogeropoulou, i. tsota, d. karnabatidis, p. angele, d. siablis, i. demitrios; patras/gr purpose: we evaluate the efficacy of a specially designed guide-stylet for percutaneous chest procedures. in 50 patients, who underwent percutaneous procedures (lung biopsies n = 30, mediastinal n = 10, spinal and rib lesions n = 10), a new specially designed guide stylet set was used. the set is composed of a guide stylet and a 22 chiba needle. the guide stylet is made of stainless steel with a diameter of 0.41 mm (william cook europe a/s, bjaeverskov, denmark) the 22 g chiba needle is initially introduced in the thoracic wall and the guide stylet is advanced through the needle towards the lesion. when the stylet has been correctly directed, the needle is also advanced towards the same direction. the stylet serves either as a guide for the biopsy needle, or as an exchange wire in cases where a larger (core or tru-cut) needle is required for adequate sampling. the stylet can be curved before its insertion inside the 22 g needle in order to reach lesions which require redirection of the needle. for this purpose a torque device is used to point to the direction of the curved stylet results: the technique was easy to perform in all cases. five patients with lung biopsies developed pneumothorax (16.66 %). the stylet was advanced beyond the lesion in 4 cases, without clinical consequences and without this affecting the biopsy outcome. conclusions: our results suggest that the guide-stylet is a useful tool for percutaneous procedures, especially biopsies, in cases were the lesions to be reached, are difficult in approach. new technique for ct guided lung biopsy k. ishii 1 , y. wada 2 , j. kanekawa 2 , t. takahashi 2 ; 1 moriya/jp, 2 ibaraki/jp purpose: we establish both new device and a biopsy method to localize lesions in term of three-dimensions by ct in order to perform the biopsy of small nodular lesions in the lung field confidently, safely, and rapidly. material and method: we carried out ct guided lung biopsy in 20 cases of nodular lesions more than 5 mm in diameter. none of them contained calcifications. we simulated a targeting line by using a new device for ct guidance, "targ" developed by us. we then confirmed the right track of the guiding needle. the biopsy needle that we used was "pro-mag". the average time needed to complete the procedure was fifteen minutes. results: ten lesions were primary lung cancer. one of them was ground glass opacity lesion measuring 7 mm in diameter. this is what we call carcinoma of type a classified by noguchi. one case was lung metastasis from colon cancer (8 mm in diameter). conclusion: this technique enables us to obtain enough material by pinpointing and shooting peripheral lung lesions approximately 10 mm in diameter. we strongly believe that this technique contributes significantly to the progress of lung cancer treatment. ct guided percutaneous fine needle biopsy of small (≤ 15 mm) lung lesions in outpatients: safety and efficacy of the procedure compared to inpatients m. romano, m. gentile, m. midulla, m. salvatore; naples/it objective: compare safety and efficacy of ct guided fine needle biopsy (fnab) of small (≤ 15 mm) lung lesions in inpatients and outpatients. materials and methods: 100 consecutive inpatients (65 m, 35 f, mean age 56) and 100 consecutive outpatients (72 m, 28 f, mean age 50) who underwent ct guided fnab of small lung lesions at our institution from may 1999 to july 2001 were included. lesion size, thickness of aerated lung traversed, number of needle passes, presence of emphysema were recorded for the two patient groups. 22 g chiba needles and the roll-over technique (1 hour) were used for all patients; in the absence of significant pneumothorax at a subsequent expiratory chest roentgenogram, outpatients were allowed to go home and instructed to return in case of dyspnea or unusual symptoms. incidence of pneumothorax and other complications, need for tube insertion and percentage of diagnostic samples were noted for both groups. results: no statistical differences were observed in lesion size, thickness of aerated lung traversed, needle passes, presence of emphysema between the groups. we had 15 pneumothoraces in inpatients, two requiring a small caliber chest tube, and 12 in outpatients, all within 1 hour from the procedure, none requiring a tube. citology diagnosed a malignant lung neoplasm in 58 outpatients and 64 inpatients, absence of malignant cells in 33 and 25; the sample was inadequate in 9 and 11 respectively. all differences are nonsignificant. conclusion: ct guided fnab of small lung lesions in outpatients is a safe and reliable procedure, allowing significant decrease in hospitalization costs. tuesday b a c d e f 317 nant, 3 % adh. the highest rate of malignancy was seen in indication c (47 %) > a (32 %) > b (28 %) > e (20 %) > d (18 %). in the 71 % benign cases mrvb was able to avoid diagnostic surgery. conclusion: mrvb is a reliable tool, which allows to solve problems of mr-detected lesions. good training is a prerequisite. radiological or surgical port device implantation: a 4-year institutional analysis of procedure performance, quality of life and cost for breast cancer iv chemotherapy p.y.r. marcy sr. 1 , c. bailet 1 , n. magne 1 , j.c. machiavello 1 , j.c. gallard 2 ; 1 nice/fr, 2 caen/fr purpose: to evaluate the safety, efficacy, quality of life and cost of percutaneous radiologic arm port (pap) and surgical subclavian port (ssp) devices. this study involved a retrospective review of 200 port device implantation procedures (100 pap, 100 ssp) performed over a 4-year period, in breast cancer patients. parameters analyzed included technical success, procedure duration, complications, analysis of the quality of life and cost evaluation for both techniques. the success rate for port implantation was higher for pap than for ssp (96 % versus 91 %). pap was subsequently performed in 2 of ten patients in whom ssp procedure had failed; and ssp in 1 of the three pap failure cases. ssp and pap were performed without conscious sedation (i.e., local anesthesia only). mean implant duration time was 168/222 days, the overall complication rate (r/s) was 9 % versus 13 % (0.24 -0.4/1000 patient-days). mean implant duration time (r vs s), without any complication or death, was 193 d vs 233 d. median number of chemotherapy courses was 6 (r = s). 6 %, and 7 % respectively of the devices had to be removed prematurely. complication rate was 7 % and 9 %. febrile neutropenia cases occurred in 2 % of each file, without occurrence of any port infection. direct costs (r/s) were respectively 230.8 vs 219.1 • . conclusion: advantages of r over s include higher success rates, shorter procedure duration, higher cosmetic result despite a 5 % relative overcost for r placement. to study functional recovery after surgical damage to the supplementary motor area (sma). patients and methods: 6 patients were studied before and after resection of a medial frontal tumor, and compared to 7 healthy subjects. all of the patient group presented with an sma syndrome. fmri examinations (1.5 t) included functional axial eg-epi and anatomical images. tasks consisted of flexion/extension of the fingers and a complex digital sequence of both hands. data analysis was performed using spm99 software. results: in control subjects and in patients during movements of the hand ipsilateral to the tumor, activation was present in the contralateral hemisphere in the primary sensorimotor cortex, postsma, and both secondary sensory cortex. during complex movements, additional activation was present in the contralateral presma, anterior cingulum, ipsilateral primary sensory cortex, lateral premotor area, and both parietal lobules. before surgery, activation patterns were similar. however, during simple movements contralateral to the tumor, additional activation was present in the ipsilateral postsma and lateral premotor area. during complex movements, activation was also present in the ipsilateral presma and anterior cingulum. after resection of the sma, the presma and the anterior cingulum of the healthy hemisphere were also activated during simple movements. during complex movements, activation in the ipsilateral presma and lateral premotor area was stronger. these results suggest that there is a dysfunction of the sma ipsilateral to the tumor before surgery, and that the sma, anterior cingulum, and lateral premotor area in the healthy hemisphere are involved in the functional recovery following surgery. purpose: first experiences in comparing different methods for localization of brain function in the setting of neuronavigated guided surgery. maximum accuracy by using the same mri-3d-dataset for fmri and neuronavigation. material and methods: 12 patients suffering from intracerebral tumours in the central region performed a finger-tapping task of the contralateral hand in fmri. activated areas were superimposed on a 3d-ge-mri dataset including the surface fiducials for neuronavigated surgery. the coordinates of the voxel with highest statistical threshold was determined and compared with the coordinates of maximal response using intraoperative monopolar cortical stimulation. results: in all patients central sulcus and postcentral gyrus were identified by fmri. in 2 patients intraoperative stimulation was not possible. in 10 patients coordinates of maximum activation differed by 12 mm mean euclidean distance (stdev. 7 mm, range 2 to 28 mm). the distance was mainly dependent on the different approach of cortical stimulation to the surface of the brain and fmri activation in the depths of central sulcus (hand motor area). larger distances were due to a distorted morphology of central area from invading tumour. there was no influence of tumour histology, had motion during the fmri experiment or hemiparesis. conclusion: in the setting of neuronavigated guided surgery and intraoperative monitoring, fmri may help to guide the placement of a cortical electrode on the surface of the brain. maximum stimulation or activation points differ due to the approach, especially if morphology of the central area is distorded by tumour. this has to be considered in preoperative planning. 3d mr reconstruccion for neurosurgical planning f. matute, a. saiz ayala, j. arrazola garcía, j. jimenez del rio, c. saldaña; madrid/es purpose: we used three-dimensional reconstructed magnetic resonance images for planning the operations of 40 patients with intraxial brain tumors and vascular malformations. we studied the cases of these patients to determine the advantages and current limitations of our computer assisted surgical planning technique as it applies to the treatment of neurosurgical cases. we assessed the cortical surface-anatomy combining with vascular images using a single volumetric acquisition scanning technique from 3d spgr. in collaboration with neurosurgeons, we obtained tangential and oblique views, that simulates as properly as possible, the real surgical field. this technique, allow us not only to localize the lesion and define the tumor margins but to identify the superficial cortical veins related to the tumor. this multiplanar superficial venography will be used as a road map by the neurosurgeons at the moment of the craniotomy. in addition, a introperative electrical stimulation mapping was performed to assesses eloquent cortices. results: in each case neurosurgeons could easily reach the lesion with minimum risk because the combination of our multiplanar reconstruction model and intraoperative electrical mapping. conclusion: our technique is a easy and useful way of proper determining brain surface and venous anatomy over the tumor. this technique can be used to choose the best method of intervention, to minimize the surgical risk and to select the best surgical approach. intraoperative mri in a stereotactical operation unit: report on the first two cases v. hesselmann, r. girnus, u. von smekal, m. hoevels, b. krug, v. sturm, k. lackner; cologne/de purpose: to introduce practical issues for interventional procedures controlled by mri (gyroscan intera, philips, best, the netherlands) in a stereotactical operation unit. we report on a mri-controlled stereotactically guided catheter implantation for interstitial irradiation with iodine 125 seeds (case 1) in a patient with glioblastoma and the implantation of electrodes for deep brain stimulation (medtronics, minneapolis, usa) in a patient with parkinson's disease (case a c d e f 318 2). patients were placed on a mri-compatible operation table and fixed in a mricompatible stereotactic head frame (mrc-systems, heidelberg, germany). trajectory and target planning had been done prior to the intervention with a computerized stereotactic treatment planning system (fischer leibinger freiburg, frg). anatomical (t2-tse, t1-se), real-time mri and functional imaging (fmri) using a fingertapping paradigm was performed in both cases. in case 1 fmri was performed after placement of the head frame for localizing the motor hand area. in case 2 fmri was performed during deep brain stimulation of the subthalamic nucleus. results: in case 1 the trajectory for the catheter with 125 i seed was remodeled after fmri. performance on fmri for monitoring the effect of deep brain stimulation was poor in case 2, as the localizer of the stereotactical system caused severe distortions of epi-images. monitoring the placement of deep brain electrodes could easily be controlled mri with t2-weighted spin echo sequences. conclusion: intraoperative mri revealed decisive information during operation procedures such as brain shift, fmri and placement of catheters and electrodes. for fmri metallic materials are to be removed out of the scanner. neurovascular compression of the rostral medulla as a cause of essential hypertension? prospective mr study in hypertensive and normotensive subjects j. zizka 1 , j. ceral 1 , p. elias 1 , l. klzo 1 , m. solar 1 , j. tintera 2 ; 1 hradec kralove/cz, 2 prague/cz purpose: several experimental studies assigned vascular compression of the left rostral ventrolateral medulla (lrvm) as a possible cause of essential hypertension. published data concerning the mr imaging of the neurovascular compression of the medulla oblongata widely differ, particularly due to methodological incongruities. the aim of the study is to conduct a blinded prospective mr study of normotensive and hypertensive individuals. methods and materials: we examined 32 patients with severe essential hypertension (mean age 52 ± 9 years) and 40 normotensive subjects (mean age 53 ± 10 years). mr imaging protocol consisted of transverse and coronal t2 tse (slice thickness 3 mm), transverse 3d tof mra (0.8 mm) and 3d ciss (1 mm) sequences. the presence and degree of vascular compression at the lower brain stem and particularly at the lrvm were evaluated together with the conspicuity of the anatomical structures on different mr imaging sequences. results: among 32 hypertensive patients, 24 (75 %) showed neurovascular contact of the medulla at any location and 6 (19 %) at the lrvm. in the control group of 40 normotensive subjects, 32 (80 %) showed neurovascular contact of the medulla and 13 (32 %) of the lrvm. compared to t2 tse and 3d tof imaging sequences, 3d ciss offered better contrast resolution of neural and vascular structures and superior delineation of the outer vascular contours. conclusion: neurovascular compression of medulla oblongata is a frequent finding in both hypertensive and normotensive subjects. our results do not support the hypothesis of neurovascular compression at the lrvm as an etiological factor of essential hypertension. volume estimation of the fornix in patients with organic amnesias using co-axial mr images q.y. gong 1 , d. montaldi 1 , a.r. mayes 1 , j. aggleton 2 , j.r. hanley 3 , d. scutt 1 , n. roberts 1 ; 1 liverpool/gb, 2 cardiff/gb, 3 essex/gb purpose: the fornix is the principal tract that links the hippocampus with the midline diencephalon. the relative contribution of the fornix in human memory is debated and a reliable method is required for estimating the extent of fornix damage in patients with anterograde amnesias. this study aims to develop an efficient and unbiased approach for estimating the fornix volume on 3d mr images. subjects and methods: 12 healthy subjects and four patients with organic amnesia (one or other of the fornices were severed) were recruited. mr examinations were performed using a 1.5 t signa (general electric, milwaukee), and t1weighted images were obtained using a 3d spgr pulse sequence (tr/te 34/ 9 ms, flip angle 30°, slice thickness 1.6 mm). image analysis was performed using analyze tm (mayo foundation, rochester) and mri3dx (http://www.liv.ac.uk/mariarc/mri3dx). analysis algorithm using reformatted co-axial mr images was developed based on the pappus method of modern design stereology. results: the measurement reliability for fornix volume were obtained (cv = 3.61 %; r = 0.98). fornix volume in controls obtained was 0.46 ± 0.07 ml on the right and 0.46 ± 0.10 ml on the left. in three of four patients with radiological evidence of severance of either the left and/or right fornix, significant atrophy of the ipsilateral fornix was demonstrated, and ranged between 46.1 % and 22.3 %. the developed technique provides an efficient and mathematically unbiased approach for estimating the fornix volume on co-axial sections, and can be applied to assess the extent of fornix atrophy in patients with organic amnesia. voxel based morphometry in narcolepsy s.c.a. steens 1 , s. overeem 1 , c.d. good 2 , g.j. lammers 1 , m.a. van buchem 1 ; 1 leiden/nl, 2 london/gb purpose: recently, a close association between narcolepsy and hla-dqb1*0602 was found and linked to loss of hypocretin production by hypothalamic neurons, as suggested by lack of csf-hypocretin-1 levels. the aim of this study was to assess potential (autoimmune-based) hypothalamic degeneration using voxel-based morphometry. methods/materials: 15 narcoleptics and 15 age-and sex-matched controls were included. all patients were hla-dqb1*0602-positive, had typical findings during polysomnographic and multiple sleep latency testing, and had no csf-hypocretin-1. to optimize gray-white matter contrast, we used a 3d-t1 gradient-echo sequence with tr/te 26/12 ms, flip-angle 45°, 256 × 256 matrix, 100 slices of 1.5 mm and fov 250 mm. vbm was used for brain extraction, spatial linear and non-linear normalization, segmentation and smoothing. univariate analyses for gray and white matter were performed with statistical parametric mapping, employing the general linear model. regionally specific increases or decreases in gray or white matter between groups were assessed, with age, sex and global mean voxel values as confounders (p < 0.05, corrected for whole brain volume). because of our strong prior hypothesis for structural change in the hypothalamic region, we also assessed uncorrected data with a small volume correction for the hypothalamus (p < 0.001). results: no significant regional differences in gray and white matter could be detected between narcoleptics and controls, even with reduced thresholds and small volume corrections for the hypothalamus. conclusion: no evidence is found for structural changes in the hypothalamic region. either the hypothalamic changes are too subtle to be detected using vbm, or the hypocretin neurons remain intact but are not able to synthesize hypocretin. evaluation of requests for mri performed during 2 weeks in stockholm, sweden b. isberg, o. flodmark, l. zachrisson, h. jorulf, y. palmquist; stockholm/se over the last years the use of mri as a diagnostic tool has increased as the method has been recognised as the most efficient way to investigate a variety of clinical conditions. in stockholm the use of mri has rapidly increased to a level of today 40 mr examinations per 1000 inhabitants and year. a study was undertaken as an attempt to answer the question whether or not the indications for the mr study, were considered appropriate or not. method: all requests for 3205 mri studies performed during two weeks in the county of stockholm were collected and reviewed. material: the information on each request form was evaluated by one of four experts. two experts were clinicians two were radiologists. these experts were asked to evaluate whether the indications for study were considered appropriate or not. results: in the total material 43 % of the studies were thought to be indicated, 21 % probably indicated, 16 % were thought probably not to be indicated, while 17 % were clearly not indicated. in another 3 %, the material was inadequate and the indication was not possible to evaluate. there were clear differences between different groups of referring physicians in their pattern of using mri as a diagnostic tool. as an example, specialists in hospital practice had the highest proportion of indicated requests. the results of this study strongly supports the need for ongoing education and precise guidelines in the proper utilisation of mri as a diagnostic tool. standardized virtual endoscopic approach to the intracisternal course of the cranial nerves v -viii r. klingebiel, c. heine, r. lehmann; berlin/de purpose: standardization of a virtual endoscopic (ve) approach to the intracisternal course of cranial nerves (cns) v -viii. methods and material: magnetic resonance imaging (mri) of the basal cisterns was performed in 6 healthy volunteers on a 1.5 t unit, using a ciss sequence with an isotropic voxel size of 0.5 3 mm. data post processing (pp) was effected by means of direct volume rendering (vr), comprising the cn's complete intracisternal course as well as their root-entry-zone. subsequently, 12 patients with clinical signs of intracisternal pathology were investigated by standardized ve approach. the pp time was recorded and image quality and diagnostic value were rated by consensus reading of two neuroradiologists, using a five-point score (1 = insufficient, 5 = excellent). results: 2 -4 virtual endoscopic views were needed per cn for comprehensive intracisternal visualization (n. v = 4, n. vi = 2, n. vii/viii = 4). the pp time per cn varied between 7 -15 minutes. the average scores for image quality and diagnostic value amounted to 4.5 and 4.4, respectively. in 11/12 patients comprehensive ve cn imaging was achieved. in 4/11 patients variations of the ve views were required. different intracisternal pathologies were assessed by the ve approach such as neurovascular conflicts between the aica and the cns v, vi and viii at the rez level. conclusion: comprehensive intracisternal 3d visualization of the cns v -viii is possible within a routine imaging setting, provided direct volume rendering and standardized pp protocols are used. the value of the sagittal median nerve diameter in the diagnosis of median nerve entrapment o.j. sommer 1 , h. czembirek 2 , h. gruber 1 , p. kovacs 1 , m. stiskal 2 , f. kömürcü 2 ; tuesday b a c d e f 321 material and methods: 23 patients with suspected carotid artery stenosis were examined with mscta after bolus administration of 80 ml of nonionic contrast agent at 4 ml/s. before the acquisition, a test bolus of 16 ml of c.a. at 4 ml/s was performed. real time interactive 3d evaluation was performed on a dedicated workstation. the degree of stenosis and the morphology of the plaque (qualitative and quantitative) were evaluated. in all patients dsa was also performed and considered the standard of reference. results: excellent image quality was obtained in all cases, without venous filling. good correlation with dsa was achieved using the interactive 3d assessment. a perfect correlation between mscta and dsa regarding the degree of stenosis was seen in 42 carotids. in 3 carotids, with moderate stenosis at dsa, mscta showed a severe stenosis, due to the presence of calcification. the values of sensitivity, specificity and accuracy were respectively 96 %, 82 % and 91 %. regarding the morphology of the plaque, mscta correctly demonstrated all ulcerated plaques, also showing the presence, by means of quantitative assessment, of calcium, fibrous stroma and lipid. conclusion: mscta surpasses all the limitations of single slice spiral cta regarding venous overprojection and volume coverage. real time 3d interaction is necessary to correctly demonstrate the degree of stenosis. mscta appears to be particularly valid in the assessment of plaque morphology and therefore in treatment planning. carotid artery stenosis: accuracy of noninvasive imaging g. mansueto, m. d'onofrio, a. guarise, p. tamellini, c. procacci; verona/it purpose: this study prospectively compared contrast-enhanced magnetic resonance angiography (cemra) and doppler ultrasound (dopplerus) with digital substraction angiography (dsa) and endarterectomy findings to determinate the accuracy in assessing carotid artery stenosis. in this prospective study from january to may 2001, 32 patients underwent carotid endarterectomy, 21 studied by cemra, dopplerus and dsa and 11 with cemra and dopplerus. the degree of stenosis of 41 carotid arteries (21 patients) was analyzed with cemra and dopplerus and compared with dsa (reference standard) by using the spearman rank correlation coefficient (rs). in 9/32 patient eversion endarterectomy was performed; thus it was possible to compare cemra, dopplerus and dsa with the specimen measurement (reference standard). in 23/32 standard endarterectomies the presence of ulcers was documented to determinate the accuracy of noninvasive imaging. results: there was a significant correlation between cemra and dsa (rs = 0.81; p < 0.001) and between dopplerus and dsa (rs = 0.086; p < 0.001) for degree of stenosis. overestimation was the most frequent error, even in the specimen comparison. there was one false positive for cemra and dopplerus and one false negative for dsa, so the accuracy was the same at 89 %. ulcers were most frequently seen at cemra. conclusion: there was a significant correlation between cemra, dopplerus and dsa for estimating the degree of stenosis with the same accuracy by comparing with specimen measurements. ce mra was shown to be the best imaging modality to detect plaque ulceration. multislice spiral ct angiography vs. time resolved contrast enhanced mr angiography in the assessment of carotid artery stenosis f. pediconi, c. catalano, a. laghi, f. fraioli, a. napoli, m. danti, r. ferrari, r. passariello; rome/it purpose: to compare multislice ct angiography (mscta) with contrast enhanced mr angiography (mra) in patients with suspected carotid artery stenosis. material and methods: 23 patients with suspected carotid artery stenosis were examined with mscta and mra using a 1.5 t magnet. mscta was performed after bolus administration of 80 ml of c.a. at 4 ml/s. before the acquisition, a test bolus with 16 ml of c.a. at 4 ml/s was performed. ce-mra was performed using a multiphase sequence after bolus administration of 15 ml of gd-dtpa at 2 ml/s. a real time interactive 3d assessment of mscta images was performed in all cases, while mra images were assessed using a standard mip algorithm on the magnet console. the two techniques were compared for degree of stenosis and plaque morphology, using dsa as standard of reference. results: with both techniques images were diagnostic and free from artifact. no significant difference between the two techniques, regarding the degree of stenosis, was seen; the values of sensitivity, specificity and accuracy were respectively of 96, 100 and 97 % for both techniques. qualitative assessment allowed us to correctly demonstrate the morphology of the plaque in all cases with cta, while mra correctly demonstrated 10 of 12 ulcerated plaques. although multislice spiral ct encompasses most of the problems of spiral cta, contrast enhanced mra provides a simpler way to achieve comparable results, using well tolerated contrast agents and without ionizing radiation, regarding the degree of stenosis. nevertheless, mscta also appears accurate in the assessment of plaque morphology. power doppler screening for carotid artery stenosis in cardiac surgery patients v. beslagic, f. dalagija, z. merhemic; sarajevo/ba purpose: to assess the use of power doppler imaging as a screening tool for detecting carotid stenosis in the preoperative evaluation of cardiac surgery patients admitted at university clinical center sarajevo in an eighteen month period. material and methods: we surveyed 174 patients admitted for cardiac surgery. imaging methods were power doppler, duplex doppler, mra, dsa. a two phase study was performed. in the first, a prospective preoperative evaluation of 60 patients was performed using duplex doppler. during the second phase, prospective preoperative evaluation of 114 patients was performed using power doppler imaging. in patients with 50 % stenosis or more, duplex doppler was performed. imaging methods for further evaluation of 70 % and above stenosis, in both groups, were mra and intravenous dsa. results: stenosis of 60 % or more was found in 11 (19 %) patients in the first phase group and in 27 (24 %) of patients in the second phase group. stenosis of 70 % and above was found in 4 (7 %) patients in the first phase group, and in 12 (11 %) patients in the second phase group. adequate images were achieved in 89 % of the power doppler studies compared with 82 % in the duplex doppler studies. conclusion: power doppler, as a screening tool for carotid stenosis, is a low risk, fast, cost effective method with accurate results. using power doppler to screen patients and duplex doppler to evaluate significant carotid stenosis with possible further diagnostic evaluation with mra and dsa, an optimal imaging approach is achieved. cerebral hemodynamics in patients with vertebral artery hypoplasia comparing those with and without atherosclerotic lesions of carotid arteries s.g. mazur, i.i. glazovska, v.v. kuznetsov; kiev/ua purpose: comparison of brain hemodynamics in patients with vertebral artery hypoplasia (vah) comparing those with and without atherosclerotic lesions of the carotid arteries. methods and materials: we examined 102 patients, 51 males and 51 females, mean age 51 ± 15 years: (1) 58 patients without a history or signs of cerebrovascular diseases and without carotid stenosis: 31 with vah (1a) and 27 without vah (1b); (2) 44 patients with ischemic stroke caused by atherosclerotic lesions of the carotid arteries: 19 with vah (2a) and 25 without vah (2b). we used a sonography system "elegra" (siemens) to measure the vessel diameters, volume blood flow velocities (vf), total cerebral volume blood flow (tvf) -vf through both internal carotid arteries (vfica) + vf through both va (vfva). results: group 1a had lower vfva than 1b (p < 0.05), but there was no significant difference between tvf in these two groups. tvf in group 2a (0.36 ± 0.038 l/min) was lower compared with group 2b (0.51 ± 0.071 l/min, p < 0.001) resulting in a lower vfva in group 2a (0.028 ± 0.087 l/min and 0.12 ± 0.051 l/min respectively, p < 0.001). gropp 2b versus 2a patients had a higher severity of arterial narrowing. conclusions: patients with vah without stenotic lesions of the carotid arteries have brain hemodynamic compensation through the carotid arteries and contralateral vertebral artery. vah in patients with cerebral atherosclerosis evokes decompensation of cerebral hemodynamics and causes ischemic stroke with a lower grade of carotid stenosis than in patients without vah. purpose: the aim of this work was to retrospectively evaluate with mri, 45 patients affected by anterior knee pain without clinical symptoms of meniscal or ligamentous injuries. materials and methods: 45 patients with anterior knee pain entered this study. mr examination was performed using 0.2 t dedicated and 1.5 t whole body mr units. in 5 cases dedicated mr unit was performed to have kinematic evaluation of the knee at three different degrees of flexion. in 25 cases i.v. injection of contrast media was performed. all the patients underwent arthroscopy. results: in all the cases mri revealed a region of altered signal intensity in the superior aspect of the hoffa's body. arthroscopy revealed the presence of synovial thickening in 25 cases and enlargement of the patello-femoral synovial recess in 20 cases. in 14 cases of synovial thickening infusion mri showed in 8 cases contrast enhancement suggesting an acute stage of inflammation and in 5 cases no enhancement was found due to the presence of synovial fibrous tissue. in 9 cases of histologically proved villonodular synovitis se t2w sequences showed a region of non homogeneous high signal intensity because of the presence of spots of low signal intensity. in all the 5 patients showing synovial thickening of the hoffa's body apex, kinematic mr revealed impingement with the femoro-patellar joint. conclusion: our experience demonstrates that mri, eventually completed by kinematic study and/or infusion of contrast media, may be considered the method of choice in the evaluation of the synovial impingement of the knee. cystic degeneration of the lateral meniscus of the knee: mri evaluation before and after arthroscopic treatment using radiofrequency energy a. barile, a.v. giordano, m. caulo, m. sabatini, f. iannessi, g. bonanni, v. calvisi, c. masciocchi; l'aquila/it purpose: to assess by mri the potential benefit and efficacy of selective meniscectomy combined with radiofrequency ablation in cystic degeneration of the lateral meniscus treatment. materials and methods: forty-one patients entered this study. 18 patients underwent arthroscopic selective meniscectomy and 23 patients underwent arthroscopic selective meniscectomy and radiofrequency treatment (arthocare, sunnyvale, ca) of the meniscal remnant. all of them were submitted to mri and clinical evaluation three months later. in 9 cases mri examination was performed before and after the surgical treatment. mri examination was performed using a dedicated 0.2 t (artoscan esaote italy) and a 1.5 t superconductive unit (ge signa horizon usa) employing se t1-w, se t2-w and ge t2-w sequences in axial and longitudinal scan planes. in 3 cases arthro-rm was also performed. clinical evaluation included physical examination and a questionnaire. results: clinical evaluation did not show statistically significant discordance between the two groups. mri follow-up after 3 months demonstrated a very clear decrease of the degenerative spots inside the meniscal remnant in the group with combined treatment respect to the group treated only with selective meniscectomy. conclusion: in conclusion our experience considers mri the method of choice in the evaluation of post surgical treatment of degenerative cystic diseases of the lateral meniscus of the knee. bone-patellar tendon-bone (btpt) acl reconstruction of the knee: diagnostic imaging evaluation of failures compared to second-look arthroscopy a. barile, g. cerone, a.v. giordano, l. zugaro, a. catalucci, g. bonanni, v. calvisi, c. masciocchi; l'aquila/it purpose: to evaluate the capability of diagnostic imaging in cases of failed anterior cruciate ligament (acl) reconstruction. materials and methods: 16 patients submitted to acl surgical reconstruction with patellar tendon entered this study. all the patients were clinically suspected of acl reconstruction failures and had diffuse pain and/or joint swelling or instability. all of them underwent mri from 3 to 12 months after surgery. all patients underwent plain film and 11/16 ct. in all cases mr study was performed using dedicated 0.2 t or whole-body superconductive 1.5 t units; in 12 cases arthro-mri was also employed. results: in 5 patients enlargement of the graft related to the presence of significant synovial reaction were found. in 4 cases synovial reaction was exuberant (cyclops syndrome). in 3 patients the bone tunnels were not perfectly aligned and standard examination revealed the presence of osteophytosis of the intercondylar fossa. in 2 cases the bad positioning of the interference screws was found. in the last 2 cases mri documented a tear of the graft. in all the cases arthroscopic second look confirmed the mri diagnosis. conclusion: mr manifested very accurate evaluation of the acl autograft because of its high contrast resolution and multiplanarity. in our experience mri eventually completed with arthro-mri may be considered the method of choice in the evaluation of acl reconstruction failures. dynamic mri in the evaluation of femoro-patellar disorders m. mastantuono, e. bassetti, m. francone, m. valeo, r. passariello; rome/it purpose: alterations of femoro-patellar biomechanics in the young and sports practicing subjects may determine the development of early involutive process and painful conditions. in our experience we verified the potential offered by kinematic studies of the extensor complex on sagittal and axial planes and we developed an innovative method of study that permits a correct clinical assessment of in this particular problem. we used a 0.2 t dedicated magnet, dedicated to the study of limbs with an adapting device holding the knee at different angles of flexion. we obtained images in different positions of flexion-extention acquired with a se t1 and t2 and g.e. sequences slice thickness 2 -3 mm without gap. we studied 27 healthy volunteers and 39 patients with anterior pain or instability of knee joint of which was suspected of being femoro-patellar in origin. in 29 patients the correlations between cartilage and subchondral bone sufference and the cinemotion findings were significant. conclusion: our method differs from the other dynamic studies described in the literature in that it allows volume study and permits a clear evaluation of the dynamic relationship between rotule and troclear articular surfaces. postoperative meniscus: assessment with dual-detector spiral ct arthrography of the knee c. mutschler, b. vande berg, f.e. lecouvet, p. poilvache, j.e. dubuc, b. maldague, j. malghem; brussels/be purpose: to evaluate dual-detector spiral computed tomography (ct) arthrography of the knee in the assessment of the postoperative meniscus. materials and methods: two observers retrospectively determined in consensus the meniscal changes observed in 20 consecutive patients who have had partial meniscectomy (n = 23) and who underwent dual-detector spiral ct arthrography of that knee for recurrent symptoms before second-look arthroscopy. at ct arthrography, postoperative menisci were considered to be either stable (no tear or small partial tear) or unstable (meniscal separation, complete tear, large partial tear, displaced meniscal fragment). the sensitivity, specificity, and positive and negative predictive values for the detection of unstable meniscal tears among all postoperative menisci at spiral ct arthrography was determined with second-look knee arthroscopy as the standard. the sensitivity and specificity for the detection of unstable postoperative meniscal tears among all postoperative menisci were 85 % and 90 %, respectively, with positive and negative predictive values of 92 % and 82 %, respectively. conclusion: dual-detector spiral ct arthrography of the knee is an accurate method for detecting unstable meniscal tears among postoperative menisci. purpose: to analyze mr-pathology of the knee joint at different stages of osteoarthritis and to find out whether pain, stiffness and limited function assessed clinically correlate with the degree of pathology assessed on mr images and radiographs. material and methods: the study population consisted of 50 patients with varying degrees of osteoarthritis. osteoarthritis was graded clinically using the womac (western ontario and mcmaster university) score and on the basis of conventional radiographs using the standard kl (kellgren-lawrence) score. in addition mr imaging was performed at 1.5 t using a high-resolution fat-suppressed, spoiled gradient echo sequence, t2-weighted fat suppressed fast spin echo sequences and t1-weighted spin echo sequences. all images were analyzed by two readers concerning cartilage lesions, bone marrow edema and pathology of the ligaments and menisci. these findings were correlated with the clinical and the radiological score. results: 13/16 joints with a kl-score of 4 showed full thickness cartilage lesions and bone marrow edema. meniscal tears were found in all joints with kl-score 4. cruciate ligament pathology was found in 5/13 kl-score 3 and 9/16 kl-score 4 joints. the kl-score correlated well with the extent of mr-pathology. correlations between the extent of cartilage damage, bone marrow edema and the kl-score versus the womac-score were non-significant (p > 0.05). conclusion: a high percentage of meniscal and ligamentous pathology, bone marrow edema and severe cartilage lesions could be shown in advanced osteoarthritis. however, a significant correlation between clinical findings and the extent of mr-and x-ray pathology could not be demonstrated. purpose: evaluation of real-time mri in the assessment of velopharyngeal closure in comparison to videofluoroscopy. materials and methods: 1 healthy volunteer and 7 patients with suspected velopharyngeal insufficiency (age from 5 -19 years, mean 9 years) underwent videofluoroscopy and real-time mri, using a tse "zoom" sequence (tr = 170 ms, te = 21 ms, slice thickness 6 mm) with 6 images per second. images were acquired in the midsagittal, the coronal and axial plane at the level of maximal velopharyngeal closure during phonation of test words. results were analysed by 2 radiologists in comparison to videofluoroscopy as the standard of reference concerning overall quality of the depiction of the velopharyngeal isthmus and the pattern of velopharyngeal closure in all three image planes. results: in all cases, real-time mri could correctly depict the pattern of velopharyngeal closure in correspondence to videofluoroscopy: velopharyngeal insufficiency with wide open velopharyngeal portal (n = 2), with moderate open velopharyngeal portal (n = 1), close approximation of the velum (n = 1) and velum touching the dorsal phayryngeal wall (n = 3). in 3 cases, the coronal plane was not of diagnostic quality in mri due to motion-artefacts. in one case, mri showed an asymmetric movement of the pharyngeal walls which could not be seen in videofluoroscopy. conclusion: real-time mri can successfully analyse the pattern of velopharyngeal closure even in small children. being a non-invasive method, it avoids the radiation exposure of videofluoroscopy and the discomfort of nasoendoscopy. evaluation of the movements of the oro-pharyngeal cavity during phonation with different voice intensity using mri m. di girolamo, g. ruoppolo, e. iannicelli, m. mattei, a. carriero, v. david; rome/it purpose: the aim of this study was to evaluate with mri the movements of the different anatomical structures of the oro-pharyngeal cavity during the utterance of the fundamental vowels with different voice intensity. we have evaluated 30 volunteers using a 0.5 t mr superconductive unit (philips medical system). we performed turbo-field-echo sequence (tr: 12 ms; te: 6 ms; ti: 900 ms; n.ex.: 4; acq. time: 6 s) with midsagittal scan (slice thickness: 8 mm). the volunteers were asked to perform a prolonged emission of the fundamental vowels [a], [i], and [u] with normal voice intensity (50 db) and with loud voice (70 db). the voice intensity was measured with a phonometer before the mr acquisitions. on each midsagittal scan we measured the area of the mouth and pharyngeal lumen. results: in all the patients we accurately evaluated the movement and the activity of the lips, tongue, soft palate and the pharynx. comparing the utterance of loud voice to normal voice, we found an increase of the area of the mouth lumen for the vowels [a] and [u] and an increase of the area of pharyngeal lumen for the vowel [i]. the intensity of voice emission depends not only on subglottic air pressure and glottic rim dimension but also upon the oro-pharyngeal cavity volume. the three-dimensional ct study of the upper airway of obstructive sleep apnea syndrome: an oral appliance can change the volume of oro-and nasopharynx w. zhang; beijing/cn purpose: to evaluate the utility of the spiral ct and post processing with 3d remodelling to review the anatomical abnormalities and the changes of the upper airway of osas with or without an oral appliances in the mouth. method/materials: 15 osas patients who had ideal oral appliance therapy have undergone spiral ct with or without the oral appliances in the mouth. scan was obtained with 3 mm collimation at 1.0:1 pitch from nasopharynx to the level of the hyoid bone at end expiration. 3d remodeling, raysum and mpr were performed at the workstation. luminal area at naso-, oro-and hypopharyngeal levels, the distance of the posterior airway space and distance between the hyoid bone and mandibular plan were measured. results: oro-and nasopharyngeal areas were significant larger in patients with oral appliances than in patients without oral appliances, p < 0.05 (478.07 mm 2 ± 104.29 vs 374.86 mm 2 ± 135.72 and 175.50 mm 2 ± 80.30 vs 112.50 mm 2 ± 65.05). no differences of hypopharyngeal areas were found between patients with or without oral appliances, p > 0.2 (202.36 mm 2 ± 119.78 vs 250.43 mm 2 ± 103.78). the three dimensional spiral ct scanning had many advantages in the evaluation and measurment the upper airway in patients with osas, these results show the main therapeutic mechanism of oral appliance use is the volume change of the oro-and nasopharynx. static and dynamic evaluation with mri of larynx and oro-pharingeal cavity in professional opera singers m. di girolamo, g. ruoppolo, f. assael, m. minnetti, e. iannicelli, v. david; rome/it purpose: to assess the anatomical configuration of phonetic organs by mri in singers with different vocal range. methods and materials: 26 professional opera singers (7 tenors, 5 basses, 8 sopranos, 6 mezzosopranos) underwent mri, performing both static and dynamic studies. we performed tse t2-weighted axial scans at the level of larynx in order to evaluate the area of superior surface of vocal cord. in the dynamic study, the singers were asked to perform a prolonged vocalization at a confortable tonality of the foundamental vowels a. we performed a midsagittal turbo-field-echo scan (acq.time: 6 s) at the level of the oro-pharyngeal cavity measuring the area of the mouth and pharyngeal lumen. these data underwent statistical evaluation using the mann-whitney u-test. results: we determine the average size of the vocal cord (sopranos: 0.71 cm 2 ; mezzosopranos: 1.20 cm 2 ; tenors: 1.58 cm 2 ; basses: 2.88 cm 2 ) and of mouth and pharyngeal lumen (sopranos: 15.8 cm 2 ; mezzosopranos: 14.6 cm 2 ; tenors: 23.6 cm 2 ; basses: 32.2 cm 2 ). the differences in vocal cord size between sopranos and mezzosopranos (p: 0.0641) and between tenors and basses (p: 0.0833) are tendentially statistically significant. the variation in vocal tract size during the uttuesday b a c d e f 327 terance of the vowel a between tenors and basses is considered tendentially statistically significant (p: 0.0833) while the difference between sopranos and mezzosopranos is not statistically significant (p: 0.6434). we demonstrate a correlation between the vocal cord's surface and the vocal tract configuration and the vocal tessitura of a singer. long vocal cord and wide vocal tract are characteristic of singers with low-pitched voice types (bass, baritone, contralto, mezzosoprano) while short vocal cord and narrow vocal tract are characteristic of singers with high-pitched voice types (tenor, soprano). correlation between arnold chiari malformation and sleep apnea syndrome (sas) i. thomassin 1 , k. marsot-dupuch 1 , v. stahl 2 , f. bourquin 2 , f. parker 1 , p. lasjaunias 1 ; 1 le kremlin bicêtre/fr, 2 bobigny/fr purpose: to identify morphologic abnormalities on cervical mri exams among patients suffering from sleep apnea syndrome and arnold chiari malformation. materials and methods: 13 patients suffering from severe apnea syndrome (30/ mn) were referred for surgery of arnold chiari malformation. pre-and post-contrast sagittal and axial t1 w and t2w images of the brainstem and cervical cord were performed. were studied the cerebello-medullary cistern, the angle between posterior border of brainstem and cerebellum, location of cerebellar tonsilla, cervicooccipital junction and ventricular system. results: three brainstem abnormalities were detected (two isolated and one in a complex malformative syndrome). 9 patients have filling of cerebello-medullary cistern and a diminution of the angle between the posterior border of the brainstem and the cerebellum. anterior translation of cerebellar tonsils was noticed. discussion: many authors have studied obstructive sas which was partially explicated by laryngo-pharyngeal abnormalities. associated transient ischemic attacks are well known. our study suggested that abnormalities of brainstem may alter the function of respiratory nuclei and tracks and that brainstem abnormalities may be linked with sleep apnea syndrome. conclusion: sleep apnea syndrome may be associated with lesions of brainstem. further larger study should be done to prove a statistical correlation. however radiologists should look for posterior fossa abnormalities when exploring patients referred for sleep apnea syndrome. comparison of differently viscous iodinated and barium-containing contrast agents in the detection of pharyngeal perforation m. keberle, g. wittenberg, a. trusen, w. baumgartner, d. hahn; würzburg/de purpose: unlike in the investigation of esophageal perforation, the more radiopaque barium-suspensions are not as important as iodinated aqueous contrast agents for the detection of pharyngeal perforations. this study was performed to find out whether the highly different viscosities (of iodinated and barium-containing contrast agents with comparable radiopacities) are a reason for this. methods: viscosity, subjective difference in contrast, and ct-density of an iodinated aqueous (telebrix) and a 50 wt/vol% barium-containing contrast agent (micropaque) were determined. moreover, to exclude postoperative perforation, 104 patients were prospectively examined by pharyngography using both contrast media. pharyngographies of patients with perforation were later compared by two independent readers. all patients with perforation were followed up clinically to exclude complications due to barium-application. results: in-vitro comparison showed comparable radiopacity but the 50 wt/vol% barium-suspension was much more viscous than the iodinated contrast agent. in total, during pharyngography, 14 perforations were clearly delineated with the iodinated aqueous contrast agent. however, two of them were not detected with the barium-suspension. all the other perforations presented equally. conclusions: given a sufficient radiopacity, a low viscosity appears to be essential for a contrast agent to detect pharyngeal perforation. thus, we recommend the sole use of an iodinated contrast agent (if suspicion of aspiration as isoosmolar variant) for this purpose. percutaneous tracheostomy (pt) in the intensive care unit (icu) using ultrasound guidance t. geroukis, d. voultsinou, a. papachillea, v. kalpakidis, p. palladas; thessaloniki/gr purpose: to present the ultrasonographic investigation of anterior neck structures conducted prior to the performance of pt in the icu patients. to determine whether the ultrasonographic contribution could reduce the complication rate and improve the outcome of these patients. we studied 26 icu patients, with an average age of 58. the ultrasound investigation was performed with bedside portable equipment in the icu immediately prior to the tracheostomy, indicating the point of puncture and analyzing the relationships of the anatomic structures lying in the vicinity of the tracheostomy site. results: in 13 patients the thyroid isthmus was extending over the tr1-tr2 space and in 8 patients a considerable isthmus thickness was calculated. goiter was found in 7 patients whereas clinical examination indicated this in only in 3 patients. the clinical examination was unable to evaluate by palpation the guiding points in 6 patients. in all these cases the anatomical relations required were satisfactorily visualized ultrasonographically. complications included hemorrhage, pneumothorax and subcutaneous emphysema. the ultrasonographic results influenced the decision for surgical or transcutaneous tracheostomy or surgical management during the procedure in three cases but small complications were unavoidable. with increased practice and better evaluation of the ultrasound results, the radiologist can play a vital role in the management of the icu patient. possibilities of spiral ct in differential diagnosis of carotid chemodectomas g. results: chemodectomas were revealed in 23 pts, neurinomas in 8 pts and carotid arteries aneurysms in 11 pts. among 23 chemodectomas there were 19 carotid and 4 vagal tumours. the analysis of data revealed that chemodectomas were characterised with early (arterial phase) pronounced (up to 200 hu) contrast enhancement. sometimes a low density area can be seen in center of the lesion. neurinomas are characterized with less pronounced enhancement (up to 100 hu). "spots" and "stripes" of the contrasted vessels stand out against parenchyma in neurinomas. there was no significant difference (p < 0.01) of average density rates in arterial phase in chemodectomas (171.2 ± 45.8 hu) and in neurinomas (64 ± 26 hu). there was a simultaneous contrast enhancement both in aneurysm and in surrounding vessels. additional information valuable for differential diagnosis was not obtained in venous and delayed phases. simultaneous enhancement of tumor and carotid arteries gives the possibility of evaluat ing their relationship and the involvement of carotid arteries in pathological processes. conclusion: spiral ct with bolus contrast enhancement allows differentiation of tumors and other pathological masses of the carotid space and allows a 3d-image of the area of interest to be obtained for surgery planning. acute calcific retropharyngeal tendinitis m. tassart, c. le breton, f. bahlouli, n. szelei, j. bigot; paris/fr purpose: to improve early diagnosis of an under recognized cause of cervical pain and stiffness whose initial misdiagnosis can lead to parenteral administration of antibiotics or even open biopsy materials and methods: two patients (1 male, 33 a; 1 female: 47 a) were admitted to the hospital with dysphagia, severe neck discomfort and fever. lateral radiographs of the cervical spine were performed in 2 patients, computed tomography in 2 and mri in 1. using non-steroidal anti inflammatory medications (interruption of antibiotics), the two patients had complete resolution of the symptoms within one week results: the calcification of the prevertebral muscle was demonstrated by ct for 2 patients and identified on lateral radiography in one case only. the soft tissue swelling was seen both on ct and mri. conclusion: acute calcific tendinitis is an under recognized disease which can be initially misdiagnosed as retropharyngeal or nasopharyngeal abscess, leading to unecessary parenteral injection of antibiotics or even biopsy. the radiologist has an important role to ensure the definitive diagnosis: in our 2 cases, specific imaging led to the correct diagnosis. to test the hypothesis that the single testicular artery is low resistance, and two further arteries (cremasteric/differential) are high resistance, we examined the spermatic cord vessels in a cohort of healthy men. materials and methods: patients presenting for a scrotal us with normal testes and epididymis were recruited. the right and left spermatic cords were examined from the superior aspect of the testis to the external inguinal ring to locate the testicular, cremasteric and differential arteries. a 15l8w multifrequency (8 -13 mhz) linear array probe (acuson ca) identifed each artery as separate, allowing a spectral doppler waveform to be recorded. the resistive index (ri) from each artery was measured three times (right and left) and the mean calculated. the three arteries were labelled a (lowest ri) b and c (two higher ri). results: 51 patients (median age 39 a, range 18 -79 a) were examined. the ri's on the right were: a: 0.71 ± 0.05, b: 0.82 ± 0.05, c: 0.84 ± 0.04 and the left a: 0.70 ± 0.06, b: 0.84 ± 0.04, c: 0.83 ± 0.05. a paired t-test demonstrated a significant difference of the combined right and left measurements for avsb (p < 0.001) and avsc (p < 0.001) but not bvsc (p = 0.3). conclusion: although it is not possible to identify a named artery in the spermatic cord, consistently one artery has a significantly lower ri than the other two arteries. this finding may be useful as an adjuvant parameter in the assessment of scrotal pathology in particular with patients with spermatic cord torsion. age related changes in testicular perfusion and serum androgen levels o.j. sommer 1 , k. kharom 2 , t. zils 2 , h. czembirek 2 , h. pflüger 2 , e. plas 2 ; 1 innsbruck/at, 2 vienna/at purpose: to assess the peripheral vascular resistance of small testicular arteries in different age groups and to match these results with age and androgen levels. materials and methods: 94 testicular units of 47 healthy subjects were investigated. according to age, patients were divided into 5 groups (30 -39, 40 -49, 50 -59, 60 -69, > 70 a). using high resolution sonography (7 -13 mhz, elegra, siemens, erlangen) including power mode we measured resistive indices (ri) and pulsatility indices (pi) of centripetal testicular arteries of the lobuli testes excluding mediastinal arteries. androgen (testosterone, free testosterone, fsh, lh, sexual hormone binding globulin) levels were evaluated between 8 and 10 a.m. and correlated with age, ri and pi values. results: there was a significant difference of 0.58 to 0.67 in ri (p = 0.025) and 0.94 to 1.29 in pi (p = 0.0005) values between young and old men. with the exception of sexual hormone binding globulin (sbhg) (4.8 versus 2.5; p < 0.05) no significant correlation of androgens with age, ri or pi was shown. our study suggests a positive correlation of age with the peripheral resistance of small testicular arteries, i.e. an increase of peripheral resistance with increasing age. sbhg showed a positive correlation with age, ri and pi values. the other androgens do not seem to relate with ri and pi values of the aging male. consecutive patients referred for scrotal us were examined with a 15 l8w multifrequency (8 -13 mhz) linear array probe (acuson, ca). if a focal testicular lesion was identified, the abnormality was re-examined with colour doppler mode (7 -12 mhz). vascularity was classified into, (a) no intra lesional vessels, (b) surrounding but no intra lesional vessels, (c) intra lesional vessels present and subdivided into (1) vessels crossing (2) disordered vessels (3) complete infilling. us findings were correlated with histology or findings confirmed on follow up. results: over a 24 month period a total of 2032 patients were examined. 53 focal abnormalities in 44 patients were found. the distribution of vascular patterns was as follows: a: 9, b: 5, c1: 26, c2: 9, c3: 2. 27 primary testicular tumours (seminomas, teratomas) were detected, 26 showing a c1 (criss cross pattern), a single well differentiated teratoma showing no intra lesional vessels (a). nine patients demonstrated a c2 vascular pattern; 4 secondary tumours, 3 acute myeloid leukaemia, 1 chronic fibrosis and a single patient without histological diagnosis. the c1 pattern was present in 95 % of primary testicular tumours. the presence of the 'criss cross' vascular pattern allows confident diagnosis of primary testicular tumours, although not differentiating seminomas from teratomas. with the improvement in probe technology, vascularity of testicular tumours proves to be an important differentiating feature. varicocele and venous insufficiency in left low extremity s. deftereos, g. alexiadis, g. kafetzis, s. touloupidis, j. manavis; alexandroupolis/gr the aim of our study is to compare the rate of incidence of varicocele and venous insufficiency in left low extremity. the flow pattern of left common iliac, external iliac, common femoral and great saphenous veins were evaluated with colour doppler sonography (cds) in 32 patients (19 -22 years old) with clinically suspected left varicocele. the same veins were also evaluated with cds in 15 clinically healthy voluntaries (same age range). in 24 patients of the first group (32 patients with clinically suspected varicocele) when valsalva manouvre was performed with the patient in upright position, flow reversed for a period of one or more seconds in left common femoral vein. thus the diagnosis of venous insufficiency was made. in the second group (15 clinically normal males) normal findings were found in thirteen persons. in the remaining two young men the diagnosis of venous insufficiency were made. the cds of scrotum revealed varicocele in both of them. on the basis of these findings and also because of the anastomotic branches between the veins of scrotum and low extremity we believe that a cds evaluation of the flow pattern in left common iliac, left common femoral and great saphenous veins must be performed before surgical treatment of varicocele in order to avoid the possibility of varicocele recurrence. quantitative evaluation of scrotal veins by colour doppler us in healthy population a. cina, t. pirronti, r. foschi, g. restaino, g. oliva, a. pignatelli, d. ribatti; rome/it scrotal varicocele (sv) is a very common disease of the male population. presently, colour doppler ultrasonography (cdus) represents the most frequently adopted technique for confirming the clinical diagnosis of sv and for a treatment indication. despite for several years cdus has been clinically used in cases of sv, discriminating quantitative values are not reported in medical literature. this is at least partly due to the lack of studies of cdus patterns obtained from large populations of healthy subjects. the aim of this paper is to investigate the role of ecd in instrumental diagnosis of male varicocele, the implications in selecting the patients who require treatment, and to investigate the risk of sv overdiagnosis by adopting the qualitative criteria currently used. we prospectively examined by cdus a population of 150 healthy and symptomless subjects, with a negative clinical examination for varicocele, and a normal spermiogram. we found that the normal value range of the maximum diameter of scrotal veins (2.62 ± 0.53 mm) widely overlaps with the values currently accepted for a varicocele diagnosis (1.5 -3.0 mm). furthermore, a venous reflux, a criteria adopted also for the diagnosis of subclinical varicocele, was present in 53 % of the normal population. these data show that there is a risk of sv overdiagnosis by using cdus. the quantitative criteria for sv diagnosis should be re-examined. the patients group included 10 uvjs with grade i -ii and 5 uvjs with grade iii -iv of reflux. urethra-to-orifice distance (uod) and length of intravesical and submucosal parts of the ureter were measured. results: in normal cases ureteral orifices were situated on equal distances from urethra, the uod was 11.2 ± 2.8 mm (mean ± sd). the length of intravesical part of the ureter was 24.9 ± 6.6 mm and correlated with height (correlation coefficient 0.78), body surface area (0.73) and weight (0.66). the length of submucosal part of the ureter was 12.6 ± 3.1 mm and correlated with body surface area (0.85), weight (0.83) and height (0.82). the ratio between intravesical and submucosal parts of the ureter was 1.95 ± 0.3. there was no significant difference between the normal values and values of all 10 low-grade reflux uvjs. in 4 from 10 spontaneous resolution of the reflux during 1-year period was found. the grade iii -iv reflux orifices (5 uvjs) were situated farther from urethra than opposite normal orifices, the uod was 14.2 ± 2.5 mm. the intravesical part of the ureter was short (9 ± 2.1 mm) and submucosal part was absent in all cases. none high-grade reflux was resolved spontaneously. conclusion: ultrasonography is a valuable noninvasive method of examination of uvjs and may be useful in vesicoureteric reflux assessment. comparison of contrast-enhanced colour doppler targeted biopsy to conventional systematic biopsy: impact on prostate cancer detection f. frauscher, a. klauser, l. pallwein, a.h. schuster, h. volgger, g. helweg, d. zur nedden; innsbruck/at purpose: we investigated whether a limited biopsy approach with contrast-enhanced colour doppler ultrasound (cdus) targeted biopsy of the prostate could detect cancers as well as grey-scale us guided systematic biopsy with a larger number of biopsy cores. we examined 230 men (psa ≥ 1.25 ng/ml). two independent examiners evaluated each subject. one investigator performed contrastenhanced targeted biopsies (≤ 5) into hypervascular regions in the peripheral zone during intravenous infusion of levovist™. subsequently, a second examiner performed 10 systematic biopsies of the prostate. cancer detection rates for the two techniques were compared. results: cancer was detected in 69/230 subjects (30 %). cancer was detected in 56/230 subjects (24.4 %) by contrast-enhanced targeted biopsy, and in 51/230 patients (22.2 %) with systematic biopsy. cancer was detected with targeted biopsy alone in 17 subjects, and with systematic biopsy alone in 13 subjects. overall cancer detection rate was not significantly different for targeted and systematic biopsy (p = 0.53). the detection rate of targeted biopsy cores (118/1139) was significantly better than that of systematic biopsy cores (123/2300) (p < 0.001). targeted biopsy in a patient with cancer is 2.6 times more likely to detect prostate cancer than a systematic biopsy. conclusions: contrast-enhanced cdus targeted biopsy detected as many cancers as systematic biopsy with fewer than half the number of biopsy cores. although an increase in cancer detection rate is obtained by combining targeted and systematic techniques in this screening population, contrast-enhanced targeted biopsy alone is a reasonable approach to reduce the number of biopsy cores. intermittent contrast-enhanced ultrasonography for prostate cancer detection a. klauser, l. pallwein, w. horninger, f. frauscher, g. helweg, d. zur nedden; innsbruck/at purpose: we investigated the usefulness of intermittent contrast-enhanced ultrasonography (us) in the detection of prostate cancer. methods and materials: nineteen patients with an elevated prostate-specific antigen level (³ 2.5 ng/ml) or an abnormal digital rectal examination were enrolled in the study. we used an i.v. us contrast agent (levovist™). continuous greyscale, intermittent grey-scale, phase inversion grey-scale, and colour and power doppler us of the prostate were performed. sonographic findings were correlated with biopsy results. after us contrast agent administration we found significant enhancement on both grey-scale and doppler images (p < 0.01). in 3 isoechoic tumours we only detected focal enhancement using intermittent imaging. focal areas of enhancement were identified in only 1 patient (5 %) without cancer. conclusions: intermittent contrast-enhanced us of the prostate seems to be useful for selective enhancement of malignant prostatic tissue. therefore, this technique may be useful for targeted biopsies. power doppler sonography was more sensitive in detection of tumoral vessels than pd sonography and increased ppv from 81 % to 87 %, npv from 75 % to 82 %, sensitivity from 80 % to 90 %, specificity from 77 % to 86 %. conclusion: 3d pd trus examination is more informative than pd trus examination in detection prostate cancer. 3d pd trus examination can be used as a diagnostic tool in addition to complex examination of patients with suspected prostate cancer. trus cd and mri with contrast agent in the evaluation of local recurrence after radical prostatectomy f.m. drudi, s. petta, a. carbone, a. righi, f. trippa, p. ricci, r. passariello; rome/it purpose: aim of our study was to evaluate sensitivity of us cd and mri, both with contrast medium enhanced, in the detection of local recurrence of prostate cancer after radical prostatectomy and to evaluate the specificity of these techniques in distinguishing recurrent cancer from fibrosis. materials and methods: twelve patients (mean age 64.4; range 56 -73) presented with increasing psa values (> 0.4 ng/ml) in a variable period (mean time elapsed 3 years; range 3 months to 4 years). all patients underwent bone scintigraphy before and after treatment, mri (gadolinium dtpa i.v. 2 ml/s, 20 ml total. flash t1 fat sat 20 slice thickness 5 mm), grey scale trus, trus cd (levovist shu508) and trus guided multiple biopsy. presence of colour signal and mr signal after c.a. was considered sign of recurrence, absence of recurrence or fibrosis. the results were compared to biopsy showing a sensitivity of 100 % and a specificity of 100 % for cd, while mr showed a sensitivity of 100 % and a specificity of 80 %. cd showed colour signal after c.a. in all positive patients. mr showed local recurrence in 5 patients: 4 cases had been evidenced already at cd examination and confirmed by biopsy, while one was a false positive. scintigraphy detected distant bone metastases in 2 patients, one of which also presented local recurrence. conclusions: from this early experience we can say that trus cd with c.a. is a valid tool as compared to mr in the evaluation of local tumour recurrence in patients with rising serum levels of psa after radical prostatectomy. contrast media in mr, ct and dsa angiography mdct in emergency radiology: is a standardized chest and/or abdomen protocol sufficient for evaluation of thoracic and lumbar spine trauma? percutaneous image-guided radio-frequency thermal ablation of the lung bilateral inferior petrosal sinus sampling in cushing's syndrome; final solution? 16:20 multi detector ct in morphological assessment of pulmonary veins in patients with focal atrial fibrillation j. baqué, v. huart, a. azzarine, a. hakime, b. cauchemez, e. mousseaux; paris/fr purpose: to analyse the appearances of the pulmonary veins (pv) in patients with focal atrial fibrillation (faf) initiated by ectopic beats in the pv, compared to a control group. material and methods: the appearances of the pv were assessed in 10 patients with faf originating from the pulmonary veins (confirmed by electrophysiological study) and in 10 asymptomatic patients screened for coronary artery disease. the patients were paired by age and gender (age 50 ± 14 years). each patient underwent ecg-gated contrast-enhanced multi-detector ct (mdct) of the heart. the diameter of each of the four pv was calculated on 1 mm thick slices on axial and coronal oblique images and was measured at the ostium and at 5, 10 and 15 mm. both the mean and the maximal diameters were calculated for each vein. any anomalies or abnormalities of the heart and vessels were also described. results: the maximal diameters of proximal portions of the right superior (18.6 ± 3 vs 16.2 ± 2 mm; p < 0.05) and the left inferior (18.1 ± 2 vs 15.4 ± 2 mm; p < 0.05) pv were significantly dilated in faf patients compared to controls. the right inferior (19.9 ± 4 vs 17.4 ± 2 mm) and left superior veins (19.1 ± 3 vs 16.8 ± 2 mm) were not significantly different between the two groups. conclusion: on mdct images, the proximal portion of right superior and left inferior pv was significantly dilated in patients with faf initiated by ectopic pulmonary beats. accurate evaluation of the appearances of the pv gives valuable and complementary information before selective cannulation of each vein and percutaneous radiofrequency ablation procedure. evaluation of left atrium and pulmonary veins with mri and mra before and after circumferential radiofrequency ablation of pulmonary vein ostia for atrial fibrillation f. de cobelli, f. gugliotta, r. mellone, m. venturini, c. pappone, a. del maschio; milan/it purpose: the pulmonary veins (pvs) and surrounding ostial areas frequently house focal triggers or reentrant circuits critical to genesis of atrial fibrillation (af). a new anatomic treatment aimed at isolating each pvs from the left atrium (la) by circumferential radiofrequency (rf) lesions around their ostia has been developed. we evaluated the role of mri and mra in assessment of pvs and la before and after rf ablation. method and materials: 12 patients with resistant af underwent mri and mra before and the day after rf ablation. 8 patients underwent mr after 3 months. mr images were obtained with black-blood ecg-triggered breath-hold multishotfse sequences with double and triple inversion pulses (ir-msfse) (tr/te 2 × rr/ 40, etl = 32). mras were acquired with efgre 3d sequences (tr/te/ti/fa 5.4/ 1.6/30/30°) with gadolinium injection and reconstructed with mip and volume rendering. results: at the pretreatment mra, all four pvs were visualized in all patients. in four patients, the following anatomic variants were found: a common ostium of the left superior (ls) and inferior (li) pvs; a single ostium of the ls and lipvs; a posterior ostium of lipv; 3 separate right pvs ostia. at the post-treatment mri, high signal intensity was detected in the atrial wall around veins ostia due to oedema; in three patients a thin pericardial effusion was evident. at three months follow-up, the la volume was significantly decreased in patients who had sinus rhythm restoration; with mra, a left inferior pv stenosis was found. conclusion: mr is useful in evaluation of patients with af before and after rf ablation of pvs ostia. preoperative planning and intraoperative navigation with 3d-reconstructions and image guidance for robotically-assisted coronary artery bypass grafting t.c. mamisch 1 , g. zuend 2 , j. gruenenfelder 2 , s.p. hoerstrup 2 , f.a. jolesz 3 , r.m.m. seibel 1 , m. turina 2 , r. kikinis 3 ; 1 mülheim a. d. ruhr/de, 2 zürich/ch, 3 boston, ma/us objective: closed chest coronary artery bypass grafting with the use of a telemanipulator is still performed in very few numbers worldwide. preoperative planning and intraoperative navigation might be a helpful tool for increasing success rates of this procedure. the most promising accomplishments of image guidance in surgery are three-dimensional image-processing algorithms.methods: creation of 3d-visualization involves segmentation from acquired crosssectional images. in 15 patients cross-sectional images were obtained by ct and mr. images were digitally transferred via an internal high-speed to the surgical planning laboratory. we primarily used high-end computer workstations sparcand ultrasparc (sun microsystems, mountain view, ca), equipped with specially developed software, the 3d-slicer for registration, filtering of regions of interest, semi-automated segmentation and 3d-visualization. results: preoperative planning with identification of target vessels and assessment of wall quality, as well as the possibility of image-guided surgery can be obtained by the 3d-visualization of the acquired data-sets. together with the development of semi-automated segmentation, 3d rendering, integration of different image-modalities, intraoperative navigation and tracking can be provided for closed chest coronary artery bypass grafting. conclusion: 3d-image guidance in surgical procedures for preoperative planning in a non-invasive fashion, as well as intraoperative navigation and tracking of robotically-assisted instruments are very helpful in detecting complex coronary lesions and calcified vessels which facilitate closed chest coronary artery bypass procedures. patients with intramural vessels or severe calcification can be identified and the operative technique can be chosen adequately. subsecond spiral ct angiography for detection of small pulmonary thrombi: an experimental study k. li, y. li, x. du; beijing/cn purpose: to evaluate the diagnostic value of subsecond spiral ct pulmonary angiography (ctpa) to detect the small pulmonary thrombi (diameter 2.5 -3.5 mm) in canine models. methods: ctpa was performed as a control study in 23 dogs. pulmonary embolism was induced in the dogs by administation of autologous blood clots, after that ctpa and x-ray pulmonary angiography (xpa) were performed. the images were evaluated on a workstation by two radiologists and the image findings were compared with that of pathological dissections. results: 22 pulmonary embolism models were successfully made in this study and 110 thrombi were found during pulmonary artery dissection. the thrombi were same number as that injected and the all thrombi located in segment and subsegment of pulmonary arteries, of which 107 thrombi were in field of the view (fov). 90 thrombi were found with ctpa, whereas 82 thrombi with xpa. the sensitivity of the ctpa and xpa was 84.1 %, 76.6 %, respectively, and the positive predictive values were 93.8 % and 94.3 % respectively for the diagnosis of small pulmonary thrombi. conclusion: subsecond spiral ct angiography has a high diagnostic value for detection of small experimental pulmonary thrombi in dogs, and the result is superior to routine x-ray pulmonary angiography. diagnosis of acute pulmonary embolism with contrast-enhanced mr angiography: an experimental study k. li, y. li, x. du; beijing/cn purpose: to assess the diagnostic value of three-dimentional gadolinium-dtpa contrast-enhanced magnetic resonance angiography (3d cemra) in acute pulmonary embolism (pe). methods: seventeen dogs with acute pulmonary embolism induced by autologous blood clots were examined with 3d cemra using a fast spoiled gradient echo technique and with x-ray pulmonary angiography (xpa). the findings were compared with pathological dissection. results: the 3d cemra signs of acute pe were: total vessel occlusion (30/41), pulmonary perfusion defect (23/41), partial filling defect (3/41) and central filling defect (1/41) in the pulmonary artery. the sensitivity of 3d cemra was 82.9 %, specificity 98.2 %, whereas the sensitivity and specificity of xpa was 75.6 %, 97.9 %, respectively. conclusion: 3d cemra for the detection of acute pe demonstrated high sensitivity and specificity, but the technique needs to be confirmed by further clinical trials. cardiac imaging in infants with aortic isthmus stenosis: a comparison of contrast enhanced mra and a high-resolution 3d double slab technique u. kramer, f. dammann, j. breuer, l. sieverding, c.d. claussen; tübingen/de purpose: comparison of a three-dimensional gradient-echo (ge) sequence with interleaved double-slab excitation versus a contrast enhanced (ce) mra for the assessment of the great thoracic vessels in children with congenital heart disease.purpose: to assess applicability and diagnostic performance of a standardized multidetector row spiral ct (mdct)-trauma imaging protocol for evaluation of the thoracic and lumbar spine in patients with blunt or spinal trauma. material and methods: mdct (siemens somatom volumezoom, germany) was performed in 86 patients with a history of thoracoabdominal blunt or spinal trauma. all imaging was acquired using a collimation of 4 × 2.5 mm and a pitch of 6. based on this raw data set, the spine was targeted reconstructed using the following parameters: slice width 3 mm; reconstruction interval 1.5 mm. two readers separately assessed imaging quality of spinal structures for diagnostic purposes. in addition, both readers evaluated all patients for spinal fractures using five-point confidence scale (1 = fracture definitely absent, 2 = fracture probably absent, 3 = equivocal, 4 = fracture probably present, 5 = fracture definitely present). the number and level of fractures were categorized and correlated to thin section mdct and/or surgical findings. results: visibility of spinal anatomic details for diagnostic purposes was rated as excellent by reader 1/2 in 927/862 levels and good in 119/184 levels of 1046 spinal levels using a standardized trauma protocol. scoring on the confidence scale was 4.6 (± 0.8) in case of fractures and 1.1 (± 0.1) in case of no fractures, respectively. 20 of 21 spinal fractures (98 %) were correctly depicted by each reader. an anterior wedge fracture was missed by both readers. conclusion: evaluation of the thoracic and lumbar spine is possible with targeted image reconstruction based on standardized chest and/or abdomen mdct data sets. whole body spiral ct in trauma patients -part ii: spinal injuries j. von schlippenbach, t. albrecht, k.-j. wolf; berlin/de purpose: to assess the accuracy of a standardized "whole body" spiral-ct protocol in the initial work-up of spinal injuries in trauma patients and to compare spiral-ct with conventional radiography. methods: 46 trauma patients with potentially life threatening injuries underwent a spiral-ct of the skull base and neck (collimation/table speed/reconstruction interval: 3/5/3 mm), chest, abdomen and pelvis (5/7.5/5) immediately after resuscitation. additional dedicated axial and sagittal high-resolution reconstructions of the entire spine were performed from the data sets. 40 patients also had conventional radiographs of the spine. ct findings were compared with conventional radiographs and final diagnoses at discharge or death. results: image quality was adequate in 39/46 cts and 28/40 conventional spine series. in the remaining cases at least one area of the spine was not adequately imaged, in ct this was most often the thoracic spine. the final diagnoses included 8 cervical, 19 thoracic and 23 lumbar spine fractures; 7 fractures were instable. on ct, all but 1 thoracic and 3 lumbar spine fractures were diagnosed. none of the undetected fractures were instable. on conventional radiography, 1 cervical, 2 thoracic and 5 lumbar spine fractures were missed; again none of these were instable. conclusion: ct was superior to conventional radiography and allowed fast and accurate diagnoses of the vast majority of spinal fractures, and missed no instable fractures. in our experience, time consuming conventional spine radiography is dispensable with this approach in most cases. part i: b-0206 (ss 401) magnetic resonance imaging in traumatized intervertebral discs n.a. ghanem, p. uhrmeister, c.a. müller, c. altehoefer, m. markmiller, m. langer; freiburg/de purpose: evaluation of mri in trauma patients with traumatized adjacent discs of fractured vertebrae before dorsoventral stabilization material and methods: a prospective diagnostic study with 15 trauma patients (12 male, 3 female, mean age: 40 years, range: 17 -72) was performed on a 1.5 t magnetom. the preoperative mri using sagittal t1-w-se and t2-w-tse was compared to the gold standard being intraoperative discography which was carried out on both intervertebral dics adjacent to the fractured vertebrae within a time frame of 1.8 days (0 -3). beside signal alterations of adjoining intervertebral disc, morphological changes such as disc herniation and annular tears were evaluated in 30 adjacent discs. in 12/15 (80 %) patients and in 24/30 of the intervertebral adjacent discs, the results of both imaging findings were concordant. in 3 patients the adjoining discs were normal. concerning the positive concordant imaging findings, mri and discography both revealed traumatized adjacent dics in 6 cases. in 3 cases the upper adjacent disc was injured. among these 15 traumatized discs, 9 intraosseous herniations into the fractured vertebrae and 6 anuluar tears were depicted. in 3 patients the findings were discordant. in one case mri was false positive whereas discography as the gold standard demonstrated no lesion. in 2 cases mri failed to detect a disc injury. conclusion: mri as a noninvasive method may detect traumatized adjacent intervertebral dics, but mri is inferior compared with intraoperative discography although it does reveal additional preoperative information prior to dorsoventral stabilization. mri in traumatic lesions of cervical intervertebral disc b.w. raab, u. fischer, g. kernbach-wighton, k.s. saternus, e. grabbe; göttingen/de purpose: to demonstrate typical findings on mri in traumatic lesions of the intervertebral discs of the cervical spinal column. materials and methods: mri was evaluated in 2 groups of patients with traumatic changes of the cervical spine column including an injury of intervertebral discs. the study included 6 in-vivo examinations of patients and 8 postmortem examinations (neck specimen, c1 -t1 including the skull base) of human corpses after an acceleration trauma. disc lesions were verified intraoperatively in all patients or macroscopically by sections of the specimen. results: the variability of traumatic lesions of the intervertebral disks ranged from small detachments of the disc from the vertebral body to large ruptures of the entire disc. extensive lesions of the disks were correlated with other injuries such as fractures or ligamental trauma. mri depicted all lesions with extension to the entire disk due to morphologic changes in hr-sequences and/or signal alteration in inversion recovery and t2-wi sequences. most of the circumscribed disc detachments seen macroscopically were not detectable with mr imaging. conclusions: mri allows a reliable diagnosis of significant lesions of the cervical intervertebral discs after trauma. small detachments of the disc, however, will be missed. cervical spine injuries in elderly patients: does trauma mechanism and age influence the distribution, type and stability? f. lomoschitz 1, 2 , c.c. blackmore 2 , k.f. linnau 2 , s.k. mirza 2 , f.a. mann 2 ; 1 vienna/at, 2 seattle, wa/us purpose: the purpose of our study was to describe types and distribution of cervical spine injuries in consecutive elderly patients in regard to causative trauma mechanism and to patient age. materials and methods: the distribution and type of 225 cervical spine injuries in 149 consecutive subjects older than 65 years over a 5-year interval were retrospectively assessed. for each subject, initial imaging studies were reviewed and injuries were classified. the trauma mechanism (falls from standing or seated height vs. mechanisms consistent with higher energy) and initial clinical and neurological status were recorded. data were correlated according to patients' age (65 -75 a and > 75 a) and causative trauma mechanism. results: the majority of patients (64 %) had injuries to the upper cervical spine. multilevel injuries were frequent (40 %). the main causes for cervical spine injuries were motor vehicle crashes in "young elderly" (65 -75 a) (61 %) and falls from standing or seated height in "old elderly" (> 75 a) (40 %). fracture patterns at risk for neurologic deterioration were common (> 50 %), even in absence of acute myelopathy or radiculopathy. subjects older than 75 years, independent of causative mechanism, and patients who fell from standing height, independent of age, were more likely to have injuries of the upper cervical spine (p = 0.026 and p = 0.006, respectively). conclusion: injuries of the cervical spine in elderly patients tend to involve more than one level with consistent clinical instability. "old elderly" patients (> 75 years) and subjects who fall from standing height are more prone to injuries of the upper cervical spine. the fluid sign in acute vertebral compression fractures on mri a. baur, a. stäbler, r.h. dürr, m.f. reiser; munich/de purpose: to evaluate the occurrence of the linear fluid sign in acute osteoporotic and neoplastic vertebral compression fractures in magnetic resonance imaging (mri). methods and materials: the study group comprised 87 consecutive patients with a total of 107 acute vertebral compression fractures due to osteoporosis or neoplastic infiltration. the mri protocol included unenhanced t1-weighted spin echo (se) and short-tau inversion recovery sequences (stir) on a 1.5 t system. confirmation of diagnosis was made by surgery, follow-up mri examinations, clinical follow-up or unequivocal imaging findings. results: all fractures showed hypointensity on t1-weighted se images and hyperintensity on stir images, both in cases of osteoporosis (n = 65) and tumor (n = 41). in the fractured vertebral bodies the fluid sign appeared as a circumscribed lesion adjacent to the fractured end-plates, which exhibits signal intensities isointense to cerebrospinal fluid. the shape of the fluid sign was linear (n = 18), triangular (n = 5) or focal (n = 2). it was associated statistically significant with the osteoporotic cause of a fracture (n = 23, 35 %; chi square p < 0.001). the fluid sign occurred also in 2 cases of neoplastic compression fractures (5 %). at the site of the fluid sign histologic correlation demonstrated osteonecrosis, edema and fibrosis. the linear fluid sign represents areas of osteonecrosis with accumulation of fluid in acute vertebral compression fractures. it is highly indicative of osteoporosis, but may occur rarely in metastatic fractures. to compare the efficacy and stability of two different, "second generation", self-expandable, metallic, covered stents in patients with lower third malignant oesophageal strictures. material and methods: 53 patients with inoperable disease were randomised to receive either an ultraflex (n = 31) or flamingo (n = 22) covered oesophageal stent. all procedures were performed in the interventional radiology suite using fluoroscopic guidance. dysphagia before and after stent placement was scored on a 5-point scale and the incidence of early and late complications was compared between the two groups. the initial technical success rate was 100 % in both groups. improvement in the dysphagia score was demonstrated in both groups at both short and long-term follow-up (mean reduction of 2 points). no significant difference was seen in the improvement of dysphagia between the two groups. the early (migration, severe reflux and perforation) and late (haematemesis, tumour ingrowth) complication rates were similar in both groups. purpose: mnd is characterized by bulbar symptoms resulting in reduced respiratory function, diaphragmatic paralysis and weakening of respiratory muscles. nutrition is a prognostic factor for survival but surgical or percutaneous endoscopic gastrostomy is not well tolerated. rig can be safely deployed with severe respiratory compromise. we describe our experience using a modified technique, which allows safe percutaneous puncture of a high subcostal stomach. mnd patients requiring nutritional support were selected for a rig as a consequence of a sub-normal vital capacity. the standard procedure for rig insertion was adhered to, with use of four t-fastners to secure a gastropexy. to take account of the high subcostal site of the stomach, the procedure was modified, performed with lateral screening, allowing for constant visualization of the puncture needles, to confidently puncture the anterior stomach wall.results: this modified technique has been employed in 35 patients (20 male and 15 female, median age 62 years, range 31 -81 years) over a 36 month period. 28 standard radiological gastrostomy tubes were inserted, six were self retaining devices. the procedure was successful in 34 patients (97 %), 7 tubes were reinserted (20 %, all standard radiological tubes) over a variable period of time. the one failure was due to a high subcostal stomach in an obese patient. conclusion: diaphragmatic palsy presents a technical challenge for insertion of a rig in the conventional manner. using lateral screening a high success rate is achieved, and should be the method of choice for mnd patients with respiratory compromise. nineteen uncovered sems (diameters: 18 -22 mm, length: 60 -90 mm) were placed using fluoroscopic guidance in fifteen patients via a per-oral route using standard catheter and guidewire techniques. nine patients had pyloric obstruction. four had undergone gastric pull-up operations or partial oesophagogastrectomy for oesophageal or gastric carcinoma with pyloric dysfunction not responsive to balloon dilatation. five patients had inoperable oesophageal or gastric carcinoma involving the pylorus. six other patients had duodenal obstruction secondary to pancreatic carcinoma. in only three patients were two stents required to cover the obstruction. four patients with malignant obstructive jaundice required concurrent stenting of the common bile duct. results: all patients were able to consume an adequate diet after stent insertion. one stent migrated proximally into the fundus at day two in a patient with gastric carcinoma involving the pylorus. he presented five days later with perforation of the antrum at the tumour site which required emergency surgery. of the remaining patients (seven have died) none have experienced recurrent symptoms of gastric outflow obstruction over a mean follow-up period of fourteen weeks. conclusion: non-endoscopic placement of sems under fluoroscopic guidance is a safe and effective treatment for outflow obstruction secondary to pyloric and duodenal stenosis. it is particularly effective in the palliation of malignant disease, but also has a role on the treatment of pyloric dysfunction after gastric pull-up surgery. purpose: reference methods in diagnosis of crohn's disease are endoscopy and small bowel follow-through. involvement and activity of proximal segments of the small bowel is difficult to evaluate. our purpose was to develop a sensitive, easy to perform and minimal-invasive method for imaging small bowel without radiationexposure for mostly young patients. methods: 30 patients (18 female/12 male) with suspected or established crohn's disease were included. the patients were administered orally 1.5 l of a mannitolsolution (2.5 %) within 1 hour before imaging. a rectal mannitol-solution filling was also employed. butylscopolamin was administrated i.v. haste and fs-hastesequences and t1-bh-sequences were acquired before and after (fs-t1-bh) i.v. gd-dtpa administratio in transversal and coronal orientation. the images were analysed by three radiologist using a standardised protocol. in all cases a correlation with ultrasound-imaging was performed. results: alterations indicative of crohn's disease were noted in 21 patients. in these cases we detected wall thickening with an average of 7.1 mm and with a mean length of 11.5 cm. the signal-intensity-increase after contrastmedium-application of thickened bowel wall was 58 %. normal bowel-wall had an increase of signal-intensity after gd-dtpa of 38 %. 11 relevant stenoses (37 %), 2 abscesses (6 %) and 9 fistulas were visualized (31 %). in ultrasound imaging we detected 9 cases with a stenosis (31 %), 5 fistulous tracts (17 %) and 1 case (3 %) with an abscess. conclusion: mannitol-mri of small bowel in patients with suspected or established regional enteritis is a promising method for the diagnosic work-up and renders results superior to ultrasound imaging. inflammatory bowel disease in children: helical ct with water enema administration g. tognini 1 , f. ferrozzi 1, 2 , g. zuccoli 3 , a. patti 1 , p. bini 1 , p. pavone 1 ; 1 parma/it, 2 cremona/it, 3 reggio emilia/it purpose: the diagnosis of inflammatory bowel disease (ibd) in children, due to the non specificity of the clinical picture, is often difficult and delayed. the aim of our study was to assess the role of helical ct after water enema administration in the diagnosis and staging of ibd.materials and methods: 33 consecutive patients with ibd (19 ff, 14 mm) age range: 4 -15 years underwent spiral ct examination of the abdomen. the imaging protocol consisted of 5 mm collimation, pitch: 1.5, injection of iodinated contrast medium with high flow rates: 3 -3.5 ml/s. the lumen of the colon was filled with water (300 -1000 ml); oral administration of water was also performed in order to obtain gastric and duodenal distension. we evaluated: wall thickening, structure and enhancement of the involved segments, polypoid components, mesenteric fat involvement, adenopathies, extraparietal flogistic complications. 33/33 underwent endoscopic and bioptic confirmations: 23/33 were crohn's disease, 8 ulcerative colitis, 2 showed non specific features. results: wall thickening (26/33), increased contrast enhancement (23/33), endoluminal pseudopolyps (7/33), fibrofatty proliferation (9/33), edematous infiltration (6/33), mesenteric adenopathies (19/33), abscesses and flogistic complications (10/33). in 21/33 patients ct allowed for the correct differentiation between crohn's disease and ulcerative colitis. the morpho-structural evaluation which can be obtained by means of helical ct and water enema administration allow for an accurate study of the intra and extraluminal manifestations of the ibd. ct was found useful in the diagnosis but mainly in the follow-up of patients with ibd giving important differentiating criteria. ct signs of active crohn's disease and comparison with immunscintigraphy g. tóth, g. szentmártoni, t. györke, e.k. makó; budapest/hu purpose: this retrospective study compares the diagnostic yields of ct and immunscintigraphy in the assesment of activity in crohn's disease. we evaluated which ct signs are the best predictive signs of the activity in crohn's disease. we examined 126 patients with clinically suspected crohn's disease. we studied patients first with bifasic dinamic helical ct and 4 -7 days later with immunscintigraphy. ct studies were performed in a standard fashion: collimation 8 mm, feed 12 mm, reconstruction 5 mm. i.v. contrast administration flow 3.5 ml/s, volume 1.5 ml/kg, delay 40 s in a caudo-cranial direction. we were searching for bowel wall thickening, increased contrast enhancement of the bowel wall, mesenterial signs, fistulas and abscesses, and enlarged lymph nodes. ct results were correlated with immunscintigraphy. results: in cases where the affected loops showed thickened wall (4 -10 mm) increased contrast enhancement and sourranded by a fibrofatty proliferation, with increased vascularisation the immunscintigraphy was positive in 91 % of patients. when we saw fistulas, abscesses and a thickened bowel wall (10 mm or more), with increased vascularisation in mesentery with prominent enlarged lymph nodes the immunscintigraphy was always positive. conclusion: contrast enhanced spiral ct study is a goog tool in the assesment of the activity in crohn's disease. results: in 10 cases the embolization failed: no bronchial artery catheterized in the bleeding side (n = 6), pulmonary artery bleeding due to central excavated lung tumors (n = 2), right intercosto-bronchial trunk with a doubt about a spinal radicular artery (n = 1), unstable catheter position (n = 1) and technical problem (n = 1). among the 61 patients with at least one bronchial artery selectively catheterized (included 4 non-embolized), bronchial hypervascularisation was visualized in 58 associated with nonbronchial systemic hypervascularisation in 4 cases. the immediate control of bleeding was obtained in 72 %. long-term result was 61.4 %.complications were: acute but transient renal failure (n = 1), cerebral ischemia (n = 2) with one definitive deficit. conclusion: tumours are responsible of 12.3 % of massive hemoptysis and embolization is efficient. the bleeding is rarely due to nonbronchial arteries. stroke incidence is increased in comparison with other hemoptysis probably because of elderly patients. intraoperative transthoracic ultrasonography for the localization of hidden pulmonary nodules during thoracoscopy f. coppola, m. piolanti, f. gruppioni, s. papa, m.p. di simone, s. mattioli, g. gavelli; bologna/it purpose: the purpose of this study was the evaluation of intraoperative sonography in the detection of hidden pulmonary nodules during thoracoscopic resection. to perform intrathoracic ultrasonography an endosonographic linear probe with high-frequency transducer (7.5 -10 mhz) was employed. 40 patients, entered the study; 52 pulmonary nodules were preoperatively detected, 50 by spiral ct and 2 by pet. 10 sonographic examinations were performed during thoracotomic surgery and 30 examinations during thoracoscopy. the times needed to reach lung collapse and to localize the targets were recorded. complete lung collapse is essential to perform intraoperative sonography and can be accomplished by single controlateral lung intubation and ventilation for 45 minutes at least. the sonographic examination is quick, unless the patient is affected by obstructive disease (such as centrilobular emphysema): retained air in the parenchyma reduces the explorability. all but one preoperatively detected lesions and two further unknown lesions were localized by intraoperative sonography. there were no complications related to the procedure. conclusions: intraoperative sonography proved itself to be a reliable and satisfactory localization technique of lung nodules during thoracoscopy. in comparison with the other localization techniques it shows several advantages, such as the capability of revealing preoperative occult lesions and determining the surgical borders. the aim of this study was to assess the accuracy of wire-versus carbon-localization in slobb. a total of 1115 slobbs were performed from 01/94 until to 12/97 in 1068 female patients (age range, 22 -90 years) on a prone stereotactic unit (mammotest, fisher imaging, denver, co). in 1007 cases successful slobb was verified either by a malignant histopathological diagnosis or by immediate mammographic evaluation after surgery. in 582 (58 %) of these cases the lesion was localised with a wire, in 410 cases (41 %) carbon was used. both methods were used to localise the remaining 15 cases (1.4 %). a lesion was considered as missed when it could be still seen on mammography performed after surgery. results: ten out of 1007 cases (1 %) were missed at slobb. of these, 5 lesions had been localised by wire (5/582; miss-rate 0.9 %) and 5 lesions with carbon (5/410; miss-rate 1.2 %). there were no significant differences in miss-rates between wire-and carbon-localisation (p > 0.05). conclusion: both wire-and carbon-localisation are reliable and accurate in the localisation of non-palpable breast lesions undergoing slobb. considering the fact that wire-localisation is far more expensive than carbon-localisation the routine use of carbon should be considered. histological results of scnb and surgical biopsy were compared and scored for each needle size on a three point scale (1 = no agreement, 2 = partial agreement, 3 = complete agreement) to determine the optimal needle size for such lesions. results: there were 69 masses, 13 asymmetric densities, and 53 calcifications. a diagnosis of malignancy was found at surgery in 1/135 (0.7 %) [1 ductal carcinoma in situ]. the remaining 134 lesions revealed a benign histology. the 11 g needle (score = 3) scored better than both 14 g needles (score = 2.9). conclusion: based on the relative low rate of carcinomas scnb should not be performed routinely in probably benign lesions (bi-rads 3). mammographical short time follow-up seems to be more useful to confirm the benign nature of such lesions. if scnb will be performed 11 g needles seem to be advantageous. does the preoperative core-cut biopsy influence the appearance of local recurrence of breast cancer? u.g. aichinger, j. ammon, r.w.s. schulz-wendtland, i. kuchar, w.a. bautz; erlangen/de objective: retrospective study to evaluate if preoperative core-cut biopsy influences the incidence of local recurrence of breast cancer and metastasis -a phenomenon described after puncture biopsies of the pancreas and the prostate. material and methods: core-cut biopsy was performed on 150 patients with suspected breast cancer before undergoing surgery in the period 1992 -1993. the clinical outcome of 123 patients was analysed retrospectively over a period of seven years. 66 had undergone breast conservation therapy, 57 had had a mastectomy. adjuvant therapy was also included in the evaluation. results: local recurrence after breast conserving therapy (bct) was seen in 11 of 66 patients (17 %), and in 7 of 57 (12 %) after mastectomy (mrm). the rate of local relapse after radiotherapy (rt) was 8 of 43 (19 %) (bct) and 4 of 25 (16 %) (mrm) respectively. in the group without rt 3 of 23 (13 %) (bct) and 3 of 22 (9 %) (mrm) relapsed respectively. in two cases the biopsy site was not removed. one of those, not having had a rt, had a local recurrence. conclusion: according to the literature, breast cancer local recurrence rate is 1 -2 % per year. in our group recurrence rate reached 1.9 % totally per year and 2.1 % after bct being in agreement with international studies. no higher rate of local recurrence or metastasis was seen after core-cut biopsy. nevertheless the biopsy site should be removed in any case. multicenter evaluation of stereotactic vacuum biopsies of mammographically indeterminate or suspicious lesions the consensus was achieved based on the existing literature and on the experience of the participants using a consensus process as suggested by sackett. results: complete standard imaging work-up is the prerequisite before a decision for percutaneous breast biopsy is made. the following lesions are considered well-suited for vacuum biopsy: microcalcifications, small non-palpable masses with or without microcalcifications. the following indications may not be (as well) suited: architectural distortion (suspected radial scar) or lesions close to the skin. needle access (angulation of compression, stroke margin) must be exactly documented. acquisition of > 20 cores (11 g) shall be routinely attempted (goals: radiologic removal of lesions < 10 mm to increase diagnostic reliability, decrease of underestimates). documentation of scout, pre-, post-fire and post-biopsy images and another orthogonal view after biopsy are required. final diagnosis must consider imaging-histopathologic correlation, and for certain histopathologic entities the result on an interdisciplinary conference. standard documentation of the examination and of 6 months-follow-up is required. the closed architecture of 1.5 t whole body magnets makes it difficult to perform mr-guided biopsies at high-field strength. the aim of this study was to investigate the feasibility of mr-guided breast biopsy using a remote-controlled robotic system which assists the localisation of the target and runs inside the magnet. materials and methods: 13 patients with suspicious lesions (diameter 19 ± 11 mm) in mr-mammography were biopsied using the robotic system. after localisation of the lesion the robotic system positioned the troacar at the correct position. the troacar was inserted into the breast in front of the lesion. the specimen was taken in coaxial technique using a 13 g core needle biopsy gun. in case of malignant histology the patient underwent open surgery afterwards. the biopsy procedure inside the magnet was completely performed without interruption in all cases. operation proved the histological finding of breast cancer in 5 cases. one patient was diagnosed as atypical ductal hyperplasia by biopsy and turned out to have an invasive ductal carcinoma. 8 patients showed benign lesions and will be followed up. the current study demonstrated the feasibility of breast biopsies inside the magnet of a whole body scanner using a robotic system. one false negative case was due to tissue shift during the insertion of the troacar into the breast. first experiences with two patients show the procedure to be feasible. in future imaging, biopsy and therapy of breast lesions should be possible in one single patient session using this robotic system. to assess the feasibility of assessment of stent-graft patency by contrast-enhanced magnetic resonance angiography (ce-mra). materials and methods: 9 bifurcated abdominal aortic aneurysm (aaa) stentgrafts made of nitinol, elgiloy or stainless steel were investigated regarding their appearance on mr imaging. the stent-grafts were positioned in a phantom filled with aqueous gadolinium solution. coronal and axial three-dimensional gradient echo sequences were performed (tr 8.1 ms/te 1.3 ms, fa 40°). relative signal intensity reduction within the stent-grafts and the difference of the real stent-graft lumen as compared to the lumen measured on the mr images were calculated. results: the graft lumen was depicted in 6 prosthesis. the lumen of stainless steel stent-grafts showed a complete signal void. relative narrowing of the inner diameter on the mr images was less in the proximal part of the stent-grafts than in their legs. with a gadolinium dilution of 1:20 the artifical narrowing of the proximal lumen ranged from 7.1 % to 59.5 %, whereas in the stent-graft legs the visible lumen was diminished between 2.4 % to 71.8 %. only three stent-grafts presented with a lumen reduction on the mr images of less than 33 % along the whole prothesis length. conclusion: to differentiate between artifacts and stenoses, a knowledge of the degree of signal intensity reduction and artificial lumen narrowing within aaa stentgrafts is essential. stent-graft composition and the design of the stent-graft influence the artifact behaviour and lumen visibility as displayed on ce-mra. only a minority of the investigated stent-grafts is suitable for imaging by ce-mra. 28 patients with aaa undergoing endovascular stent grafting were included in the study. helical ct scanning was performed within one week following stent graft implantation with a scanning protocol of collimation 5 mm, pitch 1.0, and a 2 mm reconstruction interval. contrast enhancement was given to all patients with a total volume of 100 ml, flow rate of 2 ml/s and scan delay of 30 seconds. all patients received a zenith tm /aaa endovascular graft with uncovered supra renal struts of 2.5 cm length placed above the level of the renal arteries. vie images were created for each patient with analyze avw 1.0. the follow-up period ranged from 1 to 12 months (mean 6.9 ± 4.7 months). results: 4 of 28 coeliac arteries, 23 of 28 superior mesenteric arteries, 28 of 28 right renal arteries and 28 of 29 left renal arteries were covered by the stent struts to different extents. vie demonstrated the number of metal wires crossing the arterial ostia and the configurations of these stent struts relative to the arterial ostia such as partial or complete encroachment. follow up ct scanning showed that all of these aortic arterial branches were patent. conclusion: vie is a novel imaging technique to visualise the 3d intraluminal relationship of the aortic stent struts to the arterial ostia in patients with aaa following suprarenal stent grafting. the current system consists of a see-through, headmounted display, a tracking system, computer system and tracked surgical instruments. interaction with the system is provided through voice and speech. the system produces a computer-generated image overlaid onto the real world, using virtual "hanging windows" for information display eg. ct/mr scans, real-time fluoroscopy, vital signs, live video (endoscopy), step-by-step instructions, dictation/ communication screens, 3d models of anatomy and other information to assist the radiologist during a procedure. in addition, different pre-and post-stent 3d models of the aortic aneurysm from recent procedures can be brought up within the environment for comparison or as a reference. the user can also bring up patient-specific 3d reconstructions. results: this system provides assistance during aaa stent placement. all previous imaging series (ct-, mr-, and conventional angiography), stent planning data, 3d models of the aneurysm and monitoring of vital signs are shown on one screen (head mounted display) in "hanging windows" on request. the software provides a real-time surgical simulation system with integrated monitoring and information retrieval and a voice input/output subsystem. the system provides all the important information on one screen (head mounted display) and can be used to assist various tasks such as stent implantation procedures in the interventional radiological theater. a.m. grozaj, t. pfammatter, m. lachat, u. wolfensberger, p.r. hilfiker; zürich/ch purpose: to report midterm results of endovascular stent-grafting in patients with ruptured abdominal aortic aneurysm (raaa). materials & methods: 17 patients (3 women, 14 men; mean age 73 years) with raaa were treated with bifurcated endoprostheses. preinterventional assessement was performed with ct (n = 17), ultrasound (n = 5) and angiography (n = 1). all patients were a high operative risk. the procedure was performed under local (n = 12) or general anesthesia (n = 5). follow-up cta was performed after 1 day, 6 weeks, 3, 6, 12 months and annually. results: immediate technical success was achieved in 16 patients (94 %). one needed conversion to open surgery due to progressive hemodynamic instability. mean intervention time was 149 min (± 114), mean icu stay 2.6 days (± 0.85) and mean hospitalization 10 days (± 5). the 30 day mortality was 0 %. within 30 days one patient was treated with hemofiltration and three had reinterventions: replacement with homograft of an infected iliac-femoral extension sg; sg extension for retroperitoneal bleeding; embolization of the inferior mesenteric artery. in the mean follow-up period of 8 months two endovascular reinterventions were performed: sg implantation for graft disconnection; thrombolysis of a stent leg. one patient needed elective late conversion to open surgery after 14 months due to sg migration. aneurysmal diameter decreased in 58.8 %, increased in 5.8 % and remained unchanged in 35.3 % of patients. conclusion: endovascular treatment of raaa with sg implantation is feasible and safe. this technique seems to be a valuable alternative to emergency open surgery with its high mortality. emergency treatment of thoracic aorta diseases by endovascular stentgraft l. lovato, r. fattori, g. napoli, c. grazia, f. settepani, g. gavelli; bologna/it purpose: surgery of the thoracic aorta is characterized by high mortality and morbidity, especially if performed in an emergency. we report our experience in emergency endovascular treatment of the thoracic aorta. material and methods: from 1997, 58 patients underwent endovascular treatment of the thoracic aorta. seven (2 type b acute aortic dissections, 2 traumatic aortic ruptures, 1 penetrating aortic ulcer and 2 surgical prosthesis dehiscences) presented with clinical and/or imaging findings of impending aortic rupture and were treated as an emergency. before the procedure, spiral ct was used to define the lesion's anatomy. all procedures were performed in the operating theatre and monitored by a radiographic c-arm system and by echocardiography. results: stent-graft implant was successful in all patients. no death nor complication occured during the procedure. in 4 cases, immediately after stent-graft implant, surgical drainage of a hemothorax was performed. the average time of intensive care unit stay was 12 ± 8 hours, while hospital stay was 7 ± 5 days. in 6 patients a spiral ct follow-up showed complete aneurysmal exclusion; 1 case showed persisting leakage over 3 months and was converted to surgery. conclusion: endovascular stent treatment of thoracic aorta diseases could be a very effective option and an alternative to surgery, especially in an emergency. endovascular treatment of thoracic aortic aneurysms and dissections with the use of customized stent-grafts q. sénéchal, f. koskas, p. cluzel, j.-d. singland, e. kieffer, p.a. grenier; paris/fr purpose: to describe our experience with the use of a custom stent-grafts for the treatment of thoracic aortic aneurysms and dissection. patients and methods: from january 1996 to september 2001, 28 aneurysms and 11 dissections were selected to be treated using custom stent-grafts of different designs. custom stent-grafts were constructed with gianturco z stents sutured together and covered with polyester (twillweave, vascutek, innachinnan, scotland). endovascular treatment of aortic arch aneurysms combined with extraanatomic bypasses were used in 7 cases. elephant trunk in one step was used in three cases. endovascular bypasses were used as the second step of the elephant trunk technique in two cases. results: four patients (10 %) were converted to standard open repair. no leakage of the thoracic aneurysms was observed at follow-up. sealing of the primary tear in thoracic aortic dissections was successful in all cases. three patients died of myocardial infarction in the perioperative period. conclusion: custom stent-grafts in the endovascular treatment of thoracic aortic pathology achieve good results. endovascular treatment of aortic arch aneurysms combined with extra-anatomic bypasses provides a less invasive method of treatment of these complex lesions. longer follow-up is needed for the device. percutaneous treatment of thoracic aortic dissections and thoracic aortic aneurysms using commercially available stent-grafts f. fanelli, f. salvatori, m. rossi, g. marcelli, f. venditti, p. rossi; rome/it purpose: to report our preliminary experience in the treatment of thoracic aortic dissections and thoracic aortic aneurysms (taa) using endovascular stent-grafts. methods and materials: from november 2000, 16 patients were treated using a commercially available stent-graft: thoracic excluder (w.l. gore). twelve cases were performed in the angio-suite and 4 in the or under general or epidural anesthesia. taa: 6 patients. in two patients (12.5 %), with a short proximal neck, the origin of the left subclavian artery was covered by the stent-graft. dissection: 10 patients with type b dissection, 7 acute and 3 chronic. two cases were complicated by renal ischemia treated by renal stenting. the primary entry tear was located in 7 patients less than 2 cm from the origin of the left subclavian artery which was covered by the stent-graft. results: after a follow-up ranging from 2 to 10 months, all patients are in a good condition with no signs of paraplegia. taa: technical success with complete exclusion of the aneurysm was evident in all cases. dissection: 1 technical complication (6.25 %) was observed during stent-graft deployment of antegrade extension of the dissection into the ascending aorta which was surgically managed. in all cases progressive thrombosis of the false lumen developed. in 3 cases (18.7 %) an endoleak was detected: one, originating from the subclavian artery was treated by coil embolization; one sealed spontaneously after 6 months; the last one is under control. conclusion: endovascular techniques represent a new frontier in the treatment of thoracic dissection and taa, but further evaluations are mandatory to clearly understand its efficacy. the probability of location of breast mass margin in digitised mammograms f. sendra-portero, e. ristori-bogajo, e. nava-baro, m. martínez-morillo; málaga/es purpose: most of the computational problems in segmentation are originated by tortuous contours which are difficult to be perceives by either a computer segmentation program or a human observer. a method based on successive interactive segmentations of breast masses in digitised mammograms to plot the probability of location of the mass contour is introduced. materials and methods: 81 digitised mammograms including 94 masses with different type of margins were studied. the border of each mass was outlined with a digitising tablet, encompassing all densities considered as part of the mass, including spicules and tracts. two observers repeated the same procedure three times. the segmentations of the same image were split off between them 48 hours at least. a labelled image was obtained from the six binary images of each case, giving seven possible values per pixel, from 0 to 6. thus, the lines that delimit each value are considered "isoprobability lines", encompassing a probability equal to 1.00; 0.83; 0.67; 0.50; 0.33 and 0.17 to find the mass margin. results: when the margin is well defined the isoprobability lines are closed one each other, with a sharp transition from 1 to 0. in exchange, when the margin is illdefined obscured or speculated, the isoprobability lines spread out. in those combined type margins the uncertainly zones are clearly distinguished from well defined zones. conclusion: images obtained by this method can be used as a reference pattern to evaluate automatic or semi-automatic computer-aided segmentation systems of breast masses. results: mfe-vq employing 16, 25, and 36 cvs identified clusters that could be attributed to known suspicious regions in each case. the lesions could clearly be differentiated from surrounding tissue. in 7 patients, single clusters could be related to motion artifacts based on signal dynamics and cluster localization. in addition, a self-organized subclassification within the lesions could be achieved in up to 4 clusters w.r.t. fine-grained local differences in mri signal amplitude and dynamics. conclusions: mfe-vq by deterministic annealing is a useful strategy for the analysis of contrast-enhanced mri mammography time-series without time-consuming interactive contour tracing of regions of interest by human observers, thus enabling segmentation of suspicious lesions. in addition, a subclassification w.r.t. local differences of perfusion patterns inside suspicious regions can be achieved. in three dimensional medical imaging like ct or mri, knowledge based approaches foster the robustness and reliability of segmentation processes. a robust fully automatic segmentation is a desirable task to acquire objective and accurate results. a 3d surface model for the segmentation of parenchymal organs in ct-and mri-datasets has been developed. the necessary training set for the surface model is computed from semiautomatically generated triangulated surface meshes of the objects. the generated surface model is capable of learning the characteristic shape and gravalue information stored in the training set. the optimization is performed by iteratively moving the surface points of the model towards fitting image structures under the constraints of its grayvalue and shape information. two different 3d surface models have been developed: a milt model consisting of 10 training objects, and a kidney model generated from 7 left kidneys. the two models have been tested on 6 unknown milt-and also 6 unknown kidney-datasets. the total cover between the model and the organs varied between 70 % and 80 %, which is a respectable result in the face of the small training set. further the computing time of the model optimization lies in contrast to other approaches within a few minutes on a standard pc. to overcome the problems segmenting organic structures in 3ddatasets using arbitrary methods, a knowledge-based 3d surface model has been successfully introduced. the segmentation accuracy of the model can be actually improved by using a larger training set in future. use of the dicom file format for quantitative analysis of brain images m. larobina 1 , a. prinster 1 , m. quarantelli 1 , a. ciarmiello 1 , j.p. hornak 2 , b. alfano 1 ; 1 naples/it, 2 rochester, ny/us purpose: to review our experience using dicom format files to perform quantitative analysis of mr brain images. we use an algorithm that performs segmentation and volumetric measurement of normal and abnormal brain tissue. the method is based on calculations of relaxation rates of brain tissues from spin-echo images. the algorithm reads dicom format images, and requires repetition time, magnetic field strength, slice location, fov, and matrix size information from the dicom file header. we have applied the algorithm to studies performed on ge signa 1.0 t, ge signa 1.5 t, philips gyroscan nt 1.5 t, picker eclipse 1.5 t, and siemens magnetom 1.5 t. results: images generated by these scanners reveal that the manufacturers interpret dicom standard differently. in some cases, the file header reports fields with structure or content not always in accordance with the dicom standards. deviations in the dicom header usually do not render the file unreadable, but do hinder the use of the header information and the dicom format. the dicom format has the potential to become the format of choice for all the post-processing of mr images. unfortunately, the standard is too complex to easily discover if violations of the standard are present. we emphasize the need to have a way to know if a set of dicom files is totally compliant with the dicom standard. the use of images that are labeled dicom-compliant except for information reported in one field represents a non-negligible problem. multi-slice ct for visualization of pulmonary embolism using perfusion weighted color maps j. purpose: to evaluate the feasibility of a new technique for perfusion weighted colour display of lung parenchyma density derived from multi-slice ct (msct) data sets for visualization of pulmonary embolism (pe). methods and materials: imaging of patients with suspected pe was performed on a msct (somatom volume zoom; siemens, forchheim, germany) after intravenous application of 120 cm 3 of contrast-medium. based on thin collimation axial slices (slice thickness 1.25 mm, reconstruction increment 0.8 mm), a new image processing technique was deployed. automated 3d-segmentation of the lungs was performed followed by threshold based extraction of major airways and vascular structures. the filtered volume data were color encoded and finally overlayed onto the original ct-images. this color encoded display was presented in axial, coronal and sagittal plane orientation. in ten patients with excluded pe as well as in ten patients with proven pe the new technique was performed. results: in ten patients, considered negative regarding pe on msct, lung densitometry showed a homogeneous distribution of color encoded densities without circumscribed decreased or increased areas. in the patient group with proven pe, low density values on perfusion weighted colour maps were found distally to the occluded pulmonary arteries. conclusion: our initial experience indicates that lung densitometry with an optimized colour encoded display of the density distribution within the lung parenchyma may provide additional information in patients with suspected or proven pe. the evaluation of first-pass myocardial perfusion imaging in patients with coronary artery disease (cad) represents a complex issue in the field of cardiac mri. commercially available software allows placement of regions of interests (rois) into the myocardium and calculation of mean signal-intensity over time. however, small perfusion defects may be missed using this method due to the large area of rois. purpose of our study was to develop a pixel-based evaluation software for mr myocardial perfusion images. we chose a matlab based approach (matlab version 6.1). our software enables the user to register the images using a least-square-algorithm. images can be filtered with a median filter. maximum upslope and area-under-the-curve for the signal intensity are calculated for each pixel and shown graphically. myocardial perfusion measurements were performed in 10 patients with angiographically proven cad using a sr-truefisp-2d-sequence (tr 2.4 ms/te 1.2 ms). mr perfusion data were analysed with our software and compared to spect and angiography. results: data of 8 patients could be adequately analysed with the software developed. parameters upslope and area-under-the-curve in regions found to be malperfused by mri correlated well with angiographically detected coronary artery stenosis and hypoperfused areas detected by spect. main limitation of the method was incorrect registration of images, caused by cardiac arrhythmia and patient motion. conclusions: semi-automated pixel-based analysis of perfusion measurement is possible and the promising results of this analysis correlate with angiographic and spect findings. artefact free data acquisition and image registration are important and the limiting factor for pixel-based analysis. purpose: to evaluate clinical feasibility of spatial domain filtering as an alternative to additional image reconstruction using different kernels in chest ct. methods and materials: 40 adult patients with clinical suspicion of pulmonary embolism were examined utilizing multi-slice ct (somatom volume zoom, siemens, germany). two sets of images were reconstructed using lung (b50) and soft tissue (b30) kernels, respectively. additionaly, b50 images were filtered in the spatial domain, producing images largely equivalent to b30 images. diagnostic images were compared to the initially reconstructed b30 images. subjective image quality was rated using a three point scale (1 = excellent, 2 = fair, 3 = nondiagnostic). results: filtered images provided 10 central emboli, 19 segemental thrombi and 20 emboli on the subsegmental level in the 20 patients with proven pulmonary embolism. latter was excluded in the other 20 subjects. subjective gradings of image quality, based on soft tissue settings were comparable for all data sets. therefore, these filtered images provided exactly the same diagnostic accuracy for central, segmental and subsegmental pulmonary embolism compared to conventional soft tissue reconstructions. the new method presented has proven to be clinically feasible and should therefore be implemented in the clinical enviroment. online modifications of image sharpness and pixel noise in real time will potentially replace the need for multiple reconstructions using different kernels. volume rendering of portal liver vessels: correlation between 3d recostruction parameters and image quality g. luccichenti, f. cademartiri, p. pavone; parma/it purpose: aim of this work is to determine the influence of the scan, density values and opacity curves in three-dimensional reconstruction with volume rendering technique of portal liver vessels. methods and material: 22 patients with previous abdominal ct or us underwent spiral ct during the arterial and the portal phase with the following parameters: collimation 3 mm, feed 4.5 mm, pitch 1.5, increment 1 mm. images were sent to a workstation running on a nt platform equipped with vitrea 2.0 (vital images, usa) allowing 3d reconstructions in order to generate volume rendering of the liver vascular supply. density values of portal vessel (p) and liver (l) parenchyma were measured and correlated with 3d image quality and the parameters of three different opacity curves (oc) utilized to obtained them. results: better visualisation of arterial supply was obtained with non linear opacity curves. it was possible to clearly demostrate them up to the fifth generation. significative correlations were observed between curve parameters (c), liver and portal vessels densities ratios (c vs p and c vs l r = 0.67 and r = 0.82 respectively for linear oc and r = 0.82 and r = 0.84 for non linear oc). correlation between c and 3d image quality was r = 0.22 and r = 0.52 for linear and non linear oc respectively. p/l density ratio poorly correlate with 3d image quality r = 0.34. wilcoxon test was significant between the three opacity curves. conclusion: 3d image quality mainly depends on scan quality. density ratios are important to set the opacity curve parameters. direct volume rendering based interactive virtual colonoscopy d. jocha, j. koloszár; budapest/hu purpose: virtual colonoscopy is a non-invasive computerized medical examination method for examining the interior of the human colon, aiding the detection of polyps. nowadays systems for this purpose are slow and/or require special hardware and have many other problems making their usage difficult and limited. our purpose was to develop a system without these disadvantages. materials and methods: in our study we have examined the most popular approaches to the problems of high-speed rendering and navigation within the colon, and devised our own algorithms. our intent was to make them fast and reliable enough to be implementable on a consumer-end pc system, instead of state of the art high-end workstations, and to minimize off-line calculations. results: we present our idea of a real-time direct volume rendering based visualization technique combined with an on-line computer aided interactive navigation method for the examination of volume data constructed from ct scans. these algorithms have been implemented in our colon visualization program called colvis. the results are promising, the current implementation is close to the real-time requirements (∼ 2 seconds/frame). conclusion: according to our results virtual colonoscopy can be an everyday diagnostic method in the near future. results: acl and pcl avulsion fractures occurred with equal frequency. acl avulsion fractures occurred more commonly in adults than previously believed. about one half of acl avulsion fractures are partial, involving the anteromedial bundle only, one quarter are comminuted and one-half are extended. pcl avulsion fractures occur in an older age group than acl avulsion fractures. the majority are complete, half are comminuted (between the individual pcl bundles) and half are extended. when compared to anteroposterior and lateral radiographs, ct is helpful at delineating the fracture margins (for type i and ii fractures) and at delineating comminution and extent in pcl injuries. three-dimensional ct allows good perception of the fracture type and tibial bony defect as a prelude to operative reduction. this study leads to a better understanding regarding the relative freqeuncy and variablity of cruciate avulsion fractures and indicates those areas where ct is particularly helpful. dual-detector spiral ct arthrography of the knee: assessment of anterior cruciate ligament and associated meniscal tears b. vande berg, f.e. lecouvet, p. poilvache, j.e. dubuc, b. maldague, j. malghem; brussels/be purpose: to assess dual-detector spiral computed tomography (ct) arthrography of the knee in the evaluation of anterior cruciate ligament (acl) and associated meniscal tears. the acl and meniscal abnormalities in 125 consecutive patients who underwent dual-detector spiral ct arthrography of the knee were evaluated based on both initial interpretations and retrospective review of ct images and were compared with arthroscopic findings. the sensitivity and specificity of ct arthrography for the detection of acl tears and of meniscal lesions in aclabnormal knees were determined. results: the sensitivities and specificities for the detection of acl tears were 90 % and 96 %, respectively, at initial interpretation and 95 % and 99 %, respectively, at retrospective interpretation. the sensitivities and specificities for the detection of meniscal tears in acl-abnormal knees were 92 % and 88 %, respectively, at prospective initial interpretation and 96 % and 94 %, respectively, at retrospective interpretation. conclusion: dual-detector spiral ct arthrography of the knee is an accurate method for detecting acl and associated meniscal tears. 2d-tof unenhanced and 3d-spoiled-gre contrast-enhanced images were acquired. unmatched, randomized evaluation of images was performed on-site and by two off-site blinded reviewers. the change from unenhanced mra in total diagnostic quality (tdq) was evaluated for each patient using a five-point scale. evaluation was made also of the number of vascular lesions detected, confidence in lesion characterization and the ability to grade stenoses. a full safety assessment was performed. results: both off-site reviewers noted a significant improvement in tdq from preto post-contrast at all but the lowest dose. the increase appeared to plateau at 0.1 mmol/kg. the dose effect was significant for the on-site reviewers (p = 0.005) and off-site reviewer 2 (p < 0.001) but not for off-site reviewer 1 (p = 0.06). for all but the lowest dose group, gd-bopta increased (a) the number of patients with lesions detected and, for most patients, the number of lesions detected per patient; (b) confidence in lesion characterization; (c) the percentage of stenoses visualized clearly enough for grading (reviewers 1 and 2 graded 80 -95 % and 57 -80 % of stenoses, respectively). all doses were well tolerated. the overall incidence of adverse events was 9.6 %. conclusion: ce-mra of the pelvic arteries using gd-bopta is safe and significantly more effective than tof-mra. a dose of 0.1 mmol/kg appears the most suitable. purpose: the purpose of this study was to evaluate the feasibility of blood pool contrast-enhanced mra to visualize the arterial and venous vessel tree and to detect deep venous thrombosis (dvt) of the lower extremities. material and methods: nine consecutive patients with pulmonary embolism (mean age: 46 ± 9) were randomized to various doses of nc100150 (between 0.75 and 6 mg fe/kg b.w.). a t1w 3d-gre sequence (te: 2.0 ms, tr: 5.0 ms) was used. two observers blinded to the dose of contrast agent assessed image quality, contrast attenuation and appearance of thrombi.results: qualitative assessment of overall mra image quality and semiquantitative vessel scoring revealed good to excellent delineation of venous and arterial vessel segments independent of the dose of nc100150. however, quantitative roi analysis revealed a significantly higher s/n ratio in the high dose group compared to the mid and low dose groups of nc100150 (p < 0.01). between dose groups, s/n ratio was independent of vessel type (artery or vein) and vessel segment localization (proximal or distal). all seven venous thrombi (mean length 7.2 ± 0.95 cm) were characterized by a very low si, which was only 16.6 ± 7 % of the si in adjacent venous segments (p < 0.0001).conclusions: high quality mr angiograms of the lower extremities can be obtained using low concentrations of nc100150 in combination with a strong t1w 3d-gre sequence. the obvious delineation of venous thrombi suggest that this technique may be potentially used as a non-invasive "one-stop shopping" tool in the evaluation of thromboembolic disease. injection-associated pain in femoral arteriography: a european multicentre study comparing safety and efficacy of iodixanol and iomeprol c. manke 1 , f. cognet 2 , a. page 3 , j. pueyo 4 , n. batakis 5 ; 1 regensburg/de, 2 dijon/fr, 3 winchester/gb, 4 mallorca/es, 5 athens/gr purpose: to evaluate injection-associated pain, safety, and efficacy with the isoosmolar contrast medium iodixanol (visipaque 270 mg i/ml) compared with iomeprol (iomeron 300 mg i/ml) in femoral arteriography. materials and methods: a prospective, multicentre, double-blind, randomised, parallel-group clinical trial was performed in nine hospitals in europe. of the 352 patients evaluated, 176 received iodixanol and 176 received iomeprol during peripheral arteriography (automated stepping intra arterial dsa). the first injection was standardised (volume 80 ml, flow rate 10 ml/s), additional injections were performed according to needs. the injection-associated pain, safety and efficacy were evaluated. adverse events were recorded during the procedure and up to 72 hours after the examination. the iodixanol group reported statistically significant less injection-associated pain than the iomeprol group after first injection (7.4 % vs. 18.5 %; p = 0.002), and after all injections (10.9 % vs. 20.2 %; p = 0.02). no significant differences were found between the iodixanol group and the iomeprol group for frequency of contrast-related adverse events (1.7 % vs. 1.7 %). overall diagnostic utility was excellent/good for 86.8 % of the patients in the iodixanol group and 86.7 % in the iomeprol group (p = ns). in this trial iodixanol 270 mg i/ml caused significantly less injectionassociated pain during femoral arteriography than iomeprol 300 mg i/ml and was shown to be equally safe. despite lower iodine concentration, iodixanol provided similar diagnostic efficacy to iomeprol. optimizing vessel enhancement for cta of the pulmonary vasculature by contrast media dilution c.w. bader 1 , j.m. froehlich 1 , h.a. lohr 1 , j. sousa 2 , c.l. zollikofer 1 , k. wentz 1 ; 1 winterthur/ch, 2 wädenswil/ch purpose: to verify the hypothesis, that with a defined amount of 27 g iodine a higher injection rate of diluted contrast media (iobitridol) results in higher vessel enhancement in pulmonary cta methods and materials: after bolus timing, ctas were performed in 93 patients divided into 2 groups: group a with 48 patients (age 66 ± 17 a, 90 ml cm, 300 mg/ml concentration, flow 3 ml/s) and group b with 45 patients (age 66 ± 15 a, 108 ml cm, 250 mg/ml concentration, flow 4 ml/s). each cm application was followed by a saline flush of 30 ml. contrast enhancement of the pulmonary trunc, right and left main arteries and lower lobe arteries was measured. results: contrast enhancement of the pulmonary trunc was 342 ± 87 hu in group a and 382 ± 80 hu in group b, the right pulmonary artery 321 ± 86 and 352 ± 79 hu, the left pulmonary artery 311 ± 83 and 353 ± 78 hu. in the right lower lobe artery contrast enhancement was 309 ± 88 hu in group a and 335 ± 83 hu in group b, the left lower lobe artery 310 ± 88 and 342 ± 84 hu and the mean enlancement values of all measured pulmonary arteries was 319 ± 86 and 353 ± 82 hu. the effect on vascular enhancement was most significant (z-test, two sided p-values) in the pulmonary trunc (p = 0.002) followed by all measured pulmonary arteries (p = 0.05) and least remarkable in the lower lobe arteries. with an identical amount of 27 g iodine, the higher injection rate of diluted cm leads to a significant increase of vessel enhancement in cta of the pulmonary vasculature. with exact bolus timing this can be used to reduce the applied dose of iodine per patient. purpose: to evaluate prospectively diagnostic accuracy of 1 mol gadobutrol as a contrast agent for intraarterial x-ray digital subtraction angiography (dsa) in comparison to iodinated, non-ionic contrast media and 0.5 mol gadolinium-dtpa. methods: flush arteriograms (ascending, descending, abdominal aorta, iliac and femoral arteries) and selective angiograms (carotid, renal and visceral arteries) were obtained from bilateral femoral arterial access (5 f sheaths) in ten domestic pigs (70 kg body weight). digital subtracted angiograms were obtained during injecton of non-diluted 1 mol gadobutrol, 300 mg i/ml iopromide, or 0.5 mol gadopentetate. injection parameters (volume and velocity) were similar for all three contrast agents. in paired arteries, two different contrast media were used during the same angiographic run. diagnostic quality and accuracy of the angiograms were evaluated on a three-step scale by three independent blinded investigators. results: sufficient angiographic images were obtained in 90 % of cases using iodinated contrast material. gadobutrol achieved sufficient non-selective angiograms in 64 %. selective angiograms were sufficient in 98 % using iodinated contrast material, 90 % using 1 mol gadobutrol and 48 % using 0.5 mol gd-dtpa. adverse reactions to any of the used contrast agents were not noted. conclusion: 1 mol gadobutrol solution allows x-ray digital subtraction angiography with an diagnostic accuracy equivalent to 300 mg/ml iodinated contrast media, if selective injections are performed. flush aortograms are of inferior image quality to iodinated contrast material. 1m gadobutrol achieves significantly better image quality compared with 0.5 mol gadolimium solutions. high iodine concentration non ionic contrast agent. comparison with lower iodine concentration in peripheral multislice ct angiography c. catalano, a. laghi, r. brillo, f. fraioli, f. pediconi, a. napoli, i. reitano, r. passariello; rome/it purpose: multislice ct (msct) has already proved a very accurate technique in the assessment of different vascular pathologies. purpose of this study was to determine whether high iodine concentration non ionic contrast agent could improve vascular enhancement and vessel conspicuity. material and methods: 60 patients referred for msct angiography (mscta) of the aorta and peripheral arteries were included in the study. all examinations were performed with a msct using a fixed delay time of 28 seconds. in all patients a standard amount of 40 g of iodine was administered at 4 ml/s. all patients were randomly assigned to one of three following groups: group 1 consisting of 100 ml of 400 mg i/ml; group 2 consisting of 113 ml of 350 mg i/ml; group 3 consisting of 133 ml of 300 mg i/ml. rois were obtained at different. a qualitative assessment was also made by two observers, regarding image quality and vessel conspicuity. results: no significant difference was detected between the 350 and the 400 mg i/ml contrast agents at the level of the aorta, pelvic and thigh arteries; a 13 % difference was found between the 300 and 400 mg i/ml in the same districts. with the 400 mg i/ml a significant increase in vascular enhancement of respectively 16 and 21 % was seen in the popliteal and infrapopliteal arteries. conclusion: a significant improvement in contrast enhancement and vessel conspicuity can be achieved in mscta by means of high iodine concentration contrast agents. key: cord-022888-dnsdg04n authors: nan title: poster sessions date: 2009-08-19 journal: eur j immunol doi: 10.1002/eji.200990224 sha: doc_id: 22888 cord_uid: dnsdg04n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx3, the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx3 facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th1-oriented adaptive response. actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx3, which does not show direct activity on fungal cells. finally, ptx3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx3-mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx3 amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx3-opsonised conidia, cd11b activation, internalization, recruitment to the phagocytic cup and cd11b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd11b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx3, fcgriia/cd32 mediates inside-out activation of cd11b and consequently phagocytosis of c3b-opsonised conidia. in vivo phagocytosis experiments performed with c1q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx3) and the cellular arm (fcgrs, cr3). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx3 binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx3-/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx3. we found high concentration of ptx3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( x 2 ng/ml). the presence of ptx3 in human colostrum seems to be due to the secretion of ptx3 by human mammary gland since we report the production of ptx3 by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx3 production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx3 supplying by breast feeding: (i) soluble ptx3 in colostrum (ii) hmc that can secrete ptx3 upon stimulation in the specific tissue, (iii) an increase of ptx3 production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx3 may participate to the beneficial role of breast feeding on the newborn health. a. m. baru 1 , j. stephani 2 , h. wagner 2 , t. sparwasser 1 1 twincore, institute for infection immunology, hannover, germany, 2 technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of 10 receptors in humans (tlr 1-10) and 12 in mice (tlr 1-9, 11-13). as transmembrane receptors, tlrs are expressed on the cell surface (tlr 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (tlr 3, 7, 8 and 9) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about 5 years later to this, toll-like receptor 9 (tlr9) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr9 (hutlr9) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr9 (mutlr9) is also expressed on conventional dendritic cells (cdcs). consequently, tlr9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr9 (henceforth called as hut9 mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr9 knock-out background. we expect that hut9-mutlr -/mice mimic the human specific expression pattern of tlr9, i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr9. by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr9 ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c1q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. the main inducers of il-12p40 are microbial components activating toll-like receptors with the magnitude of il-12p40 induction depending on the specific tlr engaged. differential induction of il-12p40 upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il-12p40 promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il-12p40 promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. acetylation of h4 showed a specific distribution pattern and occured mainly in regulatory elements within the il-12p40 promoter, whereas acetylation of h3 took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue 4 on h3 turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue 1 , w. ellmeier 1 1 medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi 1 , m. buxadé 1 , j. minguillón 1 , r. berga 1 , j. aramburu 1 , c. lópez-rodríguez 1 1 universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat5 is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat5 participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat5 is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat5 could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat5 is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat5 is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat5. our work indicates that nfat5 is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr3, tlr4, tlr7 and tlr8 and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p50 and p65 nfxb subunits. interestingly, we observed that tlr3 stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr4, tlr7 and tlr8 stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl-2 expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr3, tlr4, tlr7 and tlr8. finally, in vivo tlr7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr4, tlr7 or tlr8 triggering can directly favor tumor development whereas tlr3 signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms-354825) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd8 + and cd4 + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr 4 ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd4 + and cd8 + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd4 + and cd8 + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: 1. innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr 4 stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . 2. adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni 1 , r. oatuni 1 , m. collini 2 , m. caccia 2 , p. castagnoli 3 , g. chirico 2 , f. granucci 1 1 university of milano-bicocca, biotechnology and bioscience, milan, italy, 2 university of milano-bicocca, physics, milan, italy, 3 singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin-2 (il-2) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il-2 production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca2+/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca2+ influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr4 engagement, depending instead exclusively on cd14. we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase-2 (cox-2) that, by generating prostaglandins (pgs), such as pge2, regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd14. given the essential involvement of cd14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd14 represents a major step towards the development of potential treatments with modes of action involving interference with cd14 functions. we have examined the interaction of cd55, a 80-kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd14. human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd14 antibody coupled to biotin visualised by qd-525-streptavidin and anti-cd55 antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd55 and cd14, suggesting a new functional role of cd55 as a member of a multimeric lps receptor complex. l. lundvall 1 , r.r. schumann 1 1 charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase-1 induction leads to an increased release of mature il-1b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d39) was established for assessing il-1b induction during this disease. the murine raw 264.7 cell line, the human astroglial u-87 mg and the murine microglial cell-line bv-2 were stimulated with the tlr4 ligand lps, the tlr2 ligand pam 2 cys, the nod2 ligand mdp, or the nod1 ligand c12-ie-dap, and, as control, atp alone or in combination. we assessed il-1b by elisa and caspase-1 and pro-il-1b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il-1b induction pathway. s. pneumoniae (d39) infected mice showed a significant increase in il-1b release after 24 hours. in vitro, an increase in il-1b levels after costimulation with lps or pam 2 cys, and mdp or c12-ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il-1b by tlr-and nlr-ligands was observed in raw cells, bv-2 cells, u-87 cells and primary astrocytes. active caspase-1 (p10) was induced by mdp or c12-ie-dap, corresponding with high il-1b responses when lps or pam 2 cys was added. sirna experiments show that a knock-down of nod2 leads to a diminished il-1b release after lps-and mdp-stimulation. the precursor forms of il-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il-1b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il-1b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il-1b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg 1 1 toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd3 and cd28 activation model system -tlr4 presence on cd4+ cells is found in mouse t cells, human t cells and jurkat cell lines. following cd3/cd28 activation for 48 hours we have identified a small but distinct populationof tlr4+ cells. further characterization indicates these cells to be cd4+cd25+ cells. further characterization of the expression and functional acitvity of the tlr4+ t cells indicates co-expression of tlr4 with md-2 indicating a functional tlr4 receptor. in addition lps activiation did not lead to upregulation of tlr4 expression in t-cells. the data indicate that tcr activation leads to tlr4 expression. the expression appears to be associated with cd4+cd25+ cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann 1 , d. kaul 1 , c. krüger 1 , f. zipp 1 , r. nitsch 2 , s. lehnardt 1 1 charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, 2 charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr7 plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr7 (and human tlr8) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr7 is expressed in these cells. incubation of microglia with all three of the above mentioned tlr7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il1-b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr7 knock out (ko) microglia by realbecause neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin-1 is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin-1-transfected plb985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd11b labeling. concerning apoptosis, increased coronin-1 expression in dmf-differentiated plb985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin-1 significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or bid truncation suggesting that coronin-1 interfered with mitochondria-related events. to validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin-1 immunolabeling. we concluded that coronin-1 could constitute a potential target in resolving inflammation. p.-n. hsu 1 1 national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw264.7 macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p38 map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf-6 sirna and traf-6 decoy peptide, indicating this pathway is traf-6 dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b7-h1 and b7-dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il-6 was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il-2 and ifn-g which could not be overcome by restimulation with acd3. this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd4 t cells into t helper 17 (th17) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th17 differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th17 cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il-1 receptor-related protein st2 could be implicated in lps tolerance. the natural ligand of st2 was recently identified as interleukin-33 (il-33), a new member of the il-1 family. in this study, we investigated whether il-33 triggering of st2 was able to induce lps desensitization of mouse macrophages. we found that il-33 actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il-33 enhancing effect of lps response is mediated by the st2 receptor since it is not found in st2 ko mice. the biochemical consequences of il-33 pretreatment of mouse macrophages were investigated. our results show that il-33 increases the expression of the lps receptor components myeloid differentiation protein-2 (md2), cd14 and tlr-4 and the myeloid differentiation factor 88 (myd88) adaptor molecule. in addition, il-33 pretreatment of macrophages enhances the cytokine response to tlr-2 but not to tlr-3 ligands. thus, il-33 treatment preferentially affects the myd88-dependent pathway activated by the tlr. c. klotz 1 , b. lenz 1 , r. lucius 1 , s. hartmann 1 1 humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., 2008) . the cystatin effect was blocked by the application of anti-il-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin-10 (il-10). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il-10 in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il-10 production after avcystatin stimulation. application of specific inhibitors revealed that the il-10 induction was p38 and erk dependent, and inhibitor titration indicated a higher sensitivity towards p38. western blotting experiments confirmed the phosphorylation of p38 and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il-10 is also regulated by the phosphatidylinositol-3-kinase (pi3k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il-10 and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose-7-phosphate (s-7p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose-5p and xylulose-5p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p38/jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s-7p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts 1 , y. morias 1 , p. de baetselier 1 , a. beschin 1 1 vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m1) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m1 in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd88, ifng, il-10, ccr2 and nf-kb to the development and/or recruitment of pathogenic m1 in the liver was investigated using knock-out mice or by delivering il-10 in infected mice. results: we established that cd11b+ly6c+cd11c+ tnf and inos producing dcs (tip-dcs) represent the major m1 liver subpopulation. tip-dcs differentiated in an ifng/myd88-dependent manner from cd11b+ly6c+ inflammatory monocytes in the liver of infected mice. ccr2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr2 ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il-10 in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p50 was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p50 and il-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m1, in particular tip-dcs. the inflammatory activity of liver m1 is controlled by il-10 and/or p50 nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov 1 , j. driesen 1 , z. abdullah 2 , a. niño castro 1 , t. chakraborty 3 , m. krönke 4 , o. utermöhlen 4 , c. wickenhauser 5 , j.l. schultze 1 1 limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, 2 institute of molecular medicine and experimental immunology, bonn, germany, 3 institute of medical microbiology, university of giessen, giessen, germany, 4 institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, 5 institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and cd25 and production of prostaglandin e2 (pge 2 ) and interleukin 10. all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd25, expressed by regulatory myeloid cells, acts as an il-2 scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge 2 or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd25 + ido + cox-2 + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin 12 (il-12) and il-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il-12 and il-18 or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il-12 and il-18. in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il-12 and il-18. therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel 1 , m. rodriguez gomez 2 , m. niedermeier 2 , y. talke 2 , n. göbel 2 , k. schmidbauer 2 , m. mack 2 1 unversity hospital regensburg, internal medicine ii, regensburg, germany, 2 university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il-4 and il-6. activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp-1 cells infected with m. bovis bcg, the avirulent m. tuberculosis h37ra strain and the virulent m. tuberculosis h37rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva 1 , l.d. miteva 1 , s.a. stanilova 1 1 trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il-23 is a heterodimeric cytokine composed of a p19 subunit associated with the il-12/23p40 subunit. like il-12, il-23 is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il-12p40-related cytokines controls the appearance of normal th 1 and pathological th 17 mediated immune responses. in this study, we examined the dynamics of inducible il-12p40 and il-23p19 mrna expression and protein production in purified human monocytes and how jnk and p38 mapks inhibitors influenced il-12p40 and il-23 production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il-23p19 gene expression was higher than those observed for il-12p40 gene at all time-points. the level of il-23p19 mrna increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for c3bgp and 22.7 fold for lps). c3bgp and lps triggered il-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. the higher level of il-12p40 gene expression was detected at 6 th h in lps compared to c3bgp stimulated monocytes (8.1 vs. 4.9 fold). however, il-12p40 and il-23 protein production was increased in the highest level after c3bgp stimulation. the inhibition of p38 led to the statistical significant augmentation of c3bgp stimulated il-12p40 production. the inhibition of the same map kinase enhanced lps stimulated il-12p40 production without significant difference. the inhibition of jnk and p38 mapks significantly decreased c3bgp stimulated il-23 production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c3bgp and lps to induce the expression of il-12p40 and il-23p19 mrnas in purified human monocytes. we showed that inhibition of p38 mapk down regulated il-23 and upregulated il-12p40 production in stimulated monocytes. we concluded that in human monocytes p38 map kinase activation has an opposite effect on the il-12p40 and il-23p19 expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd83, cd86 and cd40, and secrete il-12, thus acquiring the ability to induce proliferation and th1 polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd1c, bdca-3, and cd16. by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd16+ subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd16+ dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd16+ dc, including their survival and their ability to produce il-12p70. besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il-6, il-8 and mcp-1 involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg 1 , s. wolke 2 , a. brakhage 2 , m. gunzer 1 1 otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, 2 hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in 2004 nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to 3 hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor 7 (irf7), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr9. in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr9 to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens 1 , s. vander beken 1 , p. bogaert 1 , j. grooten 1 1 ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th1-driven immunological counterpart of the th2-driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd45 bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th1-and th2-driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd45.1 + ram remained constant in cell number for the first 2 days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker 1 , a. stojanovic 1 , a. cerwenka 1 1 german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr-1 + cd11b + f4/80 + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas 1 , f. marchesi 1 , m. fabbri 1 , s. schiarea 2 , c. chiabrando 2 , a. mantovani 1,3 , p. allavena 1 1 istituto clinico humanitas, rozzano, italy, 2 istituto mario negri, milano, italy, 3 università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m1 and m2. m1 macrophages act as a first line of defence against pathogens whereas m2 cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd16, cd68 and low levels of mhc class ii. tc-macro produced il-10, il-6, tnf but not il-12, even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl2, cxcl8, ccl17 and cxcl10. the transcriptional profile of tc-macro revealed that several genes in line with an m2 polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g 100.000 kda). il-3 and il-6 were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl-2h3 rat mast cell line. we demonstrate that gr nuclear translocation begins within 5 minutes and completes after 30 minutes in dx treated rbl-2h3 cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within 5 minutes as non-genomic. studying gc-caused apoptosis, rbl-2h3 cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl-2h3 cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. 5 minutes of dx treatment inhibited ca 2+ -signaling in antigen stimulated rbl-2h3 cells in the concentration range of 100nm -10mm. moreover, 5 minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl-2h3 cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box 1 (hmgb1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . extracellular hmgb1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb1 exerts antibacterial functions in human adenoid and testis (2) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity (3). we asked whether hmgb1 from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for 40 or 120 minutes with 25 nm phorbol ester (pma), 100 ng/ml interleukin 8 (il-8), or 100 ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h2a-histone h2b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb1 is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb1 localizes on nets. we hypothesize that net-bound hmgb1 might exert a direct antimicrobial function, or that nets might concentrate hmgb1 locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation (37°c, 3 h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr-1 and anti-vegfr-2 antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il-18. altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd1 molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd1a and cholesterol-dependent lipid rafts impact on cd1a surface expression and cd1a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd1-restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd1 group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd1a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd1 molecules in multiple sclerosis. we first analyzed cd1 expression on monocytes from 16 ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd1a was not expressed on ms patient monocytes, while the other members of the cd1 family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd1 molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd1 molecules in this context. c. ohnmacht 1 , d. voehringer 1 1 ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th2 cells. these results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il-1beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il-6 and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il-6 and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant 143038). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, mph1), and during resolution of inflammation (type-2 macrophages, mph2). methods: mph1 / 2 were generated by culturing cd14 + human monocytes for 6 days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph1 / 2 of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph1 / 2 were stimulated with lps and secreted il-6, il-8, il-10, il-12p40 and tnf were quantified by elisa. results: mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas mph1 are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph1 and mph2 which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il-6, il-12 and tnf production is suppressed while il-10 secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp70 in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). intracellular ros and hsp70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (invitrogen) and anti-hsp70 antibody (brm-22, sigma), respectively. intracellular level of hsp70 was also estimated in neutrophils after heat shock (hs) performed at 43°c for 10 min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at 1-15 min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for 1 min at 42°c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp70 (hsp70 basal ) and ros level, both intracellular and extracellular. at the same time increased hsp70 level immediately after hs (hsp70 (0 min)) correlated negatively with intracellular ros (initial and after hs). the hsp70 increase value (hsp70 (0 min) -hsp70 basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp70 increase was registered in 60 min after hs (hsp70 (60 min) -hsp70 basal ). thus it was found that within this age group the alteration in hsp70 induced by hs in neutrophils but not basal hsp70 itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant #3303. d. goyeneche-patiño 1 , z. orinska 1 , f. mirghomizadeh 1 , s. bulfone-paus 1 1 forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd8 + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type 1 ifn. two novel genes, receptor transporter protein (rtp4) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp4 and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd88 and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd8 -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during 8 and 48 h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp4 and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine 2000. stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp4 in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd88 knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr 3 and 9, as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp4 and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd88 and trif and the pathways that they represent are not relevant in the expression of rtp4 and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano 1 , e. erba 2 , r. frapolli 2 , m. d'incalci 2 , a. anselmo 1 , s. pesce 1 , p. allavena 1 , a. mantovani 1, 3 1 humanitas clinical institute, rozzano, italy, 2 mario negri institute, milan, italy, 3 institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et-743) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl2, cxcl8 and the inflammatory protein pentraxin3 (ptx3) either at transcriptional and protein level, especially after tnfa/il1b stimulation. down-regulation of ccl2, cxcl8, ptx3 and also of il6 and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin-4 (il-4) is a key cytokine of the t helper 2 cell response. il-4 has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il-4 production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il-4 producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day 3-6 after birth, a small population of il-4 producing cells was observed in the absence of any stimuli. the il-4 + population was not detectable at 6 or 12 weeks of age. other cytokine producing cells (ifn-g, il-10) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il-4 + cell population. phenotyping of the neonatal il-4 + cells showed that they were ige + /mhcii -/cd4cells. the occurrence of cd4 + il-4 producing cells after pma stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il-4 at day 5 after birth. neonatal pbmc collected before colostrum uptake did not produce il-4 in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il-4 responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il-4 response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel 1 , d. voehringer 1 1 ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat6-dependent manner when stimulated with the th2 cytokines il-4 or il-13. alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat6-dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat6-deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat6 expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat6 expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat6-deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il-4 exposed macrophages from wild-type and stat6-deficient mice. candidate genes will be expressed using retroviral transfections of stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. the following human malignant cell lines were tested: a549, ht29, hct116, sw60, mcf7, mda-mb231, jurkat and hl60. results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m1, and the tams isolated from the malignant effusions similar to m2. among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms4a4a and ms4a6a: highest trascriptional level after 18h of stimulation with 10-6m dexamethasone. ms4a murine genes are differently expressed respect to the human counterpart and only the homologs of ms4a6a (ms4a6b, 6c and 6d) have a similar regulation. finally, egfp-tagged ms4a4a, ms4a6a, and ms4a7 expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms4a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms4a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (mcp-1)/ccl2 and the gpcr ligand fmlp or the tlr4 agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il-1b or ifn-g, significantly and dose-dependently synergized with ccl2 in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin-8/cxcl8, but not of the ccl2 receptor ccr2 in monocytic cells. in turn, the induced cxcl8 synergized with ccl2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr1/cxcr2, because cxcl8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl2 intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, 2004) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for 20 min at 37°c with gentle shaking, followed by spinning down of pmns for 5 min, at 4°c and 500g. the supernatant was sedimented at 15000g for 10 min, 4°c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd56 has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd56 low cd33+ blood monocytes (mo) in healthy individuals and the increase in cd16+cd56+ blood mo in patients with inflammation has been reported. the functional activity of human cd56+ blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd56 expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd33 and activation markers such as hla-dr and trem-1. percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (p x 0.0001) in the total number of circulating cd56 low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd33 and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd56+ blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd33+ blood mo expressed increased levels of cd56 on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem-1+) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd56 on cd14 high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m1-and m2-polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m1 and m2 macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m1 type and were cd14 negative but cd80 and mhcii positive and produced high levels of il-8, tnfalpha and il-6 following lps stimulation. culture in the presence of mcsf resulted in induction of the m2 phenotype. these cells were cd14 positive with intermediate expression of cd80 and mhcii expression and produced high levels of il-10, il-6 and il-8 following lps stimulation. interestingly, already baseline il-8 production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m1-and m2-polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr4 ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma (50 ng/ml) treatment for 48h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl60 cells expressed antigen receptors cd14, tlr2, tlr4 and cd68 , and displayed enhanced phagocytic activity and production of ros. expression of cd13, cd33 and cd15 was also maintained however the cells were hla-dr and cd1a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl60 cells to differentiate into macrophages in response to pma. hl60 may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta 1 , n. ferrer 2 , a. gómez 2 , j. gonzalo 2 , a. arbués 2 , a. anel 1 , c. martín 2 , j. pardo 1 , apoptosis, immunity and cancer 1 university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, 2 university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so2, a m. tuberculosis phop mutant strain that was shown (perez et al 2001) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al 2006) . in the present study, we compare the time course and phenotype of cell death induced by so2, bcg and wild type m. tuberculosis on the murine macrophage cell line j774 and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type 1 receptor for vip (vipr1) gene is highly conserved through species and, in humans, is highly polymorphic. vipr1 has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from 53 blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr1 in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr1 after 3, 6, 9 and 12 h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g 50 %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from 53 donors that had been typed for the relevant vipr1 snps. the down-regulation of vipr1 correlates with the presence of a t at rs896 mapping in the 3'-end of the gene (p= 0.004). the vipr1 protein level was decreased about 40 % in monocytes of 3 subjects typed as t/t at rs896 whereas 3 subjects typed as c/c at rs896 maintained a high level of expression after 9h of lps treatment. the data show that different haplotypes of the vipr1 gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska 1 , t. vavrochova 1 , d. filipp 1 , immunobiology 1 institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd14 in the early mouse embryo (me). facs analysis of cell suspension prepared from 10.5 day me showed that about 0.7-1 % of cells were positive for cd11b. these cells exclusively were also positive for cd14, tlr2, and cd45 antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e7,5), embryo-derived mhcii + macrophages start to appear in the embryo around day 13. multicolor facs analysis of cd11b, cd45, cd14, f4/80, tlr2, tlr4, c-kit and mhcii surface markers revealed differential expression of tlr2 and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd11b + tlr2 + cells isolated from the e10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il12/il23-dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several ifng pathway genes, other candidate genes are being investigated for the 2 other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp1. we did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: tlr2 is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il-10 cytokine. using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to 70-80 % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, 2009. hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp70 have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp70 on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp70 were used. exogenous hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp70 on their surface. results demonstrating effect of exogenous hsp70 on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp70 on amplitude of respiratory burst. the cells expressing surface hsp70 impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp70 might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp70 to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd14/cd86 and cd14/cd163 double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd14/86 subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd14/163 subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m2 polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević 1 , i. pilipović 1 , s. stanojević 1 , k. mitić 1 , k. radojević 1 , v. pešić 2 , g. leposavić 1,2 1 institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, 2 faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h 2 o 2 ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both no and h 2 o 2 production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h 2 o 2 production. an increase in h 2 o 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h 2 o 2 production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd14 ++ cd16 -('classical') and cd14 + cd16 + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd14 + cd16 + monocytes display higher tlr2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd14 ++ cd16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr2 stimulation. methods: cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for cd14 and cd16 and intracellular il-12p70 and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il-12p70 (% positive cells and geomfi) was significantly higher for cd14 + cd16 + cells than for cd14 ++ cd16cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd14 ++ cd16cells were positive for tnf to a significantly higher extent than the cd14 + cd16 + cells. in particular the tnf response to tlr2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd14 ++ cd16monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. after 2 months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf1 gene concerns the well-known gt deletion in the second exon of ncf1 gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after cci. levels of tnf-a, il-1b, il-6, il-10 and il-1 receptor antagonist were analyzed in brain tissue using real time rt-pcr at 4 and 72 hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. data were analyzed with the mann-whitney u test and a two-tailed p x 0 .05 was considered significant. all cytokines were highly up-regulated 4 hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il-10 were significantly lower in the ubiquitin treated animals, whereas the levels of il-6 and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight (38) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while 18 were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p=0.02), tpp (p=0.00), osi (p = 0.00), mda (p = 0.00) were significantly raised but taa (p = 0.01) was significantly reduced in ptb patients compared with controls. the levels of mda (p = 0.04), neopterin (p=0.00) and tpp (p=0.00) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p=0.00), mda (p=0.00), neopterin (p=0.02), osi (p=0.00) were significantly reduced while taa (p=0.01) was significantly raised in c+m after 4 weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after 4 weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas 1 , e.m. cunha 1 , m.j. oliveira 1 1 icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap-70 is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap-70 regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., 2006; sivalenka and jessberger, 2004) . swap-70-/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., 2002; sivalenka and jessberger, 2004; sivalenka et al., 2008) . crucial regulators of these processes are members of the rho family of small gtpases such as rac1 and rhoa. swap-70 interacts with rac1 in vitro and preferentially binds the active gtp-bound rac1. swap-70 supports the increase of active rac1 in vitro by a yet to be defined mechanism (shinohara et al., 2002) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap-70 and rac1. it was found that fulllength swap-70 preferentially binds to constitutively active rac1 (rac1q61l) but not to its dominant negative form (rac1t17n). binding assays with swap-70 truncated mutants showed interaction of swap-70's n-terminus with gtpgs rac1 or rac1 depleted of guanine nucleotide, whereas swap-70 central or c-terminal regions do not bind to any form of rac1. preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire swap-70 sequence, mapped the rac1 binding site near the n-terminus of swap-70. full-length swap-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap-70 with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap-70. v. c. barbosa 1 , c. d. polli 1 , m.c. roque-barreira 1 , m.c. jamur 1 , c. oliver 1 , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl-2h3 cells were sensitized with ige anti-tnp and stimulated with dnp 48 -hsa or artinm. artinm binding to rbl-2h3 cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp-1 and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp-1 release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand 1 (psgl-1), b1 and b7-integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd44 (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il-1b, il-18 and tnf-a. herpes simplex virus 1 (hsv-1) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il-18 and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv1. methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) (10ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv-1 for 18 h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot 2.0 software. results: in the first itraq experiment over 300 human proteins were identified in the hsv1 infected cell supernatants. from these proteins 119 had at least 3 fold increase after poly(i:c) + hsv1 infection compared to the uninfected cells. hsv1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of 2.7 fold increase in the protein amount in the poly(i:c) + hsv1 infected cell supernatant and a 2.6 fold increase in the hsv1 infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand 10, il-6, tnf-a induced protein 6, complement factor b, galectin-1 and mxa. at present, further experiments are on-going for more detailed analysis of the hsv1 infected macrophage secretome. h. p. prakash 1,2 1 german cancer research centre, translational immunology, heidelberg, germany, 2 max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein-1 (ciap-1) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal3, laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal3 or rpmi and monocytes (1x10 5 ) were added into each insert. when necessary, hrgal3 was pre-incubated with 10mm lactose or sacarose. mcp-1 (100ng/ml) was used as positive control. we observed that hrgal-3 binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal-3, we observed that the association between these glycoproteins and hrgal-3 resulted in a 60 % increase in the number of migrating cells. both n-and c-terminal domains of hrgal-3 are involved in the association between laminin or fibronectin and hrgal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal-3 induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for 3 weeks in the presence of bmp4,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd14+cd45+ cells. the sorted cd14+cd45+ cells were further cultured for 7 -10 days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd14, m-csf receptor cd115 and the scavenger-receptor cd36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd14 and cd16 (fcgammariii) : the cd14lowcd16-and cd14+ cd16+. trancscriptional, phenotypic and functional assays suggest the alternative (m2) polarization of cd14+cd16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m2 -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi 1 , p. kropf 1 1 imperial college london, immunology department, london, united kingdom the balance between t helper (th) 1 and th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th1 or th2 responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd4 + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg9vd2 t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f1-atpase) is expressed on many cell types and is a possible specific ligand for the vg9vd2 tcr. the present study aims at understanding the role of f1-atpase in antigen regognition. using video microscopy calcium imaging in single vg9vd2 t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f1-atpase. purified f1-atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg9vd2 cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f1-atpase. thus the f1-atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg9vd2 t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f1-atpase. by using purified f1-atpase and peptides derived from vg9vd2 tcr sequences, interaction sites between f1-atpase and tcr were identified on both ligands. based on these findings a generalized model for vg9vd2 t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg2d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg2d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg2d activation in vivo. by transiently overexpressing the nkg2d ligand rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma5vdelta1 gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg2d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg2d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg2d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae1 induction. the primary systemic th2 response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg2d receptor suggest that different ones may play unique roles. a novel nkg2d-ligand, h60c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae-1 induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg2d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd1d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th1 (ifn-g, tnf) and th2 (il-4, il-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type 1 diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd4 -nk1.1 -inkt cells producing high levels of the pro-inflammatory cytokine il-17 has been identified (inkt17 cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt17 cells in type 1 diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il-17 as compared to c57bl/6 mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt17 cells present in nod mice were mainly cd4 -nk1.1 -, express the ror-g transcription factor and il-23 receptor, both molecules being usually associated with th17 commitment. we are currently analyzing, using co-transfer experiments, whether these inkt17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in nod mice. j. s. dodd 1 , r. muir 1 , s.s. affendi 1 , p.j. openshaw 1 1 imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd1d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd1d-deficient mice with poor nkt cell responses have inefficient induction of cd8 t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th2 immunity (increasing il-5 and il-10), promoting pulmonary eosinophilia and ablating cd8 t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd8 t cell activity (as measured by cd25 expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d4) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d7) weight loss by a cd8 t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd4 + ab t cells, are regarded as immature or t h 2 biased. vg9 + vd2 + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg9 + vd2 + t cells towards these activators. because il-23 is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg9 + vd2 + t cells. herein, we observed that zoledronate induced neonatal vg9 + vd2 + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h 1-like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h 2-like cytokines such as il-4 and il-5 were not induced. addition of il-23 to zoledronate selectively costimulated ifn-g production from neonatal vg9 + vd2 + t cells. furthermore, zoledronate/il-23 treatment resulted in neonatal vg9 + vd2 + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il-23 (il-23r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il-23 resulted in a further increase of t-bet expression in neonatal vg9 + vd2 + t cells. these changes in the expression of il-23r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il-23 treatment. of note, in contrast to adult peripheral blood vg9 + vd2 + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg9 + vd2 + t cells. altogether, these observations show that neonatal vg9 + vd2 + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il-23 is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e3 ligases such as hhv8 encoded k3, k5 or mhv68 encoded mk3. these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e3 ligases manipulate them. we discovered that both the cellular march and viral e3 ligases ubiquitinate cd1 molecules. however, whereas viral molecules inhibit cd1-antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd1 molecules. in contrast mhc class ii was only targeted by cellular and not by viral e3 ligases. furthermore cd1 molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e3 ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd2 negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht29 colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd2 neg clones in vivo, we generated hypodermal ht29 tumors in immunodeficient mice. concomitant injections of vd2 neg clones, in contrast to vd2 + cells, prevented the development of ht29 tumors. vd2 neg clones expressed chemokine c-c motif receptor 3 (ccr3) and migrated in vitro in response to chemokines secreted by ht29 cells, among which were the ccr3 ligands macrophage inflammatory protein (mip)-1d and monocyte chemoattractant protein (mcp)-4. more importantly, a systemic intraperitoneal (i. p.) treatment with vd2 neg clones delayed the growth of ht29 subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr3 antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd2 neg clones were not able to inhibit the growth of a431 hypodermal tumors. our findings suggest that cmv-specific vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht29 cells expressing the luciferase and realized orthotopic injection of ht29-luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint1, currently the only known determinant of a canonical iel compartment, that is selectively required for vg5vd1 + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint1 expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint1 function; for example, we demonstrate that the constitutive expression of wild-type skint1 fully restores detc development in a skint1 mutant mouse, but does not rescue normal detc function. thus, skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd1d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd19 + cd21 hi cd23 lo cd11c -) and splenic conventional dendritic cells (cdcs) (cd11c hi cd8a +/-cd11b +/-b220 -) from wt and cd1d -/mice as apcs for nkt cells from va14-ja18 transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd1d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd1d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il-4 by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il-4 producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h 2 response, whereas cdc-primed nkt cells rather favor a t h 1 response. objectives: il-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il-18 give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd1d -/-) mice. we set out to investigate the activated b cells in il-18 injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il-18 (2 mg) for 10 days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. results: mice injected with il-18 developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il-18 was increased in inkt cell deficient (cd1d -/-) mice, in contrast to published data. an increased response to il-18 was also observed in ja281 -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il-18 induced antibody responses. further characterization of the recruitment of b cells in il-18 injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il-18 induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd1d, are prone to autoantibody production and often involved in early immune responses. the il-18 induced antibody response in mzb deficient (cd19 -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il-18 induced antibody responses. we conclude that the role for inkt cells in il-18 induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c57bl/6 mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha 18-/-and cd1d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il-4. moreover, ifn-g production required the presentation of ehlppg by cd1d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il-12. similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd16 in cmv-infected individuals. cd16 is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that 71.9 ± 15.9 % of gamma-delta t cells from 21 cmv-infected ktr expressed cd16, when compared with only 19.8 ± 16.7% in 11 non cmv-infected ktr (p x 0.0005). similarly, 51.9 ± 25.7 % of gamma-delta t cells from 13 cmv-seropositive blood donors expressed cd16 compared to 27.1 ± 25.1 % in 15 cmv-seronegative donors (p x 0.01). cd16+ gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd16-dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il-12 and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd16 mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd107a on cd16+ gamma-delta t cell lines. cd16 is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd16+ gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a431 skin carcinoma cell line pre-incubated either with rituximab (anti-cd20) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd16-dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k5mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il-6, ifn-g, tnf-a, osm, ccl2, cxcl8, cxcl10, and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd40, cd86, hla-dr, and dc-sign, and lost cd14, ccr2, ccr5, and cxcr4. addition of further microbial stimuli (lps, peptidoglycan) induced ccr7 and enabled these inflammatory dcs to trigger antigenspecific cd4 + effector ab t cells expressing ifn-g and/or il-17. importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg9/vd2 t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg9/vd2 t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg9/vd2 t cells stimulated with hmb-pp in the presence of il-21 express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il-21 regulate expression of the b cell attracting chemokine cxcl13/bca-1, its receptor cxcr5, and co-stimulatory molecules involved in b cell help. purified peripheral vg9/vd2 t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to 4 days with and without hmb-pp, in the absence or presence of il-2 or il-21, or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl13 protein were detected in co-culture supernatants only when both il-21 and hmb-pp were provided, implying an il-21-dependent and tcr-dependent expression. vg9/vd2 t cells were confirmed as producers of cxcl13 by flow cytometry and immunofluorescence. under the same conditions, activated vg9/vd2 t cells expressed cd27, cd28, cd40l, cd70, icos and ox40. in contrast, neither cxcr5 nor ccr7 changed markedly by il-21 stimulation of peripheral vg9/vd2 t cells. conclusion: our findings confirm on the protein level that stimulation of vg9/vd2 t cells with hmb-pp and il-21 induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto 1 , m. emoto 1 1 gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)-12. although downmodulation of surface t cell receptor (tcr)/nkr-p1c (nk1.1) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il-12 stimulation. to determine whether failure to detect inkt cells after il-12 stimulation is caused by dissociation/internalization of tcr and/or nkr-p1c or by block of de-novo synthesis of these molecules, and to examine the role of il-12 in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p1c by inkt cells after stimulation with a-galcer or il-12, and influence of il-12 neutralization on down-modulation of stcr/snkr-p1c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il-12 neutralization. whereas s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of il-12, s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p1c remained unaffected by a-galcer or il-12 treatment, despite the down-modulation of ctcr and/or cnkr-p1c protein expression. in contrast, ctcr + cnkr-p1c + stcr -snkr-p1c -inkt cells and cnkr-p1c + snkr-p1c -inkt cells were detectable after in-vitro stimulation with a-galcer and il-12, respectively. our results indicate that tcr and nkr-p1c expression by inkt cells is differentially regulated by signaling through tcr and il-12r. they also suggest that il-12 participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p1c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg9-gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region 3 d1 (cdr3d1) and cdr3d2 repertoire, with a striking enrichment in a specific germline-encoded cdr3d1 sequence. differentiated gd t cells and the enriched cdr3d1 sequence were detected as early as after 21 weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd1-jd1 and vgi-jg1.3/2.3 rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd2-jd1 and vg9-jg1.2 selection was seen in pb tcrgd cells. detailed analysis of the cdr3 motifs revealed selection determinants in both vg9-jg1.2 (canonical length and cdr3 motif) and vd2-jd1 (minimal cdr3 length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚4) and tcrgd cb cells (6-7) to tcrgd pb cells (˚10 or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type 1 diabetes (t1d). we have previously shown that transgenic expression of a cd1d-restricted, va3.2-vb9 tcr in nod mice lead to an increase in cd1d-restricted type ii nkt cells (24abnkt cells), and prevention of the development of t1d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by 24abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc2.5 cd4+ t cells, in the presence or absence of selected cells from 24abnkt cell transgenic mice. results: in 24ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, 24abnkt cells expressed a high level of cxcr3 and a low level of ccr7 and cd62l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd4+ bdc2.5 t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with bdc2.5 cd4+ cells resulted in the prevention of diabetes development. the protection from disease required a minor cd4+ subset of 24ab+ nkt cells, but was independent of cd25+ t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd1d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs44, induces in vitro inkt cell expansion and ifng and il-4 secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th2 profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il-10 with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr1 cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th17 immune response is characterized by the secretion of il-17a and il-17f. the il-17 locus encodes the highly conserved il-17a and il-17f cytokines that are syntenic in 44kb distance to each other. besides cd4 + th17 and nkt cells, approximately 50 % of the il17a producers are gd t-cells. like cd4 + th17 cells, il-17 producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il-17 production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th17 cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il-17 or ifn-g. combined with the well known classification of il-17 producing gd t-cells along the markers cd27 and cd122, our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th17-type immune responses in vivo. in this context, the potential redundancy of il-17a and il-17f may complicate the analysis. so far, most studies were carried out with il-17a single-deficient or il-17f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il-17a and il-17f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg9vgd2 t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b1 and il-15 differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd2 tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp3) and, similarly to ab tregs, suppress the proliferation of anti-cd3/anti-cd28 stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd2 tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il-17 was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd11b in the liver following a-galcer treatment, numbers of cells lacking cd11b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma9/vdelta2 t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma9/vdelta2 t cells expansion upon stimulation with zoledronic acid (za) and interleukin-2 (il-2). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in 43 patients (49 %) (responders, r), whereas 44 patients (51 %) were non-responders (nr). vgamma9/vdelta2 t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt2, whereas temra of r patients had an higher expression of the costimulatory molecule nkg2d. the proliferative response of vgamma9/vdelta2 t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, 82 % of r patients were m, whereas 77 % of um patients were nr (p x 0.001). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c57bl/6 mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il-12 p40 (clone r2-10f6; atcc) or anti-cd-1d (clone 1b1) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at 18 hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il-12p40, a component of both il-12 and il-23; pretreatment with anti-cd1d mab had no effect. il-12rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il-12 was not a critical factor. this suggestion is supported by the increased expression of il-23p19 and il-12p40 (but not il-12p35) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il-23 production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb1) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing il-17. this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd27-defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine 123. cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to 40 % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, 40-60 % of gd 27+ cells were positive for pgp activity, although their gd 27counterparts remained largely negative (p x 0.01). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd27. as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma9vdelta2 t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma9vdelta2 t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma9vdelta2 t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma9vdelta2 t cells from healthy donors were stimulated with different compounds (zol, ipp) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma9vdelta2 t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma9vdelta2 t cells. moreover, mcp-2 depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma9vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma9vdelta2 tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma9vdelta2 t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg9vd2 t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg9vd2 t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg9vd2t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg9vd2 t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg9vd2 t cells and their effects on the viability of glioma cells, we expanded in vitro vg9vd2t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg9vd2t cell lines to kill three different glioma cell lines (t70, u251, u373) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg9vd2t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg9vd2 t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg9vd2 t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg9vd2 t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd1d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e7-expressing transplanted skin is dependent on interactions between populations of cd1d-expressing cd11c+/f480+ myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e7 graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il-2 and a-galcer. expression of cd1a, cd1d and the costimulatory molecules cd86 and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il-4) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd1d expression on the cell surface in comparison with control cells. in contrast cd1a expression was decreased. cd86 and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd1d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd1a, cd1b and cd1c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il-4 producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd1d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, 0.5-10 % of circulating lymphocytes express a vg9vd2 t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg9-enriched genes compared to conventional mhc-restricted cd8 + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf4 & crtam in gd and ab t cells respectively. because igsf4 binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd8 + ab t cells express crtam, and that engagement of igsf4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf4/crtam. we therefore sought to answer: 1. what is the function of igsf4/crtam on gd t cells? 2. how is the igsf4-crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf4 enrichment on resting gd t cells, with expression also detected on˚10 % of ab t cells. the properties of those cells are being examined. however, igsf4 generally correlates with markers of activation/antigen experience such as cd45ro. thus, igsf4 cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg9 + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf4 and crtam within 48 hours. however, engagement of igsf4 by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg2d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf4 interactions are sufficient for cd8 t cells to kill gd t cells and/or vice versa. instead, crtam-igsf4 interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd3z is a 16 kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately 3-6 % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd2 tcr variable segment associated with the vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg9vd2 t cells without antigen presentation. in vitro stimulated vg9vd2 t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg9vd2 t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg9vd2 t cell anergy observed in hiv+ patients. to this aim, cd3z expression and ifn-g production by vg9vd2 t cells from hiv+ and hiv-subjects were analyzed. we show that vg9vd2 t cells from hiv-infected patients expressed lower level of cd3z compared with healthy donors. a direct correlation between cd3z expression and ifn-g production capability by vg9vd2 t cell was found. however, pkc activation by pma is able to restore cd3z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg9vd2 t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner 1 , m. swamy 1 , s.l. clarke 1 , a. hayday 1 1 king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd8aa or cd8 -cd4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. the cells are initially activated by plate-coated acd3 antibody and a cytokine cocktail and maintained further in medium containing low levels of il-2. after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg2d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il-6) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after 6 hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd94 in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg2 molecules in heterodimer with cd94, we screened the presence of these receptors on t cell subsets in bd. the expression of nkg2 a/c/d molecules on gd and cd8+ t cells were analyzed in 17 active and 9 inactive patients with bd and 21 healthy controls. expression of nkg2 molecules was evaluated on cd8+, gd t and cd56+ nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls (4.4 vs. 2.8 %, p=0.001). in addition to the increase of gd t cells, increased expression of activating nkg2c molecules was also observed on gd t cells (20 % vs. 11 %, p= 0.01). nkg2a expression on gd t cells was found to be higher than nkg2c expression in patients and controls; but nkg2a expression on the t cells was not statistically different in both groups (33.5 vs. 40 %). nkg2d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd8+ cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th1 and th2 cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg9/vd2+ lymphocytes. we have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg9/vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two vg9/vd2+ resistant ("non-target") cell lines (under 20 % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs 697 (non-target), and validated the results by rt-qpcr quantification. results: we identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp1, ifitm1 and prame, for example, are up-regulated, whereas cd274 and clec2d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg9/vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd1d, in the rat. mice and rats have very similar cd1d and inkt tcr genes, with the exception of the va14 gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd1d revealed a very similar pattern of cd1d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il-4 production was 10-100 fold lower in the best responder rat strain (f344) compared to mouse (c57/bl6). since nkrp1a (rat homologue of mouse nk1.1) and tcr are not appropriate markers for rat inkt, cd1d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd1d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd1d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd1d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd1d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g9d2 t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g9d2 t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g9d2 t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g9d2 t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp-1. a significant (p x 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of pbmcs cultured with picostim+il-2 in comparison with pbmcs cultured with il-2 or media alone. picostim+il-2 caused significant expansion and activation of gd t cells following culture of pbmcs for 10-14 days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers 100-fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il-2, reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma9/vdelta2 (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than 5 % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: 1) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; 2) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); 3) to establish whether the same issues could be solved using a simplified protocol of dc generation. 1) dc were generated from cd14 + cells of healthy donors/mm patients; immaturedc on day 6 were induced to fully mature by incubation for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za. after 7 days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; 2) idc generated from cd14 + cells of hla-a*0201 + healthy donors/patients were pulsed with sv-peptide and stimulated for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za; after 2 rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd8 + t cells was determined by svpentamers staining; 3) the same experiments were performed both with dc generated following a standard protocol and a 48h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [6-3 h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd4 + cd25 + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp3 negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd1 subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg2d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd8+ have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n=8) and inactive (bdna) (n=20), versus healthy controls (hc) (n=30) and patients with recurrent oral ulcerations (ru) (n=14). to determine gd cytokine profile and surface markers treg-related in bd (n=9) and hc (n=11). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta1, vdelta2, cd8alpha, cd8beta, nkg2d, nkg2a and cd103. -intracellular expression of ctla-4, and foxp3. -intracellular expression of il-2, il-4, ifngamma, il-10 and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta2+ cells were significantly increased in ru. vdelta1+ and gdcd8+ lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg2d was slightly increased in gd from bda. -nkg2a expression by gdcd8+ was not different in bd versus hc. -most of gdcd8+ presented cd8alpha-alpha homodimers in bd and hc and were negative for cd103, foxp3 and ctla-4. gdcd8+ and gdcd8-subsets were (in bd and hc): -high ifngamma-producers without differences. -low il-2-producers: il2+ cells were lower in gdcd8+ than in gdcd8-. -low il-10-producers: il10+ cells were lower in gdcd8+ than in gdcd8-. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd8+ than in gdcd8--very low producers of il4 in most of cases. the hallmark in bd was the increase of gdcd8/vdelta1+. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg2a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd8+, except a lower percentage of il-2+ cells than in the gdcd8-subset. gdcd8+ from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd56 in humans. a subgroup, the invariant nkt cells (inkt), expresses the va24vb11 tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd3 + cd56 + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with ifn-g (50 ng/ml), anti-cd3 (25 ng/ml) and il-2 (500 ui/ml). these cells were cultured for 21 days and the expanded cd3 + cd56 + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd3 + c56 + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x 0,05 was considered significant. results: we could significantly expand cord blood cd3 + cd56 + nkt cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). table 1 shows the percentage (mean±sd,n=10) of phenotypic markers in cd3 + cd56 + cells at baseline (day 0) and after 21 days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb4 and vb8 families (p x 0,001). conclusion: our results show that cord blood-derived nkt cells are mainly cd8 + and cd94 + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb4 and vb8 families in these cells. l. marischen 1 , d. wesch 1 , p. rosenstiel 2 , a. till 2 , d. kabelitz 1 1 institute of immunology, kiel, germany, 2 institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod2. peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod2 gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp1 19 and pvama1 proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day 5 culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd69 + and cd25 + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day 3 after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd107a + (lysosomal associated membrane proteins: lamp-1) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th1 and th2 cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th1-like and th2-like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-cd3 and monoclonal antibody specific for the cdr3 loop of the invariant tcr a chain of inkt cells (clone:6b11). furthermore, after pma/ionomycin stimulation for 4 hours, intracellular ifng and il-4 cytokines were detected in cd4+cd8-, cd4-cd8-(dn), cd4-cd8+ and cd4+cd8+ subsets of inkt cells by five colour flow cytometry in patients with ad (n=10) and healthy controls (n=10). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x 0.01) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x 0.01). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r=0.726 and p x 0.001) and healthy controls (r=0.693 and p x 0.001). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il-4 level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x 0.05). the frequency, the number of inkt cells and the cytokine producing capacity of the cd4/cd8 inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il-4 that promotes the th2 differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells (1.8 %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd4 + nkt and cd4 -cd8 -nkt cells. nk1.1 + nkt cells may release large amounts of il-2, il-4, ifn-g and il-10 after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il-2 had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il-2. there was a significantly increase in the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd69 molecule existed in 68.95 % of the cells from large-scale selection. the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. under a light microscope at x400 magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. the differentiation pathway of the seb-activating cd8 + and tcrvb8 + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd8 + nkt cell and tcrvb8 + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl3 which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo-1am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h2begfp reporter mice. stimulation with anti-gd clone gl3 or anti-cd3 clone 2c11 elicited activation of gd t cells suggesting that tcr gd and cd3 molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl3 in vitro produced ccl4, ifng and tnfa. therefore, we were interested whether the ccl4 production of gd iiel influenced the homing of ccr5 cells such as lamina propria (lp) cd4 + foxp3 + cells (tregs). to test this, wt mice were i. p. treated with gl3 mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr5 + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr5 + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr5 expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il-4 production in g4 reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il-4 gene. in the absence of any manipulation gfp was expressed from the il-4 locus in populations of immature thymic nkt cells (predominantly cd4+cd44lotcrhi cells on a balb/c background, and cd4+cd44lonk1.1-on a c57bl/6 background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il-4 production was induced predominantly in cd4+ nkt cell subsets of the liver and spleen, and after i. n. administration, in cd4+ nkt cells of the airways. spontaneous and a-galcer-induced expression from the il-4 locus occurred in the absence of stat6 signalling, and did not require initial exposure to il-4 protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin-17 (il-17) plays an important role in neutrophil recruitment. herein, we investigated the role of il-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c57bl/6 (wt) and il-17 receptor gene-deficient (il-17r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. the ability of il-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il-17r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il-17 induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il-17r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il-17receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il-17 also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin-1 receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il-1r associated kinases to activate nfxb and p38 mitogen-activated protein kinase. the il-33-induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il-33 in different mast cell subsets. methods: different mast cells subsets (hmc-1, human cbmcs and murine bmmcs) were stimulated with il-33. the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk1/2, pakt, pnfxb, p38 and pjnk). additionally, we studied the signal transduction of il-33 in il-33r transfected hek293t cells. results: we found, that a tir family member, il-33r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il-33-induced cytokine production depends on c-kit transactivation. il-33r and il-1r accessory protein (il-1racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il-1r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il-33-induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il-33 in mast cells in absence of iger activation. (1) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease-1 (ho-1) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho-1 expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap1 small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap1 activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. rna interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in rap1 activation and that the oscillations in the formation of rap1-gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c3g (rap1gef) and spa1 (rap1gap): preliminary results suggest that spa-1 has probably a role in the early activation peak. since this oscillatory chemokine-induced rap1 activation is present on other myeloid cell lines (hl60, 32d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different sh3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)-3 and il-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il-3 and il-5 receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il-3 , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn 328 are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: (1) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna-146 expression is induced by activation of the toll-like/interleukin-1 receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna-146a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (casp8/3). in line with this, mirna-146a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna-146a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna-146a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna-146a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il-15, il-7r a-chain and il-15r a -chain genes in hiv disease progression. methods: we studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (ltnp). we analyzed 2 snps in the il-15 gene, 5 snps in the il-7r gene and 3 snps in the il-15r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il-15r aa 182 and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il-7r and il-15r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd4 and cd8 t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art1, and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art1 in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art1 transcripts by rt-pcr analysis in human blood leukocytes. soluble art1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek293 cells with art1 and tnf resulted in modification of tnf at at least 2 distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r32, a site that has previously been implicated in binding to tnfr2. binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit225 than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art1 resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik 1 , t.v.poroshina 1 1 institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and 11 control men were assessed for slpi, tnf-alpha, il-8 and free tgf-b1 in ejaculate by elisas using stat-fax 303 plus. a 3 to 5 day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p x 0.001) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p x 0.05). the il-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p x 0.05) in seminal fluid. free tgf-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b1 concentrations were 36.2 ± 6.2 pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il-8 and free tgf-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi 1 , e. a. elgaaeid 1 1 faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th1 or th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod2/card15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod2/card15 gene in cd. we performed a cases /controls study upon 75 cd patients and 90 healthy controls. this study suggests that in northen tunisian population, 3020insc mutation in nod2/card15 gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il-10 polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il-10 cytokine genetic profile in patients with ibd. we examined the contribution of il-10 gene promoter polymorphisms (-627 and -1117) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card15/nod2 gene mutations in cd presentation and location. in tunisian population, the 3020insc insertion in nod2/card15 gene is a marker of susceptibility to cd, while the a allele at position -627 in the il-10 promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il-10 gene polymorphisms and card15/nod2 gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il-2-dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il-2-driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il-2 molecule by ganglioside. but gangliosides apparently can also form complexes with il-2r; such complexes influence on the signal transduction through il-2r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il-2r subunits. in our work we use il-2-dependent cytotoxic t-cell murine line ctll-2. two different approaches for study of possible interaction types between exogenous gangliosides and il-2r subunits were applied: antibody staining of il-2r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 i-dcp-gm1 followed by immunoprecipitation of il-2r subunits. the fluorescence intensity of the antibody-labeled il-2r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside gm1, and by 10.7 % after incubation with gm2. labeling of the cells with antibodies to the il-2r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm1 (2.8 %) or with gm2 (5.9 %). to determine the mode of this impressive masking influence of ganglioside gm1, photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm1 with subunits of il-2r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il-2r, but not for il-2r a-subunit. these results demonstrate that exogenous ganglioside gm1 can interact with a-and bsubunits of il-2r in different modes. interaction of il-2r b-subunit with ganglioside gm1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa 1 , j. bukur 1 , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak2 and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak2. results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other ifn-g signal transduction components like stat1 and jak1 were not affected. jak2 blockade by two jak2-specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak2 leading to a lack of jak2 expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak2 inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of 506 controls of the spanish population. methods: two il18 polymorphisms located on the il-18 promoter region, snps -607 a/c (rs1946518) and -137 g/c (rs187238) were genotyped using taqman snp genotyping assays. the functional il-18 gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il-18 -607 p=0,318. il18 -137 p=0,740) but may contribute to disease onset and aggressiveness: the genotype il-18 -607 cc genotype, which is associated with higher il-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), t (p=0,001), m (p=0,012) and stage (p=0,002). the influence of the il-18 -103 gg genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), t (p=0,026) and stage (p=0,011). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il-18 polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il-18 gene (il-18 -607 and -137) may be associated with an worse prognosis of rcc. high levels of il-18 production can play an important role in grow, invasion and metastasis of renal cancer. interleukin-18 (il-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il-18 is its ability to induce the production of ifn-gamma in presence of il-12. moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l0. then it seems that il-18 has a crucial role in immunity against brucella infection. since the expression of il-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il-18 gene polymorph isms and brucellosis. methods: a total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il-18 polymorphisms at position -137 g/+113t/+127c (correlated with high production of il-18), codon 35/3c and -656g/-607c (correlated with higher production of il-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). discussion: as data revealed genotypes that have correlation with higher production of il-18 are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il-18 at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor 2 (ifngr2) mouse mutant. we have generated a mouse line carrying a conditional ifngr2 allele using the 2 loxp 2-flp recognition target (frt) approach. a targeting vector with 2 loxp sites flanking exons 4-6 and 2 frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat1 following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat1 activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd4 cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr2 on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez 1 , r. carretero 2 , p. saenz-lopez 2 , j. cantón 2 , j. carretero 2 , f. ruiz-cabello 2 , l.m. torres 1 , cts-143 1 hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, 2 hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and 130 normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca)12 allele of ifgn is significant more frequent in healthy control than in cin patient (p=0,030) in contrast homozygosis for (ca)12 allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). conclusions: our study suggest that ifng (ca)12 allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin-15 (il-15), a th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il-15 single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il-15 (c267t, g367a, c13687a and a14035t) polymorph isms using pcr-rflp. results: at position 267, the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p=0.025 and p=0.042, respectively). at position 13687, the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p=0.050, p=0.015). discussion: as shown the frequency of cc genotype and c allele at position 267 were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position 13687 is lower in patients than the healthy controls and the frequency of c allele at position 13687 is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf-7 and skbr-3, as well as their ability to respond to several cytokines and to the g-1 gpr30 agonist. the mcf-7 and skbr-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il-2ra, il-2rg, il-3/il-5/gm-csfr, il-4/il-13r, il-5ra, il-6ra, il-6r (gp130), il-7ra, il-10r, tnfr i, tnfr ii, ifngra, cxcr1, cxcr2, cxcr3, cxcr4, cxcr5. cells were incubated with il-10, ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr30 agonist. cytosolic ca 2+ concentrations [ca 2+ ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr-3 cells were found positive for a larger number of receptors than the mcf-7 cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf-7 cells with il-10 reduced the calcium response to g-1, while pretreatment with ifn-g and tnf-a potentiated the calcium response to g-1. tnf-b had no effect on mcf-7 g-1 stimulation. no direct effect on basal [ca 2+ ] i stimulation could be noticed when administering the cytokines alone. in skbr-3 cells, pretreatment with il-10 or tnf-b had no effect on basal [ca 2+ ] i and did not significantly alter the calcium response to g-1, while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g-1. pretreatment with tnf-a produced calcium oscillations and reduced the response to g-1. conclusions: mcf-7 and skbr-3 cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr30 stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd8a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd8a -pdcs and cdcs were infected with mcmv, whereas after 24h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd8 t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag1-deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag1 and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd8 t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg 1668, respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd8a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt3-l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f4/80 + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd8 t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr7 and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd11c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd8 t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd4 t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd4 t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd8 t cells in response to an adova infection due to an impaired cd4 t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than 500 passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il-12. to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp33 and vacv-gp33 expressing the gp33 epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p14 t cell receptor recognizing the gp33 epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p14ifnar -/mice. upon adoptive co-transfer of p14ifnar -/and p14ifnar wt/wt t cells and subsequent challenge with mva-gp33, a massive expansion of p14ifnar wt/wt t cells was observed, whereas p14ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp33 induced a rather similar expansion of ifnar competent and ifnar deficient p14 t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h5n1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h5n1 induced an exceptionally nf-kb dependent antiviral response. irf3 is essential part of this interferon-response of human endothelia. furthermore, we identified hmga1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h5n1. finally, nfatc4 was found to be a transcriptional regulator for specifically h5n1-induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h5n1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns1, delta-ns1 influenza virus (a recombinant influenza virus lacking the ns1 gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered t cell priming in vivo. a direct comparison of delta-ns1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns1 protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns1 protein. thus, we propose that the virally encoded ns1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin 18 is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin 18 is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of 14-3-3 target proteins. thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections. i. wessels 1 , d. fleischer 1 , l. rink 1 , p. uciechowski 1 1 institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il-1b expression is not elucidated, yet. it is known that the il-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il-1b when stimulated. b-cells which are il-1b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il-1b promoter and the impact of methylation on il-1b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl-60 cells were differentiated into monocytic cells after dihydroxyvitamine d 3 treatment. the monocytic phenotype was confirmed by flow cytometry. the il-1b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with 5-aza-2-deoxycytodine (aza) and changes in il-1b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl-60 cells, differentiated cells displayed upregulation of cd14 antigen and acquired the ability to express il-1b. by comparing the accessibilities of il-1b promoter we detected that the il-1b promoter was not accessible in undifferentiated hl-60 cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl-60 cells, demonstrating that the chromatin remodeling of the il-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il-1b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il-1b expression were found. our data indicate that the il-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg 1 , g. wetzel 1 , h. arnold 1 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p65 family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il-1b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp2, 3, 4, 5 and 6 mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd88 was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. (2)). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd8 + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. 2008 aug 1;181 (3)). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. 2008 dec; 82 (24) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il-1b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory 4t1 breast cancer cells over expressing il-1b (4t1/il-1b) tumor cells, but rarely in untransfected 4t1 cells. secretion of il-1b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg2d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il-4ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used 130 healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array 5.0 having p x 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of il-6, il-8, il-10, tnf-alfa and il1-ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed 150 snps with p x 1,0*10 -4 . to identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. a combined analysis revealed 10 snps with p x 9,1*10 -5 .these results are not genome wide significant. the 10 snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find 10 nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp60 on adipocytes. for the first time we could show a hsp60-stimulated release of the proinflammatory cytokines il-6, cxcl1 and mcp-1 in a time-and concentration-dependent manner from murine 3t3-l1 adipocytes. analyses of hsp60-signalling in these adipocytes revealed that members of the mapk-family (erk1/2, p38) and the transcription factor nfkb are involved in hsp60-mediated induction of the mediators il-6, cxcl1 and mcp-1. binding-studies with fluorescence-labelled hsp60 demonstrated that the interaction of hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mm. binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of hsp60-binding. further analyses to characterize hsp60-receptor structures on adipocytes revealed the presence of toll-like receptor (tlr)4 on adipocytes. tlr4 has been found to be expressed on macrophages and to interact with hsp60, therefore suggesting tlr4 as a potential receptor candidate for hsp60 on adipocytes. in order to identify the responsible binding-epitope of hsp60 we investigated the effect of specific antibodies directed against different epitopes of the hsp60-molecule. incubation with antibodies directed against the n-terminus of hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the hsp60-binding to adipocytes (47-80 %) indicating that the n-terminal region of hsp60 is involved in receptor binding. our experiments demonstrate that hsp60 stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf3. we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf3 knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with 3 % of fetal calf serum at 37°c and 5 % co2. bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd10, cd29, cd73, cd21 and stro-1 and were negative for cd45. intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost 100 % of msc. interleukin-2, ifn-gamma and tnf (th1 cytokines) increased msc contractility, whereas il-10 (a th2 cytokine) decreased msc contractility. by immunofluorescence, we observed that il-2, ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il-10 decreased that incorporation. our results suggest that th1 and th2 cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca 2+ -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as 40 nm and 400 nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents (10 mm) at the 24-h culture interval reached the values of approximately 3 ng/ml and 2 ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of 2-6 h in rat pecs. the 24-h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p38 and erk1/2. it was not suppressed by the calcium chelating agents bapta-am and tmb-8. the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr 305/07/0061. pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin1 substrates and targeting sites. recent data show that pin1 interacts with apo-bec3g (a3g). the pin1/a3g interaction results in a reduced a3g expression and a diminished a3g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin1 gene (-842 g/c and -667 t/c) modulate pin1 expression; in particular, the -842 gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, 28; 69-74, 2007) . the -842 c/g and -667 t/c polymorphisms in the promoter of pin1 gene as well as pin1 protein levels were analyzed in 30 exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; 40 hiv-infected patients (hiv) and 40 healthy controls (hc). the genotype and allele distributions of the -842 snp was skewed in esn (genotype: p= 0.008; allele: p= 0.013). in particular esn showed a significantly lower frequency of the -842 gg genotype compared to hiv and hc (p=0.017 and p=0.019, respectively) and consequently a lower g allele frequency (p=0.026 and p=0.028, respectively). no significant differences were found for the -667 snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, 9-(r)[2-(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. secretion of cytokines was determined after the 5-h culture by elisa. production of no was assayed at the interval of 24 h using griess reagent. approximately 300 compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately 30 compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip-1a and cytokines tnf-a and il-10. although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n 6 -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mm. the remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant 1m0508. host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and 10 million units of ifn-a treatment three times a week for 6 months was initiated. before and after treatment: percentages of the il-2 and ifn-g in cd4+ t cells were assessed to determine intracellular t helper cell 1 (th1) type cytokine expression. similarly, percentages of intracellular il-2 and ifn-g were detected to verify cytotoxic t cell 1 (tc1) type cytokine expression in cd8+ t cells. percentage of th2 and tc2 type cytokine expression, (il-4 and il-13) were determined in cd4+ and cd8+ t cells, respectively. six (50 %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th2 cells with respect to healthy controls before treatment. tc percentages, both tc1 and tc2, were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il-4 expression was higher and the percentages of th1 type cells were significantly low. il-13 expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency (29,8 %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt 1 , f. juengerkes 1 , b. schumak 1 , g. gielen 1 , j. kalff 2 , p. knolle 1 , b. holzmann 3 , a. limmer 1 1 university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, 2 university hospital bonn, department of surgery, bonn, germany, 3 department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il-12, the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh 1 1 rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il-17 and il-23 in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, 45 h. pylori-infected du patients (23 patients were positive for anti-caga antibody and 22 patients were negative for anti-caga antibody), 30 h. pylori-infected as carriers (15 subjects were positive for anti-caga antibody and 15 subjects were negative for anti-caga antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il-17 and il-23 was measured by elisa method. the mean serum levels of il-17 in total du patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total as subjects (5.19 pg/ml ± 3.75, p x 0.001) and healthy control group (3.55 pg/ml ± 3.76, p x 0.0001). in du group, it was found that the mean serum levels of il-17 in subjects with positive test for anti-caga (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-caga (7.65 pg/ml ± 4.74; p x 0.05). the mean serum levels of il-23 in du (8.66 pg/ml ± 8.41) and as groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, p x 0.02 and p x 0.03, respectively). however, no significant difference was observed for mean serum levels of il-23 between du and as groups. moreover, in both du and as groups the mean serum levels of il-23 was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il-17 and il-23 in h. pylori-infected subjects as compared with control group. in du group the expression of il-17 influenced by the bacterial caga factor. a. aral 1,2 , a. atak 1 1 gazi university faculty of medicine, department of immunology, ankara, turkey, 2 gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il-6 levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il-6 elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il-6 levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the 1st week and started to decrease at the 3rd week. il-6 reached to its peak at the 3rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic 1 , m. jurisic 2 1 university of kragujevac, school of medicine, kragujevac, serbia, 2 university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd3, cd20 and cd68. tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x 0.05) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. 1) drawings blood from 20 patients and 20 healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. 2) detection of the gene expression of prolactin and tlr-2 in cd14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr-2 and peripheral prolactin expression in cd14+ monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u937 culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table-2 ). when tb household contacts and healthy controls were compared, cfp10 and esat6 seemed to be more useful than tst in tb contacts for displaying ltb (table-2) . although cfp10 spot numbers were much more than esat6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: 0,069)( table-3 ). both esat6 and cfp10 spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip-10, il-6 and il-10. notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type 1 ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/1/68 (h3n2) (hk) differs from its putative avian precursor by 7 amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk-5aa-i62r, n81d, k92n, s193n, g144a, human (2-6); rhk-r2-l226q, s228g and rhk-7aa-i62r, n81d, k92n, s193n, g144a, l226q, s228g, avian (2-3)). among these variants, the double mutant rhk-r2 and the seven mutant (rhk-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l226q and s228g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip-10 and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r2 and rhk-7aa as compared to rhk and rhk-5aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il-8, shedded adhesion molecules (cd25, vcam-1, icam-1), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of 98 newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il-10, and neutrophilic cd64 levels, procalcitonin and il-10 were measured by elisa technique while, neutrophilic cd64 by single colour flowcytometric technique. of these 49 "infected" infants, 16 had positive blood culture (subgroup ia: culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another 49 newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il-10, cd64, and crp. il-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/ml. for pct, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/ml. neutrophilic cd64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il-10 and neutrophilic cd64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and npv 86 %. the combination of il-10 and crp had high sensitivity and moderate specificity, 93 % and 79 %, respectively. conclusions: il-10 and neutrophilic cd64 levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp3, naip5 or ipaf and the adaptor asc are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of il-1b and il-18. recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi3k, vps34 and can be inhibited with the pi3k inhibitors wortmannin and 3 methyladenine (3ma). in contrast, activation of akt, via class i pi3k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il-1b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with 3ma, wortmannin or an akt inhibitor. supernatants were analysed for il-1b by elisa. results: 3ma enhanced il-1b secretion by bmdc treated with the tlr3 and tlr4 ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il-1b secretion was greatly reduced in bmdc from nalp3 -/mice compared to wild type c57/bl6 controls. treatment with the akt inhibitor had no effect on lps-induced il-1b secretion by bmdc. tlr-dependent secretion of il-1a was also enhanced by treatment with 3ma. conclusions: these data demonstrate that il-1b secretion by bmdc in response to treatment with pi3k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp3 inflammasome. this response is limited to tlr3 and tlr4 agonists. inhibition of akt had no effect on lps-induced il-1b production, suggesting that the effect of wortmannin and 3ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi3k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (s1p) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the s1p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s1p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s1p, and later analyzed by flow cytometry. a pharmacological analysis of the s1p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam-1 expression was increased following lps and s1p concomitant stimulation in both venous and arterial cells, suggesting that tlr4 and s1p receptors cooperate in the expression of icam-1. conversely, no cooperation was observed when tlr2 ligands were used. in order to elucidate which s1p receptor subtype was involved in the increase of icam-1 expression, we used a pharmacological approach with s1p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam-1 after lps and s1p challenge was significantly reduced by blocking s1p receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s1p receptors 2 and 3, which suggest that s1p receptor 1 might be involved in the effect. conclusions: altogether these data demonstrate that tlr4 and s1p receptors can interact to increase adhesion molecules such as icam-1 in human endothelial cells, and the s1p receptor subtype involved in the effect differs between arterial and venous cells. 15 with ssc without pah) and a pool of 12 sera of healthy controls (hc) were tested. results: in 1 dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in 2 dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. twenty one protein spots were recognized by more than 80 % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam-1 and vcam-1 were assesed by an enzyme immunoassay (elisa). results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). reference value for icam-1 was 130-299 ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients (40 %) having increasd levels of icam-1. high icam-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). mean values were found 735,5 ng/ml and 400 ng/ml accordingly. those findings verify the positive correlation between icam-1 and inflammatory diseases and tissue damage but not for vcam-1. colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: 16 cases of ulcerative colitis (urc), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. immunohistoobjectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). samples of outlying veined blood were extracted to the 24 hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd18 in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in 3 degrees: degree 0. without expression. degree 1. weak; degree 2. moderate; degree 3. intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. the placebo group: mixed pattern, degree 1-2. group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree 2-3. the percentage of cells that expressed cd 18 was greater in the group study (40 mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd18 answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam10 and adam17. we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam17. these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam17 activity in the tissue. in the presence of the adam17 inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam17 as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf-460/ vegf+405 is associated with rcc risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). on the other hand, the polymorphism vegf -2578 a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. therefore, pten is one of the main antagonists of the pi3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il-6 as well as il-8 levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il-6 as well as il-12 and il-23 mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th-17 t cells, we measured th-17 cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il-17 and a strong reduction of il-22 mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il-1ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il-10. based on our previous observation that support a direct role of il-10-activated stat3 in the enhancement of il-1ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il-10. quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il-1ra promoter. crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after il-10 addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il-1ra promoter. chip assays showed that the pol ii recruitment to the il-1ra promoter induced by lps is significantly increased by il-10, further strengthening the concept that the rapid enhancement of lpsinduced il-1ra gene expression by il-10 initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat3 immunoprecipitated dna showed statistically significant levels of stat3 binding to the il-1ra promoter only in cells stimulated with lps in the presence of il-10. surprisingly, anti-p65 and anti-p50 chip assays revealed enrichment of both p65 and p50 recruitment to the il-1ra promoter when il-10 was added to lps-stimulated cells, suggesting that il-10 enhances the recruitment of nf-kb to the il-1ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il-10-treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il-1ra promoter site is dependent on il-10-activated stat3, since it is greatly reduced when stat3 activation by il-10 is impaired. the molecular mechanism through which il-10-activated stat3 promotes the recruitment of nf-kb to the il-1ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mmcp-2). we examined the mechanisms underlying the repression of mmcp-2 gene expression. our data show that the "repressor" effects of il-15 on mmcp-2 promoter activity are still operating on the mmcp-2 591 bp long minimal promoter. moreover, il-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy1. furthermore, chromatin immunoprecipitation revealed that il-15 promoted specific reciprocal recruitment of c/ebpb but not yy1 to the mmcp-2 promoter. finally, il-15 deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp-2. thus, we proposed that the expression of mmcp-2 and possibly other immunoregulatory genes may be regulated by il-15 through epigenetic modification and by balancing the content and binding of c/ ebpb and yy1 in mast cells. i. nagy 1 , k. filkor 1 , a. vörös 1 , l. kemény 2 , a. szász 1 1 bzaka, baygen, szeged, hungary, 2 university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir-203, mir-146a and mir-155, which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir-203 expression; in contrast, pgn re-stimulation had no further effect on mir-146a and mir-155 expression. next, we investigated the correlation between the expression of mir-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (socs-3). although the gene-expression profile of neither p63 nor socs-3 changed, we found that the expression of mir-203 reversibly correlates with both p63 and socs-3 protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir-203 prior to pgn-treatment completely abolished both p63 and socs-3 down-regulation, revealing the involvement of mir-203 in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir-203 expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb1+/-had a lower incidence and burden of benign papillomas when compared to tgfb1+/+ animals. however, more scc developed in the tgfb1+/-mice. after acute and chronic promotion, tgfb1+/-skin showed a reduced proliferative response with no increase in epidermal tgfb1 or nuclear p-smad2 compared to tgfb1+/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb1 gene dosage. further, pharmacological inhibition of alk5 suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb1 are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb1+/-skin, tgfb1+/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb1+/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb1+/+ but not tgfb1+/-keratinocytes, indicating that tgfb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb1 +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of 132 patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp-1) and fibroblast (mrc-5) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of 132 ra patients and 82 controls the levels of survivin correlated to urokinase (upa) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that 30/132 ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc-5 and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type b (itpkb) phosphorylates inositol (1, 4, 5) trisphosphate (ins(1,4,5)p 3 ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca 2+ responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h1 and h2 receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of il-1b by thp-1 cells after a 24h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il-1b secretion. tissue transglutaminase (tg2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg2 in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg2 expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg2 can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg2 not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg2 inhibitor, in a transgenic mouse model cf and in the taz10 transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz10 tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg2 in generating inflammation in two very different pathologies. this work underlines the critical role of tg2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge 2 stimulated a-dc. preliminary results indicate no accumulation of hif-1alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif-1alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p38 but not erk1/2 or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il-12 and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt5a -but not wnt3a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp130 in te. after infection with t. gondii, mice lacking neuronal gp130 (synapsin-cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. death of synapsin-cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp130 expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il-1b, il-6 and il-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il-4, il-10 and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb1 further may generate more effective therapies. as tnfa mrna 3' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb1 suppresses tnfa protein production by upregulating the rnabinding protein fxr1, which can bind to tnfa mrna and inhibit translation. methods: using raw 264.7 cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb1, we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 3'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb1 and luciferase expression was quantified. cells treated with lps and tgfb1 were also examined for fxr1 expression using pcr and western blot. following fxr1 knockdown using sirna, the influence of tgfb1 and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb1. using the luciferase-tnf-3'utr vector we show that tgfb1 targets the 3'utr of tnfa. furthermore, tgfb1 and il-10 both upregulate fxr1 mrna and protein; and treatment with tgfb1 and lps can synergistically upregulate mrna expression, more than tgfb1 alone. following sirna inhibition of fxr1, tgfb1 can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp-1 and mmp-3 secretion from saecs, nhbes and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. interleukin-17 augmented comtb-stimulated up-regulation of mmp-1 and mmp-3 secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin-17 down-regulated mmp-9 secretion from saecs by 50 %. interleukin-22 up-regulated mmp-1 and mmp-3 secretion from fibroblasts but not from saecs. timp1 secretion from saecs was enhanced by interleukin-17 but there was no effect of interleukin-22. mmp up-regulation by interleukin-17 and comtb was inhibited by the pi3kinase inhibitor ly294002 and on western analysis akt (protein kinase b) was phosphorylated at 30 minutes. chemical inhibition of the p110d isoform of pi3kinase with ic87114 abrogated the il-17 and comtb driven secretion of mmp-3 from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome 10) accentuated mmp-3 secretion. these inhibitory effects were confirmed with sirna. mmp-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. in summary, interleukin-17 and interleukin-22 drive transcription dependent mmp-1 and mmp-3 secretion from airway epithelial cells and fibroblasts. interleukin-17 also increases timp but down-regulates mmp-9 gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi3kinase pathway is central in interleukin-17 driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto 1 , l.s. moreira 2 , e. gonzalez-rey 2 , m. delgado 1 1 institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, 2 university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within 15 years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper 1 response. cholesterol metabolism is regulated by factors such as pparg1 (proliferator activated receptor g), srb1 (a class b scavenger receptor), cd36 or abca1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th1 immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg1, srb1, cd36, and abca1. we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to 60 % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il-6 in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il-6 and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il-6, tnf-a, il-1b, il-8 and il-12, and the anti-inflammatory cytokine il-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il-23 was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il-6 and il-10, and in some cases, of il-23. the maximal plasma levels of il-6 and il-10 were found at 24 hours and of il-23 between 48-72 hours. modest plasma levels of il-8 were also observed, with maximal production at various time points (4-24 hours). by contrast, production of tnf-a, il-1b and il-12 did not occur to a significant extent, while production of il-17 occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il-6, il-8, il-23 and il-10 also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il-6, il-10, il-8 and il-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving 165 patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il-6, activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il-33 is a novel il-1 cytokine family member that is expressed as an intracellular precursor (pro-il-33) and is thought to be cleaved by caspase-1 to yield a mature bioactive form of the molecule (mat-il-33). to date however, evidence of cell-associated proteolytic processing and caspase-1 dependent secretion of mat-il-33 has not been reported. here we show that pro-il-33 but not mat-il-33 is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il-33 to the il-33r and also il-33r-dependent bioactivity of pro-il-33 on mast cells. we propose a previously unrecognized role for pro-il-33 as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il-23 p19 mrna expression in myeloid cells, and markedly increase secretion of il-23, but not il-12, by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il-23 promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop10. chromatin immunoprecipitation (chip) assays using anti-chop10 and isotype control mab were performed using nuclear lysates from u937 cells and il-23 promoter dna measured by qpcr. chop10 binding on the il-23 promoter was detected following stimulation of u937 cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il-23 promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop10 in il-23 gene transcription, u937 cells expressing shrna's specific for chop10 or non-specific gene target were tested for their ability to express il-23 following tlr and er stress stimulation. u937 expressing three independent shrna targets for chop10 exhibited significant reductions in il-23p19 mrna (up to 87 % reduction of the response to lps+tp) compared to u937 expressing a control shrna. chop10 shrna expression did not affect the expression of other lpsresponsive genes, including il-1, il-8, ccl3 and sod2. to identify if er stress induction of il-23 mediated by chop10 expression plays a role in a more physiological setting, we examined the role of chop10 in the induction of il-23p19 gene expression following chlamydia trachomatis (ct) infection. infection of u937 cells with live but not g-irradiated ct induced expression of er stress response genes, including chop10. u937 infected with live ct exhibited increased il-23p19 mrna expression compared to u937 infected with nonviable bacteria. chop10 silencing significantly reduced the ability of live ct to induce il-23p19mrna, confirming the important role of chop10 in this response. these data suggest that er stress induction of chop10 could contribute significantly to the pathogenesis of diseases in which il-23 plays an major role, through induction of il-17 and il-22 producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd45b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd45b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd45b maintained a sustained activation of p38 kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd5 and cd69 were elevated on gadd45b-deficient thymocytes. thus, we provide evidence that gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo 1 1 institut pasteur, paris, france, 2 monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic il-7 niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il-7 promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il7 transcripts (il-7 hi cells). il-7 hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl19, ccl25, cxcl12) and cytokines (il15) that are critical for normal thymopoiesis. in the adult thymus, il-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il-7 levels. conversely, the frequency of il-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il-7-expression by tecs. > together, our temporal-spatial analysis of il-7-expressing cells in the thymus suggests that thymic il-7 levels are dynamically regulated under distinct physiological conditions. this novel il-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il-7 expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l5 and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl2-tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl2 and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl2-tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl2-tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg2 plasma cells and serum igg2 levels were˚5-10 fold increased. importantly, serum from young slc-tg;em-bcl2-tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in 2/3 and 6/6 cases, respectively. these values were increased when compared with control groups: 1/6 in fcgriib -/and 0/6 in bcl2-tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd27 + cd135 + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd117 hi cells, representing multipotent progenitors, or cd127 + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb2 and ephb3 expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b1 and/or ephrin b2, the ligands of ephb2 and ephb3 receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb1 or ephrinb2 genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb1/b2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb1 and ephrinb2 in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink 1 , d. vanhecke 1 1 university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op9-dl1 cell culture system (1) . using this in vitro assay we obtain large numbers of human cycd3 + and cd4 + cd8 + double positive thymocytes starting from umbilical cord blood (ucb) derived cd34 + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il-7 and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd34 + cells upon notch signaling is cd7 followed by cd127. t cell specification is accompanied by the induction of cd1a and loss of cd34 on cd7 + cd127 + cells. these cd34 -cd1a + cd7 + cells become dependent on continuous il7 and notch signaling for sustained survival and further differentiation into cd4 + cd8ab + dp thymocytes. we found that flt3l is not essential for the differentiation of cd4 + cd8 + human thymocytes but that addition of exogenous flt3l in the co-cultures increases the number of cd34 + precursors and consequently result in higher yields of developing cd4 + cd8ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd3 + cd4 + cd8ab + dp subset in this in vitro assay suggesting that op9 stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn3 to dn4 stage, where bdnf and its receptor p75 are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga 1 , c. lópez-rodríguez 1 1 pompeu fabra university, barcelona, spain nfat5 is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat5 are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat5 participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat5 deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd8 + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat5-null mice are unable to mount cd4+-and cd8 + -immune responses. data from our laboratory indicate that nfat5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat5 during the development of t lymphocytes, we developed mouse models that delete nfat5 at early (lck-cre + /nfat5 flox/flox ) or late (cd4-cre + /nfat5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat5 is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat5 at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p53-/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p53 has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic t-cell tumors. s. myrczek 1 , r. pardi 2 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, 2 vita-salute san raffaele university school of medicine, milano, italy jab1 is the catalytic subunit of the highly conserved cop9 signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab1 regulates the activity of ap1 transciption factors. to date jab1 is thought to be essential for every cell type as jab1 knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab1. to investigate the function of jab1 in b cells we established a mouse strain deficient for jab1 selectively in b cells. mice with floxed alleles of jab1 kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb1-locus (m. reth, freiburg). ablation of jab1 expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b1 and b2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl2 under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab1-deficient, bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab1 in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab1 when apoptosis is prevented. t. nitta 1 , s. murata 2 , k. tanaka 3 , y. takahama 1 1 university of tokushima, tokushima, japan, 2 university of tokyo, tokyo, japan, 3 rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd8 t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd8 t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd8 t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th1 and th2 antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd4+ t cells or functional deficits that selectively interfere with th1 or germinal centre responses. in this talk i will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn1 c-kit high (etp), dn2 and dn3 thymocyte populations was hybridised to affymetrix mouse 430a-2.0 genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as acvr1, bmpr1, fzd7, chemokine receptors cx3cr1, cxcr6 and integrins a2, a4, a9, ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata-3, tcf-1, notch-1, rag-1, rag-2 and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn2 stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn3 population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd8aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e14 thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day 18 of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd34 + cd1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd34 + cd1precursors with recombinant wnt3a (100 ng/ml) or with licl (10 mm) for 12hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab (8e7) under conditions of phosphatase activity inhibition. wnt3a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il-15 and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st2 cells suplemented with il-7 and flt3l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt3a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt3a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il-7 and flt3l and modify the transcription factor profiles of cd34 + cd1thymocytes mainly increasing hes-1 and id3 expression levels. human th17 clones and circulating th17 cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th1 and th2 clones or circulating th1oriented t cells, respectively. accordingly, human th17 cells exhibited lower expression of clusterin, and higher bcl-2 expression and reduced apoptosis in the presence of tgf-beta, in comparison with th1 cells. umbilical cord blood naï ve cd161(+)cd4(+) t cells, which contain the precursors of human th17 cells, differentiated into il-17a-producing cells only in response to il-1beta plus il-23, even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd161(+) t cells but it increased the relative proportions of cd161(+) t cells differentiating into th17 cells in response to il-1beta plus il-23, whereas under the same conditions it inhibited both t-bet expression and th1 development. these data suggest that tgf-beta is not critical for the differentiation of human th17 cells, but indirectly favors their expansion because th17 cells are poorly susceptible to its suppressive effects. m. irla 1 , w. reith 1 1 university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd4(+) or cd8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd4(+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd40 on mtecs by cd40l induced on the positively selected cd4(+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd4(+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu 1 , i. ravens 1 , g. bernhardt 1 1 hannover medical school, institute of immunology, hannover, germany cd155 is originally identified as human poliovirus receptor (pvr) and as rodent tage4, which is overexpressed in rodent colon carcinoma. cd155 is also known as necl-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd155 is overexpressed in transformed cells and promotes the cell cycle. thus, cd155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd4 and cd8; the dn subset is further subdivided into four stages (dn1-4) by differential expression of cd44 and cd25. thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd4+ sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd8 lineage. here we show that the frequency of terminally matured cd8+ sp cells but not that of cd4+ sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd155, a selective deficiency of mature cd8+ sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd155 is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd8+ sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd8+ t cells. in cd155 deficient animals, a shift in the tcr repertoire displayed by the pool of cd8+ sp cells was found demonstrating that cd155 is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. 2005 , 202, 1599 . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e9 (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd71 + ter119 + erythroid and cd45 + cd11b + myeloid cells are simultaneously cycling (s/g2/m) in the post-gastrulation mouse embryo (e10-12). the peak of lymphohematopoietic cell proliferation occurs at e13 in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd40-cd154 interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd40, we hypothesised a role for cd40-cd154 interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd40 expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd154 message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd154-/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd154 on reconstitution of b cell numbers following depletion. we show that cd40 is expressed by pro-b cells, and these cells proliferate in response to cd40 signalling in vitro. pcr identified a source of cd154, negative for cd3eta, in the bm of wt mice showing this cd154 is not provided by activated re-circulating t cells. we have shown that when cd154 -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn1 (cd4 -/cd8 -/cd44 + /cd25 -) to the dp (cd4 + /cd8 + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn1 stage on. in the bone marrow we found yfp + /b220 + and yfp + /b220populations. thus these pta expression analyses show closely similar pattern to those observed with hucd25 preta-reporter transgenic mice (gounari f. et al. 2002 , martin et al. 2003 . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. 3, 489-496 (2002 ) martin c. h. et al., nat. immunol. 4, 866-873 (2003 . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz 1 , g. klein 1 1 zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm-332 which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x 70kda.mmp-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm-332. in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp-19. by western blotting the zymogen and the activated form of mmp-19 can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm-332 and mmp-19 can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp-19 which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp-19, timp-2 and timp-3, are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd4/cd8 expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd4+cd8+ double positive (dp) tcrab low cells entering selection, and their cd4+cd8+ dp tcrab-immediate precedents followed by underrepresentation of the selected cd4+cd8+ dp tcrab high and the most mature cd4-cd8+ and, particularly, cd4+cd8-single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd4-cd8-double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd4+cd8-/cd4-cd8+ sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a 1 -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a 1 -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd4/ cd8/tcrab), in adult wistar rats subjected to 14-day-long treatment with a 1 -ar blocker urapidil (0.20mg/kg body weight/day s. c.). the a 1 -ar immunoreactivity was found in both thymocytes (mainly less mature cd3and cd3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed1-postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd4/ cd8/tcrab expression were observed. these changes comprised of an increase in the percentage of cd4+8+ tcrabthymocytes, which was accompanied by the reduction in that of cd4+8+ tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd4+8-tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd4-8+ tcrab high was reduced. in addition, the percentage of cd4+ t regulatory and cd161+tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a 1 -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c57bl/6 mice infected with m. avium (10 6 cfus, iv) were sacrificed at different time points after infection (5, 16 and 25 weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il-10) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at 16 weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at 24 weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il-10 in this organ. in the spleen, ifn-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm29935 and ai067906 from nih and grant i-823 from the robert a. welch foundation to wtg, and by grants hl067256 and hl61897 from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. 2001) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c57bl/6 x balb/c)f1 mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f1 mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski 1 , s. lang 1 , m. stein 1 , t. winkler 1 1 friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h3ac) or methylation of h3 on lysine 4 (h3k4me2/3). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h3ac and h3k4me2/3) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar #3) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 5'rlm race at the ivar #3 element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo2 locus, is thought to be accessible only during the double-negative (dn) 1 and 2 thymocyte stages based on mrna expression, implying that translocations between lmo2 and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx1 (hox11), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed 1) to evaluate lmo2 and tlx1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for lmo2 or tlx1 translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo2 accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo2 and tlx1 loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn1 and dn2 development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn1, dn2, dn3 and immature single positive (isp) stages for both lmo2 and tlx1. conclusion: our findings show that both the lmo2 and tlx1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn1 and dn2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e10-12), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag2 -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd4 cell depletion. however, cd8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd8 t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd8 cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd8 t cells has important implications for vaccine development against neonatal infections. (2008) showed that irf8 knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf8 as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf8 in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf8 were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs17444416. conclusions: although recent findings indicated that irf8 function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf8 that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez 1 , t. boehm 1 1 max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l1 expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd4 and cd8 t cells from tuberculosis patients highly express pd-1 when compared to healthy uninfected individuals. in addition, analysis of pd-1 expression in lung biopsies from tuberculosis patients revealed that pd-1 is expressed on cd4 and cd8 t cells confined to lung granulomatous lesions. finally, blocking of the pd-1/pd-l1 axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd-1/pd-l1 pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p100 to p52. among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly 1 wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th17 and th1 cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd4 + t cells, nfatc1 and c2 are predominantly expressed. nfatc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc1/a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc1/c. as demonstrated by y2h screen and co-ips, nfatc1/c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc1/c -but not the unsumoylated nfatc1/a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc1 target gene interleukin-2. other lymphokines like ifng and il13 are reversely regulated. interestingly, ntreg cells which do not express il2 exerted only nfatc1/c, but no nfatc1/a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc1 function. therefore, especially ntreg cells and anergized cd4 + t cells might be regulated by the long sumoylatable isoform nfatc1/c. lnk/sh2b3 and aps/sh2b2, two members of the lnk/sh2b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b-1 cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il-7 signalling in pre-b cells overexpression of lnk dramatically inhibits il-7-dependent growth demostrating that lnk negatively regulates il-7 pathways. furthermore, we showed that il-7 stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e3 ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav1. to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd3 complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd3 complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd4 + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh2 or sh3 domains known to mediate protein-protein interactions are key players in these processes. sly2 (sh3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a sh3 and a sam domain as well as a bipartite nls. sly2 belongs to a family of three molecules: sly1, sly2 and sash1.in humans, the sly2 gene is located on chromosome 21, in mice on chromosome 16. sly2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly2 we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin3-associated polypeptide p30 (sap30) as a putative interaction partner of sly2. sap30 is a conserved member of the sin3a-hdac corepressor complex that contains histone deacetylase 1 (hdac1) and histone deacetylase 2 (hdac2) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293t cells. in addition, we could show a direct interaction between sly2 and hdac1. to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly2 increases the activity of hdac1 in whole cell lysates and, more precisely, in nuclear extracts of 293t cells. the interaction of sly2 with sap30 and hdac1 indicates a transcriptional function of this protein. within the sin3a-hdac corepressor complex sly2 might act as a switch for the activity of hdac1. cd46-cyt1 and cyt2 are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd19 (b cell marker) fused to the transmembrane and intracellular domain of cd46-cyt1 or cyt2 in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt1 coengagement controlled il-10 secretion, while cyt2 coligation inhibited ifng production. moreover, our preliminary data suggest that cd46-cyt2 inhibits the phosphorylation of several molecules known to be activated by cd46 stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd46 cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh2 domain and three sh3 domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut78 cells with gst fusion proteins containing full length nck, the three sh3 domains or the individual sh3 and sh2 domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp76 and cd3epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam68) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip55 once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g4, results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g4) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g4) degranulation as measured by cd107a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi3k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the vps10p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla-4 (sctla-4) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla-4 in immune responses. using a specific anti-human soluble ctla-4 monoclonal antibody, jmw-3b3 that selectively binds the soluble isoform but not membrane bound ctla-4, or cleaved fragments of it, we demonstrate that sctla-4 plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla-4, secreting increased amounts of cytokines including interferon-g, il-17 and tnf-a, but lower amounts of il-10. soluble ctla-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla-4 induced secretion of the immunoregulatory cytokine il-10 by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sctla-4. it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla-4 it is crucial to t cell inhibition. membrane-bound ctla-4 exists as a homo-dimer on t cells but sctla-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b7 ligands on antigen presenting cells. a third important observation from this study is that sctla-4 exists both in serum and culture supernatants as a natural 46kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il-10 dependent regulation is most critical, boosting sctla-4 secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin-1/efhd-2, in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin-1/efhd2 is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin-1/efhd-2 in the immature murine b cell line wehi231 enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin-1/efhd-2 impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g1 cell cycle arrest. to understand how swiprosin-1/efhd2 enhances pro-apoptotic bcr signals, we analyzed whether swiprosin-1/efhd2 is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin-1/efhd2 enhanced bcr-induced calcium flux in wehi231 cells, whereas shrna-mediated down-regulation of swiprosin-1/ efhd2 impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin-1/efhd2 interacts with phospholipase cg2 (plcg2) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg2 and swiprosin-1/efhd2 was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin-1/efhd2 silenced wehi231 cells with swiprosin-1/efhd-2 was inhibited by the syk inhibitor bay 61-3606. in analogy, swiprosin-1/efhd2 regulated syk activity positively. moreover, swiprosin-1/efhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg2 and of syk tyrosine residue 352, which is involved in syk activation. finally, reconstitution of swiprosin-1/efhd2 knock-down cells with swiprosin-1/efhd2 mutants revealed that the n-terminal putative sh3-binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi231 cells. interestingly, swiprosin-1/efhd2 re-expression in swiprosin-1/efhd2-silenced cells induced already in unstimulated cells raft partitioning of syk, plcg2 and the bcr, which was reversed after 2 min of bcr stimulation. in summary, swiprosin-1/efhd2 is an accelerator of proximal bcr signalling and acts through syk and plcg2 by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e3 ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc1 tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd28 co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e3 ligase dead mice can spontaneously reject tc1 tumors. conclusion: cbl-b e3 ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e3 ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e3 ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd8 t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd8 + cd45ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip10 and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-cd3/cd28 antibodies. gene expression patterns were compared for cells stimulated with anti-cd3/cd28 beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip10, itac). the enhanced expression of granzyme-b, ifn-g, trail and ip10 were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd3-coated p815 cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal-3 to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd3 + foxp3 + and cd4 + cd25 high t cells. moreover, t cell receptor activation with combined anti-cd3 and anti-cd28 stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk-506, but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv-1 infection leads to immune dysfunction owing to a successive loss of the cd4 + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv-1 virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd4 and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk1/2. this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk1/2 directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap-1, nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk1/2 is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal 1 , t. brdicka 1 , v. horejsi 1 1 institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd45 are key regulators of src-family kinases in leukocytes. while cd45 is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh2 domain of csk binds phosphotyrosine 317 of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd25 and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd69 upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd25-csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd8 t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd8 t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie-2kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd8 t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd8 t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd8 t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd8 t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa-1 integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git2 in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi 1 , s. parusso 1 , b. frossi 1 , g. gri 1 , c. pucillo 1 1 university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il-6 -/-mcs and anti-il-6 receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il-6 derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd40 on naï ve b cells and the interaction of cd40 on b cell surface and cd40l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd40l e cd40 on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves 1 , n. bercovici 1 , a. caignard 1 1 inserm u567, paris, france inhibitory killer ig-like receptors (kir2dl1-2/3) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd8 + t cell subsets. these receptors suppress cd8+ t cell activation through recruitment of the src homology 2 domain-containing protein tyrosine phosphatase 1 (shp-1). to further analyse the yet largely unclear role of inhibitory kir receptors on cd4+t cells, kir2dl1 transfectants were obtained from a cd4 + t cell line and primary cells. the transfection of cd4 + t cells with kir2dl1 dramatically increased the t cell receptor (tcr)-induced production of il-2 independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir2dl1-itim phosphorylation, shp-2 recruitment, zap-70 and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp-2 and p-pkc-v but not of shp-1. in contrast, the kir2dl1/hla-cw4 interaction led to a strong synaptic accumulation of kir2dl1 and the recruitment of shp-1/2, inhibiting tcr-induced il-2 production. kir2dl1 may induce two opposite signaling outputs in cd4 + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir2dl1 receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg1 is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg1 antibody during these conditions. the observed igg1 switching behaviour mimics that of b cells responding to lps and il-4, but is mediated by a different, stat6-independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana 1 , c. schwindling 1 , m. pasche 2 , c. junker 1 , c. kummerow 1 , u. becherer 2 , e.c. schwarz 1 , j. rettig 2 , m. hoth 1 1 saarland university, biophysics, homburg, germany, 2 saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai1 channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than 150 nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd4 + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd4 + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. under tolerogenic conditions (ova alone), cd4 + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, 4-1bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd4 + t cell expansion and contraction kinetics in the early phase of the t cell response (days 1-6). in the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic cd4 + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il-2, ifn-g, and il-17a, and only few ova-specific foxp3 + regulatory t cells ( x 10 %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd4 + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells (30 %) was substantially increased. on day 14, both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells (50 %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd4 + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il-17a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd8 t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd8 tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by cd8 tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd8 tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd8 tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog 35-55 protein. our results provide direct evidence that mc contribute to cd8-specific priming in eae and show that the tc proliferation failure is specific for cd8 tc from mog 35-55 -immunized w/w sh mice. the role of mc-cd8 tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd8 tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd8 tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd8 tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd28 engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd28 costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd3complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla-4, but not via any conventional motifs in this region. overexpression of trim augments ctla-4 surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla-4 localisation, mainly restricted to the tgn. ctla-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla-4 trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla-4 expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla-4 transport to the cell surface. it is imperative to reveal the mechanisms by which ctla-4 is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd1 molecules, nkt cells are activated and release cytokines, including ifn-g, il-2 and il-4. nkt cells are efficiently recruited to the liver via cxcr6-dependent chemotaxis toward cxcl16 and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd8 t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd8 t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd8 t cell tolerisation via interaction with lsec. to this end we analysed cd1d expression on lsec and their ability to activate nkt cells by presentation of the cd1d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd1d, as agalcerpresenting-lsec were capable to induce tnf-a, il-2, il-4 and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd54 and b7-h1 on lsec. as naï ve cd8 t cell tolerisation by lsec critically depends on b7-h1, we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg9, jg1.2, and vd2 gene products. they recognize nonpeptide antigens like (e)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd-1. one of the inhibitory receptors, pd-1, is a member of cd28/ctla-4 family and contains a single ig v-like domain in its extracellular region. pd-1 can bind to two b7 homologue molecules, pd-l1 and pd-l2. it has been reported that interaction of pd-1 with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd-1 is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus il-2 to obtain gd t cells. pd-l1+ and pd-l1-human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l1 mabs, the pd-l1 extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd-1 in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd-1/pd-l1 interaction. results: gd t cells expressed pd-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l1. we first examined whether or not the engagement of pd-1 receptor could modulate the cytotoxic activity of gd t cells. pd-l1-expressing tumor cells tempered cytotoxic activity of pd-1+ gd t cells, and cytokine production such as tnf-a was down-regulated by pd-1 engagement. in addition, inclusion of anti-pd-l1 mab reversed cytotoxic activity and cytokine production when pd-l1-expressing tumor cells were challenged by pd-1-expressing gd t cells. conclusion: pd-1 delivers inhibitory signals in gd t cells upon engagement with pd-l1. peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il-2 production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e3 ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd4 + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova 323-339 peptide)-specific t cells from do11.10 tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e3 ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e3 ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler 1 , p. aichele 1 1 immh, university freiburg, immunology, freiburg, germany interleukin 12 (il-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il-12 has a direct influence on cd 8 + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal 1) and costimulation (signal 2). we analysed direct il-12 signaling to cd8 t -12 signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd8 t cells lacking il-12 signaling failed to up-regulate klrg1 and to down-regulate cd127 in the context of listeria but not viral infections. thus direct il-12 signaling to cd8 t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd8 + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd8 + t cells are tightly controlled in their effector functions by cd152 (ctla-4). we demonstrate that signals induced by cd152 reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd8 + t cells. for this novel function cd152 specifically represses the transcription factor eomes, but not t-bet. a cd152 mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd152-mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd8 + t cells differentiated in the absence of cd152 signaling could be demonstrated in vivo. the novel insights that cd152-mediated signal transduction in vivo indeed alters cd8 + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd8 + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p2 receptors. p2x 1-7 receptors open to non-selective ion channels, whereas p2y1, 2, 4, 6, 11-14 are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p2 receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr9 but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca(2+)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek 1 , a. brouckova 1 , d. filipp 1 1 institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih3t3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y505flck. comparative 2-d gel analyses followed by ms/maldi identified rack1 as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih3t3 cells of ha-tagged rack1 with either a wild type lck or constitutively active y505flck revealed a significantly enhanced complex formation between y505flck and rack1 compared to that of wtlck. ectopic expression of y505flck with its domain-inactivating mutations showed that lck-rack1 interaction depends on functional sh2, sh3 and the c-terminal tail sequence of lck. lck-rack1 interaction is readily detectable also in primary cd4 + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack1 with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack1 in the context of lck translocation to lr is further strengthen by the observation that rack1 is associated with elements of cytoskeleton. these results are the first to characterize rack1 as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (il-21) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd19+cd20+cd27-cd38-igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th2 response to alumova inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to igg1. the plasma cells up-regulate cxcr4, but not cxcr3, a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th1-associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚5%), igg2a (˚30 %), igg2b (˚30 %) or igg1 (˚30 %). in addition to cxcr4, some 70 % of these plasma cells strongly express cxcr3. the induction of cxcr3 in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg2a switching during th1 responses. this is functionally significant for oti-dependent cxcr3 expression, as well as induction of switching to igg2a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg2b. t-bet is known to be induced in b cells exposed to ifng or tlr9 stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd88. objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd272) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd4+ t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd4+ t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla-4 pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa-1 and cd2 in the psmac of untransformed human t-cells. in marked contrast, tcr/cd3 accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl-1 regulates interclonal t cell competition during acute and chronic immune activation. we found p53-independent noxa gene induction and mcl-1 downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl-1, which was delayed in noxa -/cells. using ot-1 cells and altered peptide ligands we observed that the level of mcl-1 downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl-1 -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np366) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen 9 (ebag9) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag9 confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-adaptin in t cells. both interactions suggested an involvement of ebag9 in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag9-/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag9-deficient ctls. these data imply a role for ebag9 in regulating the formation of mature ctl granules and identify ebag9 as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag9 defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd8 t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd80/86 and il-12. in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd3/acd28 or pma/ionomycin could not significantly activate cd4 and cd8 t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca 2+ influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena 1 , s. carrasco 1 , i. merida 1 1 centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp1-ras-erk signal intensity is critical to determine the final cell outcome. rasgrp1 is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. 1. analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. 2. develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd8 cells expressing gfp-c1 domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c1 domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h4, cd278), a cd28-like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th2 biased responses and high production of the anti-inflammatory cytokine il-10, and was essential to the development of germinal centres. however, icos can also help in the il-21-dependent differentiation of inflammatory th17 cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd28 a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p85 subunit of class ia pi-3 kinases. these can complex with one of the three 110 kda catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate pip 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi3k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi3 kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi3-k regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) pi3-kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p85a g p53a g g p85b and p110a g p110d g g p110b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd3-induced secretion of il-10 and il-4. during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap450, a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap450 construct that acts as a dominant negative, or sirna knockdown of endogenous akap450 expression in t cells prevents the correct organization of cd3z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa-1 localization was also analyzed to assess p-smac architecture and, interestingly, confocal 3d reconstruction revealed that lfa-1 ring was not clear in the akap450-disrupted cells. moreover, akap450 was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap450 decrease the phosphorylation of molecules such as lat, plcg1 and pkcv. these defective activation events as reflected in a reduction of il-2 production. together, our results underscore a key role for akap450 in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin85/cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh3 domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca2+ mobilization and cell activation involving the pi3k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc1d10c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr 1) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as 2) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. 3) we then studied the phenotype of carabin kd (shrna expressing) a20 b cells after bcr stimulation. 1) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t1 tot2 b cells and to follicular mature b cells. 2) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. 3) bcr simulation, but not lps stimulation, of carabin kd a20 b cells shows an acceleration of ras target erk1/2 phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk1/2 pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor 3 (tlr3) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd4 + t cells express tlr3 and respond to the well characterized synthetic tlr3 ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd4 + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr3. tlr3 stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd4 + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor 3 (irf3) as revealed by realtime-rcr analysis of ifn b and irf7, whose transcription depends on the activity of irf3. combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr3 signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd4 + t cells. this study was supported by dfg spp 1110 "innate immunity" (ka 502/8-3). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd3e cytoplasmic domain (cd3e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd3e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd3e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd3e cd and a fluorescent membrane dye (r18). with this assay, we show that the cd3e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd3e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd3e cd to lipid bicells. membrane binding by the cd3e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia 1 , y. ge 1 , u. quitsch 1 , p. beckhove 1 1 german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd4+ t cell help is believed to contribute to optimal cd8+ memory expansion via cd40l on cd4+ t cells binding cd40 on dendritic cells. however, a few reports suggest that cd40l-cd40 engagement may mediate direct cell-cell contacts between cd4+ and cd8+ t cells. in this study, we investigated the importance of cd4-cd8 co-operation and cd40l-cd40 interactions for t em proliferation. methods: we isolated human cd4+ and cd8+ t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd4+ and cd8+ populations were activated in vitro using anti-cd3/cd28 beads. proliferation was measured by [ 3 h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd40 or cd40l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd4+ or cd8+ t em cells, demonstrating that optimal t em expansion requires direct cd4-cd8 interactions. surprisingly, not only cd8+ but also cd4+ t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd4+ t em proliferation depends on signals from cd8+ t em cells. activation induced the expression of cd40 on both populations and cd40l on subsets of cd4+ and cd8+ t cells. blocking of cd40l on cd8+ t em cells impaired significantly cd4+ t em proliferation, which confirms that the improved expansive potential of cd4+ t em cells in mixed populations depends on cd40l co-stimulation by the cd8 t em subset. conclusions: our data demonstrate for the first time that activated cd8+ t em cells deliver help to the cd4+ t em subset via cd40l-cd40 signalling and may play an important role for cd4+ t em expansion upon stimulation. the t cell surface glycoprotein cd5, a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd5 localization upon t cell:apc conjugation. we have questioned which domains of cd5 mediate the localization within the is, and for this we have expressed cd5 mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd5 mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd5 by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd5 depends on sequences within the cytoplasmic domain, as a cd5 deletion mutant lacking most of the cytoplasmic tail, cd5.k384 stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd5 translocation was mapped within amino acids glu 418 and his 449 since the cd5.h449 stop mutant, just short of 22 aa is still able to translocate to the is, whereas cd5.e418 stop , that lacks 2 important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu 418 -his 449 ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna-2 is missing, but lmp-1 is still expressed. using a hl derived cell line, we have shown that the cytokine il-4 can induce lmp-1 expression in vitro and can replace ebna-2. we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is 2, 3 or 4. a high affinity stat6 binding site is spaced by 4 nucleotides. we found three potential stat binding sites in the lmp-1 promoter, which we named lrs, tr and edl1. they were spaced by 4, 3 and 2 nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il-4-treated or non-treated kmh2-ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat6 binding site, or lrs-stat6. a stat6 complex binding to the gl-epsilon promoter and lrs-stat6 was induced by il-4. the specificity of the stat6 complex was shown by supershift experiments with anti-stat6, but not anti-stat5 antibodies. when gl-epsilon or lrs-stat6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat6 contains a functional stat6 binding site. oligonucleotides, corresponding to lrs in which the stat6 site had been mutated, could not compete for stat6 binding. interestingly, the unlabeled lrs-tr with 3 nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by 2 nucleotides (lrs-edl1), it could not compete. thus, expression the transforming protein lmp-1 can be induced directly by the t cell derived cytokine il-4 in a stat6 dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp-1 and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd8 t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji 2007 (ji . 179:2250 . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd4 t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd4+ t cells primarily affect the proportion of cd4+ t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd40, cd80 and cd86 on parameters of cd4+ t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd40l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd40 leads to an enhancement of ig and cytokine production. the current dogma postulates that these 2 signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd40l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd40l (rmcd40l) can increase proliferation induced by tlr3 (poly ic) and tlr4 (lps) agonists. by contrast, we never observed any synergy between rmcd40l and tlr1/2 (pam3csk4) or tlr2/6 (pam2csk4) agonists. to go further in the study of cd40l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd40l, named mini-cd40ls, based on a c 3 -symmetry core holding cd40-binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd40ls bind to immobilized human cd40 and compete with the binding of cd40l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd40l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd40l, mini-cd40ls synergize tlr4 (lps), tlr3 (poly ic) and tlr7/8 (r848) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd40ls and tlr 1/2 (pam3csk4), tlr 2/6 (pam2csk4) and tlr9 (odn 2395) agonists. synergy between cd40l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd40l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd8 + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd8 + t cell tolerance. we have defined a phenotypic profile for cd8 + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd25 and cd69, and high levels of expression of cd62l and ly6c. whereas, cd8 + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd25 and cd69, and loss of cd62l and ly6c expression. ly6c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly6c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly6c, expressed on naïve cd8 + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd8 + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to 60 days after infection. results: daily injection of paraoxon induced g 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. mice exposed to paraoxon exhibited g 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th1 and th17 cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide 3-kinases (pi3k) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi3k are divided into two types: class ia p85/p110 heterodimers, which are activated by tyr kinases, and the class ib p110g (p110gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p110g deletion affects tcr-induced t cell stimulation. mice lacking p110g show a partial defect in t cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd4+/cd8+ t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi3kg-specific inhibitor causes a reduction in the number of cd4+ memory t cells that mediate renal injury. similarly, pi3kg deletion in p65 pi3k transgenic mice also reduces the numbers of cd4+ memory t cells. there is therefore evidence that pi3kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi3kg regulates this process is not well understood. we studied the specific role of p110g in t cell activation. methods: we studied whether the tcr activates p110g and the consequences of interfering with p110g expression or function on t cell activation. results: we found that after tcr engagement, p110g interacts with and forms a complex with ga q/11 , lck and zap70. tcr stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p110g controls rac1 activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p110g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p110g-/-t cells. our observations clarify the activation mechanism and mode of action of p110g in the control of t cell activation, confirming a crucial role for p110g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a4b2 and a7, expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c57bl/6j mice. they were stained with fluorescently labeled igm-, cd40-or cd23specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by 3 h-thymidine incorporation upon stimulation with anti-cd40 and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a7 nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a4 or b2 nachr subunits inhibited binding of igm-and cd23-specific antibodies but facilitated that of cd40-specific antibody. in contrast, antibody against a7 subunit prevented binding of anti-cd40 but not of anti-igm or anti-cd23 suggesting that a7 nachrs are located close to cd40, while a4b2 ones are close to bcr/cd23. consequently, anti-cd40-induced b lymphocyte proliferation was increased by mla much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd40-mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd4 + t h -lymphocytes to antigen presenting cells or of cd8 + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim1 and trpc3), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about 96 %. down-regulation of stim1 was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd4 + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, 2008) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein 70 (hsp70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd8 + cytotoxic t-lymphocyte (ctl) and cd4 + t-helper cell (th1) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k562 cells as well as targetindependent cytotoxicity. cd4 + cells exhibited a strong increase in proliferation after stimulation with hsp70, with rates of up to 29 %. in the presence of target cells, a 35-fold up-regulation of granzyme b mrna was observed after stimulation of cd4 + t-helper cells with hsp70 in combination with il-7, -12 and -15. the target cell-independent secretion of granzyme b by cd4 + cells was greatly augmented after stimulation with hsp70 plus il-2 or il-7, -12 and -15. in this study, we have shown that hsp70 is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd4 + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd3 + and cd8 + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp70 on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd40, which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd150 receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk1 overexpression in a model system was achieved by transfection. pjnk1/2 expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd150 on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk2, but not jnk1 activation. cd40 ligation on primary tonsillar b cells also resulted in jnk2 activation. however, bcr crosslinking did not affect the level of jnk1/2 phosphorylation. cd150-mediated jnk2 activation was independent from sh2d1a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd150 and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase 1 (hpk1) was associated with cd150 in primary b cells as well as in b cell lines. cd150-hpk1 association was independent from cd150 tyrosine phosphorylation and sh2d1a expression. overexpression of hpk1 in a model system significantly enhanced cd150mediated jnk2 phosphorylation. it is known that tnf family receptors such as cd30, cd40, rank trigger survival signals in hrs cells. we observed the expression of pjnk1/2 in hrs cells of primary classical hl. cd150 could be involved in sustained jnk2 activation in primary hrs cells, and this may reflect the role of cd150 receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk2 is activated via cd150 in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk1 is involved in cd150-mediated jnk2 activation. objectives: cd5 has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd5 upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd5 are involved in c-cbl phosphorylation and association. methods: el4 thymoma cell line was stably transfected with wild-type human cd5 or hcd5 cytoplasmic tail mutants: cd5.k384stop (maintaining only a pseudo itim); cd5.h449stop (lacking the distal s and y in the carboxy-terminal region); cd5. ¿ e418-l444stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y700 in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd3 in combination or not with anti-human cd5 biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py700) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x 0.05). in murine thymocytes, co-crosslinking of cd3 with cd5 induces an increase in c-cbl phosphorylation compared to cd3 alone. analysis of the el-4 transfectants showed that mutants cd5.k384stop and cd5.h449stop lost the ability to costimulate cd3-mediated phosphorylation of c-cbl. in contrast, cd5. ¿ e418-l444stop mutant, was able to efficiently costimulate cd3-mediated c-cbl phosphorylation, similarly to the hcd5wt. our results indicate that the absence of the pseudo itam in cd5 does not interfere with c-cbl phosphorylation in response to cd3 plus cd5 crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd5 appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd5 cytosplasmic tail, but rather, may indirectly associate with cd5 through the interaction with other sh2-sh3 domain-containing molecules, that may be recruited to cd5 through its carboxy-terminal region. l. kolly 1 , s. narayan 1 , j. tschopp 2 , a. so 1 , n. busso 1 1 chuv, rheumatology, lausanne, switzerland, 2 unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent il-1b and il-18 production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd4 + and cd8 + t cells were activated in vitro through anti-cd3 stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd3 ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd3 activated asc -/-t cells predominantly displayed a more th 2 phenotype, producing more il-10 (199 vs. 692 pg/ml; asc +/+ vs. asc -/-t cells respectively; p=0.0074) and less ifn-g (15,831 vs. 6 ,921 pg/ml; asc +/+ vs. asc -/-t cells respectively; p = 0.0021). when asc +/+ and asc -/-t cells were purified into cd4 + and cd8 + t cell fractions and activated individually using anti-cd3, no inhibition in proliferation was observed amongst activated asc -/-cd4 + and cd8 + t cells. interestingly, the activated asc -/-cd4 + t cell fraction produced significantly more il-10 when compared to activated asc -/-cd8 + t cells and asc +/+ cd4 + and cd8 + t cells (asc -/-cd4 + t cells = 380 pg/ml il-10; asc -/-cd8 + t cells = undetectable il-10; asc +/+ cd4 + t cells = 11 pg/ml il-10; asc +/+ cd8 + t cells = undetectable il-10). cd4 + and cd8 + t cell mixing experiments revealed that asc -/-cd4 + t cells are able to inhibit the proliferative ability of asc -/-cd8 + t cells, asc +/+ cd4 + and cd8 + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd4 + t cells. collectively, these results demonstrate that the absence of asc drives cd4 + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd4 + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge 1 and pge 2 suppress some t-cell functions including proliferation, activation and cytokine production. pge 2 signals through four types of gpcrs called the ep receptors. at low concentrations, pge 2 is believed to be necessary for t cell function, whereas at higher concentrations, pge 2 inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge 2 and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge 2 diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd4 + t cells with ep receptor antagonists was found to impair cell surface expression of cd71, cd69, cd25 and ox40 but not cd44. suppression of t cell proliferation by pge 2 has already been widely studied. however, blocking ep receptors in cd4 + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd4 + t cells to the chemokine sdf-1b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd44 + cd4 + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd4 + t cells. our results also suggest that considering pge2-mediated camp signaling in cd4 + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd4 + human t-cells was examined. oxidation affects several ca 2+ signalling pathways by altering the activity of ip 3 receptors, trp channels and store operated ca 2+ channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca 2+ signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd147 (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd147 knockout mouse possess enhanced mixed lymphocyte reactions and cd147 monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd147 signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd147 in jurkat t cells augments the secretion of the t cell growth-factor interleukin-2 (il-2) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il-2 promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd147, we identified the immunomodulatory sub-domain of cd147. supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd40 and il4 (simulates t cell-dependent activation). microarray assays identified about 104 mirnas in unstimulated b cells. 35 of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor-4 (irf-4). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf-4 transcripts differs from that observed for irf-4 protein abundance after b cell stimulation. further analysis identified the irf-4 transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk592 and the dfg forschergruppe for832. objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on 11 different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey 1 , f. hauck 2 , i. berberich 1 , f. berberich-siebelt 2 , gk 520 -immunomodulation 1 institute for virology and immunobiology, university of wuerzburg, würzburg, germany, 2 university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd4+ t lymphocytes, the transcription factor is predominantly expressed in t helper 2 (th2) compared to t helper 1 (th1) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g1 phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp-1 is expressed in differentiated effector t cells where it is higher in th2 than th1 cells. the regulation of the blimp1 expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm1 promoter and activates blimp-1 expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp-1 in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp-1 in b cells using cre recombinase. moreover we found a new putative blimp-1 isoform lacking exon 2. currently, we analyze the expression of c/ebpb and blimp-1 in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening 53 cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd21low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd4 + t cells but not transitional b cells or cd8 + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (mip-1a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy2 revealed constitutively high background levels in cd21low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas 1 , v. courtois 1 , k. de luca 1 , r. sodoyer 1 1 sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il-2, il-4 or il-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il-2, il-4 and il-6. furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a-136-0033-01835). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen-4 (ctla-4) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla-4 during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il3) in the tissue and the released eggs and first stage larvae (l1) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd3 and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla-4 during the infection, a neutralysing antibody (a-ctla-4; 4f10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with s. ratti il3. the in vivo neutralisation of ctla-4-signalling by applying a-ctla-4 during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th2 cytokines, such as il-4 and il-5 and a reduction of the proinflammatory cytokines ifn-g and il-17. the investigation of the humoral response showed a remarked increase of the igg1-titer in the serum during secondary infection in mice that had been treated with a-ctla-4 during primary infection. furthermore, the blockade of ctla-4 resulted in a diminished worm burden as indicated by reduced release of l1 in the faeces. these results suggest that the blockade of ctla-4 during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg1 during secondary infection also reflects the induction of a potent th2 response. objectives: cd36 is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd36 is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd36 on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd36 expression. cd36 knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd36 ko compared to heterozygous mice. since reduced levels of cd36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd36 ko mice. after one injection of apoptotic cells, cd36 ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd36 on b-cells are involved in setting this threshold. conclusion: our data suggest that cd36 is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd152 acts as a major check-point of immune responses, but the mechanism by which cd152 controls peripheral t cell responses is unknown. the consequences of cd152 signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd152 in th1 cells reduced migration towards ccl4, cxcl12 and ccl19. crosslinking of cd152 together with cd3 and cd28 stimulation on activated th1 cells increased expression of the chemokine receptors ccr5 and ccr7, which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd152 expression and cd152-mediated migration-enhancing signals. importantly, migration of cd152 positive th1 lymphocytes in in vivo experiments increased, as compared to cd152 negative counterparts, showing that indeed cd152 orchestrates specific migration of selected th1 cells to sites of inflammation and antigenic challenge in vivo. these data show that cd152 signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd152 adds to the already significant role of cd152 in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd152 signaling on the longevity of cd28 null t cells. using a sensitive staining method for cd152, we show that human cd4 + cd28 null and cd8 + cd28 null t cells rapidly express surface cd152. serological inactivation of cd152 using specific fab fragments or blockade of cd152 ligands using ctla4ig in cd4 + cd28 null and cd8 + cd28 null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd152 crosslinking on activated cd28 null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd152 is mediated by pi3'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd152 act directly on activated cd28 null t lymphocytes and, due to its exclusive expression as a receptor for cd80/cd86 on cd28 null t cells, prevention of cd152 mediated signaling is likely a major target mechanism taking place during therapy with ctla4ig. objectives: cd45 is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd45 -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd148 (ptprj, dep-1) which acts as a positive regulator of sfk in cd45 -/-b cells and macrophages and can compensate for cd45 deficiency in these cells. indeed, combined deficiency of cd45 and cd148 in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd148 and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd148 -/mice were reported so far. however, in human t cells the role of cd148 may be different since naive human t cells express cd148 at a level comparable to b cells. using cd45 -/-/cd148 -/human t cell line (jurkat-derived js-7 cells) we tested the ability of cd148 to complement cd45 deficiency in t cells. we used retroviral transduction to express human cd45 or cd148 in js-7 cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd148 to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js-7 cells. expression of wild type cd45 or cd148 in js-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd69 upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js-7 cells expressing cd148 when compared to control cells. finally, cd148 substrate trapping mutant expressed in js-7 cells interacted with lck in vivo suggesting that lck is a direct substrate of cd148 in js-7 cells. the results suggest a level of redundancy between cd45 and cd148 in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam1.6) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr 505 and tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native 2d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca2+ release-activated ca2+) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa-1 prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al.2006) we describe a non-viral vector based approach (vockerodt et al. 2008) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in 9 independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd40 signaling cascade was conducted. after activating this particular signaling cascade (cd40) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. 2005 ). a rat thymic epithelial cell (tec) line (r-tnc.1) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc.1 cell line in vitro. it was found that a number of adhesion molecules, such as cd2, cd4, cd8, cd11a, cd18, cd54, cd90 was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc.1 line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il-2 activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc.1 cells. we found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mab to cd2 and cd8 molecules (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells). early adhesion was inhibited by mab to cd90, abtcr, mhc class i molecule (ifn-g stimulated r-tnc.1 cells) and cd4 molecule (ifn-g unstimulated r-tnc.1 cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc.1 cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mab to cd2, cd4, cd8, cd90 molecule (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc.1 cells). our results also suggest the involvement of cd11a/cd18 dependent -cd54 independent pathway in adhesion and cd11a/cd18 dependent -cd54 dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin-27 (il-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il-27 favours naï ve cd4 t cell differentiation into th1 cells to the detriment of th17 or th2 differentiation. the il-27 receptor (il-27r) is a heterodimer composed of tccr, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. il-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd8 t cells, but the potential impact of il-27 on human cd8 t cells has not been elucidated. our goal is to investigate the impact of il-27 on human cd8 t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il-27r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd8 than cd4 t cells expressing the complete surface il-27r (gp130+tccr). however, we detected high amounts of intracellular tccr in both, cd4 and cd8 t cells, but only polyclonal activation (anti-cd3) of cd8 t cells led to an actual increase of il-27r surface expression. purified cd8 t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il-27 activated stat1 and stat3 signalling with rapid kinetics in both cd8 and cd4 t cells, indicating the capacity of il-27 to signal through these molecules. addition of il-27 to anti-cd3 activated cd8 t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il-27 on human cd8 t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd-1/pd-l1 pathway is associated with production of the immunoregulatory cytokine il-10, the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l1 and pd1, as well as myelin basic protein (mbp)-stimulated il-10 production, pakt inhibition, and apoptosis (annexin v), in 50 ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); 24 had a diagnosis of stable disease (sms). results showed that: 1) pd-l1 -expressing cd14+ and cd19+ cells are reduced in ams compared to sms individuals (p=0.04); and 2) pd1 expression is increased in cd4+ t cells of sms individuals and is comparable on cd8+ t cells of ams and sms patients. this is associated with a significant decrease in il-10 production by mbp-stimulated cd14+ and cd19+ cells of ams patients (p=0.03). additionally, cd8+ anexin v+ (av+) cells were diminished and cd8+ pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd4+av+ and cd4+ pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l1/pd-1 pathway seen in ams patients result in a reduced mbp-specific il-10 production by cd14+ and cd19+ cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd8+ t. the pd1/pdl1 pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti-4-1bb in cd8 cells. this difference could be due to down regulation of cd28 by activated lymphocytes and possible preferential response of cd8 cells to anti-4-1bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin7 (stx7) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx7. rna interference technique was also used to down regulate stx7 expression in primary human ctls. results: we identified stx7 in ctls by pcr and immunocytochemistry. stx7 accumulates at the is after ctl/target cell contact. when stx7 function was blocked by overexpression of a dominant negative stx7 mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx7 is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the 20 years history of mouse t h 1 and t h 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h 0, t h 1 or t h 2 phenotypes, based on their cytokine production (il-2, ifng or il-4). a comparative analysis of t-bet, ifng and il-4 mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca 2+response, membrane raft expression/organization, k + -and ca 2+ -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h 1 g t h 0 g t h 2, although the membrane cholesterol level (detected with anti-cholesterol ab, ac8) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h 1 cells upon activation than in t h 2 cells. t h 1 cells produced a more sustained calcium response with higher amplitude than t h 2 cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav1.2 and kv1.3 ion channels, major functional determinants of the sustained calcium influx. t h 2 cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd19-dynabeads. cd19 + isolated cells were facs sorted into cd19 + cd27naïve b or cd19 + cd27 + memory b cells. dna synthesis was measured by 3h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd19 + cd27naï ve b cells, of which bmp-6 and -7 were most efficient (40 % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd19 + cd27 + memory b cells by 40 -50 %. to induce differentiation, both naï ve and memory b cells were stimulated with cd40l and il-21. this increased the production of igm, iga and igg 10 -100-fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad1/5/8 in cd19 + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd40l/il-21-induced production of igs in mature human b cells. s. gutenberger 1 , k. warnatz 1 1 university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (erk 1/2). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd86 upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of 20 hd and 25 cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk1/2. to increase the signal intracellular phosphatases were inhibited by h2o2. as markers of activation and initiation of proliferation, cd86 and ki67 expression were measured after 2 days of in vitro stimulation. k. theil 1 , p. aichele 1 1 immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd8 t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p14 cd8 t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b6-recipient mice and compared their expansion with wild-type (wt) p14 t cells after viral infection. we could demonstrate a severe impairment in the capacity of p14 t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p14 t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd8 t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p14 t cells into h8 mice. h8 mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp33-41. therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h8 mice are infected with lcmv8.7. this is a lcmv variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the p14 t cells. s. frischbutter 1 , r. baumgrass 1 1 deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap1 which are important for expression of cytokines such as il-2, ifng and il17. it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl-10 was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl-10 phosphorylation but rather an inhibition of bcl-10 dephosphorylation. furthermore, calcineurin and bcl-10 co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl-10/malt1 signaling complex and dephosphorylates bcl-10 and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop 1 , j. lamoureux 1 , c. fortin 1 1 université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: 25 healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla-4 has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla-4 expression was higher after stimulation in t cells of elderly. there was differences between cd4 and cd8 t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd95l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh3 and ww domain proteins. since fasl surface expression is regulated by adam10-mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl2a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh3 domain proteins that potentially interact with the riped fasl prd, we used a sh3 domain phage display library containing all 288 sh3 domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conseoptimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b7 family members on antigen-presenting cells with cd28 on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd28 co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound12, with classical gcs regarding their suppressive effect on cd28-costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd3 and anti-cd28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd28-costimulated human memory/effector cd4+ t cells by compound12 than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound12 was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound12 and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound12 than prednisolone. when evaluating possible mechanism for the increased activity of compound12 in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound12 which was not prevented by the partial gc receptor antagonist, ru-486, in vitro. moreover, in vivo we observed less induction of il-1beta and tnf-alpha by pre-treatment with compound12 than with prednisolone. our data indicate that the non-steroidal segra, compound12, may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b6 mice elicits robust cd8+ t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il-2+ cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa 224 -specific cells also express tnfa, but only about half of the ifng+ np 366 -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd8+ t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd8+ t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova 257-264 peptide shows a close correlation with division in vivo. early after antigen encounter (0-2 divisions) the vast majority of cells express only tnfa. after 3-4 divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd4+ t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd8+ t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd8+ t cells, expansion plays a very important role in the regulation of cd8+ t cell effector function. in addition to its chemo-attractant function, sdf-1a (stromal-cell derived factor-1a, cxcl12) has been described to costimulate cd4 + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd4 + t cells was increased by immobilized sdf-1a to a level similar to that obtained with the costimulatory molecule cd28. as visualized by real time confocal microscopy, t cells entering in contact with sdf-1a formed a tether and displayed an active scanning activity. since sdf-1a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd3 mabs, it is conceivable that the sdf-1a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf-1a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf-1a in the context of cd4 + t activation by antigen-presenting cells secreting sdf-1a. this study should help us to better define how sdf-1a modulates cd4 + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd4 + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd3 and anti-cd28 antibodies and cape for 2 days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by 3h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il-5 production and lymphoproliferation in cd4 + t cells stimulated by anti-cd3/cd28. cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd4 t cells express a variety of molecules on their surface, such as mhc-class ii, cd80, cd86, cd70, whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd4 t cells might induce t cell proliferation and differentiation from cd4 resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd4 t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd4 memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd25, cd30, and cd69. analysis of the cytokine profile of these cells revealed the differentiation of il-10-and ifn-g-double-producing cells in response to contact with th1 effector cells, and il-4-producing cells in response to contact with th2 effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd3/cd28 stimulation. whereas neutralization of ifn-g or il-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd70, cd80, and cd86 could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. conclusion: interaction of cd4 memory t cells with activated t cells resulted in the production of the cytokines il-4, il-10, and ifn-g. given the immunomodulatory capacity of il-4 and il-10, these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir 1 , s. thompson 1 , j.j. murphy 1 1 king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il-2 and il-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp36l1 by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp36l1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl1 differentiation was observed to be associated with downregulation of zfp36l1 protein. in an attempt to determine whether zfp36l1 downregulation was directly linked to bcl1 differentiation, a zfp36l1 shrna expressing lentivirus (psicor) was employed to knockdown zfp36l1 expression. this reagent downregulated zfp36l1 expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp36l1 shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp36l1 downregulation in promoting bcl1 plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of 32 patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all 32 isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd95 (fas, apo-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd3/cd28-triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo1 mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd95l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il-2 and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk1/2 phosphorylation, the expression of various activation markers, the il-2 production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb415) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb1,3galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd4+ and cd8+ t cells. in addition, mitogenic stimulus increase 3-fold the all binding to cd4+ t cells. previous studies in human pbmc showed that all binds to human cd4+ t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd4+ t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd3-dependent activation of purified cd4+ t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd25 and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd25 expression, but enhanced the anti-cd3-dependent proliferation and cd25 expression of purified cd4+ t cells. analisis of calcium influx showed that all enhanced anti-cd3 dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd4+ t cells by up-regulating t cell activation mediated by anti-cd3 stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in214609) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd3 complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd95 receptor. it has been previously demonstrated that cd95 ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp-76, slap-130 and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il-1r, il-2r, il-3r, il-7r, il-12r, il-18r, cd27, cd28, cdw137( 4-1bb), cd 95(fas) and cd178 (fasl) on hpbls in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. 1. the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) the expressions of membrane immune molecules before cultured. 2) expressions of the membrane molecules on hpbls during culturing. 5% fbs rpmi 1640 group (1640 group), il-2 group, pha group... (1) mtt assay. (2) proliferative times and growth curves of hpbls... 1. during cultured in vitro, there are expression changes of the il-1rs (il-1ra, il-2ra, il-2rg, il-3r, il-7r, il-12r, il-18r), co-stimulatory molecules (cd27, cd28, 4-1bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in 1640 group, il-2 group and pha group, but the rests are different. 2. our data also suggest that the hpbls cultured in cd3mcab+cd28mcab+il2+il1a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. 3. celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun 1 , r. tauber 1 1 zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the ras/rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-jun n-terminal kinase (1), which leads to an enhanced binding of l-selectin to soluble ligands (3). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues (4). here we show that the protein phosphatase 2a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp2a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp2a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer 1 , e. glasmacher 1 1 helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago1-4 genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm1 and rck or expression of dominant-negative gw182. interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd4 + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd8 + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd8 + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd8 + t-cell activation and differentiation. naïve cd62l hi cd44 lo cd8 + t cells were sorted and stimulated by anti-cd3 and anti-cd28 antibodies. results: firstly, we show that the activation and differentiation of cd8 + t cells require il-2 provided by activated cd4 + t cells at the initial priming stage after stimulation. secondly, this critical il-2 signal is delivered through il2rbg of cd8 + cells and is independent of il-2ra. besides promoting cell proliferation, il-2 stimulation increases the amount of ifng and granzyme b produced by cd8 + t cells. conclusion: therefore, our studies demonstrate that a full cd8 + t-cell response is elicited by a critical temporal function of il-2 released from cd4 + t cells, providing mechanistic insights into the regulation of cd8 + t cell activation and differentiation. most antigenic peptides recognized by cd8 t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a3168-176 is a tumor antigenic peptide presented by hla-a1 and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a3168-176 by tumor cells. by electroporating hla-a1 cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart-1/melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart-1/melan-a 26-35 specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart-1/melan-a 26-35 ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart-1 expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart-1/melan-a 26-35 epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart-1/melan-a 26-35 ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd8 + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd4 t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev 207: 293-313, 2005) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h2-m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity 11: 453-462, 1999) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. furthermore, also reduction of tap1 and tap2 transcription was observed contrasting to the elevated levels of erp57 and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their ifng-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about 50% of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a*0201) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a*0201 binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a*0201 from tapasin, but only ttp-ha dissociated hla-a*0201 from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl-721.220-a2 showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a*0201 from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a*0201. m. basler 1,2 , c. lauer 2 , m. groettrup 1,2 1 biotechnology institute thurgau, kreuzlingen, switzerland, 2 university of constance, division of immunology, department of biology, konstanz, germany two lmp7-dependent antigens have been described that relied on the 'structural presence' of lmp7 in the proteasome but not on the activity of lmp7. here we have investigated processing of the h-2d b -restricted uty 246-254 epitope of the male minor antigen uty reported to be lmp7-dependent. using splenocytes from lmp7 -/-, lmp2 -/and mecl-1 -/mice we found that the uty 246-254 epitope requires lmp7 and lmp2 but not mecl-1. curiously, a selective lmp2 inhibitor did not interfere with uty 246-254 presentation. objective: we investigated why the deletion but not the inhibition of lmp2 interferes with uty 246-254 presentation. we hypothesized that the 'structural' requirement for lmp2 is based on replacement of the caspase-like activity of b1 in the proteasome. methods: it was determined if t1a mutants of lmp2 and/or b1 can rescue the uty 246-254 epitope. we used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. finally we determined by mass spectrometry if the uty 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by lmp2-deficient and proficient immunoproteasomes in vitro. results: we found that t1a mutants of lmp2 and b1 rescue presentation of uty [246] [247] [248] [249] [250] [251] [252] [253] [254] . also inhibition of cells with a b1-selective inhibitor preserves uty [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. an aspartate in position 7 of the uty 246-254 sequence wmhhnmdli is preferentially used as a cleavage site by lmp2-deficient but not half as frequently by lmp2-proficient immunoproteasomes. the generation of the uty 246-254 epitope relies on the replacement of the caspase-like activity of b1 by lmp2 because the b1 activity destroys the uty 246-254 epitope. this is the first example for the 'structural' requirement of lmp2 for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp2 and perhaps also lmp7 and mecl-1 exert their function in antigen processing. a. linnemann 1 , a. musiol 2 , r. lindner 1 1 hannover medical school, cell biology, hannover, germany, 2 hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf6) were overexpressed in 3t3 fibroblasts. we show that antibody-mediated oligomerization of mhc i in 3t3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf6: endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf6-regulated to a novel, arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv40 virus and since sv40 binding triggers mhc i oligomerization, this novel pathway may be involved in sv40 uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of 57 rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab3b, 3c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab3b and rab3c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab3b or 3c positive vesicles. furthermore, the rab3b, 3c positive compartment were colocalizd with a fraction of rab27a at a juxtaposition of phagosomes. together these data demonstrate that rab3b and rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over 90 % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd8+ t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf2a and bglf5 have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf1, has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens 2009). this represents a novel function for a virally-encoded gpcr. bilf1 is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf1 displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf1 is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf1 can hinder the recognition of virally-infected cells by cytotoxic cd8+ t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg1 is transcribed in thymus from both male and female individuals. finally, we detected the semg1 mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). deficiency in beta5i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta5i-deficient cells could be restored by treatment with d3t, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr2, 4 and 5, which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd4 + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd4 + t cells. the presence of exogenous tlr3 and tlr8 ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james 1 , i. bailey 1 , t. elliott 1 1 university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct26 colon carcinoma. this response is long lasting and mediated by both cd4 and cd8 t cells. importantly, the treg depleted ct26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a20, c26, bcl1, renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw11, which is h2-d d restricted. analysis of the generation of gsw11 in ct26 revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct26 cells substantially increased the amount of gsw11 present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct26 and immunised balb/c mice. greater than 80 % of mice injected with eraap kd ct26 were found to reject the tumour. analysis of t cell responses revealed the presence of gsw11-specific t cells, however, these responses were small (0.5-1 %). this compared to a much larger response to ct26 (˚5 %). preliminary results indicate that the majority of the t cell responses (non-gsw11-specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [1] . the di-allelic hla-a2 restricted minor histocompatibility antigen ha-1 locus codes for the highly immunogenic ha-1 his and the non-immunogenic ha-1 arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha-1 arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for ha-1 his and less than 5/ cell for ha-1 arg [2] . as ha-1 his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha-1 his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha-1 mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha-1 allelic peptides, we investigated the impact of the ha-1 his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha-1 his and ha-1 arg peptides were performed. moreover, the crystal structures of hla-a2 in complex with either ha-1 his , ha-1 arg or a ha-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the hla-a2/ha-1 peptide complexes. our results exclude a role for antigen processing in preventing ha-1 arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha-1 arg allele in the hla-a2 peptide binding groove [3] . they provide a rationale for the lack of ha-1 arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd8 t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd8 t cell response. on the other hand, immunosubunit expression is not essential for development of cd8 t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp7 (ib5) and mecl-1 (ib2) develop a variety of autoimmune responses, including a latent form of t1d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd8 but not cd4 t cells. a cd8 t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd8 t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd8 t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, 162 autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr3 and dr4. analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type 1 (hsv-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd4 + and cd8 + t cells in hsv-1 immunity have yet to be clearly elucidated. to better understand the role of hsv-1-specific cd4 + t cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein d of hsv-1. following flank infection, gd-specific cd4 + t cells were first detected in the draining brachial and axillary lymph nodes (ln) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gd-specific cd4 + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day 6 pi and peaked at day 9. while hsv-specific t cells were first observed in the draining ln at day 5 pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd4 + t cells was observed up to 23 days post-infection in the brachial ln. ex vivo analyses suggest that only cd11c + cells were involved in gd antigen presentation at days 2, 5 and 15 post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd8a + dcs can present the gd antigen to cd4 + t cells at day 2 pi, whereas by day 5 pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap1 and erap2 unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap1/erap2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp57. upon assembly of the heavy chain with b 2 m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp57. both crt and erp57 are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp57 to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp57 or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp57 must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp57 point mutant that fails to bind to cnx or crt was just as effective as wild type erp57 in normalizing rates of disulfide formation. we conclude that erp57 does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp57 point mutant, class i heavy chains, crt and the tapasin-erp57 disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp57 and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp57 have no effect on plc stability or class i surface expression, suggesting that erp57 plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb1*0405/dqb1*0401 haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr*0405 transgenic ab0 nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd4+ and cd8+ t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr*0405 transgenic mice (cd4+ t cell competent) develop aip even unprovoked, similar to ab0 nod mice (cd4+ t cell deficient), while hla-dr*0401, hla-dq8 or hla-dr*0405/dq8 (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr*0405 fails to protect from aip, likely due to defects in the induction of cd4+ regulatory t cells. our results identify hla-dr*0405 as a prominent risk factor for aip on the hla-drb1*0405/dqb1*0401 haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan 1 , c. britten 1 , h. overkleeft 2 , g. van der marel 2 , k. melief 1 , d. filippov 2 , f. ossendorp 1 1 leiden university medical center, section tumorimmunology, leiden, netherlands, 2 leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd8 and cd4 t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi 1,2 , x. hu 1,2 , a. kawanatachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping 8-mer and 10-mer epitopes (rypltfgwcf (nef138-10) and rypltfgw (nef138-8)) were found to be presented by hla-a*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y2f, y2w, t5c, f6l, w8r, f10r, f10y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef138-10 (wild type) and its four mostly common immune escape mutants (y2f, t5c, y2f&t5c, f6l), and also nef138-8 (wild type). we found that there was little difference between the nef138-10 (wild type) and nef138-10 (y2f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y2 or f2. interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (t5c) and nef138-10 (y2f&t5c), which may affect the binding of peptide and the recognition of t cell receptor. for nef138-10 (f6l), the side chain of l6 was more flexible compared to the nef138-10 (wild type). alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of f6 was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd8+ and cd4+ t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a2 restricted cmvpp65 derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. this pattern was seen for 5/9 epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to 2-5 log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b7 restricted cmvpp65 rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t2 cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd8+ t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp110, and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor-5 (fgf-5) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf-5 spliced peptide by cells transfected with mutant constructs encoding fgf-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b*4402, are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b*4402 and b*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us3 protein. to address this hypothesis, us3 was stably coexpressed in b lymphoblastoid cell lines expressing hla-b*4402 or hla-b*4405. in the presence of us3, the surface expression of hla-b*4402 was substantially reduced whereas hla-b*4405 expression was relatively unaffected. although us3 was able to form complexes with both hla class i allotypes, only hla-b*4402 was retained intracellularly in an immature form whereas hla-b*4405 was transported to the cell surface. accordingly, in the presence of us3, hla-b*4405, but not hla-b*4402, constitutively presented a hla-b44 restricted alloantigen to reporter t cells, suggesting that us3 binds hla-b*4405 without interfering with peptide loading. us3 has been reported by others to bind the plc but surprisingly we have not detected such us3-plc complexes in our system. rather, in the presence of us3 we identified a pool of class i molecules distinct from the plc and only present in us3 expressing cells, implying that us3 may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa 1 , b. seliger 1 1 martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il12p70. since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for 24h with the gold standard of maturation (tnfa, il1b, il6 and pge2) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase-3 (lap3) had a more than 20-fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap1 and erap2 and of the immunoproteasome subunits lmp2 and lmp10 was enhanced between 2 and 5-fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr4 ligand mpla in comparison to the tlr-3 and 7/8 ligand polyi:c and r848. these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd4 + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il-4 induced surface expression of mature mhc class ii molecules and cd86. when ifn-g/il-4 primed pcmc were used as antigen presenting cells for cd4 + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd69 up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b15-23 to specific cd8+ t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd8 + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th1 immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd8+ t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd8+ tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd8+ t cell clones, faure, 2009, eur j immunol 39 (2): 380-90). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd8+ t cells after their in vitro differentiation into dc, 6 days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv-1 genome contains arfs within gag, pol and env genes encoding for a panel of hla-b*07 restricted epitopes. qprsdthvf (q9vf/5d) is one such epitope but its parental epitope qprsnthvf (q9vf/5n) has a significant higher frequency among hiv-1 isolates. strikingly, q9vf/5d-or q9vf/5n-specific ctls recognize apcs infected with hiv strains encoding for q9vf/5d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad8 ) encoding for q9vf/5n do not activate ctl responses raising the possibility that q9vf/5n epitope is not presented by infected cells. we asked whether introducing mutations within q9vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q9vf/5n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q9vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q9vf mhc-i presentation. we performed in vitro proteasomal digestions of 28mer peptides encompassing q9vf/5d or q9vf/5n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q9vf/5n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q9vf/5n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q9vf/5d or q9vf/5n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q9vf/5d peptide. we cloned and sequenced hiv-1 genomes from the three donors. surprisingly, out of 20 hiv proviral genomes isolated from pbmcs of q9vf/5d reactive donors, we could not find any virus bearing the q9vf/5d sequence. the isolated hiv sequences either encoded for q9vf/5n or had a stop codon within the epitope. in contrast, viruses encoding for q9vf/5d were isolated from pbmcs of the q9vf/ 5d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv-1 seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch 1 , y. wang 1 , d.c. tscharke 1 1 the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd8 + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd8 + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd8 + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h-2 bxd f 1 mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f 1 progeny, with some peptides being almost equally immunogenic in f 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in f 1 mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f 1 mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f 1 mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd8 + t cells with a restricted vb usage in f 1 mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr2 agonist, s-[2,3-bispalmitoyiloxy-(2r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp-2), as an adjuvant for cross-priming against cellular and soluble antigens. malp-2 has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd8 + and cd8 -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr2/6 results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap1) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to 90 % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, 50 and 10 % as compared to control rma cells, were injected s. c. in the flank of c57bl/6 syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to 45 days after injection. all mice injected with rma clone with a 50 % level of eraap expression developed a tumor and died within 23 days after injection. surprisingly, any animal injected with rma clone with a 10 % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd4 + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd4+ t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor (207 bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd20 therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor (2.5 bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg4 levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb1*0701-restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with 20-mer synthetic fviii peptides to stain epitope-specific cd4+ cells.cd4+ t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a2 domain: fviii 405-424 , fviii 421-440 and fviii 653-672 , as well as the c1 domain peptide fviii 2093-2112 , but not any c2 domain peptides. the c1 domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost.85:123-33, 2005 ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni 1 , s. albini 1 , m.z. limongi 1 , l. cifaldi 1 , d. fruci 1 1 ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p50 nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap1 and erap2, we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap1, erap2 and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap1 and erap2. this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p65 nf-kb subunit, we demonstrated that nf-kb is involved in erap1 and erap2 expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap1 and erap2 and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap1, erap2 and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch 1 , s. tenzer 1 , h. schild 1 , e. von stebut-borschitz 1 1 uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c57bl/6 mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th1 immunity, whereas ifng secretion of both cd4 + th1 and cd8 + tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il-12-dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd8 + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd8 + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm7 (lmp7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c57bl/6 mice. in addition, ex vivo co-cultures with infected dc from either lmp7 -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc1 cell ifng secretion and the dc restimulatory capacity of cd8 + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd8 + t cells, isolated from low dose infected c57bl/6 wildtype or lmp7 -/mice, were not detected. in summary, our data indicate that despite the fact that cd8 responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd8 + t cells against l. major is independent of the lmp7 subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a20, generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd4 + t cell epitope (84-103) from the g1 domain of aggrecan 10,000 times more efficiently than non-specific b cells and over 100 times more efficiently than the macrophage line j774. however, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by j774. treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b27 is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b*2705, peptide loading is relatively independent of this chaperone. we analyzed the effect of b27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b27 subtypes. the association of b27 heavy chain with tapasin was analyzed in c1r cells transfected with hla-b27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta-1, which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me1 monoclonal antibody that recognizes b27properly folded b27/peptide complexes. the formation of fully assembled b27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc10, which recognizes mhc class i free heavy chains (hc), or with me1. maturation was analyzed by pulse-chase analysis, immunoprecipitation with me1 and treatment with endoglycosidase h (endo h). hla-b27 polymorphic positions other than 116, both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b27 subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd4 + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd8 + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c57bl/6 mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately 15 % of vacv global presentation in the context of h-2 b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd8+ and cd8-dc, which differ in their mhci antigen presentation capacities. cd8+ dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd8-dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd8+ and cd8-dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd8+ and cd8-dc was determined. surprisingly, cd8+ dc exhibit rapid surface mhci turnover compared to cd8-dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd8+ and cd8-dc were stabilized and no longer underwent rapid turnover. this suggests that cd8+ and cd8-dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd8+ and cd8-dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h-2kb loaded with the ova-derived peptide, siinfekl. cd8+ and cd8-dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd8+ dc, but not the cd8-dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd8+, and not cd8-dc. this data further validates the role for cd8+ dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp-1 cytosol using gelatinase b/mmp-9 as a model enzyme. in the first dimension, ion exchange chromatography separated the thp-1 proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp-9. in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp-1 cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp-9-cleaved or intact thp-1 cytosol. proliferation was assessed by measuring incorporated 3 hthymidine. results: 100-200 mmp-9 candidate substrates were isolated, of which 69 were identified, revealing many novel mmp-9 (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp-9 decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e3-19k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue 56 in hla-a11 and residue 93 in e3-19k are highly critical for association of both proteins. this defines a putative interaction surface between e3-19k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e3-19k and the class i antigen presentation pathway. moreover, because soluble e3-19k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap1 and tap2. tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap1 and tap2 were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap1 on tap2 was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap1 mutants suggested that the lack of tap1 protein expression is associated with a strong reduction of tap2 protein levels, which could be restored by tap1 gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap1 on tap2 different shrna plasmids specifically targeting tap1 or tap2, respectively, were stably transfected into constitutively tap1 and tap2 expressing hacat keratinocytes and colo794 melanoma cells. in both cell types the shrna-mediated tap1 and tap2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap1 caused not only an almost complete loss of tap1, but also a strong decrease of tap2 protein expression. in contrast, the tap2 knock down exhibited no influence on the tap1 expression. specific inhibition of the proteasome prevented the degradation of tap2 in the tap1 knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap1 on tap2 protein expression is not restricted to rare tap1 mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of uv. blister roofs were then collected before and 1, 4 and 10 days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd14 + cd36 + and of a macrophagic cd14 -cd36 + cell subsets that emerge 1 and 4 days post exposition, respectively. more importantly whereas classical cd1a hi cd207 + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd1a low cd207and cd1a low cd207 + that emerged 1 and 4 days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd86 and hladr. finally, 10 days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey 1 , e. reeves 1 , t. elliott 1 , e. james 1 1 university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd4+ cd25+ regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct26, stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct26-derived cross-protective antigen, gsw11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. this protein has previously been shown to encode ct26-specific cd8 and cd4 antigens, implicating it as a 'hot-spot' for ct26 tumour antigens. interestingly, we have identified a truncated version of gp70 which may be responsible for generation of gsw11. expression studies have revealed increased gp70 expression in ct26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw11) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p4, p6 and p9 tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp2 molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp85-99, bound to hla-dp2 with high affinity (10-30 nm). binding studies of mbp85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f91a, f92a and k93a) were affected, and only by a 20-50 fold reduction in affinity for hla-dp2. the observation that none of the substitutions resulted in a complete loss of binding to hla-dp2 indicates that 1) hla-dp2 binding to peptides does not depend on a large hydrophobic residue accomodated in p1, or 2) mbp85-99 can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp2 molecule binds the immunodominant epitope in ms, mbp85-99, possibly in more than one register. additional studies are required to resolve the hla-dp2 peptide binding properties, and to determine whether expression of hla-dp2 affects the disease course in ms patients. results: depletion of mncs for cd14+ cells abrogated the tg-induced cytokine production and proliferation of cd4+ t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g 98 % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd4+ t cell proliferation was significantly reduced in cd14/cd19-depleted mnc cultures, as compared to cultures depleted for cd14+ monocytes alone. the same applied to the production of il-2, il-6 and tnf-a. production of ifn-g and il-10 was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa 1,2 , p. pala 1 , g. miiro 3 , j. todd 3 , p. kaleebu 1,2 , n. imami 2 , f. gotch 2,3 1 mrc uganda, basic science, entebbe, uganda, 2 imperial college, london, united kingdom, 3 mrc uganda, entebbe, uganda background: hiv-1-specific t-cell responses are preserved in hiv-1 infected individuals with non-progressing hiv-1 disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv-1 for g 8 years, maintaining cd4+ t-cell counts g 500, and with minimal cd4+ decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: 17 art naï ve hiv-1 infected patients from the entebbe cohort in uganda were recruited and stratified by cd4+ t-cell count, cd4+ decline slopes, and time of enrolment, into 2 groups -10 ltnp and 7 rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd4 t-cell count, and ifn-g, il-2 and il-4 elispot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). peptides were based on consensus sequences of gag and nef from hiv-1 clades a1, a2 and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il-4 responses were significantly higher in the ltnp than in rp (p=0.02, ifn-g responses to gaga2 pool 1; p=0.04, il-4 responses to gaga1 pool 2). il-2 responses were low and not significantly different between ltnp and rp. there was a positive correlation between il-4 responses to gaga1 pool 2 and cd4 t-cell counts (r 2 =0.502, p=0.04), but no correlation between either il-4 or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv-1 specific responses were higher in ltnps than in rps confirming previous results. non-specific il-4 responses were high possibly reflecting baseline th2 responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her2/neuand her2/neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using 7-aad for dna analysis and specific antibodies directed against the proliferation marker ki-67, the m-phase specific phistone h3 as well as for the h-2l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her2/neuversus her2/neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g 0 /g 1 -, s-and g 2 /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g 0 /g 1 -phase, which continuously raised and peaked within the s-phase. however, h-2l d surface antigen expression was not altered in her2/neucells during cell cycle progression. in contrast to her2/neufibroblasts the her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h-2l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd207) and ccr6 were cultured from cd34+ stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd34+ derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il-4-dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mø). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to 45 min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mø than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mø have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, 1999) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, 1992, 1999) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x 0,0001), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, 2252 patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for 2207 of them. for 538 of these patients hla-drb1 data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p=0.02). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb-1a preparations, whereas no differences were shown for ifnb-1b. an association between hla-drb1*11 and nab-negativity was detected (p=0.028). the known associations between hla-drb1*15 and csf-positive ms and hla-drb1*04 and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb-1a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb-1b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart-1 and gp100 more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni 1 , s. lorenzi 1 , f. mattei 1 , f. spadaro 1 , l. gabriele 1 1 istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd8 t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd8a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd8 t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd8 t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg7 thymoma line, we show that type i ifn promote the ability of cd8a + dc to capture apoptotic eg7 cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd8a + dc and the persistence of apoptotic eg7-cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg7-derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd8a + dc. as a result, eg7-loaded dc become competent at inducing ot-i cd8 t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd8a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. (2). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd8+ dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam3csk4, and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd8+ dc but not cd8-dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm1, which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a549 human lung cancer cell line. methods: dm1 induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a549 cells. we examined the morphological change using optical microscope. and gfp-lc3 punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm1 was showed high cytotoxicity on various cancer cell line, especially a549 cells. dm1 treated a549 cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc3 and beclin-1 protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc3 was increased concentration and time-dependently. beclin-1 also was increased. and then, we examined gfp-lc3 punctation on a549/ gfp-lc3 stable cells using confocal. a549 cells were formed gfp punctuation after dm 1 treatment. to confirm these data, we measured cell death ratio using 3-methyladenine (3-ma), an autophagocytosis inhibitor. after 3-ma treatment, dm1 induced cell death was decreased comparing with dm1 treatment. conclusion: dm1 did not induce apoptotic cell death. but dm1 showed several characteristics of autophagic cell death. taken together, dm1 induced autophagocytosis on a549 cells. these finding indicated that therapeutic potential of dm1 by triggering autophagic cell death. s. b. rasmussen 1 , s. r. paludan 1 1 university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr9 recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv-1 infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv-1, we found that tlr9 dependent ifn regulatory factor 3 activation was abrogated. in addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr9 dependent recognition. in the ongoing project we are examining how hsv-1 triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko 1 , i. berberich 1 , gk 520 -immunomodulation 1 universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a1 and its human homologue bfl-1 are anti-apoptotic members of the bcl-2 family. a1 is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a1. unless the c-terminal ends of other bcl-2 proteins the tail of a1 does not function as a strong membrane anchor. nevertheless, the last amino acids of a1 are important for the protein. in fact, the c-terminus of a1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a1 undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl-2 factor bimel results in increased stability of a1. this is due to reduced ubiquitination of a1 after binding of bimel. we conclude that the cterminus of a1/bfl-1 serves as a docking site for e3 ubiquitin ligase(s) that control the stability of a1 by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e3 ubiquitin ligase that interacted with a1 in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs-1 in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs-1 is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs-1 significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs-1 was manifest not only by the suppression of jaks acting upstream of p38 mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak 1/2 activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs1 which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs-1 overexpressing cells. the results strongly suggest that socs-1 acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl12/sdf-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl12 effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u-87, and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p53 and fas-l expression was carried out by western-blot. results: cxcl12 induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p53 pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p53-ser46 levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after 24h of tnf-a treatment. conclusions: cxcl12 induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p53function/levels. s. y. demiroglu 1 , r. dressel 1 1 universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp70 does not protect against cell death mediated by ctl. acute overexpression of hsp70 can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res 63: 8212) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp70 on granzyme b-induced apoptosis. methods: hsp70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp70 induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. results: hsp70 did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp70 in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g1 peak measurements (p=0.01). in contrast, a partial protection of ge-tet cells was observed after acute hsp70 overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp70 does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood 102:1788) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk1034 and dr394/2-3. the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr1 and tnfr2, both belonging to the tnf receptor superfamily. tnfr1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr2-associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr2, resulting in depletion of traf2 from the cytoplasm. here we confirm that tnfr2 induced depletion of cytosolic traf2 by the use of tnfr1-and tnfr2-specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf2 in the alternative nfkb pathway, we show that tnfr2 induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr2 and membrane tnf. j. c. morales 1 , d. ruiz-magaña 1 , d. carranza 1 , c. ruiz-ruiz 1 1 universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase-8. in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r2 and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp36l1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp36l1 may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl-2 protein is an important cell survival protein at different stages of b cell development. bcl-2 mrna also contains are regions that could possibly be targeted by zfp36l1 protein. in the present study, we have tested the ability of zfp36l1 protein to bind to a bcl-2 mrna are probe and to degrade bcl-2 mrna. recombinant bacterially expressed zfp36l1 protein was shown to bind specifically to a bcl-2 are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp36l1 also provided evidence that endogenous zfp36l1 in b cells could bind to the bcl-2 are. in order to examine whether zfp36l1 binding to bcl-2 are resulted in bcl-2 mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp36l1 knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl-2 mrna was expressed in both wild-type and knockout mefs. the half-life of bcl-2 mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp36l1 does play a role in degradation of bcl-2 mrna. overall, our data are consistent with a role for zfp36l1 in degradation of bcl-2 mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp1. since lmp1 is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp1 function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi3-kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te1) and lymphocytic (nc5) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes1 or tes2 (transforming effectors site) derived from the c-terminal intracellular part of lmp1, in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp1's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase-8. using this nc5 tumorigenic model in scid mice, we showed that induction of the dn lmp1-tes2 prevent development of tumours and mouse death. these dominant negative derived from lmp1 could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs1 and socs3 differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38mapk activities, while socs3 promoted apoptosis with an increase in p38 activities. in contrast, both socs1 and socs3 display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs1 or socs3 induced a slight decrease in g1 or s phase cells and a prominent increase in g2/m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g1/s transition and g2/m arrest induced by socs1 or socs3. socs1 promoted dna damage-induced p21 induction and g2/m cyclin b expression, while socs3 induced decrease in g1 cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein 90 (hsp90) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp90 has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp90 can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp90 is released during necrotic cell death and proinflammatory effects of extracellular hsp90 have been observed. thus, we asked whether apoptozing cells cleave hsp90 during apoptosis or how hsp90 is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp90 protein content. we observed that hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp90 (hsp90 alpha and hsp90 beta) we could show that the 55 kda fragment is formed after degradation of the alpha isoform of hsp90. further, we were able to show, that hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp90 and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp90 was inhibited or if exogenous hsp90 was added. these results demonstrate that cleavage of hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc3-i is processed to lc3-ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf-7 gfp-lc3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl-2/beclin-1 control of autophagy. recently, jnk was shown to be necessary for beclin-1 upregulation, and jnk-mediated phosphorylation of bcl-2 is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf-7 cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol 3-angelate (pep005), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep005 provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb4 turned their response to pep005 from an increased to decreased rate of apoptosis. furthermore, our data show that pep005 inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl-1 and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p75, trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch11 and analysed by flow cytometry. expression of the caspase 8 inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch11 treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor 1 (tnf-r1), and tumor necrosis factor (tnf)-a receptor 2 (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd95 independent of its ligand fasl/cd95l. fas/cd95 is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl2-family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo 201, sw1116, lovo, caco-2, ht-29) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki-67, pcna, p53, bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl-2, bax, caspase 3, 6, 9, nfkb, parp1/89kda, tnfr1/cd120a and cd95/fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl-2, bax, nfkb, parp1 was significantly lower and expression of caspases, tnfr1 as well as percentage of cd95 cells significantly higher as compared with control group. caspase 3 expression was significantly higher as compared with nfkb, parp1 and tnfr1. in comparison to the standard nutrition preoperative immunonutrition increased bcl-2 and nfkb expressions and decreased caspases and parp1 expressions. in addition, we found a significant down-regulation of bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x 0.01). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x 0.007). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either la1 (10 9 cfu/day) or placebo, during 57 days prior to uv (2 × 1.5 med). blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p53). while a similar decrease of lc for both groups was observed on day 1 after uv exposure compared to placebo, la-1 group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd1a low cd207 -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il-10 and a tendency to inhibit il-6 was observed in la-1 group compared to placebo. on day 4, la-1 group presented a greater recruitment of early lc precursors and a trend to increase cd1a low cd207 + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il-6, tnfa, il-8, and il-10) was observed in la1 group compared to placebo. we show that la1 limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la1 supplementation for photoprotection at a lower dose (1 log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around e11.5. previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn1 in a nude (foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn1. if true, it should be possible to target this cell type by use of foxn1-promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either 19 or 82 glutamine residues under the control of foxn1 promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei 1,2 , y. toriumi 3 , k. kimura 4 1 european university viadrina, frankfurt (oder), germany, 2 shimane institute of health science, izumo, japan, 3 shimane university faculty of medicine, department of pediatrics, izumo, japan, 4 japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp2). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r=0.83, p x 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r=0.77, p x 0.05) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd4 (r=0.96, p=0.0001). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd4, within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd4 + cd25 + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox40l expressed on mcs and the constitutive expression of ox40 (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox40l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca 2+ responses and degranulation. the cross-talk between t reg cells and mcs through ox40-ox40l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th1 or th2 effector cells and the induction of antigen specific foxp3+ regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th2 cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th2 and treg subset, gata3 and foxp3 respectively, counter regulate each other (mantel y et al., 2008) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp3+ tregs and high gata3+ th2 cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost 100 million allergic patients are sensitized to the major birch pollen allergen bet v 1, which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v 1 recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v 1 molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g 90 %) of patients' ige binding to bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (p2) and aa 74-104 (p6) of bet v 1. cross-reactive ige epitopes between bet v 1 and related pollen allergens and plant food allergens involved primarily p2. p2 and p6 are not adjacent peptides in the bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of bet v 1. as determined by surface plasmon resonance, monoclonal antibody mab2 specific for p2 and mab12 specific for p6 showed high affinity, i. e., dissociation constants, k(d) = 8.35e-11 m and k(d) = 1.05e-9 m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p2 and anti-p6 antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v 1 allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th1 cells as well as tc1 cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th2 immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th1/tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd4 + and cd8 + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd4 (gk1.5) or anti-cd8 (53.6.72) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr-1 monoclonal antibody rb6-8c5 or monoclonal antibody nimp-r14. one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd8 + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd4 + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd8 + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd8 + t cells, cd4 + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd4 + or cd8 + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th1 and tc1 cells operative in the elicitation of airway inflammation. conclusions: robust type 1 immune responses, although highly effective in the counter-regulation of local th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h292. the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p 1, we were able to show that the major allergen of phleum pratense, phl p 1, although sharing molecular similarities with der p 1, does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p 1. chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il-6 and il-8 from nci-h292 cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p 1. in contrast to hdm extract grass pollen allergens induce the release of mcp-1 from respiratory epithelial cells, as well as moderate levels of il-12. detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr22) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, 3 distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): 7.2±5.7) and of il-13 (mean±sd: 1157±909 pg/ml), and reduced il-10 (mean±sd of il-10-spot forming units/2x10 5 cells (sfu): 912±510), compared to healthy subjects (3.4±2.7 si, p x 0.05; 355±396 pg/ml, p x 0.05; 1272±623 sfu, for proliferation, il-13 and il-10, respectively); children with non-ige mediated cma had a significant reduction of il-13 (192±362 pg/ml), compared to ige patients (p x 0.0004) and an increased, although not statistically significant, production of ifn-g (33.7±54.6 sfu) compared to control (9.5±9.5 sfu) and to ige-allergic patients (0.6±0.8 sfu). finally, tolerant patients showed reduced il-13 (636±1048 pg/ ml, p x 0.05) and proliferation (3.9±3.5 si), compared to acute ige-cma children. interestingly, the high level of il-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il-4 was undetectable in all patients even blocking the il4-receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th2-like response with il-13 and proliferation is dominant in ige-mediated cma patients; by contrast a th1-skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang 1 , b. chua 1 , f.c. lew 1 , a. ho 1 , k. wong 1 , k.l. wong 1 , d. m. kemeny 1 1 national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd4 th2 t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd8 t cells inhibits the induction of the th2 response. here we have investigated the effect of cd8 t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd8 t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of 3 airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd8 + t cells (36.7 %±4.1 % to 17.6 %±2.7 %). when ifn-gamma -/-ot-i cd8 t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for cd8 t cell ifn-gamma. cd11c + cd103 + cd11blung dcs from cd8 transferred mice secreted higher levels (500pg/ml) of il-12p70 following ex-vivo stimulation as compared with animals that were not given cd8 t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd8 t cells can subsequently divert the local lung environment to one that favors th1 immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in 8 % of patients with hiv infection. this reaction is strongly associated with hla-b*5701 and appears to be mediated by cd8+ t cells producing inflammatory cytokines. we show that cd8+t cell responses can be primed in vitro, in normal blood donors who are hla-b*5701+, but not in non-b*5701+ donors. cd8 t cells, but not cd4 t cells, are expanded by abacavir pulsed autologous apc over a13-day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b*5701. similar responses were detected in abacavirhypersensitive hlab*5701+ patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b*5701 since they were undetectable using apc expressing closely related hla-b57 or b58 allotypes. responses to apc expressing mutants of hla-b*5701 demonstrated a crucial role for residue 116. isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b*5701 triggering cd8 t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b*5701 may be relevant to the role of other disease-associated class i allotypes such as hla-b27 and b51. a. jenckel 1 , s. bulfone-paus 1 , n. föger 1 1 research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin1a (coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro1a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro1a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro1a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro1a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro1a during mast cell activation. cell fractionation experiments demonstrated that the association of coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro1a phosphorylation. a functional correlation between coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro1a. thus, coro1a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin1a (coro1a) deficient mice have demonstrated that coro1a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin1b (coro1b) is a closely related homolog of coro1a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro1b deficient mice and crossed them with coro1a deficient mice to obtain coro1a/coro1b double deficient mice. analysis of t lymphocytes from coro1a/coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro1a/coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro1a/1b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro1a/1b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn1 (also known as hacs1 or sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology 3 (sh3) domain and a predicted bipartite nuclear localization signal. the samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn1 in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn1 in all tested hematopoietic cell types. the highest expression level of samsn1 mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd4+ and cd8+ t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly1 (hacs2) and sly3 (sash1) -were expressed only at a very low level in mast cells. the high level of samsn1 mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn1 deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn1 deficient mast cells. in additional studies we are now investigating the effects of samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., 2008). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg1 abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st8siai-v; st6galnac i-iv, st3gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg1 abs. results: we observed that the expression of st3gal i, iii and v coding genes was similar in both hybridomas, while the st3gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg1. in addiction, the expression levels of st8sia and st6galnac genes in the hybridoma producer of anaphylactic igg1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg1. conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg1 abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h 2 cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after 2 times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il-5 and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf1 mediates the differentiation into th1 cells by activating multiple genes which are independently crucial for the development of naive t cells into th1 cells. because irf1 is expressed in many different tissues, it can be considered as a master switch factor for th1 cell differentiation. methods: here, we tested mice deficient in irf1 in the murine acute asthma model to evaluate its importance in this th2 cell-mediated disease. the protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype cd8+ t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf1 deficient mice with the help of adoptively transferred cd8+ t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by 3.5 fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. 1) were also significantly higher in irf1 deficient mice. conclusion: interferon regulatory factor 1 plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd8+ t cells. mechanistically, a potential counterregulation of asthma by th1 cells is not available in irf1 knockout mice. together with our previous report that irf1 represents a susceptibility gene for allergy in the human, our data highlight irf1 as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c57bl/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for 24hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk1-receptor and ppt1 mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd11c expressing apc substets characterized by cd4 and cd8 expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th-1 cytokine profile and increased levels of il-2 mrna. further the number of cd25 + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: 1) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase3, 2) asthma and ee are prototypic th2 diseases in which the cytokines il-4 and il-13 play a principal role through the activation of the transcription factor stat6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate stat6 by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat6, we analyzed its effect in the activation of stat6 by il-4 and il-13. we found that treatment of cells with ppis inhibited the ability of il-4 and il-13 to signal stat6 activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th2 mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied 12 asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd4 and cd25 high (treg)), that might inhibit the development of a th2 response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for 48 hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd69 or cd25 at 48 hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h4r. the level of intracellular ccl2 production in human lc was reduced after stimulation with h4r agonists and basal production could be restored when h4r was blocked with the specific antagonist jnj7777120. moreover histamine and a h4r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h4r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h4r in allergic skin diseases and encourage further exploration of the h4r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g1p3, an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g1p3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g1p3 which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd20 antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th2 to chronic th1-predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g 5,000; g 6,000; g 19,000 mg/dl, respectively). all patients underwent prior treatment with omalizumab for 6 months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up 1 year), ige levels decreased from 5,512 to 1,500 mg/dl. the other two patients are in the 3 and 1-months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd20 antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) 1 and th2 cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either 1 %dncb, 25 %tma or to vehicle alone for 0.5-6h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il-2 (mean=131pg/ml, n=12, p x 0.001), il-17 (mean=69pg/ml, n=12, p x 0.001) and il-1a (683pg/ml, n=12, p x 0.001) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant il-2 or of the known regulator of lc migration; il-1b. interleukin-1b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p x 0.001). in contrast, il-2 or control injections were without effect. however, il-2 administration caused an increase in cutaneous il-17 production (347pg/ml, n=12, p x 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). in addition, systemic treatment with anti-il-2 antibody failed to impact on lc migration provoked by dncb (33 % reduction; n=6, p x 0.001). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il-1a (987pg/ml, n=10, p x 0.001) and il-17 (248pg/ml, n=6, p x 0.01) expression was reduced to baseline levels by anti-il-2 treatment. these data demonstrate that il-2 is not involved in the regulation of lc migration, unlike il-1b and tnf-a. however, il-2 is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il-2 may influence lc and dermal dc maturation, via the expression of il-1a and il-17. allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey 2005), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p38-mapk-pathway related protein (p38prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p38 mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct-2005 -018681, www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, 15 % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il-1 receptor antagonist (il-1ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il-1b and of il-1-like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il-1-related genes by real-time pcr on mrna from blood cd14+ monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il-18, il-18bp, and caspase-1, both before and after therapy with kineret. il-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il-1b, tnfa, il-12) nor anti-inflammatory cytokines (il-10, tgfb) were detected. as expected, il-1ra was only detectable after therapy. il-18 was detectable in schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pm) and decreased after therapy (13.2 pm). the circulating il-18 inhibitor il-18bp was lower than in controls and not affected by therapy. thus, free il-18 levels were increased in schnitzler's patients as compared to controls (17.6 pm vs. 7.2 pm in controls) and decreased after therapy (9.9 pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il-1-related cytokines (il-1b, il-18), nor of the il-1/il-18-converting enzyme caspase-1 in blood monocytes. however, the high circulating levels of il-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around 20 % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr3 signal via myd88 to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd88 signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c57bl/6 myd88-deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg1-titers in ova-treated myd88-/-mice compared to wildtype mice, although the nacl-treated myd88-/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = 4) or from non-infested contra-lateral sites. expression of mip-1a, igf-1, mcp-1 and ip-10 genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd3, cd4, cd8, cd21, mhc class ii, and p46 than that of holsteins. lymphocytes expressing wc1 and cd21 antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x 0.05). infested skin of nelores contained significantly more message for mip-1a, igf-1, mcp-1 and ip-10 than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th2 response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd3+ t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il-1 plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa-1 in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam-1/lfa-1 binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th1 than a th2 local response pattern by virtue of il-1 secretion and icam-1/lfa-1 interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th2 like micromilieu in acute towards a th1 dominated milieu in chronic lesions. the genotyping of ccl4l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi07/0329). psoriasis is an inflammatory dermatosis with 2 % prevalence among caucasians. hla-cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in -308 and -238 positions. strong association was found between polymorphisms in the -238 region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b27, and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. hla-a -b -c -dr -dq alleles and tnf-238 and -308 snp were differentiated by pcr/ssp. analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. in the male group, the mean age at disease onset was 16,3 years. severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw*06 allele was positive in 10 cases (47,62 %), tnf 238 g/a genotype was found in 5 (23,8 %) and tnf 308 g/a in 4 (19,1 %). in the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. cw*06 allele was positive in 26 cases (54,2 %), tnf 238 g/a genotype was presented in 16 (33,3%) and tnf 308 g/a in 8 (16,6 %). severe disease was seen in male patients. there was no difference in frequency of cw*06 allele between male and female groups, but there was a tendency of significant difference in tnf 238 g/a genotype. we found that c57bl/6 mice were more susceptible than balb/c and dba/2 mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il-6 and mrp8/14, among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that 1)the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. 2) in particular, in many cases it is proved that there is a personal but also a family history of atopy. 3) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd86 we could observe dose dependent upregulation of programmed death ligand 1 (pdl-1) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a 60 year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since 35 years. in march 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters (120 mg) every second day. in april 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the auricle. the histological examination showed an hhv-8 positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th1 type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs1, socs3 and foxp3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for socs1, socs3 ve foxp3 mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd3, cd4, cd25, foxp3, cd4 + cd25 high , cd4 + foxp3 + . expression of socs3 and foxp-3 mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs1 was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd25, foxp3, cd4+cd25 high , cd4 + foxp3 + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs1, socs3 and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs1 and socs3 molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing fcs (exposed), and 43 healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than 0.05 was considered significant.neopterin level was significantly elevated in exposed (3,2±0,8ng/ml) compared to controls (1,8±1,3ng/ml; p=0,0001). moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls (81,9±22,7au vs 64,3±16,7au p=0,0001) and to silicosis patients (69,6±20,0au p=0,002). in contrast, igmcic was significantly elevated in silicosis than in exposed (70,2±18,8au vs 62,1±24,2au; p=0,03).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p=0,001 and 0,02 respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a*01, b*08, drb1*03 (these of good prognosis) *12/*13/*14/*15) or tbc (drb1*02/*05/*14/*16 and associations with dqa1*02/*03/*05) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a*01, b*08 and drb1*03 (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a*01, drb1*14. as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd4 + , cd25 ++ , foxp3 + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein 60 (hhsp60). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd4 + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il-17, il-6, il-8, tgf-b and ifn-g production prevailed, pointing to a th17/th1 weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp60. importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th17/th1 cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp60 might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices (1) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a 384 well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c3-complement, myeloid related protein 14 and two new proteins, integrin-ß4 and fibrinogen. data from more than 100 patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least 3 mice per group with distinct cytokine pattern: th1 (c57bl/10, c57bl/6) and th2 (balb/c). muscular injury was performed by injection of bupivacaine. both th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd3 bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at 4 dpi. balb/c mice showed increased collagen expression and decrease of mmp-9 activity associated with more mrna for tgf-b1. this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th1 cytokines) or myonecrosis and collagen deposition (th2 cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß1-adrenergic receptor (anti-ß1ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß1ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß1ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß1ec ii -specific rat monoclonal antibody (clone 13f6), and showed by elisa that it binds to the linearized ß1ec ii peptide. additionally, we confirmed with flow cytometry that 13f6 also binds the ß1ec ii in its native conformation, i. e. directly labeled circular ß1ec ii (dyl649-ß1ec ii ) peptide. moreover, we demonstrated activation of the ß1-adrenoreceptor by 13f6 using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß1ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß1ec ii -specific peptide-based therapy, by intravenously applying a circular ß1ec ii peptide in the dcm lewis rat model to neutralize the anti-ß1ec ii antibodies. while the peptide therapy strongly reduced the anti-ß1ec ii titers in the serum by up to 80 % and consecutively lead to clinical remission, elispot assays for the detection of ß1ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß1ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß1ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b3 or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca 614-629 epitope elicits autoreactive cd4 + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca 614-629 peptide. in a first step, hybridoma cells were generated by fusing bw5147 tcra -cd8lymphoma cells with myhca 614-629 -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle 1 , a. schwarting 1 , p.r. galle 1 1 university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase 3 (pr3), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr3-positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il-8. pr3-mrna from the murine cancer cell line wehi-274 was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr3-promoter in the second intron of the mouse pr3 gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr3 transcript and its promoter indicates that the murine pr3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th1 mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd1d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th2 cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n=13) or pbs (n=11) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr4 activation, in the presence/ absence of och. subsequently we transferred 1.5x10 6 mdcs (n=11) or och-primed mdcs (n=11) (3 times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a 70.6 % reduction in plaque size compared to mice treated with mdcs (p x 0.05). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p x 0.05) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with och-primed dcs (p x 0.05). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il-10. discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th2 phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c57bl/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c57bl/6 and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr3 regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg2c were isolated only from fcgriib deficient mice, but not from c57bl/6 control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box 1 protein (hmgb1), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb1. we investigated if hmgb1-containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb1 remains associated with ncs released from late apoptotic cells in vitro. hmgb1-ncs complexes were detected also in the blood of patients with sle. hmgb1 containing ncs from apoptotic cells induced secretion of il-b, il-6, il-10, and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd88 and toll-like receptor 2. neither hmgb1-free ncs from living cells nor from apoptotic hmgb1-or hmgb1/2-deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb1 activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il-10 release by macrophages. immunizations with hmgb1-containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr2-dependent manner. in conclusion, hmgb1 in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher 1 , k.p. hoefig 1 , e. kremmer 1 , v. heissmeyer 1 stretches and helps in the selection of the correct splicing borders. a allele of (r61h) creates a strong binding site for a splicing enhancer protein srp40 according to bioinformatics. our findings indicate that, the putative branch point, r61h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank1. finally, we believe that bank1 delta 2 protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta 2 protein may contribute to the observed reduction in sle susceptibility. s. beermann 1 , r. seifert 1 , d. neumann 1 1 hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h 1 receptor (h 1 r), h 2 r, h 3 r, and h 4 r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd3 antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or 4-methylhistamine, a h 4 r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and 4-methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c57bl/6 mice induced by either a-cd3 antibodies or cpg-odn. this histamine effect was completely inhibited by the h 2 r-specific antagonist famotidine, while h 4 r-, h 1 r-, and h 3 /h 4 r-selective antagonists had no or only moderate effects. interestingly, the h 4 r-selective reagent jnj7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd3 induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h 4 r, jnj7777120 may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h 2 r and, to a much lesser extend, the h 4 r. using this assay system, cells obtained from control c57bl/6 mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of 20 ng/ml resolvin e1(rve1) on the release of tgfb, il-6 and il-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (pma) (1nm), and the combination of pma and ionomycine (3 mg/ml) for 72 hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = 0.010) and sjögren's (p=0.017) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve1 significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p=0.041) or pma+ionomycin (p=0.021), however rve1 was ineffective at reducing tgfb release in the sle and ss patients. rve1 caused a non-significant decrease in il-6 release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il-17 was not significantly modified by rve1 in any of the groups tested. the release of tgfb by 20 ng/ml of rve1 can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve1 tested, il-6 and il-17 release were not significantly affected in healthy or autoimmune patients. omega-3 fatty acid derived rve1 may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis 1 , a.k. tsirogianni 1 , m. herrmann 2 , h. m. moutsopoulos 1 , m.n. manoussakis 1 1 university of athens, dpt pathophysiology, athens, greece, 2 university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included 29 with primary ss (american-european criteria 2002) and 14 with sle (acr criteria 1997). age-and sex-matched healthy blood donors to the ss and sle groups (13 donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd68-staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p=0.0001). in ss patients, defective mb-phagocytosis involved only monocytes (p x 0.0001) and significantly correlated with the presence of extraglandular manifestations (p=0.02). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x 0.0001) and of ss (p=0.001). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: 30 healthy persons have been included in our study and 71 sle patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b 2glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in 36,6 %, aphl of igg class -in 45,1 % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x 2 =6,4; p x 0,02). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x 2 =3,9; p x 0,05). the increased levels of anti-ada were revealed in 11 of 18 women with hnp, and the combination of anti-ada and aphl (9/18) was found out more often than isolated anti-ada (2/18, x 2 =6,5; p x 0,02) or isolated aphl (3/18, x 2 =4,5; p x 0,05). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc1 and mdc2, and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc1, mdc2 and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from 31 pss patients fulfilling the american european consensus group criteria (aecc) and 28 gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p=0,0002) and mdc2 (p x 0,0001) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd5+ b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin 21 (il-21) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd5+ b cells and increased il-21 levels. we therefore hypothesized that il-21 may have similar biological effects on cd5+ b cells in autoimmune diseases. here we demonstrate that the amount of il-21 in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to 14 % of cd5+ b cells in sle individuals expressed grb. in-vitro experiments revealed that il-21 was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor 9. importantly, il-21 significantly decreased the cd5+/cd5-b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd5+ b cell death. these results suggest that il-21-induced grb may play a regulatory role for cd5+ b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il-21 and grb levels in sle and showing that il-21 reduces the cd5+/ cd5-b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il-21 may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il-21 in sle and other autoimmune diseases. r. de palma 1 , e. d'aiuto 1 , s. vettori 1 , g. abbate 1 , g. valentini 1 1 second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd4+ t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il-1beta, il-6 and il-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed 169 igg antibodies from single facs purified cd19+cd27+cd38+cd138+ bone marrow plasma cells of 3 patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran 1 , e. moazemi godarzi 1 , e. kamali sarvestani 1 , e. aflaki 1 1 shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin 6 (il-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il-6 gene at positions -572 g/c and -174 g/c have been described that are key regulators of il-6 gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il-6 gene polymorphism at -174 position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the -572 position significantly differed in sle patients and controls. at this position gg genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p x 0.014). the frequency of -572 g allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x 0.04). at -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. in conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to sle and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained 161 patients with ibd (89 with cd, 41 with uc, 31 with gluten sensitive enteropathy, gse) and 33 healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of 7 asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd4-ve cd8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd4(+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling-1 (rgs-1) protein, we have now found substantial over-expression (10-100 fold) of rgs-1 in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs-1 decreases primary t cell responses to cxcl12 and ccl19, strongly implying that it may regulate t cell localisation. thus, rgs-1 may be a novel target for modulating t cells in ibd, consistent with which snps in rgs-1 have been associated with both coeliac disease and type 1 diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c57bl/6j mice were divided in control (c) and experimental (e) groups. c1-c3 received peanut protein immunization, animals of the control groups c4 were sham immunized, and control group c5 received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a 30 day peanut containing cd, the experimental group was divided into 5 groups (e1-e5). ova feeding began 7 days prior cd (e1) on day 0 (e2), 7 (e3), 14 (e4) and 21 (e5) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e1) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive il1-b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il1-b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (dss) in the drinking water for 8 days to wildtype mice. when ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl 1 , n. schneiderhan-marra 1 , m. blum 2 , g. stein 2 , m. schmolz 2 , t. joos 1 1 nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, 2 edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [1] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h 1-associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h 2-like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il-17 production and t h 17 polarization and decreases recruitment of cd4 + cd25 + foxp3 + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd11c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd11c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h 17 and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen 1,2 , g. van assche 3 , p. rutgeerts 3 , a. liston 1 , j. l. ceuppens 2 1 k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, 2 k. u. leuven, experimental immunology lab, leuven, belgium, 3 k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp-1 allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced 3 types of colitis dss (th1/th17), tnbs (th1) and oxazolone (th2) in hp ko mice. neutralizing anti-il-17 mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th17/th1 cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. 1) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; 2) in dss but not in oxa colitis mice, il-17, ifn-g, tgf-b and il-6 were significantly increased (p x 0.01, dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x 0.05, ko vs wt). in tnbs colitis, we found elevated il-12 and ifn-g (p x 0.01, tnbs vs control). although not significant, il-17 was also somewhat upregulated; 3) in dss colitis we observed that il-23 enhanced differentiated th17 cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il-6 and il-21, more mln-t cells from hpko mice differentiated into th17 cells; 4) anti-il-17 mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il-17 showed reduced il-6, il-17 and ifn-g in both mln and lp (p x 0.05, anti-il-17 vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il-17, supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann 1 , g. talabér 1 , g. süt" o 2 , p. németh 1 , t. berki 1 1 university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, 2 university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that 2.2 million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd4+cd25hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th1 and th2 cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd4+ and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd4+ inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells (74.5 ± 3.5 %, mean ± sem), while cd4+ inkt cells ratio was moderate (25.4 ± 3.5 %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: 38.0 ± 7.1 %; cd: 34.0 ± 5.2 %, mean ± sem), while the percentage of cd4+ inkt cells was elevated (uc: 62.0 ± 7.1 %; cd: 65.9 ± 5.2 %, mean ± sem) in both disease groups. proportions of foxp3+ treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b1 gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b1 adenovirus (tgf-b1-exo). the t cell inhibitory function of tgf-b1-exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b1-exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b1-exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd4 + foxp3 + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b1-exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b1-exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd4 + foxp3 + regulatory t cells (treg) revealed that the relative numbers of cd4 + foxp3 + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b1 gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd4 + foxp3 + treg. tgf-b1-exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd8+ t-cells was similar in the four vaccinees as observed by ifng, il-2 production-profiles. however, the killing capacity of expanded cd8+ t-cells was distinct as observed by the competence to kill ns3-peptide presenting transfectants in vitro. as depicted in figure 1 , cd8+ t-cells cells from both vac1 (cleared ) and vac2 (chronic) produced il-2 and ifng after stimulation with ns3-peptide59. however, specific killing of the peptide loaded transfectants was only observed in vac 1, who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure 1 ] killing of ns3 peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd8+ t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd8 t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns3 from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr4-expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns3 might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns3 or eda-ns3 under the control of cmv promoter were prepared. recombinant ns3 and the fusion protein eda-ns3 were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd40 agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns3 rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns3. results: immunization of mice with the plasmids expressing eda-ns3, but not ns3 alone, induced strong t cell responses against the main hla-a2 restricted cytotoxic t cell determinants from ns3. the recombinant eda-ns3 fusion protein, but not ns3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp-1 monocyte cell line. immunization of hhd mice with eda-ns3 fusion protein induced both cd4+ and cd8+ t cell responses against ns3 and, when immunized with poly(i:c) and anti-cd40 antibodies, was able to down-regulate the intrahepatic expression of hcv-ns3 rna. the recombinant eda-ns3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even 25 years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e2 protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd207+cd1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd8+ responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to 3 years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd1a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd8+ t cells by mature dcs transduced with melan-a-recombinant human coronavirus 229e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin 2 (il2) and interleukin 15 (il15) will be involved, and fms-like tyrosin kinase 3 ligand (flt3l) will be expressed to modulate dendritic cells. il2 is known to enhance early t cell expansion and limits t cell overshoot, whereaes il15 guarantees survival of high affinity t cells during memory phase. on the other hand flt3l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik 1 , ag waisman 1 uniklinik mainz, 1. med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd8 t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il-10 production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd8 t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response 7 days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il-10 produced by b cells by conditional deletion of the il-10 gene in these cells. we found that b cell secreted il-10 has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb490 to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for 10 days in the presence of il-2 and il-7 with exposure to overlapping peptide pools of latent ebna-1 and lytic bzlf-1 antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd3, cd4, cd8, ccr7, cd45ra, il-2, tnfa, ifng and cd107a. the majority of ex vivo ebv-reactive cd4+ t cells as well as ebna-1-reactive cd8+ t cells were il-2 and tnfa-producing memory cells, the later being more frequent in bone marrow (cd4+, median, ebna-1: bm 0.69 %;pb 0.12 %; bzlf-1: bm 0.37 %;pb 0.01 %, p=0.039). after in vitro expansion a major subset of ebv-specific cd4+ and cd8+t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd4+ and cd8+ t cells retained, however, a tnfa single, tnfa/il-2 or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd4+ and cd8+ t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr7/cd45ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il-2 delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd4 + and cd8 + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd154 or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd4 + and cd8 + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd8 + and cd4 + t cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to 29-90 % in the t cell lines and the absolute numbers of both hexon-specific cd4+ and cd8+ t cells were 2 to 3 log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd8 + and cd4 + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype 5 (ad5) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad5 vaccine vectors expressing whv core protein was first examinated in c57bl6 mice. groups of mice were immunized with a dna prime-ad5 boost regimen or with dna and ad5 alone. ad5 was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd8+ t cell responses against peptide pools 1 and 3 in spleens of dna and dna-ad5 immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd8+ t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad5 very weak cd8+ t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad5 level of antibodies did not change. those antibodies were only igg2a subclass what indicates th1 t helper type of response. ad5-immunized mice had over 3-fold lower level of anti-whc: both igg2a and igg1 subtypes were detected. the weak response induced by ad5 may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m2e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal 2007) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m2e serum ab titers against peptide and native m2e. this correlated with a large number of m2-specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h1n1), which contains a variant m2e-sequence different in 3 amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x31 (h3n2) and the highly pathogenic pr/8 (h1n1) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m2e as a potential "universal" influenza vaccine. this research is supported by a nih t32 fellowship ca09171-32, the nih grant ai 46457 and a grant from the commonwealth of pa. l. yu 1 1 zhejiang university, zhangzhou, china interleukin-18 (il-18) is a cytokine produced by stimulated mononuclear macrophage system. in this report, 18-day-old chicken embryos were vaccined with the plasmid dna (pci-chil-18) encoding chicken interleukin-18 and the copy numbers of chil-18 in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil-18 could accelerate high concentrations of chil-18 in nonage peripheral blood, accelerate high expression of chil-18 in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil-18 could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil-18 (p x 0.05). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd8 cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on 12 volunteers and a phase i study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd8 cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv-1 neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) 120 and the transmembrane protein gp41. two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (v3) is recognized by the human monoclonal antibody 447-52d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies 4e10 and 2f5. in order to elicit anti-hiv-1 neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp120-v3 region or the gp41-mper. the vlps are based on the acyltransferase component (e2 chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e2 chain self-assembles into a 24 nm protein scaffold resembling a vlp and that contains 60 copies of e2. efficient display and refolding of the v3 and mper regions in e2 vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for e2. the priming and boosting with a combination of vlps and specific hiv-1 envelope dna were used to immunize mice and rabbits. results: the v3-e2 and mper-e2 vlps were purified as stable 60mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of 447-52d, 4e10 and 2f5 antibodies to hiv-e2 monomers was confirmed by western blot. we obtained high titers of hiv-1 gp140-specific antibodies in mice immunized with a combination of vlps plus dna (hiv-1 sf162 gp160). these antibodies generated a low (20 %-27 %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e2 vlps plus dna elicited a low titer of hiv-1 gp140 specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e2 vlps are able to display antigenic determinants of hiv and to induce high titers of hiv-1-specific antibodies. the e2 vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h5n1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h5n1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h5n1 whole virus wild-type vaccine, could induce an immune response and protect mice against h5n1 influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h5n1 antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h5n1 virus. the protective efficacy of the trivalent vaccine derived mainly from the h1n1 component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h5n1 virus, suggesting that antibodies are the main contributor to protection. h1n1 specific serum did not inhibit neuraminidase activity of h5n1 virus suggesting that protection was not mediated by neuraminidase n1-specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h5n1 whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h5n1 vaccine enhanced anti-h5n1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h5n1 vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h5n1 candidate vaccine. [1] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of 335 children in the first grade of primary school in the area of central and west macedonia. there were 234 greek and 101 foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following 1) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%. 2) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). 3) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type 1 diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om-85, a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd28 -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om-85. cytokine secretion, activation and proliferation of b cells and foxp3+ tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr2, tlr4 or the myd88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om-85. two synthetic tlr4 agonists used as adjuvant in human (om-174-dp and om-197-mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om-85-induced protection of diabetes required natural tregs, as nod-cd28 -/mice were not protected. remarkably, om-85 activated b cells and not t cells, promoting their proliferation and il-10 secretion, two phenomena that were tlr4-and myd88-dependent. om-174-dp and om-197-mp-ac two synthetic murine tlr4 agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd4 + cd25 + foxp3 + t cells and the proliferation of il-10 secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr4 signaling in the protective effect of om-85 on development of diabetes and show that two other tlr4 agonists induce proliferation of b cells and their secretion of il-10 as well as stimulation of regulatory cd4 + cd25 + foxp3 + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit 1 , a. legat 1 , s. thomas 1 , m. van mechelen 2 , p. hermand 2 , m. goldman 1 1 institute for medical immunology/université libre de bruxelles, gosselies, belgium, 2 glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide 4-phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor 4 (tlr4). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd14 (scd14) for its tlr4 ligand activity, we investigated the involvement of both forms of cd14 in the responses elicited by crx-527, a prototypical agp. first, we found that crx-527 efficiently induces nf-kb and irf3 activation in hek cells transfected with tlr4 and md-2 genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd14. likewise, crx-527 efficiently induces the synthesis of nf-kb and irf-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd14 prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd14 respond to crx-527 but not to lps in serum-free medium. the addition of the soluble form of cd14 did not modify the levels of il12 and tnf produced by crx-527 stimulated dc but increased the levels of interferon-b (ifn-b). when scd14 was added to hek cells expressing tlr4/md-2, nf-kb activity was not modified but irf3 activity was increased in a dose-dependent manner in response to crx-527. we will further compare the responses induced by crx-527 in wild-type and cd14 deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h-2d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled 14 and 28 days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin-15 gene polymorph isms (c267t, g367a, c13687a and a14035t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype infg+/cd69-, p=0.02) and activated (subtype infg+/cd69+, p=0.028) cbmc, producing ifn-g, as compared with newborns from urban mothers (n=79) exposure to pets and the risk of allergic symptoms during the first 2 years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc09/16 to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6) tb has the highest diagnostic value in children n 4 years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp-10 antigen is more sensitive for detection of tb-specific t cells compared to esat-6 antigen. 4. in children with tst 5-14 mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr2 and tlr4 in the development of spontaneous lupus disease by creating tlr2 or tlr4 deficient c57bl/6 lpr/lpr mice. methods: tlr2 and tlr4 deficient lupus prone mice have been generated by crossing c57/bl6-tlr2 -/-or c57/bl6-tlr4 -/-mice with c57/bl6 lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr2 or tlr4 deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr2 or tlr4 in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr4 deficient mice suggesting an important role of tlr4 in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr4 also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr4 pc14/13 expression of full length mcl-1 and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl-1, an anti-apoptotic protein. a splice variant of mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl-1 (mcl-1l) and its splice variant (mcl-1s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl-1. method: neutrophils were isolated from children (diagnosed x 17 years) with jsle (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, jsle serum alone or with jsle serum plus 20pg/ml gm-csf. quantitative real time pcr was used to assess mcl-1l and mcl-1s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl-1s to mcl-1l was also higher in jsle patients compared to controls (p x 0.05). the addition of gm-csf to jsle serum was associated with an increase in mcl-1l (1.66 ± 0.31) and a decrease in mcl-1s (2.56 ± 1.1) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl-1s, and less anti-apoptotic full length mcl-1 cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd4 + cyld ex7/8 t cells were transferred into rag1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex7/8 cd4 + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex7/8 mice are capable to control inflammatory responses. for this purpose cd4 + cd25 + cells were co-transferred with naïve wt cd4 + t cells into rag1 -/-recipients. interestingly, rag1 -/-recipients of cyld ex7/8 t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc17/16 the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a*03 in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb1*13, *01 and *0103 alleles, and a decreased allele frequency of drb1*15 in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb1*0103 is associated with the development of severe disease and positive association of cd with drb3*0301 and drb1*13. indeed, drb1*15 was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb1*07 frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb1*04 in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb1*15 were strongly increased with respect to cd and controls. however, the frequency of drb1*04 was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb1*15 is associated with uc in european, north american, japanese and korean populations methods: a total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 x 5 years of age, 12 5-10 years of age and 2 g 10 years of age) and 23 girls (10 x 5 years of age, 6 5-10 years of age and 7 g 10 years of age). pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). the lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis g 20.000 in 9 cases (17,6 %). the most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and 3) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il-13ra2, previously believed to be a decoy for il-13 only, is able to transmit a signal via il-13. our results support this and may suggest that il-13/ il-13ra2 signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il-4ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il-13 or il-13ra2 and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd4 + cd25 -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il-6 and il-1b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd4 + cd25 -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi-98, l. acidophilus ncfm tm and b. bifidum bi-504) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi-98 strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il-6 or il-1b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 1 1 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed 21 pediatric cd patients (13 active, 8 remission), 24 pediatric uc patients (17 active, 7 remission), and 37 age-matched non-ibd controls. nkg2d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x 0.05 was considered significant. results: nkg2d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd3+cd56+ nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg2d ligands on circulating monocytes is also observed. the dramatic increase of nkg2d+ lymphocytes, and the strong upregulation of nkg2d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg2d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc17/26 peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp3+ t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd4+ t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp7 knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than 200 million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec-205 antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa 1-191) or hcv-ns3 (aa 1027-1218) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns3 and core we successfully optimized culture and protein purification conditions. briefly, ns3 was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec-205 to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec-205/ hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd01/2 mva-nef vaccination induces polyfunctional cd4 t-cells and increases the proliferative capacity of cd8 t-cells in hiv-1 infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv-1 protein nef (mva-nef) was safe in hiv-1 infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd4 t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il-2 and mip-1b, the expression of the activation marker cd154 and the differentiation marker cd45ra in nef-specific cd4 and cd8 t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il-2 and mip-1b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd4 t-cell response and a significant increase of polyfunctional nef-specific cd4 t-cells, simultaneously expressing ifn-g, il-2 and cd154. using the standard ics no increase of nef-specific cd8 t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd8 t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il-2 and cd154 expressing cd4 t-cells and the increase of proliferating cd8 t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd4 t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd8 t-cells in hiv-1 infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv-1 mediated membrane fusion and p24-detected replication. db81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with 25mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd4 + and cd8 + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns4 (1677-1756 aa) and rns5a (2061-2302 aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/2j mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns4 protein. immunization with rns5a-immunomax conjugate and rns5 in cfa (1.4 mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns5a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns5a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd01/21 degree of cross-genotype reactivity of hcv-specific cd8 t cells directed against ns3 the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd8 t cell response targeting hcv genotype 1 (gt1) and genotype 3 (gt3) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: 53 subjects (17 with gt1, 22 with gt3 and 14 anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns3 from gt1 or gt3. individual reactive peptides and the degree of cross-reactivity between the gt1 and gt3 variants were determined by ics. complete ns3 is sequenced from all viremic patients pd01/22 anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes 1, 4, 6 or 9 suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. decreased splenic viral load after ifna1 treatment correlated with an expansion of activated fv-specific cd8 + t cells and nk cells in the spleen, whereas in ifna4-and ifna9-treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna2, 5 and 11 are under investigation pd01/23 elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd8 t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd8 t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd8 t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a*0201-or k b -restricted cd8 t cell responses by these dna vaccines differed. k b /ova 257-264 -and k b /s 190-197 -specific cd8 t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd4 + and cd8 + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of 5 peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, 3 hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd8 + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd8 + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il-12-exposed cd8 + t cells showed at least five-fold increase of survival rate in vivo than il-2-exposed cd8 + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd8 + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il-12-exposed cd8 + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd8 + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il-12 and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x 0.01) . there was a significant elevation of t ada and ada1 activities in iga deficient patients as compared to healthy individuals (p x 0.01) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd01/61 hiv-1 sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after 2-6 weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in 12 out of 17 patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, 10-day-old ross 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/79 strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/79 and h120 strains, via the drinking water at 10 days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd4 t cells from 25 patients with active tb and 28 patients with successfully treated tb were analysed for simultaneous expression of ifng and il2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with ppd. moreover, cells were stimulated with esat-6 and cfp-10 receiver operator characteristics analysis revealed that a percentage of ifng /il2 dual positive cells x 56 % served as an accurate marker for active tb patients (specificity 100 %, sensitivity 65 %), while frequencies g 56 % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd03/7 necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in 1993 and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, 12 out of 14 children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently 6.12 to 100 000 inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd03/8 novel analogues of thalidomide inhibit cd80 expression and production of tnf-a, il-12, ifn-g, cxcl-9 this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta1-dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta1r7k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd80 and cd86. these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il-1a and il-1b, while il-6 levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il-1b production. using bmdc from nlrp3 -/-mice we examined il-1b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il-1b production is dependent on nlrp3. collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho-1 expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from 20 % in wild type (hmox1 +/+ ) mice to 87 % in ho 1 deficient (hmox1 -/-) mice. hmox1 -/-but not hmox1 +/+ mice developed end-stage multiorgan failure. mortality of hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h 2 o 2 or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb1. similarly, circulating and cytoplasmic hmgb1 was increased in hmox1 -/-relative to hmox1 +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b-1,2-linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and 2-4 weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were 7.7 and 9.4 iu/ml and those of dtwp-pasteur were 8.2 and 8.6 iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were 30.2 eu/ml for dtwp-local and 47.9 eu/ml for dtwp-pasteur vaccines, respectively (p x 0.001). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd05/18 united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b:2 , a gram-negative coccobacillus. jrmt12, an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 cfu of jrmt12. a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b:2 on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for 1 h before adding concanavalin-a (cona) pd06 -vaccination and immunotherapy against parasitic diseases pd06/1 evaluation of simian adenoviral vector adch63 expressing msp-1 as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype 5 (adhu5) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch63 is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp-1 and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm128 while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho-1 expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox1 locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho-1 is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp63 expression was confirmed by sds-page and elisa using monoclonal antibody against gp63. discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a5-180recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a5-180recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at -20°c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and 2 seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r=0.216, p=0.641) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il-17 on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il-17 to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il-17 and il-17-related inhibition of cfu-e growth were dependent on p38 mapk activity, since the p38 mapk inhibitor, sb203580, markedly downregulated il-17-induced activation of nos and reversed the growth inhibitory effects of il-17 on cfu-e. the in vivo stimulating effect of il-17 on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il-17 effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il-17 acts on bone marrow cells and also revealed a link between the il-17, no and hematopoiesis. further studies on il-17-mediated induction of both inos and enos methods: a total of 1785 blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of 68 samples (3,8 %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x 100 iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had 100 iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 iu/l anti-hbs in all the blood units, which also goes for our study pd14/20 neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen 1,2 1 national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa-1a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa-1a iggs we monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. in 11 of them all was diagnosed for the first time. 1 subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. 2005, nature 435, 452-8). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 3'utr of icos, in which a 47bp sequence, containing a possible mir-101 binding site, was sufficient (yu et al. 2007, nature, 450, 299-303). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. 2000, j biol chem 275, 33655-62). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia-1-and ago2-deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r01 ai 42269. we have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw 264.7 macrophages. in this report, we investigated the effect of 6-hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin-8 (il-8), monocyte chemotactic protein-1 (mcp-1) in ht-29 human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed 6-hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated 6-hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. 6-hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p65. in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of 6-hma significantly reduced the mrna levels of il-8 and mcp-1 stimulated by tumor necrosis factor-a (tnf-a) in the ht-29 cells. pretreatment of 6-hma also significantly blocked the ixba degradation and nf-xb p65 nuclear translocation stimulated by tnf-a in the ht-29 cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of 6-hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, 6-hma could be new potential therapeutic agent for inflammatory bowel disease.cd4 serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd4 igg immune response in hiv-1-exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd4 domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human cd4, in the absence of the target cells expressing the co-receptor ccr5. the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (ccr5 expressing) cells. thus, in the absence of ccr5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv-1 infections. a conventional protocol for the generation of monoclonal antibodies was used. db-81 (igg1, x), one of the anti-cd4 antibodies obtained, recognized preferentially cd4 complexed to gp120, as compared to cd4 alone, not competed for the gp120 binding site on cd4 and was specific for the second extracellular domain of cd4. g. röder 1 , l. geironson 2 , a. darabi 3 , m. harndahl 1 , c. schafer-nielsen 4 , k. skjödt 5 , s. buus 1 , k. paulsson 2 1 copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, 2 lund university, immunology bmc d14, lund, sweden, 3 lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, 4 schafer-n, copenhagen, denmark, 5 department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first 87 n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a*0201. to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn 1-87 /80 , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn /80 was demonstrated to be located on tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first 87 n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd8+ t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region 301-498 of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b*2705-restricted np-flu epitope (aa 383-391) was replaced by hla-b27 or hla-a2-restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard 51 cr release assay and through the ifn-g production. results: using hla-b27 or hla-a2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a2 molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a2 restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b27 as a remarkable hla-class i molecule that, differently from hla-a2, can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b27 molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv1. consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p17-gag and p24 region was determined in vitro. 25mer peptides were digested with i20s and c20 proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv-1 p17-and p24-gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers 1 , g. koster 1 , o. landsverk 1 , f. skjeldal 1 , o. bakke 1 1 university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi3 kinases and thus ptdins(3)p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier 1 , l. visvabharathy 1 , j. fu 1 1 university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e3-19k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e3-19k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type 2 e3-19k and class i molecules.results: we showed that e3-19k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a11 molecules, with the mature form being more tightly associated. we also provided evidence that e3-19k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e3-19k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e3-19k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il-12, il-6, tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands 2 and 4, but also with the t-celldependent stimulus cd40l (cd154) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd40, cd83, and cd86 was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p38 and erk-1/2. strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd4+ t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th2 (il-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th1 (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th1 / th2 balance and to identify potential t cell target antigens (ags), we analyzed circulating cd4+ t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il-4 expression in peripheral blood cd3+cd4+ lymphocytes were measured in 9 ccs patients and 7 healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il-4/ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il-4 and lower ifn-g intracellular expression were detected in cd4+ t cells from css patients, as compared to hs (il-4: 1.3±0.3 % vs. 0.50±0.21 %, p x 0.05; ifn-g: 14.2±4.5 % vs. 27.0±4.8 %, p x 0.025). similar results were obtained by elisa (il-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p x 0.05; ifn-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p x 0.05). elispot counts of hexavalent vaccine-stimulated cd4+ cells were positive for il-4 in 4/5 (80 %) css patients and in 5/5 (100 %) hs, and for ifn-g in 2/9 (22 %) css patients and 7/7 (100 %) hs. mpo stimulation determined significant ifn-g release in 5/8 (62 %) css patients, but not in hs (0/5) no il-4 response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd4+ cells from css patients show a th2-polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd4+ t cells, and responses towards it are instead th1-polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd4+ t cells in css. a. voigt 1 , e. opitz 1 , k. savvatis 2 , k. klingel 3 , k. stangl 1 , u. kuckelkorn 1 , p.-m. kloetzel 1 1 charité -universitätsmedizin berlin campus mitte, berlin, germany, 2 charite -campus benjamin franklin, berlin, germany, 3 universität tübingen, tübingen, germanymurine models of coxsackievirus b3 (cvb3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp2 of the stress-induced ip, were infected with 1x10e5 pfu cvb3 nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp7 as early as day 4 p. i. in lmp2-deficient mice. however, lmp2-deficiency was linked to less severe myocarditis at day 8 p. i. (he stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. lmp2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p x 0.001). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x 0.05), there was no difference in lmp2-deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb3 infection in lmp2-deficient mice. likewise, diastolic function was preserved in lmp2-deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp2-immunosubunit function in regulatory processes of viral replication. absence of lmp2 confers host protection in enterovirus myocarditis. h. w. liao 1 , j. xu 1 , j.q. huang 1 1 sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype 2 (den-2) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr1 and tnfr1-2 were constantly very low whereas trailr2-4 decreased after den-2 infection. fasl was expressed at similar levels on huvecs throughout den-2 infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by den-2 infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for 7 days into wis rat paw web tissue in saline containing 7.5x10 7 cells. group ii. s.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day 8.they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p x 0.05). immunohistochemical pictures showed increase in percentage of ox62 (migrating dendritic cell), mhc ii, his48 (granulocytes), ox7 (stem cells) and cd54 (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed1 (macrophages) and ox62 cells. popliteal lymph nodes contained evidently less of ox62, his48 and mhcii cells than in group i (p x 0.05). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega-3 fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption. the results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega-3 fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising 1 % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e2f2 mutation, and other affecting b cell apoptosis control, such as bcl-2 over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c57bl/6 (b6) genetic background. e2f2-/-bcl-2tg were obtained backcrossing e2f2-/-and e2f2+/+hbcl-2tg mice. e2f2-/-bcl-2tg, e2f2-/-, e2f2+/+hbcl-2tg and control mice (e2f2+/+) were followed up to 18mo-old. serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h3]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl-2tg in b lymphocytes of e2f2-/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e2f2-/-bcl-2tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e2f2-/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e2f2 or the overexpression of a bcl-2 tg in the b6 genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor 9 (tlr9) abolishes the generation of antinucleosome igg2a and igg2b autoantibodies but exacerbates lupus disease. however, the tlr9-dependent tolerance mechanism is unknown. here we show that loss of tlr9 in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th1 and th17 t cells. transfer of a synthesized monoclonal polyreactive igm to tlr9 deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr9-dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n=115) (vitali et al. 2002) . ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g 1 (fs+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x 0.05). interleukin (il) 17, il-1ra, il-1beta, il-12p40, il-15, macrophage inflammatory protein (mip) 1alpha, mip-1beta, eotaxin, interferon (ifn) alpha, and il-4 levels were significantly increased in the gc+ patients (n=27) compared with the gc-patients (n=70). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il-4 found in these patients (gao et al. 2006) . increased titers of th17-associated cytokines, il-17, il-1beta and the il-23 subunit il-12p40, may indicate a higher activity of these cells in gc+ patients (nguyen et al. 2008) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il-6. the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg2. production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g 50 %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd88-dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd88-dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/-1 baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il-8. objectives: increased levels of il-18, an innate and inflammatory cytokine of the il-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il-18 and other genes of the il-1/il-1r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il-18 and il-18bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il-18 are higher than in controls. serum il-18 correlates with disease activity and decreases upon remission. monocyte expression of the receptor il-18rb is increased and correlates with disease severity, while expression of tir8/sigirr (a down-regulatory receptor of the il-1r/il-18r family) is reduced. in mrl lpr/lpr mice, expression of il-18, caspase-1 and il-18rb genes precedes disease onset in lymph-nodes. in other organs, changes in il-1-related genes (il-33 and tigirr-1 up-regulation, tir8/sigirr down-regulation) occur after disease onset. free il-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il-18 levels correlate with autoimmune lupus both in mice and humans. free il-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il-18rb is a marker of pathology, suggesting increased il-18-dependent activation. both in mouse organs and human monocytes, tir8/sigirr expression decreases with disease, suggesting impaired control of il-18r activation. thus, il-18 may be involved in autoimmune lupus pathology, and il-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f4 (amino acid (aa) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f4 was recognized by all patients. fragment f1 (aa 5-30) was recognized by 9 of 34 dcssc patients. fragment f8 (aa 350-400) was recognized by 4 of 8 sle patients. analysis of clinical data revealed a significant difference between the f1 negative and f1 positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f1 and f8 could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd4 + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd4 + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril-2. by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd4 + t cells enriched in tregs were co-cultured with activated cd4 + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd4 + t cells anergy was also evaluated based on cbl-b, grail and foxp3 mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd4 + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd4 + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b-1 cells through targeting p110d by shrnas strategy. methods: we used the drugs, ly294002 and wortmannin, pan-specific inhibitors against pi3ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. we then introduced either pan-specific inhibitors against all pi3ks or p110d-targeting shrnas into an sle-prone animal model, nzb/w f1 mice, for therapeutic purposes. the results suggested that pi3ks are not only important for the development of b-1 cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p110d. either pan-specific inhibitors against pi3ks or p110d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f1 mice. one inhibitor, ly294002, and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi3ks are critical for the maintenance of b-1 cell populations might shed light on future treating other diseases associated with b-1 cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool 1 , m. alarcón-riquelme 1 , s. kozyrev 1 1 institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank1 gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs10516487, r61h). we identified that bank 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank1 isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs17266594), which is in complete ld with r61h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia1, which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a 57-year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti-2gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow 1 1 cnrs 9021, strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below 2. b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd19 surface protein for all patients. this cd19 lower expression is associated with cd45 lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf2 expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf2 lacking an exon in the c2-domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf2 in t-cells. smurf2 expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf2 expression was upregulated by tgf-beta stimulation in t-cells and smurf2 was markedly upregulated in tumor infiltrating cd4+ lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day 42, smurf2 transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf2 showed an upregulation of the tgfbrii and an activation of smad2 and 3 as compared to wild-type t-lymphocytes, which were previously described as typical smurf2 targets for degradation. in addition the transfection of smurf2 and a caga-luc plasmid into cos-cells for smad3-promotor studies yielded the same effect as shown by upregulation of the smad3 activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf2 splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf2 has beneficial effects on mucosal inflammation and tumor development. smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat3 has important functions in cytokine signalling in a variety of tissues. however, the role of stat3 in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat3 activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat3 activation in dss colitis is dependent on il-22 rather than il-6. il-22 was secreted by colonic cd11c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat3 activity were highly susceptible to experimental colitis, indicating that epithelial stat3 regulates gut homeostasis. stat3 iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il-22 and epithelial stat3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat3 activation regulates immune homeostasis in the gut by promoting il-22-dependent mucosal wound healing. stat3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd40 and tlr4 expression at day 3 and day 7 after infection. mycobacterial infection did not result in differential tlr2 expression as compared to uninfected cells. cd40 is involved in stimulating th1 pro-inflammatory responses, although map may interfere with cd40 signalling (1) . tlr4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd40 and tlr4 receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd8 t cells by exogenous antigen leads to colitis. methods: eight million naïve cd8 + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h2-kb, were transferred i. v. into b6 mice. at day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day 2 after the injection of ot-i cells. the phenotype of effector cells was evaluated at day 5 by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd8, the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd8 + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd8 + t cells. our study demonstrates that the local activation of antigen-specific cd8 t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b6 mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x 2 and t-test programmes. results: in 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. on the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: 1) helicobacter pylori infection is relatively common in the general population (30,5%).2) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova 1 , t. ulmannova 1 , k. stechova 1 , k. tesarova-flajsmanova 1 , v. stavikova 1 , j. nevoral 1 1 charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il-4, il-6, il-10, il-17, il-23, ifn-gamma, tgf-beta1, egf and eotaxin. man-whitney u test was used for statistical analysis, p x 0.05 was considered statistically significant.results: il-10 as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). higher concentrations of il-4 and lower concentration of tgf-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. 8310-5. background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il-8 (murine homologs kc and mip-2) and its receptor cxcr2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. anti-kc antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. results: facs analysis revealed 80 % neutrophil purity, 35 % of which were cxcr2 + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, 4 h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model (1.5 % dss in drinking water for 1 week, followed by pure drinking water for 1 week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, 2 % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell 2004), treatment of commensal-depleted mice with the tlr4 ligand lps in drinking water protected against the lethality of 2 % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. at 2 % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by 1.5 % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il-13 production which is an important pathological factor. neutralizing il-4 or il-13 prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th2 cytokines in colitis remain undefined, we used mice deficient in il-4/il-13 or the key receptor through which they signal, il-4ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il-4ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il-4/il-13 double deficient mice which were protected from colitis. removing il-13 production from il-4ra -/mice, by using il-4ra/il-13 double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il-13 mediates susceptibility in an il-4ra independent manor. recent evidence pc17/33 introduction: the activation of cd4 + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th1-like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th1 cytokines, such as il-5,-6 and il-13. however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc2 was found in uc and cd colonic tissue compared to control specimen. transmitted to the th2-mediated oxazolone-induced colitis model, nfatc2-production is significantly increased in both diseases, too. nfatc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc2 deficient mice compared to control mice, which can be observed by tunel assays, caspase3 and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl-2 and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il-6, ifn-gamma, il-13 and il-17 by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc2 regulates il-23/il-17 in an indirect way. last, administration of il-6 blocked the protective effects of the nfatc2 deficiency in experimental colitis, suggesting that nfatc through il-6 signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc2 in colitis by controlling mucosal t cell activation in an il-6 dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd4cd45rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd4cd45rb high t cells also regulatory cd4cd45rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at 3 different time points during colitis progression. after 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. microrna was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from 11 micrornas that demonstrated an induction during the development of disease we selected 4 micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd4cd45rb high transfer model. objective: the purpose of this clinical trial (id: nct00287677 of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in 3 groups: group a (n=8) receiving haart+ gh (for 6 months) + tt+hva/b vaccines (at month 6 post gh adminsitration); group b (n= 6) receiving haart+gh but not vaccines; and group c (hiv control group, n=7) with haart+vaccines (at month 6) but without gh. all patients are followed up 6 months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after 24 weeks of administaring hormone the absolute numbers of cd4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (mean and sem; p x 0.0025). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p x 0.02). viral load remained undetectable in all patients. despite the increase in cd4 counts the percentage of recent thymic emigrants (as assessed by the expression of cd31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv (80,000-160,000), hepatitis c virus (hcv; 2.3-4.7 million) and hepatitis b virus (hbv; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv-1 infected patients with g 350 cd4 + t-cell counts and plasma hiv-rna levels of x 1.7 log 10 copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from 4 timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g 50 and when the responses were g 2 times the standard deviation above the mean of replicate negative controls (mock electroporated dc). 16/17 patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in 4/16 patients before and 11/16 patients after vaccination. for rev, 7/16 patients showed a pre-existing rev specific response and 14/16 patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus 71 (ev71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev71 is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev71 inactivated virus. methods: mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after 0, 2, and 4 weeks. blood samples were collected at 0, 21, 35, and 45 days. the total serum anti-ev71 igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il-5 from splenocytes was also measured. results: immunization with ev71/ps-g showed that the anti-ev71 igg levels were significantly increased compared with ev71 alone or ev71/cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev71/ps-g group compared to ev71 alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev71/ps-g-immunized mice compared to those of ev71 or ev71/cfa/ifa-immunized mice, indicating a th1 cells response elicited by heat-inactivated ev71 vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd8+ t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd8+ t-cell responses against hcv-ns3, studying induction of il-2, ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il-2 and ifng production by cd8+ t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv-1-recognizing cd8 + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv-1 replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd8 + t cells by electroporation, and chose tcrs which were able to recognize the hla-a2 restricted hivpol-peptide iv9, or the hivgag-peptide sl9. results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il-2, tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd25, after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd4 + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd8 + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd8 + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt3l pretreated balb/c mice were incubated for 3 hrs with hcv ns5-coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g 80 % dcs and was used for immunization. dc expression of the maturation markers cd40, cd80 and cd86 was determined before and after ingestion of ns5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns5 expressing sp2/0 cells). results: in immunized animals, the ctl activity was increased 3-fold compared to mock immunized mice. accordingly, tumor challenge with ns5 expressing tumor cells showed a significant reduction in tumor growth. the number of cd4 + ifn-g + cells was increased g 3-fold and the number of cd4 + il-2 + increased g 5fold in the dc-ns5-bead immunization group. these results paralleled the proliferative response of splenocytes to ns5 protein obtained from immunized animals with the most significant response in the cd4+ population of dc-ns5-bead immunized animals. the use of ns5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th1 biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec-205 induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec-205 antibody can be developed. we screened the anti-dec-205 sequence computationally for putative hla dr4-restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec-205 antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec-205 as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih 1r21ai078800 a live oral vaccine based on human adenovirus (ad)4 has proved safe and effective in us military recruits for nearly 50 years. in these experiments, we have investigated whether replication-deficient ad4 can be an efficient potential vaccine carrier for oral vaccination. ad5 vectors were used throughout to provide a benchmark for efficacy. we generated novel ad4 and ad5 vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad4 than ad5. ad5 routinely infected and provided transgene expression in˚10 % of human cd4+ and cd8+ t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to 25 -30 % of cd8+ t cells present, showing that ad5 infected a surprisingly large proportion of t cells. in comparison, ad4 provided egfp expression in x 2 % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad4 and ad5 vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad4 and ad5 induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. interestingly, when delivered together ad4 and ad5 vectors targeted the same subset of cells. together, these data show that ad4 is a viable alternative to ad5-based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; g 300 in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the 667 mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, 2005; gros et al, 2008; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd8 + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp60) of rhdv at two different locations: 1) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp-2), and 2) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp-306). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd8 + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp-2) was more immunogenic than insertion at position 306 for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp-2 at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr-2 ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd8+ t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd8+ t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np 147-155 specific cd8+ t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr-2 targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l929cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during 7 days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd2+, cd4+, cd8+, cd16+ t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns4 and ns5a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h5n1 influenza virus inhibits the ifn-g synthesis (mibayashi et al., 2007) and causes a decrease in cd4 + and cd8 + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., 2000) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a 10 amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions 129-131 (tat'kov c. i. et al., 2000) . deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 iu once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast rna preparation (5 mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., 1983) . results: when deltaferon was administered in doses of 2-20*10 4 iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of 2*10 4 iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon (2*10 4 iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd8 responses (tetramer+ hiv-1 gag p24 cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd8 responses after pla-p24 immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p24 were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas 1 , c. prego 2 , s. vicente 2 , m.j. alonso 2 , a. gonzález-fernández 1 1 university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, 2 university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to balb/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease (100 miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigenbackground: infection with human immunodeficiency virus type 1 (hiv-1) is characterized by dysfunction of hiv-1-specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from hiv-1 and the tlr3 ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p24 microcapsules containing p24 and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for 10 days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p24 peptides. intracellular staining of interferon-gamma (ifn-g), interleukin-2 (il-2) and cd107a after p24 stimulation was also performed. results: mddc from hiv(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p24 containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il-2, upon p24 peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd16b between our model cytokine il-2 and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p30gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il-2 dependent proliferation assays of il-2 variants. results: murine il-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il-2 dependent cell line ht-2. we found that the membrane anchors comprising one and four ig-like domains (il-2::1iggpi and il-2::4iggpi) resulted in severely reduced vlp production by 293 producer cells and despite of an increased targeting of il-2::4iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il-2 fused to the minimal gpi-anchor acceptor sequence of hcd16b (il-2::gpi). il-2::2iggpi, however, showed comparable particle production and biological activity in vitro when compared to il-2::gpi. furthermore, il-2 fused to 2ig::gpi showed an increased capacity to co-stimulate primary p14 tcr transgenic t-cells specific for lcmv-gp 33-41 in the context of h2-d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. 2ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il-2::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f1816-b13 of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant 812079 & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild 1 , interdisciplinary transplant laboratory 1 charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with 6 different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from 2,5 month to 16 days. t cell lines are composed of cd8 and cd4 cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker 1 , m. assenmacher 1 , a. richter 1 1 miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp65-specific cd4 + and cd8 + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd45ro -cd25 -) cd4 + and cd8 + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp65 peptide pool and cd3-depleted autologous pbmc as feeder cells in the presence of il-2, il-7, and il-15. already 9-13 days after primary activation pp65 495-503 /a2-tetramer + cd8 + t cells were detectable for 3 hla-a2 + blood donors. to analyze cd8 + t cells having other specificities than for the peptide pp65 495-503 as well as probably primed cd4 + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to 3.9% of the cd8 + t cells and up to 3.8% of the cd4 + t cells after restimulation with pp65 peptide pool, but not with either irrelevant ie-1 peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd154 and cd137 only if restimulated with the pp65 peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il-2, indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd4 + and cd8 + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf 1 , k. mozdzanowska 1 , l. otvos 2 , j. erikson 1 1 the wistar institute, philadelphia, united states, 2 temple university, philadelphia, united statesthe influenza virus a matrix protein 2 ectodomain (m2e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m2e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine 2003; virology journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-m2e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd8 + t cells, have been considered. clinical data confirm a crucial role for antiviral cd8 + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a*0201 and hla-b*0702 previously. considering other frequent hla alleles cmv specific cd8 + t cells were monitored longitudinally in 30 hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd8 + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd8 + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a*2402/pp65 (341-349) (7.6x10 6 cells/l) and hla-b*3501/pp65 (123-131) (4.4x10 6 cells/l) specific cd8+ t cells are significantly lower than those for hla-a*0101/pp50 (245-253) (17.2x10 6 cells/l) and hla-a*0201/pp65 (495-503) (13.2x10 6 cells/l) specific cd8 + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. (2,5 -50 mcg) . no adverse effects were indicated during trials (up to 25 month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of vichrepol.no differences were detected in cd4+ and cd8+ t cell counts and cd4+/cd8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp70 or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp70 or gp70 alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb6f1 hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb6f1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd4 + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd4 + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf01 exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf01 did not show any increase in their levels of ifn-gamma. conclusion: caf01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from 13 children aged 1-2 years old (6 boys and 7 girls) -group 1, and 11 children (6 boys and 5 girls) 6-7 years old -group 2 were isolated on a gradient of density before vaccination ( or revaccination) with priorix, 1 week, and 2 months after and incubated with cfse. then 2 million/ ml pbl were incubated in rpmi-1640 supplemented with 10 % fcs (the negative control), at presence of 5 mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % cî 2 at 37°c within 7 day. intensity of a fluorescence estimated on fl1 by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control 90 % pbl in both groups did not enter mitosis. in the positive control 90 % of cells have passed one and more mitoses. in group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. in group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. in a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. production of the interleukin (il) 6, ifn-g, tnf-a, il-4, il-1 was more informative than il-5, il-7, il-8, il-12. measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp120 cd4-binding site (cd4bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv-1 neutralizing polyclonal iggs directed against the cd4bs. the mabs were shown to react with an anti-cd4bs human neutralizing mab (b12), to elicit antibodies that recognize the gp120 molecule and an anti-hiv-1 neutralizing response in rabbits, confirming them as cd4bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd4bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd4bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp120 titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b12 in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p1 and p2) reacted in elisa only with the cd4bs-directed igg fraction. the clones were also recognized in elisa by b12. p1 and p2-immunized rabbit sera showed a strong anti-gp120 titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb2 strain glycoproteins. in particular, 3/5 rabbits in the p1 group and 1/5 in the p2 group showed an 80 % hiv neutralization at dilutions ranging from 1:20 to 1:150. the immunoenzymatic assay used, allowed to detect a p1 and p2 reactivity in hiv-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines (17d and 17dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a 23-year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following 17d-204 vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd16, cd32 and cd64) along with increased levels of nkt-cells (cd3 + cd16 +/-cd56 +/-/cd3 + ratio) and activated t-cells (cd4 + and cd8 + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il-6, il-17, il-4, il-5 and il-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il-4 + ), cd8 + t-cells (il-4 + and il-5 + ) and b-lymphocytes (tnf-a + , il-4 + and il-10 + ). the analysis of cd4 + t-cells revealed a complex profile with increased frequency of il-12 + and ifn-g + and decreased percentage of tnf-a + , il-4 + and il-5 + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester 1 , h.-g. rammensee 1 , s. stevanović 1 1 eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd8 t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd8 t cell epitopes for the frequent mhc class i alleles a*01, a*02, and a*24 from the proteins pii (hexon), pviii, and e1a of adv strains ad2 and ad5 by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a 12-day recall stimulation of pbmcs from at least 16 healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd8 t cells. we could identify 27 new peptides eliciting ifn-g responses, several of which were confirmed as novel cd8 t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle (5 ara criteria) was diagnosed in a 40-year-old african female patient with hiv-1 (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd4 t-cell count x 200 cells/mm 3 . initiation of 500 mg mmf bid was associated with biological and clinical remission of sle and cd4 t-cell increase. no opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to art. hiv-1-specific cd4 and cd8 t-cell responses were analyzed after 18 months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il-2 production following stimulation with a panel of 192 hiv-1-derived optimal epitopes (9/10-mers) covering various hiv regions and a pool of 105 hiv-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. all peptides are derived from hiv-1 consensus strain iiib. results: highly polyfunctional hiv-1 specific cd4 and cd8 t-cell responses against gag were detected. 11 epitope-specific cd8 t-cell responses were identified: except for one response restricted by hla a*23 and another one by hla cw*07, all the others were restricted by hla-b alleles and mostly by b*58 (n = 5). seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il-2+) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv-1 specific cd4 and cd8 t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv-1 t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of 10 6 dl50/ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the 7 th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt-46767, cofaa and cgpi-20090305. . state of vaccine-induced measles immunity was determined by means of elisa in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in 4-6 years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with mt conversion. in 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with mt conversion in 1-3 years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic mt igg level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il-17 producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein 65 (dnahsp65). methods: balb/c female mice were infected by intra-tracheal route with 10 5 h37rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. each dose consisted of 100 micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd4+, cd8+ and gamma-delta t cells from lungs were determined 10 and 50 days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x 0.05 were considered significant (t test). results: at day 10 after the end of the immunotherapy, dnahsp65 treated mice exhibit increased numbers of absolute cd8+ and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il-17 producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp65 treated mice showed more ifn-gamma producing cells in both cd8+ and cd4+ cell populations. at day 50 after the end of the therapy, the main observation in mice which received dnahsp65 treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd4+ and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd8+ cells, together with a more frequency of gamma-delta t cells producing il-17. finally, the immunotherapeutic effects of dnahsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd8+ cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il-17, are the main effects of dnahsp65 immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il-17 and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as 60 loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput (96-well format), requires only 100 ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that 5 candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls 71 % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all 5 m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards 4 of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to 100 % in pb, 75 % in rx and 93 % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd40l, a co-stimulatory molecule preferentially expressed on activated cd4+ t cells, is the ligand of cd40. cd40-cd40l interaction induces the production of il-12 and the initiation of a th1-type immune response. several studies show that cd40l is required for the activation of macrophages and the maturation of dcs. moreover, cd40l enhances the capacity of cd8+ t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd40l. we have constructed the recombinant bcg strain expressing cd40l (rbcg2) by electroporation of bcg with pgfm11/signalsequenceag85b-cd40lec and an another recombinant strain with empty vector pgfm11 (rbcg1) as a control. the expression of cd40l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd40l 2 weeks after vaccination but not at 4 and 8 weeks. rbcg2 seems to be more protective against paratuberculosis than rbcg1, but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd40l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il-1a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x 0.05) of tnfa, ifng and il-1a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x 0.05) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd40 mab and ag85a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd40 binding on cd40 transfected l929 fibroblasts. the conjugates were tested in vivo in wild type and cd4 + cell-depleted mice for the induction of specific anti-ag85a serum antibodies. splenocytes were challenged ex vivo with ag85a and were examined for their ability to produce th1-related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il-2. we developed a method to successfully crosslink anti-cd40 mab to ag85a. serum antibodies against ag85a were detected after immunisation with this conjugate vaccine in both wild type and cd4 + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag85a alone. production of two other th1-related cytokines, tnfa and il2, was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd40 conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd4 + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer 1 , r. burger 2 1 robert koch-institute, cellular immunology, berlin, germany, 2 robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd4-positive and cd8-positive t-lymphocytes. cd8-positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd8+ t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for 5 days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to 30 % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd4+ t-lymphocytes in vivo. similarly, the cd4-positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd4+ and the cd8+ subpopulation depended on the presence of apc. stimulation oft cd8+ cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd8-positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target 6 (esat-6) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, 83 iranian and afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of esat-6 and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of 4 spot forming unit ( g 20 spots per million), we found detectable response to esat-6 in almost 80 % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable esat-6 specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th1 immune response, against esat-6, in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal 1 , p.k. upadhyay 1 1 national institute of immunology, pdc-1, delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl 2 solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c57bl/6 mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: 2.4 % alginate, 0.012 % pva concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % pvp (poly vinyl pyrollidone). there was 6 fold increase in proliferation index of spleenocytes and releases 600 pico-gram of interferon gamma after 4 week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by 10 % increase in cd86 and 10.5 % in ccr7 expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b100 is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b100s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b100s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b100s. by cell cycle analysis we determined that b100s is able to induce apoptosis in up to 80 % of jurkat and molt-4 t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b100s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b100s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that b100s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b100s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b100s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd40, cd80 and cd86, and of the inflammatory cytokines il-6, il-23 and mcp-1. using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd4 t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th2 and th1/th17 skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd4 t cell responses. microcapsule based vaccination resulted in a marked induction of il-17 secreting th17 cells, without inducing strong th1 (cfa) or th2 (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg1, igg2c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd4 + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd80, cd86 and cd40 and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il-6 and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr4-dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr4-defective c3h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il-1b secretion by dc, through its effects on caspase-1 processing, which is also tlr4-independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr4 signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc2461 (st11) or lactobacillus rhamnosus ncc4007 (lpr), each at 5x10 8 cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week 1 to week 7 of life. fresh feces were collected at 3, 5 and 7 weeks of life for determination of iga levels (assessed by elisa). pups were bled 2 and 4 weeks after immunization for determination of measles-specific igg1 and igg2a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st11-fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from 200 kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at 3:1 ration and washed 3 times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw264.7 by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc6/propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that 15 up to 24 euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd3+ cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on 35 th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il-5 and ige serum levels were increased on animals from group i when compared to control.the enhancement on th2 immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd8+ ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd4+t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg2a and igg2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren 1,2 , n. almqvist 2 , a. lönnqvist 1 , s. östman 1 , c. rask 1 , e. telemo 2 , a.e. wold 1 1 university of göteborg, department of clinical bacteriology, göteborg, sweden, 2 university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. examination of gut sections from sea treated donor mice revealed increased density of cd8a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp21, proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna 1826, a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp21, or groel together with cpg-dna 1826 reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp21 together with cpg-dna 1826. the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp21 microspheres induced protective ifng-secreting cd4 + t-cells and raised pulmonary pmp21-specific iga levels in vivo. also, pmp21 microspheres caused lower il-6 serum levels upon administration than the injection of pmp21 together with cpg-dna 1826, indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in 44 cases of s. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised 44 iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen 2006).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter 2007) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st59 strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st59 strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in 15 and 7 ds children, respectively. samples were taken before and 3-4 weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg 1 -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at 4 years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg 1 , igg 2 and igg 4 ). at 9 years, ds children had lower postvaccination geometric mean igg 4 anti-tt-titers only. post-vaccination igg 1 -anti-tt avidity levels were decreased in 8/15 and 4/7 ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. 2 kda; 30, 6 kda; 23, 9 kda; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kda and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of 100 families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an 8-month period. sera were obtained from healthy subjects from the same community (2 months to 88 years of age). the highest incidence of salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. the lowest incidence, 10/1000, was observed in the population aged 10 to 59. whereas serum from individuals ranging 15-70 years of age showed maximum igg1, igg3 and iga anti-s. typhimurium titres, children less than 5 years-old did not show detectable igg1 and igg3 titres and had weak igg2, iga and igm antibody levels; only their igg4 levels were comparable to those detected in adults. moreover, the levels of igg2 and igg3 antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková 1 , s. bystrický 1 1 institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o135 using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd19, expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age 18-55 years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg1, igg2 and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present 5 years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after 5 to 7 days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg2 response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers 1:3200). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones (1:800 vs. 1:3200). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv 0032-06 and vega 2/7029/27 grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked d-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs (250g-350g) were treated orally with 50 mg respivax five consecutive days. after the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. the populations of cd8, cd4 and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd8 positive cells, which number reached maximum at the end of the second week. on day 14 b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd4 and cd8 positive cells. on days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta 1 , s. majumdar 1 1 bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein-10 (ip-10) and interleukin-8 (il-8) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (inos2) expression and was linked to the mapk signaling pathway via antagonistic regulation of p38mapk and erk1/2. further, ip-10 was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th1 cytokines and no. thus this study strongly demonstrates that ip-10, like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th1/th2 cytokines mediated by cd8+ t cells and activating cd4+ t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x 0.05) following immunization and after challenge with leishmania major. il-4 values were increased in all groups, but there was no statistical difference between the groups(p g 0.05) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x 0.05) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x 0.05) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x 0.05). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at 3 week intervals. three weeks after booster injection, 5×10 5 stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg2a/igg1was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type 1 immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin 10 (il-10), and cellular expression of cd4 and cd25. results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd4+ lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with 1,600 infective n. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg1 and igg2a). this was independent of level of me supply. feeding regime did not affect levels of the type-2 cytokines il-4 and il-13. conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp63 is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp63 of l.major strain mrho/ir/75/er. methods: l.major promastigotes were grown in rpmi1640 supplemented with 10 % fcs. l.major rna extraction and cdna synthesis were carried out. gp63 gene segment was amplified by specific primers and cloned into ptz57r to construct ptz57r/gp63. the presence of gp63 into ptz57r was confirmed by pcr. then, ptz57r/gp63 was sent to determine the sequence of its nucleotides. after that the gp63 gene segment was sub-cloned into pet32 a (+) expression vector and transformed into e.coli bl21 (de3) plyss and gp63 protein was expressed in presence of 1mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, 2007) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. subjects received virosomal formulations containing 50 mg of ama 49-c1 (pev301t), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (pe)-peptide conjugate and 10 ug of uk39 (pev302t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days 0, and 90. results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma9vdelta2 t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il-10 responses, to epitopes on the dominant rbc autoantigen, anion exchanger-1 (ae-1, or band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla-4 + regulatory t cells or soluble forms of ctla-4, may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =-0.81) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd8 + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b 1 (r medium =-0.74) and il-10 (r medium =-0.65) in isolated splenic hscs ex vivo. analysis of effector cd4 + t cells in spleen showed decrease of t h 1 cells quantity and simultaneous t h 2 cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd4 + cd25 + ctla-4 + -and cd4 + cd25 -il-10 + il-4regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h 1, t h 2 and regulatory t cells were correlated (r th1 =-0.74, r th2 =0.86 and r th3 =0.80 and r tr1 =0.68) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th3 =0.84 and r tr1 =0.76) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b1-and il-10-produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p38, jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il-17, as compared to non-infected controls. the infection also altered the effect of il-17 on mapk activation by preventing its stimulating effect on p38 mapk. moreover, in s.obvelata-infected animals il-17 markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il-17 induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r 2 g 0.98. the analytical range was from 1 g/l to 70 g/l. the coefficient of variation (cv) was x 10 % for [mc] n 1 g/dl, and x 20 % for [mc] x 1 g/dl. this procedure was successfully implemented to quantify the mc in 1100 serum samples between march 2008 and february, 2009. among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: 1. we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. 2. the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. moreover, cd34 + cd38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b 1 , il-10, pge 2 and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd40-ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd40l is cleaved to soluble (s)cd40l. we sought to examine the levels of scd40l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded 5 atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd40l -along with other products -were assayed by quantitative elisa. levels of il8, cd62p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd40l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l . to examine if scd40l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il-6 secretion. the il-6 concentration was consistently below 5-10 pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il-6. the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il-6 production over control (p x 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l (p x 0.05). pre-incubation of b cells with cd40-blocking antibodies substantially abrogated il-6 secretion, unlike isotype-matched control. the partial blocking of cd40 binding on cd40 + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd40l (under investigation) and indicates a sustained role for pc-derived scd40l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd40l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule-1 (dnam-1) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from 30 aml patients before specific anti-leukemia therapy and 47 healthy donors. all results were analyzed statistically using spss version 15. results: aml patients under 65 years showed a significant reduction in the expression of dnam-1, nkp30 and nkp46 compared with age-matched controls. both healthy individuals and aml patients older than 65 years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp44 expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam-1 on nk cells and its ligand cd112 on aml blasts has been found in aml patients under 65 years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam-1 expression on aml patients and confirm previous reports showing a significant decrease of nkp30 and nkp46 on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel 1 , e. gorska 1 , u. demkow 1 , m. wasik 1 1 medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved 10 children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 m. analysis of: p-gp surface expression, p-gp function (rhodamine 123 test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was 14,52 %±11,32. after 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine 123 efflux, which characterized p-gp activity, was 2,62 %±3,15. after 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). average phi in lymphoblasts was 7,78±0,19. acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . rest of lymphoblasts showed alkalization (phi -8,00).the percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-v test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd49d/cd29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd29 on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd38 and anti-cd29 (clon huts21) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures (18 hours). results: cd29 active form was expressed in the majority of normal and tumoral bm pc from healthy subjects (67.3±6.6, n=9) and mm patients in the early stages of the disease (32.6±7.5, n=17). in these cells, huts21 epitope was clearly upregulated by mn 2+ . in contrast, circulating pc were almost all huts21 negative, and levels did not significantly augment when these cells were exposed to mn 2+ . moreover, not only pb but also bm malignant cells from pcl patients were also huts21 negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index (2.35±0.9 brdu+ cells, n=3) in comparison to pc from mm patients in the first stages of disease (1.3±0.2 brdu+ cells, n=5). these results suggest that the active form of cd29 must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either 10 % fetal bovine serum (fbs) with and without fgf2,or 10 % human platelet lysate (hpl), 5 %hpl, (10 % fbs + 10 % hpl), (10 % fbs + 5 % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day 18). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p53 dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: 1) ten cases with fa diagnosed on the basis of dna breakage analysis, 2) ten cases with aaa, and 3) ten normal control cases. the presence of p53 dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p53 dna was demonstrated in bone marrow of 90 % of children with fa, compared to 10 % in children with aaa (p x 0.001), while, no p53 dna was seen in normal control. a positive correlation between dna breakage and presence of p53 dna was seen in bone marrow from fa (p x 0.02, r0.81). the presence of p53 tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients.